ChEg 6226: Analytical Methods for Environmental

Monitoring

By Dr. Tariku Bekele

(tarchem2112@gmail.com)

12/26/2024 1
 General principles of sample collection and data
analysis

1. Sampling
▪ Sampling is the process of selecting a
portion of a material, in some manner, to
represent or provide information about a
large body of a material.
▪ Sampling includes deciding on sampling
points and choosing a method to get
appropriate amounts of samples.
▪ A sample is collected such that it represents
the original object.
▪ Sampling constitutes the most important
aspect of environmental analysis , without
effective sampling all of the subsequent
data generated are worthless.
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 Cont…
▪ Sampling procedures are determined by the objectives of the
analysis and the nature of the target analytes.

▪ Any sampling protocol involves the selection of the sample

points, the size and the shape of the sample area, and the
number of sampling units in each sample. Before this can be
done, information regarding the likely distribution of the
contaminants under investigation is required.
John R. Dean

12/26/2024 3
❖The key Questions to be asked before sampling begins
include the following:
✓ Have arrangements been made to obtain samples from the site
(e.g. permission).
✓ Availability of sampling equipment.
✓ How many samples & replicates are required?
✓ Are the samples required for qualitative or quantitative analysis?
✓ What chemical or physical tests are required?
✓ What analytical methods and equipments are needed?
✓ What mass/volume of sample is required for the analytical
techniques to be use?
✓ What type of containers are required to store the samples and do
you have enough available?
✓ Is any sample preservation required and do you know what it is?
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 Errors in sampling

• Sampling is the most difficult step and the greatest source

of error during analysis.

• In a qualitative analysis the sample’s composition does not

need to be identical to that of the substance being analyzed,
provided that enough sample is taken to ensure that all
components can be detected.

• In a quantitative analysis, however, the sample’s

composition must accurately represent the target
population. The focus of this section, therefore, is on
designing a sampling plan for a quantitative analysis.
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 Cont…

• A proper sampling strategy ensures that samples are

representative of the material from which they are taken.
• Biased or non representative sampling and contamination of
samples during or after their collection are two sources of
sampling error that can lead significant errors.
• It is important to realize that sampling errors are completely
independent of analysis errors.
• As a result, sampling errors cannot be corrected by evaluating a
reagent blank.

• Home work: What is reagent blank? What about field blank?

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 Cont…

• Random errors introduced when collecting samples are

characterized by a standard deviation for sampling, ss.

• The standard deviation for the analytical method, sm, accounts

for random errors introduced when executing the method’s
procedure.

• The relationship among S0 , Ss , and Sm is given by a

propagation of random error
S20 = S2m + S2s

where so is the overall standard deviation for an analysis.

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 Designing A Sampling Plan
• A sampling plan must support the goals of an analysis.

• In characterization studies a sample’s purity is often the most

important parameter.

• For example, a material scientist interested in the surface

chemistry of a metal is more likely to select a freshly exposed
surface, created by fracturing the sample under vacuum, than a
surface that has been exposed to the atmosphere for an
extended time.

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 Cont…
• Five questions should be considered when designing a sampling
plan:
1. From where within the target population should samples be
collected?
2. What type of samples should be collected?
3. What is the minimum amount of sample needed for each analysis?
4. How many samples should be analyzed?
5. How can the overall variance be minimized?

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 Types of sampling
• Random Sampling: The ideal sampling plan provides an
unbiased estimate of the target population’s properties. This
requirement is satisfied if the sample is collected at random
from the target population.
• The best method for ensuring the collection of a random
sample is to divide the target population into equal units,
assign a unique number to each unit, and use a random number
table.
• In general, random sampling is a sample collected at random
from the target population.

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 Cont…
• Judgmental Sampling: The opposite of random sampling is
selective, or judgmental sampling, in which we use available
information about the target population to help select samples.
• Because assumptions about the target population are included in
the sampling plan, judgmental sampling is more biased than
random sampling; however, fewer samples are required.
• For example, a researcher studying the bioaccumulation of
polychlorinated biphenyls (PCBs) in fish may choose to exclude
fish that are too small or that appear diseased.
• Generally, judgmental sampling is samples collected from the
target population using available information about the analyte’s
distribution within the population.
• What are PCBs?
• What are POPs?

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 Cont…
• Systematic Sampling: Random sampling and judgmental
sampling represent extremes in bias and the number of
samples needed to accurately characterize the target
population.
• Systematic sampling falls in between these extremes.
• In systematic sampling the target population is sampled at
regular intervals in space or time.

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 Cont…
• Convenience Sampling: In convenience sampling, sample
sites are selected using criteria other than minimizing sampling
error and sampling variance.
• In a survey of groundwater quality, for example, samples can
be collected by drilling wells at randomly selected sites, or by
making use of existing wells.
• The latter method is usually the preferred choice. In this case,
cost, expedience, and accessibility are the primary factors used
in selecting sampling sites.
• Generally, convenience sampling is a sampling plan in which
samples are collected because they are easily obtained.

