Extraction Technologies For Medicinal and Aromatic Plants

Extraction Technologies for
Medicinal and Aromatic Plants

Coverpage insets include pictures of:
Catharanthus roseus (L.) G. Don
Taxus baccata L.
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for Science and High Technology, 2008
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Extraction Technologies
for Medicinal and Aromatic Plants
Scientifc Editors:
Sukhdev Swami Handa
Suman Preet Singh Khanuja
Gennaro Longo
Dev Dutt Rakesh
INTERNATIONAL CENTRE FOR SCIENCE AND HIGH TECHNOLOGY
Trieste, 2008
Contributors
Chapter 1
An Overview of Extraction Techniques for Medicinal and Aromatic Plants
Sukhdev Swami Handa
Senior Specialist, Industrial Utilization of Medicinal and Aromatic Plants
Earth, Environmental and Marine Sciences and Technologies, ICS-UNIDO,
AREA Science Park, Bldg. L2, Padriciano 99, 34012 Trieste, Italy
Chapter 2
Role of Process Simulation to Extraction Technologies for Medicinal and
Aromatic Plants
Maurizio Fermeglia
DICAMP-CASLAB, University of Trieste and Scientifc Consultant for Process
Simulation, ICS-UNIDO, AREA Science Park, Bldg. L2, Padriciano 99, 34012
Trieste, Italy
Chapter 3
Maceration, Percolation and Infusion Techniques for the Extraction of Me-
dicinal and Aromatic Plants
Janardan Singh
Scientist E II, Botany and Pharmacognosy, Central Institute of Medicinal and
Aromatic Plants, P. O. CIMAP, Lucknow, India
Chapter 4
Hydrolytic Maceration, Expression and Cold Fat Extraction
Anil Kumar Singh
Scientist F, Essential Oil Analysis Laboratory, Central Institute of Medicinal
and Aromatic Plants, P. O. CIMAP, Lucknow, India
Chapter 5
Decoction and Hot Continuous Extraction Techniques
Sudeep Tandon and Shailendra Rane
Scientist EI, Chemical Engineer, Process and Product Development Division,
Central Institute of Medicinal and Aromatic Plants, P. O. CIMAP, Lucknow, India
CONTRIBUTORS
4
Chapter 6
Aqueous Alcoholic Extraction of Medicinal and Aromatic Plants by Fermentation
Chander Kant Katiyar
Director, Herbal Drug Research, Ranbaxy Research Labs, R&D-II, Plot 20,
Sector 18, Udyog Vihar Industrial Area, Gurgaon, India
Chapter 7
Distillation Technology for Essential Oils
Sudeep Tandon
Scientist EI, Chemical Engineer, Process and Product Development Division,
Central Institute of Medicinal and Aromatic Plants, P. O. CIMAP, Lucknow, India
Chapter 8
Microdistillation, Thermomicrodistillation and Molecular Distillation Techniques
Vishwas Govind Pangarkar
Professor, University Institute of Chemical Technology, Nathalal Parekh Marg
Manunga (East) Mumbai 400 019, India
Chapter 9
Solid Phase Micro-extraction and Headspace Trapping Extraction
Rama Kant Harlalka
Director, Nishant Aromas 424, Milan Industrial Estate, Cotton Green Park,
Mumbai 200 033, India
Chapter 10
Supercritical Fluid Extraction of Medicinal and Aromatic Plants: Fundamen-
tals and Applications
Alberto Bertucco
1
and Giada Franceschin
2
1
Professor, Dipartimento di Principi ed Impianti di Ingegneria Chimica I. Sor-
gato, University of Padova, Via Marzolo 9, 35131 Padova, Italy
2
DIPIC - Department of Chemical Engineering, University of Padova, via Mar-
zolo 9, 35131 Padova, Italy
Chapter 11
Process-scale HPLC for Medicinal and Aromatic Plants
Madan Mohan Gupta
1
and Karuna Shanker
2

