CHROMATOGRAPHY

Competency Number BI: 7.4

CORE COMPETENCY

Dr. Vishnu Kumar

Professor, Department of Biochemistry,
SRMSIMS, Bareilly
vkawasthi@hotmail.com
madhwapur1976@gmail.com
 Contents

• Introduction
• History
• Principle
• Types of chromatography
• Conclusion
• References
Introduction

By
DR VISHNU KUMAR
 Introduction
• Chromatography
• Chroma Color
• Graphein To write
 Chromatography
• Chromatography is used to separate mixtures
of substances into their components.
 History
• It was first employed in Russia by
the Italian- scientist Mikhail
Tsvet in 1900.

• The separation of plant

pigments such as Chlorophyll.

• New types of
chromatography developed.
 Principle
• All forms of
chromatography work on
the same principle.

• Stationary phase (a solid,

or a liquid supported on
a solid)

• Mobile phase (a liquid or a

gas)
Thin layer chromatography
Paper chromatography
 Definition
• Paper chromatography is a type of chromatography.

• It is an analytical method used to separate colored

chemicals or substances.

• A method of separating components of a mixture by

differential movement through a two-phase system: the
mobile phase and the secondary phase.

• The separation of an unknown substance is mainly

carried out by the flow of solvents on the specially
designed chromatographic paper
 Principle
• Paper chromatography works on the principle of partition, i.e., it
is based upon continuous differential distribution of the various
components of the mixture between the stationary and the mobile
phase.

• Cellulose layers in filter paper contains moisture which acts as

stationary phase & organic solvents/buffers are used as
mobile phase.

• In paper chromatography the results are represented by Rf value

which represents the movement of component of the mixtures
relative to the solvent front.
Rf value = Distance traveled by the solute
Distance traveled by the solvent
 Procedure

Solvent Beaker
front

Separated
Filter dyes
paper

Ink spots

Solve
nt
Observing the Chromatograms

0 20 50 70 100
% % % % %
Co ncentrat ion
of Isopropanol
 Uses of Paper chromatography
• To Analyze – examine a mixture, its components, and their
relations to one another

• To Identify – determine the identity of a mixture or

components based on known components

• To Purify – separate components in order to isolate one of

interest for further study

• To Quantify – determine the amount of the a mixture and/or

the components present in the sample
High performance liquid
chromatography
 Introduction
• HPLC is a form of liquid chromatography used to separate
compounds that are dissolved in solution.
• HPLC instruments consist of a reservoir of mobile phase, a
pump, an injector, a separation column, and a detector.
• Compounds are separated by injecting a sample mixture onto the
column.
• The different component in the mixture pass through the column at
differentiates due to differences in their partition behavior between
the mobile phase and the stationary phase.

• The mobile phase must be degassed to eliminate the formation of air

bubbles.
What is Liquid Chromatography?
• Liquid chromatography is a separation technique that involves:

• the placement (injection) of a small volume of liquid sample

• into a tube packed with porous particles (stationary phase)

• where individual components of the sample are transported along the

packed tube (column) by a liquid moved by gravity.

• The components of the sample are separated from one another by the
column packing that involves various chemical and/or physical interactions
between their molecules and the packing particles.

• The separated components are collected at the exit of this column and
identified by an external measurement technique , such as a
spectrophotometer that measures the intensity of the color , or by another
device that can measure their amount.
Principles of Liquid Chromatography
 What is HPLC?
• HPLC is a separation technique that involves:
•the injection of a small volume of liquid sample
•into a tube packed with tiny particles (3 to 5 micron ( μm ) in
diameter called the stationary phase)
•where individual components of the sample are moved down the
packed tube (column) with a liquid (mobile phase) forced through
the column by high pressure delivered by a pump.

• These components are separated from one another by the column

packing that involves various chemical and/or physical interactions
between their molecules and the packing particles.
• These separated components are detected at the exit of this tube (column)
by a flow-through device (detector) that measures their amount. An
output from this detector is called a “liquid chromatogram”.
Gas chromatography

Muhammad Jawad
Reg # 636/BSBT/F14
Ion exchange chromatography
 inTro

 This technique was developed in 19th century and was used to

purify the drinking water.

 Reversible exchange of ions in the solution with ions

electrostatically bound to some sort of insoluble matrix or
stationary phase
.
 It is extremely useful in the separation of charged compounds
like proteins differing from only one charged amino acid.
 Ion exchanged is performed in the column.
 Principle

The principle of separation is by reversible

exchange of ions between ions present in the
solution and those present in the ion
exchange
resin.
 Ion exchangers

 There are two types of ion exchangers.

 Cations exchanger.
 Anions exchanger.
Cation exchange chromatography

Positively charged molecules are attracted to a

negatively charged solid support. Commonly used
Cation exchange resins are S-resin, sulfate
derivatives; and CM resins, carboxylate derived ions
Anion exchange chromatography

Negatively charged molecules is attracted to a

positively charged solid support. Commonly used
anion exchange resins are Q-resin, a Quaternary
amine; and DEAE resin, Di-EthylAminoEthane.
CATION AND ANION
EXCHANGER
 Uses

 Used in the analyses of aminoacid

 To determine the base composition of the nuclic
acid.
 Proteins are also successfully seprated by
this technique
 Used for sepration of vitamens
 This is most effective method for water
purification. Complete deionization of water (or)
a non-electrolyte solution is performed by
exchanging solute cations for hydrogen ions and
solute anions for hydroxyl ions. This is usually
achieved by method is used for softening of
drinking water.
Column chromatography
 Column chromatography
An impure sample is loaded onto a column
of adsorbant, such as silica gel
. An organic solvent or a mixture of solvents
(the eluent) flows down through the column.
Components of the sample separate from
each other by partitioning between the
stationary packing material.
Column
Precautions
Affinity chromatography
 Affinity Chromatography

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.
 Principle

Affinity chromatography is based on highly specific biological

interactions between two molecules, such as interactions
between enzyme and substrate, receptor and ligand, or
antibody and antigen.

These biological interactons are typicaly reversible

these reversible interactions are used for
purification .
 Procedure
Purification is done by placing one of the
interacting molecules, referred to as affinity ligand, onto a
solid matrix to create a stationary phase while the target
molecule is in the mobile phase. Successful affinity
purification requires a certain degree of knowledge and
understanding of the nature of
interactions between the target molecule and the ligand to help
determine the selection of an appropriate affinity ligand and
purification procedure.
 Conclusion
• Separation technique
• Many types of chromatography
• Working depend on stationary and mobile
phase
• Many applications in the field of biochemistry,
organic chemistry, etc