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 What Type of Sample to Collect

• After determining where to collect samples, the next step in

designing a sampling plan is to decide what type of sample to
collect.
• Three methods are commonly used to obtain samples: grab
sampling, composite sampling, and in situ sampling.
• The most common type of sample is a grab sample, in which a
portion of the target population is removed at a given time and
location in space.
• A grab sample, therefore, provides a “snapshot” of the target
population.

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 Cont…
• A composite sample consists of a set of grab samples that are
combined to form a single sample.
• After thoroughly mixing, the composite sample is analyzed.
Because information is lost when individual samples are
combined, it is normally desirable to analyze each grab sample
separately.
• One approach is to analyze a series of individual grab samples,
collected using a systematic sampling plan, and average the
results.
• Alternatively, the individual grab samples can be combined to
form a single composite sample.
• Analyzing a single composite sample instead of many
individual grab samples, provides an appreciable savings in
time and cost.
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 Cont…

• Composite sampling is also useful when a single sample

cannot supply sufficient material for an analysis. For example,
methods for determining PCBs in fish often require as much as
50 g of tissue, an amount that may be difficult to obtain from a
single fish. Tissue samples from several fish can be combined
and homogenized, and a 50-g portion of the composite sample
taken for analysis.

❖ Composite sample is several grab samples combined to form

a single sample.

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 Cont…

• In situ sampling
• A significant disadvantage of grab samples and composite
samples is the need to remove a portion of the target population
for analysis.
• As a result, neither type of sample can be used to continuously
monitor a time-dependent change in the target population.
• In situ sampling, in which an analytical sensor is placed directly
in the target population, allows continuous monitoring without
removing individual grab samples.
• For example, the pH of a solution moving through an industrial
production line can be continually monitored by immersing a pH
electrode within the solution’s flow.

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 Implementing the Sampling Plan

• Implementing a sampling plan normally involves three steps:

physically removing the sample from its target population,
preserving the sample, and preparing the sample for analysis.
• Except for in situ sampling, the analysis of a sample occurs
after removing it from the target population. Since sampling
exposes the target population to potential contamination, the
sampling device must be inert and clean.
• Once a sample is withdrawn from a target population, there is a
danger that it may undergo a chemical or physical change. This
is a serious problem since the properties of the sample will no
longer be representative of the target population.
• For this reason, samples are often preserved before transporting
them to the laboratory for analysis. Even when samples are
analyzed in the field, preservation may still be necessary.
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 Cont…
• The initial sample is called the primary, or gross sample and
may be a single increment drawn from the target population, or
a composite of several increments.
• In many cases the gross sample cannot be analyzed without
further treatment.
• Processing the gross sample may be used to reduce the
sample’s particle size, to transfer the sample into a more
readily analyzable form, or to improve its homogeneity.

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 Sample Containers
• The surface of the sample container may interact with the analyte.
For example, metals can adsorb irreversibly on glass surfaces, so
plastic containers are chosen for holding water samples to be
analyzed for their metal content. These samples are also acidified
with HNO3 to help keep the metal ions in solution.

• Organic molecules may also interact with polymeric container

materials. Plasticizers such as phthalate esters can diffuse from the
plastic into the sample, and the plastic can serve as a sorbent (or a
membrane) for the organic molecules.

• Consequently, glass containers are suitable for organic analytes.

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 Preservation of samples

• The sample must be representative of the object under

investigation.
• Physical, chemical, and biological processes may be involved in
changing the composition of a sample after it is collected.
• Physical processes that may degrade a sample are volatilization,
diffusion, and adsorption on surfaces.
• Possible chemical changes include photochemical reactions,
oxidation, and precipitation. Biological processes include
biodegradation and enzymatic reactions.
• Once again, sample degradation becomes more of an issue at low
analyte concentrations and in trace analysis.

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 Cont…

• Common steps in sample preservation are the use of proper

containers, temperature control, addition of preservatives, and the
observance of recommended sample holding time.
• The holding time depends on the analyte of interest and the sample
matrix. For example, most dissolved metals are stable for months,
whereas Cr(VI) is stable for only 24 hours.
• Holding time can be determined experimentally by making up a
spiked sample (or storing an actual sample) and analyzing it at
fixed intervals to determine when it begins to degrade.

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 SAMPLE PREPARATION TECHNIQUES FOR METALS
ANALYSIS
By Dr. Tariku Bekele
(tarchem2112@gmail.com)

12/26/2024 27
 1. SAMPLE PREPARATION?
• Sample preparation is the series of steps needed to convert a
representative bulk sample into a form suitable for chemical
analysis.

• Or sample preparation is a process required for the

transformation of a sample to make it amenable for the
chemical analysis or to improve the analysis.

• This is necessary when a given sample cannot be direct

analyzed or when directly analysis generates poor results.

• Sample preparation is usually needed to eliminate

interferences and to increase sensitivity.

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 Cont…

• Sample preparation is very often the slowest and the most

costly part of the analytical process particularly when multi-
step procedures are utilized.

