Chromatography 1

PRINCIPLES
Chromatography usually consists of mobile phase and

stationary phase.
 The mobile phase refers to the mixture of substances to

be separated dissolved in a liquid or a gas.
The stationary phase is a porous solid matrix through

which the sample contained in the
mobile
. phase percolates.
The interaction between the mobile phase and the

stationary phase results in the separation of the
compound from the mixture
APPLICATIONS OF CHROMATOGRAPHY
 The chromatographic technique is used for the

separation of amino acids,proteins & carbohydrates.
 It is also used for the analysis of
drugs,hormones,vitamins
 Helpful for the qualitative & quantitative analysis of
complex mixtures.
 The technique is also useful for the determination of
molecular weight of proteins.
Paper chromatography
 Paper chromatography is a technique that involves

placing a small dot or line of sample solution onto a
strip ofchromatographypaper. The paper is placed in a
jar containing a shallow layer of solventand sealed. As
the solvent rises through the paper, it meets the
sample mixture, which starts to travel up the paper
with the solvent.


 Rf= Distance travelled by the substance

Distance travelled by the solvent front
The Rf value helps for the identification of unknown.

Thin layer chromatography
 Thin layer chromatography (TLC) is a widely employed

laboratory technique and is similar to paper
chromatography. However, instead of using a
stationary phase of paper, it involves a stationary phase
of a thin layer of adsorbent like silica gel, alumina,
or cellulose. Compared to paper, it has the advantage
of faster runs, better separations, and the choice
between different adsorbents.
THIN LAYER CHROMATOGRAPHY
ADSORPTION TLC
 Adsorbents such as activated silica gel, alumina,

kieselguhr are used.
ADSORPTION COLUMN
CHROMATOGRAPHY
 Column chromatography is a separation technique in

which the stationary bed is within a tube.Adsorbents
are packed into a column in a glass tube.This serves as
the stationary phase,leaving an open unrestricted path
for the mobile phase in the middle of the tube.
 Adsorbents such as silica gel, alumina, charcoal

powder & calcium hydroxyapatite are used.

 The sample mixture in a solvent is loaded on this
column.The individual compounds get differentially
adsorbed on to the adsorbent.
 The elution is carried out by the buffer system which is

the mobile phase.
 The individual compounds come out of the column at

different rates which may be collected separately &
identified.
COLUMN CHROMATOGRAPHY
ION EXCHANGE CHROMATOGRAPHY
 Ion exchange chromatography (usually referred to as

ion chromatography) uses an ion exchange
mechanism to separate molecules on the basis of their
electrical charges.Ion exchange chromatography uses a
charged stationary phase to separate charged
compounds including anions, cations, amino
acids, peptides, and proteins.
 Cation exchangers & anion exchangers are used as ion
exchange resins.

In conventional methods the stationary phase is
an ion exchange resin that carries charged functional
groups that interact with oppositely charged groups of
the compound to retain.
ION EXCHANGE CHROMATOGRAPHY
GEL FILTRATION CHROMATOGRAPHY
 Size-exclusion chromatography (SEC) is also known

as gel permeation chromatography (GPC) or gel
filtration chromatography and separates molecules
according to their size, shape & molecular weight.
 It is also referred to as molecular sieving or molecular

exclusion chromatography.


Smaller molecules are able to enter the pores of the
media and, therefore, molecules are trapped and
removed from the f low of the mobile phase. However,
molecules that are larger than the average pore size of
the packing are excluded and thus suffer essentially no
retention; such species are the first to be eluted. This is
how the molecules are separated.
 It is generally a low-resolution chromatography
technique and thus it is often reserved for the final,
"polishing" step of a purification. It is also useful for
determining the tertiary structure and quaternary
structureof purified proteins, especially since it can be
carried out under native solution conditions.
GEL FILTRATION CHROMATOGRAPHY
AFFINITY CHROMATOGRAPHY
 Affinity chromatography is based on selective non-

covalent interaction between an analyte and specific
molecules, referred to as ligands.
 The immobilized ligands act as molecular fish-hooks

& selectively pick up desired proteins while the
remaining protein pass through the column.
HIGH PERFORMANCE LIQUID
CHROMATOGRAPHY (HPLC )
 Liquid chromatography (LC) is a separation technique in
which the mobile phase is a liquid. Liquid chromatography
can be carried out either in a column or a plane. Present
day liquid chromatography that generally utilizes very
small packing particles and a relatively high pressure is
high performance liquid
referred to as
chromatography (HPLC).
 Since the chromatographic techniques are slow & time
consuming,hence the separation can be greatly improved
by using high pressure in the range of 5000-10000
psi(pounds per square inch),hence this technique is also
referred to as high pressure liquid chromatography.
 In HPLC the sample is forced by a liquid at high
pressure (the mobile phase) through a column that is
packed with a stationary phase composed of
irregularly or spherically shaped particles.
 The interaction between the mobile and the stationary

phase leads to the separation of the mixture.
HIGH PERFORMANCE LIQUID CHROMATOGRAPHY

Chromatography 1

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PRINCIPLES

Chromatography usually consists of mobile phase and

 The mobile phase refers to the mixture of substances to

The stationary phase is a porous solid matrix through

The interaction between the mobile phase and the

 The chromatographic technique is used for the

 Paper chromatography is a technique that involves

 Rf= Distance travelled by the substance

The Rf value helps for the identification of unknown.

 Thin layer chromatography (TLC) is a widely employed

 Adsorbents such as activated silica gel, alumina,

 Column chromatography is a separation technique in

 Adsorbents such as silica gel, alumina, charcoal

 The elution is carried out by the buffer system which is

 The individual compounds come out of the column at

 Ion exchange chromatography (usually referred to as

 Size-exclusion chromatography (SEC) is also known

 It is also referred to as molecular sieving or molecular

 Affinity chromatography is based on selective non-

 The immobilized ligands act as molecular fish-hooks

 The interaction between the mobile and the stationary

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