1
Head, Analytical Chemistry Division, Central Institute of Medicinal and Aro-
matic Plants, P. O. CIMAP, Lucknow, India
2
Scientist, Analytical Chemistry Division, Central Institute of Medicinal and
Aromatic Plants, P. O. CIMAP, Lucknow, India
Chapter 12
Flash Chromatography and Low Pressure Chromatographic Techniques for
Separation of Phytomolecules
Sunil Kumar Chattopadhyay
Scientist F, Process and Product Development Division, Central Institute of
Medicinal and Aromatic Plants, P. O. CIMAP, Lucknow, India
5
EXTRACTION TECHNOLOGIES FOR MEDICINAL AND AROMATIC PLANTS
Chapter 13
Counter-current Chromatography
Santosh Kumar Srivastava
Scientist E II, Phytochemistry, Central Institute of Medicinal and Aromatic
Plants, P. O. CIMAP, Lucknow, India
Chapter 14
Quality Control of Medicinal and Aromatic Plants and their Extracted Prod-
ucts by HPLC and High Performance Thin Layer Chromatography
Karan Vasisht
Professor of Pharmacognosy, University Institute of Pharmaceutical Scienc-
es, Panjab University, Chandigarh 160 014, India
7
Preface
Medicinal plants are the richest bioresource of drugs for tra-
ditional systems of medicine, modern medicines, nutraceuticals, food sup-
plements, folk medicines, pharmaceutical intermediates and chemical enti-
ties for synthetic drugs. Aromatic plants are a source of fragrances, favors,
cosmeceuticals, health beverages and chemical terpenes. Medicinal and
aromatic plants (MAPs) are traded as such in bulk from many developing
countries for further value addition in developed countries. The frst step
in the value addition of MAP bioresources is the production of herbal drug
preparations (i.e. extracts), using a variety of methods from simple tradi-
tional technologies to advanced extraction techniques.
Extraction (as the term is pharmaceutically used) is the separa-
tion of medicinally active portions of plant (and animal) tissues using selec-
tive solvents through standard procedures. Such extraction techniques sep-
arate the soluble plant metabolites and leave behind the insoluble cellular
marc. The products so obtained from plants are relatively complex mixtures
of metabolites, in liquid or semisolid state or (after removing the solvent) in
dry powder form, and are intended for oral or external use. These include
classes of preparations known as decoctions, infusions, fuid extracts, tinc-
tures, pilular (semisolid) extracts or powdered extracts. Such preparations
have been popularly called galenicals, named after Galen, the second cen-
tury Greek physician. The purpose of standardized extraction procedures for
crude drugs (medicinal plant parts) is to attain the therapeutically desired
portions and to eliminate unwanted material by treatment with a selective
solvent known as menstruum. The extract thus obtained, after standardiza-
tion, may be used as medicinal agent as such in the form of tinctures or fuid
extracts or further processed to be incorporated in any dosage form such as
tablets and capsules. These products all contain complex mixture of many
medicinal plant metabolites, such as alkaloids, glycosides, terpenoids, fa-
vonoids and lignans. In order to be used as a modern drug, an extract may
be further processed through various techniques of fractionation to isolate
individual chemical entities such as vincristine, vinblastine, hyoscyamine,
hyoscine, pilocarpine, forskolin and codeine.
The industrial processing of MAPs starts with the extraction of
the active components using various technologies. The general techniques
of medicinal plant extraction include maceration, infusion, percolation, di-
gestion, decoction, hot continuous extraction (Soxhlet), aqueous-alcoholic
extraction by fermentation, counter-current extraction, microwave-assisted
extraction, ultrasound extraction (sonication), supercritical fuid extraction,
and phytonic extraction (with hydrofuorocarbon solvents). For aromatic
plants, hydrodistillation techniques (water distillation, steam distillation,
water and steam distillation), hydrolytic maceration followed by distillation,
expression and enfeurage (cold fat extraction) may be employed. Some of
PREFACE
8
the latest extraction methods for aromatic plants include headspace trap-
ping, solid phase micro-extraction, protoplast extraction, microdistillation,
thermomicrodistillation and molecular distillation.
With the increasing demand for herbal medicinal products, nu-
traceuticals, and natural products for health care all over the world, medicinal
plant extract manufacturers and essential oil producers have started using
the most appropriate extraction technologies in order to produce extracts
and essential oils of defned quality with the least variations from batch to
batch. Such approach has to be adopted by MAP-rich developing countries
in order to meet the increasing requirement of good quality extracts and es-
sential oils for better revenue generation within the country, as well as for
capturing this market in developed countries.
The basic parameters infuencing the quality of an extract are
the plant parts used as starting material, the solvent used for extraction, the
manufacturing process (extraction technology) used with the type of equip-
ment employed, and the crude-drug:extract ratio. The use of appropriate
extraction technology, plant material, manufacturing equipment, extraction
method and solvent and the adherence to good manufacturing practices
certainly help to produce a good quality extract. From laboratory scale to pi-
lot scale, all the conditions and parameters can be modelled using process
simulation for successful industrial-scale production.
With the advances in extraction technologies and better knowl-
edge for maintaining quality parameters, it has become absolutely necessary
to disseminate such information to emerging and developing countries with
a rich MAP biodiversity for the best industrial utilization of MAP resources.
The experts at the South-East Asian (SEA) Regional Workshop
entitled Extraction Technologies for Medicinal and Aromatic Plants, held in
2006 in Lucknow, India, agreed to prepare a publication on extraction princi-
ples, technologies and analytical techniques for quality control of raw materials
and processed products in the form of extracts and essential oils for medicinal
and aromatic plants. This book focuses on different techniques of hydrodistil-
lation, steam distillation, cohobation and fractional distillation of volatile oils
from aromatic plants and on water-solvent extraction and supercritical fuid
extraction for medicinal plant extracts. It also discusses general, specifc and
advanced technologies for preparing extracts of medicinal plants and the ex-
traction of volatile oils and fragrances from aromatic plants. This book is in-
tended to equip emerging and developing countries with techniques of extrac-
tion that can help them to produce economical and globally competitive quality
extracts.
Gennaro Longo
Chief of Environment Area
Special Adviser on Technology Development
9
Contents
1 An Overview of Extraction Techniques for Medicinal and Aromatic Plants, S. S. Handa . . . . . .
Abstract . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
1.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
1.2 Medicinal Plant Extracts . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
1.2.1 General Methods of Extraction of Medicinal Plants . . . . . . . . . . . . . . . . . . . . . . . . .
1.2.1.1 Maceration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
1.2.1.2 Infusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
1.2.1.3 Digestion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
1.2.1.4 Decoction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
1.2.1.5 Percolation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
1.2.1.6 Hot Continuous Extraction (Soxhlet) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
1.2.1.7 Aqueous Alcoholic Extraction by Fermentation . . . . . . . . . . . . . . . . . . .
1.2.1.8 Counter-current Extraction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
1.2.1.9 Ultrasound Extraction (Sonication) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
1.2.1.10 Supercritical Fluid Extraction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
1.2.1.11 Phytonics Process . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
1.2.1.11.1 Advantages of the Process . . . . . . . . . . . . . . . . . . . . . . . . . . .
1.2.1.11.2 Applications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
1.2.1.12 Parameters for Selecting an Appropriate Extraction
Method . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
1.2.2 Steps Involved in the Extraction of Medicinal Plants . . . . . . . . . . . . . . . . . . . . . . . .
1.2.2.1 Size Reduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
1.2.2.2 Extraction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
1.2.2.2.1 Cold Aqueous Percolation . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
1.2.2.2.2 Hot Aqueous Extraction (Decoction) . . . . . . . . . . . . . . . . . .
1.2.2.2.3 Filtration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
1.2.2.2.4 Spray Drying . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
1.2.2.3 Solvent Extraction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
1.2.2.3.1 Cold Percolation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
1.2.2.3.2 Hot Percolation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
1.2.2.3.3 Concentration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
1.3 Aromatic Plant Extracts . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
1.3.1 Concrete . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
1.3.2 Absolutes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
1.3.3 Resinoids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
1.3.4 Pomades . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
1.3.5 Essential Oils . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
1.3.5.1 Sources of Natural Essential Oils . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
1.3.5.2 Essential Oil Constituents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
1.3.5.3 Methods of Producing Essential Oils . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
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CONTENTS
10
1.3.5.3.1 Hydrodistillation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
1.3.5.3.1.1 Mechanism of Distillation . . . . . . . . . . . . .
1.3.5.3.1.1.1 Hydrodiffusion . . . . . .
1.3.5.3.1.1.2 Hydrolysis . . . . . . . . . . .
1.3.5.3.1.1.3 Effect of Heat . . . . . . .
1.3.5.3.2 Three Types of Hydrodistillation . . . . . . . . . . . . . . . . . . . . . .
1.3.5.3.2.1 Water Distillation . . . . . . . . . . . . . . . . . . . . . .
1.3.5.3.2.1.1 Traditional Method
of Producing
Attar Using
Hydrodistillation . . . .
1.3.5.3.2.1.2 Disadvantages of
Water Distillation . . .
1.3.5.3.2.2 Water and Steam Distillation . . . . . . . . .
1.3.5.3.2.2.1 Cohobation . . . . . . . . .
1.3.5.3.2.2.2 Advantages of
Water and Steam
Distillation over
Water Distillation . .
1.3.5.3.2.2.3 Disadvantages of
Water and Steam
Distillation . . . . . . . . . .
1.3.5.3.2.3 Direct Steam Distillation . . . . . . . . . . . . . .
1.3.5.3.2.3.1 Advantages of
Direct Steam
Distillation . . . . . . . . . .
1.3.5.3.2.3.2 Disadvantage
of Direct Steam
Distillation . . . . . . . . . .
1.3.5.3.3 Essential Oil Extraction by Hydrolytic Maceration
Distillation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
1.3.5.3.4 Essential Oil Extraction by Expression . . . . . . . . . . . . . . . .
1.3.5.3.4.1 Pelatrice Process . . . . . . . . . . . . . . . . . . . . . .
1.3.5.3.4.2 Sfumatrice Process . . . . . . . . . . . . . . . . . . .
1.3.5.3.5 Essential Oil Extraction with Cold Fat (Enfeurage) . . .
1.3.5.3.5.1 Enfeurage and Defeurage . . . . . . . . . . . .
1.3.5.3.5.2 Hot Maceration Process . . . . . . . . . . . . . .
1.3.6 Modern (Non-traditional) Methods of Extraction of Essential Oils . . . . . . . . . .
1.4 Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Bibliography . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
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2 Role of Process Simulation to Extraction Technologies for Medicinal and Aromatic Plants,
M. Fermeglia . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Abstract . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2.2 Process Simulation Goals and Defnitions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2.3 Biotechnological and Phytochemical Processes Studied at ICS-UNIDO . . . . . . . . . . . . . .
2.3.1 Brief of Biotechnological Processes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2.3.1.1 Alcohol Production from Biomass . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2.3.1.2 Soybean Oil Refnery Process and Treatment of the Waste . . . . . .
2.3.1.3 Production of Synthetic Hydrocarbon Fuels Starting from
Biomass . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2.3.1.4 Production of Bio-ethanol from Corn . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2.3.2 Brief of Phytochemical Processes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2.3.2.1 Citral Recovery by Distillation of Lemon Peel Oil . . . . . . . . . . . . . . . . .
2.3.2.2 Menthol Recovery by Crystallization of Mentha Oil . . . . . . . . . . . . . . .
2.3.2.3 Carvone Recovery from Spearmint Oil . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2.3.2.4 Peppermint Oil Extraction by Steam Distillation . . . . . . . . . . . . . . . . . .
2.3.2.5 Multiple-effect Evaporation of Milk Serum . . . . . . . . . . . . . . . . . . . . . . . .
2.3.3 Case Study: Turpentine Oil Batch Distillation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2.3.4 Case Study: Menthol Recovery by Crystallization of Mentha Oil . . . . . . . . . . . .
2.4 Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Bibliography . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3 Maceration, Percolation and Infusion Techniques for the Extraction of Medicinal and Aromatic
Plants, J. Singh . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Abstract . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.2 General Principles and Mechanism Involved for Crude Drug Extraction
by Maceration, Percolation and Infusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.3 Factors Affecting Choice of Extraction Process . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.3.1 Nature of the Crude Drug . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.3.2 Stability of the Crude Drug . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.3.3 Cost of the Crude Drug . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.3.4 Solvent . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.3.5 Concentration of the Product . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.3.6 Recovery of Solvent. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.4 Quality Assurance of Extracts: The Extraction Process and Solvent . . . . . . . . . . . . . . . . . .
3.5 Maceration Processes (Steady-state Extraction) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.5.1 General Procedure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.5.2 Maceration Process for Organized and Unorganized Crude Drugs . . . . . . . . . .
3.5.3 Modifcations to the General Processes of Maceration . . . . . . . . . . . . . . . . . . . . .
3.5.4 Large-scale Extraction Procedures . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.5.4.1 Circulatory Extraction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
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3.5.4.2 Multistage Extraction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.5.4.2.1 Advantages . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.5.4.2.2 Procedure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.5.4.2.3 Extraction Battery . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.5.4.2.4 Equipment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.6 Percolation (Exhaustive Extraction) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.6.1 General Process of Percolation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.6.2 Modifcations to the General Process of Percolation . . . . . . . . . . . . . . . . . . . . . . . .
3.6.2.1 Reserved Percolation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.6.2.2 Cover and Run Down Method . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.6.3 Percolators . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.6.3.1 Small-scale or Laboratory-scale Extraction . . . . . . . . . . . . . . . . . . . . . . .
3.6.3.1.1 Soxhlet Apparatus . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.6.3.1.2 Offcial Extractor . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.6.3.2 Limitations of the Offcial Extractor . . . . . . . . . . . . . . . . . . .
3.6.3.3 Large-scale Extractor . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.7 Infusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.7.1 General Considerations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.7.2 General Method for Preparing Fresh Infusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.7.3 Preparation of Concentrated Infusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.8 Evaporation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.9 Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Bibliography . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
4 Hydrolytic Maceration, Expression and Cold Fat Extraction, A. K. Singh . . . . . . . . . . . . . . . . . . . . . . . . .
Abstract. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
4.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
4.2 Hydrolytic Maceration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
4.3 Expression Extraction of Essential Oils . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
4.3.1 Process of Expression . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
4.3.1.1 Hand Process . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
4.3.1.2 Ecuelle Process . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
4.3.1.3 Hand Machine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
4.3.1.4 Sfumatrici and Pelatrici . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
4.3.1.4.1 Special Sfumatrice . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
4.3.1.5 Modern Machines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
4.3.1.6 FMC Whole Fruit Extractor . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
4.4 Cold Fat Extraction (Enfeurage) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
4.5 Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Bibliography . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
5 Decoction and Hot Continuous Extraction Techniques, S. Tandon and S. Rane . . . . . . . . . . . . . . . .
Abstract . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
5.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
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5.2 Solid Liquid Extraction Process . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
5.3 Process Parameters Affecting Solid-liquid Extraction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
5.3.1 Post-harvest Processing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
5.3.2 Matrix Characteristics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
5.3.3 Choice of Solvent . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
5.3.4 Condition of Extraction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
5.4 Method of Solid-liquid Extraction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
5.5 Solid-liquid Extraction Equipment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
5.5.1 Continuous Extraction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
5.5.1.1 Percolation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
5.5.1.2 Immersion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
5.5.2 Continuous Extraction Equipment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
5.5.2.1 Continuous Horizontal Extractor . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
5.5.2.2 Hildebrandt Extractor . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
5.5.2.3 Bonotto Extractor . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
5.5.2.4 Bollmann Extractor . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
5.5.2.5 Kennedy Extractor . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
5.6 Conventional Solvent Extraction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
5.6.1 Principles and Mechanisms . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
5.6.2 Advantages and Disadvantages of Soxhlet Extraction . . . . . . . . . . . . . . . . . . . . . . .
5.7 Accelerated Solvent Extraction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
5.7.1 Principles and Mechanisms . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
5.7.2 Advantages and Disadvantages of Accelerated Solvent Extraction . . . . . . . .
5.8 Important Factors for Designing a Solvent Extraction Plant for Medicinal Plants . . .
5.9 Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Bibliography . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
6 Aqueous Alcoholic Extraction of Medicinal and Aromatic Plants by Fermentation, C. K. Katiyar .
Abstract . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
6.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
6.2 Ayurvedic Dosage Forms . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
6.2.1 Swarasa (Fresh Juice) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
6.2.2 Kalka (Wet Bolus) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
6.2.3 Kwatha (Decoction) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
6.2.4 Hima (Cold Infusion) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
6.2.5 Phanta (Hot Infusion) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
6.2.6 Solids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
6.2.7 Semisolids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
6.2.8 Liquids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
6.2.9 Fumes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
6.3 Shelf Life of Dosage Forms . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
6.4 Asava and Arishta: Self-fermented Products . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
6.4.1 Self-fermentation Process for Preparing Asava Arishta . . . . . . . . . . . . . . . . . . . . . .
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6.4.2 Merits of the Process . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
6.5 Application of Asava Arishta Technology in New Drug Discovery . . . . . . . . . . . . . . . . . . . . .
6.6 Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Bibliography . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
7 Distillation Technology for Essential Oils, S. Tandon . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Abstract . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
7.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
7.2 Principles of Distillation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
7.3 Methods for Distillation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
7.3.1 Hydrodistillation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
7.3.2 Water and Steam Distillation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
7.3.2.1 Improved Field Distillation Units . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
7.3.3 Direct Steam Distillation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
7.3.3.1 Comparison of Boiler-operated Unit with Directly Fired Type . . . . .
7.3.4 Distillation with Cohobation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
7.4 Hydrodiffusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
7.5 Parameters Affecting Yield and Quality of Essential Oils . . . . . . . . . . . . . . . . . . . . . . . . . . . .
7.5.1 Mode of Distillation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
7.5.2 Proper Design of Equipment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
7.5.3 Material of Fabrication of Equipment. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
7.5.4 Condition of Raw Material . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
7.5.5 Time for Distillation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
7.5.6 Loading of Raw Material and Steam Distribution . . . . . . . . . . . . . . . . . . . . . . . . . . . .
7.5.7 Operating Parameters . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
7.5.8 Condition of Tank and Equipment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
7.6 Purifcation of Crude Essential Oils . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
7.6.1 Continuous Steam Distillation. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
7.7 Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Bibliography . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
8 Microdistillation, Thermomicrodistillation and Molecular Distillation Techniques, V. G. Pangarkar . .
Abstract . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
8.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
8.2 Process Intensifcation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
8.2.1 Multifunctional Equipment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
8.2.2 Process-intensifying Equipment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
8.3 Solvent Extractions of MAPs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
8.3.1 Thermodynamics of Solvent Extraction and Choice of Solvent . . . . . . . . . . . . . .
8.3.2 Solid-liquid Mass Transfer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
8.3.3 Microwave-assisted Extraction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
8.3.3.1 Principle of Microwave Heating . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
8.3.3.2 Mechanism of MAE . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
8.3.3.3 Literature on MAE . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
111
112
112
113
115
115
115
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116
116
118
119
120
121
121
123
123
123
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129
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15
8.3.3.4 Industrial-scale MAE . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
8.4 Microwave-assisted Hydrodistillation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
8.5 Molecular Distillation or Short Path Distillation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
8.5.1 Principle of MD. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
8.5.2 Advantages of MD . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
8.5.3 Separation Effciency of MD . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
8.5.4 Parameters that Affect the MD Process . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
8.5.5 Typical Applications of MD . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
8.6 Recovery of Dissolved Essential Oils from Steam Distillation Condensates . . . . . . . . .
8.6.1 Polymeric Adsorption Process . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
8.6.2 Pervaporation Process . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
8.7 Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Bibliography . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
9 Solid Phase Micro-extraction and Headspace Trapping Extraction, R. Harlalka . . . . . . . . . . . . . . . .
Abstract . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
9.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
9.2 The SPME Device . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
9.3 Calibration, Optimization, Precision and Suitability of SPME . . . . . . . . . . . . . . . . . . . . . . . . .
9.3.1 Selection of Fiber Coating . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
9.3.2 Selection of the Extraction Mode . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
9.3.3 Selection of the Agitation Technique . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
9.3.4 Selection of Separation or Detection Technique . . . . . . . . . . . . . . . . . . . . . . . . . . . .
9.3.5 Optimization of Desorption Conditions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
9.3.6 Optimization of Sample Volume . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
9.3.7 Determination of the Extraction Time . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
9.3.8 Optimization of Extraction Conditions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
9.3.9 Determination of the Linear Dynamic Range of the Method . . . . . . . . . . . . . . . .
9.3.10 Selection of the Calibration Method . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
9.3.11 Precision of the Method . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
9.3.12 Suitability . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
9.4 Headspace Trapping Extraction and GC-FID/MS Analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
9.4.1 History of Headspace . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
9.4.2 The Aura . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
9.4.3 What is Diffusivity? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
9.4.4 Application of Headspace Trapping . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
9.4.4.1 Jasmine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
9.4.4.2 Yellow Tea Rose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
9.4.4.3 Passion Flower . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
9.4.4.4 Lotus . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
9.4.4.5 Lavender . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
9.4.4.6 Chamomile . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
9.4.4.7 Shefali . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
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149
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CONTENTS
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9.4.4.8 Spearmint . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
9.4.4.9 Cinnamon Bark . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
9.4.4.10 Ginger . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
9.4.4.11 Peach . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
9.4.4.12 Pineapple . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
9.4.5 Classical Perfumes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
9.4.6 Need for Headspace . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
9.5 Types of Headspace Trapping . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
9.5.1 Static Headspace Trapping . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
9.5.2 Dynamic Headspace Trapping . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
9.6 Principles of Static Headspace-GC Systems . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
9.6.1 Trace Analysis by HS-GC . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
9.7 Headspace Trapping Techniques . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
9.7.1 Static Headspace Trapping . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
9.7.2 Dynamic Headspace Trapping . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
9.7.3 Recovering the Adsorbed Volatiles by Thermal or Liquid Solvent
Desorption . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
9.7.4 Some Practical Examples Using Headspace Technique Use . . . . . . . . . . . . . . . .
9.7.4.1 Tomato Juice . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
9.7.4.1.1 Preparation of Traps . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
9.7.4.1.2 Thermal Desorption . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
9.7.4.2 Headspace of Hedychium coronium . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
9.7.4.3 Volatiles of White Hyacinths Isolated by Dynamic Headspace
Trapping . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
9.7.4.4 Medical Materials Testing by Headspace Trap-GC/MS . . . . . . . . . .
9.8 Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Bibliography . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
10 Supercritical Fluid Extraction of Medicinal and Aromatic Plants: Fundamentals and Applications,
A. Bertucco and G. Franceschin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Abstract . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
10.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
10.2 Supercritical Fluids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
10.3 SFE Processes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
10.4 The SFE Process and Equipment Development . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
10.5 SFE Applied to Medicinal and Aromatic Plants . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
10.6 Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Bibliography . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
11 Process-scale High Performance Liquid Chromatography for Medicinal and Aromatic Plants,
M. M. Gupta and K. Shanker . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Abstract . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
11.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
11.2 Theoretical Aspects of HPLC . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
157
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160
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161
162
163
163
163
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164
165
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166
166
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17
11.2.1 Chromatography Classifcation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
11.2.2 Important Factors that Infuence HPLC Separation . . . . . . . . . . . . . . . . . . . . . . . . . .
11.2.3 Main Components of HPLC . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
11.2.4 HPLC Classifcation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
11.2.5 Advantages of HPLC . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
11.3 Preparative HPLC . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
11.3.1 Strategy for Preparative Separation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
11.4 Practical Consideration in Preparative HPLC Scale-up . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
11.4.1 Sample Loading . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
11.4.2 Separation Time . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
11.4.3 Solvent Composition . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
11.4.4 Washing Steps . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
11.4.5 Recycling . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
11.5 Stepwise Operations in Process-scale HPLC . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
11.6 Problems Encounter in Preparative Scale-up . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
11.6.1 Purity of Crude Extract . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
11.6.2 Removal of Chromatographic Solvent . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
11.6.3 Temperature Variation from Laboratory to Pilot Scale . . . . . . . . . . . . . . . . . . . . . . .
11.6.4 Increase in Pump Pressure Due to Accumulation of Impurities
on the Column . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
11.7 Summary: Scale-up Strategy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
11.8 Applications: Natural Products Isolation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
11.9 Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Bibliography . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
12 Flash Chromatography and Low Pressure Chromatographic Techniques for Separation of
Phytomolecules, S. K. Chattopadhyay . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Abstract . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
12.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
12.2 Flash Chromatography . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
12.2.1 Theory of Flash Chromatography . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
12.2.2 Converting TLC to Flash Chromatography . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
12.3 Isocratic versus Gradient Chromatography . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
12.4 Adsorbent Selection and Mode of Separation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
12.4.1 Isolute Flash Columns . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
12.4.2 Method Development Using Isolute Flash Columns . . . . . . . . . . . . . . . . . . . . . . . . .
12.4.2.1 Column Equilibration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
12.4.2.2 Typical Equilibration Solvents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
12.5 Sample Application . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
12.5.1 Wet Loading . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
12.5.1.1 Practical Tips for Wet Loading . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
12.5.2 Dry Loading . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
12.5.2.1 Practical Tips for Dry Loading . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
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12.6 Elution . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
12.6.1 Step Gradient Elution . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
12.6.2 Linear Gradient Elution . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
12.6.3 Method Development using Gradient Elution . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
12.6.4 Practical Tips for Gradient Elution . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
12.6.5 Optimizing Flow Rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
12.7 Fraction Collection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
12.7.1 Off-line Flash Chromatography . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
12.7.2 On-line Flash Chromatography . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
12.8 Low Pressure Liquid Chromatography . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
12.8.1 Gel Filtration Chromatography . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
12.8.2 Ion Exchange Chromatography . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
12.8.3 Affnity Chromatography . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
12.9 Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Bibliography . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
13 Counter-current Chromatography, S. K. Srivastava . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Abstract . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
13.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
13.2 Principles and Development of Counter-current Chromatography . . . . . . . . . . . . . . . . . . . .
13.2.1 Liquid-liquid Extraction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
13.2.2 Partition Coeffcient . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
13.2.3 Droplet Counter-current Chromatography . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
13.2.4 Applications of Droplet Counter-current Chromatography . . . . . . . . . . . . . . . . . . .
13.2.5 Limitations of DCCC . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
13.2.6 Modern Counter-current Chromatography . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
13.3 HSCCC Instrument and Mechanism . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
13.4 CPC Instrument and Setup . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
13.4.1 Various Kinds of CPC Instruments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
13.5 How to Achieve Nice Separation of Various Kinds of Natural Products Using
HSCCC and CPC . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
13.5.1 Search for a Suitable Solvent System . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
13.5.1.1 Solvent Systems for the Separation of a Large Variety of
Natural Products . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
13.5.1.2 Retention of the Stationary Phase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
13.5.1.3 Preparation of Sample Solution . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
13.5.1.4 Separation Column . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
13.5.1.5 Choice of the Mobile Phase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
13.5.1.6 Flow rate of the Mobile Phase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
13.5.1.7 Revolution Speed . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
13.5.1.8 Filling the Column with the Stationary Phase . . . . . . . . . . . . . . . . . . . . .
13.5.1.9 Sample Loading . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
13.5.1.10 On-line Monitoring of Effuent . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
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13.5.1.11 Measurement of Stationary Phase Retention . . . . . . . . . . . . . . . . . . . .
13.5.2 Applications of HSCCC-CPC Technologies in Natural Products Isolation . . . .
13.5.2.1 Purifcation of Coenzyme Q
10
from Fermentation Extract: HSCCC
versus Silica Gel Column Chromatography . . . . . . . . . . . . . . . . . . . . . . .
13.5.2.2 Preparative Separation of Gambogic Acid and its C-2 Epimer
by HPCCC . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
13.5.2.3 Separation and Purifcation of 10-Deacetylbaccatin III
by HSCCC . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
13.5.2.4 Large-scale Separation of Resveratrol and Anthraglycoside
A and B from Polygonum cuspidatum by HSCCC . . . . . . . . . . . . . . . .
13.5.2.5 Separation of Andrographolide and Neoandrographolide from
the Leaves of Andrographis paniculata using HSCCC . . . . . . . . . . . .
13.5.2.6 Separation of WAP-8294A Components, a Novel Anti-methicillin-
resistant Staphylococcus aureus Antibiotic, using HSCCC . . . . . . .
13.5.2.7 Other Examples of Separation of Phytoconstituents by CCC . . . . .
13.6 Advantages of CCC . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
13.6.1 Advantages of HSCCC-CPC Technologies over HPLC . . . . . . . . . . . . . . . . . . . . . . . .
13.7 Manufacturers of CCC Instruments. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
13.7.1 Manufacturers of HSCCC Machines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
13.7.2 Manufacturers of CPC Machines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
13.8 Selected Reviews on CCC. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
13.9 Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Bibliography . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
14 Quality Control of Medicinal and Aromatic Plants and their Extracted Products by HPLC and
High Performance Thin Layer Chromatography, K. Vasisht . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Abstract . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
14.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
14.2 Quality Control of Medicinal Plants and their Products . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
14.3 Biological and Chemical Standardization of Drugs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
14.3.1 Chemical Standardization and Markers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
14.3.2 Analytical Techniques for Quantifying a Marker . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
14.3.3 Validation of Analytical Procedures . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
14.3.3.1 Specifcity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
14.3.3.2 Linearity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
14.3.3.3 Range . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
14.3.3.4 Accuracy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
14.3.3.5 Precision . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
14.3.3.5.1 Repeatability . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
14.3.3.5.2 Intermediate Precision . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
14.3.3.5.3 Reproducibility . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
14.3.3.6 Detection Limit . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
14.3.3.7 Quantitation Limit . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
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14.3.3.8 Robustness . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
14.4 Thin Layer Chromatography in Quality Control of Plant Products . . . . . . . . . . . . . . . . . . . . .
14.4.1 Sample Preparation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
14.4.2 Selection of Chromatographic Layer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
14.4.3 TLC versus HPTLC Layers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
14.4.4 Selection of the Mobile Phase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
14.4.5 Application of Sample . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
14.4.6 Developing the Chromatogram . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
14.4.7 Drying the Plate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
14.4.8 Derivatization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
14.4.9 Evaluation of the Chromatograms . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
14.4.10 Improving the Effciency of TLC . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
14.5 High Performance Liquid Chromatography . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
14.5.1 Pumps . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
14.5.2 Injector . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
14.5.3 Columns. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
14.5.4 Detectors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
14.5.5 Data Processing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
14.5.6 Factors Affecting the HPLC Analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
14.5.7 HPLC in Quality Control of Plant Products . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
14.6 TLC versus HPLC . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
14.7 Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Bibliography . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
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1 An Overview of Extraction Techniques
for Medicinal and Aromatic Plants
S. S. Handa
Abstract
A wide range of technologies is available for the extraction of active components and
essential oils from medicinal and aromatic plants. The choice depends on the eco-
nomic feasibility and suitability of the process to the particular situation. The various
processes of production of medicinal plant extracts and essential oils are reviewed in
this paper.
1.1 Introduction
Asia is the largest continent and has 60% of the worlds popu-
lation. It has abundant medicinal and aromatic plant species, well docu-
mented traditional knowledge, a long-standing practice of traditional medi-
cine, and the potential for social and economic development of medicinal
and aromatic plants (MAPs). Asia is one of the largest biodiversity regions
in the world, containing some of the richest countries in plant resources.
The continent has diverse plant fora but species richness is concentrated
mainly in tropical and subtropical regions. Six of the worlds 18 biodiversity
hot spots, namely eastern Himalaya, North Borneo, Peninsular Malaysia,
Sri Lanka, Philippines and the Western Ghats of South India, lie in Asia.
The countries of the region have large fora: China has 30,000 species of
higher plants; Indonesia, 20,000; India, 17,000; Myanmar, 14,000; Malay-
sia, 12,000; and Thailand, 12,000. The total numbers of plant species and
the endemics in the region are given below:
Region Species Endemics
South East Asia 42-50,000 40,000
China and East Asia 45,000 18,650
Indian Subcontinent 25,000 12,000
South West Asia 23,000 7,100
Sustainable industrial exploitation of such a valuable biore-
source, through use of appropriate technologies, can substantially contrib-
ute to the socio-economic growth of Asian countries. The International Cen-
tre for Science and High Technology (ICS-UNIDO) has thus organized this
regional workshop on extraction technologies for medicinal and aromatic
plants for South East Asian countries.
1 AN OVERVIEW OF EXTRACTION TECHNIQUES FOR MEDICINAL AND AROMATIC PLANTS
22
1.2 Medicinal Plant Extracts
Extraction, as the term is used pharmaceutically, involves the
separation of medicinally active portions of plant or animal tissues from
the inactive or inert components by using selective solvents in standard
extraction procedures. The products so obtained from plants are relatively
impure liquids, semisolids or powders intended only for oral or external use.
These include classes of preparations known as decoctions, infusions, fuid
extracts, tinctures, pilular (semisolid) extracts and powdered extracts. Such
preparations popularly have been called galenicals, named after Galen, the
second century Greek physician.
The purposes of standardized extraction procedures for crude
drugs are to attain the therapeutically desired portion and to eliminate the
inert material by treatment with a selective solvent known as menstruum.
The extract thus obtained may be ready for use as a medicinal agent in the
form of tinctures and fuid extracts, it may be further processed to be incor-
porated in any dosage form such as tablets or capsules, or it may be frac-
tionated to isolate individual chemical entities such as ajmalicine, hyoscine
and vincristine, which are modem drugs. Thus, standardization of extraction
procedures contributes signifcantly to the fnal quality of the herbal drug.
1.2.1 General Methods of Extraction of Medicinal Plants
1.2.1.1 Maceration
In this process, the whole or coarsely powdered crude drug is
placed in a stoppered container with the solvent and allowed to stand at
room temperature for a period of at least 3 days with frequent agitation until
the soluble matter has dissolved. The mixture then is strained, the marc
(the damp solid material) is pressed, and the combined liquids are clarifed
by fltration or decantation after standing.
1.2.1.2 Infusion
Fresh infusions are prepared by macerating the crude drug for
a short period of time with cold or boiling water. These are dilute solutions
of the readily soluble constituents of crude drugs.
1.2.1.3 Digestion
This is a form of maceration in which gentle heat is used during
the process of extraction. It is used when moderately elevated temperature
is not objectionable. The solvent effciency of the menstruum is thereby
increased.
23
1.2.1.4 Decoction
In this process, the crude drug is boiled in a specifed volume
of water for a defned time; it is then cooled and strained or fltered. This
procedure is suitable for extracting water-soluble, heat-stable constituents.
This process is typically used in preparation of Ayurvedic extracts called
quath or kawath. The starting ratio of crude drug to water is fxed, e.g.
1:4 or 1:16; the volume is then brought down to one-fourth its original vol-
ume by boiling during the extraction procedure. Then, the concentrated ex-
tract is fltered and used as such or processed further.
1.2.1.5 Percolation
This is the procedure used most frequently to extract active
ingredients in the preparation of tinctures and fuid extracts. A percolator
(a narrow, cone-shaped vessel open at both ends) is generally used (Figure
1). The solid ingredients are moistened with an appropriate amount of the
specifed menstruum and allowed to stand for approximately 4 h in a well-
closed container, after which the mass is packed and the top of the percola-
tor is closed. Additional menstruum is added to form a shallow layer above
the mass, and the mixture is allowed to macerate in the closed percolator
for 24 h. The outlet of the percolator then is opened and the liquid contained
therein is allowed to drip slowly. Additional menstruum is added as required,
until the percolate measures about three-quarters of the required volume
of the fnished product. The marc is then pressed and the expressed liquid
is added to the percolate. Suffcient menstruum is added to produce the
required volume, and the mixed liquid is clarifed by fltration or by standing
followed by decanting.
Figure 1: Percolator
1.2.1.6 Hot Continuous Extraction (Soxhlet)
In this method, the fnely ground crude drug is placed in a porous
bag or thimble made of strong flter paper, which is placed in chamber E of
the Soxhlet apparatus (Figure 2). The extracting solvent in fask A is heated,
24
and its vapors condense in condenser D. The condensed extractant drips
into the thimble containing the crude drug, and extracts it by contact. When
the level of liquid in chamber E rises to the top of siphon tube C, the liquid
contents of chamber E siphon into fask A. This process is continuous and is
carried out until a drop of solvent from the siphon tube does not leave residue
when evaporated. The advantage of this method, compared to previously de-
scribed methods, is that large amounts of drug can be extracted with a much
smaller quantity of solvent. This effects tremendous economy in terms of
time, energy and consequently fnancial inputs. At small scale, it is employed
as a batch process only, but it becomes much more economical and viable
when converted into a continuous extraction procedure on medium or large
scale.
Figure 2: Soxhlet apparatus
1.2.1.7 Aqueous Alcoholic Extraction by Fermentation
Some medicinal preparations of Ayurveda (like asava and arista)
adopt the technique of fermentation for extracting the active principles. The
extraction procedure involves soaking the crude drug, in the form of either a
powder or a decoction (kasaya), for a specifed period of time, during which
it undergoes fermentation and generates alcohol in situ; this facilitates the
extraction of the active constituents contained in the plant material. The al-
cohol thus generated also serves as a preservative. If the fermentation is to
be carried out in an earthen vessel, it should not be new: water should frst
be boiled in the vessel. In large-scale manufacture, wooden vats, porcelain
jars or metal vessels are used in place of earthen vessels. Some examples
of such preparations are karpurasava, kanakasava, dasmularista. In Ayurveda,
this method is not yet standardized but, with the extraordinarily high degree of
25
advancement in fermentation technology, it should not be diffcult to standard-
ize this technique of extraction for the production of herbal drug extracts.
1.2.1.8 Counter-current Extraction
In counter-current extraction (CCE), wet raw material is pulverized
using toothed disc disintegrators to produce a fne slurry. In this process, the
material to be extracted is moved in one direction (generally in the form of a
fne slurry) within a cylindrical extractor where it comes in contact with extrac-
tion solvent. The further the starting material moves, the more concentrated the
extract becomes. Complete extraction is thus possible when the quantities of
solvent and material and their fow rates are optimized. The process is highly
effcient, requiring little time and posing no risk from high temperature. Finally,
suffciently concentrated extract comes out at one end of the extractor while the
marc (practically free of visible solvent) falls out from the other end.
This extraction process has signifcant advantages:
A unit quantity of the plant material can be extracted with i)
much smaller volume of solvent as compared to other meth-
ods like maceration, decoction, percolation.
CCE is commonly done at room temperature, which spares ii)
the thermolabile constituents from exposure to heat which
is employed in most other techniques.
As the pulverization of the drug is done under wet condi- iii)
tions, the heat generated during comminution is neutralized
by water. This again spares the thermolabile constituents
from exposure to heat.
The extraction procedure has been rated to be more eff- iv)
cient and effective than continuous hot extraction.
1.2.1.9 Ultrasound Extraction (Sonication)
The procedure involves the use of ultrasound with frequencies
ranging from 20 kHz to 2000 kHz; this increases the permeability of cell
walls and produces cavitation. Although the process is useful in some cas-
es, like extraction of rauwolfa root, its large-scale application is limited due
to the higher costs. One disadvantage of the procedure is the occasional but
known deleterious effect of ultrasound energy (more than 20 kHz) on the ac-
tive constituents of medicinal plants through formation of free radicals and
consequently undesirable changes in the drug molecules.
1.2.1.10 Supercritical Fluid Extraction
Supercritical fuid extraction (SFE) is an alternative sample prep-
aration method with general goals of reduced use of organic solvents and
increased sample throughput. The factors to consider include temperature,
pressure, sample volume, analyte collection, modifer (cosolvent) addition,
26
fow and pressure control, and restrictors. Generally, cylindrical extraction
vessels are used for SFE and their performance is good beyond any doubt.
The collection of the extracted analyte following SFE is another important
step: signifcant analyte loss can occur during this step, leading the analyst
to believe that the actual effciency was poor.
There are many advantages to the use of CO
2
as the extract-
ing fuid. In addition to its favorable physical properties, carbon dioxide is
inexpensive, safe and abundant. But while carbon dioxide is the preferred
fuid for SFE, it possesses several polarity limitations. Solvent polarity is
important when extracting polar solutes and when strong analyte-matrix in-
teractions are present. Organic solvents are frequently added to the carbon
dioxide extracting fuid to alleviate the polarity limitations. Of late, instead
of carbon dioxide, argon is being used because it is inexpensive and more
inert. The component recovery rates generally increase with increasing pres-
sure or temperature: the highest recovery rates in case of argon are ob-
tained at 500 atm and 150 C.
The extraction procedure possesses distinct advantages:
The extraction of constituents at low temperature, which i)
strictly avoids damage from heat and some organic sol-
vents.
No solvent residues. ii)
Environmentally friendly extraction procedure. iii)
The largest area of growth in the development of SFE has been
the rapid expansion of its applications. SFE fnds extensive application in
the extraction of pesticides, environmental samples, foods and fragrances,
essential oils, polymers and natural products. The major deterrent in the
commercial application of the extraction process is its prohibitive capital
investment.
1.2.1.11 Phytonics Process
A new solvent based on hydrofuorocarbon-134a and a new
technology to optimize its remarkable properties in the extraction of plant
materials offer signifcant environmental advantages and health and safety
benefts over traditional processes for the production of high quality natural
fragrant oils, favors and biological extracts.
Advanced Phytonics Limited (Manchester, UK) has developed
this patented technology termed phytonics process. The products mostly
extracted by this process are fragrant components of essential oils and bio-
logical or phytopharmacological extracts which can be used directly without
further physical or chemical treatment.
27
The properties of the new generation of fuorocarbon solvents
have been applied to the extraction of plant materials. The core of the sol-
vent is 1,1,2,2-tetrafuoroethane, better known as hydrofuorocarbon-134a
(HFC-134a). This product was developed as a replacement for chlorofuoro-
carbons. The boiling point of this solvent is -25 C. It is not fammable or
toxic. Unlike chlorofuorocarbons, it does not deplete the ozone layer. It has
a vapor pressure of 5.6 bar at ambient temperature. By most standards this
is a poor solvent. For example, it does not mix with mineral oils or triglycer-
ides and it does not dissolve plant wastes.
The process is advantageous in that the solvents can be cus-
tomized: by using modifed solvents with HFC-134a, the process can be
made highly selective in extracting a specifc class of phytoconstituents.
Similarly, other modifed solvents can be used to extract a broader spectrum
of components. The biological products made by this process have extreme-
ly low residual solvent. The residuals are invariably less than 20 parts per
billion and are frequently below levels of detection. These solvents are nei-
ther acidic nor alkaline and, therefore, have only minimal potential reaction
effects on the botanical materials. The processing plant is totally sealed
so that the solvents are continually recycled and fully recovered at the end
of each production cycle. The only utility needed to operate these systems
is electricity and, even then, they do no consume much energy. There is no
scope for the escape of the solvents. Even if some solvents do escape, they
contain no chlorine and therefore pose no threat to the ozone layer. The
waste biomass from these plants is dry and ecofriendly to handle.
1.2.1.11.1 Advantages of the Process
Unlike other processes that employ high temperatures, the
phytonics process is cool and gentle and its products are
never damaged by exposure to temperatures in excess of
ambient.
No vacuum stripping is needed which, in other processes,
leads to the loss of precious volatiles.
The process is carried out entirely at neutral pH and, in the
absence of oxygen, the products never suffer acid hydrolysis
damage or oxidation.
The technique is highly selective, offering a choice of operat-
ing conditions and hence a choice of end products.
It is less threatening to the environment.
It requires a minimum amount of electrical energy.
It releases no harmful emissions into the atmosphere and
the resultant waste products (spent biomass) are innocuous
and pose no effuent disposal problems.
The solvents used in the technique are not fammable, toxic
or ozone depleting.
The solvents are completely recycled within the system.
28
1.2.1.11.2 Applications
The phytonics process can be used for extraction in biotechnol-
ogy (e.g for the production of antibiotics), in the herbal drug industry, in the
food, essential oil and favor industries, and in the production of other phar-
macologically active products. In particular, it is used in the production of top-
quality pharmaceutical-grade extracts, pharmacologically active intermediates,
antibiotic extracts and phytopharmaceuticals. However, the fact that it is used
in all these areas in no way prevents its use in other areas. The technique is
being used in the extraction of high-quality essential oils, oleoresins, natural
food colors, favors and aromatic oils from all manner of plant materials. The
technique is also used in refning crude products obtained from other extrac-
tion processes. It provides extraction without waxes or other contaminants. It
helps remove many biocides from contaminated biomass.
1.2.1.12 Parameters for Selecting an Appropriate Extraction Method
Authentication of plant material should be done before per- i)
forming extraction. Any foreign matter should be completely
eliminated.
Use the right plant part and, for quality control purposes, record ii)
the age of plant and the time, season and place of collection.
Conditions used for drying the plant material largely depend iii)
on the nature of its chemical constituents. Hot or cold blow-
ing air fow for drying is generally preferred. If a crude drug
with high moisture content is to be used for extraction, suit-
able weight corrections should be incorporated.
Grinding methods should be specifed and techniques that iv)
generate heat should be avoided as much as possible.
Powdered plant material should be passed through suitable v)
sieves to get the required particles of uniform size.
Nature of constituents: vi)
a) If the therapeutic value lies in non-polar constituents, a
non-polar solvent may be used. For example, lupeol is the
active constituent of Crataeva nurvala and, for its extrac-
tion, hexane is generally used. Likewise, for plants like
Bacopa monnieri and Centella asiatica, the active con-
stituents are glycosides and hence a polar solvent like
aqueous methanol may be used.
b) If the constituents are thermolabile, extraction methods
like cold maceration, percolation and CCE are preferred.
For thermostable constituents, Soxhlet extraction (if non-
aqueous solvents are used) and decoction (if water is the
menstruum) are useful.
c) Suitable precautions should be taken when dealing with
constituents that degrade while being kept in organic sol-
vents, e.g. favonoids and phenyl propanoids.
29
d) In case of hot extraction, higher than required tempera-
ture should be avoided. Some glycosides are likely to
break upon continuous exposure to higher temperature.
e) Standardization of time of extraction is important, as:
Insuffcient time means incomplete extraction.
If the extraction time is longer, unwanted constituents
may also be extracted. For example, if tea is boiled
for too long, tannins are extracted which impart astrin-
gency to the fnal preparation.
f) The number of extractions required for complete extrac-
tion is as important as the duration of each extraction.
The quality of water or menstruum used should be specifed vii)
and controlled.
Concentration and drying procedures should ensure the safety viii)
and stability of the active constituents. Drying under reduced
pressure (e.g. using a Rotavapor) is widely used. Lyophiliza-
tion, although expensive, is increasingly employed.
The design and material of fabrication of the extractor are ix)
also to be taken into consideration.
Analytical parameters of the fnal extract, such as TLC and x)
HPLC fngerprints, should be documented to monitor the
quality of different batches of the extracts.
1.2.2 Steps Involved in the Extraction of Medicinal Plants
In order to extract medicinal ingredients from plant material,
the following sequential steps are involved:
Size reduction 1.
Extraction 2.
Filtration 3.
Concentration 4.
Drying 5.
1.2.2.1 Size Reduction
The dried plant material is disintegrated by feeding it into a
hammer mill or a disc pulverizer which has built-in sieves. The particle size
is controlled by varying the speed of the rotor clearance between the ham-
mers and the lining of the grinder and also by varying the opening of the
discharge of the mill. Usually, the plant material is reduced to a size be-
tween 30 and 40 mesh, but this can be changed if the need arises. The
objective for powdering the plant material is to rupture its organ, tissue and
cell structures so that its medicinal ingredients are exposed to the extrac-
tion solvent. Furthermore, size reduction maximizes the surface area, which
in turn enhances the mass transfer of active principle from plant material
to the solvent. The 30-40 mesh size is optimal, while smaller particles may
become slimy during extraction and create diffculty during fltration.
30
1.2.2.2 Extraction
Extraction of the plant material is carried out in three ways:
Cold aqueous percolation i)
Hot aqueous extraction (decoction) ii)
Solvent extraction (cold or hot) iii)
1.2.2.2.1 Cold Aqueous Percolation
The powdered material is macerated with water and then
poured into a tall column. Cold water is added until the powdered material
is completely immersed. It is allowed to stand for 24 h so that water-soluble
ingredients attain equilibrium in the water. The enriched aqueous extract is
concentrated in multiple-effect evaporators to a particular concentration.
Some diluents and excipients are added to this concentrated extract, which
is then ready for medicinal use.
1.2.2.2.2 Hot Aqueous Extraction (Decoction)
This is done in an open-type extractor. The extractor is a cylin-
drical vessel made from type 316 stainless steel and has a diameter (D)
greater than the height (H), i.e. the H/D ratio is approximately 0.5. The bot-
tom of the vessel is welded to the dished end and is provided with an inside
false bottom with a flter cloth. The outside vessel has a steam jacket and a
discharge valve at the bottom.
One part powdered plant material and sixteen parts deminer-
alized water are fed into the extractor. Heating is done by injecting steam
into the jacket. The material is allowed to boil until the volume of water is
reduced to one-fourth its original volume. By this time the medicinal ingredi-
ents present in the plant material have been extracted out.
1.2.2.2.3 Filtration
The extract so obtained is separated out from the marc (ex-
hausted plant material) by allowing it to trickle into a holding tank through
the built-in false bottom of the extractor, which is covered with a flter cloth.
The marc is retained at the false bottom, and the extract is received in the
holding tank. From the holding tank, the extract is pumped into a sparkler
flter to remove fne or colloidal particles from the extract.
1.2.2.2.4 Spray Drying
The fltered extract is subjected to spray drying with a high
pressure pump at a controlled feed rate and temperature, to get dry powder.
The desired particle size of the product is obtained by controlling the inside
31
temperature of the chamber and by varying the pressure of the pump. The
dry powder is mixed with suitable diluents or excipients and blended in a
double cone mixer to obtain a homogeneous powder that can be straighta-
way used, for example, for flling in capsules or making tablets.
1.2.2.3 Solvent Extraction
The principle of solid-liquid extraction is that when a solid material
comes in contact with a solvent, the soluble components in the solid material
move to the solvent. Thus, solvent extraction of plant material results in the
mass transfer of soluble active principle (medicinal ingredient) to the solvent,
and this takes place in a concentration gradient. The rate of mass transfer de-
creases as the concentration of active principle in the solvent increases, until
equilibrium is reached, i.e. the concentrations of active principle in the solid ma-
terial and the solvent are the same. Thereafter, there will no longer be a mass
transfer of the active principle from plant material to the solvent.
Since mass transfer of the active principle also depends on its
solubility in the solvent, heating the solvent can enhances the mass transfer.
Moreover, if the solvent in equilibrium with the plant material is replaced with
fresh solvent, the concentration gradient is changed. This gives rise to differ-
ent types of extractions: cold percolation, hot percolation and concentration.
1.2.2.3.1 Cold Percolation
The extraction of plant material is carried out in a percolator
which is a tall cylindrical vessel with a conical bottom and a built-in false
bottom with a flter cloth. The percolator is connected to a condenser and a
receiver for stripping solvent from the marc.
The powdered material is fed into the percolator along with a
suitable solvent (ethyl alcohol or another non-polar solvent). The material
is left in contact with the solvent until equilibrium of the active principle is
achieved. The solvent extract, known as miscella, is taken out from the bot-
tom discharge valve of the percolator. Fresh solvent is added into the per-
colator and the miscella is drained out after acquiring equilibrium. Overall,
the plant material is washed four to fve times until it gets exhausted. All
washes from the percolator are pooled and concentrated.
The solvent in the marc is stripped out by passing steam from
the bottom of the percolator. The solvent and steam vapors rise and are con-
densed in a tubular condenser. The condensate, which is a mixture of alcohol
and water, is collected in a receiver and then subjected to fractional distillation
to get 95% pure ethyl alcohol which is again used as a fresh solvent.
This type of percolation is not effcient as it takes a long time
to reach equilibrium due to the slow mass transfer rate. The mass transfer
32
rate can be enhanced if some sort of movement is created between the
particles and the solvent.
This can be achieved either by providing inside agitation with a
mechanical stirrer or by repeated circulation of the extract back to the per-
colator. The frst method is cumbersome and power intensive whereas the
latter has been successful. A circulation pump that continuously circulates
the miscella back to the top of the percolator gives a better mass transfer
rate and reduces the equilibrium time considerably. Still, this type of percola-
tion is energy-consuming as large amounts of miscella from multiple washes
must be concentrated to remove the solvent.
To overcome this problem, a battery of percolators can be con-
nected in series. If three washes are required for completion of the extrac-
tion, four percolators are connected in series with their respective miscella
storage tanks.
At a particular time, one percolator is out of circuit, for charging
and discharging the material and also for stripping solvent from the marc,
whereas the other three percolators are in operation. Material is fed into
all the percolators and the solvent is fed into the frst percolator. When the
equilibrium in the frst percolator is reached, the extract from the frst perco-
lator is sent to the second percolator. The frst percolator is again flled with
fresh solvent. The extract of second percolator is transferred to the third,
the extract of frst is transferred to second, and fresh solvent is added to the
frst. The extract of the third percolator is transfered to the fourth percolator.
After attaining equilibrium, the extract from the fourth percolator is drained
off. The extract of the third percolator goes to fourth, the extract of second
goes to third, and the extract of frst goes to second percolator. The mate-
rial of the frst percolator, which has received three washes, is completely
exhausted. This percolator is taken out of the system for stripping the sol-
vent and discharging the extracted marc. This is again flled with fresh plant
material and the sequence is repeated with other percolators. In this way,
solvent of each percolator comes in contact three times with solid material
and gets fully enriched with active principle. The enriched extract is sent for
solvent recovery and concentration. Thus, instead of concentrating three vol-
umes of solvent, only one volume has to be concentrated; this saves energy
and the process is effcient.
1.2.2.3.2 Hot Percolation
Increasing the temperature of the solvent increases the solu-
bility of the active principle, which increases the concentration gradient and
therefore enhances the mass transfer of active principle from solid material
to the solvent, provided the active principle is not heat sensitive. This is
achieved by incorporating a heat exchanger between the circulation pump
and the feed inlet of the percolator. The extract is continuously pumped into
33
a tubular heat exchanger which is heated by steam. The temperature of the
extract in the percolator is controlled by a steam solenoid valve through a
temperature indicator controller. This sort of arrangement can be incorpo-
rated in single percolators or in a battery of percolators as needed.
The percolators that are tall cylindrical towers must be housed
in sheds of relatively great height. Tall towers are diffcult to operate, espe-
cially when charging material and discharging the marc from the top and bot-
tom manholes, which are time-consuming and labor-intensive procedures.
Tall towers have been replaced by extractors of smaller height for which the
H/D ratio is not more than 1.5.
These extractors have perforated baskets in which the material to
be extracted is charged. These perforated baskets, when loaded outside, can
be inserted into the extractor with a chain pulley block and, after the extraction,
they can be lifted out from the extractor for discharging the marc. Some extrac-
tors have an electrical hoist for the charging the material and discharging the
marc, which makes the operation less labor-intensive, quick and effcient.
The other type of instrument for extraction of medicinal ingre-
dients from plant material is the Soxhlet apparatus, which consists of an
extractor, a distillation still, a tubular condenser for the distillation still, a tu-
bular condenser for the recovery of solvent from the marc, a receiver for col-
lecting the condensate from the condenser, and a solvent storage tank. The
plant material is fed into the extractor, and solvent is added until it reaches
the siphon point of the extractor. Then, the extract is siphoned out into the
distillation still, which is heated with steam. The solvent vapors go to the
distillation condenser, get condensed and return to the extractor. The level
of the solvent in the extractor again rises to the siphon point and the extract
is siphoned out into the distillation still. In this way, fresh solvent comes in
contact with the plant material a number of times, until the plant material is
completely extracted. The fnal extract in the distillation still, which is rich in
active principle, is concentrated and the solvent is recovered.
1.2.2.3.3 Concentration
The enriched extract from percolators or extractors, known as
miscella, is fed into a wiped flm evaporator where it is concentrated under
vacuum to produce a thick concentrated extract. The concentrated extract is
further fed into a vacuum chamber dryer to produce a solid mass free from
solvent. The solvent recovered from the wiped flm evaporator and vacuum
chamber dryer is recycled back to the percolator or extractor for the next
batch of plant material. The solid mass thus obtained is pulverized and used
directly for the desired pharmaceutical formulations or further processed for
isolation of its phytoconstituents.
34
1.3 Aromatic Plant Extracts
The types of volatile isolates that are obtained commercially
from aromatic plants are essential oils, concretes, absolutes, pomades
and resinoids. Essential oils are isolated from plant material by distillation
whereas other volatile isolates are obtained by solvent extraction.
1.3.1 Concrete
This is an extract of fresh fowers, herbs, leaves and the fower-
ing tops of plants obtained by the use of a hydrocarbon solvent such as bu-
tane, pentane, hexane and petroleum ether. Concrete is rich in hydrocarbon-
soluble material and devoid of water-soluble components. It is generally a
waxy, semisolid, dark-colored material free from the original solvent.
In practice, concretes are produced in static extractors. These
extractors are ftted with numerous perforated trays so that the fowers do
not get compressed by their own weight. Each perforated tray has a spacer
so the number and distance between them are predetermined.
The set of perforated trays can be within a removable cylindri-
cal basket. In the centre of the lower tray, there is a rod on which the spac-
ers and the perforated trays are ftted while at the top there is a ring or a
hook so that the entire contents of the extractor can be readily removed by
a chain pulley block.
While stacking the fowers on these trays, care should be taken
to minimize bruising and damage of the fowers, because such damage can
result in the release of enzymes in the fower juice which deteriorates the
quality of concrete. The basket stacked with fowers is inserted into the
extractor and the solvent of choice is introduced from the bottom into the ex-
tractor until the material on the perforated disc assembly is completely im-
mersed. Four to fve such washes are given until the material is exhausted.
The enriched solvent from the extractor is pumped into an
evaporator for solvent recovery and the solvent content is reduced to about
one-tenth the original volume. The recovered solvent is pumped to the sol-
vent tanks to be used again. The concentrated material from the evaporator
is pumped into a vacuum evaporator where the solvent is removed more
carefully under high vacuum and the recovered solvent is returned to the
solvent tanks for repeated use. The resultant concrete has an odor similar
to but stronger than the material from which it was extracted.
In concrete manufacturing, it is a normal practice to circulate
fresh solvent through a battery of extractors. At each cycle, the solvent be-
comes more enriched with the fower volatiles until extraction is complete.
35
The number of extractors has to be synchronized with the number of solvent
washes.
1.3.2 Absolutes
Concretes are not widely used in perfumery in their native
form but are generally converted into an alcohol-soluble volatile concentrate
known as an absolute, i.e. they have to be extracted with alcohol.
To make an absolute, the concrete is mixed with absolute al-
cohol and agitated thoroughly in a vessel with an agitator. During agitation,
the temperature is kept at 40-60 C and the concrete is immersed in the
solution. The solution is cooled down to -5 to -10 C to precipitate out the
wax, since waxes are normally insoluble in alcohol below -1 C. The pre-
cipitated wax is removed by passing the solution through a rotary flter. The
fltrate from the rotary flter is pumped into a primary evaporator, where it is
concentrated to about 10% alcohol content. Finally, the concentrated extract
is pumped into an agitating-type evaporator, where the alcohol is carefully
removed under high vacuum.
1.3.3 Resinoids
Resinoid is an extract of naturally resinous material, made with
a hydrocarbon solvent. Resinoids are usually obtained from dry materials.
The extraction process is same as that of concrete production, except that
perforated discs are not used for stacking the material; instead powder from
dry plant material is fed into the extractor.
1.3.4 Pomades
Pomades are obtained by a process known as enfeurage,
which is a cold fat extraction method. The fat is spread out on glass plates
contained in wooden frames, leaving a clear margin near the edges. The
absorptive surface of the fat is increased by surface grooves made with a
wooden spatula.
Fresh fowers are spread out on the surface of the fat and the
frames are stacked in piles. After the perfume oils have been absorbed from
the fowers, the spent fowers are removed by hand. Fresh fowers are again
spread on the fat surface. This is repeated until the fat surface is completely
enriched with perfume oils. The pomade so obtained is ready for cold alco-
holic extraction.
1.3.5 Essential Oils
Essential oils are used in a wide variety of consumer goods such
as detergents, soaps, toilet products, cosmetics, pharmaceuticals, perfumes,
36
confectionery food products, soft drinks, distilled alcoholic beverages (hard
drinks) and insecticides. The world production and consumption of essential
oils and perfumes are increasing very fast. Production technology is an es-
sential element to improve the overall yield and quality of essential oil. The
traditional technologies pertaining to essential oil processing are of great sig-
nifcance and are still being used in many parts of the globe. Water distillation,
water and steam distillation, steam distillation, cohobation, maceration and
enfeurage are the most traditional and commonly used methods. Maceration
is adaptable when oil yield from distillation is poor. Distillation methods are
good for powdered almonds, rose petals and rose blossoms, whereas solvent
extraction is suitable for expensive, delicate and thermally unstable materials
like jasmine, tuberose, and hyacinth. Water distillation is the most favored
method of production of citronella oil from plant material.
1.3.5.1 Sources of Natural Essential Oils
Plant organs containing natural essential oils are illustrated in
Figure 3. Essential oils are generally derived from one or more plant parts,
such as fowers (e.g. rose, jasmine, carnation, clove, mimosa, rosemary,
lavander), leaves (e.g. mint, Ocimum spp., lemongrass, jamrosa), leaves and
stems (e.g. geranium, patchouli, petitgrain, verbena, cinnamon), bark (e.g.
cinnamon, cassia, canella), wood (e.g. cedar, sandal, pine), roots (e.g. an-
gelica, sassafras, vetiver, saussurea, valerian), seeds (e.g fennel, coriander,
caraway, dill, nutmeg), fruits (bergamot, orange, lemon, juniper), rhizomes
(e.g. ginger, calamus, curcuma, orris) and gums or oleoresin exudations
(e.g. balsam of Peru, balsam of Tolu, storax, myrrh, benzoin).
Specialized plant structures that produce and store essen-
tial oils are shown in Figure 4. Depending upon the plant family, essential
oils may occur in specialized secretary structures such as glandular hairs
(Labiatae, Verbenaceace, Geraniaceae), modifed parenchymal cells (Piper-
aceae), resin canals (conifers), oil tubes called vittae (Umbelliferae), lysig-
enous cavities (Rutaceae), schizogenous passages (Myrtaceae, Graminae,
Compositae) or gum canals (Cistacae, Burseraceae). It is well known that
when a geranium leaf is lightly touched, an odor is emitted because the long
stalked oil glands are fragile. Similarly, the application of slight pressure
on a peppermint leaf will rupture the oil gland and release oil. In contrast,
pine needles and eucalyptus leaves do not release their oils until the epi-
dermis of the leaf is broken. Hence, the types of structures in which oil is
contained differ depending on the plant type and are plant-family specifc.
Unfortunately, not enough is known even today about these oil secretary
structures to carefully categorize them. From the practical standpoint, they
can be categorized into superfcial and subcutaneous oils. Based on the
currently available information, it may be inferred that oils of the Labiatae,
Verbenaceae and Geraniaceae families are the only superfcial oils known;
consequently, the others are considered subcutaneous oils.
37
During handling, some fowers continue to produce aroma while
other quickly loose their odor. Flowers collected at different times may also give
different perfumery values. Regarding the rose, half-open fowers with plump
anthers give higher oil yield than fully opened fowers with shrivelled anthers.
Humidity, wind, rain and surface temperature also affect the oil yield consider-
ably. Harvesting schedule affects both quantity and quality of the oil.
1.3.5.2 Essential Oil Constituents
Major constituents of essential oils are shown in Figure 5, from
which it is clear that most essential oils consist of hydrocarbons, esters, terpe-
nes, lactones, phenols, aldehydes, acids, alcohols, ketones, and esters. Among
these, the oxygenated compounds (alcohols, esters, aldehydes, ketones, lac-
tones, phenols) are the principal odor source. They are more stable against
oxidizing and resinifying infuences than other constituents. On the other hand,
unsaturated constituents like monoterpenes and sesquiterpenes have the ten-
dency to oxidize or resinify in the presence of air and light. The knowledge of
individual constituents and their physical characteristics, such as boiling point,
thermal stability and vapor-pressure-temperature relationship, is of paramount
importance in technology development of oxygenated compounds.
1.3.5.3 Methods of Producing Essential Oils
Methods for producing essential oils from plant materials are
summarized in Figure 6. Regarding hydrodistillation, the essential oils indus-
try has developed terminology to distinguish three types: water distillation;
water and steam distillation; and direct steam distillation.
Originally introduced by Von Rechenberg, these terms have be-
come established in the essential oil industry. All three methods are subject to
the same theoretical considerations which deal with distillation of two-phase
systems. The differences lie mainly in the methods of handling the material.
Some volatile oils cannot be distilled without decomposition
and thus are usually obtained by expression (lemon oil, orange oil) or by oth-
er mechanical means. In certain countries, the general method for obtaining
citrus oil involves puncturing the oil glands by rolling the fruit over a trough
lined with sharp projections that are long enough to penetrate the epidermis
and pierce the oil glands located within outer portion of the peel (ecuelle
method). A pressing action on the fruit removes the oil from the glands, and
a fne spray of water washes the oil from the mashed peel while the juice is
extracted through a central tube that cores the fruit. The resulting oil-water
emulsion is separated by centrifugation. A variation of this process is to
remove the peel from the fruit before the oil is extracted.
Often, the volatile oil content of fresh plant parts (fower petals)
is so small that oil removal is not commercially feasible by the aforementioned
38
methods. In such instances, an odorless, bland, fxed oil or fat is spread in
a thin layer on glass plates. The fower petals are placed on the fat for a few
hours; then repeatedly, the oil petals are removed, and a new layer of petals is
introduced. After the fat has absorbed as much fragrance as possible, the oil
may be removed by extraction with alcohol. This process, known as enfeurage,
was formerly used extensively in the production of perfumes and pomades.
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Figure 4: Family-specic plant tissues responsible for producing or storing essential oil
39
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Figure 6: Methods of producing essential oils from plant materials
In the perfume industry, most modern essential oil production is
accomplished by extraction, using volatile solvents such as petroleum ether
and hexane. The chief advantages of extraction over distillation is that uniform
40
temperature (usually 50 C) can be maintained during the process, As a result,
extracted oils have a more natural odor that is unmatched by distilled oils, which
may have undergone chemical alteration by the high temperature. This feature
is of considerable importance to the perfume industry; however, the established
distillation method is of lower cost than the extraction process.
Destructive distillation means distilling volatile oil in the ab-
sence of air. When wood or resin of members of the Pinaceae or Cupressace-
ae is heated without air, decomposition takes place and a number of volatile
compounds are driven off. The residual mass is charcoal. The condensed
volatile matter usually separates into 2 layers: an aqueous layer containing
wood naptha (methyl alcohol) and pyroligneous acid (crude acetic), and a
tarry liquid in the form of pine tar, juniper tar, or other tars, depending on the
wood used. This dry distillation is usually conducted in retorts and, if the
wood is chipped or coarsely ground and the heat is applied rapidly, the yield
often represents about 10% of the wood weight used.
1.3.5.3.1 Hydrodistillation
In order to isolate essential oils by hydrodistillation, the aro-
matic plant material is packed in a still and a suffcient quantity of water
is added and brought to a boil; alternatively, live steam is injected into the
plant charge. Due to the infuence of hot water and steam, the essential oil
is freed from the oil glands in the plant tissue. The vapor mixture of water
and oil is condensed by indirect cooling with water. From the condenser,
distillate fows into a separator, where oil separates automatically from the
distillate water.
1.3.5.3.1.1 Mechanism of Distillation
Hydrodistillation of plant material involves the following main
physicochemical processes:
Hydrodiffusion i)
Hydrolysis ii)
Decomposition by heat iii)
1.3.5.3.1.1.1 Hydrodiffusion
Diffusion of essential oils and hot water through plant mem-
branes is known as hydrodiffusion. In steam distillation, the steam does not
actually penetrate the dry cell membranes. Therefore, dry plant material can
be exhausted with dry steam only when all the volatile oil has been freed
from the oil-bearing cells by frst thorough comminution of the plant material.
But, when the plant material is soaked with water, exchange of vapors within
the tissue is based on their permeability while in swollen condition. Mem-
branes of plant cells are almost impermeable to volatile oils. Therefore, in
41
the actual process, at the temperature of boiling water, a part of volatile oil
dissolves in the water present within the glands, and this oil-water solution
permeates, by osmosis, the swollen membranes and fnally reaches the
outer surface, where the oil is vaporized by passing steam.
Another aspect of hydrodiffusion is that the speed of oil vapori-
zation is not infuenced by the volatility of the oil components, but by their
degree of solubility in water. Therefore, the high-boiling but more water-sol-
uble constituents of oil in plant tissue distill before the low-boiling but less
water-soluble constituents. Since hydrodiffusion rates are slow, distillation
of uncomminuted material takes longer time than comminuted material.
1.3.5.3.1.1.2 Hydrolysis
Hydrolysis in the present context is defned as a chemical re-
action between water and certain constituents of essential oils. Esters are
constituents of essential oils and, in the presence of water, especially at
high temperatures, they tend to react with water to form acids and alcohols.
However, the reactions are not complete in either direction and the relation-
ship between the molal concentrations of various constituents at equilib-
rium is written as:
(alcohol) x (acid)
K =
(ester) x (water)
where K is the equilibrium constant.
Therefore, if the amount of water is large, the amounts of alcohol and acid
will also be large, resulting in a decreased yield of essential oil. Further-
more, since this is a time-dependent reaction, the extent to which hydrolysis
proceeds depends on the time of contact between oil and water. This is one
of the disadvantages of water distillation.
1.3.5.3.1.1.3 Effect of Heat
Almost all constituents of essential oils are unstable at high
temperature. To obtain the best quality oil, distillation must be done at low
temperatures. The temperature in steam distillation is determined entirely
by the operating pressure, whereas in water distillation and in water and
steam distillation the operating pressure is usually atmospheric.
All the previously described three effects, i.e. hydrodiffusion,
hydrolysis and thermal decomposition, occur simultaneously and affect one
another. The rate of diffusion usually increases with temperatures as does
the solubility of essential oils in water. The same is true for the rate and
extent of hydrolysis. However, it is possible to obtain better yield and quality
of oils by: (1) maintaining the temperature as low as possible, (2) using as
42
little water as possible, in the case of steam distillation, and (3) thoroughly
comminuting the plant material and packing it uniformly before distillation.
1.3.5.3.2 Three Types of Hydrodistillation
Three are three types of hydrodistillation for isolating essential
oils from plant materials:
Water distillation 1.
Water and steam distillation 2.
Direct steam distillation 3.
1.3.5.3.2.1 Water Distillation
In this method, the material is completely immersed in water,
which is boiled by applying heat by direct fre, steam jacket, closed steam
jacket, closed steam coil or open steam coil. The main characteristic of
this process is that there is direct contact between boiling water and plant
material.
When the still is heated by direct fre, adequate precautions
are necessary to prevent the charge from overheating. When a steam jacket
or closed steam coil is used, there is less danger of overheating; with open
steam coils this danger is avoided. But with open steam, care must be taken
to prevent accumulation of condensed water within the still. Therefore, the
still should be well insulated. The plant material in the still must be agitated
as the water boils, otherwise agglomerations of dense material will settle
on the bottom and become thermally degraded. Certain plant materials like
cinnamon bark, which are rich in mucilage, must be powdered so that the
charge can readily disperse in the water; as the temperature of the water
increases, the mucilage will be leached from the ground cinnamon. This
greatly increases the viscosity of the water-charge mixture, thereby allowing
it to char. Consequently, before any feld distillation is done, a small-scale
water distillation in glassware should be performed to observe whether any
changes take place during the distillation process. From this laboratory trial,
the yield of oil from a known weight of the plant material can be determined.
The laboratory apparatus recommended for trial distillations is the Clev-
enger system (Figure 7).
During water distillation, all parts of the plant charge must be
kept in motion by boiling water; this is possible when the distillation material
is charged loosely and remains loose in the boiling water. For this reason
only, water distillation possesses one distinct advantage, i.e. that it permits
processing of fnely powdered material or plant parts that, by contact with
live steam, would otherwise form lumps through which the steam cannot
penetrate. Other practical advantages of water distillation are that the stills
43
are inexpensive, easy to construct and suitable for feld operation. These
are still widely used with portable equipment in many countries.
The main disadvantage of water distillation is that complete
extraction is not possible. Besides, certain esters are partly hydrolyzed and
sensitive substances like aldehydes tend to polymerize. Water distillation
requires a greater number of stills, more space and more fuel. It demands
considerable experience and familiarity with the method. The high-boiling
and somewhat water-soluble oil constituents cannot be completely vapor-
ized or they require large quantities of steam. Thus, the process becomes
uneconomical. For these reasons, water distillation is used only in cases in
which the plant material by its very nature cannot be processed by water and
steam distillation or by direct steam distillation.
Figure 7: Clevenger-type laboratory-scale hydrodistillation apparatus
1.3.5.3.2.1.1 Traditional Method of Producing Attar Using Hydrodistillation
Floral attars are defned as the distillates obtained by hydrodis-
tillation of fowers (such as saffron, marigold, rose, jasmine, pandanus) in
sandal wood oil or other base materials like paraffn.
Attar manufacturing takes place in remote places because
the fowers must be processed quickly after collection. The apparatus and
equipment used to manufacture attar are light, fexible, easy to repair, and
have a fair degree of effciency. Keeping in view these facts, the traditional
deg and bhapka process has been used for centuries and is used even
now with the following traditional equipment (Figure 8).
Deg (still)
Bhapka (receiver)
Chonga (bamboo condenser)
44
Traditional bhatti (furnace)
Gachchi (cooling water tank)
Kuppi (leather bottle)
Figure 8: Traditional process of water distillation for making attar
1.3.5.3.2.1.2 Disadvantages of Water Distillation
Oil components like esters are sensitive to hydrolysis while
others like acyclic monoterpene hydrocarbons and aldehydes
are susceptible to polymerization (since the pH of water is
often reduced during distillation, hydrolytic reactions are fa-
cilitated).
Oxygenated components such as phenols have a tendency
to dissolve in the still water, so their complete removal by
distillation is not possible.
As water distillation tends to be a small operation (operated
by one or two persons), it takes a long time to accumulate
much oil, so good quality oil is often mixed with bad quality
oil.
The distillation process is treated as an art by local distill-
ers, who rarely try to optimize both oil yield or quality.
Water distillation is a slower process than either water and
steam distillation or direct steam distillation.
1.3.5.3.2.2 Water and Steam Distillation
In water and steam distillation, the steam can be generated
either in a satellite boiler or within the still, although separated from the
45
plant material. Like water distillation, water and steam distillation is widely
used in rural areas. Moreover, it does not require a great deal more capital
expenditure than water distillation. Also, the equipment used is generally
similar to that used in water distillation, but the plant material is supported
above the boiling water on a perforated grid. In fact, it is common that per-
sons performing water distillation eventually progress to water and steam
distillation.
It follows that once rural distillers have produced a few batches
of oil by water distillation, they realize that the quality of oil is not very good
because of its still notes (subdued aroma). As a result, some modifcations
are made. Using the same still, a perforated grid or plate is fashioned so
that the plant material is raised above the water. This reduces the capacity
of the still but affords a better quality of oil. If the amount of water is not suf-
fcient to allow the completion of distillation, a cohobation tube is attached
and condensate water is added back to the still manually, thereby ensuring
that the water, which is being used as the steam source, will never run out.
It is also believed that this will, to some extent, control the loss of dissolved
oxygenated constituents in the condensate water because the re-used con-
densate water will allow it to become saturated with dissolved constituents,
after which more oil will dissolve in it.
1.3.5.3.2.2.1 Cohobation
Cohobation is a procedure that can only be used during water
distillation or water and steam distillation. It uses the practice of returning
the distillate water to the still after the oil has been separated from it so
that it can be re-boiled. The principal behind it is to minimize the losses of
oxygenated components, particularly phenols which dissolve to some extent
in the distillate water. For most oils, this level of oil loss through solution
in water is less than 0.2%, whereas for phenol-rich oils the amount of oil
dissolved in the distillate water is 0.2%-0.7%. As this material is being con-
stantly re-vaporized, condensed and re-vaporized again, any dissolved oxy-
genated constituents will promote hydrolysis and degradation of themselves
or other oil constituents. Similarly, if an oxygenated component is constantly
brought in contact with a direct heat source or side of a still, which is consid-
erably hotter than 100 C, then the chances of degradation are enhanced.
As a result, the practice of cohobation is not recommended unless the tem-
perature to which oxygenated constituents in the distillate are exposed is
no higher than 100 C.
In steam and water distillation, the plant material cannot be
in direct contact with the fre source beneath the still; however, the walls of
the still are good conductors of heat so that still notes can also be obtained
from the thermal degradation reactions of plant material that is touching the
sides of the still. As the steam in the steam and water distillation process is
wet, a major drawback of this type of distillation is that it will make the plant
46
material quite wet. This slows down distillation as the steam has to vaporize
the water to allow it to condense further up the still. One way to prevent the
lower plant material resting on the grid from becoming waterlogged is to use
a baffe to prevent the water from boiling too vigorously and coming in direct
contact with the plant material.
1.3.5.3.2.2.2 Advantages of Water and Steam Distillation over Water Distillation
Higher oil yield.
Components of the volatile oil are less susceptible to hy-
drolysis and polymerization (the control of wetness on the
bottom of the still affects hydrolysis, whereas the thermal
conductivity of the still walls affects polymerization).
If refuxing is controlled, then the loss of polar compounds
is minimized.
Oil quality produced by steam and water distillation is more
reproducible.
Steam and water distillation is faster than water distillation,
so it is more energy effcient.
Many oils are currently produced by steam and water distilla-
tion, for example lemongrass is produced in Bhutan with a rural steam and
water distillation system.
1.3.5.3.2.2.3 Disadvantages of Water and Steam Distillation
Due to the low pressure of rising steam, oils of high-boiling
range require a greater quantity of steam for vaporization -
hence longer hours of distillation.
The plant material becomes wet, which slows down distil-
lation as the steam has to vaporize the water to allow it to
condense further up the still.
To avoid that the lower plant material resting on the grid
becomes waterlogged, a baffe is used to prevent the water
from boiling too vigorously and coming in direct contact with
the plant material.
1.3.5.3.2.3 Direct Steam Distillation
As the name suggests, direct steam distillation is the proc-
ess of distilling plant material with steam generated outside the still in a
satellite steam generator generally referred to as a boiler. As in water and
steam distillation, the plant material is supported on a perforated grid above
the steam inlet. A real advantage of satellite steam generation is that the
amount of steam can be readily controlled. Because steam is generated in
a satellite boiler, the plant material is heated no higher than 100 C and,
consequently, it should not undergo thermal degradation. Steam distillation
47
is the most widely accepted process for the production of essential oils on
large scale. Throughout the favor and fragrance supply business, it is a
standard practice.
An obvious drawback to steam distillation is the much higher
capital expenditure needed to build such a facility. In some situations, such
as the large-scale production of low-cost oils (e.g. rosemary, Chinese ce-
darwood, lemongrass, litsea cubeba, spike lavender, eucalyptus, citronella,
cornmint), the world market prices of the oils are barely high enough to
justify their production by steam distillation without amortizing the capital
expenditure required to build the facility over a period of 10 years or more.
1.3.5.3.2.3.1 Advantages of Direct Steam Distillation
Amount of steam can be readily controlled.
No thermal decomposition of oil constituents.
Most widely accepted process for large-scale oil production,
superior to the other two processes.
1.3.5.3.2.3.2 Disadvantage of Direct Steam Distillation
Much higher capital expenditure needed to establish this
activity than for the other two processes.
1.3.5.3.3 Essential Oil Extraction by Hydrolytic Maceration
Distillation
Certain plant materials require maceration in warm water be-
fore they release their essential oils, as their volatile components are gly-
cosidically bound. For example, leaves of wintergreen (Gaultheria procum-
bens) contain the precursor gaultherin and the enzyme primeverosidase;
when the leaves are macerated in warm water, the enzyme acts on the
gaultherin and liberates free methyl salicylate and primeverose. Other sim-
ilar examples include brown mustard (sinigrin), bitter almonds (amygdalin)
and garlic (alliin).
1.3.5.3.4 Essential Oil Extraction by Expression
Expression or cold pressing, as it is also known, is only used
in the production of citrus oils. The term expression refers to any physical
process in which the essential oil glands in the peel are crushed or broken
to release the oil. One method that was practiced many years ago, par-
ticularly in Sicily (spugna method), commenced with halving the citrus fruit
followed by pulp removal with the aid of sharpened spoon-knife (known as
a rastrello). The oil was removed from the peel either by pressing the peel
against a hard object of baked clay (concolina) which was placed under
a large natural sponge or by bending the peel into the sponge. The oil
48
emulsion absorbed by the sponge was removed by squeezing it into the
concolina or some other container. It is reported that oil produced this way
contains more of the fruit odor character than oil produced by any other
method.
A second method known as equaling (or the scodella method),
uses a shallow bowl of copper (or sometimes brass) with a hollow central
tube; the equaling tool is similar in shape to a shallow funnel. The bowl is
equipped with brass points with blunt ends across which the whole citrus
fruit is rolled by hand with some pressure until all of the oil glands have
burst. The oil and aqueous cell contents are allowed to dribble down the
hollow tube into a container from which the oil is separated by decantation.
Obviously, hand pressing is impractical because it is an extremely slow proc-
ess, e.g. on average only 2-4 lbs oil per day can be produced by a single per-
son using one of these hand methods. As a result, over the years a number
of machines have been designed to either crush the peel of a citrus fruit or
crush the whole fruit and then separate the oil from the juice.
1.3.5.3.4.1 Pelatrice Process
In the pelatrice process, citrus fruits are fed from a hopper into
the abrasive shell of the machine. The fruits are rotated against the abra-
sive shell by a slow-moving Archimedian screw whose surface rasps the fruit
surfaces causing some of the essential oil cavities on the peel to burst and
release their oil-water emulsion. This screw further transports the fruit into
a hopper in which rollers covered with abrasive spikes burst the remaining
oil cavities. The oil and water emulsion is washed away from the fruit by a
fne spray of water. The emulsion next passes through a separator where any
solids are removed, after which it passes through two centrifugal separators
working in series to yield the pure oil. Most bergamot oil and some lemon
oil are produced this way in Italy.
1.3.5.3.4.2 Sfumatrice Process
The sfumatrice equipment consists of a metallic chain that is
drawn by two horizontal ribbed rollers. The peels are conveyed through these
rollers during which time they are pressed and bent to release their oil. As
in pelatrice, the oil is washed away from the sfumatrice rollers by fne sprays
of water. Again, the oil is initially passed through a separator prior to being
sent to two centrifuges in series, so that purifed oil can be produced. At one
time, sfumatrice was the most popular process for citrus oil isolation in Italy;
however, today the pelatrice method appears more popular.
1.3.5.3.5 Essential Oil Extraction with Cold Fat (Enfeurage)
Despite the introduction of the modern process of extraction
with volatile solvents, the old fashioned method of enfeurage, as passed on
49
from father to son and perfected in the course of generations, still plays an
important role. Enfeurage on a large scale is today carried out only in the
Grasse region of France, with the possible exception of isolated instances
in India where the process has remained primitive.
The principles of enfeurage are simple. Certain fowers (e.g.
tuberose and jasmine) continue the physiological activities of developing
and giving off perfume even after picking. Every jasmine and tuberose fower
resembles, so to speak, a tiny factory continually emitting minute quantities
of perfume. Fat possesses a high power of absorption and, when brought
in contact with fragrant fowers, readily absorbs the perfume emitted. This
principle, methodically applied on a large scale, constitutes enfeurage. Dur-
ing the entire period of harvest, which lasts for eight to ten weeks, batches
of freshly picked fowers are strewn over the surface of a specially prepared
fat base (corps), let there (for 24 h in the case of jasmine and longer in the
case of tuberose), and then replaced by fresh fowers. At the end of the
harvest, the fat, which is not renewed during the process, is saturated with
fower oil. Thereafter, the oil is extracted from the fat with alcohol and then
isolated.
The success of enfeurage depends to a great extent upon the
quality of the fat base employed. Utmost care must be exercised when pre-
paring the corps. It must be practically odorless and of proper consistency.
If the corps is too hard, the blossoms will not have suffcient contact with
the fat, curtailing its power of absorption and resulting in a subnormal yield
of fower oil. On the other, if it is too soft, it will tend to engulf the fowers
and the exhausted ones will adhere; when removed, the fowers will retain
adhering fat, resulting in considerable shrinkage and loss of corps. The
consistency of the corps must, therefore, be such that it offers a semihard
surface from which the exhausted fowers can easily be removed. The proc-
ess of enfeurage is carried out in cool cellars, and every manufacturer must
prepare the corps according to the prevailing temperature in the cellars dur-
ing the months of the fower harvest.
Many years of experience have proved that a mixture of one
part of highly purifed tallow and two parts of lard is eminently suitable
for enfeurage. This mixture assures a suitable consistency of the corps in
conjunction with high power of absorption. The fat corps thus prepared is
white, smooth, absolutely of uniform consistency, free of water and practi-
cally odorless. Some manufacturers also add small quantities of orange
fower or rose water when preparing the corps. This seems to be done for
the sake of convention. Such additions somewhat shade the odor of the
fnished product by imparting a slight orange blossom or rose note.
50
1.3.5.3.5.1 Enfeurage and Defeurage
Every enfeurage building is equipped with thousands of so-
called chassis, which serve as vehicles for holding the fat corps during
the process. A chassis consists of a rectangular wooden frame. The frame
holds a glass plate upon both sides of which the fat corps is applied with a
spatula at the beginning of the enfeurage process. When piled one above
the other, the chassis form airtight compartments, with a layer of fat on the
upper and lower side of each glass plate.
Every morning during the harvest the freshly picked fowers ar-
rive, and after being cleaned of impurities, such as leaves and stalks, are
strewn by hand on top of the fat layer of each glass plate. Blossoms wet
from dew or rain must never be employed, as any trace of moisture will turn
the corps rancid. The chassis are then piled up and left in the cellars for
24 h or longer, depending upon the type of fowers. The latter rest in direct
contact with one fat layer (the lower one), which acts as a direct solvent
whereas the other fat layer (beneath the glass plate of the chassis above)
absorbs only the volatile perfume given off by the fowers.
After 24 h, the fowers have emitted most of their oil and start
to wither, developing an objectionable odor. They must then be removed
from the corps, which process, despite all efforts to introduce labor-saving
devices, is still done by hand. Careful removal of the fower (defeurage) is
almost more important than charging the corps on the chassis with fresh
fowers (enfeurage) and, therefore, the persons doing this work must be
experienced and skilled. Most of the exhausted fowers will fall from the fat
layer on the chassis glass plate when the chassis is struck lightly against
the working table, but since it is necessary to remove every single fower and
every particle of the fower, tweezers are used for this delicate operation.
Immediately following defeurage, that is, every 24 h, the chassis are re-
charged with fresh fowers. For this purpose the chassis are turned over and
the fat layer, which in the previous operation formed the top (ceiling) of the
small chamber, is now directly charged with fowers. In the case of jasmine,
the entire enfeurage process lasts about 70 days: daily the exhausted fow-
ers are removed and the chassis are recharged with fresh ones. At the
beginning of, and several times during, the harvest, the fat on the chassis is
scratched over with metal combs and tiny furrows are drawn in order change
and increase the surface of absorption.
At the end of the harvest, the fat is relatively saturated with
fower oil and possesses the typical fragrance. The perfumed fat must then
be removed from the glass plates between the chassis. For this purpose,
it is scraped off with a spatula and then carefully melted and bulked in
closed containers. The fnal product is called pomade (pomade de jasmine,
pomade de tuberous, pomade de violet, etc.). The most highly saturated
pomade is pomade no. 36, because the corps on the chassis have been
51
treated with fresh fowers 36 times during the whole process of enfeurage.
At the beginning of the harvest, every chassis is charged with about 360
g fat corps on each side of the glass plate, in other words, with 720 g per
chassis. Every kilogram of fat corps should be in contact with about 2.5 kg
(preferably with 3.0 kg) of jasmine fowers for the entire period of enfeurage,
which lasts from 8 to 10 weeks. The quantities differ somewhat for different
fowers. At the end of enfeurage, the fat corps has lost about 10% of its
weight because of the various manipulations.
1.3.5.3.5.2 Hot Maceration Process
In this process, the long enfeurage time is reduced by the im-
mersion of petals in molten fat heated at 45-60 C for 1 to 2 h, depending
upon the plant species. After each immersion, the fat is fltered and sepa-
rated from the petals. After 10 to 20 immersions, the fat is separated from
waste fowers and water. Absolute of maceration is then produced from fat
containing oil through the process of extraction and concentration under
reduced pressure. It is mainly used for highly delicate fowers whose physi-
ological activities are lost rapidly after their harvest, such as lily of valley.
1.3.6 Modern (Non-traditional) Methods of Extraction
of Essential Oils
Traditional methods of extraction of essential oils have been
discussed and these are the methods most widely used on commercial
scale. However, with technological advancement, new techniques have been
developed which may not necessarily be widely used for commercial pro-
duction of essential oils but are considered valuable in certain situations,
such as the production of costly essential oils in a natural state without any
alteration of their thermosensitive components or the extraction of essential
oils for micro-analysis. These techniques are as follows:
Headspace trapping techniques
Static headspace technique -
Vacuum headspace technique -
Dynamic headspace technique -
Solid phase micro-extraction (SPME)
Supercritical fuid extraction (SFE)
Phytosol (phytol) extraction
Protoplast technique
Simultaneous distillation extraction (SDE)
Microwave distillation
Controlled instantaneous decomposition (CID)
Thermomicrodistillation
Microdistillation
Molecular spinning band distillation
Membrane extraction
52
Some of these techniques are discussed in other chapters.
Here, a few important, relevant references are provided.
1.4 Conclusions
Some of the major constraints in sustainable industrial exploi-
tation of medicinal and aromatic plants (MAPs) are due to the fact that
the countries of South East Asia have poor agricultural practices for MAPs,
unscientifc and indiscriminate gathering practices from the wild, poor post-
harvest and post-gathering practices leading to poor quality raw material,
lack of research for the development of high-yielding varieties of MAPs, poor
propagation methods, ineffcient processing techniques, poor quality control
procedures, lack of research on process and product development, diff-
culty in marketing, non-availability of trained personnel, lack of facilities and
tools to fabricate equipment locally, and fnally lack of access to the latest
technologies and market information. This calls for co-operation and co-
ordination among various institutes and organizations of the region, in order
to develop MAPs for sustainable commercial exploitation.
The process of extracting MAPs determines how effciently we
add value to MAP bioresources. In the case of essential oils, the extraction
process affects the physical as well as internal composition. External ap-
pearance, at times, can result in rejection of the batch even if the analytical
results are within acceptable limits. Furthermore, essential oils are evalu-
ated internationally for their olfactory properties by experienced perfumers
and these olfactory qualities supersede analytical results. Variations in the
chemical constituents of the extracts of medicinal plants may result by us-
ing non-standardized procedures of extraction. Efforts should be made to
produce batches with quality as consistent as possible (within the narrowest
possible range).
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53
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55
2 Role of Process Simulation in
Extraction Technologies for Medicinal
and Aromatic Plants
M. Fermeglia
Abstract
This paper illustrates the role of process simulation in the eld of extraction technolo-
gies for medicinal and aromatic plants. The paper starts with a brief introduction to
process simulation fundamentals and the role of process simulation in the industry
today. It describes procedures to follow in simulating a process and the benets of
process simulation. In the second part of the paper, phytochemical processes that
have been simulated at ICS-UNIDO are listed, followed by two case studies to illustrate
the applicability of the methodology proposed: (i) turpentine oil batch distillation and
(ii) menthol recovery by crystallization of mentha oil. At the end, recommendations are
given advocating the importance of process simulation for developing countries.
2.1 Introduction
Developing countries are rich in medicinal and aromatic plants
(MAPs) but, due to diffculty in accessing effcient extraction technologies,
value addition to this rich bioresource is diffcult. In most cases, and par-
ticularly in very poor countries, the technologies used are inappropriate and
not economical. The crucial problem is related to the quality of the product:
primitive extraction technologies do not guarantee a stable and high-quality
product and, in some cases, inappropriate technologies and procedures re-
sult in producing contaminated product which has low market value.
In order to assist developing countries to achieve the objective
of using rich MAP resource for producing value-added products, dissemina-
tion of knowledge of existing extraction technologies and of the latest devel-
opments in these technologies is essential.
Commercial process simulation software can be used to predict,
on a computer, the real plant and consequently is a useful tool for optimizing
the process conditions and enhancing the capacity of managing the phyto-
chemical processes. In particular, process simulation can assist developing
and emerging countries in optimizing an advanced process rather than man-
aging a primitive process, which should be substituted by more effcient and
standardized procedures. The focus in this case is more related to practical
problems such as the quality of the materials and of the water to be used for
the extraction. In most cases, developing countries face problems in the type
of vessel, quality of water and stability of the product during the processing.
2 ROLE OF PROCESS SIMULATION IN EXTRACTION TECHNOLOGIES FOR MEDICINAL AND AROMATIC PLANTS
56
This paper describes the use of process simulation software
in the extraction and purifcation of essential oils at both pilot and industrial
scales. Such processes have been developed and are in operation in devel-
oping and emerging countries. The goal of this paper is to illustrate a proce-
dure for obtaining better knowledge of the extraction process and, therefore,
for optimizing the process in terms of energy use, raw material consumption
and environmental impact.
2.2 Process Simulation Goals and Defnitions
Simulation is the act of representing some aspects of the real
world by numbers or symbols which may be manipulated to facilitate their study.
A process simulator is an engineering tool that performs several tasks, includ-
ing automated calculations, material and energy balances, physical property
estimations, design or rating calculations, and process optimization. A process
simulator is not a process engineer, and a process engineer is always needed to
analyze the problem and the output of a process simulator. A process simulator
solves material and energy balances by means of computer code.
In principle, a process simulator for the study of a chemical proc-
ess goes through the procedure outlined in Figure 1. One starts from the
defnition of the problem (problem analysis) and then develops the process
model, i.e. the system of equations (algebraic or differential). Furthermore,
one collects the necessary additional data and solves the model with a suit-
able method, depending on the system of equations. Finally, the process en-
gineer analyzes the results and perhaps starts over again to develop a more
realistic model.
The same picture applies to steady-state simulation, dynam-
ic simulation and optimization problems; only the process model and the
method of solution change. Solution of the system of material and energy
balance equations is not an easy task because it must be solved consider-
ing many components, complex thermophysical models for phase equilibri-
um calculations, a large number of subsystems (equipment), rather complex
equipment (e.g. distillation columns), recycle streams and control loops.
0ivider
Mixer Reactor 5eparator
0efine
problem
Analyze
results
0evelop
process
model
Collect
additional
data
5olve model
equations
$IFNJDBM1MBOU
Figure 1: Fundamental steps in running process simulation software
57
A typical process simulation scheme, with the most important
elements and their connections, is shown in Figure 2. Clearly, a process
simulator includes cost estimation as well as economic evaluation. The im-
portance of the database is shown in the fgure as a necessary source of
information for different objects in the structure.
fnery and Material
alances
fquipment 5izin
Cost fstimation
fconomic fvaluation
humerical
5ubroutines
5ystem
Manaer
0tilities
fditor
0raphics
Reports
forms
0ata ase
(physical
properties,
costs, etc.)
Figure 2: General scheme of a steady-state process simulator
The following approaches are available in process simulation:
Steady state simulation, which considers a snapshot in time
of the process.
Dynamic simulation, which considers the evolution in the
time domain of the equations describing the process.
Integrated steady-statedynamic simulation, which com-
bines the previous two approaches.
These three approaches may be used in different ways when deal-
ing with process simulation. One possibility is to perform process analysis, in
which an existing process is studied and alternative conditions as well as dy-
namic behavior are investigated in the appraisal of effectiveness of the design.
The second is process synthesis, in which different process confgurations are
compared in order to identify the best choice of units and the connections be-
tween them. The third possibility is process design and simulation, which aims
at establishing the optimal operating conditions of a given process.
In all these possibilities, impact on industry is pervasive rath-
er than restricted to a single moment in the development of the process.
Process simulation has strongly affected the way engineering knowledge
is used in processes. The traditional way of using process simulation was
mainly focused on designing fowsheets and on defning critical equipment
parameters, such as distillation column stages and column diameter. Today,
58
engineers are oriented to a more comprehensive use of process simulation
in the entire life of the plant, as in designing control strategies, optimiz-
ing process parameters, studying process time evolution for understanding
startup and shutdown procedures, performing risk analysis, training opera-
tors, and defning procedures to reduce non-steady-state operations.
The main benefts gained from such a comprehensive use of
process simulation are the partial or total replacement of pilot plants (reduc-
tion of the number of runs and planning), the reduction of time to market for
the development of new processes, and the fast screening of process alter-
natives to select the best solution in terms of economics, environmental as-
pects, energy consumption and fexibility. Due to the high complexity of chemi-
cal processes, to get these benefts one must critically simplify the process
and apply process simulation techniques in the entire life cycle of a process.
Steady-state simulators are considered the core products
of process simulation and are used for designing processes, evaluating
process changes and analyzing what-if scenarios. Steady-state simulation is
normally performed before all other kinds of simulation: dynamic simulation,
process synthesis with pinch technology, detailed equipment design, off-line
and on-line equation-based optimization, and application technologies for
vertical markets (e.g. polymers).
The problems involved in a process simulation run are the def-
nition of an accurate thermodynamic model (equations of state or excess
Gibbs energy model), the necessity of defning dummy operations (not al-
ways easy to identify), and the tear streams identifcation to achieve rapid
convergence.
The logical procedure for performing a simulation is as follows.
First, one defnes all the components to be used in the simulation, including
conventional and non-conventional components. Next, the most important
step in the defnition of the simulation is the selection of physicochemical
properties to be used in the calculations. Having done this, one proceeds to
fowsheet connectivity and to the defnition of the feed conditions. The next
step is the unit operation internal defnitions. At this stage, it is possible to
run a base case and check that the system is converging.
Process specifcation defnition, control parameters, and
equipment hold-up defnition are added later to refne the simulation and
to obtain results similar to the reality. Various different results are obtained
from a simulation run. The most important are the validation of phase equi-
libria models for the real system to be used in similar conditions, the veri-
fcation of the process operating conditions, information on intermediate
streams and enthalpy balance, verifcation of plant specifcations, and infu-
ence of the operative parameters on the process specifcations.
59
All this information is useful for de-bottlenecking the entire
process, or part of it, for identifying process control strategies, and for tun-
ing the instrumentation. This is important since it allows one to verify the
behavior of security systems when process conditions are varied.
As far as dynamic simulation is concerned, applications can
be found in continuous processes, concurrent process and control design,
evaluation of alternative control strategies, troubleshooting process oper-
ability, and verifcation of process safety.
The most important benefts of dynamic modeling are: the
capital avoidance and lower operating costs through better engineering de-
cisions; the throughput, product quality, safety and environmental improve-
ments through improved process understanding; and the increased produc-
tivity through enhanced integration of engineering work processes.
2.3 Biotechnological and Phytochemical
Processes Studied at ICS-UNIDO
This section summarizes ongoing work involving the simulation
of important biotechnological and phytochemical processes.
2.3.1 Brief of Biotechnological Processes
2.3.1.1 Alcohol Production from Biomass
The goal of the process is the steady-state simulation of the
production of ethanol from biomass fermentation. The process is divided
into two parts: (i) biomass fermentation that produces a mixture of ethanol,
water and other components, and (ii) separation using a distillation column
that concentrates the ethanol. The modelled reactors are continuous stirred
tank reactors in series or parallel.
2.3.1.2 Soybean Oil Refning and Treatment of the Waste
The goal of this work is to simulate the soybean oil refning
process. This is a complex biotechnological process that involves many re-
actions and the treatment of solids. The process is divided into three sec-
tions: (i) degumming and neutralization, (ii) bleaching, and (iii) deodorization.
The main objective of the simulation is to reduce the consumption of steam
by applying the pinch technology concept. Steam is consumed in the heat
exchangers to heat the oil, in the bleacher equipment, and in the deodorizer.
The diffculty of this simulation lies in the large number of undefned com-
ponents that must be characterized in order to obtain a reliable simulation.
In addition, an alternative way of reducing the free acids, by using extraction
60
with ethanol, is being examined. Key features are the achievements of the
required product quality by minimizing the capital and operating costs.
2.3.1.3 Production of Synthetic Hydrocarbon Fuels from Biomass
Starting from natural gas, coal, or wood, a syngas of suitable
composition can be produced by gasifcation. Then, water-gas shift reaction,
Fischer-tropsch synthesis and hydrocracking can be applied to the syngas to
obtain a mixture of liquid hydrocarbons that can be used as synthetic fuel.
This is a complex process that can be simplifed by neglecting the kinetics
of the chemical reactions involved. The purpose of this project is to quan-
tify the mass and energy consumption and the emission of carbon dioxide.
Key features are: the selection of the thermodynamic model to achieve a
realistic simulation; the heat integration among different process sections
to minimize the environmental impact during recovery and recycling of the
entrainer; and the reduction of energy duties.
2.3.1.4 Production of Bio-ethanol from Corn
By fermentation of sugar cane, corn or wheat, ethanol (bio-eth-
anol) can be easily produced. This process includes the steps of liquefac-
tion, cooking, fermentation, distillation, dehydration, evaporation and drying
of the solid by-product. By a careful simulation of the operations involved,
the needs for water and energy can be minimized, and the use of fossil fu-
els can be avoided. Key features are the energy balance starting from the
content in the feedstock biomass, and the water saving.
A second problem can be addressed by accurately simulating
the distillation and dehydration aspects, which have the highest energy de-
mand of this process. Key feature is the use of pressure as an operating
variable.
2.3.2 Brief of Phytochemical Processes
2.3.2.1 Citral Recovery by Distillation of Lemon Peel Oil
The goal of this process is the production of citral. Lemon peel
oil is fractioned by a traditional method of separation to get an oxygenated
substance (citral). A simulation model of the distillation helps identify the
optimal operating conditions. The objective of this simulation is to separate
the oxygenated compounds from terpenes.
2.3.2.2 Menthol Recovery by Crystallization of Mentha Oil
Mentha oil contains menthol, a commercially important prod-
uct. Menthol is separated from the other components on the basis of differ-
ences in melting temperatures. Crystallization from solution is an industrially
important unit operation due to its ability to provide high purity separations.
61
The crystal growth and nucleation kinetic parameters must be determined
experimentally before systematically designing a crystallizer and computing
optimal operations and control procedures.
2.3.2.3 Carvone Recovery from Spearmint Oil
Spearmint oil contains the major component carvone that must
be separated from other components. The separation of carvone is done by
continuous distillation and the process is optimized after identifcation of
the relevant parameters worked out by sensitivity analysis. The objective of
this simulation is to obtain carvone at 95% purity or more.
2.3.2.4 Peppermint Oil Extraction by Steam Distillation
For the steam distillation of peppermint, the peppermint
leaves are placed at the bottom of a distillation fask and steam is perco-
lated through. The peppermint oil evaporates, and the emerging mixture of
vaporized water and oil moves through a coil, usually cooled with running
water, where the steam condenses. The mixture of condensed water and
essential oil is collected and separated by decantation or, in rare cases,
by centrifugation.
2.3.2.5 Multiple-effect Evaporation of Milk Serum
Evaporation is a widely used operation for the recovery of valu-
able products from dilute aqueous mixtures, such as milk serum. In this
case, a four-effect process helps minimize the energy consumption and
makes this process economically attractive. Key features are the effects of
both pressure and heat transfer coeffcients on the overall performance of
the apparatus.
2.3.3 Case Study: Turpentine Oil Batch Distillation
Turpentine oil obtained from species of Pinus (family Pinaceae)
is mainly used in paint and soap manufacturing industries, and in the phar-
maceutical industry its use is limited to balms and oil bases. Semi-fuid
mixtures of resins remain dissolved in the volatile oil, thus it is produced
by fractional distillation. The objectives of the process simulation are to: (i)
develop the process simulation base case, (ii) understand how to obtain
complete fractionation of the oil, (iii) optimize the composition of pinene,
carene and longifolene in the product streams, and (iii) optimize the time
and energy consumption of the process.
Figure 3a shows the fractional composition profles of pinene,
carene and longifolene versus time obtained in the top of the distillation col-
umn. Figure 3b shows the instantaneous energy consumption of a constant
refux operation.
62
-c.z
c
c.z
c.o
c.6
c.8
1
1.z
c 1c 1 zc z c
1ime (hr)
Carene
Pinene
C
e
m
p
e
s
i
t
i
e
n
B
e
a
t