• In most of the analytical procedures, sample preparation takes

50 - 75% of the total time of the analysis.

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 Figure 1: Major analytical chains within sample preparation
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• Metals contained in samples are determined by a wide variety
of analytical methods.
• FAAS
• GFAAS
• AES
• ICP-AES
• ICP-MS
• XRF
• Ion chromatography (IC)
Require the sample to be presented as a dilute aqueous
solution, usually in acid. Except XRF which analysis solid
sample.

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 Cont...

Some of the major functions of sample preparation are:

To degrade and solubilize the matrix to release all

metals for analysis.
To extract metals from the sample matrix.
To concentrate metals present at very low levels to
bring them into a concentration range suitable for
analysis.
To separate a single analyte or group of analytes from
other species.
To dilute the matrix sufficiently.
To separate different chemical forms of the analytes
for individual determination of the species present.

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 Figure 2: Plan for sample preparation for metals determination

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 2. SAMPLE PREPARATION METHODS FOR METAL ANALYSIS FROM
SOLID MATRICES

2.1 Digestion Methods

Digestion means the complete destruction of the sample

matrix.
It involve the liberation of the analyte (metal of interest)
from an interfering matrix by using:
➢ Reagent like: mineral/oxidizing acid , fusion flux and heat.

2.1.1. WET DIGESTION METHODS

•Samples to be analyzed for elemental metal content are
prepared by digesting the matrix with HNO3.

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 Cont...

• HNO3 is commonly used, because there is no chance of

forming insoluble salts as might happen with HCl or H2SO4.

• Unfortunately, the carbon contained in organic materials is

only partly converted to CO2 by HNO3 at temperatures up to
200 °C.

• HNO3 never be used for the digestion of highly aromatic

compounds because of highly explosive.

• Inorganic samples, soils, sediments, ores, rocks, and minerals

may be digested in dilute or concentrated acids or mixtures of
acids, which may be sufficient to leach out the analytes.

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 Cont...

Table 1: Reagents Commonly Used in Sample Dissolution or Digestion

12/26/2024 36
 Cont...

If total dissolution is required, HF can be used as a final digestion

step to dissolve silicates.
The acid extraction effectiveness strongly depends on:
– the chemical solubility of individual metals.
– their concentration, and the kind of matrices.

Noble metal exhibits a high resistance to single material acids.

Palladium and rhodium are the only metals attracted by hot nitric
acid and boiling sulfuric acid respectively.

Aqua-regia is used for dissolution of palladium, platinum and gold.

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Aqua regia (Latin and Ancient Italian, lit. "regal water"), aqua regis (Latin, lit. "king's water"), or nitro-
hydrochloric acid is a highly corrosive mixture of acids, a fuming yellow or red solution. HNO3+3HCl

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 2.1.2 Acid Digestion-Wet Ashing

❖ Most commonly, carried out in open glass vessels using a hot-

plate or multiple-sample digester.

• Sample is placed in an open container.

• The digestion reagent is added.
• The beaker is covered with a watch glass and placed on a hot
plate.
• The sample is allowed to boil very gently to avoid spattering.
• More solution may be added from time to time to prevent the
sample from drying out.
• H2O2 may be added at a point during the digestion to oxidize
organic materials.
• When digested completely, it is evaporated to near dryness and
taken up in a dilute acid solution and diluted to volume for
analysis.
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 Figure 3: Open digestion can be done on a hotplate in a loosely covered beaker

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 Cont...

• Multiple samples can be simultaneously digested

Figure 4: Schematic of a commercial acid digestion system

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Figure 5: The EPA procedure for the acid digestion of sediments, sludges and soils using a hot-
plate
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 Generally,

Figure 6: Acid mixtures used for digestion

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 2.1.3 Microwave Digestion

• A microwave sample digestion system consists of:

- a microwave oven
- a rotating carousel holding several sample digestion bombs
- a system for venting these in a controlled fashion
• It may also provide monitoring and recording of both temperature
and pressure in the containers.
The sample containers:
• high-temperature-resistant polymers.
- Often polycarbonate for strength or PTFE for chemical
resistance.

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• There are two types of laboratory microwave units, one uses closed
extraction vessels under elevated pressure; the other uses open
vessels under atmospheric pressure.

Figure 7: Schematic diagram of an open Figure 8: Schematic diagram of a closed-

focused-microwave system for extraction vessel microwave system for extraction

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Cont...
• Microwave energy consists of an electric field and a magnetic field,
only the electric field transfers energy to heat a substance.
• Dipole rotation and ionic conduction are the two fundamental
mechanisms for transferring energy from the electric field of the
microwave to the substance being heated.
• Dipole rotation is an interaction in which polar molecules try to align
themselves with the rapidly changing electric field of the microwave.
• Ionic conduction happens when there are free ions or ionic species
present in the substance being heated.

• The dipole rotation is related to alternative movement of polar

molecules which try to line up with the electric field.

• Multiple collisions from this agitation of molecules generate energy

release and therefore a temperature increase.
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 Figure 9: Schematic of sample heating by (A) conventional heating and
(B) microwave heating.

