Chromatography

The Russian botanist Mikhail Tswett coined the term chromatography in 1906.
Chromatography (from Greek chroma "color" and graphein "to write") is the collective term
for a set of laboratory techniques for the separation of mixtures.

Chromatography is an important technique that enables the separation, identification, and

purification of the components of a mixture for qualitative and quantitative analysis. It is a
powerful separation tool that is used in all branches of science and is often the only means of
separating components from complex mixtures.

Applications of Chromatography:
Chemical industry

 In testing water samples and also checks air quality.

 HPLC and GC are very much used for detecting various contaminants such as
polychlorinated biphenyl (PCBs) in pesticides and oils.
 In various life sciences applications

Pharmaceutical sector

 To identify and analyze samples for the presence of trace elements or chemicals.
 Separation of compounds based on their molecular weight and element composition.
 Detects the unknown compounds and purity of mixture.
 In drug development.

Food Industry

 In food spoilage and additive detection

 Determining the nutritional quality of food

Forensic Science

 In forensic pathology and crime scene testing like analyzing blood and hair samples of
crime place.

Molecular Biology Studies

 Various hyphenated techniques in chromatography such as EC-LC-MS are applied in the

study of metabolomics and proteomics along with nucleic acid research.
 HPLC is used in Protein Separation like Insulin Purification, Plasma Fractionation, and
Enzyme Purification and also in various departments like Fuel Industry, biotechnology,
and biochemical processes.
Principle of Chromatography:
Chromatography usually consists of mobile phase and stationary phase. The mobile phase refers
to the mixture of substances to be separated dissolved in a liquid or a gas. The stationary phase is
a porous solid matrix through which the sample contained in the mobile phase percolates. The
interaction between the mobile phase and the stationary phase results in the separation of the
compound from the mixture.

Classification of Chromatography:
 Classification on the basis of physical state/basis:

Chromatography

Mobile Phase; Solvent Stationary Phase; Sorbent

Gas Liquid Solid Liquid

Mobile Phase Solid Phase Chromatography Type
Gas Liquid Gas- Liquid Chromatography
Gas Solid Gas- Solid Chromatography
Liquid Liquid Liquid-Liquid Chromatography
Liquid Solid Liquid- Solid Chromatography

 Classification on the basis Stationary Phase:

Chromatography

Planner Column

Paper Thin Layer Packed Open Pore

Chromatography Chromatography Column Column

 Classification on the basis Molecular Interection:

1. Partition
2. Adsorption
3. Ion-Exchange
4. Affinity
5. Size Exclusion
Different Types of Chromatography:
There are several types of Chromatography. Amongst them following are mostly important;
 Mobile Basis of
Technique Stationary phase Notes
phase separation
Paper polarity of Compound spotted directly on a
solid (cellulose) liquid
chromatography molecules cellulose paper
Thin layer Glass is coated with thin layer
solid (silica or polarity of
chromatography liquid of silica on which is spotted the
alumina) molecules
(TLC) compound
Liquid column solid (silica or polarity of Glass column is packed with
liquid
chromatography alumina) molecules slurry of silica
Small molecules get trapped in
the pores of the stationary
phase, while large molecules
flow through the gaps between
solid the beads and have very small
Size exclusion size of
(microporous liquid retention times. So larger
chromatography molecules
beads of silica) molecules come out first. In this
type of chromatography there
isn’t any interaction, physical or
chemical, between the analyte
and the stationary phase.
Molecules possessing the
opposite charge as the resin will
bind tightly to the resin, and
Ion-exchange solid (cationic or ionic charge of
liquid molecules having the same
chromatography anionic resin) the molecules
charge as the resin will flow
through the column and elute
out first.
If the molecule is a substrate for
the enzyme, it will bind tightly
solid (agarose or binding affinity to the enzyme and the unbound
porous glass of the analyte analytes will pass through in the
beads on to which molecule to the mobile phase, and elute out of
Affinity
are immobilized liquid molecule the column, leaving the
chromatography
molecules like immobilized on substrate bound to the enzyme,
enzymes and the stationary which can then be detached
antibodies) phase from the stationary phase and
eluted out of the column with an
appropriate solvent.
Samples are volatilized and the
gas
molecule with lowest boiling
(inert
Gas liquid or solid boiling point of point comes out of the column
gas like
chromatography support the molecules first. The molecule with the
argon or
highest boiling point comes out
helium)
of the column last.
Paper Chromatography:
Chromatography technique that uses paper sheets or strips as the adsorbent being the stationary
phase through which a solution is made to pass is called paper chromatography. It is an
inexpensive method of separating dissolved chemical substances by their different migration
rates across the sheets of paper. It is a powerful analytical tool that uses very small quantities of
material. Paper chromatography was discovered by Synge and Martin in the year 1943.