d
u
t
y
,

c
a
|
{
s
LengiIe|ene
-z1occ
-z1c
-z1cc
-z1zc
-z1zcc
-z11 c
-z11 cc
-z1cc
-z1ccc
-zcc
c 1c 1 zc z
1ime (hr)
Figure 3: a) Fractional composition versus time, and
b) Energy consumption versus time for the base case
Figure 4 shows the same process in which the refux ratio is
varied in order to obtain a constant composition at the top of the column. It
is interesting to note that the distillation time as well as energy consump-
tion are greatly reduced.
c
c.z
c.o
c.6
c.8
1
1.z
c 1c 1 zc
1ime (hr) 1ime (hr)
-z1c
-z1cc
-z1zc
-z1zcc
-z11c
-z11cc
-z1cc
-z1ccc
-zcc
c 1c 1 zc
Carene
Pinene
C
e
m
p
e
s
i
t
i
e
n
B
e
a
t

d
u
t
y
,

c
a
|
{
s
LengiIe|ene
b) Energy consumption versus time for the constant composition case
Figure 5 shows that a total separation of the oil constituents
is achievable.
1ime (hr) 1ime (hr)
-c.z
c
c.z
c.o
c.6
c.8
1
1.z
c 1c zc c oc c 6c ;c -zoccc
-zccc
-zzccc
-z1ccc
-zcccc
-1ccc
-18ccc
-1;ccc
c 1c zc c oc c 6c
Carene
Pinene
C
e
m
p
e
s
i
t
i
e
n
LengiIe|ene
B
e
a
t

d
u
t
y
,

c
a
|
{
s
b) Energy consumption versus time for the total fractionation case
These simulations show that, in the base case with a refux
ratio of 15 and a high consumption of energy (Figure 4), the distillate ac-
cumulator collects a high percentage of pinene (93%). If a PID controller
is introduced to maintain the concentration constant, the composition of
pinene is around 90% and it takes only 12 h and a refux ratio of 5 to achieve
the desired value, thus saving time and energy. In the case of complete frac-
tionation, we can collect 93% of pinene, 88% of carene and 5.7% of dump
products at the end of the process.
63
2.3.4 Case Study: Menthol Recovery by Crystallization
of Mentha Oil
Crystallization from solution is an industrially important opera-
tion due to its ability to provide high-purity separations. The menthol crystal-
lization process using menthe oil is rather simple, and consists of a cascade
crystallization as shown in Figure 6.
The objective of the simulation is to optimize the menthol crystalli-
zation process. The oil, composed of 75% menthol and also containing menthyl
acetate, limonene and menthone (Table 1), is fed into the frst crystallizer where
the temperature is 35 F. The menthol crystals produced here are separated by
decantation. The decanted liquid is passed to the second crystallizer and the
crystals obtained in this second stage are also separated from the liquid by
decantation. Thus, the assumptions made are: (i) limonene is present in all the
fractions, and (ii) the separation of solid material from the liquid portion is com-
plete. Furthermore, the thermophysical properties of menthone and menthyl
acetate are included in the softwares database. The feed stream conditions
are: temperature, 80 F; pressure, 1 atm; and fow rate, 50 lb mol/h.
Table 1: Major constituents of mentha oil
Constituent Concentration
Menthol 75%
Menthyl acetate 11%
Limonene 8%
Menthone 6%
CR1
SO1
SO2
CR2
S1
S3
S2
S7
S8
S4
S5
S6
S9
Figure 6: Flowsheet for the menthol crystallization process
64