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 2.2. Dry Ashing

• The goal of dry ashing is the removal of organic material, leaving

behind an inorganic residue, or ash, that can be further analyzed.

• Any C present in the sample is oxidized to CO2.

• H2, S , and N are removed as H2O, SO2 and NO2.

• Crucibles used for ashing are usually made of silica, porcelain,

platinum, or Pyrex glass.

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 ✓ The major drawbacks of the method are :
• possible loss of some elements by volatilization.
• contamination of the sample by airborne dust.

✓ Losses from volatilization of the analyte can be

minimized by restricting the temperature at which
ashing takes place.
✓ Suitable for nutritional elements in foods, such as Fe,
K, Ca, Mg, and Mn.

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 2.3 Extraction with Supercritical Fluids

✓ A pure supercritical fluid is a substance above its critical

temperature and pressure.
✓ Above its critical temperature, it does not condense or
evaporate to form a liquid or a gas but is a fluid with
properties changing continuously from gas-like to liquid-like
as the pressure increases.
✓ Above the critical temperature Tc and pressure Pc, increasing
the pressure increases the density and thus the solvating power
of the fluid.
✓ Supercritical fluids can replace liquid solvents in many
processes such as extractions from solids. E.g. CO2,N2O,H2O

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 Figure 10 : T/P Phase Diagram

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 Figure 11: Schematic of the layout of a typical supercritical fluid extraction system

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 Cont…

– SFE are effective cleaning agents.

– Have ability to rapidly penetrate substrates and small
interstitial spaces.
Table 2.: Critical properties of some selected materials

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 2.4 Organic Extraction of Metals
➢ The formation of metal chelates is the most common extraction
technique for metals.
➢ A complex formed between a metal and a chelating agent is
hydrophobic in nature and soluble in organic solvents.
➢ The most common chelating agents used to extract metals from
water samples are ammonium pyrolidine dithiocarbamate
(APDC) and 8- hydroxyquinone.
➢ Methyl isobutyl ketone (MIBK) is generally used as a solvent.
➢ In a typical extraction, 1 mL of APDC is added to 50 to 100 mL
of aqueous sample in a volumetric flask.

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 Cont…

➢ Then, 10 mL of MIBK is added and the mixture is vigorously

shaken for 30 seconds.

➢ The metal chelate partitions into the organic phase, which

floats on the water.

➢ More water can be added to raise the organic level into the
neck of the flask so that it can be aspirated directly into the
analytical instrument.

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 Figure 12: Structures of some commonly used chelating agents

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 CONCLUSION
❖ Sample preparation is a process required for the transformation
of a sample to make it amenable for the chemical analysis or to
improve the analysis.
❖ Sample preparation may be done on the analytes, on the
matrix, or on both to perform dissolution, cleanup,
concentration, or chemical modifications of the sample for
obtaining better results.
❖ The Common methods of preparing a sample for metal
analysis are digestion methods such as wet digestion such as
acid digestion-wet ashing, and dry-ashing, microwave
digestion, organic extraction, extraction with supercritical
fluids, ultrasonic sample preparation.
❖ Metals contained in samples are determined by AAS, AES,
ICP-MS, XRF, EDTA, and GRAVIMETRY.
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 SAMPLE PREPARATION TECHNIQUES
FOR ORGANIC COMPOUNDS ANALYSIS
by Dr. Tariku Bekele
(tarchem2112@gmail.com)

12/26/2024 58
 Sample preparation techniques for solid matrices

Classical techniques
Shaking Soxhlet Sonication
1879 1960

SFE MAE PLE

1976 1986 1995

Recent techniques

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 Table 1: Methods Accepted as Standards for the Extraction of Semi-volatile Organics
from Solid Matrices

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 Cont…
• Classical methods include Solvent-shake extraction, Soxhlet
extraction, automated Soxhlet extraction, and ultrasonic
extraction.

• Operated under atmospheric pressure, with heating or

ultrasonic irradiation. These methods consume relatively large
volumes of organic solvents, and the extraction may take a
long time.

• Other group consists of SFE, ASE, and MAE, which are

performed under elevated pressure and/or temperature.

• The extraction is faster, efficient, relatively less consumption

of organic solvents and environmentally friendly.

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Classical Sample Preparation Methods for Organic analysis
Solvent – shake extraction techniques
• Conventional liquid-solid extraction, in the form of shake-
flask extraction is the simplest extraction technique.
• It is performed by mixing the sample with a suitable solvent,
and then agitating or shaking at ambient temperature for a pre-
specified time-period.
• After extraction, the solvent containing the analytes need to be
separated from the matrix by means of centrifugation and /or
filtrations.
• Extract clean-up, concentration and re-constituting in an
appropriate solvent compatible with final analysis, are the last
step in the procedure.

