CHROMATOGRAPHY

Dr. Anita Motiani

MBBS, MD
Assistant Professor
AIIMS, Rajkot
Introduction

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◦ Tswet- Russian Botanist- Father of Chromatography
◦ Principle: Differential affinities (strength of adhesion) of the
various components of the analyte towards the stationary and
mobile phase results in the differential separation of the
components. In turn, Affinity is dictated by the molecule’s
properties: ‘Adsorption’ and ‘Solubility’.
◦ We can define adsorption as the property of how well a component
of the mixture sticks to the stationary phase, while solubility is the
property of how well a component of the mixture dissolves in the
mobile phase.

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 • The higher the adsorption to the stationary phase, the slower the
molecule will move through the column.
• The higher the solubility in the mobile phase, the faster the
molecule will move through the column.
Term Definition
Mobile phase or carrier solvent moving through the column
Stationary phase or adsorbent substance that stays fixed inside the column
Eluent fluid entering the column
Eluate fluid exiting the column (that is collected in flasks)
the process of washing out a compound through a column using a
Elution
suitable solvent

mixture whose individual components have to be separated and

Analyte
analyzed 08/06/2024
◦ The components that adhere more strongly to the stationary phase travel
more slowly compared to those with a weaker adhesion.
◦ Analytical chromatography can be used to purify compounds ranging from
milligrams to gram scale.
◦ The adsorption and solubility of a molecule can be manipulated by choosing
the appropriate stationary phase and mobile phase.
◦ Now, the question arises why do different compounds possess different
affinities toward the stationary and mobile phases? “Polarity” of the
compounds dictates their affinities towards the stationary and mobile
phases.

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 TYPES OF CHROMATOGRAPHY
◦ Normal-phase chromatography - stationary phase is polar (hydrophilic) in nature; the mobile phase is
non-polar (hydrophobic) in nature.
◦ Reverse-phase chromatographic techniques where the scenario is reversed i.e. the stationary phase is non-
polar while the mobile phase is polar.

Basis of
Technique Stationary phase Mobile phase Notes
separation
compound spotted
*Paper polarity of
solid (cellulose) liquid directly on a
chromatography molecules
cellulose paper
glass is coated with
*Thin layer
solid (silica or polarity of thin layer of silica
chromatography liquid
alumina) molecules on which is spotted
(TLC)
the compound
glass column is
*Liquid column solid (silica or polarity of
liquid packed with slurry
chromatography alumina) molecules
of silica
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 small molecules get
trapped in the pores of the
stationary phase, while
large molecules flow
through the gaps between
the beads and have very
Size exclusion solid (microporous small retention times. So
liquid size of molecules
chromatography beads of silica) larger molecules come out
first. In this type of
chromatography there isn’t
any interaction, physical
or chemical, between the
analyte and the stationary
phase.

molecules possessing the

opposite charge as the
resin will bind tightly to
Ion-exchange solid (cationic or ionic charge of the the resin, and molecules
liquid
chromatography anionic resin) molecules having the same charge as
the resin will flow through
the column and elute out
first.
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 if the molecule is a
substrate for the enzyme, it
will bind tightly to the
enzyme and the unbound
solid (agarose or porous analytes will pass through
binding affinity of the in the mobile phase, and
glass beads on to which
Affinity analyte molecule to the elute out of the column,
are immobilized liquid
chromatography molecule immobilized leaving the substrate
molecules like enzymes
on the stationary phase bound to the enzyme,
and antibodies) which can then be
detached from the
stationary phase and eluted
out of the column with an
appropriate solvent.

samples are volatilized and

the molecule with lowest
boiling point comes out of
gas (inert gas like argon boiling point of the
Gas chromatography liquid or solid support the column first. The
or helium) molecules molecule with the highest
boiling point comes out of
the column last.
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• Gel-permeation (molecular sieve) chromatography

• Dye-ligand chromatography

• Hydrophobic interaction chromatography

• Pseudoaffinity chromatography

• High-pressure liquid chromatography (HPLC)

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Retention factors (Rf)

◦ Retention factor (Rf)- quantitative parameter that can be calculated

for every individual component

◦ The distance traveled by the individual component is divided by the

total distance traveled by the solvent. ‘Lower the Rf, the more polar
the component.’

