What is chromatography?

•Chromatography is an important biophysical technique that enables the

separation, identification, and purification of the components of a mixture
for qualitative and quantitative analysis.
•The Russian botanist Mikhail Tswett coined the term chromatography in
1906.
•The first analytical use of chromatography was described by James and
Martin in 1952, for the use of gas chromatography for the analysis of fatty
acid mixtures.
•A wide range of chromatographic procedures makes use of differences in
size, binding affinities, charge, and other properties to separate materials.
•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.
•Chromatography is derived from the Greek
word ‘chroma’ means ‘color’ and ‘graphein’
means writing or recording.
•Mikhail Tsvet, a Russian Italian Botanist
invented an earliest form of true
chromatography technique for the
separation of plant pigmentation.
•Chromatography separates a component of
mixture which is dissolved in a substance called the
mobile phase and is carried out by a second
substance called the stationary phase.
•It separates a chemical mixture into an individual
component and helps in analysis of the particular
compound.
Three components thus form the basis of the chromatography technique.
Stationary phase: This phase is always composed of a “solid” phase or “a layer
of a liquid adsorbed on the surface solid support”.

Mobile phase: This phase is always

composed of “liquid” or a “gaseous
component.”

Separated molecules
The type of interaction between the stationary phase, mobile phase, and
substances contained in the mixture is the basic component effective on the
separation of molecules from each other.
Basic working principle of Chromatography
•Chromatography is based on the principle where
molecules in mixture applied onto the surface or into
the solid, and fluid stationary phase (stable phase) is
separating from each other while moving with the aid
of a mobile phase.
•The factors effective on this separation process
include molecular characteristics related to
adsorption (liquid-solid), partition (liquid-solid), and
affinity or differences among their molecular weights.
•Because of these differences, some components of
the mixture stay longer in the stationary phase, and
they move slowly in the chromatography system,
while others pass rapidly into the mobile phase, and
leave the system faster.
•Several key factors are responsible on the separation process
like partition between liquid-liquid, affinity between molecular
weight and characteristics related to liquid-solid adsorption.
•An interaction between the molecules are physical and involves
weak chemical bonds like dipole-dipole interaction and
hydrogen bond formation and adhere to the stationary
components.
•Components that adhere strongly to the stationary phase
moves slowly than those who adhere weakly.
 Types of Chromatography
Commonly used chromatographic technique differs in the kind of
stationary and mobile phase they use:

1.Paper chromatography
2.Thin layer chromatography
3.Liquid column chromatography
4.Size exclusion chromatography
5.Ion exchange chromatography
6.Affinity chromatography
7.Gas chromatography
8.High performance liquid chromatography
 How it works?
In a paper chromatography,

separation of the mixture is

performed on a paper strip

which is a stationary phase

and a liquid solvent acts as a

mobile phase.
•A drop of mixture is placed on one end of the paper and dried.
Then the paper is dipped into the solvent up to the spot.
•In the paper chromatography, component separates in two ways:
In paper adsorption chromatography, stationary phase and mobile
phase molecules act based on the degree of interaction. Higher
affinity molecules are adsorbed for a long time where movement of
speed is decreased.
However, low affinity molecules move faster thus, molecules are
separated.
In paper partition chromatography, the moisture on the cellulose layers
acts as a stationary phase where the mobile phase molecules are strongly
absorbed hence, the molecules gets separated.

During the separation of molecules, retention factor is determined as;

Retention factor (Rf) = Distance travelled by solute
Distance travelled by solvent
 Thin layer chromatography (TLC):
Thin layer chromatography works on the basis of solid-liquid partition
where silica or alumina plate used is a stationary phase and liquid as a
mobile phase.

Basically, TLC is a technique that is used to separate non- volatile mixtures.

•A thin layer of silica gel or cellulose is used as an adsorbent and the solvent
mixture as a mobile phase.
•The mixture of the solution is placed at a distance of 2cm above one end of
the plate. Then the plate is placed in a jar containing a fluid solvent.
•Moving phase is drawn up by the capillary action where the mixture rises up
the plate carrying different components.
•The plate is taken out after the mobile solvent reaches the top and its
retention factor is calculated.
 Liquid column chromatograph
Liquid column chromatography is a technique used to separate when a
molecules or ions are dissolved in a solvent, it separate the components
using a column of adsorbent packed glass tube.

How it works?
•The slurry or powdered silica acts as a stationary phase which is
loaded on a column or a paper and the sample is added to the
liquid in the chromatographic system which acts as a mobile
phase.
•The degree of the adsorption depends upon the affinity of the
molecules. If the components have a high affinity, the molecule
moves down the column faster but low degree affinity molecules
move slowly and remains at the top.
•High performance liquid chromatography (HPLC) is a modified
version of the liquid column chromatography.
 Size exclusion chromatography

From its given name, size exclusion chromatography is a method where

the molecules in a solution are separated in terms of their size or

weight. It is also known as gel filtration, gel permeation and molecular

sieve chromatography.
•The semi permeable porous polymer gel bead is a
stationary phase molecules that is filled in a column and the
sample is mixed with mobile phase liquid.
•The molecules are partitioned on the basis of their
respective sizes. Molecules with small size moves fast out of
the column while the large size molecules stay at the top.
•If the aqueous solution is used in mobile phase then it is gel
filtration. Similarly, if an organic solvent is used, it is gel
permeation chromatography.
 Ion exchange
chromatography:
In ion exchange chromatography,
the separation is between the
charged molecules where solid
polymeric ion exchange resins are
used. This technique is mostly
used to purify the drinking water.
•In a column, charged ion resins are packed which is
taken as stationary phase. The mixture with the
charged particles when passed through the column
binds to the oppositely charged resins.
•If a negative charged molecule is used, it binds to the
positively charged resins and vice versa.
•Then an appropriate buffer is used to separate the
complex charged molecules and resins.
 Affinity chromatography:
The mixture is separated on the
basis of affinity between the
absorbent and the ligand.

Absorbent is the separation

material, whereas the ligand is
the desired components in a
mixture

How it works?
Gas chromatography:
Gas chromatography is a technique of
separation of chemical components
which are usually organic molecules
or gases and determines their
presence.

How is works?

•It is different from other chromatography because the gas acts as a mobile
phase and the separation is vapor.
•The sample is injected into the column which is either gas or liquid.
•Helium is usually used as a carrier gas as a mobile phase which moves in the
analyte through the column at their own rate.
•The components are collected and their retention time is determined.
 Applications of Chromatography
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.

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
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.