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Soxhlet Extraction technique (SOX)
• Soxhlet extraction apparatus was designed by a German
agricultural chemist Franz von Soxhlet in 1879 for the
extraction of fat from dried milk.
• Soxhlet extraction is used as the benchmark against which any
new extraction technique is compared.
• The basic Soxhlet extraction apparatus consists of a Solvent
reservoir, an extraction body, a heat source (e.g. an isomantle)
and a water-cooled reflux condenser.
• A Soxhlet uses a range of organic solvents to remove organic
compounds, primarily from solid matrices.

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 Cont…

• The apparatus is then fitted to a round-bottomed flask of

appropriate volume containing the organic solvent of
choice, and to a reflux Condenser.
• The solvent is then boiled gently using an isomantle – the
solvent vapour passes up through the tube marked (A), is
condensed by the reflux condenser, and the condensed
solvent falls into the thimble and slowly fills the body of
the Soxhlet apparatus.
• When the solvent reaches the top of the tube (B), it
siphons over into the round-bottomed flask the organic
solvent containing the analyte extracted from the sample
in the thimble. The solvent is then said to have completed
one cycle.

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 Figure (2): The basic Soxhlet extraction system
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 Cont…
• Since the extracted analytes have higher boiling points than the
extraction solvent, they accumulate in the flask while the
solvent recirculates.
• Consequently, the sample is always extracted with fresh
solvents in each cycle.
• Because the sample is extracted with cooled and condensed
solvents, Soxhlet is slow and can take between 6 to 48 hours
and large solvent consumption.
• The extract volume is relatively large, so a solvent evaporation
step is usually needed to concentrate the analytes prior to
extract cleanup and analysis.

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Ultrasonication extraction technique (USE)
➢ Sonication involves the use of sound waves to agitate a sample
immersed in an organic solvent.

➢ The sample is placed in a suitable glass container and enough

organic solvent is then added to cover the sample.

➢ The system is then sonicated for a short time-period, typically

3 min, using the sonic bath or probe.

➢ After extraction, the solvent containing the analyte is separated

by filtration.
➢ The whole process is repeated three times and all of the
solvent extracts are then combined for analytical step.
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➢The sample is placed in a suitable glass
container

➢The system is then sonicated

for a short time-period, typically
3 min, using the sonic bath or
probe.

➢It is relatively fast, but the

extraction efficiency is not as high as
the Other techniques and ultrasonic
Anhydrous
irradiation may decompose some sodium sulfate
compounds (organophosphorus)
Figure (3): The Ultrasonic extraction

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Solid phase extraction (SPE)
SPE is an extraction process whereby an aqueous sample is filtered
through a thin bed of sorbent particles, the analytes of interest are
removed from the liquid matrix, and concentrated onto the sorbent.
Once concentrated, the analytes are removed by an eluting solvent.
Solid-phase extraction refers non-quilibrium, exhaustive removal of
chemical constituents from a flowing liquid sample via retention on
a contained solid sorbent and subsequent recovery of selected
constituents by elution from the sorbent.

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 Cont…
Steps in SPE

Figure (4): Solid phase extraction

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 Cont…
• Generally, SPE consists of four steps (Figure 4 above): column
preparation, or prewash, sample loading (retention or
sorption), column postwash, and sample desorption (elution or
desorption), although some of the recent advances in sorbent
technology reduce or eliminate column preparation
procedures.
• The prewash step is used to condition the stationary phase if
necessary, and the optional column postwash is used to remove
undesirable contaminants.
• Usually, the compounds of interest are retained on the sorbent
while interferences are washed away.
• Analytes are recovered via an elution solvent.

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 Table (2) : SPE Sorbent–Analyte Interaction Mechanisms

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 Two common designs of SPE device; cartridge and disk formats

(A) cartridge (B) disk formats

Figure (5): SPE device

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 Modern Sample Preparation Methods for Organic analysis

Supercritical Fluid Extraction

• The term supercritical fluid is used to describe any substance
above its critical temperature and pressure.
• When a substance is above the critical point , it exists in a
single phase called supercritical phase , which is neither a
liquid nor a gas.
• The densities of supercritical fluids are close to that of a liquid,
whereas their viscosities are gas like. The diffusion
coefficients are in between.
• Due to these unique properties, supercritical fluids have good
solvating power (like liquid), high diffusivity (better than
liquid), low viscosity, and minimal surface tension (like gas).
• With rapid mass transfer in the supercritical phase and with
better ability to penetrate the pores in a matrix, extraction is
fast in SFE, along with high extraction efficiency.
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 SFE
properties intermediate between gas and liquid

Low viscosity implies

•Higher mass transfer
•Fast penetration of the matrix
•Wetting of the sample matrix

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 Cont..
• The density of a supercritical fluid is relatively close to that of
a liquid and can easily be adjusted by simply changing the
pressure.
• Especially at temperature close to Tc a small increase in
pressure leads to dramatic density increases.
• This great compressibility of supercritical fluids allows the
solvent strength, which is related to density, to be easily
adjusted.
• This is in contrast to liquid extraction where the solvent
polarity is modified by changing the extraction liquid.
• Supercritical fluid extraction is thus the technique that
employs this fluid phase to effect solubilization of solutes.