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 Thin layer chromatography (TLC)
◦ Performed on a piece of glass plate, coated with a thin layer of silica.
◦ Silica  stationary phase, and the solvent  mobile phase.
◦ The stationary phase  polar in nature, mobile phase  less polar
compared to silica.
◦ The polar components of the analyte will adhere to the silica tightly and
thus travel slowly up the plate, while the less polar or non-polar
components will not adhere that strongly to the silica and travel up the plate
relatively fast with the solvent
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◦ So just by looking at a TLC plate, we can tell which component is
more polar and which component is less polar.

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COLUMN CHROMATOGRAPHY

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 ION-EXCHANGE CHROMATOGRAPHY
◦ Based on electrostatic interactions between charged protein groups, and solid
support material (matrix).
◦ Matrix has an ion load opposite to that of the protein to be separated, and the
affinity of the protein to the column is achieved with ionic ties.
◦ Proteins are separated from the column either by changing pH, the
concentration of ion salts, or the ionic strength of the buffer solution.
◦ Positively charged ion-exchange matrices are called anion-exchange
matrices and adsorb negatively charged proteins. While matrices bound with
negatively charged groups are known as cation-exchange matrices and
adsorb positively charged proteins 08/06/2024
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 GEL PERMEATION (MOLECULAR SIEVE)
CHROMATOGRAPHY
◦ We use dextran containing materials to separate macromolecules based on their
differences in molecular sizes.
◦ Used to determine molecular weights of proteins, and to decrease salt concentrations of
protein solutions
◦ Stationary phase  inert molecules with small pores.
◦ The solution containing molecules of different dimensions are passed continuously with a
constant flow rate through the column.
◦ Molecules larger than pores can not permeate into gel particles, and they are retained
between particles within a restricted area.
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◦ Larger molecules pass through spaces between porous particles and move rapidly
through inside the column.
◦ Molecules smaller than the pores are diffused into pores, and as molecules get smaller,
they leave the column with proportionally longer retention times

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 AFFINITY CHROMATOGRAPHY
◦ Used for the purification of enzymes, hormones, antibodies, nucleic acids, and specific
proteins.
◦ A ligand that can make a complex with a specific protein (dextran, polyacrylamide,
cellulose, etc) binds the filling material of the column.
◦ The specific protein which makes a complex with the ligand is attached to the solid
support (matrix), and retained in the column, while free proteins leave the column.
◦ Then the bound protein can be freed from the column by means of changing its ionic
strength through the alteration of pH or the addition of a salt solution

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PAPER CHROMATOGRAPHY
◦ In paper chromatography support material consists of a layer of
cellulose highly saturated with water.
◦ In this method, a thick filter paper comprises the support, and water
drops settled in its pores make up the stationary “liquid phase.”
◦ The mobile phase consists of an appropriate fluid placed in a
developing tank.
◦ Paper chromatography is a “liquid-liquid” chromatography

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 HPLC
◦ Possible to perform structural, and functional analysis, and purification of many
molecules within a short time,
◦ Yields perfect results in the separation, and identification of amino acids,
carbohydrates, lipids, nucleic acids, proteins, steroids, and other biologically active
molecules,
◦ In HPLC, mobile phase passes throuıgh columns under 10–400 atmospheric
pressure, and with a high (0.1–5 cm/sec) flow rate.
◦ In this technique, use of small particles, and application of high pressure on the rate
of solvent flow increases separation power, of HPLC and the analysis is completed
within a short time.

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◦ HbA1c estimation
◦
◦ Hemoglobinopathies- Hb variants

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 APPLICATIONS IN MEDICINE
• Pharmaceutical industry - analyze and identify the presence of any trace amounts of
chemicals and elements in a given sample.
• Separation of certain chemical compounds based on their molecular masses (and
sometimes on the basis of the elements that constitute them).
• Development of new drugs. For example, the presence of impurities and other unknown
compounds can be detected in the drug sample with the help of chromatography.
Furthermore, the purity of the drug sample can also be analyzed.
• The study of proteomics and metabolomics often involves the use of various hyphenated
chromatographic techniques
• Nucleic acid research
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