a b
Figure 7: Variation of menthol ow rate (Y axis, lb mol/hr) as product of both crystallizers
(S6 + S7) versus T (X axis, F). a) Crystallizer 1. b) Crystallizer 2
Figures 7a and b show on the Y axes the total fow rate (lb mol/
hr) of menthol crystal produced in both crystallizers (the combination of S6+S7
of Figure 6). It is evident from the sensitivity analysis that the temperature of
the crystallizers has an effect on the total amount of pure product obtained,
and consequently on the product yield. In fact, the base case (Table 2) reports
a fgure of 28.469 lb mol/hr and this amount can be raised linearly if the tem-
perature of the crystallizers is lowered. The sensitivity analysis helps the engi-
neer to select the right temperatures of the crystallizers for a given production.
The material and energy balance of the menthol extraction plant
for the base case is listed in Table 2. This is an example of the simulators
output and these values may change if the process conditions are changed. In
the frst crystallizer, the menthol produced is equal to 5.633 lb mol/hr (purity
one) and in the second crystallizer the amount is 22.836 lb mol/hr (purity
one). If we compare the total amount of menthol produced by the two crystal-
lizers (22.836 + 5.633 = 28.469 lb mol/hr) with the total amount of menthol
fed to the process (37.5 lb mol/hr), we obtain a recovery (amount of pure
menthol produced/total amount of menthol fed to the process) of 75.91%,
which is rather satisfactory. Table 2 also reports the temperature, pressure,
total fow and composition of each single stream considered in the process.
Table 2: Material and energy balance for the menthol crystallization process.
The stream names refer to Figure 6
Stream name S1 S2 S5 S6 S7 S8 S9
Phase Liquid S/L S/L Solid Solid Liquid Liquid
Temperature F 80 35 18 18 35 35 18
Pressure PSIA 14.696 14.696 14.696 14.696 14.696 14.696 14.696
Molecular weight 159.170 159.170 161.608 156.270 156.270 161.608 163.004
Total lb mol/hr 50.000 50.000 27.164 5.633 22.836 27.164 21.531
Component mole fractions
D-limene 0.0800 0.0800 0.1472 0.1473 0.1858
Menthol 0.7500 0.7500 0.5399 1.000 1.0000 0. 5398 0.4195
Menthyl acetate 0.1100 0.1100 0.2025 0.2025 0.2554
Menthone 0.0600 0.0600 0.1104 0.1104 0.1393
65
2.4 Conclusions
There are two important benefts of the application of process
simulation to phytochemical processes of industrial interest.
The frst is to improve process knowledge. This is achieved by
verifying in silico the operating conditions and the estimates of data for
intermediate streams, which are diffcult to measure. Process knowledge
also includes: (i) enthalpy balance information, (ii) verifcation of plant speci-
fcations, (iii) infuence of operative parameters on process specifcations,
(iv) validation of phase equilibrium models for the real system to be used in
similar conditions, and fnally (v) process de-bottlenecking for each section.
The second important beneft is process optimization, in terms
of: (i) consumption of energy and raw materials, (ii) identifcation of process
control strategies, and (iii) clarifcation of security system behavior when
process conditions are varied.
Running process simulation software requires: (i) availability of
thermodynamic properties for all components involved, (ii) defnition of an
accurate thermodynamic model (equations of state or excess Gibbs energy
model) for binary and multi-component mixtures, (iii) availability of all neces-
sary interaction parameters, (iv) availability of all necessary unit operation
modules, and (v) identifcation of tear streams to achieve rapid convergence
in case of recycles. Furthermore, sometimes it is necessary to defne and
develop user models and user thermodynamic models.
It is necessary to stress some important principles. First, the
program is an aid in making calculations and decisions: the process en-
gineer must ensure that it is ft for purpose and is responsible for the
results generated and for any use which is made of the results. Second, it is
the professional, ethical and legal responsibility of the process engineer to
take care and to exercise good judgment. Process simulation is, in essence,
a program.
Nonetheless, process simulation is important since it: (i) has
high accuracy, (ii) allows one to focus on the interpretation of the results
rather than on the methods for obtaining the results, (iii) allows a global vi-
sion of the process by assembling theories and models, (iv) is essential in
the design of new and existing processes, (v) is essential in the analysis of
existing plants in terms of environmental impact, and (vi) is a simple tool for
treating real cases.
Process simulation is a well established tool in the chemical
industry and has been used for a decade in the petrochemical industry.
Process simulation is now applicable in many different felds besides the
66
petrochemical and fne chemical industries, and is particularly interesting
for biotechnological and phytochemical processes.
In summary, process simulation may play an important role in
the optimization of phytochemical processes and, therefore, application of
process simulation can assist in the development of advanced processes.
This paper showed that it is possible to achieve energy reduction and maxi-
mization of product yield.
Process simulation perhaps is not a good tool for countries
that are using primitive techniques, since they should aim at reaching a
stable quality of the product rather than an optimization of energy consump-
tion and environmental issues. Moreover, they should focus on practical
problems such as the quality of the materials and the water to be used for
extraction.
Bibliography
Hazra, P. and Kahol, A. P., 1990, Improved process for production of liquid menthol by
catalytic reduction of menthone, Research and Industry, 35: 174-176
Prausnitz, J. M., Lichtenthaler, R. N. and Gomes de Azevedo, E., 1998, Molecular
Thermodynamics of Fluid Phase Equilibria, 3rd Edition, Prentice Hall
Turton, R., Bailie, R., Whiting, W. and Schweitz, J., 2003, Analysis, Synthesis and
Design of Chemical Processes, 2nd Edition, Prentice Hall
67
Maceration, Percolation and Infusion 3
Techniques for the Extraction of
Medicinal and Aromatic Plants
J. Singh
Abstract
Techniques of maceration, percolation and infusion have been traditionally used for
making galenicals and tinctures from medicinal and aromatic plants (MAPs). This ar-
ticle describes the underlying principals and mechanisms of these extraction tech-
niques, and discusses the various modications made for the small- and large-scale
extraction of MAPs, the factors affecting the extraction process, and the quality of the
extracts produced.
3.1 Introduction
Before the nineteenth century, there was no real progress in
methods of extraction of plant materials for industrial use. Nonetheless, the
various classes of preparations involving simple expression and extraction
techniques were in vogue for a long time for the preparation of medicines
used in traditional medicine and in complimentary and alternative medicine,
practiced throughout the world. The techniques available were limited to ex-
pression, aqueous extraction and evaporation; later on, the use of extraction
processes was extended by using alcohol as a solvent. Such techniques
were highly successful in the phytochemical feld and, consequently, single
pure molecules were isolated for industrial and medicinal uses. After the
nineteenth century, rapid progress was made in extraction processes which
led to the isolation and characterization of many groups of plant metabo-
lites of therapeutic importance, including both single chemical constituents
as well as standardized extracts of crude drugs. In manufacturing various
classes of medicinal plant preparations, such as decoctions, infusions, fuid
extracts, tinctures, semisolid extracts (pilular) and powdered extracts, popu-
larly known as galenicals, both simple traditional methods and advanced
technologies are used, conforming to the offcial procedures and specifca-
tions as laid down in various pharmacopoeias and codices of the world.
Maceration, percolation and infusion are the general tech-
niques used for the extraction of medicinal plants and are mostly applied
for galenical preparations. The sole purpose of such basic extraction pro-
cedures is to obtain the therapeutically desirable portion and eliminate the
inert material by treatment with a selective solvent known as menstruum.
These techniques also play a decisive role in the qualitative and quantita-
tive evaluation of the extracts. The standardized extracts thus obtained are
further processed for inclusion in other solid and semisolid herbal dosage
3 MACERATION, PERCOLATION AND INFUSION TECHNIQUES FOR THE EXTRACTION OF MEDICINAL AND AROMATIC PLANTS
68
forms. These extracts are also used as sources of therapeutically active
chemical constituents for various dosage forms of modern medicines.
Historically, galenical preparations were much more extensively
used than they are at the present time. Nonetheless, due to resurgence in
interest of herbal drugs throughout the world, these extraction procedures
are still relevant and are mentioned in offcial and unoffcial monographs
about drug preparations. The preparations involving these procedures are
primarily intended for extemporaneous dispensing and must be freshly pre-
pared, due to the fact that they rapidly produce a deposit because of coagu-
lation of inert colloidal material and readily support microbial growth due to
absence of preservatives.
This article describes the principal methods of extraction by
maceration, percolation and infusion as well as the modifcations in these
procedures for small-scale, offcial and large-scale extraction. In addition,
the paper discusses the choice of extraction method, quality assurance, and
factors affecting the extraction procedures.
3.2 General Principles and Mechanisms Involved
in Crude Drug Extraction by Maceration,
Percolation and Infusion
The general principles and mechanisms involved in macera-
tion, percolation and infusion for the extraction of the crude drugs are same
as to those for the extraction of soluble constituents from solid materials
using solvent, which is generally referred to as leaching. The processes of
leaching may involve simple physical solution or dissolution. The extraction
procedures are affected by various factors, namely the rate of transport of
solvent into the mass, the rate of solubilization of the soluble constituents
by the solvent, the rate of transport of solution out of the insoluble material.
The extraction of crude drugs is mostly favored by increasing the surface
area of the material to be extracted and decreasing the radial distances
traversed between the solids (crude drug particle). Mass transfer theory
states that the maximum surface area is obtained by size reductions which
entail reduction of material into individual cells. However, this is not possible
or desirable in many cases of vegetable material. It has been demonstrated
that even 200 mesh particles contain hundred of unbroken cells with intact
cell wall. Therefore, it is pertinent to carry out extraction with unbroken cells
to obtain an extract with a high degree of purity and to allow enough time for
the diffusion of solvent through the cell wall for dissolution of the desired
solute (groups of constituents) and for diffusion of the solution (extract) to
the surface of the cell wall.
69
3.3 Factors Affecting the Choice of Extraction
Process
The choice of the process to be used for the extraction of a
drug depends on a number of factors.
3.3.1 Nature of the Crude Drug
The choice to use maceration or percolation primarily depends
upon the nature and characteristics of the crude drugs to be extracted.
Therefore, knowledge of the type of organs and tissues of the plant matter
is essential for achieving the best result.
3.3.2 Stability of the Crude Drug
Continuous hot extraction procedures should be avoided when
constituents of the drug are thermolabile.
3.3.3 Cost of the Crude Drug
When the crude drug is expensive (e.g. ginger), it is desirable
to obtain complete extraction. Therefore, from the economic point of view,
percolation should be used. For inexpensive drugs, maceration, despite its
lower effciency, is acceptable in view of its lower cost.
3.3.4 Solvent
Selection of the solvent depends on the solubility of the de-
sired components of the material. If the constituents demand a solvent
other than a pure boiling solvent or an azeotrope, continuous extraction
should be used.
3.3.5 Concentration of the Product
Dilute products such as tinctures can be made by maceration
or percolation. For semi-concentrated preparations, the more effcient per-
colation process is used. Concentrated preparations, such as liquid or dry
extracts, are made by percolation.
3.3.6 Recovery of Solvent
Solvent is preferably recovered under reduced pressure to save
thermolabile constituents.
70
3.4 Quality Assurance: the Extraction Process
and Solvent
The type of extraction procedure also plays a decisive role in
determining the qualitative and quantitative composition of the extract. Some
important points regarding the quality of the extracts need to be considered:
The more exhaustive the extraction, the better is the yield of i)
the constituents from the herbal drugs.
If maceration is facilitated by stirring and by use of commi- ii)
nuted material, the additional stirring and shearing forces
may lead to better extraction.
Other factors determining the quality of the extracts are ex- iii)
traction time, temperature and solvent volume.
Some drugs (e.g. Hypericum spp.) are extracted very slowly iv)
so that exhaustive extraction can only be achieved by per-
colation or multistage motion extraction. In many cases,
the transfer of quality-relevant constituents from the herbal
drugs to the extract (i.e. extraction rate) can be considerably
improved by raising the temperature. Hypericin, pseudohy-
pericin and biapigenin are extracted better at higher tem-
perature and with longer extraction times.
The quality of the extracts and the spectrum of constituents v)
obtained by maceration or digestion (i.e. maceration at higher
temperature) are also infuenced by the ratio of herbal drug
to solvent. The quantity of extracted matter increases with
the volume of extraction solvent. For example, maceration of
Salvia offcinalis fowers achieves almost exhaustive extrac-
tion and thus the full spectrum of constituents obtained with
percolation can be achieved with a drug:solvent ratio of 1:20.
The composition of an herbal extract depends on the type, vi)
concentration and elution strength of the solvent. The spec-
trum of constituents may vary considerably depending on
the hydrophilic or lipophilic nature of the solvent.
3.5 Maceration Processes (Steady-state
Extraction)
3.5.1 General Procedure
The general process of maceration on a small scale consists
of placing the suitably crushed plant material, or a moderately coarse pow-
der made from it, in a closed vessel and adding the selected solvent called
menstruum. The system is allowed to stand for seven days, with occasional
shaking. The liquid is then strained off and the solid residue, called marc,
71
is pressed to recover as much occluded solution as possible. The strained
and expressed liquid thus obtained is mixed and clarifed by fltration. Plant
material in fne powder form is never used, as it makes subsequent clarifca-
tion of the extract diffcult. In the case of vegetable drugs, suffcient time
is allowed for the menstruum to diffuse through the cell wall to solubilize
the constituents within the cells and for the resulting solution to diffuse
out. As the system is static, except for occasional shaking, the process of
extraction works by molecular diffusion, which is very slow. Occasional shak-
ing assists diffusion and also ensures dispersal of the concentrated solu-
tion accumulating around the surface of the particles, thereby bringing fresh
menstruum to the particle surface for further extraction. A closed vessel is
used to prevent evaporation of the menstruum during the extraction period
and thus avoids batch to batch variation.
At the end of the maceration process, when equilibrium has
been reached, the solution is fltered through a cloth; the marc may be
strained through a special press. The concentrations of active constitu-
ents in the strained and expressed liquids, sometimes called miscella, are
the same and hence they can be combined. The expressed liquid may be
cloudy with colloidal and small particles, and suffcient time (perhaps sev-
eral weeks) is necessary for coagulation and settling. The settled matter is
fltered through a flter press or any other suitable equipment.
3.5.2 Maceration Process for Organized and Unorganized
Crude Drugs
Organized drugs have a defned cellular structure whereas un-
organized drugs are non-cellular. Bark and roots are examples of organized
crude drugs, while gum and resin are unorganized crude drugs. The proc-
esses of maceration for organized and unorganized drugs are slightly differ-
ent, as shown in Table 1.
Table 1: Four differentiating steps of the maceration process, for organized and
unorganized crude drugs
Organized drugs Unorganized drugs
(i) Drug + entire volume of menstruum
(i) Drug + four-ffths of
menstruum (in most cases)
(ii) Shake occasionally for 7 days
(ii) Shake occasionally on days 2
to 7, as specifed
(iii) Strain liquid, press the marc
(iii) Decant the liquid. Marc is
not pressed
(iv) Mix the liquids, clarify by subsidence for
fltration. Filtrate is not adjusted for volume
(iv) Filter the liquid and add
remaining menstruum through
the flter
72
During maceration of organized drugs, the marc is pressed be-
cause a considerable proportion of liquid adheres to it and cannot other-
wise be separated. Moreover, the volume is not adjusted because a variable
amount of liquid containing soluble matter is left in the marc. If the volume
is adjusted, a weak product will result. Omitting adjustment, the volume of
liquid expressed infuences the product yield and the percentage of soluble
matter, regardless of the effciency with which the marc is pressed in a hand
press, screw press or hydraulic press; the strength of the product is not
affected. Preparations made by this processes include vinegar of squill (Brit-
ish Pharmaceutical Codex, BPC), oxymel of squill (BPC), tincture of orange
(Indian Pharmacopoeia, IP), tincture of capsicum (BPC), compound tincture
of gentian, tincture of lemon, and tincture of squill (BPC).
In maceration of unorganized drugs, the marc is not pressed
because the desirable material is mostly dissolved and the remaining marc
is gummy and slimy. Thus, it is neither practicable nor necessary to press
it. Moreover, the volume is adjusted because the clear upper layer is easily
separated by fltration from the lower layer. The solution contains practically
all the soluble matter of the drug; the small amount adherent to the gummy
matter is recovered when the marc is washed by menstruum in the flter.
Therefore, adjustment of volume leads to uniformity. Preparations made by
this process include compound tincture of benzoin, tincture of myrrh (BPC),
and tincture of tolu (BPC).
3.5.3 Modifcations to the General Processes of
Maceration
Repeated maceration may be more effcient than a single mac-
eration process, as described earlier, because an appreciable amount of
active principle may be left behind in the frst pressing of the marc. Double
maceration is used when the active constituents are particularly valuable
and also when the concentrated infusions contain volatile oil. Where the
marc cannot be pressed, a process of triple maceration is sometimes em-
ployed. The total volume of solvent used is, however, large and the second
and third macerates are usually mixed and evaporated before being added
to the frst macerate.
3.5.4 Large-scale Extraction Procedures
For large-scale, industrial extraction, certain modifcations are
warranted. When the extraction vessel contains a small amount of solvent
(500-1000 ml), occasional shaking is no problem. But, for industrial work
where a large amount of solvent and huge vessels are involved, shaking the
vessels is diffcult. Obviously, there are alternative methods of agitation that
are just as effective and much simpler to put into practice. In addition, eco-
nomics become increasingly important and one of the most important objec-
tives is to improve the effciency of extraction so that less solvent is needed
73
and evaporation requirements for concentrated products are reduced. Reduc-
ing the cost of evaporation has the further advantage of minimizing the heat
damage to thermolabile constituents. Some of the modifed maceration pro-
cedures used for large-scale extraction are described in the next paragraphs.
3.5.4.1 Circulatory Extraction
The effciency of extraction in a maceration process can be
improved by arranging the solvent to be continuously circulated through the
drug, as indicated in the Figure 1. Solvent is pumped from the bottom of the
vessel (through an outlet) and is distributed by spray nozzles over the sur-
face of the drug. The movement of the solvent reduces boundary layers and
the uniform distribution minimizes local concentration in a shorter time.
Figure 1: Circulatory extraction
3.5.4.2 Multistage Extraction
In the normal maceration process, extraction is incomplete,
since mass transfer ceases when equilibrium is reached. This problem can
be overcome using a multistage process. The equipment needed for this
method is a vessel for the crude drug, a circulating pump, spray distributors
and a number of tanks to receive the extracted solution. The extractor and
tanks are connected with piping and valves as shown in Figure 2, so that any
of the tanks may be connected to the extractor for transfer of the solution.
Each batch of drug is treated several times with solvent and, once a cycle
is in process, the receivers contain solution with the strongest in receiver 1
and the weakest in receiver 3.
74
3.5.4.2.1 Advantages
The crude drug is extracted as many times as there are receiv-
ers (in Figure 2 there are three receivers). If more extraction stages are
required, it is only necessary to have more receivers. The last treatment of
the drug before it is discharged is with fresh solvent, giving maximum
extraction. The solution is in contact with fresh drug before removal for
evaporation, giving the highest possible concentration.
3.5.4.2.2 Procedure
Fill the extractor with crude drug, add solvent and circulate.
Run off to receiver 1. Refll the extractor with solvent and circulate. Run off
to receiver 2. Refll the extractor with solvent and circulate. Run off to re-
ceiver 3. Remove drug from the extractor and recharge. Return solution from
receiver 1 to the extractor. Remove for evaporation. Return solution from
receiver 2 to the extractor and circulate. Run off to receiver 1. Return solu-
tion from receiver 3 to the extractor and circulate. Run off to receiver 2. Add
fresh solvent to the extractor and circulate. Run off to receiver 3. Remove
drug from the extractor and recharge. Repeat cycle.
Figure 2: Multistage extraction
3.5.4.2.3 Extraction Battery
In the normal percolation process, the percolate is a very dilute
solution, while the ideal situation is to obtain the maximum concentration
possible. Continuous extraction devices of battery type are used when large
amounts of a single material are handled. Such devices can be achieved by
treating percolation as a multistage process. In an extraction battery proc-
ess, a series of vessels is used and extraction is semicontinuous.
3.5.4.2.4 Equipment
An extraction battery consists of a number of vessels with in-
terconnecting piping. Vessels are so arranged that solvent can be added
to and the product taken from any vessel. These vessels can, therefore, be
75
made into a series with any of vessels as the frst of the series. The use
of an extraction battery is illustrated in Figure 3, which shows the simplest
arrangement of three vessels.
2
2
2
2
2
2
1
1
3
3
3
3
3
3
4
4 S
4 S
4 S
E
4 E
4 E
P
1 P
1 P
F
1 F
1 F
A B C
A B C
A B C
A B C
A B C
A B C
2.
1.
3.
4.
5.
6.
1. Charee fresh drue to C.
2. Transfer solutlon from 8 to C.
3. Transfer solutlon from A to 8.
4. Add fresh solvent to A.
1. kemove product solutlon from C.
4. 0ump exhausted drue from A.
1. kemove product solutlon from A.
2. Transfer solutlon from C to A.
4. 0ump exhausted drue from 8.
1. kemove product solutlon from 8.
4. 0ump exhausted drue from C.
1. Charee fresh drue to 8.
4. Add fresh solvent to C.
1. Charee fresh drue to A.
4. Add fresh solvent to 8.
Arrows lndlcate transfer of materlal to or from the vessels. An asterlsk(.) ls added after
each contact staee. Key: F = fresh drue. S = solvent. P = product. F = exhausted drue.
A, 8, C, extractlon vessels. Start wlth C empty and unlts A and 8 contalnlne drue, wlth
A more nearly exhausted.
Figure 3: Extraction battery
3.6 Percolation (Exhaustive Extraction)
3.6.1 General Process of Percolation
In this process, an organized vegetable drug, in a suitably pow-
dered form, is packed in a percolator and the solvent is allowed to percolate
through it. Although some materials (e.g. ginger) may be packed directly into
the percolator in a dry state, this may cause diffculties with other drugs. With
the addition of solvent, the dry material swells and this swelling increases
with increasing aqueous nature of the solvent. This swelling reduces or blocks
the fow of the solvent, thus seriously affecting the extraction process. Fur-
76
thermore, if the dry powder is packed, fne particles may be washed down the
column and settle at the lower levels, reducing the porosity drastically, blocking
the column and making the column nonuniform. The fner particles may even
be washed out of the column. These diffculties can be prevented by a prelimi-
nary uniform moistening of the raw material with the menstruum for a period
of 4 h in a separate closed vessel; this process is called imbibition. During
this period, the crude drug is allowed to swell to the maximum extent. Hence,
when aqueous solvents are used for extraction, more menstruum is needed
during imbibition. Also, the occluded air in the drug powder is replaced by the
vapor of the solvent, thereby enabling the material to be more evenly packed
and allowing the menstruum to fow more uniformly. Uneven packing permits
more solvent to pass through channels offering less resistance to the fow of
the solvent, thus resulting in ineffcient extraction. After imbibition, the drug is
packed evenly into the percolator. The imbibed drug is packed over a loose plug
of tow or other suitable material previously moistened with the solvent. Even
packing can be achieved by introducing the material layer by layer and pressing
it with a suitable implement to give even compression; the pressure exerted on
the material depends on the nature of the material and its permeability. After
packing is over, a piece of flter paper is placed on the surface followed by a
layer of clean sand such that the top layer of the drug is not disturbed when
solvent is added for extraction. Suffcient menstruum is now poured over the
drug slowly and evenly to saturate it, keeping the tap at the bottom open to
allow the occluded gases between particles to pass out. Menstruum should
never be poured with the tap closed since the occluded air will escape from
the top, disturbing the bed. When the menstruum begins to drip through the
tap, the tap is closed; suffcient menstruum is added to maintain a small layer
above the drug and allowed to stand for 24 h. The layer of menstruum above
the surface of the bed prevents drying of the top layer, which may result in the
development of cracks on the top surface of the bed. The 24-h maceration pe-
riod allows the solvent to diffuse through the drug, solubilize the constituents
and leach out the soluble material. In this way, the extraction is more effcient
than carrying out percolation without the maceration period.
After the maceration, the outlet is opened and the solvent is
percolated at a controlled rate with continuous addition of fresh solvent. The
volume of percolate collected depends on the nature of the fnal product. In
general, about 75% of the volume of the fnished product is collected, the
marc is pressed and the expressed liquid is added to the percolate, giving
about 80%-90% of the fnal volume. After assay, the volume is adjusted with
calculated quantities of fresh menstruum. If no assay is available, the volume
is adjusted after adding the other constituents, if any. In percolation, the ex-
pressed liquid is devoid of active constituents as they are already extracted
during the percolation period; pressing the marc is only to recover the valuable
solvent. This is in contrast with maceration in which the marc is pressed.
77
3.6.2 Modifcations to the General Process of Percolation
In the general process of percolation, particularly in the manu-
facture of concentrated preparations like liquid extracts, the following prob-
lems may arise:
If the active substances are thermolabile, evaporation of
large volumes of dilute percolate may result in partial loss
of the active constituents.
In the case of alcohol-water mixtures, evaporation results
in preferential vaporization of alcohol, leaving behind an al-
most aqueous concentrate. This may not be able to retain
the extracted matter in solution and hence the substances
may precipitate.
In such cases, the general process of percolation is modifed,
as described in the next paragraphs.
3.6.2.1 Reserved Percolation
In this case, extraction is done through the general percolation
procedure. At the end, the evaporation is done under reduced pressure in
equipment like a climbing evaporator to the consistency of a soft extract
(semisolid) such that all the water is removed. This is then dissolved in the
reserved portion, which is strongly alcoholic and easily dissolves the evapo-
rated portion with any risk of precipitation.
3.6.2.2 Cover and Run Down Method
This is a process that combines the maceration and percola-
tion techniques. This process cannot be used for materials that contain
volatile principles or for those which undergo change during the evaporation
stage. This procedure is advantageous because industrial methylated spirit
may be used for extraction instead of the costly rectifed spirit.
The detailed procedure is as follows. After the imbibition stage,
the material is packed in a percolator and macerated for a few hours with a
suitable diluted industrial methylated spirit. Then, the liquid is run off and the
bed is covered with more menstruum. Maceration is done as before and the
second volume of the extract is collected. This process is repeated several
times with the later weaker extracts used for extraction of a fresh batch of the
drug. More concentrated fractions are evaporated under reduced pressure to
eliminate the toxic methanol. After the concentrate is assayed for the active
principle or for total solids content, it is diluted with water and ethanol to ob-
tain the correct concentration of alcohol and active principle.
78
3.6.3 Percolators
Different types of percolators are used for small- and large-
scale extraction.
3.6.3.1 Small-scale or Laboratory-scale Extraction
The processes for the manufacture of concentrated preparations,
maceration and percolations, are involved in extraction followed by the evapora-
tion of solvents. The two operations are combined in a continuous extraction
process.
The general procedures and apparatus used for small- or labo-
ratory-scale extraction are described in the following paragraphs.
3.6.3.1.1 Soxhlet Apparatus
On the laboratory scale, the Soxhlet apparatus is used. It con-
sists of a fask, a Soxhlet extractor and a refux condenser. The raw material
is usually placed in a thimble made of flter paper and inserted into the wide
central tube of the extractor. Alternatively, the drug, after imbibition with men-
struum, may be packed in the extractor taking care that the bottom outlet
for the extract is not blocked. Solvent is placed in the fask and brought to
its boiling point. Its vapors pass through the larger right hand tube into the
upper part of the extractor and then to the condenser where they condense
and drop back onto the drug. During this period, the soluble constituents
are extracted. When the level of the extract reaches the top of the syphon
tube, the entire volume of extract syphons over into the fask. The process
is continued until the drug is completely extracted. The extract in the fask is
then processed. This procedure is thus a series of short macerations.
Figure 4: Soxhlet apparatus for hot extraction
79
3.6.3.1.2 Offcial Extractor
An offcial extractor, as described in the offcial monographs
(e.g. IP, British Pharmacopoeia), is illustrated in Figure 5. In this case, the
extraction is a continuous percolation procedure. In this apparatus, vapors
rise through the extraction chamber passing the drug container; the vapor
condenses in the refux condenser and returns through the drug, taking the
soluble constituents to the fask.
Figure 5: Apparatus for the continuous extraction of drugs
3.6.3.2 Limitations of the Offcial Extractor
It is not useful when the raw material contains thermolabile
active constituents, because the extraction is carried out at an elevated
temperature and the extract in the fask is also maintained in the hot condi-
tion until the process is complete. It can be used only with pure solvents
or with solvent mixtures forming azeotropes. If an ordinary binary mixture is
used as the menstruum, the composition of the vapor will be different from
the liquid composition.
Similar methods can be used in large-scale production.
A typical industrial set-up for continuous extraction is shown in
Figure 6. The principle of operation resembles that of the laboratory equip-
ment.
80
Figure 6: Continuous extraction: large-scale plant
3.6.3.3 Large-scale Extractor
The drug is supported on a perforated metal plate covered with
a layer of sacking or straw. The percolator has a removable lid which contains
portholes for running the solvent in and for observing the fow of solvent.
The outlet from the percolator is ftted with a tap and pipeline. This outlet
permits removal of the percolate for subsequent processing or for use as a
menstruum in a second percolator in series, resulting in more effcient use
of the menstruum by carrying out the extraction in a counter-current man-
ner. On the small scale, copper percolators were originally used but these
are now largely replaced with percolators made of glass or stainless steel.
A slightly conical percolator is better than a cylindrical one since the sloping
sides permit the eventual expansion of the bed and also allow the solvent
to permeate the material present near the sides at the bottom, which is a
problem with a cylindrical percolator.
Figure 7: Commercial scale percolator
81
3.7 Infusion
3.7.1 General Considerations
Infusions are dilute solutions containing the readily soluble
constituents of crude drugs. Formerly, fresh infusions, prepared by macerat-
ing the drug for a short period in cold or boiling water, were used and diluted
to eight volumes but, now, infusions are usually prepared by diluting one
volume of a concentrated infusion to ten volumes with water. Concentrated
infusions are prepared by a modifed percolation or maceration process.
After dilution with water, concentrated infusions resemble in potency and
aroma the corresponding fresh infusion. Infusions are liable to fungal and
bacterial growth, and it is necessary to dispense them within 12 h of their
preparation.
3.7.2 General Method for Preparing Fresh Infusions
The coarsely powdered crude drug (50 g) is moistened, in a
suitable vessel with a cover, with 50 ml cold water and is allowed to stand
for 15 min. Then, 900 ml boiling water is added, and the vessel is covered
tightly and allowed to stand for 30 min. The mixture is strained and enough
water is passed to make the infusion measure 1000 ml. Some drugs are
supplied (accurately weighed) in muslin bags for preparing specifc amounts
of infusion and as such are used for infusion preparation. If the activity of
the infusion is affected by the heat of the boiling water, cold water should be
used. As fresh infusions do not keep well, they should be made extempora-
neously and in small quantities at the time of use.
3.7.3 Preparation of Concentrated Infusions
The offcial monographs also recognize certain concentrated
infusions in which 25% alcohol is added during or after the infusion process
and then diluted as per pharmacopoeial (offcial) requirement. Concentrated
infusions are especially prepared in cases in which the active and desirable
principles of drug are equally soluble in water and in the menstruum used
for both concentrates and infusions.
3.8 Evaporation
One quality-relevant parameter is the evaporation of the eluate
from the soft extract. The state of art is cautious vacuum evaporation, in
which evaporation temperatures do not exceed 55 C. The temperature in
relation to the evaporation time is of special importance for quality of this
step, especially if the extract contains volatile or thermolabile constituents.
82
3.9 Conclusions
The spectrum of constituents obtained by steady-state extrac-
tion (simple maceration) differs from that obtained by exhaustive extraction
(percolation). With maceration, one can achieve a spectrum of constituents
similar to that of percolation. Different extraction procedures may be con-
sidered to be equivalent if they respect critical quality parameters and if the
analysis of numerous production batches confrms their compliance with
standards.
Bibliography
Anonymous, 1955, Indian Pharmacopoeia, the Manager of Publication, Delhi, p.
273
Anonymous, 1973, British Pharmaceutical Codex, the Pharmaceutical Press, London,
p. 703-704
Anonymous, 1980, British Pharmacopoeia, VOL. II, University Press, Cambridge, Lon-
don, p. 576
Anonymous, 2002, Bentleys Text book of Pharmaceutics EA Rawlins (Ed.). Reprint,
Bailliere Tindall, London/All India Traveller Book Selter, New Delhi
Cooper, J. W. and Gunn, C., 1975, Tutorial Pharmacy, S. J., Carter Reprint, CBS Pub-
lication, Delhi, p. 251- 261
Cooper, J. W. and Gunn, C., 1985, General Pharmacy, CBS Publishers and Distribu-
tors Delhi, p. 308-333
Evans, W. C., 1998, Trease and Evans Pharmacognosy (14th Edition), W. B. Saun-
ders Company Limited, London, p. 119
Sambamurthy, K., 2002, Pharmaceutical Engineering Reprint, New Age International
(P.) Ltd., New Delhi, p. 173-194
Singh, J., Bagchi, G. D., and Khanuja, S. P .S., 2003, Manufacturing and quality
control of Ayurvedic and herbal preparations, In: Verpoorte, R. and Mukherjee,
P. K.(Eds), GMP for Botanicals, Regulatory and Quality Issues on Phytomedicine
(1st Edition), Business Horizons, New Delhi, p. 201-230
Waldesch, F. G., Konigswinter, B. S., and Blasius, H., 2003, Herbal Medicinal Prod-
ucts, Medpharm, Stuttgart, Germany and CRS Press, London, p. 48-54
83
Hydrolytic Maceration, Expression 4
and Cold Fat Extraction
A. K. Singh
Abstract
The incorporation of bioactive ingredients without loss of activity into foods, avors,
pharmaceuticals, pesticides and cosmaceutical products is very important. Extraction
of active constituents from raw materials is an important and critical step in main-
taining bioactivity. A number of methods are available for extraction, and these are
selected in such a way that the activity of the phytoconstituents is retained. This paper
discusses the processes of hydrolytic maceration, expression and cold fat extraction.
4.1 Introduction
The extraction of active constituents from plants is one of the
most critical steps in the development of natural products for commercial
use. The simplest example of extraction may be brewing a cup of coffee,
wherein caffeine and tannins are extracted from coffee beans in hot water.
All living organisms contain complex mixtures of chemicals, usually held
within cellular structural material (protein, lipid, polysaccharides etc.) of
which some are desired while others are not. Thus, taking out the desired
part from the whole crude drug is referred to as extraction and it is done in
solvents where ingredients move from one phase to another.
A number of methods are available for extraction and the choice
among them is dictated by the physicochemical properties and stability of
the phytoconstituents to be obtained. For the extraction of essential oils,
the simplest methods are hydrodistillation and steam distillation while other
methods also employed are cold fat extraction, expression, maceration and
solvent extraction. Nowadays, more advanced technologies are used, such
as supercritical fuid extraction, solid phase micro-extraction and phytonic
extraction. The present article deals with extraction by hydrolytic macera-
tion, expression and cold fat extraction.
4.2 Hydrolytic Maceration
The word maceration is derived from the Latin word macera-
tus, which means to soften. In reference to medicinal and aromatic plants,
maceration refers to the preparation of a solution by soaking plant material
in vegetable oil or water. Maceration methods are based on the immersion
of crude drug in bulk solvent, while percolation methods depend on the fow
4 HYDROLYTIC MACERATION, EXPRESSION AND COLD FAT EXTRACTION
84
of solvent through the powdered drug. The rate of extraction depends upon
the following:
The rate of transport of solvent into the mass to be leached.
The rate of solubilization of soluble constituents into solvent.
The rate of transport of solute out of the insoluble material
and from the surface of the insoluble material into the solu-
tion.
In the process of maceration, the powered solid material is
placed in a closed vessel (to prevent evaporation) and the chosen solvent
(menstruum) is added. It is allowed to stand for a long time (varying from
hours to days) with occasional shaking. Suffcient time is allowed for the
menstruum to diffuse through the cell wall to solubilize the constituent
present in the cells and for the resulting solution to diffuse out. The proc-
ess takes place only by molecular diffusion. After the desired time, the
liquid is strained off; the solid residue (marc) is pressed to recover as
much solvent as possible. When the menstruum is water and the period
of maceration is long, a small quantity of alcohol may be added to prevent
microbial growth.
Cold maceration of crushed grapes is done at room tempera-
ture before the onset of fermentation. In this process, the skin and seeds
are permitted to soak for one to two days prior to the initiation of fermenta-
tion to get more aqueous extraction without the effect of ethanol on grape
cells. In certain cases, hydrolysis is done with a suitable agent (enzyme)
prior to maceration, e.g. maceration of wine. The quality of wine is judged
by its appearance, color, aroma, taste (mouth feel) and favor. Grape-derived
aroma and favor precursors exist partially as non-volatile, sugar-bound gly-
cosides. Hydrolysis modifes sensory attributes and potentially enhances
wine quality. Flavored aglycones potentially affect the wine quality after hy-
drolysis. Cell wall-degrading enzymes help in the release of grape aroma,
when cold maceration prior to fermentation has been carried out.
4.3 Expression Extraction of Essential Oils
Most essential oils are isolated from the respective plant parts
by the process of hydrodistillation or steam distillation. A few essential oils
such as those present in the citrus fruit peel can be, and in large part are,
obtained by pressing, which yields a product of superior quality. The long
action of heat with boiling water affects some thermolabile constituents
which may decompose due to hydrolysis, polymerization and resinifcation.
Therefore, the essential oil obtained by distillation does not represent the
natural oil as it originally occurred in the plant material. In such plant ma-
terials, essential oil is extracted by the process of expression or solvent
extraction.
85
Before dealing with the process of expression in citrus oil, it is
important to review the structure of a citrus peel. The citrus peel contains
numerous oval, balloon-shaped oil sacs and glands or vesicles (0.4-0.6 mm
diameter). These are ductless glands which are irregularly distributed in the
outer colored (favedo) portion of the peel of maturing and matured fruit,
in the outer mesocarp beneath the epicarp and hypoderm and above the
inner mesocarp (albedo), respectively. Albedo is made of cellulose, hemicel-
lulose, lignin, pectin, sugars, glycosides, etc. On maturation, cells of albedo
become elongated and branched, with large intracellular spaces which give
the ripe peel its spongy texture. The spongy layer plays an important role
in the expression of oil, but it easily absorbs the oil ejected from the sacs
and causes some mechanical diffculties in oil extraction. The freshness
and stage of maturity affect the oil ejection from the peel. The total weight
of the peel is about half that of the fruit and the oil content is 0.5%-0.7%.
The cells surrounding the oil sac contain salt (colloids) in aqueous solution.
In contact with water, the higher osmotic pressure of the cell results in diffu-
sion of water into them, increasing turgor pressure and causing the oil sac
to stress from every side. If the spongy tissue is not flled with water, it will
absorb the oil as the sacs are broken and hold it with great tenacity. So the
pressure exerted yields frst aqueous fuid and later oil.
In Sicily, peels are immersed in water for several hours before
being subjected to a sponge process. A dilute aqueous solution of salt
acts as a carrier to prevent loss by spurting. The product of expression is
not a simple mixture of oil and water but a thin emulsion which is left to
stand; gradually a supernatant layer forms. Filtration through a sponge ab-
sorbs the colloidal material and leaves the mixture of oil and water.
4.3.1 Process of Expression
4.3.1.1 Hand Process
In this process, the freshly harvested fruits are cut transversely
into two halves. The pulp is removed with a sharp-edged spoon called rastrel-
lo. The peel is then immersed in water for several hours and fnally pressed
by hand. The worker holds, with the left hand, either one large or two smaller
fat sponges on top of a wooden crossbar and, with the right hand, presses
the peel against the upper sponge. Thinner peels can be pressed from in-
side. The emulsion ejected from the oil sacs is soaked by the sponge, which
retains solid matter and absorbs colloidal substances. Squeezing the con-
tent of the sponge from time to time, oil is fnally decanted and drawn off.
This process requires much labor and the yield of this method is 50%-70% of
the total oil present in the peel. The quality of oil obtained from hand press-
ing is near to the quality present in fruit peel. A large number of small units
in Sicily and Calabria were formerly responsible for the entire Italian produc-
tion of lemon and orange oil, but the process is not in use now.
86
4.3.1.2 Ecuelle Process
This process was common in the south of France. Ecuelle con-
sists of a shallow bowl of copper with a hollow central tube with which it
forms a sort of funnel. The bowl is equipped with large brass nails with blunt
ends, on which the fruit is rolled by hand with some pressure until the entire
surface of fruit yields its oil. The oil and aqueous cell contents drain into the
central tube where they are separated by decantation. The yield is only 20%
of the total oil present in the peel.
These methods were quite laborious and, with the advance of
technology, machines have been invented to do them. Nowadays, rinds are
extracted for oil using centrifugal force.
4.3.1.3 Hand Machine
For expression, the peel is placed in a hollow sponge attached
with other sponges to a plate actuated by the lever and fxed below with
additional sponges to the base. The sponge is ftted with a funnel through
which oil and aqueous material pass to the receiving vessel. The part that
comes in contact with oil is made of brass or bronze.
4.3.1.4 Sfumatrici and Pelatrici
The machines that treat only the peel after removal of juice
and pulp are called sfumatrici, while those that process the whole fruit are
known as pelatrici.
4.3.1.4.1 Special Sfumatrice
This is a specially designed roller type machine in which each
peel is bent to expel the maximum quantity of oil. Not much pressure is ex-
erted to expel other contents of the cells. The emulsion is collected and fl-
tered through wool or sponges to yield oil and water. A number of sfumatrici
have been developed and were in use in many countries.
In expression using sfumatrici, two approaches are used. In
the frst approach (used by Ramino Sfumatrice), only the peel is treated, so
halving the fruit, removing the pulp and expressing the peel are the steps
involved. In the second approach, the oil is extracted by either puncturing
the peel glands or cutting a superfcial layer of peel to expose the oil glands;
this is followed by washing away the oil with a spray of water.
In whole fruit extraction with a rasping machine, the whole fruit
is crushed and oil is subsequently separated from the aqueous phase (juice
and cell liquid). As citrus peel oil is a byproduct of the citrus juice industry,
both the oil and juice are extracted subsequently, e.g. in a rotatory juice
87
extractor or Pipkin peel oil press. Here, the fruit is washed automatically,
sorted according to size and then cut in two halves. The halved fruit is
passed between two cylinders which remove only the juice by gentle press-
ing without affecting the oil glands on the surface. The residual halved peels
are extracted for essential oil. In the Pipkin peel oil press, for example, oil is
expressed by two close stainless steel cylinders with capillary grooves run-
ning around the circumference. The expressed oil automatically comes out
of the grooves and there is no need to spray water..
4.3.1.5 Modern Machines
Nowadays, complete mechanization has been achieved and
whole fruit processing machines have been developed. These machines ei-
ther crush whole fruit and then separate the oil from the aqueous phases
by distillation or centrifugation, or they express the oil in such a way that it
does not come in contact with the juice during the process. The oil extractor
developed by Brown International, California, liberates the essential oil from
whole citrus fruit. Oil removal is achieved by lightly puncturing the entire
surface of the fruit with over three million sharp stainless steel points con-
fgured in the form of rotating rolls. An adjustable speed differential between
adjacent rolls controls the amount of pressure applied to the fruit. Whole
fruits roll across the brown oil extractor (BOE, Figure 1), which is made of
toothed rollers partially set in a fowing bed of water which propels the fruit
across the machine and simultaneously punctures the peel to release the
oil from the glands. The fruit proceeds to the extractor and the oil-water mix-
ture goes to centrifugation and to the oil recovery chamber.
Figure 1: Brown oil extractor
4.3.1.6 FMC Whole Fruit Extractor
Food Machinery Corporation (FMC) of San Jos, California, has
developed many designs and improved extractors in which both the juice
and the volatile oil are extracted without getting mixed with each other. Fur-
ther details are available at: http://fles.asme.org/ASMEORG/Communi-
ties/History/Landmarks/5549.pdf.
88
Figure 2: FMC whole fruit extractor
The FMC juice extractor has two inter-meshing jaws which encom-
pass the fruit, crushing it between them. The juice exits through a mesh screen
which penetrates the center of the fruit and the juice is separated from the
peel, pith and seeds. This crushing action is suffcient to force the oil out of the
glands on the surface of the favado. The FMC machine sprays water onto the
surface of the fruit, and the oil-water emulsion is subjected to centrifugation.
Traces of water and waxy material are separated by keeping low temperature.
About 75% of citrus oil production utilizes this technology. The odor of cold
pressed oil is true in nature and similar to that of the oil present in fruit.
4.4 Cold Fat Extraction (Enfeurage)
Certain high-quality odor-producing fowers such as jasmine,
tuberose and gardenia yield small quantities of oil and cannot be distilled
by hydrodistillation. Furthermore, the oil components are thermolabile and
such fowers, even after plucking, continue to emit small quantities of per-
fume. The oil from these types of fowers is extracted by cold fat extraction,
i.e. enfeurage.
Figure 3: Chassis for holding the fat crops
89
Fat possesses a high power of absorption of volatile oil and, if
brought in contact with fragrant fowers, it absorbs the perfume. This princi-
ple methodically applied on large scale constitutes enfeurage. The quality
of the fat base is important for the quality of the fower oil. It must be odor-
less and of proper consistency. If the crops (fat) is too hard, the blossoms
will not have suffcient contact with it, the absorption will be insuffcient,
and the quality and yield will be poor. If the crops is loose, it may engulf the
fowers so that exhausted ones are diffcult to remove and retain adhering
fat when removed. The crops must have a consistency suitable to produce
a semihard surface from which exhausted fowers can be removed easily.
Saturated fragrant fat extract is known as pomade. Enfeurage process of
cold extraction is carried out in Bulgaria, Egypt, Algeria, Sicily (Italy), and
Grasse (France). France remains the main centre of production of highly
prized so-called natural fower oil. Natural fower oil does not include the
distilled essential oil but applies only to fower oils obtained by enfeurage,
maceration or solvent extraction. The whole process is carried out in cold
atmosphere cellars (cold rooms). The mixture of one part highly purifed tal-
low and two parts lard is best suited for enfeurage.
Different bases have been used in the preparation of crops.
For example, vegetable fat, mineral oil, esters of polyhydric aliphatic alcohol
(ester of glycol, glycerol, mannitol, hexitol), and fatty acids of high molecular
weight have been tried, but the best results were from the old-fashioned
mixture of lard and tallow. To purify tallow, it is melted after cleaning, wash-
ing and removing blood and muscles; the skin is beaten and cleaned, and
benzoin (0.6%) and alum (0.15%-0.30%) are added. Benzoin is a preserva-
tive while alum causes impurities to coagulate during heating. Warm melted
fat is fltered through cloth and left to cool.
The vehicle for holding the fat crops during the process is a
specially designed chassis. These are rectangular wooden frames (2 in.
high, 20 in. long and 16 in. wide) that hold a glass plate (Figure 3). The fat
crops is applied to both sides of the glass with a spatula at the beginning of
enfeurage: 360 g crops on each side is required. When piled one above the
other, the chassis form an air-tight compartment with a layer of fat on the
upper and lower sides of each glass (Figure 4). Every morning, freshly picked
fowers are cleaned by removing the leaves and stalks and eliminating the
moisture from dew or rain. The fowers are strewn (Figure 3) by hand on top
of the fat layer of each glass plate. Traces of moisture will cause the crops
to go rancid, so precaution must be taken.
90
Figure 4: Pile of chassis
When chassis are piled one above the other, fowers remain in
contact with the lower fat crops which acts as direct solvent, whereas the
upper fat layer (beneath the glass plate of the chassis) absorbs only the
volatile perfume given off by the fowers. After 24 h, the fowers are removed
from the chassis. The time of removal, however, depends on type of the fow-
ers. Removal of the fowers from the crops is known as defeurage. Immedi-
ately following defeurage, the chassis are recharged with fresh fowers. For
this purpose, the chassis are turned over, thus the fat layer which in the pre-
vious operation formed on top (ceiling) of the small chamber is now directly
charged with fresh fowers. When the fat crops becomes saturated with
fragrance (pomade), the crops is removed from the chassis with a spatula.
The pomades are extracted with absolute alcohol in a process called extrait.
At the beginning and several times during the harvest, the fat on the chas-
sis is scratched over with a metal comb and tiny furrows are drawn in order
to change and increase the surface area available for maximum absorption.
The most highly saturated pomade is pomade no. 36, meaning the crops
on the chassis has been treated with fresh fowers 36 times during the
whole process of enfeurage. In the case of jasmine, every kilogram of fat is
charged with 3 kg fowers during the entire period. The alcohol washing of
pomade no. 36 are called extrait no. 36, which reproduces the natural fower
oil to a remarkable degree. Sometimes a fatty note is present in the extrait,
which can be removed by freezing and fltering the alcoholic washes.
91
Figure 5: Batteuses
Pomades are processed during winter in cool cellars called
batteuses (Figure 5) made of copper and equipped with stirrers. Several
batteuses are arranged serially. The alcohol driven from one batteuse is
poured in second, third and frst washings of successive batteuses. For the
last washing, fresh alcohol is used. Extrait no. 36 is cooled in a refrigerator
to separate the fat. The exhausted fat is odorless and used for the manu-
facture of soap. Complete concentration of extrait is done under vacuum at
low temperature to remove alcohol, producing absolute of enfeurage which
is semisolid in nature. The fowers removed from the chassis are extracted
with solvents (petroleum ether) and the concentrated residue is dissolved in
absolute alcohol. The fat is removed by freezing. This preparation is known
as absolute of chassis. Absolute of enfeurage and absolute of chassis
logically supplement one another because each represents only part of the
total oil present in the fowers but, due to cost difference, they are kept
separately.
4.5 Conclusions
In spite of recent technological development in the feld of
extraction, hydrolytic maceration, expression and cold fat extraction tech-
niques are inevitable for certain types of raw material. For these materials,
no substitutes are available at present, although hydrodistillation or solvent
extraction can be used for essential oils of citrus and fower oils or other
perfumery materials. Nonetheless, the real replication of essential oil or
the true natural fragrances present in these materials can only be produced
by expression and cold fat extraction. Only technological improvements for
easy extraction can be made without affecting the basic principles of these
processes.
92
Bibliography
Anonymous, 2007, Brown International Corporation, Brown Oil Extractor Model
6100, 633 N. Barranca Avenue Covina, California. 91723-1297 USA. Available
at: http://www.brown-intl.com/download/BOE.PDF.
Anonymous, 1983, Food Machinery Corporation. Available at: http://fles.asme.org/
ASMEORG/Communities/History/Landmarks/5549.pdf
Cooper, J. W. and Gunn, C., 1975, Tutorial Pharmacy, Carter S. J. (Ed.), Reprint CBS
Publication, Dehli, p. 255
Ganga, A., Pinaga, A., Quero, A., Valles, S. and Ramon, D., 2001, Cell wall degrad-
ing enzyme in release of grape aroma precursors, Food Science and Technology
International, 7: 83-87
Ghosh, D. and Laddha, K. S., 2005, Herbal drug extraction. Chemical Weekly, Ragha-
van R. (Ed.), Sevak Publications. 602-B, K. J. Somaiya Hospital Road, Sion (Ed),
Mumbai India, p. 185
Goodrich, R. M. and Braddock, R. J. 2004, Major By-products of the Florida Citrus
Processing Industry. University of Florida, Institute of Food and Agricultural Sci-
ences (UF/IFAS). Available at: http://edis.ifas.uf.edu/BODY_FS107#beginning
Guenther, E., 1949, The Essential Oils, D. Van Nostrand Company, Inc. 257 Fourth
Avenue, New York 10, a. Vol 3, p. 6-75, b. Vol 1, p. 189-200
Kim, D. H., Kim, J. H., Bae, S. E., Seo, J. H., Oh, T. K. and Lee, O. H., 2005, Enhance-
ment of natural pigment extraction using Bacillus species xylanase, Journal of
Agricultural and Food Chemistry, 53(7): 2541-2545
Mc Mahon, H. M., Zoecklein, B. W. and Jasinski, Y. W., 1999, The effects of pre
fermentation maceration temperature and percent alcohol (V/V) at press on the
concentration of cabernet sauvignon grape glucosides and glycoside fractions,
American Journal of Enology and Viticulture, 50(4): 385-390
Rotter, B., 2006, Prefermentation cold maceration. Available at: www.brsquared.org/
wine/articles/coldsoak.htm
Sambamurthy, K., 2002, Pharmaceutical Engineering (Reprint) New Age International
(P.) Ltd. New Delhi, p. 174-175
Cross, S.D., 1986, Citrus juice extractor. FMC Corporation, Chicago III U.S. 4,700,620,
Oct. 20, 1987
Tsuchiya, T. and Nakamura, C., 1979, Acetocarmine squash method for observing
sugar beet chromosomes, Euphytica, 28(2): 249-256
93
Decoction and Hot Continuous 5
Extraction Techniques
S. Tandon and S. Rane
Abstract
The chapter describes the techniques, parameters and equipment used for the extrac-
tion of plants by decoction and hot continuous extraction. Principles, mechanisms,
merits and demerits of conventional solvent extraction and accelerated solvent extrac-
tion are also discussed.
5.1 Introduction
Of the traditional methods of extraction of medicinal plant ma-
terial for making an aqueous extract, decoction is one of the most described.
Decoction is a water-based preparation to extract active compounds from
medicinal plant materials. In this process, the liquid preparation is made
by boiling the plant material with water. Decoction differs from infusion in
that the latter is not actively boiled. Decoction is the method of choice
when working with tough and fbrous plants, barks and roots and with plants
that have water-soluble chemicals. The plant material is generally broken
into small pieces or powdered. Different methods have been described for
the preparation of decoctions. In the Ayurvedic method, traditionally known
as kwatha, the crude drug in form of yavakuta (small pieces) is placed in
earthen pots or tinned copper vessels with clay on the outside. Water is
added and the pot is heated on a fre. If the material is soft, four times water
is used per 1 part drug; if the drug is moderately hard, eight times water is
used and if the drug is very hard, sixteen times water is recommended. The
mixture is then boiled on low fame until it is reduced to one-fourth starting
volume, in case of soft drugs, and one-eighth in case of moderately or very
hard drugs. The extract is then cooled and strained, and the fltrate is col-
lected in clean vessels.
5.2 Solid-liquid Extraction Process
Solid-liquid extraction is one of the most widely used unit
operations in the medicinal and aromatic plant industry. One example of
solid-liquid extraction is the solvent extraction of herbs. This process, also
referred to as leaching, is a separation technique that is often employed
to remove a solute from a solid mixture with the help of a solvent. The in-
soluble solid may be colossal and permeable; more often it is particulate
and the particles may be openly porous, cellular with selectively permeable
cell walls, or surface-activated. The stream of solids being leached and the
accompanying liquid are known as the underfow. The solid content of the
5 DECOCTION AND HOT CONTINUOUS EXTRACTION TECHNIQUES
94
stream is called marc. The stream of liquid containing the leached solute is
known as the overfow.
5.3 Process Parameters Affecting Solid-liquid
Extraction
The following parameters generally affect the rate of solid-liq-
uid extraction (leaching):
Post-harvest processing
Matrix characteristics
Choice of solvent
Method of contact
Temperature of extraction
Number of washes
Condition for extraction (e.g. agitation)
5.3.1 Post-harvest Processing
After harvesting, most herbs have a moisture content of
60%-80% and cannot be stored without drying. Otherwise, important com-
pounds can breakdown or micro-organisms may contaminate the material.
Drying of the herbs in shade in a thin layer is generally preferred. Some
medicinal plants, like pyrethrum, lose their active constituents if exposed to
direct sun light for a long period. For drying large quantities of plant mate-
rial, a hot air drying oven is used where material can be placed on a large
number of trays stacked over each other. Oven temperature must be kept
at a safe level so as not to damage the active constituents of the medicinal
plant.
5.3.2 Matrix Characteristics
Knowledge of the matrix characteristics of the carrier solid is
important to determine whether it needs prior treatment to make the solute
more associable to the solvent. Grinding of plant material means mechani-
cally breaking down leaves, roots, seeds, or other parts of a plant into small
units, ranging from large course fragments to fne powder.
Solute may exist in the inert solids in a variety of ways:
On the surface of the solid, 1.
Surrounded by a matrix of inert material, Chemically com- 2.
bined, or inside the cells.
Solute adhering to the solid surface is readily removable by
solvent. When the solute exists in pores surrounded by a matrix of inert
material, the solvent has to diffuse to the interior of the solid to capture the
95
solute. In the medicinal plant industry, generally ball mills and fuid mills are
employed for powdering, and the optimum particle size is established prior
to the large-scale extraction.
5.3.3 Choice of Solvent
The following factors should be considered when selecting a
solvent for commercial use:
Solvent power (selectivity) . Only the active, desired constit-
uents should be extracted from the plant material, which
means that a high selectivity is required.
Boiling temperature . The boiling point of the solvent is as
low as possible in order to facilitate removal of the solvent
from the product.
Reactivity . The solvent should not react chemically with the
extract, nor should it readily decompose.
Viscosity . A low viscosity of the solvent leads to low pressure
drop and good heat and mass transfer.
Safety . The solvent should be non-fammable and non-cor-
rosive, and should not present a toxic hazard; its disposal
should not imperil the environment.
Cost . The solvent should be readily available at low cost.
Vapor pressure . To prevent loss of solvent by evaporation, a
low vapor pressure at operating temperature is required.
Recovery . The solvent has to be separated easily from the
extract to produce a solvent-free extract.
5.3.4 Conditions for Extraction
Too fne particle size may result in problems with packing of
solids for extraction, preventing free fow of solvent through the solid bed. In
such a case, extraction is more diffcult, especially when fnely divided sol-
ids are treated in an un-agitated state. Dispersion of the particles in liquid
solvent by agitation facilitates contact of the solid with the solvent. Agita-
tion, while giving good extraction, may cause suspension of fne particles in
overfowing solution.
5.4 Method of Solid-liquid Extraction
The three principle types of fow used in leaching systems
are:
Single-stage system 1.
Multistage counter-current system 2.
Multistage co-current system 3.
96
EXTRACTION
STAGE
FRESH SOLVENT OVERFLOW SOLUTION
SOLID UNDERFLOW SOLID FEED
Figure 1: Single-stage solid-liquid extraction
The single-stage system represents the complete operation
of contacting the solid feed and fresh solvent. This is rarely encountered
in industrial practice because of the low recovery of solute obtained and
relatively dilute solution produced. Effciency of extraction is somewhat im-
proved by dividing the solvent into a number of smaller portions and then
carrying out multiple successive extractions instead of only one contact of
the entire amount of solvent with the solid.
FRESH SOLVENT
OVERFLOW SOLUTION
SOLID FEED
SOLID UNDERFLOW
EXTRACTION
STAGE 2
EXTRACTION
STAGE n
EXTRACTION
STAGE 1
Figure 2: Multistage counter-current solid-liquid extraction
In the continuous counter-current multistage system shown in
Figure 2, the underfow and overfow streams fow counter-current to each
other. This system allows high recovery of solute with a highly concentrated
product because the concentrated solution leaves the system after contact
with fresh solid.
FRESH SOLVENT
FRESH SOLVENT
FRESH SOLVENT
OVERFLOW SOLUTION
OVERFLOW SOLUTION
OVERFLOW SOLUTION
SOLID FEED
SOLID UNDERFLOW
EXTRACTION
STAGE 2
EXTRACTION
STAGE n
EXTRACTION
STAGE 1
Figure 3: Multistage co-current solid-liquid extraction
In the multistage co-current (parallel) system shown in Figure
3, fresh solvent and solid feeds are contacted in the frst stage. Underfow
97
from the frst stage is sent to the second stage, where it comes in contact
with more fresh solvent. This scheme is repeated in all succeeding stages.
5.5 Solid-liquid Extraction Equipment
Equipment for solid-liquid extraction is of two types:
Batch solid-liquid extractor a)
Continuous solid liquid extractor b)
The most common batch extractors employed for solid-liquid
extraction of medicinal plants are:
Pot extractor . The extractor has a volume of 2-10 m
3
and a
mixer is necessary to guarantee good mixing for treatment
of fne materials. For structured materials, the mixer is only
used for evaporation of the solvent and for emptying the
extractor.
Rotating extractor . The extractor is flled with extraction ma-
terial and solvent and starts then to rotate. The installation
of heating coils and the use of a double jacket make it pos-
sible to evaporate the solvent at the end of the extraction
cycle. A special form of heating coil can act as mixer during
the extraction period.
The advantage of batch extractors is that they are simple to
operate and are robustly constructed. Disadvantages of batch extractors are
the limited capacity and the discontinuous output of the product.
Figure 4: Pot extractor and rotating extractor
98
5.5.1 Continuous Extraction
For continuously operating extraction, percolation and immer-
sion are used.
5.5.1.1 Percolation
The solvent passes through the non-moving solid material and
extracts the soluble active constituents. One advantage of this method is
that the solid material requires little mechanical treatment because it does
not need to move in the percolator while the product passes in the solution.
Moreover, since self-fltration takes place, there is minimum content of fne
solid particles in the extract.
5.5.1.2 Immersion
In this process, the solid material dips completely into the sol-
vent and is mixed with it. Therefore, no special percolation properties of the
solid material are necessary. The disadvantage is that no self-fltration of the
extract solution takes place. Therefore, a fltration step has to be added.
5.5.2 Continuous Extraction Equipment
5.5.2.1 Continuous Horizontal Extractor
The solid material is placed in baskets and comes in contact
with the solvent by percolation. The fow of solvent through the extractor is
counter-current to the fow of solid material.
Figure 5: Continuous horizontal extractor
99
5.5.2.2 Hildebrandt Extractor
The solid material is extracted according to the immersion
method. Screw conveyors are installed in the extractor for transporting the
solid material. Again, the solvent fows counter-current to the solid materials
through the extractor.
Figure 6: Hildebrandt extractor
100
5.5.2.3 Bonotto Extractor
The Bonotto extractor is used for counter-current extraction ac-
cording to the immersion method. The solid material is transported by the
mixer on a tray until it reaches the open sector where it falls onto the next
tray. The screw conveyor at the outlet withdraws the extracted solid material
(underfow) and prevents the solution from fowing out of the extractor.
Figure 7: Bonotto extractor
5.5.2.4 Bollmann Extractor
The fresh solvent is added during the upward movement of the
baskets so that this part operates in counter current. The already preload-
ed solution is withdrawn from the bottom of the extractor and enters the
downward-moving baskets so that this part of the extractor operates in a
co-current way. The full miscella is withdrawn at the bottom of the extractor.
101
In the baskets, self-fltration takes place, so that no further treatment of the
miscella before distillation is necessary.
Figure 8: Bollmann extractor
5.5.2.5 Kennedy Extractor
The solid material is transported by paddles from one chamber
to the next, in counter-current way to the solvent. The chamber where the
miscella is withdrawn is used as a fltration unit where fne particles are
separated from the extract solution.
Figure 9: Kennedy extractor
102
5.6 Conventional Solvent Extraction
5.6.1 Principles and Mechanisms
Classic techniques for the solvent extraction of active constitu-
ents from medicinal plant matrices are based on the choice of solvent cou-
pled with the use of heat or agitation. Existing classic techniques used to
obtain active constituents from plants include: Soxhlet, hydrodistillation and
maceration with an alcohol-water mixture or other organic solvents. Soxhlet
extraction is a general and well-established technique, which surpasses in
performance other conventional extraction techniques except for, in limited
felds of application, the extraction of thermolabile compounds.
Figure 10: Soxhlet extractor
In a conventional Soxhlet system, as shown in Figure 10, plant
material is placed in a thimble-holder, which is flled with condensed fresh
solvent from a distillation fask. When the liquid reaches the overfow level, a
siphon aspirates the solution of the thimble-holder and unloads it back into
the distillation fask, carrying extracted solutes into the bulk liquid. Solute is
left in the fask and fresh solvent passes back into the plant solid bed. The
operation is repeated until complete extraction is achieved.
103
5.6.2 Advantages and Disadvantages of Soxhlet Extraction
Advantages:
1. The displacement of transfer equilibrium by repeatedly bringing
fresh solvent into contact with the solid matrix.
2. Maintaining a relatively high extraction temperature with heat
from the distillation fask.
3. No fltration of the extract is required.
Disadvantages:
Agitation is not possible in the Soxhlet device. 1.
The possibility of thermal decomposition of the target com- 2.
pounds cannot be ignored as the extraction usually occurs
at the boiling point of the solvent for a long time.
Worldwide, most of the solvent extraction units are based on the
Soxhlet principle with recycling of solvents. Basic equipment for a solvent extrac-
tion unit consists of a drug holder-extractor, a solvent storage vessel, a reboiler
kettle, a condenser, a breather system (to minimize solvent loss) and support-
ing structures like a boiler, a refrigerated chilling unit and a vacuum unit.
Table 1: Some common solvents used for the extraction of medicinal and aromatic plants
Solvent Boiling point, C Miscibility with H
2
O
Threshold limit
values, ppm
Acetone 56 1000
Acetic acid 116-117 10
Ethyl acetate 77 80% 400
Benzene 80 <0.01% 25
2-Butanol 79.5 19% 2200
Cyclohexane 80.7 <0.01% 300
Dichloromethane 39.7 1.3% 2200
Chloroform 61 8% 50
Carbon tetrachloride 76.77 0.8% 10
Hexane 69 <0.01% -
Ethanol 78 1000
Ethyl ether 34.6 1.2% 400
Petrol ether 30-50 - 500
Propanetriole 290* -
Methanol 64.7 200
1-Propanol 91 M 400
2-Propanol 82.4 M ? 400
Toluene 110.6 0.06 100
t = < 0.01%; * with decomposition; M miscible; completely miscible
104
5.7 Accelerated Solvent Extraction
5.7.1 Principles and Mechanisms
Accelerated solvent extraction (ASE) is a solid-liquid extraction
process performed at elevated temperatures, usually between 50 and 200
C, and at pressures between 10 and 15 MPa. Therefore, accelerated solvent
extraction is a form of pressurized solvent extraction. Increased tempera-
ture accelerates the extraction kinetics and elevated pressure keeps the
solvent in the liquid state, thus achieving safe and rapid extraction. Also,
high pressure allows the extraction cell to be flled faster and helps to force
liquid into the solid matrix. A typical accelerated solvent extraction system is
illustrated in Figure 11. Although the solvent used in ASE is usually organic,
pressurized hot water can also be used. In these cases, one refers to pres-
surized hot water extraction or sub-critical water extraction.
Figure 11: Accelerated solvent extraction
5.7.2 Advantages and Disadvantages of Accelerated
Solvent Extraction
Compared with traditional Soxhlet extraction, ASE presents a
dramatic reduction in the amount of solvent and extraction time. Particular
attention should be paid to ASE performed at high temperature, which may
lead to degradation of thermolabile compounds.
5.8 Important Factors for Designing a Solvent
Extraction Plant for Medicinal Plants
High effciency of extraction
Minimal solvent loss
Facilities for cold and hot extractions
Extraction with agitation
105
Multiple solvent extraction systems
Multiple fraction collection systems
On-line fltration unit
Solvent recycling and condensing unit
Vent lines with breather for minimizing solvent loss and max-
imizing safety
Brine circulation unit
Fractionating column for separation of solvent mixtures
Effcient evaporating systems like falling flm, wiped flm or
rotary evaporators to work under low pressures
Vacuum manifold system with cold traps
GMP compatible
Automation
Figure 12: Solvent extraction plant at CIMAP, India
5.9 Conclusions
Decoction, a water-based preparation, is one of the most used
traditional methodologies for the extraction of active constituents of a me-
dicinal plant. It is generally carried out by boiling the plant part for a fxed
period. Hot continuous extraction or solvent extraction technique is one of
the most widely used extraction techniques for the processing of medicinal
plants. The solvent extraction method is simple, well established and eco-
nomical. Important factors that can affect the effciency of extraction, such
as post-harvest processing, solid characteristics, choice of solvent, method
of contact, and temperature, should be optimized for best yield. The choice
of solvent especially for commercial plants and high effciency usually de-
106
pends on many factors such as selectivity, polarity, boiling point, chemical
and thermal stability, safety, fammability, and costs. Despite the economic
advantages of solvent extraction, the use of volatile organic solvents such
as hexane, acetone and methanol for processing medicinal plants has been
limited due to environmental considerations. Hot continuous extraction
technology shall always remain the method of choice for high effciency,
economical extraction and with less capital investment.
Bibliography
Dung N. X. and Dinh, T., 2005, Extraction and Distillation of essential Oils, Process-
ing, Analysis and Application of Essential Oils, 1st Edition, Har Krishan Bhalla &
Sons Book Company, p. 59
Gamse, T., 2002, Lecture on Liquid-liquid Extraction and Solid-liquid Extraction. Avail-
able at: www.iq.uva.es/separacion/archivos/SkriptumExtraction.pdf
Hazra, P. et al., 1989, Solvent extraction of Artemisia annua L. on pilot scale, Re-
search and Industry, 36: 14-16
Kahol, A. P., Tandon, S. and Singh, K. L., 1998, Developments in separation technolo-
gies for the processing of medicinal and aromatic plants, PAFAI Journal, 20(3):
17-28
Pangarkar, V. G., 1990, Liquid-liquid contact operation: review of equipment, PAFAI
Journal, 12(3): 13-17
Perry, R. H., 2003, Perrys Chemical Engineers Handbook, McGraw-Hill Company
Sambamurthy, K., 2002, Pharmaceutical Engineering (Reprint), New Age Internation-
al (Pvt.) Ltd., New Delhi, p. 173-195
Schweitzer P. A., 1979, Handbook of Separation Technique for Chemical Engineers,
1st Edition, McGraw-Hill Book Company
Tandon, S. et. al., 2003, Pilot plant processing data for the isolation of artemisinin
from the new variety of Artemisia annua Jeevanrakha, Journal of Scientic and
Industrial Research, 62: 457-461
Treybal, R. E., 1988, Mass Transfer Operations, McGraw-Hill Book Company, 3rd Edi-
tion, p. 717
Wang, L. and Weller, C. L., 2006, Recent advances in extraction of nutraceuticals
from plants, Trends in Food Science & Technology, 17: 300312
Warren, L. M., 1987, Unit Operations of Chemical Engineering, McGraw-Hill Book
Company, p. 531
107
6 Aqueous Alcoholic Extraction of Medicinal
and Aromatic Plants by Fermentation
C. K. Katiyar
Abstract
The history of the development of pharmaceutical dosage forms can be traced back to
the Vedic era. It provides insight into the innovations made during ancient times when
the crude herbs were initially used in powder form and later on they were used as de-
coctions, self-fermented products, paste, pills and other advanced dosage forms. New
drug development involves the search for novel and new pharmacophores. Combinato-
rial chemistry cannot solve the problem in a satisfactory manner nor can the reduc-
tionist approach be applied to medicinal plants. Asava arishta, a fermented Ayurvedic
product, is a good source of novel pharmacophores for new drug discovery.
6.1 Introduction
The history of development of pharmaceutical dosage forms
can be traced back to Charak Samhita, the frst systematic documentation
of Ayurveda. Ayurveda has recommended a comprehensive Materia Medica
including medicinal plants, minerals, metals, and products of marine and
animal origin. However, the use of herbs has been given priority.
Medicinal plants have been used for therapeutic purposes for
centuries. Initially, these were used in fresh or dried powder form, which
caused the problems of high dose, high volume and low shelf life. This led
to the development of extraction processes. Extracts were found to be more
useful as the necessary dose was less, the volume was low and shelf life was
higher. Initially the solvents used for extraction were either water or alcohol, or
their mixture. This evolutionary phase is continuing even today where solvents
of all kinds of polarity are tried and extraction technologies have evolved from
simple water decoction centuries ago to supercritical extraction. While this is
true for some streams of products, Ayurveda did not exactly follow the same
route but adopted a sui generis system of innovation.
6.2 Ayurvedic Dosage Forms
During ancient times, various dosage forms were developed.
The number of dosage forms that developed over time is given below:
Charak Samhita (12
th
century BC) 128 dosage forms
Sushruta Samhita (10
th
century BC) 129 dosage forms
Ashtanga Hridaya (6
th
century AD) 90 dosage forms
6 AQUEOUS ALCOHOLIC EXTRACTION OF MEDICINAL AND AROMATIC PLANTS BY FERMENTATION
108
Chakradutta (9
th
Sarngadhara (14
th
Bhaishajya Ratnavali (18
th
The evolutionary phases of dosage forms followed by Ayurveda
starting from crude plant material are mentioned in the following paragraphs.
6.2.1 Swarasa (Fresh Juice)
The evolution of liquid orals started from the administration of
freshly obtained juices of plant material. To obtain fresh juices, green herbs
are crushed and the juice is expressed by squeezing the crushed material.
The product is referred to as swarasa.
6.2.2 Kalka (Wet Bolus)
In this method, the crushed fresh plant material is adminis-
tered as such, without expressing the juices.
6.2.3 Kwatha (Decoction)
One part of coarsely powdered herb is boiled with 16 times its
weight of water in an earthen pot over a mild fre until the liquid is reduced to
one-fourth or one-eighth of the original quantity, depending upon the nature
of the plant material.
6.2.4 Hima (Cold Infusion)
The plant material is dried and coarsely powdered. As and when
required, the powder is soaked in plain water for a defned period. Then it is
fltered, the marc is squeezed, and the combined fltrate is used.
6.2.5 Phanta (Hot Infusion)
As a further advancement of hima, the phanta method was
adopted. This method uses boiled water for obtaining a hot infusion.
6.2.6 Solids
Anjana, Churna, Mansa potli, Utkarika, Kshara, Gutika, Guda,
Dhumravarti, Puplika, Prithuka, Mandura, Modaka, Rasakriya, Vati, Varti,
Shashkuli, Saktu, Bhasmas, Rasaushadhis.
109
6.2.7 Semisolids
Oral: odana (rice preparation), kalka, krishara, avaleha.
Topical: lepa, upnaha (poultice), tilapishta, patrasveda, madhuc-
chishta (beeswax).
6.2.8 Liquids
Oral: taila, ghrita, asava/arishta, arka, kwatha, kshirapaka, takra,
phanta, him, swarasa, peya, phanita, manda, manasa rasa, yusha, vesavara,
vilepi, madya
Topical: ashchyotana, karna purana.
6.2.9 Fumes
Dhumrapana, dhupana.
6.3 Shelf Life of Dosage Forms
Sarngadhara Samhita gave the shelf life of various dosage
forms. In ancient times, Ayurvedic physicians themselves prepared the
recipes for patients. In ancient times, Ayurvedic physicians themselves
prepared the recipes for patients; during the fourteenth century AD, they
became aware of the problem of poor shelf life of the botanicals in some
dosage forms, such as powder and decoction. This led to the discovery of
novel dosage forms termed asava and arishtas, which are self-fermented
preparations having approximately 10%-12% alcohol. These are similar to
medicated wines. In the preparation of asava cold infusion of unprocessed
plant material is used, whereas for the preparation of arishta decoction of
the plant material is used for fermentation.
6.4 Asava and Arishta: Self-fermented Products
This unique dosage form discovered by Ayurveda is supposed
to have indefnite shelf life and it was said that the older the better it
is. In terms of current understanding, this phrase assumes more impor-
tance because this dosage form has an inherent attribute of continuous
hydro-alcoholic extraction and probably formation of natural analogues of
the chemical compounds present in the medicinal plants. Some of the major
self-fermented Ayurvedic preparations are given in Table 1.
110
Table 1: Major self-fermented Ayurvedic preparations
Product
No. of
ingredients
Indication
Aravindasava 27 Pediatric tonic
Arjunarishta 5 Cardiotonic
Ashokarishta 14 Menstrual cycle regulator, especially to control
excessive bleeding for prolonged periods
during menstrual cycle
Ashwagandharishta 24 General tonic
Dashamularishta 67 Normalization of physiological processes after
childbirth in women; also anti-infammatory
Drakshasava 17 General tonic
Jeerakadyarishta 13 Galactogogue
Kumaryasava 46 Liver disorders
Kutajarishta 6 Diarrhea and dysentery
Lohasava 14 Anemia
6.4.1 Self-fermentation Process for Preparing Asava
Arishta
Preparation of asava arishtas involves complicated processes
where the following factors play important roles:
Ingredients and their ratios
Process
Pot
Season
Place
The method of preparing asava arishtas is known as sandhana
kalpana in Ayurveda. Briefy, a decoction or cold infusion of several herbs is
taken and a defned amount of jaggery (dried juice of sugarcane) is added
along with fowers of Woodfordia fruticosa as inoculum to initiate fermenta-
tion. It is kept for about four weeks for fermentation by anaerobic method
to obtain a particular level of self-generated alcohol. The product is then
kept for some time for maturation. Spices like cardamom and cinnamon are
added as favoring agents. A brief outline of the process of preparing asava
arishta is given in Figure 1.
111
0round herbs
Fxtractlon:
lnfuslon]decoctlon
Preparatlon of fermentatlon
medlum
|aeeery and]or honey
Fermentatlon
Powdered splces
Powdered Flower of
8PPEGPSEJBGSVJUJDPTB
Figure 1: The traditional process of preparing asava-arishtas
A crude match-box method is applied to check whether fer-
mentation has occurred. This method depends upon the release of carbon
dioxide during the process. The major role in this dosage form is played by
Woodfordia fruticosa, which is used as inoculum for fermentation but ap-
pears to play a role beyond that.
6.4.2 Merits of the Process
Prahst has mentioned some of the benefts of fermented herb-
al products which are reproduced below:
Fermentation removes most of the undesirable sugars from
plant material, makes the product more bio-available and
eliminates side effects such as gas and bloating.
Fermentation extracts a wider range of active ingredients
from the herb than any extraction method since the men-
struum undergoes a gradient of rising alcohol levels.
Yeast cell walls naturally bind heavy metals and pesticide
residues and, therefore, act as a natural cleansing system.
Not only does fermentation remove contaminants, it can
also lower the toxicity of some of the toxic components in
plants.
Fermentation actively ruptures the cells of the herb, expos-
ing it openly to the menstruum and bacteria have enzymes
that break down cell walls to further assist in the leaching
process. Fermentation also creates an active transport sys-
tem that moves the dissolved constituents from the herbal
material to the menstruum.
112
6.5 Application of Asava Arishta Technology in
New Drug Discovery
The Ayurvedic dictum with regard to asava arishtas that older
is better needs to be scientifcally evaluated. The process of preparing
asava arishtas appears to involve:
Slow hydro-alcoholic extraction at room temperature of 1.
crude plant material particles foating in the liquid. Since the particle size of
the plant material foating in the liquid is small, the effectiveness of extrac-
tion may be higher because of the larger surface area.
During the process, if the product is kept for a prolonged 2.
period, the probability of development of analogues of some of the pure
chemical compounds of the plant material is high.
With a view to enhance the success rate of isolation of pure
druggable compounds from medicinal plants, it is advised to start from
2- to 3-year-old self-fermented preparations than from solvent extracts. The
chances of successful isolation of effective therapeutic compounds using
this approach may be high and need to be evaluated.
6.6 Conclusions
Fermentation was applied hundreds of years ago in Ayurveda
to develop asava arishtas, a multiherbal product, with a view to increase the
shelf life and also to enhance the effcacy profle. The race for discovery of
new molecules is getting increasingly competitive; at the same time, lack of
new and novel pharmacophores is a big impediment which slows down drug
discovery. Nature continues to be a source of pharmacophores, although the
compounds isolated may not be druggable as such. They need to be de-
rived, mostly to enhance their potency. In asava arishtas, the self-fermented
products can undergo continuous chemical transformation which goes on
beyond hydro-alcoholic extraction of the suspended material. This may re-
sult in novel natural molecules with enhanced therapeutic activity.
113
Bibliography
Charak Samhita, 2001, Sharma & Dash (Eds.), Vol. I-VI. Pub. Chaukhambha Sanskrit
Series, Varanasi
Clardy, J. and Walsh, C. 2004, Lessons from natural molecules, Nature, 432(16):
829-837
Das, G., 1961, Bhaishajya Ratnavali. Ambikadatta Shastri (Ed.), Pub. Chowkhamba
Sanskrit Series, Varanasi
Newman, D. J. and Cragg, G. M., 2007, Natural products as sources of new drug over
the last 25 years, Journal of Natural Products, 70: 461-477
Prahst, A., 2007, Liquid Fermented Tinctures - A Natural choice. Available at:
www.vistamagonline.com
Sarngadhara, 2006, Sarngadhara Samhita. Srikantha Murthy K. R. (Ed.), Pub.
Chaukhamba Orientalia., Varanasi
115
7 Distillation Technology for Essential Oils
S. Tandon
Abstract
In many developing countries, the technology employed for extraction of essential oils
from aromatic plants is primitive and obsolete. This results in low yield and poor quality
of essential oils. Thus, there is great need for attaining adequate technological capabil-
ity in the area of processing essential oil plants. This article deals with the different
techniques of distillation, the principles of distillation, and important processing and
design aspects which affect the yield and quality of the essential oils.
7.1 Introduction
Distillation is the most popular, widely used and cost-effective
method for producing essential oils throughout the world.
Distillation of aromatic plants simply implies vaporizing or lib-
erating the oils from the plant cellular membranes in the presence of mois-
ture, by applying high temperature and then cooling the vapor mixture to
separate the oil from the water on the basis of the immiscibility and density
of the essential oil with respect to water.
7.2 Principles of Distillation
The choice of a particular process for the extraction of essen-
tial oil is generally dictated by the following considerations:
Sensitivity of the essential oil to the action of heat and water a)
Volatility of the essential oil b)
Water solubility of the essential oil c)
Essential oils with high solubility in water and those that are sus-
ceptible to damage by heat cannot be steam distilled. Also, the oil must be
steam volatile for steam distillation to be feasible. Most of the essential oils
in commerce are steam volatile, reasonably stable to heat and practically in-
soluble in water; hence they are suitable for processing by steam distillation.
Essential oils are a mixture of various aroma chemicals, basi-
cally monoterpenes, sesquiterpenes and their oxygenated derivatives, hav-
ing a boiling point ranging from 150 to 300 C. When the plant material is
subjected to heat in the presence of moisture from the steam, these oils are
liberated from the plant. For the oil to change from the liquid to the vapor
phase, it must receive latent heat that, within the tank, can only come from
condensing steam. Consequently, the temperature of the steam within the
7 DISTILLATION TECHNOLOGY FOR ESSENTIAL OILS
116
still must be higher than the temperature at which the oil boils in the pres-
ence of water on the surface of the plant material, otherwise there would
not be a temperature gradient to take the latent heat from the condensing
steam to vaporize the oil droplet. Thus, the energy from the steam in form
of heat as latent heat of vaporization converts the oil into a vapor. But, as
the boiling point of the oil is higher than that of water, the vaporization takes
place with steam on the basis of their relative vapor pressures.
It is imperative to note that a liquid always boils at the tem-
perature at which its vapor pressure equals the atmospheric or surround-
ing pressure. For any two immiscible liquids, the total vapor pressure of
the mixture is always equal to the sum of their partial pressures. The
composition of the mixture in the vapor phase (in this case, oil and water)
is determined by the concentration of the individual components multi-
plied by their respective partial pressures. For example, if a sample of
an essential oil comprised of component A (boiling point, 190 C) and
water (boiling point, 100 C) is boiled, after some time, once their vapors
reach saturation, the temperature will immediately drop to 99.5 C, which
is the temperature at which the sum of the two vapor pressures equals
760 mmHg. In other words, the oil forms an azeotropic mixture with water.
Thus, any essential oil having high boiling point can be evaporated with
steam in a ratio such that their combined vapor pressures equal the at-
mospheric pressure; the essential oil can be recovered from the plant by
the wet distillation process.
7.3 Methods for Distillation
The following four techniques for the distillation of essential
oils from aromatic plants are employed:
Water distillation (or hydrodistillation) 1.
Water and steam distillation 2.
Direct steam distillation 3.
Distillation with cohobation 4.
7.3.1 Hydrodistillation
Hydrodistillation is the simplest and oldest process available
for obtaining essential oils from plants. Hydrodistillation differs from steam
distillation mainly in that the plant material is almost entirely covered with
water in the still which is placed on a furnace. An important factor to consid-
er in water distillation is that the water present in the tank must always be
enough to last throughout the distillation process, otherwise the plant mate-
rial may overheat and char. In this method, water is made to boil and the
essential oil is carried over to condenser with the steam which is formed.
Water-distilled oil is slightly darker in color and has much stronger still notes
than oils produced by other methods. The stills based on this principle are
117
simple in design and are extensively used by small-scale producers of es-
sential oils. Care should be taken during distillation of powdered herbs, as
they tend to settle on the bottom of the still and get thermally degraded.
Also, for plant material that tends to form mucilage and increase the viscos-
ity of the water, the chances of charring are greater. For plant material that
has a tendency to agglomerate or to agglutinate into an impenetrable mass
when steam is passed through (like rose petals), water distillation is the
preferred method of oil isolation.
The primitive, traditional Indian system of essential oil distilla-
tion, bhapka method, is also based on water distillation (Figure 1). In this
process, the plant material is entirely covered with water in a distillation still,
which is made of copper and is known as deg. This deg is placed in a brick
furnace. Another copper vessel with a long neck is placed in a water tank or
natural pond to serve as a condenser. A bamboo pipe is used as the vapor
connection and mud is used to seal the various joints. The water is boiled,
the oil vapors along with steam are condensed in the copper vessel, and oil
is separated. The capacity of one deg is around 40 kg/batch. These types of
units are still being used in Kannauj in Uttar Pradesh and in the Ganjam dis-
trict of Orissa, India for the preparation of rooh and attars of gulab, kewda,
khus, rajnigandha, and bela. These units can easily be transported from one
place to another, but are not suitable for large-scale distillation of aromatic
crops like grasses and mints.
Figure 1: Traditional Indian deg bhapka method
Although hydrodistillation (water distillation) is still being used,
the process suffers from the following serious drawbacks:
118
As the plant material near the bottom of the still comes in a)
direct contact with the fre from the furnace, it may char and
thus impart an objectionable odor to the essential oil.
The prolonged action of hot water can cause hydrolysis of b)
some constituents of the essential oil, such as esters.
Heat control is diffcult, which may lead to variable rates of c)
distillation.
The process is slow and distillation times are much longer d)
than those of steam distillation.
7.3.2 Water and Steam Distillation
To eliminate some of the drawbacks of water distillation, some
modifcations were made to the distillation units. A perforated grid was intro-
duced in the still, to support the plant material and to avoid its direct contact
with the hot furnace bottom. When the water level is kept below the grid, the
essential oil is distilled by the rising steam from the boiling water. This mode
of distillation is generally termed water and steam distillation.
The feld distillation unit (FDU), also known as a directly fred-
type distillation unit, is designed according to the principle of water and
steam distillation. The FDU consists of a still or tank made of mild stainless
steel with a perforated grid and is ftted directly to a brick furnace. A chimney
is connected to the furnace to minimize the pollution at the workplace and
also to induce proper fring and draft. The plant material is loaded on the
perforated grid of the tank and water is flled below it. The tank is connected
to the condenser through a vapor line. The water is boiled and the steam
vapors pass through the herb, vaporize the oil and get condensed, mostly in
a coil condenser by cooling water. The condensate (oil-vapor mixture) is then
separated in the oil separator.
These units are simple to fabricate and can be installed in the
farmers feld. Due to their simple construction, low cost and easy opera-
tion, FDUs are extremely popular with essential oil producers in developing
countries. The furnace is always fueled by locally available frewood or straw.
This makes the unit suited for use in remote areas where the raw material
is available. This also helps in reducing transportation costs in the produc-
tion of essential oils. FDUs are currently fnding application in distillation of
patchouli oil in Indonesia, aromatic grass and mint oil in India, citronella oil
in Taiwan and many more all over the world. A local FDU currently being used
by rural farmers in India for the distillation of mint oils is shown in Figure 2.
Such feld units generally can hold 100-2000 kg plant material. Total time
for distillation with these units is about 6-8 h.
119
Figure 2: Local type eld distillation unit in India
7.3.2.1 Improved Field Distillation Units
Due to the limited heating surface available, the rate of steam
production in the FDU is always insuffcient. This results in prolonged distil-
lation periods and sometimes lower oil yields. Refuxing of oil back into the
still due to inadequate steam rate may lead to decomposition reactions and
poorer oil quality. Experimental measurements made at the Central Institute
of Medicinal and Aromatic Plants (CIMAP), India, have shown that frewood
consumption in a conventional feld still may be up to 2.5-times greater than
that of a modern steam distillation unit operated by an external boiler. This
factor may not be critical where fuel supplies are cheap and abundant but, in
many developing countries, fuel supplies are getting scarce and costly and
low thermal effciency can directly affect the cost of production.
Figure 3: CIMAPs improved eld distillation unit
120
Considering the previously mentioned demerits of FDUs, de-
signs of economical and improved units with capacities 500-2000 kg per
batch are now being preferred (Figure 3). The units are fabricated with high
quality mild stainless steel, keeping in view the plant materials to be dis-
tilled. The improved distillation unit consists of a cylindrical distillation tank
ftted on a square inbuilt boiler (calandria) having smoke pipes which reduc-
es the heating time of the water, resulting in a high rate of steam generation
and lower fuel consumption (20%-30%). Hot fue gasses of the furnace are
led through the smoke tubes where they impart heat to the water, thus rais-
ing additional steam. The tank is ftted on a specially designed furnace hav-
ing fre grate, fue ducts and fre door for proper controlling of the fring and
draft. The furnace is connected to a chimney of optimum height to maximize
the air draft and control the pollution by smoke in the workplace. A similarly
designed stainless steel shell and tube-type condenser having higher con-
densation capacity are used for cooling the vapors. It prevents loss of oil
due to improper condensation. The condensed oil-water mixture is then al-
lowed to pass through a specially designed stainless steel oil separator. The
separator has an inbuilt baffe to maximize the retention time of the mixture,
thereby resulting in no loss of oil with the outgoing water from the separator.
The unit also has a chain pulley hoist system with a support structure that
makes work easier and saves time during discharge of the distillation waste
material from the tank. CIMAP has designed, fabricated and supplied these
improved units to entrepreneurs and farmers in different parts of India.
7.3.3 Direct Steam Distillation
In direct steam distillation, plant material is distilled with steam
generated outside the tank in a steam generator or boiler. As in water and
steam distillation, the plant material is supported on a perforated grid above
the steam inlet. As already noted, the steam in an FDU is at atmospheric
pressure and hence its maximum temperature is 100 C. But, steam in a
modern pressure boiler operating at, for example, 50 psi pressure will have
a temperature correspondingly higher. Moreover, there is no limitation to the
steam generation when an external boiler is used as a source of steam. The
use of high-pressure steam in modern steam distillation units permits much
more rapid and complete distillation of essential oils.
Steam distillation is preferred when a lot of area is under cultiva-
tion and more than one unit is to be installed. Also, for distillation of high boiling
oils and hardy materials such as roots and woods like sandalwood, cedar wood
and nagarmotha, steam distillation is more effcient. Steam distillation also
reduces the time required for the extraction of oils. A charge of Java citronella,
which takes up to 5 h in an FDU, is processed within two to 3 h in a steam
distillation still. In this method of distillation, steam is generated separately in
a steam boiler and is passed through the distillation tank through a steam coil
(Figure 4). The plant material is tightly packed above the perforated grid. Steam,
containing the oil vapor, is condensed in a tube condenser and is separated in
121
the oil receiver. Fuel costs are generally lower in modern steam distillation units
due to higher thermal effciency at which most of the boilers operate. Capital
cost is higher, thus only bigger producers can afford to own such units. Still
capacities range from 1 to 3 tonne plant material per batch.
Figure 4: Boiler-operated steam distillation unit
7.3.3.1 Comparison of Boiler-operated Unit with Directly
Fired Type
It is possible to run a large number of distillation units by a a)
single steam boiler. Hence a boiler system is ideal for large-
scale production of essential oils. An FDU is more suited for
small and medium-sized farmers.
The effciency of extraction of essential oil in a well-designed b)
FDU can equal that obtained by a boiler unit. But in a poorly
designed FDU, oil recovery may be low and fuel wastage may
be heavy accompanied by smoke pollution.
Steam injection rate in a boiler-operated unit can be adjust- c)
ed with ease but steam generation rate in an FDU is limited
by the heat transfer area provided in the unit. Insuffcient
steam generation in an FDU can result in low oil yield.
Boiler-operated units require a skilled boiler man for oper- d)
ation but an FDU can be operated by relatively low-skilled
workers.
7.3.4 Distillation with Cohobation
Cohobation is a technique that can be used for water distillation
or for water and steam distillation. It uses the process of returning the distil-
late water to the still after the oil has been separated from it so that it can be
re-boiled. This is basically an improvised methodology of the directly fred type
122
steam and water distillation units for oils which have partial solubility in water.
Although most of the essential oils have fnite solubility in water, some oils
like those of rose, lavender and geranium have comparatively higher solubility.
In such extractions, the loss of oil with the outgoing water of distillation can
become alarmingly high. This problem can be solved by returning the conden-
sate water from the separator back to the still; this is known as cohobation. It
is evident that this cannot be done with steam distillation as the water level in
the still will keep building up due to continuous steam injection.
In a further improved version, a packed column is placed on top
of the column for providing mass transfer to the oil-water vapors, so as to
increase the concentration of the outgoing condensate and to coalesce the
oil droplets in the oil separator (Figure 5). The condenser is placed above the
column so that the condensate water from the separator can be recycled back
to the still by means of gravity. Additional heat, if required, can be provided by
a closed steam coil immersed in the tank bottom. The condenser is moved
above the distillation still so that condensed water from the separator can fow
by means of gravity to the still. By limiting the total quantity of water in this
closed cycle operation, it is possible to obtain increased yields of essential
oils that are more water soluble. It is relevant to point out here that prolonged
recirculation of the distillation water allows the various impurities and plant
decomposition products to build up in the system. This may sometimes affect
the quality of the oil. One must always keep this in mind when considering a
cohobation distillation system for any application.
Figure 5: Distillation unit with cohobation
123
7.4 Hydrodiffusion
This system was frst described in 1983. Unlike traditional
steam distillation, hydrodiffusion works on the diffusion principle of allowing
steam to enter the top of the plant charge and diffuse through the charge
by gravity. The process uses the principle of osmotic pressure to diffuse oil
from the oil glands. The system is connected to a steam source, and low
pressure steam is passed into the plant material from a boiler. The condens-
er, which is directly under the basket within the still, is of the tube type. The
oil and water are collected below the condenser in a typical oil separator.
Hydrodiffusion is an effcient process that is easy to use, especially regard-
ing the processes of loading and unloading the plant material. The yield of
oil is higher and the process is advantageous because of the reduced steam
consumption, shorter distillation time and absence of hydrolysis, as the raw
material does not come in contact with boiling water. However, because of
the downward fow of steam and condensate, co-extraction of other non-
volatile compounds (such as lipids, chlorophyll and fatty acids) and polar
components makes the process complicated. Although it may seem that
hydrodiffusion is a better alternative to conventional distillation processes,
the fact remains that commercial ventures based on hydrodiffusion have not
been able to take off successfully.
7.5 Parameters Affecting Yield and Quality
of Essential Oils
The yield and quality of essential oil from steam distillation is
affected by the various process parameters. It is advisable to keep them in
mind while designing such systems. Some of the important parameters are
being listed below.
7.5.1 Mode of Distillation
The technique for distillation should be chosen considering the
boiling point of the essential oil and the nature of the herb, as the heat
content and temperature of steam can alter the distillation characteristics.
For high boiling oils such as woody oils (e.g. sandalwood, cedar wood) and
roots (e.g. Cyperus), the oil should be extracted using boiler-operated steam
distillation. Since the heat content and temperature of steam depend upon
its pressure, a change in steam pressure can alter the distillation charac-
teristics. High-boiling constituents of essential oils normally require high-
pressure steam to distill over. For oil of rose and other forals, the material
is generally immersed in water, i.e. hydrodistillation, as fowers tend to ag-
gregate and form lumps which cannot be distilled using water and steam
distillation or direct steam distillation.
124
7.5.2 Proper Design of Equipment
Improper designing of tank, condenser or separators can lead
to loss of oil and high capital investments. The design of the furnace and
chimney affects the fring and heat control of the distillation rates. Tank
height:diameter ratio is important. Similarly the use of a condenser with an
improper design and without calculating the heat transfer areas based on
the steam generation areas will lead to improper condensation and loss of
oil.
7.5.3 Material of Fabrication of Equipment
Essential oils which are corrosive in nature should be prefer-
ably distilled in stills made of resistant materials like aluminum, copper or
stainless steel. The tank still can be made from a cheaper metal like mild
steel or galvanized iron, and the condenser and separator can be made
from a resistant material like stainless steel. As only vapor is present in the
tank still, the rust and other products of corrosion may not be carried over
into the oil. This can result in considerable savings in the capital cost of the
equipment. Expensive, high-value essential oils like rose, agarwood, kewda,
sandalwood and lavender should be distilled in stainless steel systems.
Although copper was the most common material of fabrication of distillation
stills since ancient times, its availability is getting reduced and with the ar-
rival of superior alloys like stainless steel, it is slowly disappearing from the
scene.
7.5.4 Condition of Raw Material
The condition of the raw material is important because some
materials like roots and seeds will not yield essential oil easily if distilled in
their natural state. These materials have to be crushed, powdered or soaked
in water to expose their oil cells. Chopping of plants will also change the
packing density of the material when placed in the distillation still. One can
pack up to 50% more plant material in the same still after chopping of some
aromatic herbs like mint. Air drying and wilting the herb prior to distillation
also has considerable effect on distillation. If required, drying of the herbs
prior to distillation should be done in shaded areas and the dried material
should not be kept in heaps.
7.5.5 Time for Distillation
Different constituents of the essential oil get distilled in the
order of their boiling points. Thus, the highest boiling fractions will be last
to come over when, generally, very little oil is distilling. If the distillation is
terminated too soon, the high-boiling constituents will be lost. In many aro-
matic plants, like vetiver, patchouli, chamomile, sandalwood and agarwood,
125
these high-boiling fractions are valuable due to the quality of their aromas.
Thus, the time of distillation must be chosen with due care.
7.5.6 Loading of Raw Material and Steam Distribution
Improper loading of the herb may result in steam channeling,
causing incomplete distillation. The herb should be evenly and uniformly
loaded in the tank without leaving any voids. Excessive flling of plant ma-
terial may also lead to formation of rat holes which may allow steam to
escape without vaporizing the oil. For powdered herbs, a proper stainless
steel wire mesh or muslin cloth should be put at the false bottom to prevent
plant material from falling into the tank base.
7.5.7 Operating Parameters
Proper control of injection rates and pressure in boiler-operat-
ed units is necessary to optimize the temperature of extraction for maximal
yield. Generally, high-pressure steam is not advisable for the distillation of
essential oils. The temperature of the condensate should not be high, as it
can result in oil loss due to evaporation. In directly fred-type FDUs, the fring
of the furnace should be well controlled as it can result in high fow rates
and high condensate temperatures.
7.5.8 Condition of Tank and Equipment
The tank and other equipment should not be rusted. If rusted,
the tank should be cleaned with dilute caustic solutions. The perforated
grids should not be corroded or have large gaps permitting the plant mate-
rial to settle to the bottom of the tank and emit a burnt odor. The distillation
tanks should be well steamed prior to distillation for multiple crop distilla-
tion.
7.6 Purifcation of Crude Essential Oils
Essential oil as obtained from the oil separator is in crude
form. It may have suspended impurities and appreciable moisture content.
It might even contain some objectionable constituents which degrade its
favor quality. The presence of moisture and impurities adversely affects the
keeping quality of oil and accelerates polymerization and other undesirable
reactions. Addition of a drying agent like anhydrous sodium sulphate to the
oil, standing overnight followed by fltration will remove the moisture and free
the oil of suspended impurities. Use of high-speed centrifugation to clarify
the essential oils is common.
Essential oils are frequently rectifed or re-distilled to remove
objectionable constituents. In order to keep the temperature of re-distillation
126
within permissible limits, the process is carried out under vacuum or with
the help of steam distillation.
7.6.1 Continuous Steam Distillation
Steam distillation units involve manual charging and discharg-
ing of plant material from the tank still. These operations are labor inten-
sive and time consuming. To overcome these problems, continuous steam
distillation plants have been developed in the Soviet Union and have been
in operation since the last couple of decades. These units are being used
for distillation of lavender and require negligible manual handing. Capaci-
ties of 2 tonnes per hour are quite common. Incoming plant material is frst
chopped with special ensilage cutters and then conveyed to the top of a tall
distillation column by means of a belt conveyor. The movement of material
inside the column is by gravity or by special helical screw conveyors. Some-
times two columns in series are used for complete removal of oil. Steam
is injected at multiple points in the column. Spent material is continuously
ejected out of the bottom of the distillation column by special screw convey-
ors with a vapor lock which does not allow steam to escape.
Fabrication and operation of continuous distillation columns is
rather complicated and these have not yet gained acceptance and popular-
ity outside the former Soviet Union. In another development, containerized
distillation is also being used for the distillation of Mentha piperita and
lavender in some parts of the United States. In this method, large capacity
containers mounted on wheels are attached to a harvester which directly
loads the plant material into the containers from the felds (these contain-
ers have inbuilt steam coils); these are then taken to the distillation area
where steam is directly connected to the coils and the top is closed and
connected through a vapor line to the condenser and subsequently to the
oil separator.
7.7 Conclusions
Distillation is the most widely used method for the extraction
of essential oils. Proper selection of the distillation technique, design and
material of fabrication of the equipment, and processing parameters all play
vital roles in determining the quality and yield of an essential oil.
127
Bibliography
Ames, G. R. and Matthews, W. S. A., 1969, The distillation of essential oils, Perfum-
ery & Essential Oil Records, 9-18
Boucard, G. R. and Serth, W. R., 1991, A continuous steam stripping process for the
distillation of essential oils, Perfumer and Flavorist, 16(2): 1-8
Denny, E. F. K., 1969, Hydro distillation of oils from aromatic herbs, Perfumer and
Flavours, 14(4): 57-63
Denny, E. F. K., 1989, The new approach to oil distillation from the herb, Indian Per-
fumer, 33(1): 70-75
Denny, E. F. K., 1991, Field Distillation for Herbaceous Oils (2nd Edition.), Denny-
McKenzie, Assoc., Lilydale, Tasmania, p. 265
Guenther, E., 1965, The Essential Oils, Vol 4, D. Van Nostrand Co., New Jersey, p.
682
Kahol, A. P., 1984, Distillation Technology. In: Practical Manual on: The Essential Oils
Industry, Wijesekera, R. O. B (Ed.), UNIDO, Vienna
Lawrence, B. M., 1995, The Isolation of Aromatic Materials from Natural Plant Prod-
ucts. In: A Manual on the Essential Oil Industry, Tuley De Silva (Ed.), United Na-
tions Industrial Development Organization (UNIDO), Vienna, Austria
129
8 Microdistillation,Thermomicrodistillation
and Molecular Distillation Techniques
V. G. Pangarkar
Abstract
Medicinal and aromatic plants (MAPs) have assumed considerable signicance in view
of their special attributes. There are many compounds of great therapeutic value which
can be obtained only from the plant kingdom. Most of the ingredients in such extracts
and oils are large bulky molecules highly sensitive to processing conditions. The proc-
esses for such extractions have been mostly based on recipes. In the recent past,
signicant advances have been made in the unit operations which are part of the rec-
ipe-based processes. It is imperative that these advances, which are essentially aimed
at achieving better yields at lower costs and are termed process intensication, are
incorporated into the processing of MAPs. This paper introduces the theme of proc-
ess intensication as applied to the processing of MAPs and presents an overview of
several new technologies which allow rapid, cost-effective extraction. The application of
such innovative technologies can yield signicant benets in terms of the quality of the
product and its yield per unit weight of the plant material processed. Recovery of dis-
solved essential oil components from steam distillation condensates is also addressed
and the two available techniques are discussed in detail.
8.1 Introduction
Phytochemicals derived from medicinal and aromatic plants
(MAPs) have been important to humans for centuries. Before the advent
of modern synthetic chemistry, many aroma and favor chemicals were de-
rived from sources of natural origin such as fowers, roots and stems. The
contemporary system of allopathic medicine, which has gained tremendous
importance in the treatment of various diseases, is mainly based on active
pharmaceutical compounds made synthetically. However, in recent years in-
creasing attention has been paid to the traditional systems of treatments
followed in Asia and Africa. The variety of medicinal plants and their con-
stituents are being discovered only recently. There are many compounds of
great therapeutic value which can be obtained only from the plant kingdom.
Thus, vincristine, perhaps better known as the chemotherapy agent Oncovin,
is only synthesized in the periwinkle plant Catharanthus roseus, and the
sole source of the compound is this plant species. There are many other
compounds which are equally valuable in other sectors such as food favors,
fragrances and cosmetics.
The processing of MAPs for obtaining the required extracts and
oils has been based on traditionally established recipes. Most of the in-
gredients in such extracts and oils are large bulky molecules highly sensi-
tive to processing conditions. Generally, relatively mild conditions are used
in such processes to protect the integrity of the valuable components. The
8 MICRODISTILLATION,THERMOMICRODISTILLATION AND MOLECULAR DISTILLATION TECHNIQUES
130
recipe-based methods are time-tested but arguably not the most effcient in
terms of yield, energy consumption per unit of product, etc. With the advent
of modern processing techniques, there is an urgent need to revisit the rec-
ipe-based processing, understand the science underlying them and develop
modern, cost-effective processes. This article deals with some important
advances made in the extraction of MAPs and the post-extraction treatment
of the products and their byproducts.
8.2 Process Intensifcation
The modern chemical industry is undergoing drastic changes
driven mainly by economic considerations. There is an upsurge of interest
in clean, energy-effcient and material-conserving processes. An entirely new
discipline, process intensifcation (PI), has become the focus of a large
and sustained effort all over the world. Stankiewicz and Moulijn have given a
precise defnition of PI as Any chemical engineering development that leads
to a substantially smaller, cleaner and more energy effcient technology.
India has not been lagging behind in developing innovative PI concepts.
PI can be broadly divided into two categories, with specifc
reference to processing of MAPs as per the defnition of Stankiewicz and
Moulijn. These are processes that employ multifunctional equipment (MF)
and those that use process-intensifying equipment.
8.2.1 Multifunctional Equipment
This category of PI employs equipment that can perform multiple
functions simultaneously. Thus, earlier process plants that required a number
of different instruments devoted to individual tasks are being replaced by such
MF equipment. A brilliant example of the use of MF equipment is the conver-
sion of a slow and polluting process for the enzymatic hydrolysis of penicillin G
to 6-amino penicillanic acid (important intermediate for semisynthetic antibi-
otics) into an intensifed and sustainable process. Since MF equipment-based
plants are smaller and consume less energy, they have become popular for
globally competitive and sustainable processes.
8.2.2 Process-intensifying Equipment
This category of PI employs equipment that specifcally focuses
on intensifying the rates of the various steps. In the case of MAP process-
ing, the main resistance in the overall extraction process is the diffusion of
the active molecules through the plant cell membrane to the surface before
extraction by the fuid. Microwave-assisted extraction (MAE) is highly use-
ful in obtaining rapid and complete extraction without signifcant damage
to the active molecules; this technique is discussed in some detail later.
Ultrasound-assisted extraction is also an alternative. However, considering
131
that ultrasound waves can produce free radicals and that many active mol-
ecules are susceptible to such highly reactive species, this approach does
not seem feasible.
8.3 Solvent Extraction of MAPs
This process entails the extraction of solid MAPs by liquid sol-
vents. This is a typical solid-liquid extraction process. Two factors that affect
the extent and rate of extraction are the thermodynamics and kinetics (rate
of mass transfer) of the process.
8.3.1 Thermodynamics of Solvent Extraction and Choice
of Solvent
Relative sorption of solutes in the solvent depends on the in-
teractions between the solutes and the solvent. Solubility or miscibility of a
component with the solvent depends on their relative solubility parameters.
For mutual solubility of two components, their free energy of mixing, G
m

should be negative. G
m
is defned as
G
m
= H
m
T S
m
(3.1)
Enthalpy of mixing, H
m
can be correlated to cohesive energy
density, i.e. solubility parameter () as:
H
m
= n
1
n
2
V
1
(
1