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 Steps in SFE
1. Sample is placed in cell
2. Supercritical CO2 is passed through the cell , solvating the
analytes.
3. After the restrictor , pressure is decreased . The CO2 becomes
a gas and is removed, while the analytes stay on the trap.
4. The trap is rinsed with organic solvent to collection vial(s).
5. Trap is rinsed to waste to remove interferents that were co-
extracted.

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 Instrumentation
An SFE system consists of six basic components, namely:
➢a supply of high-purity carbon dioxide (CO2),
➢a supply of high-purity organic modifier,
➢two pumps,
➢ the oven for the extraction cell,
➢ the pressure outlet or restrictor, and
➢ the collection vessel.

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• A number of compounds are such as CO2, N2O, NH3, SF6,
Chloroflurocarbon (freons), H2O etc have been used as super critical
fluid in SFE.
• Most of them have some kind of limitations that includes: being
reactive, toxic, explosive, high critical parameters and ozone
depletion.
• The most frequently used extraction fluid is CO2. More than 90% of
analytical applications available today have been utilizing this
substance.
• This is due to its low cost, nontoxic, non-flammability, low TC
(31oC) and PC (74.8atm) and low reactivity.
• Therefore, CO2 has become the solvent of choice for most SFE
applications. Being nonpolar and without permanent dipole moment,
supercritical CO2 is a good solvent for the extraction of nonpolar
and moderately polar compounds.
• However, its solvating power for polar solutes is rather poor.
Moreover, when the solutes bind strongly to the matrix, the solvent
strength of CO2 is often inadequate to break the solute–matrix bond.
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 Table 1. Critical properties of some selected materials

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 Supercritical solvents such as N2O and CHClF2 are more efficient
in extracting polar compounds, but their routine use is uncommon
due to environmental concerns. The extraction efficiency of polar
compounds by CO2 can be improved by the addition of small
quantities (1 to 10%) of polar organic solvents, referred to as
modifiers. This is a common practice in SFE. Table 3.4 lists some
common modifiers for supercritical CO2.

Table 2. Commonly Used Modifiers for Supercritical CO2

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 Advantages/Disadvantages and Applications of SFE
• SFE is fast (10 to 60 minutes) and uses minimum amount of
solvents (5 to 10 mL) per sample.
• CO2 is nontoxic, nonflammable, and environmentally friendly.
• Selective extraction of different groups of analytes can be
achieved by tuning the strength of the supercritical fluids with
different modifiers and by altering operating conditions.
• In addition, the extract from SFE does not need additional
filtration, as the extraction cell has frits.
• On the down side, analytical-scale SFE has limited sample size
(<10 g),and the instrument is rather expensive.
• Furthermore, SFE has been found to be matrix dependent.
• Different methods have to be developed and validated for
different sample matrices and for different groups of analytes.
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 Applications of SFE

• SFE has a wide range of applications, which include the

extraction of PAHs, PCBs, phenols, pesticides, herbicides, and
hydrocarbons from environmental samples, contaminants from
foods and feeds, and active gradients from cosmetics and
pharmaceutical products.
• Table 3. lists some examples from the literature.

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 Table 3. Selected SFE Applications

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 Cont…
Accelerated solvent extraction
• Accelerated solvent extraction (ASE) is also known as
pressurized fluid extraction (PFE) or pressurized liquid
extraction (PLE).
• It uses conventional solvents at elevated temperatures (100 to
180oC) and pressures (1500 to 2000 psi) to enhance the
extraction of organic analytes from solids.
• ASE was introduced by Dionex Corp. (Sunnyvale, CA) in
1995.
• It evolved as a consequence of many years of research on SFE.
• SFE is matrix dependent and often requires the addition of
organic modifiers.
• ASE was developed to overcome these limitations.

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 The basic principle of PLE
• The elevated pressure and temperature used in ASE affects the
solvent, the sample, and their interactions.
• The solvent boiling point is increased under high pressure, so
the extraction can be conducted at higher temperatures.
• The high pressure also allows the solvent to penetrate deeper
into the sample matrix, thus facilitating the extraction of
analytes trapped in matrix pores.
• At elevated temperatures, analyte solubility increases and the
mass transfer is faster.

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 Higher analyte solubility
Temperature Helps overcome matrix effect
Viscosity of solvent is lowered
Diffuses in to matrix more quickly

Keeps solvent liquid at higher temperature

Pressure To force the solvent into the pores of the matrix

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 Instrumentation
• ASE system consists of solvent tank(s), a solvent pump, an
extraction cell, a heating oven, a collection vial, and a nitrogen
tank.
• The sample size can be anywhere between 1 and 100 mL.

• The ASE system can sequentially extract up to 24 samples in

one unattended operation.

• The sequence of introducing and removing the cells to and

from the oven can be automated.

• Extract filtration is not required, but concentration and/or

cleanup is often necessary prior to analysis.

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 Figure 2. Schematic diagram of an ASE system

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 Advantages and Applications of ASE

• ASE has many advantages. It uses minimal amount of solvent

and is fast (about 15 minutes), fully automated, and easy to
use.
• Filtration is a built-in step, so additional filtration is not
needed.
• While operating at higher temperatures and pressures, ASE
can employ the same solvent specified by other existing
methods. Therefore, method development is simple.

• There are more solvents to choose from, because solvents that

work poorly in conventional methods may perform well under
ASE conditions.

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 Cont…

• In principle, ASE is a universal method that can be used in any

solvent extraction.
• However, majority applications so far have been in the
environmental area, such as the extraction of pesticides,
herbicides, PAHs, PCBs, base/neutral/acid compounds,
dioxins, furans, and total petroleum hydrocarbons.
• ASE has also been used to extract additives and plasticizers
from polymers, additives, and active ingredients from
pharmaceuticals, and contaminants/fat from food.
• Limitation:-Pressurised solvent extraction uses much less
solvent, but the equipment is costly.

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 Table 4. Selected ASE Applications

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 Cont…
Microwave-assisted extraction
➢ MAE utilizes organic solvent and microwave heat to extract
organic pollutants from solid matrices.
• The principle of heating using microwave energy is based on
the direct effect of microwaves on molecules by ionic
conduction and dipole rotation. In many applications these two
mechanisms take place simultaneously.
• The mechanism of microwave heating is different from that of
conventional heating.
• In conventional heating, thermal energy is transferred from the
source to the object through conduction and convection.
• In microwave heating, electromagnetic energy is transformed
into heat through ionic conduction and dipole rotation.

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 Cont…
➢ Ionic conduction refers to the movement of ions in a
solution under an electromagnetic field. The friction
between the solution and the ions generates heat.
➢ Dipole rotation is the reorientation of dipoles under
microwave radiation.
➢ A polarized molecule rotates to align itself with the
electromagnetic field at a rate of 4.9 x 109 times per
second.
• The larger the dipole moment of a molecule, the more
vigorous is the oscillation in the microwave field.

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 Advantages and disadvantages of MAE

➢ Reduction of
✓ extraction time (usually < 20 min)
✓ solvent volume
➢ Improved extraction yield (for selected matrix and
components).
• However Less efficient for thermolabile compounds.

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 Instrumentation
➢ There are two types of laboratory microwave units.

➢ One uses closed extraction vessels under elevated pressure;

➢ The other uses open vessels under atmospheric pressure.

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 Closed-Vessel Microwave Extraction Systems

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➢ Inthe oven cavity is a carousel (turntable or rotor) that can
hold multiple extraction vessels.

➢The carousel rotates 3600C during extraction so that

multiple samples can be processed simultaneously.

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 Applications of MAE
• Majority MAE applications have been in the extraction of
PAHs, PCBs, pesticides, phenols, and total petroleum
hydrocarbons (TPHs) from environmental samples.

• MAE has also been used in the extraction of contaminants and

nutrients from foodstuffs, active gradients from
pharmaceutical products, and organic additives from
polymer/plastics.

• Limitations:- Solvents used in Soxhlet extraction cannot

readily be applied to microwave extraction because some of
them do not absorb microwave.

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 CONCLUSION
❖ Sample preparation is the series of steps needed to
convert a representative bulk sample into a form suitable
for chemical analysis.
❖ Sample preparation may be done on the analytes, on the
matrix, or on both to perform dissolution, cleanup,
concentration, or chemical modifications of the sample
for obtaining better results.
❖ The Common methods of preparing a sample for organic
analysis are: Solvent-shake extraction, SPE, Soxhlet
extraction, ultrasonic extraction, SFE, ASE, and MAE.
❖ Analytes contained in organic samples are determined by
GC-ECD, GC-NPD, GC-MS, GC-TCD, LC-MS, HPLC-
MS and Electrophoresis.

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 Sample-extract clean up and pre-
concentration

By
Dr. Tariku Bekele
(tarchem2112@gmail.com)

12/26/2024 101
 1. Sample Cleanup
• Because of the complexity of samples and the low selectivity
of exhaustive extraction techniques applied, substantial
amounts of interfering substances are found in crude extracts,
and subsequent clean-up and fractionation is indispensable.
• Sample cleanup is particularly important for analytical
separations such as GC, HPLC, and electrophoresis.
• Many solid matrices, such as soil, can contain hundreds of
compounds.
• These produce complex chromatograms, where the
identification of analytes of interest becomes difficult.
• This is especially true if the analyte is present at a much lower
concentration than the interfering species.
• So a cleanup step is necessary prior to the analytical
measurements.
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 Cont…

• Another important issue is the removal of high-boiling

materials that can cause a variety of problems.

• These include analyte adsorption in the injection port or in

front of a GC-HPLC column, false positives from
interferences that fall within the retention window of the
analyte, and false negatives because of a shift in the retention
time window.

• In extreme cases, instrument shut down may be necessary due

to the accumulation of interfacing species.

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 Cont…

• Complex matrices such as, soil, biological materials, and

natural products often require some degree of cleanup.

