TOPIC:

Chromatography
Learning Objectives:
❖ Paper Chromatography
❖ Liquid Chromatography
❖ Gas 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. A wide range of chromatographic procedures makes use of differences
in size, binding affinities, charge, and other properties to separate materials.

PAPER CHROMATOGRAPHY:
Paper chromatography (PC) is a type of a planar chromatography whereby
chromatography procedures are run on a specialized paper. Paper chromatography,
in analytical chemistry technique for separating dissolved chemical substances by taking
advantage of their different rates of migration across sheets of paper.
Principle:
The principle involved can be partition chromatography or adsorption
chromatography. Partition chromatography because the substances are partitioned or
distributed between liquid phases. The two phases are water held in pores of the filter paper
and the other phase is a mobile phase which passes through the paper. When the mobile
phase moves, the separation of the mixture takes place. 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.

Applications:
➢ 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.

GAS CHROMATOGRAPHY
Gas chromatography (GC) is a common type of chromatography used in analytical
chemistry for separating and analyzing compounds that can be vaporized without
decomposition. Typical uses of GC include testing the purity of a particular substance, or
separating the different components of a mixture.

Principle:
The sample solution injected into the instrument enters a gas stream which transports
the sample into a separation tube known as the “column.” (Helium or nitrogen is used as the
so-called carrier gas).The various components are separated inside the column. The detector
measures the quantity of the components that exit the column. To measure a sample with an
unknown concentration, a standard sample with known concentration is injected into the
instrument. The standard sample peak retention time (appearance time) and area are
compared to the test sample to calculate the concentration.

Applications:
➢ GC analysis is used to calculate the content of a chemical product, for example in
assuring the quality of products in the chemical industry; or measuring toxic
substances in soil, air or water.
Gas chromatography is used in the analysis of:
➢ air-borne pollutants
➢ performance-enhancing drugs in athlete’s urine samples
➢ oil spills
➢ essential oils in perfume preparation
Gas Chromatography is used extensively in forensic science. Disciplines as diverse as
solid drug dose (pre-consumption form) identification and quantification, GC to identify and
quantify various biological specimens and crime-scene evidence.
LIQUID CHROMATOGRAPHY:
 Liquid chromatography (LC) is an analytical chromatographic technique that is useful
for separating ions or molecules that are dissolved in a solvent. High Performance Liquid
Chromatography (HPLC) is a form of column chromatography that pumps a sample mixture
or analyte in a solvent (known as the mobile phase) at high pressure through a column with
chromatographic packing material (stationary phase).

Principle:
➢ The purification takes place in a separation column between a stationary and a mobile
phase.
➢ The stationary phase is a granular material with very small porous particles in a
separation column.
➢ The mobile phase, on the other hand, is a solvent or solvent mixture which is forced
at high pressure through the separation column.
➢ Via a valve with a connected sample loop, i.e. a small tube or a capillary made of
stainless steel, the sample is injected into the mobile phase flow from the pump to the
separation column using a syringe.
➢ Subsequently, the individual components of the sample migrate through the column
at different rates because they are retained to a varying degree by interactions with
the stationary phase.
➢ After leaving the column, the individual substances are detected by a suitable detector
and passed on as a signal to the HPLC software on the computer.
➢ At the end of this operation/run, a chromatogram in the HPLC software on the
computer is obtained.
➢ The chromatogram allows the identification and quantification of the different
substances.
Applications: