Chromatography

Pharmacognosy By Sir Tanveer Khan Compiled By >>>

Lecture: Chromatography

CHROMATOGRAPHY
INTRODUCTION
Chromatography is a combination of two words;
* Chromo – Meaning color
* Graphy – representation of something on paper
DEFINITION
“ It is a physical separation method in which the components of a mixture are separated by
differences in their distribution between two phases, one of which is stationary (stationary phase)
while the other (mobile phase) moves through it in a definite direction . The substances must
interact with the stationary phase to be retained and separated by it .

CHROMATOGRAPHY TERMS
Chromatogram:
It is the visual output of the chromatograph.
Chromatograph:
It is equipment that enables a sophisticated Separation.
Stationary phase (bounded phase):
It is a phase that is covalently bonded to the support particles or to the inside wall of the
columntubing.
Mobile phase:
It is the phase which moves in a definite direction.
Analyte (Sample):
It is the substance to be separated during chromatography.
Eluate:
It is the mobile phase leaving the column.
Retention time:
It is the characteristic time it takes for aparticular analyte to pass through the system(from
the column inlet to the detector) under set conditions.
Eluent:
It is the solvent that will carry the analyte.
Retardation factor ( R ):
Fraction of an analyte in the mobile phase of a chromatographic system.

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CLASSIFICATION
According to mechanism of separation

1) Ion-exchange chromatography
2) Affinity chromatography
3) Size-exclusion chromatography
4) Adsorption chromatography
5) Partition chromatography

 ION-EXCHANGE CHROMATOGRAPHY
It is a process that allows the separation of ions and polar molecules based on their charge.

DEFINITION:
In this type of chromatography, a resin (the stationary solid phase) is used to covalently attach
anions or cations onto it. Solute ions of the opposite charge in the mobile liquid phase are
attracted to the resin by electrostatic forces. Ion exchange chromatography is performed in
columns but can also be useful in planar mode.

PRINCIPLE:
Ion - exchange chromatography retains sample molecules on the column based on ionic
interactions.The surface of stationary phase displays ionic functional groups (R-X) that interact
with analyteions of opposite charge.

MECHANISM:
Ion exchange chromatography uses a charged stationary phase to separate charged compounds
including anions, cations, amino acids, peptides, and proteins. In conventional methods the
stationary phase is an ion exchange resin that carries charged functional groups which interact
with oppositely charged groups of the compound to be retained. Ion exchange chromatography is
commonly used to purify proteins.

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TYPES:

 Cation exchange chromatography:
Cation exchange chromatography retains positively charged cationsbecause the stationary
phase displays a negatively charged functional group.

 Anion exchange chromatography:
Anion exchange chromatography retains anions using positively charged functional group.

APPLICATIONS:

It can be used for almost any kind of charged molecule including large proteins, small
nucleotides and amino acids.

Protein purification
Water analysis
Quality control

 AFFINITY CHROMATOGRAPHY
This is the most selective type of chromatography.
The method was discovered and developed by PedroCuatrecasas and Meir Wilchek for which
the Wolf Prize in Medicine was awarded in 1987 .It is a method of separating biochemical
mixtures and based on a highly specific biological interaction such as that between antigen and
antibody, enzyme and substrate, or receptor and ligand.

DEFINITION:

“ It utilizes the specific interaction between one kind of solute molecule
and a second molecule that is immobilized on a stationary phase”.

For example, the immobilized molecule may be an antibody to some
specific protein. When solute containing a mixture of proteins are
passed by this molecule, only the specific protein is reacted to this
antibody, binding it to the stationary phase. This protein is later
extracted by changing the ionic strength or pH.

PRINCIPLE:

The stationary phase is typically a gel matrix (often agarose) .The
molecule of interest has a known and defined property. The process is
an entrapment in which the target molecule becomes trapped on
stationary phase. The Stationary phase can then be removed from the

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mixture, washed and then target molecule is released from the entrapment.

