CHROMATOGRAPHY

BY
Dr.A.S.CHARAN
CHROMATOGRAPHY
• It represent one of the most important and widely used separation
methods.
• It comprises of a group of powerful separation methods that find
applications in all branches of science.
• Chromatography was invented and named by the Russian botanist
Mikhail Tswett.
• He used the technique to separate various plant pigments such as
chlorophylls and xanthophylls by passing solutions of these
compounds through a glass column packed with finely divided
calcium carbonate.
• The separated species appeared as colored bands on the column,
which accounts for the name he chose for the method (Greek chroma
meaning “color” and graphein meaning “writing”).
• Stationary Phase
• The stationary phase in chromatography is a phase
that is fixed in place either in a column or on a
planar surface
• Mobile Phase
• The mobile phase in chromatography is a phase
that moves over or through the stationary phase
carrying with it the analyte mixture. The mobile
phase may be a gas, a liquid, or a supercritical
fluid.
• Sample Mixture
Definition
• Chromatography is a separation technique in which a mixture of components are
separated in to individual components by passing them through a stationary
phase and mobile phase which are immiscible.
• Chromatography is a technique in which the components of a mixture are
separated based on differences in the rates at which they are carried through a
fixed or stationary phase by a gaseous or liquid mobile phase.
• Chromatography encompasses a diverse and important group of methods that
allow the separation, identification, and determination of closely related
components of complex mixtures; many of these separations are impossible by
other means.
Classification of Chromatography
• Based on contact between SP & MP
• Column/Closed bed
• Planar/Open bed
• Based on the principle of separation
• Adsorption
• Partition
• Ion-exchange
• Size exclusion
• Chiral
• Affinity
 Based on the nature of SP & MP

Solid LSC

Liquid

Liquid LLC

Chromatography Solid GSC

Gas

Liquid GLC
Super critical
Fluid
Classification of Chromatography
• Based on Nomenclature & Instrumentation
• Column Chromatography (CC)
• Paper Chromatography (PC)
• Thin Layer Chromatography (TLC)
• High Performance Thin Layer Chromatography (HPTLC)
• High Performance Liquid Chromatography (HPLC)
• Gas Chromatography (GC)
• Ion Exchange Chromatography (IEC)
• Gel Chromatography/Size exclusion Chromatography (SEC)
• Chiral Chromatography
• Affinity Chromatography (AFC)
• Supercritical Fluid Chromatography (SFC)
Classification of Chromatography
• Based on polarity of MP & SP

SP: More Polar

Normal Phase
(NP)
MP: Less Polar

Chromatography

SP: Less Polar

Reverse Phase
(RP)
MP: More Polar
Classification of Chromatography
• Based on composition of MP

Same
Isocratic elution
composition

Gradual
MP Changing Gradient elution
composition

Changing
Step-wise elution
polarity
Classification of Chromatography
• Based on degree of separation and analysis

Frontal Analysis

Elutional
Chromatography
Analysis

Displacement
Analysis
Classification of Chromatography
• Based on scale of operation

Analytical

Chromatography Semi-preprative

Preparative
Theories of Chromatography
• Plate Theory
• Rate Theory

1. Plate Theory
• Plate theory - older; developed by Martin L
& Synge.
• View column as divided into a number (N)
of adjacent imaginary segments called
theoretical plates
• within each theoretical plate complete
equilibration of analytes between
stationary and mobile phase occurs HETP
HETP
Significance:
• Greater separation occurs with: – greater number of theoretical
plates (N) – as plate height (H or HETP) becomes smaller
• L = N× H or H = L / N where L is the length of column, N is the number
of plates, and H is the plate height
• Band spreading - the width of bands increases as their retention time
(volume) increases.
• The smaller HETP, the narrower the eluted peak
• Not unusual for a chromatography column to have millions of
theoretical plates
• Columns often behave as if they have different numbers of plates for
different solutes present in same mixture
Rate Theory
• Based on a random walk mechanism for the migration of molecules
through a column
Takes into account:
– mechanism of band broadening
– effect of rate of elution on band shape
– availability of different paths for different solute molecules to follow
– diffusion of solute along length
Van Deemter Equation for Plate Height

• H= Height Equivalent to a Theoretical plate

• A= Eddy’s diffusion
• B= Longitudinal diffusion
• C=Non-equilibrium mass transfer
• U=Mobile phase flow rate
A= Eddy’s diffusion
 B= Longitudinal diffusion

The effect of longitudinal diffusion on a solute’s band broadening. Two horizontal

cross-sections through the column and the corresponding concentration versus
distance profiles are shown, with (a) being earlier in time. The red arrow shows the
direction in which the mobile phase is moving.
 C=Non-equilibrium mass transfer

Effect of mass transfer on band broadening: (a) Ideal equilibrium Gaussian profiles for the solute in the mobile
phase and in the stationary phase. (b, c) If we allow the solute’s band to move a small distance down the
column, an equilibrium between the two phases no longer exits. The red arrows show the movement of solute—
what we call the mass transfer of solute—from the stationary phase to the mobile phase, and from the mobile
phase to the stationary phase. (d) Once equilibrium is reestablished, the solute’s band is now broader.
Plot showing the relationship between the height of a theoretical plate, H, and the mobile
phase’s velocity, u, based on the van Deemter equation.
Resolution