CHROMATOGRAPHY

● Roll no 121 to 135

Specific Learning Objectives
📌 Chromatography introduction , principal and classification
📌 Paper chromatography
📌Thin layer chromatography
📌Gas liquid chromatography
📌 Adsorption chromatography
📌Gel filtration chromatography
📌High pressure liquid chromatography
📌Ion exchange chromatography
📌 Affinity chromatography
 Introduction
● Chromatography is analytical techhnique dealing
with the separation of closely
related compounds from a mixture

● These include proteins, peptides, amino acids,

lipids, carbohydrates, vitamins
and drugs.

It is based on differential distribution between two immiscible phases

 Historical perspective of chromatography

Chromatography (Greek). Chroma--colour

graphein-to write

● Russian botanist Mikhail Tswett described

the separation of plant leaf pigments in solution
by passing through a column of solid adsorbent in 1906

● Actually, chromatography is a misnomer, since it

is no longer limited to the separation of coloured compounds
 Definition

☆It is an analytical technique dealing with the separation of

closely related compound from mixture.

OR

☆ It is a technique or procedure for separation of similar

compounds by its continuous redistribution between 2 phases
i.e. Mobile phase & stationary phase.
Mobile phase:-
It refers to mixture of substances (to be seperated)
dissolved in liquid or gas

Stationary Phase;-
Porous solid matrix through which sample contained in a
mobile phase percolates
Eg.- Column chromatography
Stationary phase;-Silica (adsorbent material)
Mobile phase:-Solvent System
#Interaction between these 2 phases result in separation
of the compound from the mixture.
Classification of Chromatography
 Quiz Time

1) In chromatography, the stationary phase can be__

a. Solid or liquid
b. liquid or gas
c.Solid only
d. liquid only
.

2) In chromatography, which of the following can the

mobile phase be made of ?
a. Solid or liquid
b. Liquid or gas
c. Gas only
d. Liquid only
.

3)The basis of the technique of chromatography for

separating components of a mixture is ?
a. The differing movement of particles of different
mass in an electrical field
b. The interaction of the components with a
stationary and a mobile phases
c. The absorption of infrared radiation by the
cmponents
d. The deflection of charged particles in a magnetic
field
 PRINCIPLES OF
CHROMATOGRAPHY
ADSORPTION

● Analytical seperation of a chemical mixture

based on the interaction of the adsorbate
with the adsorbent
● Less soluble and strongly adsorbed
Component :- retained on the support
● More soluble and less adsorbed
Component :- flow out faster
 2. Partition

Molecules of a mixture gets partitioned

between the stationary phase and
mobile phase depending on their
relative affinity to each one of the phases.
Paper chromatography:-

Seperation of amino acids , sugars ,

sugar derivatives and peptides.
.
Rf value
= Distance travelled by the substance
Distance travelled by solvent front
 3. Gel filtration ( molecular sieving )
● Seperation of solute on the basis of molecular size
● Smaller particles pass into the pores and come out later
● Bigger particle pass between the beads and come out earlier
● Example of gel used is sephadex, agarose
 4. Ion exchange
Seperation of charged solutes
Two types of ion exchange resins -
1. Cation exchange resins
● Contains negatively charged ions
● Retain positively charged ions
● Example :-Dowax50 b
2. Anion exchange resins
● Contains positively charged ions
● Retain positively charged ions
● Example :-cellulose
 5. Affinity chromatography

based on the property of specific and non covalent bonding of proteins to other
molecules referred to as ligands

Affinity chromatography is useful for the purification of : -

● Enzymes
● Vitamins
● Nucleic acids
● Drugs
● Antibodies
● Hormone receptors
MCQ :-

1. In gel filtration chromatography, the seperation of molecules is based on

a) Electrical charges
b) Size, shape and molecular weight
c) Specific and non - covalent binding

2. Affinity chromatography is useful for the purification of

a) Enzymes , vitamins, nucleic acids

b) Drugs, hormone receptors, antibodies
c) Amino acids, sugars, sugar derivatives and peptides
d) Both a and b
PAPER CHROMATOGRAPHY

SLOs
1.Principle
2.Types
3.Procedure
4.Applications

Roll no.124
 PAPER CHROMATOGRAPHY

PRINCIPLE : Partition chromatography

Stationary phase - Water on filter paper (cellulose)
Mobile phase - mixture of immisicible solvents (organic)
e.g. butanol-acetic acid-water
Phenol-water-ammonia
Wait ✋
Solvent front -the
line indicating
TYPES : 1.Ascending
distance travelled
2.Descending
by the solvent
3.Radial
4. Two dimensional Roll no.124
1. ASCENDING PAPER CHROMATOGRAPHY

The solvet system will move in upward direction , Better resolution

As the solvent moves it takes along with it the unknown substance

Roll no.124
2.DESCENDING PAPER CHROMATOGRAPHY

Solvent system will move in downward direction

Requires less time

Roll no.124
3. RADIAL PAPER CHROMATOGRAPHY

Solvent system moves radiallly

Roll no.124
4.Two Dimensional Paper Chromatography
The mobile Phase moves in both the dimensions
The solvent System is run and then rotated by 90°

Better separation of components of sampe occurs

Roll no.124
Procedure for paper chromatography

1) Cut the whatmans filter paper

into rectangular shape

2) Prepare 10 ML of mobile phase

by mixing butanol, acetic acid
and distilled water ( 4 : 1: 5)

Roll no. 23125

3) prepare 3 sample solutions of ammino acids of 10 ML each
Solution A :- standard solution of leucine
Solution B :- standard solution of valine
Solution C :- sample mixture of solution A and B
4) Draw a starting line about 2 cm
from the bottom of filter paper by
pencil and mark 3 points on it

Also draw a end line at the top of

filter paper

5) Transfer the small volume of

solution A at mark 1 , similarly
solution B at mark 2
solution C at mark 3
6) Immerse the filter paper vertically in
the center of beaker

7) Allow the mobile phase to move over

the paper till end line

When the solvent touches end line , take

out the paper and mark the solvent
front
8) spray the filter paper with
ninhydrin reagent and allow it to
dry in hot air oven at 100 ‘C
9) outline each spot of ammino
acid and mark the center of each
spot
Measure the distance of each spot
from the starting line also ,
Measure and the distance
traveled by solvent from starting
line
calculate the Rf value
Advantages of paper
chromatography
● Simple

● Economical

● Wide range of use

 Applications
● Separation of Biochemicals ● Study of Environmental Samples

● Analysis of Inks ● Teaching Tool

● Analysis of Plant Pigments ● Food Products Analysis

● Clinical Diagnostics

● Quality Control in Pharmaceuticals

 THIN LAYER
CHROMATOGRAPHY
Defination: Thin layer chromatography is a technique
used to separate non volatile compounds on a thin layer
of adsorbent material
 PRINCIPLE OF TLC
It depends on principle of : SEPERATION
: ADSORPTION
● The separation relies on affinity of compound towards both the
phases
● The compound in mobile phase move over surface of stationary
phase
● Compound having higher affinity to stationary phase move slowly
 REQUIREMENTS FOR PROCEDURE

● Thin layer chromatography Plate

● Thin layer chromatography Chamber
● Thin layer chromatography Mobile phase
● Thin layer chromatography Filter paper
*Stationary phase: Aluminium oxide
: Cellulose
: Silica gel
● Mobile phase. : Solvent mixture
 PROCEDURE
Let stationary phase get dry and settle on plate
⬇
Make marked spots and apply sample solution to these spots
⬇
Pour mobile phase in chamber and place moistened filter in mobile phase
⬇
Place plate in TLC chamber and close lead
⬇
Spots are developed
⭐Note : Keep sample spots well above the mobile phase
⭐If the spots are not observed directly they can be
observed by following method
 ADVANTAGES OF TLC
• This is a very easy way to separate the components.

• It is feasible to visualize all components of UV light.

•The non-volatile compounds are separated by the TCL method.

• In comparison to other separation techniques, very few types of

equipment are used. The components are separated in a very short time.

•The only small sample size is required in TLC, and it can be in microlitre.

•The components there in the complex mixture of samples are able to

easily separate recovered by scratching plate.
APPLICATION OF TLC
Test the purity of the sample: Thin layer chromatography helps to
detect the purity of the sample by direct comparison with the standard
sample. Any impurity in the sample shows up as extra spots in
chromatography.

➤Identify the components: Thin layer chromatography can purify,

isolate and identify the natural products like essential oil, waxes,
terpenes, alkaloids, glycosides, steriods etc. in the test samples.
➤Biochemical analysis: Biochemical metabolites from the body fluids, blood
plasma, serum, urine etc. can be isolated using thin layer chromatography.

➤In chemistry, TLC is used to separate and identify closely related

compounds or cations and anions in inorganic chemistry.

Pharmaceutical industries utilize TLC technique for qualitative analysis or

detect impurities in various medicines like hypnotics, sedatives, tranquillisers,
anti-histaminics, analgesics, local anaesthetics, steroids, etc.

In food and cosmetic industry: Any artificial colour, preservatives, sweetening

agent, and other impurities in food and cosmetic products can be detected
and isolated by TLC technique.
 GAS-LIQUID
CHROMATOGRAPHY
✒ Definition: Method for the seperation
of volatile substances or the volatile
derivatives of certain non-volatile
substances.
✒ Stationary phase : Inert solid material
(diatomaceous earth or powdered firebrick)
impregnated with a non-volatile liquid (silicon or
polyethylene glycol)
✒ Mobile phase : Inert gas (argon, helium or
nitrogen)
Principle
partition of the components between mobile
phase (gas) and stationary phase (liquid), hence
called gas-liquid chromatography
✒The seperated compounds then identified
and quantitated by detector
✒The detector works on the principle of
ionization or thermal conductivity
 Procedure
✒Stationary phase is packed in a
narrow column ➡ maintained at
high temperature (around 200°C)
➡ mixture of volatile material is
injected along with mobile phase
✒It is sensitive, rapid and reliable.

Uses
✒Frequently used for quantitive estimation
of biological materials such as lipids, drugs
and vitamins.
Adsorption chromatography
➡separation technique based on diffrential adsorption
of components In sample onto a stationary phase
➡principle :-affinity of different components for a solid
stationary phase
➡stationary phase :-is solid material with high surface
area such as silica gel alumina
- is packed into column
-contains various functional groups that interact with
Various components in sample
➡Mobile phase:- it is a solvent or solvent mixture that carries
the sample

- it flows over the stationary phase

➡ Adsorption process:-

– Sample passes through column

–components with high adsorbing power will

adsorb more strongly and elute later

–this differential adsorption leads to separation of

components

➡Elution :- refers to process of washing the components off the

column
Detection :-various detection methods can be used to monitor the Elution

of component from the column ,such as UV spectroscopy,

fluorescence spectroscopy or mass spectroscopy

Applications :- it is widely used in various fields ,

particularly useful for separating compound

that have similar properties or polarities

Types:-there are different types of Adsorption chromatography

including column chromatography,thin layer chromatography,

paper chromatography .
 GEL FILTRATION CHROMATOGRAPHY

Also known as ➡ Size exclusion chromatography

➡ Molecular sieving chromatography
➡ Molecular exclusion chromatography
 What is gel filtration chromatography ?

It is a chromatographic method in which

molecules in a solution are separated on the basis
of their SHAPE, SIZE and MOLECULAR WEIGHT
The apparatus used to do filtration consists of two
phases:
⭐Stationary phase: Porous matrix (gel bed)
➡ Acryl amide (sephacryl)
➡Agarose(sepharose)
➡Dextran (sephadex)
⭐Mobile phase. :Buffer that flow between
matrix bed
 PRINCIPLE
* Smaller molecules and complexes that are able to move into
the pores enter the stationary phase and move through gel
filtration column by longer path through pores of the beads
* Molecules that are too large to enter the pores stay in
mobile phase and move through the column with flow of
buffer
HOW IT WORKS
 PROCEDURE
Solution mixture added to column
⬇
Washed with buffer
↙ ↘
Small molecules Large molecules
● Enter gel * Move in column
● Move slowly. * Move fast
Thus molecules of different sizes can be separated
USE OF GEL FILTRATION CROMATOGRAPHY

Gel filtration chromatography can be use

for approximate determination of
molecular weight
QUESTION TIME
1) Gel filtration chromatography is also known as all given
below except
a)size exclusion chromatography
b)shape exclusion chromatography
c) molecular sieving chromatography
d) molecular exclusion chromatography
2) Which of the following is not used as a stationary phase
in gel filtration chromatography?
a)acryl amide
b)agarose
c)water
d)dextran
 HIGH PRESSURE LIQUID
CHROMATOGRAPHY (HPLC)
Introduction:-
High Pressure Liquid Chromatography (HPLC) is a
technique widely used for separating, identifying, and
quantifying chemical compounds. It operates on the
principle of column chromatography, but with
significantly improved efficiency and speed due to the
application of high pressure. The precise control of
mobile phase flow rates and pressure allows for fast
and accurate analysis of complex mixtures.
 PRINCIPLE OF HPLC