2
)
2
(3.2)
In equation (3.2), the solubility parameter is that due to only
dispersive forces between structural units of the concerned solute and sol-
vent, since the original regular solution theory of Scatchard and Hildebrand
was restricted to non-polar, non-hydrogen bonding solute-solvent systems.
However, for many liquids and solutes, contributions from polar and hydro-
gen bonding forces need to be considered. Accordingly, equation (3.2) be-
comes:
H
m
= n
1
n
2
V
1
[(
d
)
2
+ (
p
)
2
+ (
h
)
2
] (3.3)
From equations (3.2) and (3.3), it is clear that to make G
m

negative, the difference between
i
(solvent) and
i
(solute), i.e. () for
all the three forces of interactions, should be as small as possible. It im-
plies that the solvent and the desired solute to be extracted should have
comparable polarity and hydrogen bonding capabilities to achieve similar
solubility parameter values. Grulke has given an exhaustive tabulation of
solubility parameters for the most common chemical compounds. The in-
132
dividual
h
,
d
and
p
values for compounds not listed in the tabulation can
be obtained using the group contributions due to various different groups
given by Grulke. When the individual
i
(solvent) and
i
(solute) values are
very close, a high solubility of the solute in the solvent is obtained. For
instance, it is well known that non-polar solvents dissolve terpene frac-
tions more than oxygenated compounds because both are non-polar. On
the other hand, mixed solvents of polar and non-polar compounds can yield
better results for oxygenated compounds. Bio-ethanol is a good solvent for
such oxygenated compounds on two accounts: (i) it is natural, and (ii) it is
green (renewable). However, most MAPs contain water and the complete
miscibility of ethanol with water implies dilution of the solvent after each
use. This is further complicated by the fact that ethanol forms an azeotrope
at high concentration (~95 wt%). As a result, ingress of small quantities of
water is suffcient to reach the azeotropic composition. Implementation of
the Montreal Protocol, the Clean Air Act, and the Pollution Prevention Act of
1990 has resulted in increased awareness of organic solvent use in chemi-
cal processing.
8.3.2 Solid-liquid Mass Transfer
The MAPs to be processed are in solid form. Solid-liquid extrac-
tion is a typical heterogeneous mass transfer process. In such processes,
the rate of extraction depends upon: (i) the interface area, and (ii) the mass
transfer coeffcient. Both should be high. High effective interface area can
be obtained by comminuting the solid material to be processed. During com-
minution, the ensuing friction can increase the temperature of the solid
and thereby possibly lead to degradation of thermally labile components. To
avoid this, special water-cooled roll crushers are used.
The mass transfer coeffcient depends on the diffusivity of the
solute in the solid matrix (main resistance) and the level of turbulence in
the extractor. Traditional extraction has relied upon percolation or extraction
in stirred vessels. In the case of percolation, the solid is packed in a vessel
which is flled with solvent. The latter is allowed to percolate in the solid ma-
trix under stagnant conditions. In the case of extraction in stirred vessels,
different types of agitators are used to suspend the solid in the solvent and
accelerate the mass transfer process. In both percolation and extraction in
stirred vessels, the solvent is frst sorbed by the matrix of the solid. This
sorption, which causes swelling of the matrix, is a relatively slow process.
However, once the matrix is swollen, the diffusion coeffcient increases sev-
eral fold or even by an order of magnitude as compared to the dry matrix.
Evidently the controlling step is the diffusion of the solute through the
solid matrix to the surface of the solid. Once the solute is available at the
surface, the solvent can dissolve it depending upon the rate of transport
from the solid surface into the bulk of the solvent. In percolation vessels,
this latter transport is predominantly by molecular diffusion and hence is
slow, although not as slow as the transport through the solid matrix. The
133
stirred vessels, on the other hand, provide a high level of turbulence and
hence facilitate transport into the bulk solvent phase. In both percolation
and stirred vessels, the dominant resistance is diffusion through the solid
matrix. It is then clear that even stirred vessels with high power inputs may
not intensify the mass transfer process. Therefore, instead of focusing on
the transport at the solid surface, it is desirable to increase the rate of
transport through the solid matrix by rupturing the cells which contain the
solute or oil and consequently bring the same in direct contact with the
solvent.
8.3.3 Microwave-assisted Extraction
8.3.3.1 Principle of Microwave Heating
Microwave radiation interacts with dipoles of polar and polariz-
able materials. The coupled forces of electric and magnetic components
change direction rapidly (2450 MHz). Polar molecules try to orient in the
changing feld direction and hence get heated. In non-polar solvents without
polarizable groups, the heating is poor (dielectric absorption only because
of atomic and electronic polarizations). This thermal effect is practically in-
stantaneous at the molecular level but limited to a small area and depth
near the surface of the material. The rest of the material is heated by con-
duction. Thus, large particles or agglomerates of small particles cannot be
heated uniformly, which is a major drawback of microwave heating. It may
be possible to use high power sources to increase the depth of penetration.
However, microwave radiation exhibits an exponential decay once inside a
microwave-absorbing solid.
The various industrial techniques used for heating are listed
in Table 1, which shows that microwaves have the highest effciency when
compared with the other competitive techniques.
8.3.3.2 Mechanism of MAE
In microwave-assisted extraction (MAE): 1) the heat of the mi-
crowave irradiation is directly transferred to the solid without absorption by
the microwave-transparent solvent; 2) the intense heating of step 1 causes
instantaneous heating of the residual microwave-absorbing moisture in the
solid; 3) the heated moisture evaporates, creating a high vapor pressure; 4)
the vapor pressure generated by the moisture breaks the cell; and 5) break-
age of cell walls releases the oil trapped within it (Figure 1).
134
Table 1: Relative effciencies of common heating devices
Appliance
Temperature,
C
Rating, W Time
Energy used,
kWh
Energy cost,
US$
Electric oven 177 2000 1 h 2 0.17
Convection
oven
163 1853 45 min 1.39 0.12
Gas oven 177 36 1 h 3.57 0.07
Microwave
oven
High 1440 15 min 0.36 0.03
A B
Figure 1: Mint gland: (A) before and (B) after microwave irradiation
(Microphotographs courtesy of Radient Technologies Inc.)
It is evident then that the main resistance to solid-liquid mass
transfer, the transport of the solute through cell membrane, is eliminated
because of the rupture of the cells. Besides cell breakage, the other advan-
tages of microwave heating are:
Improved existing products 1.
Increased marker recovery 2.
Increased purity of the extract 3.
Reduced heat degradation 4.
Reduced processing costs 5.
Signifcantly faster extraction 6.
Much lower energy usage 7.
Much lower (order of magnitude) solvent usage 8.
Potential for new products 9.
135
8.3.3.3 Literature on MAE
Some interesting results on MAE have recently been published.
For example, the extraction of vanillin from V. planifolia pods using MAE and
ultrasound-assisted extraction has been described. Using absolute ethanol
as the solvent at room temperature, the yield of vanillin was 1.25 wt% at each
of 3 conventional extractions performed over 24 h. Using ultrasound-assisted
extraction, the yield was 0.99 wt%, while it was 1.86 wt% using MAE. These in-
vestigations clearly showed that vanillin extraction by MAE is superior to other
techniques in terms of yield, purity of vanillin, and the time taken to extract
the same percentage of the vanillin from the pods. The extraction of vanillin
and p-hydroxy benzaldehyde (PHB) from vanilla beans using MAE has also
been studied: MAE was superior to the conventional, offcial method of extrac-
tion in Mexico, which involves maceration of the beans with ethanol for 12 h.
Specifcally, extraction time decreased 62-fold and vanillin and PHB concentra-
tions increased between 40% and 50% with respect to the Mexican extraction
method. This study also showed that extraction of commercial samples was
superior to extraction of dried and lyophilized beans. This observation illus-
trates the role played by moisture in aiding extraction, as discussed in Sec-
tion 8.3.3.2. Several other investigations have shown that MAE has gained
acceptance as a mild and controllable processing tool. MAE is a simple, rapid
and low-solvent-consuming process.
8.3.3.4 Industrial-scale MAE
As mentioned earlier, microwave radiation decays exponentially
inside a solid matrix. This aspect must be carefully weighed while designing
industrial-scale MAE. The major requirements that must be met are:
Free distribution of particles allows uniform heating of all the 1.
particles in the solid bed. This criterion also enhances the
extent and probability of proximity of the substrate to the wall
of the sample holder where the microwave exposure is high-
est. Most comminuted samples of MAPs which are used for
commercial extraction are not of the same shape and size.
Therefore, there is a strong tendency to segregate, which
must be curbed by regular renewal of the layer.
Thin and uniform spreading of the substrate layers. This per- 2.
mits complete and uniform penetration of microwave radia-
tion even at large water contents.
Low depth of the layers. Since microwaves have low penetra- 3.
tion depth (~1.5 cm in H
2
O at 2.45 GHz), the layers should be
<1.5 cm thick.
Large-scale commercial (3 tonne/hour) MAE is available for in-
dustrial use (www.radientinc.com). In view of the advantages of MAE and the
development of equipment for large-scale commercial operation, MAE has a
bright future. Figure 2 shows a fowsheet for industrial-scale MAE.
136
Figure 2: Flowsheet of microwave-assisted extraction (courtesy, Radient Technologies Inc).
8.4 Microwave-assisted Hydrodistillation
The ability of microwave radiation to heat solid material effec-
tively can also be used for obtaining essential oils. Thus, the herb is placed
in a microwave cavity and irradiated with microwaves. This process yields
essential oils consisting of relatively low volatile fractions as compared to
hydrodistillation. For instance, in coriander oil, the percentage of tetradeca-
noic and hexadecanoic acid increased whereas that of linalool decreased.
This is possibly due to the poor stability of linalool, a tertiary alcohol.
Dill seed oil obtained by microwave-assisted hydrodistillation
(MWAHD) contained greater quantities of compounds with higher boiling
points and lesser quantities of compounds of lower stability. These and
other fndings indicate that MWAHD is better for extracting stable, high-boil-
ing point components, whereas it is not suitable for recovering chemically
unstable compounds.
137
8.5 Molecular Distillation or Short Path
Distillation
Molecular distillation (MD), also known as short path distilla-
tion, is a fairly well established technique. In view of this, the discussion of
MD is restricted to its principle, advantages and applications in the process-
ing of MAPs.
8.5.1 Principle of MD
The term MD refers to a non-equilibrium process. The still used
has an evaporating surface very close to a condensing surface. Under very
low pressures, this results in a situation where the distance traveled by
the evaporating molecules is comparable to the mean free path of the mol-
ecules. The nomenclature MD is derived from this particular condition under
which the so-called distillation is carried out.
8.5.2 Advantages of MD
Operating pressures as low as 0.001 mbar can yield rela- 1.
tively low processing temperatures, thereby reducing ther-
mal degradation.
Agitated flm MD units can process high viscosity feeds with 2.
very good turndown.
Combination of low pressures and high temperatures (up to 3.
300 C) allows processing of extremely high-boiling materials
without degradation.
Short exposure to high temperature (low residence time) 4.
prevents degradation.
Very low liquid hold-up allowing use in applications involving 5.
low volume, high value materials.
Available in low (laboratory scale) to high heat transfer areas 6.
to suit the requirements.
8.5.3 Separation Effciency of MD
For high viscosity liquid flms falling under gravity, agitated flm
MD units perform far better than those without agitation of the flm. This is
due to the fact that, particularly for high viscosity liquids, the agitation of the
flm renews the surface more frequently than when there is no agitation. The
surface renewal model is useful for predicting the effciency of MD stills
without mechanical control.
138
8.5.4 Parameters that Affect the MD Process
The amount of low-boiling volatiles as well as dissolved air,
moisture, or other gasses in the feed material has a deleterious effect on
the effciency of MD. This is due to the fact that the non-condensable com-
ponents cover the condensing surface.
Higher temperature difference between the condensing and
evaporating surfaces yields higher effciency. High viscosity liquids (without
mechanical agitation) yield high liquid flm thicknesses and hence lower eff-
ciency. As a rule, the relative volatility of organics increases with decreasing
pressure, particularly in the very low pressure range common to MD. There-
fore, low operating pressure generally yields higher effciency.
8.5.5 Typical Applications of MD
Concretes obtained by solid-liquid extraction are convention- 1.
ally converted to the absolutes by dissolving in aqueous al-
cohol solvents and then precipitating the waxes by chilling to
sub-zero temperatures. This process is highly energy inten-
sive due to the electrical energy required for refrigeration.
Red palm oil (high vitamin E content). 2.
Separation of tocopherols from vegetable oil deodorization 3.
residues.
Natural vitamins A, E, K-1 and K-2 (replacing synthetics in 4.
the pharmaceutical industry).
Purifcation and separation of natural extracts into crude 5.
fractions.
Recovery of lanolin from wool grease, the soft wax from hair 6.
of sheep (cosmetic industry).
Fragrances derived from fatty acids. 7.
8.6 Recovery of Dissolved Essential Oils from
Steam Distillation Condensates
The major prerequisite of the process used for production of
essential oils is that the product obtained must resemble the natural aroma
and favor of the original source, which is a combination of different com-
pounds of varying organoleptic characteristics. Oxygenated organic com-
pounds like aldehydes, ketones, alcohols and esters are the dominant con-
tributors to the overall aroma and favor. The essential oil produced should
ideally have all these components in the same proportions as in the original
natural product in order to match the natural aroma and favor. For example,
steam-distilled rose oil contains less than 1 wt% phenyl ethyl alcohol (PEA)
whereas the solvent-extracted rose oil contains greater than 60 wt% PEA.
It is a common experience that the steam distillation condensate has an
139
odor similar to that of the oil. Thus, this condensate has some value. The
sale of rose water (otto of rose) for use in weddings in the sub-continent is
probably the only way by which the distiller realizes the value of the conden-
sate. In practically all other cases, the condensate is wasted. Table 2 gives
estimates of the values of wasted oil in the condensate in India for some
essential oils. The estimate is conservative because it does not include oil
physically carried with the condensate. Even this conservative estimate is a
mind-boggling number and is particularly important in the social context of
developing countries like India where marginal farmers are main contributors
to the overall produce. The value of the recovered oil from central distilla-
tion facilities and pro rata distribution of the value will be a big bonus to the
small farmer and can certainly stop the downslide. Table 2 shows a major
contribution from Mentha arvensis. If the relatively high value favor sector is
included, for India alone the value of wasted oil can easily reach US$ 100
million. On a rough estimate, the combined number for the South East Asian
countries can be upwards of US$ 160 million. It must be noted that these
numbers are simple statistics. They do not refect in any way the high value
that can be gained when the recovered oil is blended with the main distilled
oil fraction to obtain a premium grade of the respective essential oil.
Table 2: Loss of essential oils in distillation condensate water. Production fgures
are for India only
Essential oil
Production,
2006,
million tonnes
Unit price,
2006
Volume,
2007
Oil lost in
water, kg*
Value of oil
lost in water,
$/yr
Arvensis 28,000 $ 14/kg 35,000 MT 5.6-7x10
6
46-58x10
6
Basil 100 $ 8/kg 100 MT 1x10
4
8.2x10
4
Citrodora 100 $ 8/kg 100 MT 1x10
4
7.4x10
4
Citronella 300 $ 8/kg 300 MT 3x10
4
2.52x10
5
Peppermint 450 $ 23/kg 450 MT 4.5x10
4
1.3x10
5
Spearmint 250 $ 23/kg 300 MT 2.5x10
4
7.2x10
4
Total 29,200 36,200 MT 5.72-7.72x10
6
47-59x10
6
* Data refer to 100 kg water per kilogram oil. Solubility of oil in water is 1000 ppm.
8.6.1 Polymeric Adsorption Process
Various techniques, such as cohobation, poroplast extraction
and adsorption, which can be used to recover the dissolved substances,
have been discussed in the literature. Polymeric adsorbents can be advan-
tageously used to recover dissolved essential oil components. Several in-
vestigators have established the utility of adsorption in this context beyond
doubt. One study showed that although cis-rose oxide could not be detected
in the condensate, this valuable component was found in the recovered oil
140
in signifcant proportions. These investigations show that more than 95% of
the oil in the condensate can be recovered. The polymeric adsorbents used
are hard cross-linked macroreticular beads which can be used in adsorption-
regeneration cycles practically indefnitely. The technique is simple to use
and does not require sophisticated instrumentation as the breakthrough
can be judged from the smell of the water coming out of the adsorbent bed.
The regeneration of the spent bed can be done using low-boiling alcohols
or ketones, and the eluate can be distilled in a relatively short distillation
column to obtain a relatively high boiling oil fraction.
8.6.2 Pervaporation Process
Membrane separation processes have been receiving increas-
ing attention particularly for situations involving recovery from relatively dilute
(~1000 ppm) aqueous solutions. Pervaporation is one such process which
yields very high (~1000 ppm or more) selectivity in the very dilute solution
range. Essential oil components which have high affnity for organophilic
polymers can be recovered at very high selectivities. One study showed that
silicone rubber membranes yielded bold menthol crystals when the Mentha
condensate water was studied under the pervaporation mode. Similar re-
sults were also obtained for basil water. Subsequent studies showed that
the high selectivity of properly selected membranes results in a permeate
concentration far exceeding the solubility limit of the organics, resulting in
phase separation.
Figure 3: Recovery of dissolved organics using pervaporation
The separate oil layer can be directly recovered to blend with
the main oil fraction to obtain premium grade oil. Figure 3 shows a sche-
matic of pervaporation-based recovery of dissolved essential oils in the con-
densate. It is evident that this technique consists of a closed-loop operation
141
with only treated water going out of the battery limits. This treated water has
very low biochemical oxygen demand (BOD) and chemical oxygen demand
(COD), which is another bonus for the processor.
8.7 Conclusions
Various new technologies for effcient and cost-effective extrac-
tion of medicinal and aromatic plants have been discussed. Microwave-as-
sisted extraction (MAE) is highly effcient for obtaining extracts under mild
conditions. MAE is particularly important since the active components which
are thermally labile can be recovered without any damage. The loss of valu-
able aroma components in steam distillation condensates is estimated to
be of the order of US$ 50 million per year from aroma oils for India alone.
Two types of separation processes adsorptive and membrane-based per-
vaporation are useful in recovering practically all the oil that is lost with the
condensate water. The recovered oil can be sold as such or blended with the
main oil fraction to yield a much more natural aroma and hence a high value.
This recovered oil will be a big bonus even for the marginal farmer and hence
this approach needs to be seriously considered.
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145
9 Solid Phase Micro-extraction and
Headspace Trapping Extraction
R. Harlalka
Abstract
Solid phase micro-extraction (SPME) is a technique used in the quantitative analysis of
analytes in aqueous and gaseous phases. This novel technology captures aroma mol-
ecules surrounding ower petals without touching the ower or other part of the plant.
SPME has gained widespread acceptance as the technique of choice in many elds
of application, including forensics, toxicology, and the analysis of avors, fragrances,
and environmental and biological matrices. SPME is ideal for eld monitoring. SPME
sampling can be performed in three basic modes: direct extraction, headspace trap-
ping and extraction with membrane protection. Headspace trapping is essentially a
gas extraction technique permitting the direct analysis of volatile compounds present
in a non-volatile matrix. This technique is needed because the aromas of living plant
materials are different from those of the extracted oil. Headspace trapping permits
getting closer to the natural aroma of the living plant, and gives a clearer view of the
differences in volatile constituents between the living plant and the extracted phase.
There are two types of headspace trapping: static and dynamic, which is also called
the purge-and-trap method. A few examples of headspace trapping of well known aro-
matic owers, fruits and leaves, in comparison to the analyses of the extracted oil, are
presented in this paper. Some classical perfumes are also discussed.
9.1 Introduction
Solid phase micro-extraction (SPME) was developed in the
1990s by Professor J. Pawliszyn to provide a quick and solventless tech-
nique for the isolation of analytes from a sample matrix. The traditional
methods by which the analytes of interest were isolated are typically time-
and labor-intensive and involve multistep procedures, which could reduce
sensitivity. Also, the use of solvents can be hazardous to the operators
health and can damage the environment.
SPME was developed from the technique of solid phase extrac-
tion, but the sorbing material is permanently attached to the fber, allowing
reuse of the extracting phase. SPME uses a small volume of sorbent, typi-
cally dispersed on the surface of small fbers, to isolate and concentrate
analytes from the sample matrix. After contact with the sample, analytes are
absorbed or adsorbed by the fber phase (depending on the nature of the
coating). After the extraction step, the fbers are transferred, with a syringe-
like handling device, to the analytical instrument, for separation and quanti-
fcation of the analytes. This technique integrates sampling, extraction and
sample introduction, and is a simple way of performing on-site monitoring.
Applications of this technique include environmental monitoring, fragrance
drug analysis, and in-laboratory and on-site analyses.
9 SOLID PHASE MICRO-EXTRACTION AND HEADSPACE TRAPPING EXTRACTION
146
SPME was introduced in 1990 as a solvent-free sample prepa-
ration technique. The basic principal of this approach is to use a small
amount of the extracting phase, usually less than 1 microliter. Sample vol-
ume can be large when the investigated material is sampled directly, e.g.
the air in a room. The extracting phase can be either a high molecular weight
polymeric liquid, similar in nature to stationary phases in chromatography,
or a solid sorbent, typically of a high porosity, to increase the surface area
available for adsorption.
The confguration of SPME is a small, fused silica fber, usually
coated with a polymeric phase. The fber is mounted for protection in syringe-
like device. The analytes are absorbed or adsorbed by the fber phase until
equilibrium is reached in the system. The amount of an analyte extracted by
the coating at equilibrium is determined by the magnitude of the partition co-
effcient of the analyte between the sample matrix and the coating material.
In SPME, analytes typically are not extracted quantitatively from
the matrix. Equilibrium methods are more selective because they take full
advantage of the difference between extracting phase and matrix distribu-
tion constants to separate target analytes from interferences. Exhaustive
extraction can be achieved in SPME, and this can be accomplished for most
compounds by the application of an internally cooled fber. In exhaustive
extraction, selectivity is sacrifced to obtain quantitative transfer of target
analytes to the extracting phase.
SPME is ideal for feld monitoring. It is unnecessary to measure
the volume of the extracted sample, and therefore the SPME device can be
exposed directly to the investigated material for quantifcation of analytes of
interest. In addition, extracted analytes are introduced into the instrument by
simply placing the fber in the desorbtion unit. This convenient, solvent-free
process results in sharp injection bands and rapid separations.
9.2 The SPME Device
The commercial SPME device manufactured by Supelco (Belle-
fonte, USA) is presented in Figure 1. The fber glued into a piece of stainless
steel tubing is mounted on a special holder. The holder is equipped with an
adjustable depth gauge, which makes it possible to control repeatedly, how
far the needle of the device penetrates the sample container or the injector.
This is important, as the fber can break if it hits an obstacle. The movement
of the plunger is limited by a small screw that moves in the z-shaped slot of
the device. For protection during storage or septum piercing, the fber is with-
drawn into the needle of the device, with the screw in the uppermost posi-
tion. During extraction or desorption, the fber is exposed by depressing the
plunger. The plunger is moved to its lowermost position only for replacement
of the fber assembly. Each type of fber has a hub of a different color.
147
Figure 1: The SPME device
If the sample is in a vial, the septum of the vial is frst pierced
with the needle (with the fber in the retracted position), and the plunger is
lowered, which exposes the fber to sample. The analytes are allowed to
partition into the coating for a pre-determined time, and the fber is then
retracted back to the needle. The device is then transferred to the SPME
instrument. When gas chromatography is used for analyte separation and
quantifcation, the fber is inserted into a hot injector, where thermal desorp-
tion of the trapped analyte takes place.
For spot sampling, the fber is exposed to a sample matrix until
partitioning equilibrium is reached between sample matrix and the coating
material. In the time average approach, on the other hand, the fber remains
in the needle during exposure of the SPME device to the sample. The coat-
ing works as a trap for the analytes that diffuse into the needle, resulting in
integral concentration over time measurements.
SPME sampling can be performed in three basic modes: direct
extraction, headspace trapping, and extraction with membrane protection.
148
Figure 2: Modes of SPME operation: direct extraction (a), headspace trapping (b) and
membrane-protected SPME (c)
In direct extraction, the coated fber is inserted into the sam-
ple and the analytes are transported directly from the sample matrix to the
extracting phase. To facilitate rapid extraction, some agitation is required
to transport the analytes from the bulk of the sample to the vicinity of the
fber. For gaseous samples, natural fow of air (e.g. convection) is usually
suffcient to facilitate rapid equilibrium for volatile analytes.
In headspace mode, the analytes are extracted from the gas
phase equilibrated with the sample. The primary reason for this modifca-
tion is to protect the fber from the adverse effects caused by non-volatile,
high molecular weight substances present in the sample matrix (e.g. human
acids or proteins). Here, the amount of an analyte extracted by the fber
coating does not depend on the location of the fber, in the liquid or gas
phase; therefore, the sensitivity of headspace trapping is the same as that
of direct sampling as long as the volumes of the two phases are the same
in both sampling modes. When no headspace is used in direct extraction, a
signifcant sensitivity difference between direct and headspace trapping can
occur only for very volatile analytes. However, the choice of sampling mode
has a signifcant impact on the extraction kinetics. When the fber is in the
headspace, the analytes are removed from the headspace frst, followed by
indirect extraction from the matrix.
In general, the equilibration times for volatile compounds are
shorter for headspace SPME than for direct extraction under similar agita-
tion conditions, because of the following reasons: a substantial portion of
the analytes is present in the headspace prior to the beginning of the ex-
149
traction process; there is typically a large interface between sample matrix
and headspace; and the diffusion coeffcients in the gas phase are typi-
cally higher by four orders of magnitude than in liquids. The concentration
of semivolatile compounds in the gaseous phase at room temperature is
small, and headspace extraction rates for these compounds are substan-
tially lower. They can be improved by using effcient agitation or by increasing
the extraction temperature.
In the third mode (SPME extraction with membrane protection),
the fber is separated from the sample with a selective membrane, which
lets the analytes through while blocking the interferences. The main purpose
for the use of the membrane barrier is to protect the fber against adverse
effects caused by high molecular weight compounds when dirty samples
are analyzed. While headspace trapping serves the same purpose, mem-
brane protection enables the analysis of less volatile compounds. Use of
thin membranes and an increase in extraction temperature result in shorter
extraction times.
9.3 Calibration, Optimization, Precision and
Suitability of SPME
9.3.1 Selection of Fiber Coating
The chemical nature of the analyte of interest determines the
type of coating used. A simple general rule, like dissolves like, applies
very well for liquid coatings. Selection of the coating is based primarily on
the polarity and volatility of the analyte. Poly(dimethylsiloxane) (PDMS) is the
most useful coating and should be considered frst. It is rugged and able to
withstand high injector temperatures, up to about 300 C. PDMS is a non-
polar liquid, thus it extracts non-polar analytes very well with a wide linear
dynamic range. However, it can also be applied successfully to more polar
compounds, particularly after optimizing extraction conditions.
Both the coating thickness and the distribution constant deter-
mine the sensitivity of the method and the extraction time. Thick coatings
offer increased sensitivity, but require much longer equilibration times. As a
general rule, to speed up the sampling process, the thinnest coating offering
the sensitivity required should be used.
9.3.2 Selection of the Extraction Mode
Extraction mode selection is based on the sample matrix com-
position, analyte volatility, and its affnity to the matrix. For dirty samples, the
headspace or fber-protection mode should be selected. For clean matrices,
both direct and headspace trapping can be used. The latter is applicable for
analytes of medium to high volatility. Headspace trapping is always preferen-
150
tial for volatile analytes because the equilibration times are shorter in this
mode than in direct extraction. Fiber protection should be used only for dirty
samples in cases where neither of the frst two modes can be applied.
9.3.3 Selection of the Agitation Technique
Equilibration times in the gaseous samples are short and fre-
quently limited only by the rate of diffusion of the analytes in the coating.
When the aqueous and gaseous phases are at equilibrium prior to the begin-
ning of the sampling process, most of the analytes are in the headspace. As
a result, the extraction times are short even when no agitation is used. How-
ever, for aqueous samples, agitation is required in most cases to facilitate
mass transport between the bulk of the aqueous sample and the fber.
Magnetic stirring is most commonly used in manual SPME ex-
periments. Care must be taken when using this technique to ensure that
the rotational speed of the stirring bar is constant and that the base plate
does not change temperature during stirring. This usually implies the use
of high quality digital stirrers. Alternatively, with cheaper stirrers, the base
plate should be thermally insulated from the vial containing the sample to
eliminate variations in sample temperature during extraction. Magnetic stir-
ring is effcient when fast rotational speeds are applied.
9.3.4 Selection of Separation or Detection Technique
Most SPME applications have been developed for gas chroma-
tography (GC), but other separation techniques, including high performance
liquid chromatography, capillary electrophoresis (CE) and supercritical fuid
chromatography, can be used in conjunction with this technique. The complex-
ity of the extraction mixture determines the proper quantitative device. Regular
chromatographic and CE detectors can normally be used for all but the most
complex samples, for which mass spectrometry (MS) should be applied.
9.3.5 Optimization of Desorption Conditions
Standard gas chromatographic injectors, such as the popular
split-splitless types, are equipped with large volume inserts to accommodate
the vapors of the solvent introduced during liquid injections. As a result, the
linear fow rates of the carrier gas in those injectors are very low in splitless
mode, and the transfer of the volatilized analytes onto the front of the GC col-
umn is also slow. No solvent is introduced during SPME injection; therefore,
the large insert volume is unnecessary. Opening the split line during SPME in-
jection is not practical, since it results in reduced sensitivity. Effcient desorp-
tion and rapid transfer of the analytes from the injector to the column require
high linear fow rates of the carrier gas around the coating. This can be ac-
complished by reducing the internal diameter of the injector insert, matching
it as closely as possible to the outside diameter of the coated fber.
151
9.3.6 Optimization of Sample Volume
The volume of the sample should be selected based on the es-
timated distribution constant. The distribution constant can be estimated by
using published values for the analyte or a related compound, with the coat-
ing selected. The distribution constant can also be calculated or determined
experimentally by equilibrating the sample with the fber and measuring the
amount of analyte extracted by the coating.
9.3.7 Determination of the Extraction Time
The equilibration time is defned as the time after which the
amount of analyte extracted remains constant and corresponds within the
limits of experimental error to the amount extracted after infnite time. Care
should be taken when determining the equilibration time, since in some cas-
es a substantial reduction of the slope of the curve might be wrongly taken
as the point at which equilibrium is reached. Determination of the amount
extracted at equilibrium allows calculation of the distribution constants.
When equilibrium times are excessively long, shorter extraction
times can be used. However, in such cases the extraction time and mass
transfer conditions have to be strictly controlled to assure good precision. At
equilibrium, small variations in the extraction time do not affect the amount
of the analyte extracted by the fber.
On the other hand, at the steep part of the curve, even small
variations in extraction time may result in signifcant variations of the
amount extracted. Shorter is the extraction time, larger is the relative error.
Autosamplers can measure the time precisely, and the precision of analyte
determination can be good, even when equilibrium is not reached in the
system. However, this requires that the mass transfer conditions and the
temperature remain constant during all experiments.
9.3.8 Optimization of Extraction Conditions
An increase in extraction temperature increases the extraction
rate but simultaneously decreases the distribution constants. In general,
if the extraction rate is of major concern, the highest temperature that still
provides satisfactory sensitivity should be used.
Adjustment of the pH of the sample can improve the sensitiv-
ity of the method for basic and acidic analytes. This is related to the fact
that unless ion exchange coatings are used, SPME can extract only neutral
(non-ionic) species from water. By properly adjusting the pH, weak acids and
bases can be converted to their neutral forms, so that they can be extracted
by the SPME fber.
152
9.3.9 Determination of the Linear Dynamic Range of the
Method
Modifcation of the extraction conditions affects both the sen-
sitivity and the equilibration time. It is advisable, therefore, to check the pre-
viously determined extraction time before proceeding to the determination
of the linear dynamic range. This step is required if substantial changes in
sensitivity occur during the optimization process.
SPME coating includes polymeric liquids, such as PDMS, which
by defnition have a broad linear range. For solid sorbents, such as Carbow-
ax/DVB or PDMS/DVB, the linear range is narrower because of the limited
number of sorption sites on the surface, but it still can span over several or-
ders of magnitude for typical analytes in pure matrices. In some rare cases
when the analyte has extremely high affnity to the surface, saturation can
occur at low analyte concentrations. In such cases, the linear range can be
expanded by shortening the extraction time.
9.3.10 Selection of the Calibration Method
Standard calibration procedures such as external calibration
can be used with SPME. The fber blank should frst be checked to ensure
that neither the fber nor the instrument causes interference with the deter-
mination. The fber should be conditioned prior to the frst use by desorption
in a GC injector or in a specially designed conditioning device. This process
ensures that the fber coating itself does not introduce interference. Fiber
conditioning may have to be repeated after analysis of samples containing
large amounts of high molecular weight compounds, since such compounds
may require longer desorption times than the analytes of interest.
A special calibration procedure, such as isotopic dilution or
standard addition, should be used for more complex samples. In these
methods, it is assumed that the target analytes behave similarly to spikes
during the extraction. This is usually a valid assumption when analyzing
homogeneous samples.
9.3.11 Precision of the Method
The most important factors affecting precision in SPME are:
Agitation conditions
Sampling time (if non-equilibrium conditions are used)
Temperature
Condition of the fber coating (cracks, adsorption of high mo-
lecular weight species)
Geometry of the fber (thickness and length of the coating)
Sample matrix components (salt, organic material, humidity,
etc.)
153
Time between extraction and analysis
Analyte loss (adsorption on the walls, permeation of Tefon,
absorption by septa)
9.3.12 Suitability
SPME is well suited to the analysis of favor and fragrance com-
pounds. The typically small, volatile compounds are easily extracted by the
fbers, and the simplicity of the method allows easy coupling to analytical
instruments. Headspace trapping can reduce the potential for interference
peaks and prevent contamination of both the needle and the instrument.
Loss of these volatile compounds during sample preparation steps is mini-
mized or eliminated compared to conventional methods, and the method is
amenable to feld sampling and analysis.
SPME has been shown to be useful for semivolatile com-
pounds, even though these appeared more challenging in the early years.
With appropriate matrix modifcation, one can take advantage of headspace
trapping for these as well. SPME provides signifcant convenience for feld
and air analysis. Quantifcation is relatively straightforward, even in the pres-
ence of varying air temperature. Finally, the use of SPME for time-weighted
average sampling provides simplicity in monitoring favor and fragrance con-
centrations over time.
9.4 Headspace Trapping Extraction and GC-FID/
MS Analysis
Orange juice volatiles were extracted from the juice headspace
using a syringe-like SPME device equipped with a 75 m Carboxen-PDMS
fber (Supelco). Aliquots (25 ml) of juice were placed in 40-ml glass vials
with plastic screw caps and Tefon-coated septa, warmed to 40 C, and gen-
tly swirled to coat the walls of the vial. Juices were allowed to equilibrate
for at least 15 min prior to fber insertion and were maintained at 40 C
throughout the 35-min extraction period. The fber was then removed from
the headspace and inserted into the heated GC injector, where the volatile
compounds were thermally desorbed. Flavor extract was separated using an
HP 5890 GC instrument equipped with a 30 m x 0.32 mm i.d. DB5 capillary
column. Column temperature was initially 32 C, with a 3-min hold, and was
then increased at 6 C/min to 200 C. Helium carrier gas linear velocity
was 29 cm/s. A special narrow boar (0.75 mm) injector liner was used to
improve peak shape and chromatographic effciency; the entire separation
was conducted in the splitless mode.
154
9.4.1 History of Headspace
In November 1986, at the 10th International Congress on Es-
sential Oils, in Washington DC, USA, Dr. B. D. Mookherjee presented a paper
on the impact of live vs. dead on headspace trapping extraction, using as
example jasmine fowers.
The SPME needle which is 2- to 3-mm solid glass fber coated
with a high-boiling liquid adsorbent, is placed in close proximity to a fower
without touching it and is kept there for a period of 30-60 min depending
on the odor strength of the blossom. The aroma molecules around the pet-
als are absorbed onto the fber. Then with GC/MS, the fber is analyzed to
determine the aroma profle of that particular fower. The aroma of the living
fower was brought into space by NASA in 1998.
9.4.2 The Aura
When the Sun is totally eclipsed by the moon, the surrounding
glow is called an aura. Similarly, if we consider a drop of fragrance, the mol-
ecules surrounding the drop form an aura of that particular fragrance.
It is a common belief that one smells a fragrance, layer by layer,
from the top note of the volatile components, to the middle note of compo-
nents with boiling points in the middle range, and fnally to the bottom note
of components with the highest boiling point.
In reality, when a drop of fragrance is placed on the skin, sev-
eral different molecules, from the lowest to the highest boiling types, irre-
spective of their molecular weights, boiling points and vapor pressures, form
an aura, which eventually reaches our nose and gives us our frst impression
of the particular fragrance. The composition of this aura depends on a char-
acteristic property of each fragrance molecule, knows as its diffusivity.
9.4.3 What is Diffusivity?
Diffusivity is the inherent property of a compound to emit its
molecules into the air. One compound is said to be more diffusive than an-
other if its molecules tend to pass into the air to a greater extent than those
of other compounds. Diffusivity is independent of boiling point, molecular
weight, odor threshold or odor value.
9.4.4 Application of Headspace Trapping
Some examples of headspace trapping are discussed here.
155
9.4.4.1 Jasmine
The headspace constituents of living and picked Jasminum
grandiorum fowers are:
Compound Living fowers, % Picked fowers, %
Benzyl acetate 60.0 40.0
Linalool 3.0 30.0
Indole 11.0 2.0
Cis-jasmone 3.0 --
3,5-Dimethyl-2-ethyl pyrazine -- 0.5
Epi-methyl jasmonate 0.5 --
Methyl jasmonate 0.3 --
Differences in the volatile compounds of living fowers from
Jasminum grandiorum and Jasminum sambac are:
Compound J. grandiforum, % J. sambac, %
Methyl benzoate -- 5.0
Indole 11.0 5.0
Linalool 3.0 9.0
Epi-methyl jasmonate 0.5 --
Methyl jasmonate 0.3 --
9.4.4.2 Yellow Tea Rose
The differences in headspace constituents between living and
picked yellow tea rose fowers are:
Cis-3-hexenyl acetate 20.67 5.39
Hexyl acetate 8.40 4.26
Phenylethyl alcohol 5.73 3.30
3,5-Dimethoxy toluene 9.96 18.58
Alpha-elemene -- 4.07
Geranyl acetone 2.17 --
Dihydro-beta-ionol -- 2.62
Isocaryophyllene 0.30 2.12
Alpha-farnesene 5.83 2.96
156
9.4.4.3 Passion Flower
Volatile constituents of living passion fower (Passiora spp.) are:
Compound Living fowers, %
Methyl benzoate 90.3
Methyl salicylate 1.1
Methyl cinnamate 1.6
9.4.4.4 Lotus
The major differences in headspace constituents of living and
picked lotus (Nelumbo nucifera) are:
Sabinene 6.0 12.0
p-Dimethoxy benzene 18.0 8.0
4-Terpineol 3.0 1.5
Alpha terpineol 9.0 1.0
Cis-jasmone 0.1 --
C15 hydrocarbons 20.0 30.0
9.4.4.5 Lavender
The volatile constituents of living French lavender (Lavandula
dentata) and English lavender (Lavandula angustifolia) are:
Compound French lavender, % English lavender, %
Limonene 18 6
1-Octen-3-ol -- 7
Hexyl acetate 11 2
Eucalyptol 9 3
Linalool 7 --
Cis-3-hexenyl acetate 17 13
Borneol -- 2
Cryptone -- 6
157
9.4.4.6 Chamomile
The volatile constituents of living Roman and German chamomile are:
Compound Roman chamomile, % German chamomile, %
Ethyl 2-methyl butyrate -- 12
Cis-3-hexenyl acetate 3 22
Isobutyl methacrylate 7 --
Isobutyl angelate 18 --
Ocimene -- 1
Iso-amyl angelate 10 --
Isohexyl angelate 10 --
9.4.4.7 Shefali
The volatile constituents of living shefali (Nycanthus arbortristis) are:
Compound Living fower, %
Benzyl alcohol 11.2
Phenyl acetaldehyde 9.4
Phenyl ethyl alcohol 6.3
Methyl anthranilate 10.7
9.4.4.8 Spearmint
The major differences between living and picked spearmint are:
Compound Living plant, % Picked plant, %
Hexanal 0.5 --
Hexanol -- --
Beta-pinene 0.8 2.0
Sabinene 0.5 --
Myrcene 8.8 4.0
Alpha-phellandrene 0.7 --
Limonene 18.0 2.0
Cis- and trans-ocimene 1.0 --
Dihydrocarvone 0.6 2.6
Carvone 24.0 70.0
Alpha- and beta-elemene 5.0 0.1
Beta-caryophyllene 4.0 0.1
158
9.4.4.9 Cinnamon Bark
Comparative analysis of fresh cinnamon bark headspace and
commercial oil has revealed:
Compound Fresh cinnamon bark, % Commercial oil, %
Cis- and trans-cinnamic aldehyde 80.3 71.7
Eugenol -- 12.7
Ortho-methoxy cinnamaldehyde 0.3 --
Eugenyl acetate -- 0.5
9.4.4.10 Ginger
Comparative analysis of the headspace of fresh ginger root
and commercial oil has revealed:
Compound Fresh ginger root, % Commercial oil, %
Citral 15.3 1.2
Beta-bisabolene 3.3 6.2
Alpha zingiberene 15.2 34.4
Cis- and trans-alpha-farnesene 13.7 6.0
ar-Curcumene 11.3 4.8
Beta-sesquiphellandrene 8.0 11.8
9.4.4.11 Peach
Volatile constituents of living and picked peach (Prunus persica) are:
Compound Living peach, % Picked peach, %
Ethyl acetate 6.2 --
Dimethyl disulfde 0.6 --
Cis-3-hexenyl acetate 9.7 --
Methyl octanoate 34.2 7.1
Ethyl octanoate 7.4 11.0
6-Pentyl alpha pyrone Trace 10.6
Gamma decalactone 2.5 39.2
159
9.4.4.12 Pineapple
Volatile constituents of the interior and exterior of living pineapple are:
Compound Exterior, % Interior, %
Methyl hexanoate 13.3 24.6
Ethyl hexanoate 25.6 4.7
Methyl 3-methylthiopropionate 0.9 0.5
Ethyl 3-methylthiopropionate 1.1 --
9.4.5 Classical Perfumes
Almost all successful classical perfumes are based on foral
aromas. Perfumers created them using natural fower oil such as rose and
jasmine. Few persons are aware of the fact that fruit and fower oils that are
made by extraction of picked material exhibit different aromas from those of
the living entities. Examples of classical perfumes based on foral aromas
are Amarige (Givenchy), Joy (Jean Patou), White Linen (Este Lauder), Aura
(Hugo Boss), Anais Anais (Cacharel) and Beautiful (Estee Lauder). The differ-
ence in composition between the oil and the aura of Amarige is as follows:
Compound Oil, % Aura on skin after 60 min, %
Linalool 1.7 17.9
Styrallyl acetate 1.2 9.7
Cashmeran -- 0.5
Bacdanol 0.2 0.5
Hedione 29.9 4.9
Cedramber 1.5 4.9
Iso E super 7.1 12.1
Ambrox 0.2 0.1
Benzyl salicylate 32.5 1.1
Muskalactone 0.9 0.4
The examples include Joy by Jean Patou; White Linen by Este
Lauder; Aura of Hugo BOSS; Anais Anais by Cacharel; and Beautiful by Estee
Lauder.
160
9.4.6 Need for Headspace
When we go to a rose-feld full of bloomed roses, we detect a
pleasant smell in the atmosphere and expect the same fragrance when we
use the bottled perfume or 100% genuine essential oil extracted from the
same roses. However, this is not true. The fragrance we detect in the feld
is completely different from the bottled perfume or essential oil, for the fol-
lowing reasons:
When a fower or herb is processed to obtain the essential a)
oils, the low volatile compounds cannot always be recovered
and often evaporate. These low volatiles are important for
aroma.
During the processing of an herb, many chemical reactions b)
take place, such as saponifcation, trans-esterifcation, po-
lymerization and condensation. These reactions actually
change the character of the oil, so that its aroma no longer
resembles that of the actual plant and the product is differ-
ent in composition. Many stereoterpenes, which are highly
volatile in nature, cannot be extracted and remain in the
herbs. These stereoterpenes do not contribute directly to
the odor but, in combination with other ingredients, impart a
synergic effect to the overall odor quality.
9.5 Types of Headspace Trapping
Headspace trapping can be static or dynamic, which is gener-
ally called the purge-and-trap method. In static headspace trapping, gas ex-
traction is carried out in a single step or in a limited number of steps. On the
other hand, the purge-and-trap technique consists of two or three separate
steps, the frst of which is continuous gas extraction.
9.5.1 Static Headspace Trapping
This is a single-step gas extraction procedure (Figure 3). By
thermosetting the sample for a certain time at a preselected temperature,
equilibrium is reached between the sample phase and the gas phase of the
sample vial. Subsequently, a single aliquot of the headspace is introduced
into the carrier gas fow, which then carries it to the column where the vola-
tile compounds are separated in the usual way.
The equilibrium of the two phases in the sample vial is charac-
terized by a partition coeffcient (Ki) representing the ratio of the analytes
concentration in the sample phase and in the gas phase.
161
Figure 3: Static headspace trapping technique
9.5.2 Dynamic Headspace Trapping
In this technique (Figure 4), the sample is continuously purged
with an inert gas (the purge gas), until all volatile compounds are removed.
During this step, the gas effuent leaving the sample vessel is conducted
through a trap, either cooled to low temperature or containing an adsorbent.
This trap retards the volatile analytes purged from the sample. When gas
extraction is complete, the condensed or adsorbed analytes by rapid heating
of the trap now get purged with the carrier gas.
In Figure 4, the desorbed analytes are conducted directly into
the gas chromatograph. Thermal desorption from an adsorbent is not instan-
taneous: thus, the sample slug might be too long, creating broad peaks,
with tailing. This is particularly the case when a capillary column is used in
the gas chromatograph. For this reason, usually a second, small trap, cooled
to low temperature, is placed in the carrier gas line between the primary trap
and the column. When desorption is fnished, this small trap is then heated
very rapidly: in this way, a sharp band of the analytes enters the column.
162
Figure 4: Dynamic headspace trapping technique
This technique is being used more generally after the introduc-
tion of Tenax (poly(2,6-diphenyl-p-phenylene oxide)) as a universal adsorbent
for dynamic headspace GC by Zlatkis and his group, at the University of
Houston, in 1973. They used the technique for the investigation of biological
fuids and demonstrated the reproducibility of the purge-and-trap method.
9.6 Principles of Static Headspace-GC Systems
Gas from the headspace of a closed vessel can be sampled
simply with a gas-tight syringe. However, with such a manual method, it is
diffcult to reproduce all the conditions necessary for reliable quantitative
analysis. Therefore, today, headspace-gas chromatography (HS-GC) is car-
ried out almost exclusively with automated instruments, in which thermoset-
ting, aliquoting the headspace and introducing it into the gas chromatograph
are fully automated. In this way and using the proper calibration methods,
the required precision, accuracy and reliability are assured.
Present-day HS-GC instruments are of two types. In the frst,
the headspace aliquot is taken by an automated syringe which then is
moved above the injection port of the gas chromatograph and the sample
is injected. In essence, such systems are similar to the autosamplers used
in GC. In the second case, the aliquot from the vials headspace is not with-
drawn by suction as in the case of a syringe: instead, after equilibrium is
reached, the vial is pressurized by the carrier gas. After pressurization there
are two possibilities. The carrier gas fow can be temporary interrupted while
the pressurized gas in the vial is allowed to expand onto the column; the
transferred volume of headspace can be accurately controlled by controlling
the time of transfer and the pressure. The second possibility is to have a
gas introduced between the sample vial and the column, and fll the sample
163
loop of the valve by the pressurized headspace gas. Today, automated in-
struments based on these principles are commercially available.
9.6.1 Trace Analysis by HS-GC
HS-GC in both its dynamic and static versions permits the de-
termination of analytes at low concentrations. Usually the dynamic tech-
nique is considered to be more sensitive; however, this is not necessarily
true. For example, trace impurities in a water sample, at the parts-per-billion
level, can be determined relatively easily by static HS-GC.
9.7 Headspace Trapping Techniques
9.7.1 Static Headspace Trapping
Using the static method (Figure 5), a food sample is normally
placed in a heated vessel, which is sealed gas-tight by a septum. The food
sample stays inside the vessel for a certain period of time, so that the vola-
tile compounds evaporate to a certain concentration in the air or to certain
equilibrium. In order to determine the best conditions for the experiment, the
odor of the headspace can be checked by sniffng the vessel. Subsequently,
a distinct volume is taken out of the vessel by a gas-tight syringe and directly
injected into a gas chromatographic column, with or sometimes without prior
concentration (e.g. cryofocussing). The advantage of this method is that it
accurately assesses the composition of the odorants. An application of this
technique, called GC olfactometry of static headspace samples, has been
widely used to identify the highly volatile compounds causing the frst odor
impression of foods.
Figure 5: Static headspace trapping technique
164
However, static headspace samples are normally too small to
quantify odorants that are present only at low concentrations in the vapor
phase. In other words, one can smell them, but in many cases it is not pos-
sible to obtain a signal in a mass spectrometer.
9.7.2 Dynamic Headspace Trapping
To overcome the disadvantages of headspace trapping method,
dynamic headspace trapping can be used (Figure 6). Again, the food sample
is placed in a heated vessel but the evaporating compounds are continu-
ously swept by a stream of inert gas into a trap containing a porous polymer,
which adsorbs more or less the organic constituents. This method yields a
much higher amount of trapped volatiles so that, after desorption, it is no
longer problematic to obtain an MS signal.
Figure 6: Dynamic headspace trapping technique
However, the disadvantage of this procedure is the strong de-
pendence on the yield of the odorants, on the velocity of the carrier gas and
on the selectivity of the adsorption and desorption process for different
compounds. It is very diffcult to control these parameters precisely and
therefore, the results of such quantitative measurements might be inac-
curate.
9.7.3 Recovering the Adsorbed Volatiles by Thermal or
Liquid Solvent Desorption
Several studies have reported methods of desorption using or-
ganic solvents. Drawbacks of the use of solvent desorption include the loss
of volatile compounds during removal of excess solvent before GC analysis,
solvent selectivity and solvent impurities. We recently developed a sensi-
tive and highly reproducible dynamic headspace (DHS) protocol with thermal
165
desorption (using injector glass liners packed with Tenax-TA as adsorbent
traps for aroma collection at ambient room temperature) and desorption at
the interior of a GC injector. This DHS-type protocol was used to characterize
fresh tomato favor compounds; the results were compared with published
data from a static headspace method (Table 1).
Table 1: Concentration of selected tomato aromas from heat-processed tomato
juice by static headspace trapping (SHT) and dynamic headspace trapping (DHT),
expressed in parts per billion (ppb)
Compound SHT, ppb DHT, ppb
(E)-2-hexanal 5 340
1-Penten-3-one 61 100
2-Isobutylthiazole 2 450
2-Methylfuran 97 1,060
2-Pentylfuran 26 700
3-Methybutanal 17 750
3-Methylfuran 717 3,200
6-Methyl-5-hepten-2-one 21 1,330
Acetone 325 -
Benzaldehyde 3 30
Dimethyl disulfde 16 630
Dimethyl sulfde 5,205 2,974
Ethanol 311 -
Geranial 2 130
Hexanal 188 6,210
Pentanal 48 470
In the present study, this DHT-type protocol was used to charac-
terize fresh tomato favour compounds for comparison with related literature
methods.
9.7.4 Some Practical Examples of Headspace Technique
Use
9.7.4.1 Tomato Juice
Fresh tomato juice was made from vine-ripe fruit by Campbell
Soup Companys R&D centre in Davis, USA. Chemicals were reagent grade,
supplied from reliable sources.
166
9.7.4.1.1 Preparation of Traps
Traps were prepared using silane-treated glass tubing (79 mm
x 6 mm) packed with 13 mg 60/80 mesh Tenax-TA (2,6-diphenyl-p-phenylene
oxide) polymers held in place by silanized glass wool. The traps were initially
conditioned at 330 C for 2 h under nitrogen gas at a 20 ml/min fow rate.
The traps were regenerated at 250 C for 1 h immediately before each
purge-and-trap experiment.
9.7.4.1.2 Thermal Desorption
Adsorbed volatile compounds were recovered from the trap di-
rectly inside the GC injector. The desorption time and temperature were pre-
viously determined. The injector temperature was 200 C and a loop of the
analytical column at the injector end was immersed in a liquid nitrogen-flled
Dewar fask to cryogenically trap the desorbed volatiles. Subsequently, the
injector glass liner (insert) was replaced with the trap to desorb volatiles.
Thermal desorption was carried out for 5 min with the split vent and septum
purge closed.
9.7.4.2 Headspace of Hedychium coronium
Hedychium coronium fower has a delicate, pleasant fragrance,
but the essential oil and concrete extracted by traditional methods usually
lose this fragrance. Thus, the headspace of the H. coronium fower was ana-
lyzed. The essential oil of H. coronium fowers, which was absorbed by XAD-4
resin, eluted by organic solvent and concentrated, had a fragrance similar to
the natural fragrance of H. coronarium fowers.
9.7.4.3 Volatiles of White Hyacinths Isolated by Dynamic
Headspace Trapping
More than 70 constituents of white hyacinths can be identi-
fed by GC and GC-MS. The principal constituents are benzyl acetate and
(E,E)--farnesene. Beside these, sensorily important substances like indole,
oct-1-en-3-ol and phenylacetaldehyde were identifed. Minor traces of three
substituted pyrazines were detected by GC-sniffng. The advantages of the si-
multaneous closed-loop stripping technique using various adsorbing agents
at the same time were demonstrated. By this method, artifact formation and
discrimination of individual components can be determined
9.7.4.4 Medical Materials Testing by Headspace Trapping-GC-MS
The new technology provided by the HS-40 Trap coupled with
a sensitive detection method such as GC-MS allows volatile organic com-
pounds in medical sutures to be analyzed easily at trace levels. Individual
compounds present in the sutures can be analyzed by GC-MS and identifed
167
by a NIST library search of the acquired mass spectral data. The innovative,
patent-pending, headspace trapping technology used in this application pro-
vides sensitivity beyond the capability of traditional static headspace. This
presents a new level of detection capability for the evaluation of materials
used in medical applications, as well as in other types of material testing,
including pharmaceutical formulations and food-packaging flm.
9.8 Conclusions
Advanced technologies such as SPME and headspace trapping
extraction are well suited for the analysis of favor and fragrance compounds.
The typically small, volatile compounds are easily extracted. The simplicity
of the method allows easy coupling to analytical instruments. Loss of vola-
tile compounds during sample preparation steps is minimized or eliminated,
compared to conventional methods.
These techniques are useful for semivolatile compounds, even
though these were more challenging in the early years. With appropriate
matrix modifcation, one can take advantage of this analytical method, which
provides signifcant convenience for feld and air analyses.
Bibliography
Anonymous, 2006, On sampling, conditioning and gas handling. Available at: http://
www2.nose-network.org/members/3_Sampling.doc
Anonymous, 2006, Welcome to the SPME. Available at: http://www.Sigmaaldrich.
com/ Brands/Supelco_Home/Spotlights/SPME_central.html
Mookherjee, B. D., 1998, A novel technology to study the emission of fragrances
from skin, Perfumer & Flavorist, 23(11): 1-11
Rouseff, R. L. and Cadwallader, K. R. (eds.), 2001, Headspace Analysis of Foods and
Flavors: Theory and Practice, Kleur Publishing: New York, p. 212
Snow, M. and Grecsek, H. 2006, Medical Materials Testing by Headspace Trap-GC/
MS. Available at: http://las.perkinelmer.com/Content/ApplicationNotes/FAR_
GCMSMedical Materials.pdf
169
10 Supercritical Fluid Extraction of
Medicinal and Aromatic Plants:
Fundamentals and Applications
A. Bertucco and G. Franceschin
Abstract
The main issues related to supercritical uid extraction of medicinal and aromatic
plants are discussed in view of the development of this separation technique at indus-
trial scale. After an introduction to supercritical uid extraction, the roles of thermo-
dynamics and mass transfer properties are emphasized, and the effects of the main
operating variables on product recovery are briey examined. Fundamental concepts
about the equipment needed and basic technology are presented, including economi-
cal evaluation. Finally, a short literature survey of successful supercritical extraction
processes of medicinal and aromatic plants is reported and a future outlook is given.
10.1 Introduction
In the second half of last century, an increasing interest has
been paid to supercritical fuids as alternate solvents for the extraction of
natural bioactive molecules from plants. The main reason for the interest
in supercritical fuid extraction (SFE) was the possibility of carrying out ex-
tractions at temperature near to ambient, thus preventing the substance of
interest from incurring in thermal denaturation.
A thorough review of the results achieved up to the early 1980s
is presented in a book by Stahl et al., published in 1986. Clearly, by that
time, the fundamentals of this new extraction process were already under-
stood, even though the technical-economical assessment and the design
criteria for large-scale application of SFE were still missing. After twenty
years of research and development, it is now possible to say that such
achievements are at hand, so SFE is currently a well-established unit opera-
tion for extraction and separation. Moreover, its design and operating crite-
ria are fully understood, so that it can proftably be applied in the extraction
of medicinal and aromatic plants (MAPs).
10.2 Supercritical Fluids
A fuid at supercritical condition, also referred to as a dense
gas, is a fuid above its critical temperature (T
C
) and critical pressure (P
C
) to
a certain extent: to be supercritical, the reduced temperature T
r
(i.e. T/T
C
)
must not exceed 1.2 or 1.3, whereas the reduced pressure P
r
(i.e. P/P
C
) may
be as high as allowed by technological limits.
10 SUPERCRITICAL FLUID EXTRACTION OF MEDICINAL AND AROMATIC PLANTS: FUNDAMENTALS AND APPLICATIONS
170
At suitable conditions, any fuid can reach its supercritical
state. However, only those having a critical temperature not far from ambi-
ent temperature can be used as alternative solvents for the extraction of
MAPs. Carbon dioxide (CO
2
), with T
C
=31.06 C and P
C
=73.81 bar, is the
most attractive solvent, because of its proprieties regarding toxicity, fam-
mability and cost.
The possibility of using supercritical fuids (SFs) as extraction
solvents is directly linked to their density. In fact, according to an empirical
correlation proposed by Chrastil in 1982,
s =
a
exp
(
b
T
+ c
)
(1)
where s is the solute solubility, is the solvent density and T is the absolute
temperature; a, b and c are correlation parameters to be adjusted to experi-
mental solubility data in supercritical CO
2
.
When a fuid approaches the critical conditions, its density gets
closer and closer to that of the liquid state. This can be seen, for CO
2
, in
Figure 1, where density isotherms are plotted against the reduced pressure.
For example, at T = 35 C and P = 200 bar, = 866 kg/m
3.

