Chromatography

1. Chromatography
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Origin
 The word chromatography derived from Greek words (Chroma -
Color and Graphein - write).
 Chromatography was discovered by a Russian Botanist and Chemist,
Michael Tswett (Mikhail Semenovich Tsvett) in 1906.
 He primarily used chromatography for the separation of plant
pigments such as chlorophyll, carotenes, and xanthophylls.
 Since these components have different colors (green, orange, and
yellow, respectively) they gave the technique its name.

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 Chromatography is the term used to describe a separation technique in
which a mobile phase carrying a mixture is caused to move in contact
with a selectively absorbent stationary phase.
 IUPAC definition of chromatography (1993): Chromatography is a
physical method of separation in which the components to be separated
are distributed between two phases, one of which is stationary phase
while the other moves in a definite direction.
 Chromatography is a separation technique in which components of a
mixture are separated on the basis of their differential interaction
between the mobile phase and the stationary phase.
Difinitions

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 Mobile phase (eluant): The mobile phase is a gas or liquid which
transport the components of a mixture through the stationary phase.
The solution properties of a liquid mobile phase compete with the
retention forces of the stationary phase to determine the distribution ratio
and hence the elution time.
 Stationary phase (or absorbent): Solid or liquid immobilized on an
inert support, which attracts components in the mobile phase according
to characteristic retention forces and thus retards their progress through
the column or plate. The most common stationary phase for thin layer
chromatography and column chromatography is silica gel.
Two Phases

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 Elution is the term used in analytical and organic chemistry to describe the
extracting of one material from another by washing with a solvent.
 Three types of elutions
 Isocratic elution: The operation of chromatographic column by allowing a solvent
mixture of uniform composition (polarity) to run the column until the separation is
complete.
 Stepwise or Fractional elution: If one solvent system elutes one type of components
(some of the components) and to remove other type of components a different solvent
system can be used, this is known as stepwise elution. Sometime it may be necessary to
use several different solvent systems in fractional elution.
 Gradient elution: The continuous change in polarity of the solvent system during
chromatography is called gradient elution.
Elution

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 The separation of different components of a mixture occurs on the basis of relative
ability of each component to be moved along a stationary phase by a mobile phase.
Attraction of any component by the stationary phase causes retardation of that
component on the stationary phase. Since different components possesses different
affinity for the stationary phase which result in different migration velocities of the
components along the stationary phase.
 Different distribution of the analytes between mobile and stationary phase results in
different migration velocities.
 Components of a mixture are distributed between the two phases and equilibrium is
established.
CSP ↔ CMP
 CSP = Concentration of component in unit volume of stationary phase
 CMP = Concentration of component in unit volume of mobile phase
Theory and Principle

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 In chromatography distribution coefficient (K) is;
 K describes the relative affinity of component for two phases, hence relates to the
distance and speed with which it moves through the plate on elution.
 Large K value ensure the retardation of analyte on the stationary phase while
small K value means the components in the mobile phase.
 The equilibrium and hence the distribution coefficient (K) can be effected some
factors, which are;
i. Temperature
ii. Nature of the mobile phase
iii. Nature of the stationary phase
iv. Flow rate of the column

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Types of Chromatography
On the basis of phases
Chromatographic phases
Stationary phaseMobile phase
LiquidGas Solid
Gas Chromatography Liquid Chromatography
Gas-Liquid Chromatography Liquid-Solid Chromatography
Gas-Solid Chromatography
When both the phases (Stationary & Mobile phases) are liquid the chromatography is referred as Liquid-
Liquid Chromatography, an example is Liquid-Liquid Partition Chromatography

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On the basis of Scale (purpose)
Chromatography can be performed either on small scale or large scale depending upon
the purpose of separation, i.e.
 Analytical chromatography is done normally with smaller amounts of material to
determine the existence and possibly also the concentration of analyte(s) in
a mixture.
 Preparative chromatography is large scale chromatography and is used to purify
sufficient quantities of a substance for further use (and is thus a form of
purification).

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On the Basis of Mechanism of Separation
There are five main types depending upon the mechanism of separation
(1)Adsorption Chromatography
(2)Partition Chromatography
(3)Ion Exchange Chromatography
(4)Size Exclusion Chromatography
(5)Affinity Chromatography

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(1) Adsorption Chromatography
 Based on interaction between solute & fixed active sites on a solid stationary
phase.
 Active site in silica gel is silanol (silyl alcohol).
 In this type of chromatography the sample components are absorbed on a solid
stationary phase.
 The mobile phase may be gas, (e.g. Gas-Solid Chromatography), or liquid, (e.g.
Liquid-Solid Chromatography such as Column Chromatography, High Performance
Liquid Chromatography, Thin-Layer Chromatography and Paper Chromatography).
 Stationary phase either packed in tube, spread on plate or impregnated into porous
paper

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(2) Partition Chromatography
 Also called Bonded-Phase Chromatography
 Solute molecules distributed themselves between two immiscible phases on the basis of their
solubility.
 The liquid stationary phase is supported on an inert solid.
 The mobile phase may be a gas, (e.g. Gas-Liquid Chromatography), or liquid (e.g. Liquid-
Liquid Partition Chromatography).
 Paper chromatography is a typical example in which the stationary phase is a layer of water
absorbed on a sheet of paper.
 Two sub-types of partition chromatography are;
i. Normal phase partition chromatography: In normal mode, a polar stationary phase (e.g. water,
methanol, ethylene glycol etc) is used in combination with a non-polar mobile phase (e.g. hexane).
This favors retention of polar components and elution of non-polar components and is therefore
called "Normal-Phase Chromatography".
ii. Reversed Phase Partition Chromatography: In reversed-phase partition chromatography, non-polar
stationary phase is used in combination with a polar mobile phase, which favors retention of non-
polar components and elution of polar components. Octadecylsilane is stationary phase commonly
used in reversed phase chromatography.

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(3) Ion Exchange Chromatography
 Mobile phase is buffer solution.
 This method uses an ion exchange resin as the stationary phase. This resin is a
polymeric matrix containing ionic functional groups on its surface such as carboxylic
acids or protonated amines.
 The mechanism for separation is ion exchange equilibrium.
 As the liquid mobile phase passes over the ionic surface, causes the retention of ionic
solutes by forming electrostatic bond.
 This technique is used for the analysis of inorganic
ions (metallic ions), water soluble ionic compounds
e.g. proteins, amino acid, nucleotide etc

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(4) Size Exclusion Chromatography
 In this technique, a polymeric microporous substance is used as a stationary phase
(The stationary phase is inert).
 The mobile phase containing particles of analytes are separated according to their
size.
 This method is most suited for the separation of mixtures in which the solutes vary
considerably in molecular size.
 e.g. Size Exclusion Chromatography, Gel Permeation Chromatography

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(5) Affinity Chromatography
 This technique is highly specific between one kind of solute molecule and a second
molecule which is attached to the stationary phase.
 For example an immobilized antibody to a particular protein.
 When a crude mixture containing a large number of proteins passes through the
column, only the protein that reacts with the antibody is retained in the column.
 After washing all other proteins off the column, the desired protein is removed from
the antibody by changing the pH.