Sample Injection Mobile Phase Selection Separation Mechanism

The process begins The selection of an
Compounds interact
with the injection of the appropriate mobile
differently with the
sample into the HPLC phase is crucial in
stationary phase,
system. The sample HPLC. It can be either
leading to separation
may be dissolved in a a liquid or a
based on their
solvent and then supercritical fluid and
physicochemical
introduced into the plays a vital role in
properties such as
column to initiate the the separation of
polarity, size, and
separation process. compounds based on
charge.
their affinity to the
stationary phase.
 COMPONENTS OF HPLC
1 Pump System 2 Column 3 Detector

The pump system is The column is the heart The detector identifies
responsible for of the HPLC system, and quantifies the
generating and where the separation of separated compounds
maintaining the high compounds takes place using various
pressure required for based on their techniques such as
efficient separation, interactions with the UV-spectroscopy,
stationary phase. A fluorescence, or mass
and for delivering the
variety of columns are spectrometry,
mobile phase at a
available for different providing valuable
controlled flow rate.
separation needs. analytical data.
TYPES OF STATIONARY PHASES USED IN
HPLC
 DETECTION METHODS IN HPLC
1) UV-Visible Spectroscopy
UV-Vis detection is widely employed due to its sensitivity and ability to quantify low concentrations
of compounds based on their absorbance of UV or visible light.

2) Fluorescence
Fluorescence detection is utilized for compounds that exhibit fluorescence, offering high sensitivity
and selectivity for targeted compounds.

3) Mass Spectrometry
Mass spectrometry provides accurate identification and quantification of analytes and is essential for
the analysis of complex samples
 APPLICATIONS OF HPLC
1 Pharmaceutical Analysis

HPLC is extensively used in pharmaceutical analysis for drug purity testing, pharmacokinetics, and quality
control, ensuring the safety and efficacy of medications.

2 Environmental Analysis

It plays a pivotal role in environmental analysis by detecting and quantifying pollutants, pesticides, and other
contaminants in water, soil, and air samples.

3 Biomedical Research

In biomedical research, HPLC is used for the analysis of biomolecules, metabolites, and therapeutic
drug monitoring, contributing to advancements in healthcare and disease diagnosis
 ION EXCHANGE CHROMATOGRAPHY
● Ion exchange chromatography is a
seperation technique based on
the reversible exchange of ions
between a stationary phase and a
mobile phase
● Principle : Charged molecules in
the sample interact with
oppositely charged groups in the
stationary phase .
 Components of ion exchange
chromatography

1. Stationary phase: Consists of a solid support Matrix with

charged functional groups(anion or cation exchange
resins)
2. Mobile phase: Typically an aqueous buffer solution that
facilitates the movement of ions through the column.
3. Sample injection: introduction of the sample injection
containing analyses to be seperated.
 Mechanism and Process

1. Adsorption: Charged molecules in the sample bind to oppositely charged functional

groups on the stationary phase.
2. Elution: elution buffer with different pH or ionic strength is passed through the
column,leading to the release of bound molecules.
3. Gradient elution: gradual change in eluent composition to seperate analyte based on their
affinity.

Process: the stationary phase can be either positively or negatively charged depending on
the target molecules charges. Molecules with stronger interactions with the stationary
phase are retained longer leading to seperation.
 Applications

1. Protein seperation.
2. Enzyme purification.
3. Water treatment.
4. Pharmaceutical industry.
5. Biochemical applications.
AFFINITY CHROMATOGRAPHY
● Roll no 135
Roll no 135
 Principle

Affinity chromatography is one of the most diverse and powerful

chromatographic methods for purification of a specific molecule or a
group of molecules from complex mixtures
• It is based on highly specific biological interactions between two
molecules such as interactions between enzyme and substrate,
receptor and ligand, or antibody and antigen.
 Immobilised ligand

The ligand can be selected only after the nature of the macromolecule to
be isolated is known.
• When a hormone receptor protein is to be purified by affinity
chromatography, the hormone itself is an ideal candidate for the ligand.
• For antibody isolation ,an antigen or hapten may be used as ligand.
• If an enzyme is to be purified, a substrate analog, inhibitor, cofactor, or
effector may be used as a the immobilized ligand.
APPLICATIONS
• 1)It is used for isolation and purification of all
biological macromolecule.
• 2)It is used to purify nucleic
acid,antibodies,enzymes.etc
• 3)To notice which biological compounds bind to a
particular substance.
• 4)to reduce a amount of substance in a mixture