It is also clear from Figure 1 that, close to the critical point,
both the compressibility and expansion coeffcient of the fuid are high, so
slight changes in the operating conditions can signifcantly modify the den-
sity, i.e. the supercritical fuid solvent power. The importance of the Chrastil
equation (Eq. 1) lies in the fact that solvent density is identifed as the key
factor in a successful SFE process.
z.o
1.o
o
o.1 1.o 1o.o
R
k

=

R
R
C
CP
T
k
=

T
]
TC

=

o.S
o.
1.o
1.1
1.z
1.cc
Figure 1: Density vs. pressure diagram for carbon dioxide
When plotted against solvent density, solubility data for super-
critical CO
2
always display a regular trend such as that in Figure 2a, whereas
a more complex behavior is seen when pressure is improperly used as the
independent process variable (Figure 2b).
171
o.uo o.uc o.co o.cc o.6o o.6c o./o o./c o.So o.Sc o.o
o
1oo
zo
S
o
l
u
b
l
l
l
t
y
,

1
o
-
6

m
o
l
]
d
m
o
oo
uoo T =ocK
T =1 cK
T =oK
0enslty, e]cm
a
o co 1oo 1co zoo
o
1oo
zoo
oo
uoo
C 8 A
T=ocK
T=1cK
T=oK
Pressure, bar
S
o
l
u
b
l
l
l
t
y
,

1
o
-
6

m
o
l
]
d
m
b
Figure 2: Solubility of 1,4-bis-(n-propylamino)-9,10-anthraquinone in supercritical CO
2
: as a
function of solvent density (2a) and of pressure (2b)
Coming back to Eq. 1, is important to point out that it is not theo-
retically correct and must be applied within restricted temperature ranges only.
More importantly, the exponential form of Eq. 1 does not guar-
antee that the solubility of a solute in SF is high. The solubility depends on
parameters a, b and c; in fact, in the case of CO
2
, the solubility of a solute
of interest for MAP applications is at best in the range of 1 to 1000 by
weight or often 1 to 10,000 (Figure 2a illustrates this). This is because CO
2

is a poor solvent, even at supercritical conditions. In addition, this holds
for non-polar substances only, as supercritical CO
2
does not dissolve polar
molecules at all. Actually, CO
2
is a good solvent only for low molecular weight
solutes.
The limit on solubility is dictated by thermodynamics. Accord-
ing to the iso-fugacity criterion applied to the substance to be extracted,
between the two phases at equilibrium (the condensed one either solid or
liquid and the supercritical one), we have:
y
i
=
P
sat
P
E
i
(2)
E
i
=
i
o, v
i
v
exp
(
v
S/L
P P
sat
RT
)
(3)
where y
i
is the mole fraction of i in the supercritical phase,
i
o,v
and
i
v
are
the fugacity coeffcients of i in the standard state and in the mixture, respec-
tively, at the process conditions, P
sat
is the solute saturation (or sublimation)
partial pressure (i.e. the component volatility), and v
S/L
is the molar volume
of the condensed phase (either solid or liquid). T is the absolute tempera-
ture and R is the universal gas constant. E
i
is the so-called enhancement
factor, which accounts for the increasing solubility due to system nonideali-
ties with respect of the ideal behavior (given by Eq. 2).
172
According to Eq. 2, the solubility of i in the SF can be calculated
at the process condition, provided that the fugacity coeffcients
i
can be
evaluated accurately by means of an equation of state. However, the sub-
stance vapor pressure directly infuences the solubility when P
sat
is as usual
for MAPs, very low. Only an equally low value of the fugacity coeffcient, i.e.
a high system nonideality, can partially counteract the lack of volatility of the
pure component.
Regardless of the way its value has been obtained, i.e. from
Eq. 1 or Eqs. 2-3, the solubility is only one of two fundamental pieces of
information that must be known in order to assess the feasibility of an SFE
process for MAPs. The second one is selectivity, which is defned as the ra-
tio of the solubility of the substance i of interest with respect to a reference
substance j:

ij
=
s
i
s
j
(4)
If on one hand high solubility is desirable, to reduce the solvent
consumption per unit product extracted, on the other hand selectivity must
be as far as possible from 1, to ensure that the substance of interest is ex-
tracted as pure rather than in mixture with other components. In summary,
to develop a successful SFE process for MAPs, both solubility and selectivity
issues must be fulflled properly.
Coming back to CO
2
, it must be kept in mind that this solvent
is rather non-selective: when it is able to dissolve a group of similar sub-
stances (for example, in terms of carbon atoms), all of them are extracted
to a similar extent, provided they have similar polarities.
Therefore, it can be stated that CO
2
alone is not as selective as
a good and pure solvent. It is also noteworthy that CO
2
capacity and selectiv-
ity may be improved by using an organic solvent as the entrainer, also called
the co-solvent, with the function of modifying chemical interactions between
CO
2
and the substance to be dissolved in it.
But by doing this, the SFE process becomes more complicated,
as an extra chemical component needs to be introduced into the process.
However, the co-solvent can be easily separated from the product down-
stream, due to the high selectivity displayed by supercritical CO
2
in this
respect.
10.3 SFE Processes
An SFE process for extracting MAPs is basically composed of two
main sections (Figure 3a). The feed, containing the substance of interest, in-
dicated by A, comes in contact with supercritical CO
2
, at suitable temperature
173
and pressure, in an extraction device. In this simple scheme, component A is
selectively extracted and must be recovered from the supercritical solution,
which is usually a dilute one for the reason explained in the previous section.
Product recovery occurs in the separation section, whose temperature and
pressure can be adjusted in order to optimize the amount of A produced.
Note that, due to the low solubility in supercritical CO
2
, after
recovery of the product of interest the solvent must be recycled and pumped
back to the extractor, in order to minimize operating costs. It is also note-
worthy that the separator can be operated either at the same temperature
or at the same pressure of the extractor, the best condition resulting from
an economical analysis of the overall production costs.
If the temperature is kept constant, product separation is
achieved by depressurization (Figure 3a), and mechanical energy has to be
provided to the system to raise the CO
2
pressure from the separator to the
extractor conditions. On the other hand, extracted products can be sepa-
rated from CO
2
by increasing the temperature, and thermal energy must be
supplied in this case (Figure 3b), where the circulation of the solvent can
be done at nearly isobaric conditions. Of course, the way the separation
of products from CO
2
is achieved can be more complex: for instance, both
temperature and pressure can be varied when passing from the extractor
to the separator sectors, or a solid can be used to promote separation by
adsorption.
Figure 3: Block ow diagrams of simple SFE processes: with separation obtained by
pressure change (a) and by temperature change (b)
If, as it often occurs, many substances are extracted by CO
2
at
the extraction conditions because of lower CO
2
selectivity, their fractionation
can also be achieved in the separation section, by simply using more than one
separator, operated at different conditions. As shown in Figure 4, multiple frac-
tions with different properties can be recovered from the same extraction.
174
extractor separator
fraction 1 fraction 2 fraction 3
Figure 4: Single extractor, multiple separator scheme
Finally, a multiple extractor scheme can also be envisaged, as
represented in Figure 5 with only one separation step, for sake of clarity. This
confguration is particularly useful when, as in the case of SFE of MAPs, the sub-
stances to be extracted are embedded in a solid matrix, which is initially loaded
in the extraction vessel as a fxed bed. In this case, the extractors can be con-
nected either in parallel or in series, depending on specifc requirements.
More details on the development of SFE processes are pro-
vided in a book by Brunner listed in the bibliography. SFE of solids is a
semibatch operation, which can also be operated in a simulated moving bed
confguration to obtain a continuous production.
extract
1 2 3 4
Figure 5: Multiple extractor, single separator scheme
Typical extraction profles from solid materials (single vessel)
are shown in Figure 6 where the extractor yield, i.e. the amount of substance
of interest extracted with respect to the total amount initially contained in
the solid, is plotted against extraction time. The profles, which are steeper
if the temperature is higher, have two parts: a straight line corresponding
to the extraction of the substance readily available to supercritical CO
2
,
and an asymptotic curve representing the extraction of the part attached to
the solid matrix. In the frst case, the extraction is limited by solubility; in
the second, mass transport (diffusion) properties are important and can be
limiting and crucial for the success of SFE.
175
The effects of operating temperature are also clear in Figure 6.
Other important operating variables are pressure, CO
2
fow rate and humidity
of the material to be extracted.
o
1o
zo
o
uo
co
6o
/o
So
o
1oo
o 1 z u c 6
T1 Tz T
T Tz T1
Fxtractlon tlme 'h]
F
x
t
r
a
c
t
l
o
n

y
l
e
l
d
,

%
S
o
l
u
b
l
l
l
t
y
0
l
f
f
u
s
l
o
n
Figure 6: Extraction yield versus time at different temperatures
10.4 The SFE Process and Equipment
Development
In order to design and develop an SFE process for MAPs with
CO
2
(possibly assisted by ethanol or water as entrainers), we need to know
and optimize:
The solubility of the substance of interest 1.
The selectivity of this substance with respect to others that 2.
are extracted simultaneously
The extraction profles (such as those in Figure 6) 3.
The way to separate the substance of interest from the total 4.
extract
All this information can be obtained by simple measurements
performed in a laboratory-scale apparatus of minimum volume such as that
illustrated in Figure 7.
Figure 7: Laboratory-scale apparatus for SFE process design
176
To measure extraction profles, a small pilot-scale apparatus can
be used. Extractor and separator volumes do not need to exceed 1 liter each.
The analytical system must be suitable to measure the concentration and purity
of the products of interest. Basic requirements in terms of equipment are:
A liquid CO 1.
2
storage tank
A pump for liquid CO 2.
2
A cooler to prevent CO 3.
2
from evaporating in the pump
A heat exchanger to control the temperature of CO 4.
2
entering
the extractor
An extraction vessel 5.
A heat exchanger to control the CO 6.
2
plus solute mixture en-
tering the separator
A separation vessel 7.
Note that condensing and recycling of CO
2
after separation is
not needed at the laboratory-scale developmental level, whereas these are
essential requirements at the industrial production level.
All parts of the SFE laboratory-scale plant must be designed
in order to resist the maximum operating pressure. If this does not exceed
300-350 bar, the entire equipment (e.g. vessels, valves, fttings) is pretty
much of standard type and relatively inexpensive. If, as usual, stainless
steel is used, the thickness of any part of the plant can be easily calculated
by applying the Von-Mises equation:
P
i

k
2
k
2
1

3 <
am
(5)
with:

am
=
s
S
f
(6)
where P
i
is the internal pressure, k is the external to internal diameter ratio,
s
is the yield stress, and S
f
is a suitable safety factor (usually S
f
=1.5).
From Eq. 5, it can be seen that the thickness of a cylindrical
vessel depends on its diameter. Examples are given in Table 1 for a vessel
of 0.2 m internal diameter, with both stainless steel and carbon steel con-
struction materials.
Table 1: Thickness (s) of a thick-wall cylindrical vessel of 200 mm internal diameter
as a function of pressure (SS=stainless steel, CS=carbon steel)
P [atm] 50 100 150 200 250 300
s [mm]
SS 3.8 8.1 12.9 18.5 25.0 32.7
CS 2.2 4.6 7.2 10.0 13.0 16.2
177
Special care must be paid to closures and seals. SFE of MAPs
is mostly an extraction operation from solid materials, which is carried out in
batch or semibatch mode. Therefore, extraction vessels need to be pressu-
rized, depressurized, opened, flled, and closed again several times per day.
In order to ensure fast and safe operation procedures and reliable seals,
gaskets like O-rings are useful and closure devices have been specifcally
designed. Again, the technology needed is already fully developed. We refer
to chapter 4 of the book by Bertucco and Vetter for details. The book also
describes the machinery for moving fuids under pressure, i.e. pumps and
compressors. We conclude that setting up a laboratory-scale apparatus with
which to perform feasibility studies concerning the possibility of applying
SFE to MAPs is not really an issue, and can be done with a relatively small
capital cost.
However, this does not mean that SFE of MAPs is in itself an
economically convenient operation. An accurate evaluation of production
costs, including both capital and utility costs, must be performed before
scaling up a process whose technical feasibility has been demonstrated at
the laboratory level. Costs are also discussed in the book by Bertucco and
Vetter (chapter 8), but are only indicative. The reader should remember that
capital costs have been steadily decreasing in the last years must be taken
into account.
10.5 SFE Applied to Medicinal and Aromatic
Plants
A large number of MAPs has been considered for possible
extraction by supercritical CO
2
. The most recent developments suitable to
have industrial relevance are listed in Table 2. These examples illustrate the
great potential of SFE in this feld.
Table 2: Medicinal and aromatic plants extracted by SFE
Plant name
(part used)
Product(s) extracted Reference
Calendula
ofcinalis
(fowers)
Oleoresin Campos et al., 2005, Experimental
data and modeling the supercritical
fuid extraction of marigold (Calendula
ofcinalis) oleoresin, J Supercritical
Fluids, 34: 163-170
Danielski et al., 2007, Marigold
(Calendula ofcinalis L.) oleoresin:
solubility in SC-CO
2
and composition
profle, Chem Eng Proc, 46: 99106
178
Plant name
(part used)
Echinacea
purpurea
(whole herb)
Alkamides, polyphenolics
including chichoric acid,
carbohydrates
Catchpole et al., 2002, Supercritical
extraction of herbs I: saw palmetto,
St John's wort, kava root, and
Echinacea, J Supercritical Fluids, 22:
129-138
Eucalyptus spp.
(leaves)
Essential oil Della Porta, et al., 1999, Isolation
of eucalyptus oil by supercritical
fuid extraction, Flavour Fragr J, 14:
214218
Ginkgo biloba
(leaves)
Flavonol glycosides
(favonoids) and
terpenoids
Chun Yang et al., 2002, Extraction
of pharmaceutical components
from Ginkgo biloba leaves using
supercritical carbon dioxide, J Agric
Food Chem, 50: 846-849
Hypericum
perforatum
(herb)
Naphthodianthones,
hypericin and
pseudohypericin
Catchpole et al., 2002
Levisticum
ofcinale
(dry rhyzomes,
roots)
Essential oil Daukas et al., 1999, Supercritical CO
2

extraction of the main constituents
of lovage (Levisticum ofcinale Koch.)
essential oil in model systems and
overground botanical parts of the
plant, J Supercritical Fluids, 15: 5162
Matricaria
chamomilla
(fowers)
Oleoresin Kotnik et al., 2007, Supercritical
fuid extraction of chamomile fower
heads: comparison with conventional
extraction, kinetics and scale-up,
J Supercritical Fluids, Available online
13 February 2007 (in print)
Mentha spp.
(leaves)
Essential oil Marongiu et al., 2001, Extraction
and isolation of Salvia desoleana
and Mentha spicata subsp. insularis
essential oils by supercritical CO
2
,
Flavour Fragr J, 16: 384388
Origanum spp.
(herb)
Essential oil Leeke et al., 2002, Eng Chem Res,
41: 20332039
Piper
methysticum
(roots,
rhizomes)
Kava lactones Catchpole et al., 2002
Piper nigrum
(fruit)
Oleoresin Ferreira et al., 1999, Supercritical
fuid extraction of black pepper (Piper
nigrum L.) essential oil, J Supercritical
Fluids, 14: 235245.
179
Plant name
(part used)
Saccharum spp.
(crude wax)
Long chain n-alcohols De Lucas et al., 2005, Supercritical
extraction of long chain n-alcohols
from sugar cane crude wax,
J Supercritical Fluids, 34: 163-170
Salvia desoleana
(leaves)
Essential oil Marongiu et al., 2001
Serenoa repens
(fruit)
Free fatty acids,
phytosterols (low
concentrations), fatty
alcohols and triglycerides
Catchpole et al., 2002
Solanum
lycopersicum
(fruit)
Carotenoids, tocopherols
and sitosterols
Vagi et al., 2007, Supercritical carbon
dioxide extraction of carotenoids,
tocopherols and sitosterols from
industrial tomato by-products,
J Supercritical Fluids, 40: 218226
Taxus brevifolia
(bark)
Taxol Jennings et al., 1992, Supercritical
extraction of taxol from the bark of
Taxus, J Supercritical Fluids, 5: 1-6
Taxus cuspidate
(needles)
Paclitaxel and baccatin III Moon-Kyoon Chun et al., 1996,
Supercritical fuid extraction of
paclitaxel and baccatin III from
needles of Taxus cuspidate,
Vitis vinifera
(seeds)
Procyanidins Cao et al., 2003, Supercritical fuid
extraction of grape seed oil and
subsequent separation of free fatty
acids by high-speed counter-current
chromatography, J Chromatogr A
1021: 117124
Zingiber
ofcinale
(rhizome)
Oleoresin Badalyan et al., 1998, Extraction of
Australian ginger root with carbon
dioxide and ethanol entrainer,
10.6 Conclusions
SFE with CO
2
is a technically and possibly economically valid
technique to extract bioactive components from MAPs. Organic solvent-free
products can be obtained and the low operating temperature makes it pos-
sible to preserve all their natural properties. The feasibility study on specifc
products can be performed rather easily at laboratory scale. However, ac-
curate evaluation of production costs, including both capital and operating
ones, must be done in order to exploit SFE at the industrial level.
180
Bibliography
Bertucco, A. and Vetter, G. (Eds.), 2001, High Pressure Process Technology: Funda-
mentals and Applications, Elsevier Science, Amsterdam
Brunner, G., 1994, Gas Extraction: An Introduction to Fundamentals of Supercritical
Fluid and Application to Separation Processes, Steinkopff Darmstadt Springer,
New York
Chrastil, J., 1982, Solubility of solids and liquids in supercritical gases, Journal of
Physical Chemistry, 86: 3016-3021
Shi, J. (Ed.), 2006, Functional Food Ingredients and Nutraceuticals, Processing Tech-
nologies, Taylor and Francis, Boca Raton, USA
Stahl, E. K., Quirin, W. and Gerard, D., 1986. Dense Gases for Extraction and Refn-
ing, Springer Verlag, Berlin
181
11 Process-scale High Performance Liquid
Chromatography for Medicinal and
Aromatic Plants
M. M. Gupta and K. Shanker
Abstract
High performance liquid chromatography (HPLC) is widely used by chromatographers
and by the pharmaceutical industry for the accurate and precise analysis of chemicals
and drugs of diverse nature. The systematic scale-up from analytical to preparative
and process scale and further scale-up to industrial scale can be used in the medici-
nal and aromatic plant industry for the isolation and purication of phytomolecules of
therapeutic and commercial interest. Due to the gradual increase in the demand for
phytomolecules, the importance of process-scale HPLC as a purication tool has been
increasing. In this article, we discuss the practical aspects of process-scale HPLC and
focus on terminology, operational problems, advantages and applications of this tech-
nology to medicinal and aromatic plants.
11.1 Introduction
The term liquid chromatography (LC) refers to a range of chro-
matographic systems, indicating liquid-solid, liquid-liquid, ion-exchange and
size exclusion chromatography. Glass column chromatography is an exam-
ple of classic liquid column chromatography in which the mobile phase per-
colates under gravity through a glass column flled with a fnely divided sta-
tionary phase. Liquid chromatography has overtaken gas chromatography,
as high performance liquid chromatography (HPLC) systems now provide
features such as:
High resolving power i)
Fast separation ii)
Continuous monitoring of column effuent iii)
Qualitative and quantitative measurements and isolation iv)
Automation of analytical procedures and data handling v)
There has been tremendous growth in this technique since
1964 when the frst HPLC instrument was constructed by Csaba Horvath
at Yale University. For the isolation of compounds, preparative mode HPLC
(prep-HPLC) can be used in pharmaceutical development for trouble-shoot-
ing purposes or as part of a systematic scale-up process. The importance
of prep-HPLC in pharmaceutical production as a purifcation tool has been
increasing. Chromatographic separation can remove impurities of different
polarity and can reduce the content of an enantiomer in a racemic mixture.
In both of these instances, crystallization may be used to prepare the pure
product. Bench to pilot scale production of natural products needs some
11 PROCESS-SCALE HIGH PERFORMANCE LIQUID CHROMATOGRAPHY FOR MEDICINAL AND AROMATIC PLANTS
182
form of automation: thus, developing well-automated preparative chromato-
graphic methods is a necessary but demanding task.
Innovations in micro-analytical to preparative HPLC played an
important role in the progress of natural product chemistry. HPLC is used
routinely in phytochemistry to pilot the preparative-scale isolation of natu-
ral products and to control the fnal purity of the isolated compounds. The
development of hyphenated techniques related to this effcient separation
technique in the past 20 years has provided powerful new tools such as
LC/UV-photodiode array detection, LC/mass spectrometry (LC/MS) and LC/
NMR. The combination of high separation effciency of HPLC with these dif-
ferent detectors has made possible the acquisition of data on an LC peak
of interest within a complex mixture.
11.2 Theoretical Aspects of HPLC
Separation of chemical compounds is carried out by passing
the mobile phase, containing the mixture of the components, through the
stationary phase, which consists of a column packed with solid particles.
The cause for retention is physical and chemical forces acting between the
solute and the two phases, on the chromatographic column. The reason
for retention is the difference in the magnitude of forces; this results in the
resolution and hence separation of the individual solutes. The separation of
compounds occurs by distribution of solutes between the two phases.
11.2.1 Chromatography Classifcation
Chromatography can be classifed according to mechanism of
separation as: adsorption chromatography, partition chromatography, ion ex-
change chromatography, size exclusion chromatography and affnity chroma-
tography. In HPLC, separation is mainly governed by adsorption and partition
chromatography. In adsorption chromatography, separation is based on the
difference between the adsorption affnities of the sample components on
the surface of an active site, whereas in partition chromatography separa-
tion is mainly based on the difference between the solubility of sample
components in the stationary phase and the mobile phase.
There are two modes of analysis depending on the operation
techniques viz. isocratic and gradient. Isocratic analysis is the procedure
in which the composition of the mobile phase remains constant during the
elution process. In gradient elution, the composition of the mobile phase
changes continuously or stepwise during the elution process. HPLC can also
be classifed according to special techniques, such as reverse phase (RP)
and normal phase chromatography. Reverse phase is an elution procedure
used in liquid chromatography where the mobile phase is signifcantly more
polar than the stationary phase. On the other hand, in the normal phase
183
procedure, the stationary phase is more polar than the mobile phase. Li-
pophilic substances like oils, fats and lipids are separated by normal phase
chromatography. Commonly used mobile solvents are n-hexane, heptane,
chloroform, and alcohols. Most biomedical substances are separated by
reverse phase chromatography using aqueous mixture with methanol, ac-
etonitrile and additives (buffers, ion-pairs).
11.2.2 Important Factors that Infuence HPLC Separation
HPLC separation is infuenced by dead volume, capacity factor,
theoretical plate count and selectivity:
Dead volume (V
0
) is the volume at which an un-retained com-
ponent elutes.
Capacity factor (K
) is a measurement of the retention time

of a sample molecule, relative to column dead volume. It
changes with variations in mobile phase composition, col-
umn surface chemistry or operating temperature. Capacity
factor is calculated as follows:
K
'
=
V
1
V
0
V
0
E
i
V
1
= Retention volume of peak 1
Theoretical plate count (N) is a measure of column effciency
in terms of band-spreading of a peak. The smaller the band-
spread, the higher the number of theoretical plates, which
indicates good column and system performances.
Resolution (R
s
) is the distance between the peak centres of
two component peaks divided by the average base of the
peaks, as follows:
R
S
=
V
2
V
1
W
1
+ W
2
W
1
= width of peak 1
W
2
= width of peak 2
Selectivity () is the relative retention of two peaks in a chro-
matogram.
=
K
'
2
K
'
1
=
V
2
V
0

V
1
V
0
K
1
and K
2
= capacity factors for retention volume of peak 1
and peak 2 respectively.
184
Capacity factor (K
), selectivity () and column effciency (N) are

three fundamental parameters that infuence the resolution of a chromato-
graphic separation, as follows:
R
s
=
1
4

(
1

)

N
(
K
'

1 + K
')
11.2.3 Main Components of HPLC
An HPLC system contains the following components:
Reservoir a) . This is meant for the mobile solvents. Acetonitrile, methanol,
heptane, isopropanol and cyclohexane are the organic modifers most
commonly used. Trifuoroacetic acid, heptafuorobutyric acid, phosphor-
ic acid and triethylamine phosphate are ion-pairing reagents for better
chromatographic results. All tubing and fttings should be chemically
inert. Solvent must be fltered through a 0.45-m flter unit.
Degasser b) . In analytical operations, the mobile phase should be free of
air bubbles. For this purpose, a degasser is used.
Pumps c) . These are devices that deliver the mobile solvent at a controlled
fow rate to the separation system. HPLC uses reciprocating pumps: a
pump with a single or multiple chambers, from which the mobile phase is
displaced by reciprocating pistons or diaphragms. Binary gradients are cre-
ated by the selected mixing of two solvents, on a single-headed two-pump
system. Accurate gradient is maintained by microprocessor control.
Injector/autosampler. d) This device introduces a liquid sample into the
mobile phase or onto the chromatographic bed. An autosampler can
perform repeated functions without operator attendance, and thus is a
labor-saving device.
Column e) . Silica and modifed silica columns are available for various ap-
plications. Examples are octyl (C
8
), octadecyl (C
18
), phenyl (C
6
H
5
), and
cyno (CN) columns.
Guard column. f) This is used to protect the main column.
Detectors g) . No universal detector is available for all molecules. However,
according to the characteristic of the molecules investigated, various
detectors are used (Table 1).
Fraction collector h) . This device collects the fractions containing the mol-
ecules of interest during the chromatographic run.
Records. i) A computer is used for chromatographic data acquisition.
Table 1: Characteristics of various HPLC detectors
Detector Application Advantages and limitations
Electrochemical Responds to substances that
are oxidizable or reducible
Commercially available.
Non-specifc. High LOD
185
Detector Application Advantages and limitations
Fluorescence Detects trace-level analytes such
as afatoxins, carbamates and
polycyclic aromatic hydrocarbons
Very specifc. Low LOD.
Not everything fuoresces
Infrared Works for all molecules Many solvents are infrared-active
Mass
spectrometry
Analyte identifcation Ability to ionize analyte. Low LOD
Photodiode
array
Works for wavelengths
190-800 nm
High LOD
Refractive index Works for nearly all molecules Temperature sensitive. High LOD
Scattering Uniform response Non-specifc. LOD, 5 ng per 25
mL. Interference from solvent
Ultraviolet
and visible
Works for molecules with chromo-
phores and for complex samples
Non-specifc. All molecules that
absorb UV and visible light can
be detected.
LOD, level of detection
11.2.4 HPLC Classifcation
HPLC may be characterized depending on column diameter,
which is the governing factor for fow rate from microscale to industrial scale
chromatography. Column internal diameter (i.d.) defnes the sample load
and fow rates (Figure 1).

o.oo1 o.1 o.u z 1o o 1co(mL]mln)
Mlcro LC (o. -
-
1 mm l0)
Seml mlcro LC (1 - mm l0)
Seml - preparatlve LC (1o - zo mm l0)
Conventlon al LC (u - S mm l0)
Preparatlve LC (zo - co mm l0)
Process Scale LC co mm l0)

1oue coue zooue uome zoome 1e (sample load)
Figure 1: Classication of HPLC according to column diameter
186
11.2.5 Advantages of HPLC
The use of HPLC in the isolation and purifcation of complex
compounds is increasing tremendously due to its fexibility and effciency. It
has several advantages over traditional methods of isolation and purifca-
tion:
Variety of separating techniques. i)
Variety of column packings for different techniques. ii)
Separation optimized by alteration of the mobile phase. iii)
Mobile phase easily manipulated in gradient systems. iv)
RP technique separates very similar and very different com- v)
pounds simultaneously.
HPLC can be used as a preparative method. vi)
HPCL can be used as a purifcation technique. More than vii)
one detector can be connected in series (e.g. UV and evap-
orative light scattering detector).
Most sample analysis is carried out at room temperature. viii)
Short analysis runs. More than 70% of HPLC separations ix)
are performed on UV detectors and 15% rely on fuores-
cence without any derivatization.
11.3 Preparative HPLC
Preparative chromatography is the most powerful and versatile
method for isolation as well as purifcation of complex compounds used in
drug development studies. Prior to performing preparative HPLC, the fol-
lowing points must be taken into account to optimize the separation and
maximize the sample load on a small column:
Prior to pilot-plant scale, a systematic method for develop-
ment is required
Validated robust analytical methods are required
Scale-up of parameters from analytical method to prep-
HPLC
In prep-HPLC, buffer is not used
Stationary phase with large particle sizes to decrease costs
for prep-HPLC
11.3.1 Strategy for Preparative Separation
Selection of the appropriate mode of chromatography is fol-
lowed by the optimization of the separation, i.e. stationary phase, mobile
phase, temperature, additives. The next step is optimization of the through-
put, i.e. sample amount and column overloading. In the fnal step, stepwise
scale-up of separation is performed to obtain the desired compound.
187
It is always important to optimize the small-scale separation
(which will signifcantly impact on throughput), the size of packing material
and the column needed to obtain the desired throughput, the mobile solvent
and the instrument capability. Normal-phase methods are the frst choice
because: direct transfer from normal-phase thin-layer LC or HPLC to prep-LC
is possible; costs of RP packing materials are still high; cleaning normal-
phase silica is easier because the material is more robust; removing organic
solvents typically used in normal-phase chromatography from the fnal prod-
uct solution is easier than removing water from an RP chromatographic frac-
tion and can be achieved at lower temperatures and provides higher product
quality and lower energy costs. Particle size of the stationary phase material
also plays an important role in the isolation of the desired compounds. The
choice of 5-m particle size in a preparative column is not practical because
it not only increases the column pressure but also is extremely expensive.
Moreover, when the sample amount is increased, resolution performances
of 5-m and 15-m particles are not different.
11.4 Practical Consideration in Preparative HPLC
Scale-up
11.4.1 Sample Loading
If the tests on analytical columns with analytical loadings show
good separations, a scale-up to a larger column diameter can be performed
on prep-HPLC. Instead of jumping directly to the largest column diameter,
stepwise scale-up should be done. The frst step in the scale-up process is
the transfer of the analytical separation procedure to a 5 cm i.d. preparative
LC column. Optimization in a preparative column is required. The sample
injected onto the column usually starts at 1 g and increases to as much as
20 g, depending upon the quality (resolution) of the separation achieved,
the quality of the initial material, and the specifcations for the pure product.
Start with the 1-g injection, collect fractions and re-analyze them for purity
using the analytical method, because with an increase in sample loading
there is a decrease in resolution.
(A) Scale up factor for column size =
(Diameter
prep
)
2
x Length
prep
(Diameter
anal
)
2
x Length
anal
(B) Flow rate (prep) = Flow rate (analytical) x
(Diameter
prep
)
2
(Diameter
anal
)
2
(C) Gradient duration (prep) = Gradient duration (anal) x =
Length
anal
Length
prep
188
11.4.2 Separation Time
In preparative separations, the stationary phase is usually re-
covered and used again to purify the next batch of the same substance. Of-
ten the major operating cost in preparative LC is the solvent rather than the
packing material. Therefore, the choice of solvent is important in method
development and scale-up. As per the need for separation, isocratic mixture
or gradient elution of water with organic solvent (methanol or acetonitrile)
is used. Gradient elution has a shorter run time than isocratic elution, but
sometimes purity of the isolates is compromised.
11.4.3 Solvent Composition
Methanol is often used in preparative separations. It is an inex-
pensive and strongly polar solvent commonly used in combination with water
as a mobile phase in RP separations. Methanol can be used for fushing
normal-phase silica columns to remove adsorbed polar contaminants. It can
also be recovered easily from many mixtures. In RP applications, acetonitrile
yields better peaks but is too expensive in most situations for process-scale
separations.
An initial goal of the scale-up process is to fnd an accept-
able separation. If analysts fnd more than one set of valid conditions, then
the cost of solvents becomes a major criterion. Solvent selection is usu-
ally determined during the initial method development with the 4-mm i.d.
analytical-scale columns. Sometimes, when the overall costs of the goods is
important to a fnal product, one can perform a systematic solvent selection
even at later stages of development.
11.4.4 Washing Steps
The accumulation of impurities on the column can decrease
the resolution of the subsequent separation, and late-eluted impurities can
spoil the collected fractions of the subsequent separation. Therefore, wash-
ing steps are often implemented between chromatographic runs. Solvent
gradients and recycling steps are sometimes necessary to increase the
resolution for diffcult separation problems. Sometimes temperature pro-
gramming is used to remove strongly held impurities.
11.4.5 Recycling
Sometimes gradients and recycling steps are required for bet-
ter preparative separation of complex mixture of compounds.
189
11.5 Stepwise Operations in Process-scale HPLC
Various aspects should be taken into consideration for operat-
ing process-scale HPLC and stepwise scale-up, as follows.
Develop a robust analytical method and scale up the method
for process scale using the same stationary phase.
The main objective of method development is a simple, well-
automated and robust separation process able to run 24 h
per day.
Optimize sample loading, fow rate and column pressure.
Select the best solvent for both sample preparation and elu-
tion.
The injection solvent should be optimized because sharp
peaks and high loadability are important goals.
In process-scale separations, control the frst two runs man-
ually and observe the process. If no technical problems oc-
cur, subsequent runs can be performed automatically.
Collect fractions and re-analyze them for purity using the
analytical method.
Peak purity at three points (i.e. up slope, apex and down
slope) should be confrmed.
11.6 Problems Encountered in Preparative Scale-up
11.6.1 Purity of Crude Extract
A typical problem encountered in process scale-up is that the
plant material or enriched fraction used during method development had
been produced in analytical scale and differs in solubility and impurity from
the material that is being processed in pilot scale. The process-scale plant
material can be either of a different quality or show larger amounts of the
same impurities, and, in some instances, even new impurities can arise.
If an impurity profle shows larger amounts of the same impurities or new
impurities, the chromatographer must retest the separation method at ana-
lytical scale before starting the process-scale separation.
If during a process scale up, a compound shows higher purity,
the solubility in the weak solvent chosen during optimization may not be
good enough. In this instance, productivity can be lower than expected be-
cause the amount separated in each run will be less. Because scale-up is
linear, the chromatographic run takes the same time in preparative scale as
in analytical scale, and the substance is not stressed longer in the separa-
tion equipment.
190
11.6.2 Removal of Chromatographic Solvent
The fnal work-up after the separation step is removal of the
chromatographic solvent. The desired fraction collected is a solution which
contains the substance of interest in the range of a small percent by weight
and, therefore, large amounts of solvent must be removed. The evaporation
of solvents, especially water, takes time, so the purifed drug substance
can be changed or even destroyed during the concentration process. This
step should be performed with care considering the thermal stability of the
compound of interest.
11.6.3 Temperature Variation from Laboratory to Pilot Scale
It is useful to test the temperature stability of the substance
during analytical method development. Temperature also infuences the sepa-
ration performance. For example, the mixing of organic solvents before they
enter the column can result in a strong increase or decrease in solvent tem-
perature and can infuence the operating temperatures of the mixing unit and
column. Temperature effects at the centre of the column caused by heat dissi-
pation can also infuence the separation and ultimately the purity of isolates.
11.6.4 Increase in Pump Pressure Due to Accumulation of
Impurities on the Column
Another problem that often occurs during the frst separation
in process scale is that some impurities accumulate on the column during
a series of sequential runs. The quality of the separation deteriorates dur-
ing the sequence. Because the fraction collection is commonly controlled
by peak height, a UV detector does not detect this problem and the purity
of the fractions decreases. When impurities accumulate on the column, the
peak shapes or the retention times of the components of interest might
change, so the chromatographer can see quality problems. Unfortunately,
sometimes peak shapes and retention times show no changes. An addition-
al indicator that impurities have accumulated on the column is a pressure
increase; therefore, it is helpful to monitor column pressure. The increase in
pressure is commonly related to instrument failure.
11.7 Summary: Scale-up Strategy
Defne the problem
Find the chromatographic mode
Develop and optimize the separation
Maximize throughput
Increase sample mass and volume to the maximum while
meeting purity objectives
Determine recovery
191
Scale up to desired column size to meet throughput and
load objectives
Pool fractions of comparable purity and rerun if necessary
Check fraction purity using an analytical column
11.8 Applications: Natural Products Isolation
A few examples of prep-HPLC for the isolation of natural prod-
ucts are summarized herein. First, tannins from Guiera seregalensis can be
isolated using the following conditions:
Column RP-18e (250 x 10 mm i.d.)
Mobile phase Water:methanol:THF (90:10:0.25) and (80:20:0.25)
Flow rate 2.5 ml/min
Detection 280 nm
Compounds isolated Galloylquinic acids, namely, 3-O-, 5-O-, 1,3-di-O-, 3,4-di-O-,
3,5-di-O-, 4,5-di-O-, 1,3,4-tri-O-, 3,4,5-tri-O- and
1,3,4,5-tetra-O-galloylquinic acid
Flavonoids from Lychnophora ericoides require the following conditions:
Columns Silica (250 x 10 mm i.d.) and ODS (250 x 10 mm i.d.)
Mobile phase Water:methanol:THF
Detection 280/225 nm
Compounds isolated 7,4-dihydroxy-favonol; 5,7-dihydroxy-3-methoxy-favonol;
galangine, 7,4-dihydroxy-dihydrofavono l,5,7,4-trihydroxy-
dihydrofavonol,7-hydroxy-4-methoxy-dihydrofavonol;
pinobanksin, 5,7-dihydroxy-4-hydroxy-favanone; 7-hydroxy-
4-methoxy-favanone; 5,7-dihydroxy-favone; acacetin;
7-hydroxy-3,4-dihydroxy-isofavone; 15-desoxigoiazensolide,
2,3-dihydro-15-desoxygoyazensolide; eremantholides A and
C; 4,5-dihydroeremantholide A and lychnopholide
For the isolation of peptide components of bacitracin, use:
Column C
8
(250 x 16 mm i.d.)
Mobile phase Gradient acetonitrile, methanol and phosphate buffer
Detection 254 nm
Compounds isolated Peptides
192
The isolation and purifcation of -carotene from carrot in-
volves:
Column Shim-pack PREP-ODS (H) kit (250 x 20 mm i.d.)
Mobile phase Ethanol (99.5%)
Flow rate 10 ml /min
Detection 480 nm monitor with photodiode array (PDA)
Compound isolated
-carotene
The isolation of anti-HIV compounds from Gleditsia japonica
and Gymnocladus chinensis requires the following conditions:
Column C18-Bondpak (300 x 24.4 mm i.d.)
Mobile phase Methanol-water (varying percentages)
Detection Refractive index detector
Compound isolated Saponins
Isolation of procyanidins from Vicia faba requires these condi-
tions:
Column Sephadex LH-20 column (580 25 mm i.d.)
Mobile phase Sequential elution with ethanol, ethanol:methanol, methanol
and fnally methanol:acetone
Flow rate Varying
Detection 280 nm
Compounds isolated (+)-gallocatechin-4-phloroglucinol; ()-epigallocatechin-
4-phloroglucinol; (+)-gallocatechin; ()-epicatechin-4-
phloroglucinol; (+)-catechin-4-phloroglucinol; (+)-catechin and
()-epicatechin
Isolation of the anticancer compound taxol from Taxus yunnan-
ensis is done with:
Column D1 (4000 x 200 mm i.d.) packed with 956 polymeric resin
Mobile phase Acetone:water (58:42)
Flow rate 79 ml/min
Detection 228 nm
Compound isolated Taxol
193
11.9 Conclusions
Stepwise scale-up starting with analytical scale to process
scale is an important issue that needs to be considered. Optimization of
operating conditions is always useful for getting high purity phytomolecules.
Thus, process-scale HPLC is the choice for isolating valuable molecules with
desired purity for commercialization. Its signifcance will continue to grow
because of the increasing requirements for high-purity molecules.
Bibliography
Anonymous, 2007, Shimadzu application note SC-AP-LC-0186. Available at: http://
www2.shimadzu.com/apps/appnotes/app113.pdf
Bouchet, N., Levesque, J. and Pousset, J. L., 2000, HPLC isolation, identifcation
and quantifcation of tannins from Guiera senegalensis, Phytochemistry Annals,
11: 5256
Brandt, A. and Kueppers, S., 2002, Practical aspects of preparative HPLC in pharma-
ceutical and development production, In: LC-GC-Europe, p. 2-5
Konoshima, T., Yasuda, I., Kashiwada, Y., Cosentino, L. M. and Lee, K. H., 1995,
Anti-AIDS agents. XXI: Triterpenoid saponins as anti-HIV principles from fruits of
Gleditsia japonica and Gymnocladus chinensis, and a structure-activity correla-
tion, Journal of Natural Products, 58: 1372-1377
Lorenz, H., Sheehan, P. and Morgenstern, A. S., 2001, Coupling of simulated moving
bed chromatography and fractional crystallisation for effcient enantioseparation,
Journal of Chromatography A, 908: 201-214
Merghem, R., Jay, M., Brun, N. and Voirin, B., 2004, Qualitative analysis and HPLC
isolation and identifcation of procyanidins from Vicia faba, Phytochemistry An-
nals, 15: 95-99
Pavli, V., Kmetec, V. and Tanja, T., 2004, Isolation of peptide components of baci-
tracin by preparative HPLC and solid phase extraction (SPE), Journal of Liquid
Chromatography & Related Technology, 27: 2381-2396
Sargenti, S. R. and Vichnewski, W. 2000, Sonication and liquid chromatography as a
rapid technique for extraction and fractionation of plant material, Phytochemistry
Annals, 11: 69-73
Venn, R. F. (Ed.), 2000, Practicals and Practice of Bioanalysis, Taylor & Francis, Lon-
don, p. 44-130
Yang, X., Liu, K. and Xie, M. 1998, Purifcation of taxol by industrial preparative liquid
chromatography, Journal of Chromatography A, 813: 201-204
195
12 Flash Chromatography and Low
Pressure Chromatographic Techniques
for Separation of Phytomolecules
S. K. Chattopadhyay
Abstract
Flash chromatography is a rapid form of preparative column chromatography that
employs prepacked columns through which a solvent is pumped at high ow rate. Two
types of solvent systems are used in ash chromatography: isocratic and gradient. In
the isocratic system, a single-strength mobile phase brings about the desired separa-
tion. The gradient system, in which the solvent composition changes during the course
of elution, is suited for complex samples containing compounds that differ greatly in
column retention times. The optimum ow rate for a ash separation is related to the
particle size and dimensions of the column. Typical sorbents for normal phase ash
chromatography are polar (e.g. silica, NH
2
) and elution solvents are non-polar. In re-
verse phase chromatography, the stationary phase is non-polar (such as C18) and the
mobile phase is polar. Compounds are retained by the interaction of their non-polar
functional groups with the non-polar groups on the packing surface. Therefore, the
most polar compounds elute rst followed by other compounds in decreasing order
of polarity. To achieve a desired separation, one must select a sorbent that effectively
retains the compounds of interest under solvent conditions that are appropriate for the
samples solubility. Sample loading onto a ash column can be done with wet loading
(the liquid sample is loaded directly and allowed to percolate into the sorbent bed) or
dry loading (when samples are pre-absorbed to a small amount of sorbent which is
then loaded onto the column).
12.1 Introduction
Mikhail Semyonovich Tsvet of Russia invented the frst chro-
matographic technique in 1901 during his research on chlorophyll. He used
a liquid adsorption column containing calcium carbonate to separate plant
pigments. The method was described on 30 December 1901 at the XIth
Congress of Naturalists and Doctors in St. Petersburg. The frst printed de-
scription was published in 1903 in the Proceedings of the Warsaw Society of
Naturalists, section of biology. He frst used the term chromatography in print
in 1906 in his two papers about chlorophyll in the German botanical journal,
Berichte der Deutschen Botanischen Gesellschaft.
In 1952, Archer John Portor Martin and Richard Laurence Mil-
lington Synge were awarded the Nobel Prize in Chemistry for their invention
of partition chromatography. Since then, the technique has advanced rap-
idly. Researchers have found that the principles underlying Tsvets chroma-
tography can be applied in many ways, giving rise to the different varieties of
chromatography and allowing increasing similar molecules to be resolved.
12 FLASH CHROMATOGRAPHY AND LOW PRESSURE CHROMATOGRAPHIC TECHNIQUES FOR SEPARATION OF PHYTOMOLECULES
196
12.2 Flash Chromatography
Flash chromatography, also known as medium pressure chro-
matography, is a rapid form of preparative column chromatography that uses
optimized, prepacked columns through which a solvent is pumped at a high
fow rate. Initially developed in 1978 by W. C. Stills of Columbia University,
New York, USA, fash chromatography is now a method of purifcation and
separation using normal phases. Use of reverse phase packing materials is
opening up the technique to a wider range of preparative separations. Cur-
rently, it is considered to be a simple and economical approach to prepara-
tive liquid chromatography (LC).
Flash chromatography differs from conventional techniques
in two ways. First, slightly smaller silica gel particles (250-400 mesh) are
used. Second, due to the limited fow of solvents caused by the small gel
particles, pressurized gas (10-15 psi) is used to drive the solvent through
the column of stationary phase. The net result is rapid (over in a fash) and
high resolution chromatography.
12.2.1 Theory of Flash Chromatography
Chromatography is a separation method that exploits the dif-
ferences in partitioning behavior between a mobile phase and a stationary
phase to separate the components in a mixture. Compounds of a mixture
may interact with the stationary phase based on charge, relative solubility
or adsorption. Retention is a measure of the speed at which a substance
moves in a chromatographic system. In continuous development systems
like high performance LC (HPLC) and gas chromatography (GC), where the
compounds are eluted with the eluents, retention is usually measured as
the retention time (Rt or t
R
), i.e. the time between injection and detection.
In uninterrupted development systems like thin layer chromatography (TLC),
retention is measured as the retention factor (R
f
), i.e. the run length of the
compound divided by the run length of the eluent front:
R
f
=
Distance travelled by the analyte
Distance travelled by the solvent front
12.2.2 Converting TLC to Flash Chromatography
TLC separations can be used to help determine effective sol-
vent compositions for fash chromatography. R
f
is a common TLC unit and
R
f
is the distance between the compounds:
R
f
= R
f1
R
f2