• Highly contaminated extracts (e.g., soil containing oil

residuals) may require multiple cleanup steps.

• On the other hand, drinking water samples are relatively

cleaner (as many large molecules either precipitate out or do
not dissolve in it) and may not require Cleanup.

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The following techniques are used for cleanup and purification of
extracts:
1. Gel-Permeation Chromatography
• Gel-permeation chromatography (GPC) is a size-exclusion
method that uses organic solvents (or buffers) and porous gels
for the separation of macromolecules.
• The packing gel is characterized by pore size and exclusion
range, which must be larger than the analytes of interest.
• GPC is recommended for the elimination of lipids, proteins,
polymers, copolymers, natural resins, cellular components,
viruses, steroids, and dispersed high-molecular-weight
compounds from the sample.
• This method is appropriate for both polar and nonpolar
analytes.

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 Cont…

• Therefore, it is used for extracts containing a broad range of

analytes.
• Usually, GPC is most efficient for removing high-boiling
materials that condense in the injection port of a GC or the
front of the GC column.

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2. Acid–Base Partition Cleanup

• Acid–base partition cleanup is a liquid–liquid extraction

procedure for the separation of acid analytes, such as organic
acids and phenols from base/ neutral analytes (amines,
aromatic hydrocarbons, halogenated organic compounds)
using pH adjustment.
• This method is used for the cleanup of petroleum waste prior
to analysis or further cleanup.
• The extract from the prior solvent extraction is shaken with
water that is strongly basic.
• The basic and neutral components stay in the organic solvent,
whereas the acid analytes partition into the aqueous phase.

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 Cont…
• The organic phase is concentrated and is ready for further
cleanup or analysis.
• The aqueous phase is acidified and extracted with an organic
solvent, which is then concentrated (if needed) and is ready for
analysis of the acid analytes (Figure 1.).

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 Figure 1. Acid–base partition cleanup

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3. Solid-Phase Extraction and Column Chromatography
• The solvent extracts can be cleaned up by traditional column
chromatography or by solid-phase extraction cartridges.

• This is a common cleanup method that is widely used in

biological, clinical, and environmental sample preparation.

• It is usually used to clean up a sample before using a

chromatographic or other analytical method to quantify the
amount of analyte(s) in the sample.

• The general procedure is to load a solution onto the SPE

phase, wash away undesired components, and then wash off
the desired analytes with another solvent into a collection tube.

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 Cont…
• Some examples include the cleanup of pesticide residues and
chlorinated hydrocarbons, the separation of nitrogen
compounds from hydrocarbons, the separation of aromatic
compounds from an aliphatic–aromatic mixture, and similar
applications for use with fats, oils, and waxes.
• This approach provides efficient cleanup of steroids, esters,
ketones, glycerides, alkaloids, and carbohydrates as well.
Cations, anions, metals, and inorganic compounds are also
candidates for this method .
• The column is packed with the required amount of a sorbent
and loaded with the sample extract.
• Elution of the analytes is effected with a suitable solvent,
leaving the interfering compounds on the column.

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 Cont…
• The packing material may be an inorganic substance such as
Florisil (basic magnesium silicate) or one of many
commercially available SPE stationary phases.
• The eluate may be further concentrated if necessary.
• A Florisil column is shown in Figure 2.
• Anhydrous sodium sulfate is used to dry the sample.
• These cleanup and concentration techniques may be used
individually, or in various combinations, depending on the
nature of the extract and the analytical method used.

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 Figure 2. Column chromatography for sample cleanup
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 2. Pre-Concentration Using Solvent Evaporation
• Pre-concentration is concerned with the reduction of a larger
sample into a smaller sample size.
• It is most commonly carried out by using solvent evaporation
procedures after an extraction technique.
• The most common approaches for solvent evaporation are
rotary evaporation, Kuderna–Danish evaporative concentration,
the automated evaporative concentration system (EVACS) or
gas ‘blow-down’.
• In all cases, the evaporation method is slow, with a high risk of
contamination from the solvent, glassware and blow-down gas.

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2.1 Rotary Evaporation
• In this method, the solvent is removed under reduced pressure
by mechanically rotating the flask containing the sample in a
controlled-temperature water bath (Figure 3.).

• The (waste) solvent is condensed and collected for disposal.

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 Figure 3. A typical rotary evaporation system used for the pre-concentration
of compounds in organic solvents

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 Summary
❖ In order to achieve the lowest levels of any analyte in the
environment, we require the use of appropriate samples in
which the analyte is homogeneously distributed.
❖ Then, by careful sample pre-treatment and selection of the
most appropriate extraction/digestion procedures, we can
then go ahead and carry out the final analysis.
❖ Now, assuming that we have taken due regard to minimize
the risk of contamination, we should then expect the
analytical instrument to record an appropriate signal
(response). However, this may not always be the case.
❖ Often, the concentration levels of potential environmental
contaminants are so low that an extra step is required.
❖ This chapter has introduced the approaches available to
sample cleanup and pre-concentrate such samples prior to
analysis.

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