Pharmacognosy By Sir Tanveer

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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 column tubing.

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 a particular 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 ):
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Lecture: Chromatography

Fraction of an analyte in the mobile phase of a chromatographic

system.

CLASSIFICATION
According to mechanism of separation
1)
2)
3)
4)

Ion-exchange chromatography
Affinity chromatography
Size-exclusion chromatography
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 analyte ions 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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Pharmacognosy By Sir Tanveer

Khan

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Lecture: Chromatography

TYPES:

Cation exchange chromatography:

Cation exchange chromatography retains positively charged cations
because 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 Pedro Cuatrecasas 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:

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Pharmacognosy By Sir Tanveer

Khan

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Lecture: Chromatography

It utilizes the specific interaction between one kind of

solute molecule and a second

immobilized on a stationary phase.

molecule

that

is

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

APPLICATIONS:
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Pharmacognosy By Sir Tanveer

Khan

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Lecture: Chromatography

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:
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
analyte molecules . However, molecules that are larger than the average
pore size of the packing are excluded.

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Pharmacognosy By Sir Tanveer

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Lecture: Chromatography

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:
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
chromatography

Thin layer
chromatography

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Gas solid
chromatography

Pharmacognosy By Sir Tanveer

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Lecture: 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
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
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Lecture: Chromatography

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
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.
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Lecture: Chromatography

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, then the location is dried in
ordinary light but in case of colorless
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 for
repeated 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
Applications:
It is used for separation and identification of;
Amino acids
Peptides and proteins
Alkaloids
Carbohydrates
Fats and fatty acids
Antibiotics
Narcotic analgesics
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Lecture: Chromatography

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

PAPER
CHROMATOGRAPHY
Gas-liquid

Instrumentation:
chromatography
chromatography
Chromatography jar
Capillary tube
Stationary phase (liquid impregnated paper)
Mobile phase
Chromatography jar:

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Pharmacognosy By Sir Tanveer

Khan

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Lecture: Chromatography

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.
Location of separated components:
If the sample is separated into colored components, then the location
is dried in ordinary light. But in case of colorless components following
are used;
Uv lamp
Iodine crystals
Spraying agents
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Pharmacognosy By Sir Tanveer

Khan

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Lecture: Chromatography

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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