The ideal solvent system for TLC is one that moves the com-
pound of interest in the mixture to an R
f
of 0.15-0.35 and that separates
197
this component from the others nearest to it by a R
f
value of at least 0.15.
In contrast to TLC, fash chromatography separations are governed by col-
umn volumes.
A column volume (CV) is defned as the volume of solvent re-
quired to fll all the adsorbent pores and interstitial spaces between adsorb-
ent particles in a given column. The volume required to elute a compound
of interest from a column is expressed in terms of the number of CV. The
volume that separates the elution of two substances from the same volume
is called column volume difference (CV). The ideal fash chromatography
solvent system is one that elutes the desired compound of interest in 3-6
CV and that separates this component from others nearest to it by a CV
greater than 1.
The relationship between numbers of CV to R
f
for a given com-
pound is 1/ R
f
; therefore, for two compounds CV = 1/ R
f1
- 1/ R
f2.
For a
particular set of separation conditions, a weakly retained, fast-eluting com-
ponent with an R
f
=0.9 can be eluted in just over 1 CV, whereas a strongly re-
tained, slow-eluting component with an R
f
=0.1 requires 10 CV for complete
elution (Table 1).
Table 1: Relationship between R
f
and CV
R
f
CV
0.90 1.10
0.70 1.40
0.50 2.00
0.30 3.33
0.10 10.0
Due to factors such as change in the TLC solvent fow rate with
respect to time and interference from adhesives used to bind TLC sorbents,
solvent conditions that provide an acceptable TLC separation will not neces-
sarily work effectively for fash chromatography without modifcation.
Although some empirical experimentation may be required, the
steps below help streamline the process of converting a TLC solvent system
into a fash chromatography mobile phase:
Use matching sorbent chemistries on the TLC plate and in the 1.
fash chromatography column. Stationary phase sorbent chemistries
(including silica) can differ from one manufacturer to another. It is
important to match these sorbent chemistries if the solvent sys-
tems are expected to provide equivalent results.
198
Optimize the TLC solvent mixture so that the compound of interest 2.
has an R
f
0.15-0.35 and R
f
>0.20. These conditions will provide
the most reliable starting point for a successful fash chromatography
separation.
Adjust the solvent selectivity to provide an R a)
f
>0.20. Solvent selectivity is
defned as the ability to affect the retention of one compound in the mix-
ture relative to the others, therefore affecting R
f
and number of CV. Ex-
perimenting with different solvent combinations to obtain the desired TLC
separation usually reveals appropriate conditions for effective fash chro-
matography separation. Different solvent mixtures such as hexane:ethyl
acetate (1:1) and hexane:dichloromethane (1:2) may provide different
solvent selectivities while providing similar solvent strengths. Different
solvent mixtures can even reverse the elution order of some of the com-
ponents in the sample. It is interesting to note that R
f
and CV may vary
greatly relative to one another for a given separation. CV predicts column
capacity, i.e. the amount of material that can be effectively separated in a
single column loading (Table 2). Greater the CV, the better the effective
capacity of the column.
Adjust the solvent strength to obtain an R b)
f
between 0.15 and 0.35 (CV, 3-6).
Solvent strength refers to the solvents simultaneous effect on the reten-
tion of all compounds in the mixture; therefore, solvent strength affects R
f

and CV. Once the optimum separation has been established by modifying
solvent selectivity, it may be useful to move some or all of the compounds
off the fash column as quickly as possible by increasing solvent strength.
Often, slight changes in solvent strength can make large differences in re-
tention. In some cases, a lower-strength mobile phase provides improved
separations. It is important to remember that the sample loading solvent
should have equal or lower elution strength than the starting strength of
the mobile phase. Additional adjustments to the selectivity and strength
of the fash solvent system may be necessary to optimize the separation
and to achieve a CV3-6 and a CV>1. This can often be achieved by us-
ing a less polar solvent system or by decreasing the proportion of polar
modifer.
Table 2: Approximate capacity of a 20 g/70 ml ISOLUTE Flash Si column (Biotage)
CV Sample load, g
6 1.0
2 0.5
1 0.25
199
12.3 Isocratic versus Gradient Chromatography
Two types of solvent systems are used in fash chromatog-
raphy: isocratic and gradient. The most common is an isocratic (meaning
same solvent strength) system where a single-strength mobile phase mix-
ture brings about the desired separation.
If the mixture is complex and contains compounds that differ
greatly in column retention times, chemists may use a gradient solvent sys-
tem that changes solvent composition during the course of elution. For ex-
ample, in a normal phase system equipped with a silica column, a non-polar
solvent such as hexane is applied to elute non-polar compounds. Then, a
more polar solvent such as ethyl acetate is added to the hexane to elute the
more polar compounds. The percentage of the polar solvent in the mixture
is increased until all components of the mixture have eluted.
In a step-gradient system, the various solvent concentrations
are typically changed in large increments (or steps). Alternatively, a linear
gradient can be employed whereby a continuous linear change in the con-
centrations of the solvent (and thus mobile phase strength) is achieved.
Chemists can often achieve effective separations more rapidly by using gra-
dient solvent systems. Chemists must select miscible solvents for use in
gradient solvent systems. A common solvent system for fash separations
using polar sorbents such as silica is hexane and ethyl acetate, where ethyl
acetate is the more polar solvent.
Increasing elution
strength in normal
phase mode
Relative Solvent Strength
Hexane
Toluene
Diethyl ether
Dichloromethane
Acetone
Tetrahydrofuran
Ethyl acetate
Acetonitrile
Isopropanol
Ethanol
Methanol
Water
Increasing elution
strength in reversed
phase mode
Figure 1: Step-gradient system
200
12.4 Adsorbent Selection and Mode of Separation
Typical sorbents for normal phase fash chromatography are
polar (e.g. silica, NH
2
) and elution solvents are non-polar (e.g. hexane, hep-
tane, dichloromethane, sometimes modifed with small amounts of more
polar solvents such as isopropanol). The sample is usually applied in a weak
(very non-polar) solvent, and separation occurs when the elution solvent is
applied. In normal phase fash chromatography, the most non-polar sample
component elutes frst, followed by successively more polar compounds.
12.4.1 Isolute Flash Columns
Isolute fash columns (Biotage) are polypropylene columns pre-
packed with Isolute fash sorbents. These columns are appropriate for use
in both off-line and on-line fash chromatography. In off-line fash chroma-
tography, chemists apply the sample and elution solvent volumes to the
top of an Isolute column fxed in a FlashVac

Sample Processing Station. In
off-line fash chromatography, chemists perform isocratic or step gradient
separations. In on-line fash chromatography, an Isolute column is mounted
on a system that connects the column to an external liquid pump system to
produce a continuous fow of solvent through the column. Depending on the
capability of the pump, the solvent composition can be isocratic (a single
solvent or solvent mixture) or a gradient with an increasing proportion of
stronger solvent (either in a step gradient or a linear gradient).
12.4.2 Method Development Using Isolute Flash Columns
12.4.2.1 Column Equilibration
Prior to sample loading, the column should be prepared for the
separation by equilibration (prewetting) with a suitable solvent:
Off-line . Apply the equilibration solvent to the top of the col-
umn and allow it to fow through the column under gravity.
On-line. Equilibrate the fash column for on-line mode separa-
tion in the off-line mode using a vacuum manifold such as the
FlashVac system or by mounting the column on the on-line
apparatus and pumping a suitable volume of equilibration sol-
vent through the column.
12.4.2.2 Typical Equilibration Solvents
Suitable solvents are non-polar, e.g. hexane or pentane. For
best results, prewet the silica and NH
2
columns prior to use. A suitable vol-
ume for column equilibration is approximately two bed volumes (Table 3).
201
Table 3: Typical column solvent volumes for column equilibration
Column size,
g
Approximate column
volume, ml
Typical solvent
volume, ml
2 2.5 5
5 6.5 13
10 12.5 25
20 25 50
25 31.3 62.5
50 62.5 125
70 88 176
100 125 250
150 188 376
Normal phase fash columns can be used without prewetting,
but some column-to-column variation may be experienced.
12.5 Sample Application
There are two popular approaches to fash chromatography
sample loading: wet loading and dry loading.
12.5.1 Wet Loading
Load the liquid sample directly onto the top frit of the column,
and allow it to percolate through the top of the sorbent bed. For best results,
load the sample onto a prewetted column in a non-polar solvent.
12.5.1.1 Practical Tips for Wet Loading
Dissolve the sample in as non-polar as solvent as possible.
If compounds are not easily soluble in a non-polar solvent,
either dissolve them in a small volume of polar solvent and
then dilute with a non-polar solvent to reduce the elution
strength, or consider dry loading of the sample (discussed
in next section).
Position column on a FlashVac vacuum manifold equipped
with PTFE stopcock needles.
Apply the sample evenly over the entire area of the top frit.
To do so, seal the column by closing the stopcock.
Alternatively, load samples onto the column in on-line mode using
a Flash Master system equipped with a 3-way injection valve.
202
12.5.2 Dry Loading
Dry loading is the method of choice for loading reaction mix-
tures consisting of polar solvents onto silica or other normal phase col-
umns. Pre-absorb the reaction mixture onto a small amount of bulk material
of the chosen sorbent. Evaporate off the majority of the solvent leaving the
compounds bound to the surface of the sorbent.
Add this blend to the top of the prepacked (and prewetted)
fash column; allow settling and add a top frit to secure the blend in place.
The top frit can be placed using a suitably sized frit inserter.
A popular alternative sorbent for dry loading using the fash
sorbent is a diatomaceous earth such as Isolute HM-N. This can be used
in the same way as the fash sorbent, but has several advantages including
more effcient desorption of the compounds into the mobile phase.
12.5.2.1 Practical Tips for Dry Loading
Dissolve the sample initially in a suitable solvent, ensuring com-
plete dissolution if possible. Use the smallest volume possible.
Add the bulk material of choice. The ideal proportion of sam-
ple to bulk material ranges from 1:1 to 1:3 by volume.
Evaporate off the residual solvent using a rotary evaporator to
ensure even adsorption of the sample on the bulk material.
Pack the dry blend on top of the fash column (above the top
frit) and add another frit. Push down the new surface to pre-
vent movement of the new blend. When loading with Isolute
HM-N, ensure that the material is not crushed at this stage.
When dry loading using bulk silica, ensure that it is identical
to the material in the fash column. If this is not possible, use
a lower surface area material, ensuring that the surface pH
and moisture content are as similar as possible to the column
packing. All Isolute sorbents are available as bulk material.
Table 4: Capacity guidelines
Reaction scale, g Column size, g
0.1 1-2
0.25 2-5
0.50 5-10
1.0 10-20
2.5 25-20
5.0 50-70
10.0 70-100
203
As a general guideline, the amount of sample loaded onto the
normal phase fash column should be 5%-10% of the column size. Factors
affecting the capacity of the column include compounds, sample matrix,
concentration of reaction products and elution solvent used.
12.6 Elution
12.6.1 Step Gradient Elution
Step gradients provide controlled elution with discrete changes
in eluent strength. Each step is optimized to elute only those components
that are soluble in that eluent. This technique can be applied to both off-line
and on-line fash chromatography.
Use TLC to determine a suitable solvent strength to elute 1.
the components at discrete intervals, choosing different sol-
vent mixtures that elute each component separately with an
R
f
of 0.2-0.5.
Calculate the volume of solvent required to elute each com- 2.
ponent using CV=1/R
f .
Values of CV for different column sizes
are listed in Table 3.
Apply between 2 and 5 CV of solvent for each step, starting 3.
with the solvent with weakest strength.
Collect the eluent from each step in a separate vessel. 4.
12.6.2 Linear Gradient Elution
Liner gradients are a quick way of separating complex mixture,
reducing the complexity of the subsequent purifcation of the fractions col-
lected. This technique is suitable for on-line fash chromatography.
Use TLC to fnd both the weakest and strongest elution 1.
solvent. The weak solvent (solvent A) should give retention
of the majority of components (R
f
<0.1). The strong solvent
(solvent B) should allow elution of all of the components of
interest (R
f
>0.5).
Run a gradient starting with 100% solvent A and ending with 2.
100% solvent B.
Collect the eluent at regular intervals. 3.
Automated fash chromatography instrumentation such as the
FlashMaster II and Solo systems can monitor the signal from a UV detector,
and collect only the fractions of eluent carrying the compounds of interest.
204
12.6.3 Method Development Using Gradient Elution
Method development can be performed without TLC. Gradient
elution analysis offers a useful approach to method development, particu-
larly for non-silica-based fash chromatography (e.g. reverse phase) where
suitable TLC plates are not available.
Load the sample onto a prewetted (equilibrated) fash col- 1.
umn in as weak a solvent as possible (or use dry loading);
for example, for normal phase work use hexane for loading.
Elute the column with aliquots (2 CV each) of successively 2.
increasing solvent strength. A typical scheme for mixing the
two solvents is given in Table 5.
Collect each fraction and analyze for the presence of the 3.
components of interest.
Using these data, identify the solvent mixture that elutes 4.
the components of interest separately, and set up a step or
continuous gradient as described.
Table 5: Typical scheme of mixing solvents for method development.
Solvent A is a weak solvent (e.g. hexane) while solvent B is a strong solvent
(polar modifer, e.g. isopropanol)
Aliquot number Solvent A, % Solvent B, %
1 100 0
2 99 1
3 98 2
4 97 3
5 06 4
6 95 5
7 94 6
8 93 7
9 92 8
10 91 9
12.6.4 Practical Tips for Gradient Elution
The use of a gradient does not improve the selectivity of a 1.
separation if isocratic elution using the same solvent sys-
tem does not effectively separate the sample components.
However, a gradient can be used to decrease the time re-
quired to achieve a separation.
205
The gradient starting conditions must not cause chromatog- 2.
raphy separation. Start the gradient with a weak solvent that
matches the sample loading conditions.
The fow rate at the beginning of a gradient can be high but, 3.
for best results, it should be reduced to the optimum fow
rate at the separation area.
To speed up a gradient separation, use either a higher fow 4.
rate (most suited to samples with many components, high
sample load) or a steeper gradient (most suited to samples
with few components, low sample load).
12.6.5 Optimizing Flow Rate
The optimum fow rate for a fash separation is related to the
particle size and dimensions of the column. Theoretical optimum fow rate
for fash columns of different dimensions can be predicted. However, in prac-
tice, increasing the fow rate has not had a signifcant effect on separation
and offers important productivity advantages (Table 6). Other factors, such
as mobile phase composition and back pressure, also affect the range of
effective fow rates. For recommended fow rates, see Table 6.
Table 6: Recommended fow rates
Column diameter
(confguration)
Flow rate range
ml/min
16 mm (D) 5-25
20 mm (E) 5-25
27 mm (F) 10-30
37 mm (J) 20-50
40 mm (V, W, X) 20-50
12.7 Fraction Collection
12.7.1 Off-line Flash Chromatography
When performing fash chromatography on a vacuum manifold such
as the FlashVac system, successive fractions can be collected as follows:
Load collection rack with vials of a suitable volume in each
position.
Place a single fash column in position 1 of the collection
rack and apply the frst solvent aliquot.
Collect the aliquot in the vial in position 1 of the collection rack.
Move the column to position 2 and apply the second solvent
aliquot.
206
Collect the aliquot in the vial in position 2 of the collection
rack.
Continue until all the components of interest have been col-
lected.
Alternatively, multiple columns can be processed by replacing
collection vials at each elution step. A typical volume for each fraction is
two CV.
12.7.2 On-line Flash Chromatography
Using an automated system equipped with a fraction collector,
fractions can be collected in a variety of ways, for example:
Fixed volume fraction collection.
Individual peak fraction collection under microprocessor
control with UV detector input.
12.8 Low Pressure Liquid Chromatography
In low pressure column chromatography, a column of particu-
late material such as silica or alumina has a solvent passed through it at
atmospheric or low pressure. There are different kinds of low pressure chro-
matographic techniques:
Gel fltration chromatography (separation on the basis of i)
size)
Ion exchange chromatography (separation of the basis of ii)
charge)
Affnity chromatography (separation on the basis of specifc iii)
binding sites on the proteins)
12.8.1 Gel Filtration Chromatography
Proteins of different sizes are separated on a column in which
the stationary phase consists of polymerized agarose or acrylamide beads
with pores of particular sizes. A small protein in the mobile phase (aqueous
buffered solution) can enter the pores in the beads while a large protein
cannot due to size restriction. The result is that a smaller fraction of the
overall volume of the column is available to the large protein than to the
small protein, which thus spends a longer time on the column and is eluted
by the mobile solvent after the large protein.
12.8.2 Ion Exchange Chromatography
The material used for this type of chromatography consists of
an agarose, acryl amide or cellulose resin or bead which is derivatized to
contain covalently linked positively or negatively charged groups. Proteins
207
in the mobile phase bind through electrostatic interactions to the charged
groups on the column.
In a mixture of proteins, positively charged proteins bind to a
resin containing negatively charged groups like carboxymethyl (-OCH
2
COO
-
)
or sulfopropyl (-OCH
2
CH
2
CH
2
SO
3
-
), while the negatively charged proteins
pass through the column. The positively charged proteins are eluted from
the column with a mobile phase containing either a gradient of increasing
salt concentration or a single higher salt concentration. The most positively
charged proteins are eluted last, at the highest salt concentration.
Likewise, negatively charged proteins bind to a resin containing
positively charged groups, like diethylaminoethyl (-CH
2
CH
2
NHEt
2
+
) or a quater-
nary ethyl amino group. These proteins are separated in an analogous way.
12.8.3 Affnity Chromatography
In this technique, the chromatography resin is derivatized with
a group that binds to a specifc site on a protein of interest. It may be a
group that binds to the active site of an enzyme (such as benzamidine-
agarose used for the purifcation of trypsin) or an antibody that recognizes a
specifc amino acid sequence on a protein.
This method exploits the specifc binding of antibody to antigen
held on a solid matrix. Antigen is bound covalently to small, chemically reac-
tive beads which are loaded in the column and the antiserum is allowed to
pass over the beads. The specifc antibodies bind while all other proteins in
the serum including antibodies to other substances are washed away.
Affnity chromatography can also be used to purify antigens
from complex mixtures by using beads coated with the specifc antibody.
12.9 Conclusions
Flash chromatographic systems have been developed for the
separation and purifcation of organic molecules from natural sources and
from reaction mixtures. Method development for separations can be optimized
with proper selection of adsorbents, solvent systems and fow rate of solvents
used for the separation. Scale-up of fash chromatographic methods can eas-
ily be achieved with minimum optimization. Therefore, they are considered to
be effcient and cost-effective methods to purify compounds in little time.
208
Bibliography
Glasl, S., Gunbilig, D., Narantuya, S., Werner, I., and Jurenitsch, J., 2001, Combina-
tion of chromatographic and spectroscopic methods for the isolation and charac-
terization of polar guaianolides from Achillea asiatica, Journal of Chromatography
A, 936: 193-200
Still, C. W., Kahn, M., and Mitra, A., 1978, Rapid chromatographic technique for
preparative separations with moderate resolution, Journal of Organic Chemistry,
43: 2923
209
13 Counter-current Chromatography
S. K. Srivastava
Abstract
Counter-current Chromatography (CCC) is based on liquid-liquid partitioning and is an
excellent alternative to circumvent the problems associated with solid phase adsorb-
ents and to preserve the chemical integrity of mixtures subjected to fractionation. The
uniqueness of this technique is that it does not use a stationary phase and separation
is achieved between two immiscible liquid phases. One phase is used as a stationary
phase while the other one is used as mobile phase. The separation takes place on
the basis of partition of a sample between the two phases. In contrast to other chro-
matographic techniques, since there is no irreversible adsorption of sample on the
stationary phase, recovery is up to 100%. Centrifugal Partition Chromatography (CPC)
and High Speed Counter-current Chromatography (HSCCC) are the two variants of CCC
being used today. Separation of compounds with a wide range of polarities is possible
with the use of aqueous and non-aqueous solvent systems. Separation of crude plant
extracts, semipuried fractions and synthetic mixtures can be carried out, with sample
loads ranging from milligrams to grams.
13.1 Introduction
Natural products have been a key source for the discovery of
new drugs. Isolation of the active components from a natural product has
always been associated with complex separation problems due to the enor-
mous chemical complexity of the extracts, but recent advances in separa-
tion sciences have facilitated the isolation of these active components from
natural products.
Crude extracts of natural products which show desirable bio-
logical activity are subjected to activity-guided fractionation until an active
component is isolated and identifed. This exploratory process of fractiona-
tion typically involves suboptimal chromatographic conditions; hence, in or-
der to avoid destruction of potentially labile components, utmost care must
be taken throughout the entire process of isolation.
At present, most of the chromatographic separations of natural
products are being carried out on solid supports. However, SiO
2
, Al
2
O
3
and
reverse phase adsorbents are not chemically inert. Separation of a natu-
ral product on alumina or silica gel sometimes results in recovery of only
70%90%. Sometimes severe losses of valuable materials result because
of irreversible adsorption on a solid support. In addition, isolation of arti-
facts has also been reported due to chemical reactions of the substrates
with solid phase adsorbents.
13 COUNTER-CURRENT CHROMATOGRAPHY
210
Counter-current Chromatography (CCC) is a unique form of liquid
partition chromatography which utilizes a separation column free of solid support
matrix. Because of this support-free system, the method provides an important
advantage over other chromatographic methods by eliminating various compli-
cations such as adsorptive loss and deactivation of samples and contamina-
tion. During the 1970s, the method was steadily improved by accelerating the
separation speed and effciency. In the early 1980s, an epoch-making advance
was achieved by the advent of high-speed CCC (HSCCC) which can yield highly
effcient separation in a short period of time. Because of its high performance,
the recent research and development of the CCC technology have been almost
entirely focused on HSCCC, high performance centrifugal partition chromatogra-
phy (HPCPC), and fast centrifugal partition chromatography (FCPC).
Recent developments in CCC instruments, explaining the use
of CCC for better separation and their applications in the separation of bio-
active natural products from plants, are discussed in this paper.
13.2 Principles and Development of Counter-
current Chromatography
Anyone conversant with the technique of liquid-liquid extraction
(using a separatory funnel) can readily understand the principles of Counter-
current Chromatography (CCC), where separation is based on the partition of
solutes between two immiscible liquid phases. In CCC, one of the two phases
is retained in the column and is called the stationary phase. The second phase,
which is called the mobile phase, percolates through the stationary one.
13.2.1 Liquid-liquid Extraction
Liquid-liquid extraction is a simple means of separating large
quantities of materials, using a minimum of solvent. After dissolving the
sample in a two-phase solvent system (in a separatory funnel), the steps in
performing liquid-liquid extractions are as follows:
Shake vigorously to thoroughly mix the two phases.
Allow the mixture to settle into two phases.
Separate the phases from each other.
These steps are crucial to achieve the separation of sample compo-
nents.
The chief disadvantage of liquid-liquid extraction is that it pro-
vides only one plate of separation in the original sense.
Accordingly, either this single-step separation must be de-
signed to suit ones needs, or multiple liquid-liquid extractions must be used
to increase the separation.
211
CCC has its origin in the work of Archer John Porter Martin and
Richard Laurence Millington Synge (Martin and Synge, 1941; Synge, 1946)
carried out in Britain during World War II. For their pioneering work, Martin
and Synge shared the 1952 Nobel Prize in Chemistry. Soon after their work
appeared, Lyman Creighton Craig and Otto Post developed an apparatus
that essentially consisted of a series of separatory funnels (tubes) (Craig
and Post, 1949). The sample was automatically transferred through the
Craig-post apparatus (Figure 1). Over 1000 mixing and separation steps
could be accomplished in one day.
Figure 1: Craig-post apparatus
Individual components were separated based on their partition-
ing behavior. Craig and Post continuously improved their apparatus and were
commercially quite successful. Over 1000 publications on counter-current
distribution appeared during the period 1950-1970 citing the use of the
Craig-post apparatus.
13.2.2 Partition Coeffcient
For a given substance A, the partition coeffcient KA is defned
the concentration of A in the upper phase divided by that in the lower phase
(Figure 2).
Figure 2: KA= [A] upper phase/[A] lower phase
212
KA is a constant at any given temperature, and it is unaffected
by the presence of other substances or by the concentration of the solute.
Usually it is expressed as the amount of solute in the stationary phase di-
vided by that of the mobile phase as in conventional liquid chromatography.
For a better CCC separation, the K values of the target compounds should
be in the range of 0.5 K 1.0. A smaller K value elutes the solute closer
to the solvent front with lower resolution while a larger K value tends to give
better resolution but broader, more dilute peaks due to a longer elution time.
In CCC, one can choose either the upper or lower phase as the stationary
phase, but before deciding which phase is to be used as the stationary
phase, one should temporarily express the partition coeffcient as K
U/L
= C
U
/
C
L
, where C
U
is the solute concentration in the upper phase and C
L
is that of
the lower phase. If K
U/L
= 2, the lower phase should be used as the station-
ary phase, which gives K = 0.5. It is important that this preliminary K
U/L
be
clearly distinguished from K using the subscripts to avoid confusion. The
measurement of K
U/L
values may be performed by the shake-fask method.
Add a small amount (a few milligrams) of each target com-
pound (one at a time) to the two mutually equilibrated solvent phases
(12 ml each) in a stoppered test tube (13 mm 100 mm). Thoroughly mix
to equilibrate the contents. After settling, pipette and deliver an equal vol-
ume of the upper and the lower phases (100200 l) each into a separate
test tube, dilute each with an equal volume (2 ml) of a suitable solvent such
as methanol, and measure the absorbance with a spectrophotometer at a
suitable wavelength to obtain the K
U/L
value. If the pure sample is not avail-
able, one can subject each phase to HPLC analysis to compare the peak
heights (or areas under the peaks). If the sample does not absorb in the UV
or visible wavelengths, the K
U/L
value may be determined by other ways such
as thin layer chromatography (color reaction) or evaporative light scattering
detection (ELSD). Thin layer chromatography combined with densitometry is
effectively used for simultaneous determination of K
U/L
of multiple compo-
nents from a sample mixture.
13.2.3 Droplet Counter-current Chromatography
In the early 1970s, Ito and colleagues at the US National Insti-
tutes of Health introduced Droplet Counter-current Chromatography (DCCC).
In DCCC, as with the Craig-post apparatus, the stationary phase is held in
place only with unit gravity.
The apparatus consists of a set of vertical straight tubes seri-
ally connected with narrow transfer tubing (Figure 3). The original CCC ap-
paratus was equipped with 300 glass tubes, each 60 cm 1.8 mm i.d.
213
stationary phase mobile phase
Figure 3: Droplet counter-current chromatography
The total capacity is about 600 ml including the volume in the
transfer tubing (about 15% of total capacity). The operation of DCCC is initi-
ated by flling the entire column with the stationary phase of an equilibrated
two-phase solvent system followed by injection of sample solution. Then, the
other phase is introduced into the frst unit in such a way that the mobile
phase travels through the column of the stationary phase by the effect of
gravity, i.e. the mobile phase is introduced from the bottom if it is the lighter
phase, and from the top if it is heavier. Consequently, the solutes are sepa-
rated according to their partition coeffcients. As with all types of chroma-
tography, compounds more soluble in the mobile phase move more quickly,
while those more soluble in the stationary phase lag behind. Under optimum
fow rate, the mobile phase forms multiple droplets in the stationary phase
to divide the column space into numerous partition units; this process is
repeated in each partition unit.
DCCC necessitates a proper choice of solvent systems for pro-
ducing a droplet fow of the mobile phase in the column. The most popularly
used solvent system is composed of chloroform, methanol and water at vari-
ous volume ratios, although a sizable separation usually takes a few days.
214
13.2.4 Applications of Droplet Counter-current
Chromatography
This simple DCCC instrument has been used to perform ef-
fcient preparative separations such as:
Separation of non-polar compounds.
Isolation of virginiamycin-M1 and parthenolide.
Isolation of vitamin B12.
Pharmacognostical studies of Tabernaemontana species:
ion-pair DCCC of indole alkaloids from suspension cultures.
Chiral resolution of a carboxylic acid.
Separation of natural polar substances by reverse phase
HPLC, centrifugal thin layer chromatography and DCCC.
Effcient isolation of ecdysteroids from the silkworm, Bombyx
mori.
Analytical DCCC isolation of 20-hydroxyecdysone from Vitex
thyrsiora (Verbenaceae).
Increasing the speed of DCCC separations.
Complete resolution of isoleucine.
DCCC of anthocyanins.
Effcient isolation of phytoecdysones from Ajuga plants by
high-performance liquid chromatography and DCCC.
DCCC with non-aqueous solvent systems.
Use of DCCC in log P determinations.
Purifcation of Stevia rebaudiana sweet constituents (poten-
tial sweetening agent of plant origin).
Water-free solvent system for DCCC and its suitability for the
separation of non-polar substances.
Isolation of phorbol, 4 -phorbol and croton oil.
Purifcation of antibiotics such as gramicidins, tyrocidines
and tetracyclines.
13.2.5 Limitations of DCCC
Extremely low fow rates (sometimes solute retention is
measured in days).
Only biphasic solvents systems that form stable droplets
can be used.
Poor mixing of phases, which results in relatively low eff-
ciency.
13.2.6 Modern Counter-current Chromatography
Modern CCC has split into two basic directions. The frst, which
is called High-speed Counter-current Chromatography (HSCCC), uses an ap-
paratus with a variable gravity feld produced by a double axis gyratory mo-
tion (Figure 4). The second, termed Centrifugal Partition Chromatography
215
(CPC), employs a constant gravity feld produced by a single axis rotation,
together with rotatory seals for supply of solvent. Separation takes place in
cartridges or disks. CPC with cartridges or disks is a hydrostatic equilibrium
system. If the coil is flled with the stationary phase of a biphasic solvent
system and then the other phase is pumped through the coil at a suitable
speed, a point is reached at which no further displacement of the stationary
phase occurs and the apparatus contains approximately 50% of each of the
two phases. Steady pumping-in of mobile phase results in elution of mobile
phase alone. This basic system uses only 50% of the effcient column space
for actual mixing of the two phases.
A more effective way of using the column space is to rotate the
coil around its central axis while eluting the mobile phase. A hydrodynamic
equilibrium is rapidly established between the two phases and almost 100%
of the column space can be used for their mixing. CCC with rotating coil
instruments is an example of this latter mechanism.
The planetary motion is produced by engaging a planetary
gear mounted on the column holder axis to an identical sta-
tionary sun gear rigidly fxed to the centrifuge framework.
This 1:1 gear coupling produces a particular type of planetary
motion of the column holder, i.e. the holder rotates about
its own axis while revolving around the centrifuge axis at the
same angular velocity (synchronous) in the same direction.
This planetary motion provides two major functions for per-
forming CCC separation. The frst is a rotary-seal-free elu-
tion system so that the mobile phase is continuously eluted
through the rotating separation column.
The second and more important function is that it produces
a unique hydrodynamic motion of two solvent phases with-
in the rotating multilayer coiled column mainly due to the
Archimedean screw effect.
13.3 HSCCC Instrument and Mechanism
Figure 4: High speed counter-current chromatography instrument
When two immiscible solvent phases are introduced in an end-
closed coiled column, the rotation separates the two phases completely
216
along the length of the tube where the lighter phase occupies one end (called
the head) and the heavier phase occupies the other end (called the tail).
Although the cause of this bilateral hydrodynamic phase distribution of two
immiscible solvents is still unknown, it can be effciently used for performing
CCC. In Figure 5A the coil at the top shows bilateral hydrodynamic distribu-
tion of the two phases in the coil where the white phase (head phase) oc-
cupies the head half and the black phase (tail phase) the tail half.
This condition clearly indicates that the white phase introduced
at the tail end will move toward the head and similarly the black phase in-
troduced at the head will move toward the tail. This hydrodynamic trend is
effectively used for performing CCC (Figure 5B). The coil is frst entirely flled
with the white phase followed by pumping the black phase from the head
end (Figure 5B, top). Similarly, the coil is flled with the black phase followed
by pumping the white phase from the tail (Figure 5B, bottom). In either case,
the mobile phase quickly moves through the coil, leaving a large volume of
the other phase stationary in the coil.
A
B
Bilateral Hydrodynamic Equilibrium
in a Closed Coil
One-Way Elution Modes
Flow
Head Tail
Head Tail
Figure 5: Mechanism of HSCCC. (A) Bilateral hydrodynamic distribution of two phases
in the coiled column. (B) Elution mode of both lighter and heavier phases through the
rotating coiled column.
The motion and distribution of the two phases in the rotating
coil were observed under stroboscopic illumination, and are
illustrated in Figure 6A.
A spiral column undergoes type-J planetary motion. The area
in the spiral column is divided into two zones: the mixing zone
occupying about one-quarter of the area near the center of
revolution and the settling zone in the rest of the area.
In Figure 6B, the spiral column is stretched and arranged
according to the positions IIV to visualize the motion of the
mixing zones along the tubing.
Each mixing zone travels through the spiral column at a rate
of one round per revolution.
217
The solute in the spiral column is subjected to the repetitive
partition process of mixing and settling at an enormously
high rate of over 13 times per second (at 800 rpm).
HSCC is highly effcient.
Figure 6: (A) Motion and distribution of two phases in the rotating coil under stroboscopic
illumination. (B) Column in stretched position to visualize the motion of the mixing zones
along the tubing.
There are numerous potential variants of this instrument de-
sign. The most signifcant of these is represented by the third instrument in
the Pharma-tech product line: TCC-1000-toroidal CCC (Figure 7). In some re-
spects it is like CPC, but retains the advantage of not needing rotary seals.
In addition, it employs a capillary tube, instead of the larger-diameter tubes
employed in the helices of the other CCC models. This capillary passage
makes the mixing of two phases very thorough, despite lack of any shaking
or other mixing forces.
Figure 7: The TCC-1000-toroidal CCC
218
13.4 CPC Instrument and Setup
Figure 8: Centrifugal partition chromatography instrument
Figure 9: Centrifugal partition chromatography setup
13.4.1 Various Kinds of CPC Instruments
CPC instruments are available in various sizes depending upon
219
the rotor volume. As per the sample size and the purpose, one can choose
on the basis of the following:
Rotor volume Scale Sample amount
100 ml Analytical screening Milligrams
200 ml Analytical preparative <5 g
1000 ml Pilot scale <40 g
5000 ml Production scale 100200 g
10000 ml Production scale 200400 g
Other volumes On request Up to one kilogram
13.5 How to Achieve Good Separation of Various
Kinds of Natural Products Using HSCCC and CPC
In order to achieve the best separation of a desired natural
product, one must systematically follow the guidelines presented in this
section on choice of a solvent system.
13.5.1 Search for a Suitable Solvent System
In Table 1, sets of two-phase solvent systems are arranged from
top to bottom in decreasing order of hydrophobicity in the organic phase.
Table 1: Solvent systems arranged in decreasing order of hydrophobicity in the
organic phase
n-Hexane EtOAc MeOH n-BuOH Water
START
HERE
10 0 5 0 5

P
O
L
A
R

H
Y
D
R
O
P
H
O
B
I
C

9 1 5 0 5
8 2 5 0 5
7 3 5 0 5
6 4 5 0 5
5 5 5 0 5
4 5 4 0 5
3 5 3 0 5
2 5 2 0 5
1 5 1 0 5
0 5 0 0 5
0 4 0 1 5
0 3 0 2 5
0 2 0 3 5
0 1 0 4 5
0 0 0 5 5
220
When the polarity of the target compounds is unknown, the
search may start with the two-phase solvent system composed of hexane
ethyl acetatemethanolwater at a volume ratio of 3:5:3:5, which has a
moderate degree of polarity. If the partition coeffcient is slightly off from the
proper range, it can be adjusted by modifying the volume ratio. For example,
if K
U/L
is slightly over 2, the volume ratio may be modifed toward more hydro-
phobic such as 3.2:5:3.2:5, and if K
U/L
is slightly less than 0.5, the volume
ratio may be modifed in the opposite direction to 2.8:5:2.8:5.
If the target compound is mostly distributed in the upper or-
ganic phase, the search is directed upward along the arrow. If it is mostly
distributed in the lower aqueous phase, the search is directed downward
along the arrow. If the sample is an extract of plant material, the search
may start at any point according to the polarity of the solvent used for the
extraction.
If the sample is an ethyl acetate extract (relatively hydropho-
bic solvent), the search may start at hexaneethyl acetatemethanolwater
(1:1:1:1), whereas if the sample is a methanol extract (polar solvent), the
search may start at 1-butanolwater. The search should be continued until
a suitable range of K
U/L
values for all of the compounds of interest is ob-
tained.
13.5.1.1 Solvent Systems for the Separation of a Large Variety of
Natural Products
Table 2 presents different kinds of solvent systems that have
been used for the separation of a large variety of natural products, which
may be of immense importance for the researcher to fnd a suitable solvent
system for the desired separation of natural products.
Table 2: Solvent systems used for the separation of natural products
Sample
Solvent system
Mobile
phase
Solvents Ratio
Aryl ketones
Phenylethanone
derivatives
n-HeptaneEtOAcMeOHH
2
O 4:2:2:1 Lower
Styrylpyrones
Kava lactones n-HexaneacetoneMeOHH
2
O 4:1:3:1 Upper
Benzofurans
Dibenzofurans n-HeptaneCH
2
Cl
2
CH
3
CN 10:3:7 Lower,
upper
221
Sample
Solvent system
Mobile
phase
Solvents Ratio
Phenolic glycosides
Gastrodin EtOAc1-BuOHH
2
O 3:2:5 Lower
Phenylpropanoids
Honokiol, magnolol n-HexaneEtOAcMeOHH
2
O 5:2:5:2 Lower
Phenylpropanoid glycosides
Verbascoside EtOAcCH
3
CNH
2
O 13:13:24 Lower
Acteoside EtOAcH
2
O 1:1 Lower
Hydrolyzable tannins
Ellagic acid and corilagin 1-BuOHHOAcH
2
O 4:1:5 Lower
Catechins
Epigallocatechin n-HexaneEtOAcH
2
O 1:9:10 Lower
Tea catechins n-HexaneEtOAcMeOHH
2
O 3:10:3:10 Lower
Proanthocyanidins
Tea proanthocyanidins n-HexaneEtOAcMeOHH
2
O 1:5:1:5 Lower
Anthocyanins MTBE1-BuOHCH
3
CNH
2
O 2:2:1:5 Lower
Bilberry anthocyanins MTBE1-BuOHCH
3
CNH
2
OTFA 1:4:1:5:0.01 Lower
Flavonoids
Flavones and chalcones CHCl
3
CH
2
Cl
2
MeOHH
2
O 2:2:3:2 Lower
CH
2
Cl
2
MeOHCH
3
CNH
2
O 40:11:25:20 Lower
EtOAc1-PrOHH
2
O 140:8:80 Upper
Flavonoid glycosides n-HexaneEtOAcMeOHH
2
O 1:8:1:8 Lower
n-HexaneEtOAcMeOHH
2
O 2:12:3:15 Lower
CHCl
3
MeOHH
2
O 8:7:4 Lower
C-Glycosyl favones CHCl
3
MeOHH
2
O 6:25:29 Lower
EtOAc1-BuOHMeOHH
2
O 35:10:11:44 Lower
Icariin n-Hexane1-BuOHMeOHH
2
O 1:4:2:6 Lower
Isofavone glycosides EtOAc1-BuOHEtOHH
2
O 30:10:6:50 Lower
EtOAcEtOHHOAcH
2
O 16:4:1:20 Lower
Flavonol glycosides 1-BuOHH
2
O 1:1 Lower
Flavonolignans
222
Sample
Solvent system
Mobile
phase
Solvents Ratio
Silymarin n-HexaneEtOAcMeOHH
2
O 1:4:3:4 Lower
Lignans
Arctiin EtOAc1-BuOHEtOHH
2
O 10:1:2:10 Lower
Lignan diglucoside MTBE1-BuOHCH
3
CNH
2
O 1:3:1:5 Upper
Coumarins
Psoralen and isopsoralen n-HexaneEtOAcMeOHH
2
O 10:7:10:8 Lower
Infacoumarin A n-HexaneCHCl
3
MeOHH
2
O 5:6:3:2 Lower
Chalcones
Licochalcone A n-HexaneCHCl
3
MeOHH
2
O 3:12:6:4 Lower
Quinones
Tanshinones n-HexaneEtOHH
2
O (a) 20:11:9
(b) 10:7:3
Lower
Shikonin n-HexaneEtOAcEtOHH
2
O 16:14:14:5 Lower
Diterpene quinone n-HexaneCCl
4
MeOHH
2
O 1:3:3:2 Lower
Monoterpenes
Monoterpenes and
monoterpene glycosides
CHCl
3
MeOHH
2
O 7:13:8 Upper
Iridoid glycosides CHCl
3
MeOH1-PrOHH
2
O 5:6:1:4 Lower
Sesquiterpenes
Artemisinine, artemisitene
arteannuin B
n-HexaneEtOAcEtOHH
2
O 6:4:5:4 Lower
Diterpenes
10-Deacetylbaccatin III n-HexaneEtOAcEtOHH
2
O 2:5:2:5 Lower
n-hexaneCHCl
3
MeOHH
2
O 5:25:34:20 Lower
Trachylobanes and
isopimaranes
n-HeptaneCH
3
CNCH
2
Cl
2
10:7:3 Lower
Triterpenes
Celastrol n-HexaneEtOAcCCl
4
MeOHH
2
O1:1:8:6:1 Lower
Saponins
Ginsenosides CHCl
3
MeOH2-BuOHH
2
O 5:6:1:4 Lower
EtOAc1-BuOHH
2
O 1:1:2 Upper
223
Sample
Solvent system
Mobile
phase
Solvents Ratio
n-Hexane1-BuOHH
2
O 3:4:7 Lower
Phytolacca saponins CHCl
3
MeOH2-PrOHH
2
O 5:6:1:4 Lower
Glycyrrhizin EtOAcMeOHH
2
O 5:2:5 Lower
Carotenoids
Zeaxanthin n-HexaneEtOAcEtOHH
2
O 8:2:7:3 Lower
Lycopene n-HexaneCH
3
CNCH
2
Cl
2
20:13:7 Upper
Alkaloids
Aporphine alkaloids CH
2
Cl
2
MeOH5% HOAc 5:5:3 Lower
Naphthylisoquinoline
alkaloids
CHCl
3
EtOAcMeOH0.1 M HCl 5:3:5:3 Lower
Diterpene alkaloids n-HexaneCH
2
Cl
2
MeOHH
2
O 15:15:24:8 Lower
C
6
H
6
CHCl
3
MeOHH
2
O 5:5:7:2 Lower
Tetramethylpyrazines
Chuanxiongzine n-HexaneEtOAcEtOHH
2
O 5:5:3:7 Lower
Glucosinolates PrOHCH
3
CN(NH
4
)
2
SO
4
H
2
O 10:5:12:10 Upper
Cyclodepsipeptides n-HeptaneEtOAcMeOHH
2
O 2:8:2:8 Lower
13.5.1.2 Retention of the Stationary Phase
Successful separation in HSCCC-CPC largely depends on
the amount of the stationary phase retained in the column.
In general, higher the retention of stationary phase, better
the peak resolution.
The amount of stationary phase retained in the column is highly
correlated with the settling time of the two phases in a test tube.
Measure the settling time of the two-phase solvent system
to be used for the separation.
The procedure is as follows: the two phases are frst equili-
brated in a separatory funnel. Deliver 2 ml of each phase, a
total volume of 4 ml, into a test tube or graduated cylinder,
which is then capped. Gently invert the container for several
times and then immediately place it in an upright position to
measure the time required for the two phases to form clear
layers with a distinct interface.
If the settling time is less than 20 seconds, the solvent system will
provide satisfactory retention of the stationary phase, usually over
50% of the total column capacity, in a proper range of fow rates.
224
13.5.1.3 Preparation of Sample Solution
The sample for HSCCC-CPC may be dissolved directly in the
stationary phase or in a mixture of the two phases.
The recommended sample volume is less than 5% of the
total column capacity.
Introduction of a larger sample volume into the column will
reduce peak resolution of the analytes, especially for those
having small K values.
Ideally the analyte is injected in a small volume of the stationary
phase to preserve the sharpness of the early elution peak.
13.5.1.4 Separation Column
We must understand the headtail orientation of the separa-
tion column.
A lower (heavier) mobile phase should be introduced through
the head toward the tail, and an upper (lighter) mobile phase
in the opposite direction.
This is extremely important because the elution of either
phase in the wrong direction results in an almost complete
loss of the stationary phase from the column.
13.5.1.5 Choice of the Mobile Phase
In HSCCC-CPC, either phase can be used as the mobile phase
provided that the K value of the analyte is in a proper range.
If one has a choice, the lower phase may be used as the
mobile phase, because the system provides more stable re-
tention of the stationary phase and one can avoid trapping
air bubbles in the fow cell of the detector by introducing the
effuent from the lower end of the cell.
On the other hand, when the upper organic mobile phase (exclud-
ing the chloroform system) is used as the mobile phase, it will
facilitate the evaporation of solvent from the collected fractions.
13.5.1.6 Flow Rate of the Mobile Phase
The fow rate of the mobile phase determines the separation
time, the amount of stationary phase retained in the column,
and therefore the peak resolution.
A lower fow rate usually gives higher retention level of the
stationary phase and improves the peak resolution, although
it requires a longer separation time.
The typical fow rates for the commercial multilayer coil are
as follows:
56 ml/min for a preparative column with 2.6 mm i.d. PTFE i)
tubing (600800 rpm) (up to 1 g sample load);
225
23 ml/min for a semi preparative column with 1.6 mm i.d. ii)
PTFE tubing (8001000 rpm) (up to 500 mg sample load);
1 ml/min for an analytical column with 0.851.0 mm i.d. iii)
PTFE tubing (10001200 rpm) (up to 50 mg sample load).
These fow rates should be modifed according to the settling
time of the two-phase solvent system as well as other fac-
tors. When the settling time is around 20 s and the K value
of the analyte is small, a lower fow rate is recommended.
13.5.1.7 Revolution Speed
The optimum revolution speed (revolution and planetary rota-
tion speeds are always same) for the commercial HSCCC-CPC instrument for
preparative separation ranges between 600 and 1400 rpm.
Use of a lower speed will reduce the volume of the stationary
phase retained in the column, leading to lower peak resolution.
On the other hand, the higher speeds may produce exces-
sive sample band broadening by violent pulsation of the col-
umn because of elevated pressure.
13.5.1.8 Filling the Column with the Stationary Phase
In each separation, the column is frst entirely flled with the
stationary phase.
Before introducing the stationary phase, the column may be
fushed with a column volume of a solvent miscible with the
two phases used in the previous run (e.g. ethanol or metha-
nol) to wash out materials remaining in the column.
This will also ensure a stable, clean baseline before the sol-
vent front emerges.
Avoid trapping the air in the column, especially in a prepara-
tive column. This can be easily tested as:
If no air is present in the column, the fow from the column i)
outlet is ceased shortly after stopping the pumping.
If the solvent keeps fowing from the outlet for more than ii)
several seconds, the air trapped in the column should
be eliminated by resuming the pumping of the stationary
phase under low speed column rotation (100200 rpm)
in a tail to head elution mode to accelerate air movement
toward the outlet of the column.
13.5.1.9 Sample Loading
There are two ways to load samples; both are satisfactory for
HSCCC-CPC separations:
In the frst method, the column is entirely flled with stationary
phase and this is immediately followed by sample injection.
226
The mobile phase is then eluted through the column while
the column is rotated at the optimum rate.
In the second method, after the column is flled with station-
ary phase, the mobile phase is eluted through the column at
a desired rate until the solvent front emerges and hydrody-
namic equilibrium is established throughout the column as
evidenced by diminished carryover of the stationary phase.
The sample is then injected into the column through the
sample port.
Each method has advantages. The second method gives a
clear tracing of the elution curve because of the minimum carryover of the
stationary phase from the column.
The frst method produces a distinct solvent front and saves sepa-
ration time by eliminating the waiting period to reach hydrodynamic equilibrium.
One can conveniently use an injection valve with a sample loop
as in HPLC.
13.5.1.10 On-line Monitoring of Effuent
The effuent from the outlet of the HSCCC-CPC columns may
be continuously monitored by a UVVIS detector as in con-
ventional liquid chromatography.
An important difference between these two methods is that HSC-
CC-CPC uses the liquid stationary phase which, if carried over from
the column, tends to disturb the tracing of the elution curve.
Avoid trapping the stationary phase in the vertical fow cell
by eluting the lower mobile phase upward from the bottom;
do the reverse if the upper is used as the mobile phase.
When the upper mobile phase is eluted from the top of the
fow cell downward, it is important to prevent the formation
of air bubbles which may become trapped in the fow cell and
disturb the tracing of the elution curve.
Bubble formation can be largely avoided by degassing the
two phases in the separatory funnel before use, and also by
connecting fne PTFE tubing (typically 30 cm 0.40.5 mm
i.d.) to the outlet of the monitor so that the pressure within
the fow cell is substantially increased.
13.5.1.11 Measurement of Stationary Phase Retention
When the separation is completed, rotation is stopped and
the column contents are collected into a graduated cylinder
by connecting the column inlet to a nitrogen cylinder (ca.
50 psi; 1 psi = 6894.76 Pa).
227
After nitrogen appears at the outlet, the column is slowly rotat-
ed (100 rpm) in the tail to head elution mode so that solvent
remaining inside the column is pumped out by an Archime-
dean screw force assisted by the nitrogen fow. Measure the
retained stationary phase in a graduated cylinder.
Measurement of the retained stationary phase is useful in
efforts to improve the separation: when the peak resolution
is unsatisfactory, a measure of stationary phase retention
will serve as a guide for the next trial.
If it is less than 30%, the separation may be improved by
increasing retention, by applying a lower fow rate of the mo-
bile phase, increasing the revolution speed, or modifying the
solvent system to shorten the settling time.
If instead the stationary phase retention is over 50%, efforts
should be directed to search for a new two-phase solvent
system, which provides an improved separation factor ()
between the analytes.
13.5.2 Applications of HSCCC-CPC Technologies in Natural
Products Isolation
HSCCC-CPC technologies have applications in the following in-
dustries: nutraceuticals, fne chemicals, pharmaceuticals, biomedical, bio-
technology, fats and oils, and fermentation.
Compounds that can be isolated in high purity by HSCCC-CPC tech-
nologies include: saponins, alkaloids, chlorophylls, tannins, carotenoids, phos-
pholipids, fat-soluble vitamins, mono- and oligosaccharides, anthocyanins, lign-
ans, phenolic compounds, synthetic compounds, other active compounds present
in medicinal and aromatic plants (e.g. herbs and spices) and much more. The
numerous applications of CCC have resulted in a growth in the annual number of
publications in which this separation technology has been cited (Figure 10).
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Figure 10: Increasing number of publications on separation by CCC
228
Examples of some important natural products that have been
separated using HSCCC-CPC in the recent past are summarized herein.
13.5.2.1 Purifcation of Coenzyme Q
10
from Fermentation Extract:
HSCCC versus Silica Gel Column Chromatography
HSCCC was applied to the purifcation of coenzyme Q
10
(CoQ
10
)
for the frst time. CoQ
10
was obtained from a fermentation broth extract.
A non-aqueous two-phase solvent system composed of heptaneacetonitrile
di chloro methane (12:7:3.5, v/v/v) was selected by analytical HSCCC and
used for purifcation of CoQ
10
from 500 mg crude extract. The separation
yielded 130 mg CoQ
10
at an HPLC purity of over 99%. The results showed
the advantages of HSCCC over an alternative of silica gel chromatography
followed by recrystallization. These advantages regard purity, recovery and
yield (Table 3).
Table 3: Purifcation of coenzyme Q
10
from fermentation extract: HSCCC vs. silica gel
column chromatography with subsequent crystallization
Crude extract
CoQ
10
purifed by silica gel
chromatography
CoQ
10
purifed
by HSCCC
HPLC purity, % 89.2 96.0 99.2
Absolute purity, % 29.4 93.3 97.8
Recovery, %
*
74.3 88.0
Yield, %
*
23.4 26.4
* Recovery, amount of CoQ10 in puried product/amount of CoQ10 in crude extract. Yield,amount
of puried product/amount of crude extract (Journal of Chromatography A 1127 (1-2), 15 Sep-
tember 2006, 92-96)
13.5.2.2 Preparative Separation of Gambogic Acid and its C-2
Epimer by HPCCC
For the preparative separation of epimers, gambogic acid and
epigambogic acid, from Garcinia hanburyi, a two-phase solvent system com-
posed of n-hexanemethanolwater (5:4:1, v/v/v) was used. From 50 mg mix-
ture, 28.2 mg gambogic acid and 18.4 mg epigambogic acid were separated.
The purities of both were above 97% as determined by HPLC. The chemical
structures were then identifed by their
1
H NMR and
13
C NMR spectra.
13.5.2.3 Separation and Purifcation of 10-deacetylbaccatin III by
HSCCC
At present, the most promising approach is the semisynthesis of
paclitaxel or its analogs from 10-deacetylbaccatin III, a compound available in
a relatively high quantity from the foliage of several yew species. HSCCC was
used for the separation and purifcation of 10-deacetylbaccatin III (Figure 11).
A crude needle extract (500 mg/5 ml) from Chinese yew (Taxus chinensis) was
229
frst separated with a two-phase solvent system composed of n-hexaneethyl
acetateethanolwater (2:5:2:5, v/v). The partially purifed fraction was again
purifed with a different solvent system composed of n-hexanechloroform
methanolwater (5:25:34:20, v/v). HPLC analysis of the fnal fraction showed
that the purity of 10-deacetylbaccatin (20 mg) was over 98%.