APPLICATIONS:

Purify and concentrate an enzyme solution
Purification of recombinant proteins
Purification of antibodies

 SIZE-EXCLUSION CHROMATOGRAPHY
The technique was invented by Grant Henry Lathe and Colin R Ruthven. They later received the
John Scott Award for this invention.It is a chromatographic method in which molecules in
solution are separated by their size, and in some cases molecular weight.It is usually applied to
large molecules or macromolecular complexes such as proteins and industrial polymers.

DEFINITION:

It is also known as gel permeation or gel filtration chromatography.

This type of chromatography lacks an attractive interaction between the stationary phase and
solute. The liquid or gaseous phase passes through a porous gel which separates the molecules
according to its size. The pores are normally small and exclude the larger solute molecules, but
allows smaller molecules to enter the gel, causing them to flow through a larger volume. This
causes the larger molecules to pass through the column at a faster rate than the smaller ones.

PRINCIPLE:

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Smaller molecules are able to enter the pores of the media and, therefore, molecules are trapped
and removed from the flow of the mobile phase . The average residence time in the pores
depends upon the effective size of the analytemolecules . However, molecules that are larger
than the average pore size of the packing are excluded.

APPLICATIONS:

Purification and analysis of synthetic and biological polymers, such as;
Proteins, Polysaccharides, Nucleic acids.
It is also useful for determining the tertiary structure and quaternary structure of purified
proteins.
It is generally a low-resolution chromatography technique and thus it is often reserved for
the final, "polishing" step of a purification.

 ADSORPTION CHROMATOGRAPHY
DEFINITION:
“ It is a type of chromatography in which a mobile liquid or gaseous phase is adsorbed onto the
surface of a stationary solid phase. The equilibration between the mobile and stationary phase
accounts for the separation of different solutes.”

PRINCIPLE:

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Principle involves competition of components of sample mixture for active site on adsorbent.
These active sites are formed in molecule due to
* Cracks
* Edges
The electrostatic forces present in the molecule, which hold together the crystal lattice, are
directed outward. These forces and electrostatic forces of solute molecule cause separation.
Separation occurs because of the fact that an equilibrium is established between molecules
adsorbed on stationary phase and those which are flowing freely in mobile phase.
The more the affinity of the molecule of particular component, less will be its movement.

TYPES:

Adsorption
chromatography

Column Thin layer Gas solid
chromatography chromatography chromatography

ADSORBENTS:
“ An adsorbent is a substance, usually porous in nature and with a high surface area that can
adsorb substances onto its surface by intermolecular forces.”

AN IDEAL ADSORBENT:
The Ideal adsorbent must fulfill the following requirements:

 Insoluble in mobile phase
 Inert to solutes (adsorptive)
 Colorless especially when work with colored mixtures
 Suitable particle size enough to give good separation and reasonable flow rate
COMMON ADSORBENTS:
Hydrated silica gel
Silica gel G
Silica gel S
Silica gel GF 254
Silica gel H
Silica gel N
Silica gel HF 254

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Silica gel PF 254
Modified silica gel
Alumina
Kieselghur (Diatomaceous earth)
Cellulose MN 300
Cellulose microcrystalline

 THIN-LAYER CHROMATOGRAPHY
“ The technique which involves flowing of mobile phase over a thin layer of adsorbent,
applied on solid support, where separation of components occur by differential migration
which occurs when solvent flows along fine powder spread on glass plates, is called thin
–layer chromatography.”
Instrumentation:
Chromatography jar
Capillary tube
Thin layer chromatography plate
Stationary phase
Mobile phase

 Chromatography jar:
It is made of glass and has a lid on it.
Jar maintains proper environment that is required for separation.
 Capillary tube:
It is used to apply sample mixture on TLC plate.
 Stationary phase:
Adsorbents
 TLC plate:
Borosilicate glass plates are preferred. Most commonly used
sizes are;
20 X 20cm
20 X 10cm
20 X 5cm
Microscopic slides are also used.