Principle:
The compounds in the mixture separate themselves based on the differences in their affinity
towards stationary and mobile phase solvents under the capillary action of pores in the paper.
Adsorption chromatography between solid and liquid phases, wherein the solid surface of the
paper is the stationary phase and the liquid phase is the mobile phase. When the mobile phase
moves, the separation of mixture takes place.

Paper Chromatography Procedure:

1. Selecting a suitable filter paper: Selection of filter paper is done based on the size of the
pores, and the sample quality.
2. Prepare the sample: Sample preparation includes the dissolution of the sample in a
suitable solvent (inert with the sample under analysis) used in making the mobile phase.
3. Spot the sample on the paper: Samples should be spotted at a proper position on the
paper by using a capillary tube.
4. Chromatogram development: Chromatogram development is spotted by immersing the
paper in the mobile phase. Due to the capillary action of paper, the mobile phase moves
over the sample on the paper.
5. Paper drying and compound detection: Once the chromatogram is developed, the
paper is dried using an air drier. Also, detecting solution can be sprayed on the
chromatogram developed paper and dried to identify the sample chromatogram spots.
UV- light can also be used to detect the mixture of samples.
Retardation factor:
The retention factor, Rf, is a quantitative indication of how far a particular compound travels in a
particular solvent. The Rf value is a good indicator of whether an unknown compound and a
known compound are similar, if not identical. If the Rf value for the unknown compound is close
or the same as the Rf value for the known compound then the two compounds are most likely
similar or identical. The retention factor, Rf, is defined as;

Rf = distance the solute (x) moves divided by the distance traveled by the solvent front (y) [Rf =
x / y]

Types of paper chromatography:

1. Ascending Paper Chromatography – The techniques goes with its name as the solvent
moves in an upward direction.
2. Descending Paper Chromatography – The movement of the flow of solvent due to
gravitational pull and capillary action is downwards hence the name descending paper
chromatography.
3. Ascending–Descending Paper Chromatography – In this version of paper
chromatography movement of solvent occurs in two directions after a particular point.
Initially, the solvent travels upwards on the paper which is folded over a rod and after
crossing the rod it continues with its travel in the downward direction.
4. Radial or Circular Paper Chromatography – The sample is deposited at the center of
the circular filter paper. Once the spot is dried, the filter paper is tied horizontally on a
Petri dish which contains the solvent.
5. Two Dimensional Paper Chromatography – Substances which have the same rf values
can be resolved with the help of two-dimensional paper chromatography.

Paper Chromatography Applications:

There are various applications of paper chromatography. Some of the uses of Paper
Chromatography in different fields are discussed below:
  To study the process of fermentation and ripening.
 To check the purity of pharmaceuticals.
 To inspect cosmetics.
 To detect the adulterants.
 To detect the contaminants in drinks and foods.
 To examine the reaction mixtures in biochemical laboratories.
 To determine dopes and drugs in humans and animals.

Video link for live Paper chromatography experiment:

Separation of Pigments from the Extract of Spinach Leaves by Paper Chromatography;

https://www.youtube.com/watch?v=ej2zXOwASVI

Thin Layer Chromatography:

Thin Layer Chromatography is a technique used to isolate non-volatile mixtures. The experiment
is conducted on a sheet of aluminum foil or plastic or glass, which is coated with a thin layer of
adsorbent material. The material usually used is aluminum oxide or cellulose or silica gel. TLC
is one of the fastest, least expensive, simplest and easiest chromatography techniques.

On completion of the separation, each component appears as spots separated vertically. Each
spot has a retention factor (Rf) expressed as:

Rf = dist. travelled by sample / dist. travelled by solvent.

Thin Layer Chromatography Principle:

Like other chromatographic techniques, thin layer chromatography (TLC) depends on the
separation principle. The separation relies on the relative affinity of compounds towards both the
phases. The compounds in the mobile phase move over the surface of the stationary phase. The
movement occurs in such a way that the compounds which have a higher affinity to the
stationary phase move slowly while the other compounds travel fast. Therefore, the separation of
the mixture is attained. On completion of the separation process, the individual components from
the mixture appear as spots at respective levels on the plates.
Different components of Thin Layer Chromatography:

Before starting with the Thin Layer Chromatography Experiment let us understand the different
components required to conduct the procedure along with the phases involved.