Figure 11: Separation and purication of 10-deacetylbaccatin III (DAB) by HSCCC
230
13.5.2.4 Large-scale Separation of Resveratrol and
Anthraglycoside A and B from Polygonum cuspidatum by
HSCCC
HSCCC was successfully applied to the large-scale (5 g) sepa-
ration of resveratrol, anthraglycoside A and anthraglycoside B from a crude
extract of Polygonum cuspidatum Sieb. et Zucc (Figure 12). A two-phase
solvent system composed of chloroform, methanol and water (4:3:2, v/v)
was used. The separation yielded 200 mg to 1 g of these three compounds,
each at over 98% purity as determined by HPLC. Resveratrol is important as
it has a cancer chemopreventive activity.
Figure 12: Separation of resveratrol, anthraglycoside A and anthraglycoside B from a
crude extract of Polygonum cuspidatum Sieb. et Zucc
13.5.2.5 Separation of Andrographolide and Neoandrographolide
from the Leaves of Andrographis paniculata using HSCCC
The bioactive diterpenes andrographolide and neoandrographolide
from the leaves of Andrographis paniculata NEES (Acanthaceae) were success-
fully separated by CCC (Figure 13). A single 280-min separation yielded 189 mg
of 99.9% andrographolide and 9.5 mg of 98.5% neoandrographolide. Water
methanolethyl acetaten-hexane (2.5:2.5:4:1) solvent system was used.
231

Figure 13: Separation of Andrographolide and Neoandrographolide
from the leaves of Andrographis paniculata using HSCCC
13.5.2.6 Separation of WAP-8294A Components, a Novel Anti-
methicillin-resistant Staphylococcus aureus Antibiotic,
using HSCCC
The WAP-8294A complex was isolated from the fermentation
broth of Lysobacter sp. WAP-8294 (Figure 14). The major component, WAP-
8294A2, shows strong activity against gram-positive bacteria including me-
thicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant
Enterococci in vitro, and also exhibited a potent activity against MRSA in
vivo.
The previous separations were unsatisfactory. Hence HSCCC
was applied. n-Butanolethyl acetateaqueous 0.005 M trifuoroacetic acid
(1.25:3.75:5, v/v/v) was used as biphasic solvent. A sample size of 25 mg
yielded pure fractions of three components (16 mg). The method will con-
tribute to the clinical development of WAP-8294A2 as an anti-MRSA agent.
232

Figure 14: Separation of WAP-8294A components using HSCCC
13.5.2.7 Other Examples of Separation of Phytoconstituents by
CCC
Apart from the previously mentioned examples, isolation and
purifcation of polymethoxylated favones from tangerine peel, catechin con-
stituents from fve tea cultivars, rupestonic acid from the Chinese medicinal
plant Artemisia rupestris L., lycopene from tomato paste, spiramycin, gal-
lic acid from Cornus ofcinalis, lutein from the microalga Chlorella vulgaris,
naphthopyranone glycosides, salvianolic acid B from the Chinese medicinal
plant Salvia miltiorrhiza, dammarane saponins from Panax notoginseng, iso-
favan and pterocarpan glycosides from Astragalus membranaceus Bge. var.
mongholicus (Bge.), glycyrrhizin from the root of liquorice and active princi-
ples from the roots of Sophora avescens have been carried out successfully
using HSCCC.
13.6 Advantages of CCC
Quick (high throughput in preparative separation).
Inexpensive (only solvent costs, which are 5 times less than
for other LC techniques).
Gentle and versatile, for separation of varied compounds,
with less chance of decomposition.
Able to resolve from milligrams to tens of grams on the
same instrument.
Able to switch between normal and reverse phase at will.
A CCC machine, which is a chromatographic column with a
liquid stationary phase, can be used as a liquidliquid reac-
tor for chemical reactions involving a liquid catalyst.
233
No irreversible adsorption to a solid support (100% recovery
of sample).
Increased capacity for the same volume of stationary phase;
a CPC column gives a higher capacity than the HPLC one.
Quantity of sample depends on two factors: solubility of the
sample and properties of the solvent system.
No sample loss as a simple rinsing of the instrument allows
a full recovery of the noneluted fractions.
Easy maintenance, no costly solid phase to change.
No degradation or denaturation of compounds and no inter-
action with silica.
No polarity restriction; all biphasic mixtures can be used.
Dual mode (off-line and on-line) exchange of stationary and
mobile phase (CPC).
13.6.1 Advantages of HSCCC-CPC Technologies over HPLC
HSCCC-CPC HPLC
No column Expensive columns
High recovery Irreversible adsorption
High throughput Poor loadability
Retention of fragile compounds
(molecular integrity)
Loss of biological activity
(denaturation)
Volume ratio of stationary/
mobile very high (better resolution)
Ratio is low
13.7 Manufacturers of CCC Instruments
13.7.1 Manufacturers of HSCCC Machines
AEC S (http://www.ccc4labprep.com/)
Conway Centri Chro m (http://www.centrichrom.com/)
Dynamic Extraction s (http://www.dynamicextractions.com/)
Pharma-tech Research Corporatio n (http://www.pharma-tech.com/)
13.7.2 Manufacturers of CPC Machines
EverSeiko Corporatio n (http://www.everseiko.co.jp)
Kromaton Technologie s (http://www.kromaton.com/)
Partus Technologie s (http://www.partus-technologies.com)
234
13.8 Selected Reviews on CCC
Marston and Hostettmann, K., 1994, Counter-current Chro-
matography as a Preparative Tool, and Perspectives, J. Chro-
matog. A; 658: 315-341
Jarvis, 1992, Macrocyclic Trichothecenes from Brazilian Bac-
carin Species: From Microanalysis to Large-scale Isolation,
Phytochemical Analysis; 3: 241-249
Foucault, 1991, Counter-current Chromatography, Analytical
Chemistry; 63(10): 569-579
Marston, I., Slacanin and Hostettmann, K., 1990, Centrifu-
gal Partition Chromatography in the Separation of Natural
Products, Phytochemical Analysis; 1: 3-17
Sutherland, 1987, Counter-current Chromatography, Labora-
tory Practice
Martin, D. G., Biles, C. and Peltonen, R. E., 1986, Counter-
current Chromatography in the Fractionation of Natural Prod-
ucts, American Laboratory
Ito, Y., 1985, High-speed Counter-current Chromatography,
Critical Reviews in Analytical Chemistry; 17(1): 65-143
Conway, W. D. and Ito, Y., 1984, Recent Applications of Coun-
ter-current Chromatography, LC; 2(5)
13.9 Conclusions
Counter-current Chromatography is an excellent alternative to
circumvent the problems associated with solid-phase adsorbents and to
preserve the chemical integrity of mixtures subjected to fractionation. It pro-
vides effcient resolution of samples by a mechanism which relies solely on
partition. Separation of compounds with a wide range of polarities can be
achieved with the use of aqueous and non-aqueous solvent systems. CCC
is very fexible: solvent gradients are possible; fow rates can be varied dur-
ing a chromatographic run; lower and upper phases can be interchanged as
mobile phases during a separation, provided that the fow direction is also
changed accordingly; and instruments can be stopped during chromatogra-
phy and re-started hours later without affecting separation effciency. A wide
range of pH is tolerated in CCC, with implications in the separation of acidic
and basic samples, notably in the technique of pH-zone-refning. Separation
of crude plant extracts, semipurifed fractions or synthetic mixtures can be
carried out, with samples of any quantity ranging from 100 mg to 1500 g
(for the largest model). With these advantages, CCC is gaining popularity as
a separation method for natural products, and especially in the bioassay-
guided fractionation of natural products.
235
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239
14 Quality Control of Medicinal
and Aromatic Plants and
their Extracted Products by
HPLC and High Performance
Thin Layer Chromatography
K. Vasisht
Abstract
The interest in medicinal plants and their products has increased manifold in recent
years. The increasing public demand for natural medicines has resulted in increased
commercial activity and production of these medicines. There is also increasing con-
cern for ensuring quality and safety of plant medicines. Plant drugs, unlike active phar-
maceutical ingredients, possess some inherent limitations which deter the process
of laying standards for these drugs. This aspect has received considerable attention
from different quarters including policy planners, scientists and manufacturers. This
paper describes briey the roles of high performance thin layer chromatography and
high performance liquid chromatography in quality assurance of plant products. Some
practical aspects of these techniques are also discussed.
14.1 Introduction
The use of medicinal plants products has increased several
fold during the last decades. Individual countries are also giving increasing
emphasis to promote their use under the direction of the World Health Or-
ganization (WHO). Besides this, one fnds enormous interest in natural prod-
ucts from the public, which is attributable to several factors. These medi-
cines are affordable, safer and better tolerated by the biological system.
This has led to an increased consumption and cross-country movement of
raw materials of medicinal plants.
In some parts of the world, e.g. several places in Africa and Asia,
traditional medicines are the only affordable option. On the other hand, the
same medicines are the option of choice in developed nations like Japan and
the United States and in the European States. Despite being the more com-
mon medical option in Africa, use of traditional medicines has not matured to
the expected level. But, some countries in Asia, especially India and China,
have developed them to a level that has benefted all countries of the world.
Europe did not inherit a well-developed traditional system of
medicine but it has put in place the strongest evidence-based and scientif-
cally supported system of plant medicines. North America is the most prolifc
and famboyant market for plant products, but government regulations are such
that except for select plant drugs most are used as dietary supplements or
14 QUALITY CONTROL OF MEDICINAL AND AROMATIC PLANTS AND THEIR EXTRACTED PRODUCTS
BY HPLC AND HIGH PERFORMANCE THIN LAYER CHROMATOGRAPHY
240
nutraceuticals. South America, which has given the world some of the all-time
great plant medicines like quinine and pilocarpine, follows the medical system
of North America. Australia has a strong impact from Chinese medicines on the
continent which is otherwise dominated by the modern system of medicine.
14.2 Quality Control of Medicinal Plants and their
Products
The quality control of consumer products has become more chal-
lenging and demanding. The quality considerations of drugs are the most
stringent among all consumer products. The purity of active pharmaceutical
ingredients has been stretched to an all-time high with more and more restric-
tions on the level of the impurities. The situation is opposingly different in
the case of plant-derived medicine, where we are still striving to defne speci-
fcations to ensure consistency and safety. Therefore, the standards of plant
drugs are more relaxed and are in the process of development. The inherent
problems of plant drugs are obvious; unlike single chemical entities of mod-
ern drugs, they are combinations of infnite chemical molecules, known and
unknown; the knowledge of the active components is incomplete; the natural
variations in content and quantity of the chemical constituents are large and
exercising a precise control is impractical; and the complete chemical profling
of plant drugs is beyond scope. Therefore, laying standards for such drugs is
not an easy task and a comprehensive system of standards cannot be laid
down for such drugs. As our knowledge of plant drugs will advance, the stand-
ards for them will become more meaningful and complete.
The quality issue of plant drugs was irrelevant in ancient times
when these medicines were dispensed by the medical men for their pa-
tients. However, the issue has taken front seat with the commercialization of
plant drugs. The matter has been further complicated by the vested interest
of manufacturers who are out to exploit the loopholes in the standards and
laws governing the production and distribution of plant drugs.
Several national and international agencies have prioritized the
issue of assuring the quality of plant drugs. The effort of the World Health
Organization is outstanding: over 20 years ago it frst published Quality con-
trol methods for medicinal plant materials, which has been regularly updated
and followed by a series of monographs on globally important medicinal
plants.
The quality of a plant product cannot be assured without assur-
ing the quality of the raw material. Also required to ensure quality products
are in-process control, quality control of the fnished product, good manu-
facturing practice (GMP) controls and process validation. In this regard, it is
imperative to defne specifcations of raw materials to minimize variations
in the quality of fnished products and to achieve consistency. The specifca-
241
tions of plant materials include macro- and microscopic descriptions, tests
of identity, and analytical and physicochemical determinations. The expected
results of these tests and measurements are presented as numerical limits
or as a range or discretely observable result.
While fxing the limits of specifcations, naturally met variations
in plant drugs need to be accommodated. A plant material conforming to the
prescribed specifcations should be considered acceptable for intended use.
Several factors contribute to variation in the content and composition of raw
materials. These factors can broadly be grouped in four categories of climatic,
nutritional, collection and post-harvest factors. Climatic factors include prevail-
ing temperature, rainfall, humidity, daylight and altitude of the growing region.
The nutritional factors are those which affect the health of a growing plant and
are refected in the production of biomass and its composition; several soil fac-
tors such as availability of micro- and macronutrients, pH and cation exchange
capacity are important for optimal growth of plants. Collection factors control
the content of active components by giving due attention to the age, season,
collection time and part of the plant collected. Post-harvest factors are impor-
tant as the collected material is still live and carries out metabolic processes
and respires; the enzymatic processes continue after collection until they are
deactivated by drying or other suitable treatment; the crushing and cutting of
material leads to de-compartmentalization of reactive chemical constituents of
plants which were naturally located in intact cells; and the collected material
faces direct impact of oxidation by air and light besides physical loss of some
components.
The World Health Organization, in its volume Quality control meth-
ods for medicinal plant materials, has listed several parameters which are valua-
ble in assuring quality of plant drugs. These include identifcation, visual inspec-
tion, sensory characters, macro- and microscopic characteristics, moisture con-
tent, foreign matter, fngerprint by thin layer chromatography (TLC), ash values,
extractive values, volatile matter, microbial load, heavy metals and pesticide
residues, radioactive contaminants and, according to the nature of the drug,
one or more determinations for bitter value, tanning test, foaming, hemolytic
and swelling indices. The European Medicines Agencys Guideline on quality of
herbal medicinal products and Guideline on specifcations more precisely de-
scribe the quality-related issues of medicinal plants. These documents defne
and differentiate herbal substances (equivalent to herbal drugs), herbal prepa-
rations (equivalent to herbal drug preparations) and herbal medicinal products
(equivalent to traditional herbal medicinal products). The guidelines emphasize
the quantifcation of active or analytical markers and describe procedures to
ensure quality of raw material, semifnished and fnished products.
242
14.3 Biological and Chemical Standardization
of Drugs
All test procedures ultimately aim to determine the intrinsic po-
tency of a drug, which is attributable to the chemical constituents present.
Evaluating the biological potency of a drug provides a direct assessment
of its quality. But, the complexity of the procedures and methods forbids
implying this assessment. Moreover, it is not the practical option when one
handles large numbers of samples. The other option, besides biological
testing, is chemical testing, which uses assay procedures to determine the
quantity of chemical compounds, preferably the active ones, to assess the
quality of a product. This is complementary to recording other specifcations
like macro- and microscopic characteristics.
14.3.1 Chemical Standardization and Markers
Chemical standardization requires frst to identify and select a
chemical constituent of a drug and then to elaborate the assay procedure
for quantifcation of the chosen compound. Selection of the constituent,
called marker, is a diffcult task based on several considerations: the chemi-
cal profle of the drug, biological activity of the chemical constituents, suc-
cessful development of the assay procedure, ease of procuring or isolating
the marker, and stability of the marker.
The European Union Guidelines defne markers as Chemically
defned constituents or groups of constituents of a herbal substance, a
herbal preparation or a herbal medicinal product which are of interest for
control purposes independent of whether they have any therapeutic activity.
The WHO defnes markers as constituents of a medicinal plant material
which are chemically defned and of interest for control purposes.
Markers serve to calculate the quantity of herbal substances or
herbal preparations in the herbal medicinal product if the marker has been
quantitatively determined in the herbal substance or herbal preparations. The
European Medicines Agency differentiates an active marker from an analytical
marker: an active marker contributes to the therapeutic activity of the drug,
whereas an analytical marker serves only the analytical purposes.
The choice of a marker, in frst place, should be for a chemical
constituent of the drug responsible for the activity. More than one marker can
be employed for control purposes, separately or in combination. A second
choice of marker is for a defned chemical constituent that is uniquely as-
sociated with the drug. This is true when active constituents of the drug are
not known or active constituents are unobtainable or unstable for assay pur-
poses. As a last choice, a marker can be selected among more commonly or
ubiquitously present phytoconstituents. In rare cases, a chemical compound
243
not associated with the plant or its activity can also be used as a marker.
Such chemical compounds allow the estimation of an active constituent of
the plant, which itself is unstable and unft for the purpose of analysis. For
example, dantron is recommended by the European Pharmacopoeia for the
estimation of valerenic and acetoxyvalerenic acids in valerian root. Valerenic
and acetoxyvalerenic acids, which are active constituents of drugs, are highly
unstable and diffcult to isolate and to use as markers. The test response
measured for these acids in the drug is interpreted from the standard plot of
dantron to calculate the concentration of valerenic and acetoxyvalerenic acids
in the test solutions. It has been experimentally shown that the standard
curves of dantron and these two acids, in the range of estimation, are linear
and parallel.
Identifcation of a marker requires knowledge of the chemical
and active constituents of the plant drug. Markers are usually generated
in-house and a limited number of markers are commercially available from
different sources. Sigma Chemicals, Chromadex, Regional Research Labo-
ratory (Jammu) and Natural Remedies (Bangalore) are some commercial
sources of markers.
14.3.2 Analytical Techniques for Quantifying a Marker
After a marker has been identifed, it needs to be quantifed or
assayed in the test material for the purpose of quality control. Any of the ma-
jor analytical techniques, including high performance liquid chromatography
(HPLC), high performance thin layer chromatography (HPTLC), gas chroma-
tography (GC), radioimmunoassay, ultraviolet or infrared spectrometry, and
mass spectrometry, can be used to determine the quantities of marker in
the test samples. These techniques possess some advantages and dis-
advantages. Some are more commonly used while the others have limited
applications. The assay method requires the procedure and technique to
be simple, quick, specifc, economical and robust. The ideal method can be
used in different laboratories across the world without compromising on the
accuracy and precision, which is possible if the procedure has a minimum
number of critical variables. Whenever possible, the assay procedure should
use simple, inexpensive equipment which is affordable in most places. The
assay methods are developed through experimentation and from the exist-
ing knowledge of the drug and of techniques.
14.3.3 Validation of Analytical Procedures
Development of the assay method is followed by its validation.
The validation procedure considers issues of specifcity, linearity, range, ac-
curacy, precision, detection limit, quantitation limit, robustness and system
suitability. The International Conference on Harmonisation (ICH) has issued
guidelines on validating analytical procedures, which are widely accepted
and commonly used. Besides validation of a procedure, the instruments
244
used are also validated from time to time. Practically all manufacturers of
modern equipment supply detailed instructions and methods of equipment
validation. The validation record is maintained and, in case a deviation is
observed, a service engineer is called.
The ICH guidelines on validation are available from the internet
for comprehensive understanding of the subject. A brief explanation to the
validation procedure is provided here.
14.3.3.1 Specifcity
Specifcity indicates the extent to which an assay procedure
specifcally measures the analyte of interest. It is not always possible to
demonstrate that an analytical procedure is specifc for a particular analyte
(complete discrimination). It may require two or more assay procedures to
demonstrate the necessary level of discrimination. Spiking the sample with
the analyte or related compounds and observing the effect on the estima-
tions demonstrates assay specifcity. If spiking with compounds related to
the analyte produces no effect on the result, the procedure is considered
specifc. Specifcity is particularly valuable when analyzing an analyte among
several similar compounds present in the sample.
The issue of specifcity with respect to herbal materials has
lesser relevance as in several instances; we tend to determine the content
of total active compounds rather than one active constituent, e.g. total sen-
nosides in senna and not a particular sennoside. In such cases, a procedure
that is able to assay all sennosides together is preferred than a specifc pro-
cedure which discriminates and estimates only one sennoside.
14.3.3.2 Linearity
The linearity of an analytical procedure is its ability (within a
given range) to obtain test results which are directly proportional to the con-
centration (amount) of the analyte in the sample. A linear relationship (be-
tween amount of analyte and response) should be demonstrated across the
range used in the assay procedure. A minimum of 5 concentrations is rec-
ommended. A regression line, using appropriate statistical methods, should
be drawn to calculate correlation coeffcient, y-intercept and slope. A plot of
the data should be included in the test report. Another important feature,
which estimates the degree of linearity, is calculation of the deviation of the
actual data points from the regression line. In some cases, mathematical
transformation of data, prior to the regression analysis, may be required.
14.3.3.3 Range
The range is the interval between the lower and upper analyte
concentrations for which it has been demonstrated that the analytical pro-
245
cedure has a suitable level of accuracy, precision and linearity. It is normally
derived from linearity studies.
14.3.3.4 Accuracy
The accuracy of an analytical procedure is the closeness of
agreement between the conventional true value (or an accepted reference
value) and the value calculated. It tells how closely the analyte amount is
determined to its true amount present in the test sample. Accuracy should
be specifed across the range of analytical procedure and inferred from 9
measurements (triplicates of three concentrations in the range).
Accuracy can be demonstrated by application of the proposed
procedure to an analyte of known purity or by comparing the results of the
proposed analytical procedure with those of a second well-characterized pro-
cedure, whose accuracy is already known. Application of the procedure to
test samples after spiking at three different levels of 50%, 100% and 150%
of expected analyte concentration helps to determine the accuracy of the
procedure.
A sample containing 1.0 mg analyte may, in different analyses,
be found to contain 1.2, 0.9 and 0.8 mg. The assay procedure determining
0.9 mg is more accurate than the two other procedures.
14.3.3.5 Precision
The precision of an analytical method is closeness of results
for a series of measurements of multiple samples from the same homo-
geneous material. The precision may suffer upon varying the experimental
conditions, which are therefore assumed to be kept constant. System preci-
sion demonstrates error in recording the response and can be determined
by repeatedly analyzing a sample within a short period of time.
It is possible that the results are precise but not accurate or
vice versa. Triplicate measurements of 1.0 mg true quantity as 0.6, 1.0 and
1.4 result in an average value of 1.0 mg which is accurate but the three
measurements themselves are not precise. Similarly, triplicate measure-
ments of the same quantity as 0.6, 0.7 and 0.6 are precise but not ac-
curate. Triplicate measurements of 1.0, 0.9 and 1.0 are precise as well as
accurate.
Precision is expressed at three levels of short, medium and
long intervals, which are respectively referred to as repeatability, intermedi-
ate precision and reproducibility.
246
14.3.3.5.1 Repeatability
Repeatability is precision under the same operating conditions
over a short interval of time (intra-day precision). It is demonstrated by a
minimum of 9 determinations covering the specifed range for the procedure
(e.g. 3 concentrations/3 replicates each) or a minimum of 6 determinations
at 100% of the test concentration.
14.3.3.5.2 Intermediate Precision
Intermediate precision expresses intra-laboratory variations:
different days, different analysts, different equipment, etc.
14.3.3.5.3 Reproducibility
Reproducibility is precision at the inter-laboratory level. It is
especially important if the analytical procedure is to be used in different
laboratories, for instance, a pharmacopoeial procedure.
14.3.3.6 Detection Limit
Detection limit (DL) is the lowest amount of analyte in a sample
which can be detected, but not necessarily quantitated as an exact value.
It is determined by analyzing samples containing known concentrations of
analyte and by establishing the minimum level at which the analyte can
be reliably detected. Other methods of determining DL are based on the
signal-to-noise ratio and on the standard deviation of the response and the
slope. A signal-to-noise ratio of 3:1 or 2:1 is a fair estimate of DL. Using the
standard deviation of the response () and slope of the calibration curve
(S), DL=3.3/S.
14.3.3.7 Quantitation Limit
Quantitation limit (QL) is the lowest concentration of the ana-
lyte that can be determined with acceptable precision and accuracy under
the stated experimental conditions. It is generally determined by the analy-
sis of samples with known concentrations of analyte and by establishing
the minimum level at which the analyte can be quantifed with acceptable
accuracy and precision. QL can also be defned from the signal-to-noise ratio
(a typical signal-to-noise ratio is 10:1), or from the standard deviation of the
response () and the slope (S) of the calibration curve (QL=10/S).
14.3.3.8 Robustness
Robustness of an analytical method ensures that it performs
well and has few variables affecting its performance. For example, a robust
HPLC analytical procedure does not show variation when columns of differ-
247
ent lots or from different manufacturers are used, when there is a slight vari-
ation in pH or composition of the mobile phase, or when temperature and
fow rate vary. Steps of extraction, purifcation or enrichment of an analyte
in herbal material should be simple and time effective. Solutions used in
the analysis should be stable over a reasonable period of time. Lastly, the
procedure should be feasible in most laboratories.
If measurements are prone to variations with small changes in
the test conditions then such conditions should be suitably controlled or a
precautionary statement should be included in the procedure.
14.4 Thin Layer Chromatography in Quality
Control of Plant Products
Thin layer chromatography (TLC), also called planar chromatog-
raphy, is a widely accepted and extensively used separation technique that
is over 65 years old. The technique is simple, cost effective, versatile, and
useable in all laboratories around the globe. It can be easily adapted to any
given situation of qualitative, quantitative or preparative separation. Despite
the great variety and complete automation of the technique, it still lags be-
hind other chromatographic techniques when it comes to its use as analytical
technique. However, there is no substitute for this technique for situations
requiring qualitative analyses of plant extracts. TLC has nearly become in-
dispensable for the standardization of plant materials, be it the fngerprint
profling or analysis of a marker. The advantages of the technique over other
analytical techniques are many when handling plant materials. The samples
can be applied without undertaking tedious, time-consuming processes of
sample preparation. The loss in sensitivity is far compensated by the gain on
several fronts, including ease of assays, multiple sample analyses and low
cost per sample.
The two prominent uses of TLC in the standardization of plant
materials include fngerprint profling for the assessment of chemical con-
stituents of a drug and quantitative analysis of markers in plant drugs. A
typical TLC procedure involves sample preparation, selection of the chro-
matographic layer and the mobile phase, sample application, development
and drying of the plate, derivatization (if required) and chromatogram evalu-
ation.
14.4.1 Sample Preparation
Methods of sample preparation for fngerprinting and estima-
tion of marker differ signifcantly. Whereas in fngerprinting, only proportion-
ate quantities of components must be extracted, in assaying the marker
complete and exhaustive extraction has to be ensured. Correspondingly,
particle size of the crude drug, the solute-solvent ratio, extraction period and
248
number of extractions assume greater signifcance in marker estimations.
Normally 1-2 g of moderately fne powder (unless specifed) of plant material
is extracted with 25-50 ml solvent at room temperature, in a Soxhlet appa-
ratus or under refux on a water-bath. The extraction is repeated a number
of times to ensure complete and exhaustive extraction of the marker from
the drug matrix. The extract is fltered and solvent is removed from the com-
bined fltrate. The residue is dissolved in the solvent, fltered again, and the
volume is adjusted. The concentration of the marker is determined in the
solution.
On the other hand, in comparing fngerprint profles, the proce-
dure requires a shorter extraction scheme. A powdered specimen of pharma-
copoeial quality may be required as the reference material for comparison
of the fngerprint profles. The test and sample solutions are prepared under
identical conditions of extraction and concentration. Usually 0.1-1.0 g mate-
rial is extracted with 1-10 ml solvent for 5-30 min, by shaking at room tem-
perature or heating to boiling. The extract is fltered, concentrated and used.
Sometimes the solvent is completely evaporated and the residue is dissolved
in a small volume of solvent (typically less than 1 ml) and fltered to sepa-
rate the insoluble particles. The solution of a marker, of preferably known
strength, is required if marker presence is to be ascertained. Using known
strength of marker additionally provides semiquantitative information.
In certain cases, the extracts require further purifcation using
extraction of the residue with another solvent at different pH or using distil-
lation, sublimation or other appropriate method.
14.4.2 Selection of Chromatographic Layer
A wide variety of options is available for the adsorbent layer.
Laboratory-made plates have given way to precoated plates marketed by
several manufacturers. The precoated plates are machine-made of glass,
aluminium or plastic base coated with different adsorbents. The different
adsorbents include normal phase silica gel (most commonly used), reverse
phase silica gel (RP
2
, RP
8
, RP
18
, cyano, diol and amino plates), aluminium
oxide, cellulose, kieselguhr, hybrid (capable of being used as normal and re-
verse phase) and derivatized adsorbent layers. They come in different sizes,
from small strips to continuous rolls (20 x 20 cm
2
is most common).
The nature of the compounds defnes the choice of adsorbent
layer; a stronger adsorbent (aluminium oxide) is used for weakly adsorbed
compounds and a weak adsorbent (cellulose) is used for strongly adsorbed
compounds. Normal phase silica gel is more suited for non-polar components
and reverse phase silica gel is more suited for polar constituents, which are
eluted frst on reverse phase TLC. The silica gel plates containing fuorescent
dye (F
254
) of aluminium base are most widely used; about 80% of the analyses
are done using these plates as they are optimally effcient and cost-effective.
249
14.4.3 TLC versus HPTLC Layers
High performance TLC (HPTLC) plates use thin layers of adsorb-
ent (100 m instead of 200-250 m) and smaller particles (5-6 m versus
10-12 m) of more homogeneous size (4-8 m versus 5-20 m). Moreover,
they give better resolution (5- to 10-fold more) over shorter runs (3-6 cm ver-
sus 8-15 cm), reduce separation time (3-20 min versus 20-200 min), accom-
modate more samples per plate (more than double), use smaller sample
volumes (0.1-0.5 l versus 1-5 l) with improved detection limits (100-500
pg), and signifcantly improve the precision, accuracy and sensitivity. HPTLC
plates are substantially more expensive (4- to 6-times more) than normal
plates but are an effcient alternative when high sensitivity, accuracy and
precision are required in situations demanding high performance. More im-
provements in adsorbent layers include use of spherical particles of narrow
size distribution (reducing resolution time and size of spots while improving
the detection limit) and ultrathin layers (10 m) that improve the resolution
and sensitivity and drastically reduce the development time.
14.4.4 Selection of the Mobile Phase
Infnite combinations and a wide choice of solvents are avail-
able for TLC developments. Unlike HPLC, where choice is limited, TLC pro-
vides no or few restrictions. A mobile phase with 1-3 components is pre-
ferred over a multicomponent mobile phase. The polarity of the compounds
of interest is the key to selection of a mobile phase. Personal experience ap-
plied to existing knowledge and a trial and error method is used to select the
composition of the mobile phase. The mobile phase is freshly prepared for
each run and the constituting solvents are mixed outside before transferring
to the developing chambers. It is advised to allow the developing chamber to
saturate unless otherwise specifed. Saturation of the chamber is quickened
by lining half or more of the total area of the inside walls with flter paper
and pouring the mobile phase over it. Closing the chamber and allowing it
to stand at room temperature saturates the chambers. It is possible to use
another solvent alongside the mobile phase for chamber saturation in twin
troughs, e.g. ammonia placed in one trough and mobile phase in another.
The TLC results are sensitive to temperature and humidity vari-
ations. All operations during which the plate is exposed to the air should be
carried out at a relative humidity of 50%-60% under controlled temperature
of 20-30 C.
14.4.5 Application of Sample
Three typical options of delivering the sample solution onto the
plate are manual, semi-automatic and automatic application. Manual applica-
tion is done using a capillary, which can have a specifc volume of 1, 2 or 5 l
for quantitative purposes. The solution is applied by the technique of touch
250
and deliver. The precision and accuracy, as known to the author from personal
experience, is fairly high after a short experience. The semi-automatic applica-
tion uses devices such as Linomat 5 from Camag and Applicator AS 30 from
Desaga, which use a syringe that has to be manually cleaned and flled. The
remaining part of the application is automated through computer commands.
The solution is applied as a spot or band of predetermined size at predeter-
mined points by touch and delivery or spray-on technique. The needle touches
the surface of the adsorbent layer and delivers, whereas in spray-on technique
the predetermined volumes are sprayed onto the plate. In the fully automated
application, all steps are controlled through a computer including washing of
the delivery line.
The typical concentration of the applied samples ranges from
0.1 to 1 mg/ml for qualitative analysis but is usually much lower for quantita-
tive purposes, which further depends on the molar absorption of the marker.
The typical volume for spot application is 1-5 l, and 10 l for band applica-
tion. These volumes are drastically reduced in HPTLC plates or ultrathin TLC
plates. Bands are known to give better resolution and results than spots, as
a narrow band is better suited to the optics of the TLC scanner.
14.4.6 Developing the Chromatogram
Development of plates is carried out in chambers which are spe-
cial purpose jars or simple containers good enough to hold the solvent in an
air-tight environment. There is no doubt that special purpose chambers pro-
duce better chromatograms. Twin-trough chambers allow use of another mobile
phase in the chamber for the purpose of saturation, besides consuming smaller
quantities of solvent. The cost of the chamber, which seems high in the begin-
ning, is recovered by way of savings on the quantity of expensive solvents. Pre-
saturation of the chambers decreases R
f
values and corrects side distortions
of the solvent front. The plate is placed as nearly vertical as possible in the
chamber, ensuring that the points of application are above the surface of the
mobile phase and the sides of the plate do not touch the container walls.
The developing chamber should always be kept out of direct
sunlight. It should be protected from light during development, if the com-
ponents being investigated are suspected to be unstable. If sun rays fall di-
rectly on the developing chamber, they may be refracted to different degrees
through the glass walls, producing areas of high temperature on the plate
and resulting in erratic fow of the mobile phase.
The technique of development has been largely improved in
horizontal developing chambers and completely automated in automated
development chambers or automated multiple development chambers.
However, the cost of this equipment (except for the horizontal development
chamber) is excessively high.
251
14.4.7 Drying the Plate
After development, the plate is dried. This is an automatic pro-
cedure in automated development chambers, but it has to be accomplished
in air at room temperature, in a vacuum desiccator or by heating or blow-
ing hot-air over the surface of the plate. In all instances, the mobile phase
should be as completely removed as possible before proceeding to derivati-
zation or scanning the plate.
14.4.8 Derivatization
Derivatization involves treatment of developed chromatograms
with suitable spray reagents for locating the position of the constituents for
qualitative evaluation and for quantifying ultraviolet-insensitive markers. Two
methods are employed for derivatization of plates: spraying with a fne mist
of a reagent (a traditional method) and dip-in technique, which of late has
become more popular. The spray method does not allow the uniform wetting
of the plate, producing areas of high wetting and defcient spray. This affects
the precision and accuracy in case of quantitative determinations. The dip-in
technique produces more uniform wetting; special equipment is available for
this purpose. In most cases of derivatization, heating is required after spray-
ing the plate. Heating the plate uniformly in the open air produces better re-
sults than heating in an oven. The fumes from heating are strongly reactive
and damage the inner walls of the oven. The plate is heated at about 110
C for about 10 min or until the spots are best seen. Special purpose heating
plates are available from the manufacturers of TLC equipment.
14.4.9 Evaluation of the Chromatograms
The TLC plate is observed in daylight, under short-wave and
long-wave ultraviolet light, for comparing the chromatograms of standard and
test samples or for observing the presence of a marker or compounds of
interest in the test chromatogram. The centre of each spot is marked with
a needle. The distance from the centre of each spot to the point of applica-
tion is measured to record the R
f
value (the ratio of the distance travelled
by a given compound to that travelled by the solvent front) or the R
r
value
(the ratio of the distances moved by a compound and a stated reference
substance). R
f
values may vary depending on the temperature, degree of
saturation, the activity of the adsorbent layer and the composition of the
mobile phase.
Quantitative evaluation is done by scanning the plate in a TLC
densitometer or scanner. The densitometer uses two modes of transmit-
tance and refectance depending upon the available optics. It uses fuores-
cence mode, ultraviolet absorption or visible light for quantitation of the
marker depending upon the option exercised. Ultraviolet and visible light
absorption modes come as a standard option on a scanner and the fuores-
252
cence mode is optional. Data acquisition and analysis is through standard
PC-based software. Multi-wavelength scanning, recording and comparing ul-
traviolet spectra, and generating and acquiring spectra libraries are among
several options available on the software provided with the TLC scanner.
The determination of analyte concentration is through a stand-
ard plot or single or double point calibrations.
14.4.10 Improving the Effciency of TLC
Several precautions can be taken to improve the effciency of
TLC analysis. These include carefully selecting the range of concentrations
for analysis; using correct instrument parameters like slit dimensions, wave-
length selection, scanning speed, base line correction; using HPTLC plates
for high sensitivity and resolution; use of appropriate sorbent from a wide
range of sorption properties to optimize selectivity; use of automated sam-
ple application, development and detection; use of precise in situ recording
and quantitation of chromatograms; and avoiding derivatization in assay pro-
cedures and, if necessary, using dip-in technique of derivatization.
The following example, of one of the Ayurvedic drugs, illustrates
the use of TLC in quality control of plant material. The drug was analyzed for
one of the active compounds and the TLC fngerprint profle was used for the
purpose of positively identifying the plant material.
To prepare the fngerprint profle, about 5 g plant material was
extracted with 50 ml methanol for 30 min at 50 C in a conical fask. The
extract was fltered and the fltrate was concentrated to about 5 ml under
vacuum. One of the active constituents isolated from this plant (code name
DPH-1) was used as a reference. The solution of the reference substance
was prepared by dissolving about 5 mg in 1 ml chloroform. About 10 l of
each test and reference solution was manually applied in band form on alu-
minium base, silica gel 60 F
254
, 0.2-mm thick TLC plates (Merck). The plate
was developed using mobile phase containing 95 volumes toluene and 5
volumes ethyl acetate. The plate after development was dried and visual-
ized under 254 nm ultraviolet light (Figure 1A). The same plate was sprayed
with anisaldehyde-sulphuric acid reagent and heated for about 10 min to
visualize the spots (Figure 1B). These profles can be used to confrm the
identity of the plant material and to obtain semiquantitative information on
the amount of DPH-1 present in the drug.
253
A B
Figure 1: TLC chromatograms as visualized (A) under 254 nm UV and (B) after being
sprayed with anisaldehyde-sulphuric acid reagent
Besides developing the fngerprint profle of the drug, the quan-
tity of DPH-1 was also estimated in different samples of the plant material.
For the purposes of analysis, 5 g moderately fne powder of drug material
was extracted with methanol in a Soxhlet apparatus for 4 h. The extract was
fltered and the volume was adjusted with methanol to 50 ml in a volumetric
fask. One milliliter of this solution was diluted to 10 ml in a volumetric fask
and used for analysis. A standard solution of DPH-1 was prepared by dissolv-
ing 4.95 mg DPH-1 in 10 ml methanol and diluting 0.5 ml of this solution to
10 ml in a volumetric fask. Six different concentrations of this solution were
applied in triplicate on a precoated TLC plate, which was developed using
mobile phase containing 90 volumes toluene and 10 volumes ethyl acetate.
The developed and dried plate was scanned at 305 nm in a TLC scanner and
the standard plot was constructed (Figure 2). One microliter of test solution
was similarly analyzed using the same conditions as used for DPH-1, and
the amount of DPH-1 in the test sample was calculated from the response
obtained in a TLC scanner. The analyzed drug samples showed large varia-
tions in the content of DPH-1, ranging from below 0.3% to over 1.4%. The
method was validated according to ICH guidelines.
254
o
o
1ooo
zooo
ooo
uooo
cooo
zo uo 6o So
Amount of 0Ph-1 ln ne
A
u
C
y = c.6/x u6.1
k
z
= o.uS
Figure 2: Calibration curve of DPH-1
The TLC-based method of analysis and fngerprint development
is quick and reliable, and can be used conveniently in different laborato-
ries. Similarly, it is possible to apply this technique to other plant drugs to
develop fngerprint profles and also to estimate the percentage of marker
substances in the crude drugs or in fnished products.
14.5 High Performance Liquid Chromatography
In a period of less than 50 years, HPLC has become the most
widely used analytical tool in most laboratories of the world. The technique
has received great attention for innovations leading to its overall develop-
ment, regarding both consumables and equipment. HPLC separations are
achieved using any of the fve basic chromatographic modes: liquid-solid
(adsorption), liquid-liquid (partition), bonded-phase (partition), ion exchange,
and size exclusion chromatography. The selected mode depends on the na-
ture and properties of the analyte. Bonded-phase chromatography, in which
a stationary phase of organosilanes of varying carbon lengths is chemi-
cally bonded to silanol groups, is the most commonly used mode of sepa-
ration. In liquid-liquid chromatography, the solid support (usually silica or
kieselguhr) is mechanically coated with a flm of high boiling point organic
liquid, unlike bonded-phase chromatography where non-polar hydrocarbon
chains are chemically bonded to hydroxyls of the silica support. Liquid-liquid
chromatography, by virtue of its mechanism, is more susceptible to changes
by interaction with mobile phase than bonded-phase chromatography.
A typical HPLC operation includes pumping of mobile phase at
moderately high pressure through a narrow-bore column containing adsorb-
ent. The separation of the mixture takes place in the column and separated
components are detected by employing a suitable detector. As the mobile
phase is being pumped at high pressures, a system is required to inject the
mixture into the system without dropping the pressure and disturbing the
fow characteristics, i.e. rate and pressure. To accomplish these require-
ments, an HPLC system requires a pump to push the mobile phase against
high pressure, an injector to insert a solution of standard substance or test
255
mixture, a column to effect separation, a detector to reveal the presence of
analyte in the eluate, and a suitable data processing unit.
14.5.1 Pumps
The pump is considered a heart of the HPLC system, as all
depends on the composition of the mobile phase and its fow rate accuracy.
The pump gives a pulse-free fow of mobile phase; the expected variations
in fow rate are less than 1.0%. Online mixing of solvents is preferred to
manual mixing. However, compositions containing less than 10% of a par-
ticular solvent are better prepared by manual mixing. The composition of the
mobile phase is either constant during the analysis (isocratic mode) or it is
changed (gradient mode). The type and design of modern pumps allow low
pressure mixing of up to four solvents; else, different pumps, one for each
solvent, are required for gradient operation and the solvents are mixed at
high pressure. A typical analytical procedure uses a fow rate of about 1 ml/
min and operating pressure between 1000 and 2000 psi. Higher fow rates
generating higher pressure should always be used with justifcation, as they
decrease column life besides requiring frequent servicing of the pump.
The fow accuracy of the pumps is critical for analysis. The con-
stancy of retention time of the last eluted peak is a measure of long-term
fow accuracy of the pumps, whereas short-term fow accuracy is checked
from the average peak areas of each component and their standard devia-
tions. The mobile phase must be free of dissolved gases to ensure an accu-
rate fow and to minimize noise due to bubbles. Vacuum fltration, sonication
and helium gas purging are methods for degassing.
14.5.2 Injector
The injector allows a predetermined volume of test solution to
be introduced into the fow channel of the system, without disturbing the
fow kinetics. Typically, fxed volume injections are preferred over variable
volume injections. When using fxed volume loops, it is advisable to fush
higher volumes of the sample through the loop to ensure complete flling of
the loop with the sample solution. The mobile phase close to the inner walls
of the loop can only be assured to have pushed out after injecting volumes
larger than the loop volume, e.g. injecting 20 l test solution into a 20-l
loop cannot assure accurate injection; if the quantity of the test material
is not a problem, fush the loop with over 100 l test solution. Only the ap-
propriate needle (compatible with the injection port) should be employed
for the purpose of making an injection. It is important to select a syringe of
appropriate size when giving variable injections; a thumb rule for any analyti-
cal technique is not to use the volumetric apparatus if less than 20% of its
total volume is being used. Thus, a syringe of 25 l should not be used to
measure or inject volumes less than 5 l.
256
14.5.3 Columns
Columns come in varied sizes, structural architecture and
chemistry. The chromatographic material is usually packed in stainless steel
casing. The material is composed of porous particles which vary in nature
(inorganic ceramic, organic polymer, or hybrid), shape (irregular or spheri-
cal), size (ranging from 2 to 20 m; normally around 5 m) and surface
modifcations (silanes of different carbon lengths, aminopropyl, diol, cyano,
sulphonic acid and ammonium ions). The choice of column is based on the
type of analysis. Comprehensive information is available on the websites of
the leading manufacturers of HPLC columns, which serves as good guides
in choosing columns for analysis. Most analyses are reported on reverse
phase columns, usually C
18
, with increasing emphasis on reducing the col-
umn length, diameter and analysis time. Most HPLC separations are suc-
cessful on columns maintained at ambient temperature, but thermostatted
column maintained to 0.2 C is necessary for reproducible results. This is
because all mechanisms of separation are temperature-dependent and any
shift in temperature has remarkable bearings on the result.
14.5.4 Detectors
A wide variety of detectors is available to cater to diverse needs
of the analysts. Ultraviolet detectors of fxed wavelength, dual wavelength or
variable wavelength (photodiode array detector) are most frequently used.
Other options are refractive index detector, fuorescent detector, electro-
chemical detector, evaporative light scattering detector and chemilumines-
cence detector.
14.5.5 Data Processing
The electrical response from the detector is digitalized and fed
to a data processing module, which in present days is invariably a computer,
and computations are made using special software. Several software pro-
grams are available for data processing, from both manufacturers and third
parties. Besides computing the data, they also control the entire operation
of the machine.
14.5.6 Factors Affecting HPLC Analysis
Numerous variables affect an HPLC analysis. This topic is be-
yond the scope of this paper, but some critical variables are discussed briefy.
Increased emphasis is now paid to control the temperature of the column
within a narrow range to ensure precision of the result. This is desirable, as
factors such as solubility, solute diffusion, viscosity of the mobile phase, and
column plate number all are affected by temperature. Mobile phase compo-
sition is another vital parameter that affects the resolution, retention time
and peak area. Pumps contribute the most towards variations of results, as
257
precise composition of mobile phase and fow rate can only be assured by ac-
curate pumps. Gases dissolved in the mobile phase are a source of fow-rate
inaccuracies and errors in detector response.
Retention time variations are often discussed to know the toler-
able limits. The retention time is affected by fow rate, column temperature, mo-
bile phase composition and integration. An error in fow rate leads to changes in
the retention time to the same extent. Small variations in column temperature
have more signifcant effects on retention time. Ideally, a column is thermostat-
ted to 0.2 C. However, a high precision of 0.1% in retention time requires the
column to be thermostatted to 0.04 C. Changes in mobile phase composition
leave a stronger impact on the retention time. It is estimated that in a typical
isocratic elution, a variation of 1% in mobile phase composition occurs, which
introduces an error of 0.4%-0.7% in retention times. The observed variations in
composition of the mobile phase are more in the gradient elution. Recording
devices also introduce variations in the retention time through faulty recording,
but the effect is much smaller (in the range of 0.1% to 0.04%).
Peak area is affected by all the factors that affect retention
time. Additionally, the recorder response in marking the start and end of the
peak is crucial; this has been seen to be the main source of error in record-
ing peak areas.
Several more factors, like injection volume, connecting tubing,
end fttings and detector volume, also have bearings on the fnal results.
Large injection volume and quantity of analyte result in broadening of the
peak. Preparing the sample in the mobile phase produces the best result
and should be taken as the frst choice.
14.5.7 HPLC in Quality Control of Plant Products
HPLC is the most popular technique among all the analytical
techniques used today. It is therefore understandable that most happenings
are taking place in the modernization of this technique. As discussed in the
section on TLC, HPLC can be used for similar purposes. There are two ap-
plications of HPLC: one to generate the profle, for which TLC is preferred,
and one to estimate the quantity of markers, where HPLC is preferred. The
initial steps of sample preparation are similar to those for TLC with the ex-
ception that the samples for HPLC are fltered through a flter of 0.45 m
or less. Furthermore, it is assured that the test sample does not contain
substances which are permanently retained on the HPLC column, which
means in most cases purifcation procedures are applied to extracts before
injecting them onto the column.
After the sample has been prepared, it is injected onto the
column and the response is recorded preferably using a variable wavelength
ultraviolet detector. As the nature of all the compounds of the extract cannot
258
be known beforehand, the photodiode array detector is useful, especially
when constructing profles of plant extracts. The fngerprint profle of plant
extracts can be used for identifcation purposes and also for obtaining semi-
quantitative information if the sample preparation was not done for quantita-
tive analysis. Similarly, the profle can be generated for the fnished product
and used to record batch to batch variations. The fngerprint profle can be
used to study changes in the composition of the fnished product or, in other
terms, to indicate the stability of the product.
The most important use of HPLC is in estimation of markers in
plant drugs. The steps in HPLC analysis are fundamentally the same as used
for any other analytical technique. The response of the test sample is com-
pared to that of a known quantity of the marker to quantify the marker in the
test substance. The HPLC method is developed from knowledge of the tech-
nique and chemistry of the marker. In chemical analysis, HPLC has no parallel
and can be customized to produce the most precise and accurate results. The
HPLC analysis is vital in the analysis of a fnished product and the expected
results are superior to those from TLC, as the separations in HPLC are better.
However, run time of HPLC analyses usually varies from 15 to 30 min, which
restricts its use if large numbers of samples are to be analyzed.
14.6 TLC versus HPLC
TLC has emerged as a major tool in standardization of plant
materials. The advancement and automation of the technique has made it a
frst choice for plant drugs. Its use has become more popular in developing
countries where advancements of HPLC are not cost effcient. TLC offers
several advantages over HPLC. Sample and mobile phase preparation do
not require elaborated steps of purifcation, degassing, and fltration, which
are essential to protect expensive columns from deterioration. Several sam-
ples (up to 18) can be accommodated on a single 20 x 20 cm
2
plate. The
test samples and standards are analyzed simultaneously under the same
conditions. Several analysts can work simultaneously as each step in analy-
sis is carried out independently using separate equipment. The choice of
solvent systems is unlimited, unlike for HPLC where column chemistry disal-
lows the use of extremes of pH in mobile phase. The technique allows enor-
mous fexibility of derivatization with chromogenic spray reagents, making
possible the detection of an analyte that is transparent to ultraviolet light.
It also allows multiple evaluations of the developed chromatogram, which
is not possible in HPLC. There is no leftover from the previous analysis to
interfere in the next, as each time a fresh plate is employed. Lastly, it saves
tremendously on the time and cost of the analysis.
TLC offers many advantages but also has some disadvantag-
es. It fails to match the sensitivity of HPLC and has not kept with the pace
of developments and advancements happening in the area of HPLC. TLC is
259
an open system and hastens the degradation of compounds sensitive to
light and air, which in the case of HPLC pass through an enclosed environ-
ment. Detection of the analyte in HPLC occurs in solution, permitting high
sensitivity, whereas in TLC the solid phase interaction makes detection less
sensitive. Finally, recent advances and effcient fow kinetics of HPLC allow
more complex separations than TLC.
14.7 Conclusions
Both TLC and HPLC are vital in the analysis and quality control
of plant material and the extracted products. Each of these techniques has
its own limitations and advantages. TLC is fast, adaptable and economical,
whereas HPLC is more precise and accurate. Based on the preferences and
demand of the situation, one can opt to use one or the other for quality as-
surance of plant products.
Bibliography
Barwick, V. J., 1999, Sources of uncertainty in gas chromatography and high perform-
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EMEA, 2006, Guideline on Quality of Herbal Medicinal Products/Traditional Herbal
Medicinal Products, EMEA/CVMP/814/00 Rev 1, European Medicines Agency,
London, U.K., p. 1-11
EMEA, 2006, Guideline on Specifcations: Test Procedures and Acceptance Criteria
for Herbal Substances, Herbal Preparations, and Herbal Medicinal Products /
Traditional Herbal Medicinal Products, EMEA/CVMP/815/00 Rev 1, European
Medicines Agency, London, U.K., p. 1-21
European Pharmacopoeia 5.0, 2004, Vol. 2, European Directorate for the Quality of
Medicines, Strasbourg, France, p. 2667-2668
ICH, 1996, ICH-Harmonised Tripartite Guideline on Validation of Analytical Proce-
dures: Methodology, International Conference on Harmonization, p. 1-8
Poole, C. F., 1999, Planar chromatography at the turn of the century, Journal of Chro-
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) is a measurement of the retention time

), selectivity () and column effciency (N) are

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