 Mobile phase:
Mobile phase may be a single liquid or a mixture of liquids.
Commonly used mobile phases are;
Methanol

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Ethanol
Ethyl acetate
Diethyl ether
Acetone
Chloroform
Procedure:
 Clean and dried chromatography jar is taken.
 A paper impregnated in the mobile phase is applied to the walls to ensure that
atmosphere of the jar is saturated with solvent vapors.
 Mobile phase is added to the jar at a length of 0.5-1cm from the bottom.
 Jar is closed.
 Equilibrium is allowed to be maintained.
 Base line is marked on adsorbent.
 Procedure
 Sample is applied on TLC plate with help of capillary tube.
 Sample spot is air dried.
 TLC plate is put in the chromatography jar and lid is closed.
 The system is allowed to be static until the solvent move to a proper distance from
baseline.
 TLC plate is taken out and dried.
Location of separated components:
If the sample is separated into colored components,
thenthe location is dried in ordinary light but in case
ofcolorless components following are used;
Uv lamp
Iodine crystal
Spraying agents

Documentation:

Storage of chromatogram for TLC is difficult. It is
usually undesirable since plates are employed forrepeated use. Various methods for
separation include;
Rf value in TLC
Preservation of chromatogram by peeling off adsorbent.
Graphical copying i.e. tracing on transparent paper.
Photography

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Applications:

It is used for separation and identification of;
Amino acids
Peptides and proteins
Alkaloids
Carbohydrates
Fats and fatty acids
Antibiotics
Narcotic analgesics
Glycosides

 PARTITION CHROMATOGRAPHY

DEFINITION:
“ This form of chromatography is based on a thin film formed on the surface of a solid support
by a liquid stationary phase. Solute equilibrates between the mobile phase and the stationary
liquid.”

PRINCIPLE:
Separation of components of a sample mixture
occurs because of partition. Stationary phase is coated
with a liquid which is immiscible in mobile phase.
Partition of component of sample between sample and
liquid/ gas stationary phase retard some components of
sample more as compared to others. This gives basis
for separation. The stationary phase immobilizes the
liquid surface layer, which becomes stationary phase.
Mobile phase passes over the coated adsorbent and
depending upon relative solubility in the coated liquid,
separation occurs. The components of sample mixture
appear separated because of differences in their
partition coefficient.

TYPES:
Partition
chromatography

Liquid-liquid Gas-liquid
chromatography chromatography
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 PAPER CHROMATOGRAPHY
Instrumentation:
Chromatography jar
Capillary tube
Stationary phase (liquid impregnated paper)
Mobile phase

 Chromatography jar:
It is made of glass and has a lid on it. Jar maintains proper environment
that is required for separation.
 Capillary tube:
It is used to apply sample mixture.
 Stationary phase:
liquid impregnated paper
 Mobile phase:
Mobile phase may be a single liquid or a mixture of liquids.
Commonly used mobile phases are;
Methanol
Ethanol
Ethyl acetate
Diethyl ether
Acetone
Chloroform
Procedure:
 Clean and dried chromatography jar is taken.
 A paper impregnated in the mobile phase is applied to the walls to ensure that
atmosphere of the jar is saturated with solvent vapors.
 Mobile phase is added to the jar at a length of 0.5-1cm from the bottom.
 Jar is closed.
 Equilibrium is allowed to be maintained.
 Base line is marked on adsorbent.
 Sample is applied on paper with help of capillary tube.
 Sample spot is air dried.
 Paper is put in the chromatography jar and lid is closed.
 The system is allowed to be static until the solvent move to a proper distance from
baseline.
 Paper is taken out and dried.

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Location of separated components:
If the sample is separated into colored components, then the location is dried in ordinary
light. But in case ofcolorless components following are used;
Uv lamp
Iodine crystals
Spraying agents
Documentation:
Storage of chromatogram.
Calculating Rf values
Applications:
It is used for separation and identification of;
Amino acids
Carbohydrates
Tannins
Glycosides
Alkaloids etc.

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Chromatography

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