1. Thin Layer Chromatography Plates – ready-made plates are used which are
chemically inert and stable. The stationary phase is applied on its surface in the form of a
thin layer. The stationary phase on the plate has a fine particle size and also has a uniform
thickness.
2. Thin Layer Chromatography Chamber – Chamber is used to develop plates. It is
responsible to keep a steady environment inside which will help in developing spots.
Also, it prevents the solvent evaporation and keeps the entire process dust-free.
3. Thin Layer Chromatography Mobile phase – Mobile phase is the one that moves and
consists of a solvent mixture or a solvent. This phase should be particulate-free. The
higher the quality of purity the development of spots is better.
4. Thin Layer Chromatography Filter Paper – It has to be placed inside the chamber. It
is moistened in the mobile phase.

Thin Layer Chromatography Experiment Procedure:

The stationary phase that is applied to the plate is made to dry and stabilize.

 To apply sample spots, thin marks are made at the bottom of the plate with the help of a
pencil.
 Apply sample solutions to the marked spots.
 Pour the mobile phase into the TLC chamber and to maintain equal humidity, place a
moistened filter paper in the mobile phase.
 Place the plate in the TLC chamber and close it with a lid. It is kept in such a way that the
sample faces the mobile phase.
 Immerse the plate for development. Remember to keep the sample spots well above the
level of the mobile phase. Do not immerse it in the solvent.
 Wait till the development of spots. Once the spots are developed, take out the plates and
dry them. The sample spots can be observed under a UV light chamber.

Thin Layer Chromatography Applications:

 The qualitative testing of various medicines such as sedatives, local anesthetics,

anticonvulsant tranquilisers, analgesics, antihistamines, steroids, and hypnotics is done by
TLC.
 TLC is extremely useful in Biochemical analysis such as separation or isolation of
biochemical metabolites from its blood plasma, urine, body fluids, serum, etc.
 Thin layer chromatography can be used to identify natural products like essential oils or
volatile oil, fixed oil, glycosides, waxes, alkaloids, etc
 It is widely used in separating multicomponent pharmaceutical formulations.
 It is used to purify of any sample and direct comparison is done between the sample and
the authentic sample
  It is used in the food industry, to separate and identify colours, sweetening agent, and
preservatives
 It is used in the cosmetic industry.
 It is used to study if a reaction is complete.

What is the major difference between thin layer chromatography and paper
chromatography?

The stationary phase used in paper chromatography Cellulose filter paper containing water in its
pore whereas in TLC the stationary phase used is Glass plate coated with silica gel.

Video link for live Thin chromatography experiment:

https://www.youtube.com/watch?v=qdmKGskCyh8

Column Chromatography:
Column chromatography is a technique which is used to separate a single chemical compound
from a mixture dissolved in a fluid. It separates substances based on differential adsorption of
compounds to the adsorbent as the compounds move through the column at different rates which
allow them to get separated into fractions. This technique can be used on small scale as well as
large scale to purify materials that can be used in future experiments. This method is a type of
adsorption chromatography technique.

Column Chromatography Principle:

When the mobile phase along with the mixture that needs to be separated is introduced from the
top of the column, the movement of the individual components of the mixture is at different
rates. The components with lower adsorption and affinity to stationary phase travel faster when
compared to the greater adsorption and affinity with the stationary phase. The components that
move fast are removed first whereas the components that move slow are eluted out last.
Different components of Column Chromatography:

Before starting with the Column Chromatography Experiment let us understand the different
phases involved.

Mobile phase – This phase is made up of solvents and it performs the following functions:

1. It acts as a solvent – sample mixture can be introduced in the column.

2. It acts as a developing agent – helps in the separation of components in the sample to
form bands.
3. It acts as an eluting agent – the components that are separated during the experiment are
removed from the column
4. Some examples of solvents used as mobile phase based on their polarity are – ethyl
acetate, hexane, methanol, chloroform, ethanol, acetone, water etc.

Stationary phase – It is a solid material which should have good adsorption property and meet
the conditions given below:

1. Shape and size of particle: Particles should have uniform shape and size in the range of
60 – 200μ in diameter.
2. Stability and inertness of particles: high mechanical stability and chemically inert. Also,
no reaction with acids or bases or any other solvents used during the experiment.
3. It should be colourless, inexpensive and readily available.
4. Should allow free flow of mobile phase
5. It should be suitable for the separation of mixtures of various compounds.

Column Chromatography Experiment Procedure:

 The stationary phase is made wet with the help of solvent as the upper level of the mobile
phase and the stationary phase should match. The mobile phase or eluent is either solvent
or mixture of solvents. In the first step the compound mixture, that needs to be separated,
is added from the top of the column without disturbing the top level. The tap is turned on
and the adsorption process on the surface of silica begins.
 Without disturbing the stationary phase solvent mixture is added slowly by touching the
sides of the glass column. The solvent is added throughout the experiment as per the
requirement.
 The tap is turned on to initiate the movement of compounds in the mixture. The
movement is based on the polarity of molecules in the sample. The non-polar components
move at a greater speed when compared to the polar components.
 For example, suppose a compound mixture consists of three different compounds A, B &
C, and their order based on polarity is A>B>C. As the polarity C is less, it will move first
and fast. When it arrives at the end of the column it is collected in a clean test tube. After
this, B is collected and at last A is collected. All these are collected in separate test tubes.
Column Chromatography Applications:

 Column Chromatography is used to isolate active ingredients.

 It is very helpful in separating compound mixtures.
 It is used to determine drug estimation from drug formulations.
 It is used to remove impurities.
 Used to isolation metabolites from biological fluids.

Types of Column Chromatography:

1. Adsorption column chromatography – Adsorption chromatography is a technique of

separation, in which the components of the mixture are adsorbed on the surface of the adsorbent.

2. Partition column chromatography – The stationary phase, as well as mobile phase, are
liquid in partition chromatography.

3. Gel column chromatography – In this method of chromatography, the separation takes place
through a column packed with gel.

4. Ion exchange column chromatography – Chromatography technique in which the stationary

phase is always ion exchange resin.

Video link for live Column chromatography experiment:

https://www.youtube.com/watch?v=UmWMlKJAdSk

Ion-Exchange Chromatography
Ion-exchange chromatography is a versatile, high resolution chromatography technique to purify
ions or polar molecules or proteins from complex mixtures. In addition, this chromatography has
a high loading capacity to handle large sample volume and the operation is very simple.

Principle: This chromatography distributes the analyte molecule as per charge and their affinity
towards the oppositely charged matrix. The analytes bound to the matrix are exchanged with a
competitive counter ion to elute. The interaction between matrix and analyte is determined by net
charge, ionic strength and pH of the buffer. For example, when a mixture of charged analyte (M,
M+, M-1, M-2) loaded onto a positively charged matrix, the neutral or positively charged analyte
will not bind to the matrix where as negatively charged analyte will bind as per their relative
charge and needed higher concentration of counter ion to elute from matrix.
 Affinity of analytes (M, M+, M-1, M-2) towards positively charged matrix

The matrix used in ion-exchange chromatography is present in the ionized form with reversibly
bound ion to the matrix. The ions present on matrix participate in the reversible exchange
process with analyte. Hence, there are two types of ion-exchange chromatography:

1. Cation exchange chromatography: In cation exchange chromatography,

matrix has a negatively charged functional group with affinity towards positively charged
molecules. The positively charged analyte replaces the reversible bound cation and binds to the
matrix. In the presence of a strong cation (such as Na+) in the mobile phase, the matrix bound
positively charged analyte is replaced with the elution of analyte.

2. Anion Exchange chromatography: In anion exchange chromatography,

matrix has a positively charged functional group with affinity towards negatively charged
molecules. The negatively charged analyte replaces the reversible bound anion and binds to the
matrix. In the presence of a strong anion (such as Cl-) in the mobile phase, the matrix bound
negatively charged analyte is replaced with the elution of analyte.

List of selected Ion-exchange matrix

 Cation and Anion exchange chromatography

Operation of the technique: Several parameters need to be considered to perform ion-

exchange chromatography;

1. Column material and stationary phase: Column material should be

chemically inert to avoid destruction of biological sample. It should allow free flow of liquid
with minimum clogging. It should be capable to withstand the back pressure and it should not
compress or expand during the operation.

2. Mobile Phase: The ionic strength and pH are the crucial parameters to
influence the property of the mobile phase.

3. Sample Preparation: The sample is prepared in the mobile phase and it

should be free of suspended particle to avoid clogging of the column. The most recommended
method to apply the sample is to inject the sample with a syringe.

4. Elution: There are many ways to elute an analyte from the ion-exchange
column. (a) Isocratic elution (b) Step-wise gradient (c) Continuous gradient either by salt or pH
(d) affinity elution (e) displacement chromatography.

5. Column Regeneration: After the elution of analyte, ion-exchange

chromatography column require a regeneration step to use next time. Column is washed with a
salt solution with an ionic strength of 2M to remove all non-specifically bound analytes and also
to make all functional group in an iononized form to bind fresh analyte.
 Operation of the Ion-exchange Chromatography
(A) Chromatography system to perform gradient elution of analytes
(B) Elution profile.

Applications of Ion-exchange chromatography:

1. Protein Purification
2. Softening of water
3. Purification of rare earth metals from nuclear waste
4. Study of Protein-DNA interaction
5. Protein kinase assay
6. Concentrating a sample

Gas Chromatography………continued on next content