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Chromatography
Definition
“Chromatography refers to a group of method used for separating molecular mixtures depending upon
the differential affinities of solute between two immiscible phases.”

One phase is a fixed bed of large surface area called stationary phase.

Other is a fluid or gas which moves through over the surface of stationary phase called mobile phase.

Stationary phase may be porous or finely divided solid or liquid that has been coated as a thin layer on
an inert support material

Mobile phase may be a pure liquid or a mixture of solvents or a gas or mixture of gases.

Essentially the technique of chromatography is based on the differences in the rate at which the
components of a mixture move through a porous stationary phase under the influence of mobile phase.

Type of Chromatography On the Basis of Principle Involved

Adsorption Chromatography
This chromatographic procedure was originated by T. Swett.

If the stationary phase is solid, the process is known as adsorption chromatography.

In A.C. the mobile phase containing the dissolved solutes passes over the surface of stationary phase
(solid adsorbent).

Retention and separation depends on the ability of atoms on the surface to remove the solutes from the
mobile phase and adsorb temporarily (by electrostatic force, hydrogen bonding or Vander Waal forces).

If the mobile phase is a liquid, the process is known as liquid solid chromatography, and if is a gas than
gas solid chromatography.

Commonly used adsorbent are: Starch, silica gel, talc, charcoal, alumina, calcium phosphate etc.

Adsorption chromatography is used for the separation of vitamins, hormones, alkaloids, cardiac
glycosides, anthraquinone

Steps involved
1. Adsorption/ retention of compounds on stationary phase
2. Separation of adsorbed compound by the mobile phase (desorption or detachment)
3. Recovery of separated substances or compound by continuous flow of mobile phase, the
process is called elution.
4. Qualitative analysis and quantitative analysis of eluted substances (analytes)

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Partition Chromatography (Liquid – Liquid Chromatography)

If the stationary phase is a liquid the process is called partition chromatography. Partition
chromatography was introduced by Martin and Sunge in 1941 for separation of acetylated ammonia
acids.

IN partition chromatography solutes are separated on the basis of their relative tendency to partition
between stationary and mobile phase (on the basis of partition coefficient)

An inert solid like silica gel is used to support a thin film of a liquid usually water, which act as stationary
phase.

When the mobile phase containing solutes passes through the stationary phase (which usually water)
retention and separation occur due to relative solubility of solutes in the two liquids determined by the
partition coefficient

If the mobile phase is a liquid, the process is called liquid-liquid chromatography and if it is a gas than
gas-liquid chromatography (GLC)
𝐶1
𝑃𝑎𝑟𝑡𝑖𝑡𝑖𝑜𝑛 𝐶𝑜𝑒𝑓𝑓𝑖𝑐𝑖𝑒𝑛𝑡 (𝑘) =
𝐶2

Ion Exchange Chromatography

In ion exchange chromatography the stationary phase consists of polymeric resin matrix. Ionic functional
groups i.e. carboxylic or quaternary amines are chemically attached to the surface of this matrix.

As the mobile phase passes over this surface ionic solutes are retained by forming electrostatic bonds
with the functional group (present in matrix). Liquid mobile phase are used always. It is useful for
separation of inorganic cations or amino acids. The most frequently used ion exchange materials are
inorganic copolymers made from styrene.

Size Exclusion Chromatography

It is also known as Gel chromatography. The stationary phase is a polymeric substance containing
numerous pores of molecular dimension. Solutes with small molecular size can diffuse into the pores of
gel beats and remain in gel for longer period of time. They take longer/circuitous path to pass through
the column. While larger molecules because of their larger size cannot enter/diffuse into the gel matrix
and consequently remain in the mobile phase following with it through the spaces between the gel
beats and are eluted at a much faster rate than separation of a mixture containing solutes of different
molecular size. Mobile phase can be a liquid or a gas.

Affinity Chromatography
This method depends upon reversible binding of individual particles usually proteins with a particular
ligand. Ligand refers to a substrate, product, co-enzyme, allosteric effectors or any other substance that
interacts reversibly and specifically with the protein or other macromolecules to be separated.

The ligand is attached to a suitable carrier such as cellulose, beaded agrose or cross linked dextran
packed in a column. The protein with sufficient affinity for the bound or attached ligand is retarded or
retained and can be later eluted in pure form by changing ionic strength or pH of the column buffer.

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This technique is used for separation of enzymes, antibiotics, mRNA, antigen and for fractionation of
blood cells like RBCs and lymphocytes.

Isocratic Elution

In isocratic elution, the mobile phase consisting of a single solvent or different solvent in a fixed ration
is used throughout the process of elusion. Ratio of solvent does not change.

Gradient Elution

In gradient elution the mobile phase is changes during the elusion process. The ration of solvent
included in the mobile phase does not remain fixed.

Types of Chromatography on the Basis of Modes/ Equipment

Column Chromatography
In column chromatography the stationary phase is packed in a column and the mobile phase is allowed
to pass through it. The phase is allowed to pass through it. The mixture to be separated is placed on the
top of stationary phase in the column. If the stationary phase is adsorbent than the column
chromatography is called adsorption column chromatography.

It can also be used for other types like partition, size elusion, ion extraction etc.

Thin Layer Chromatography

TLC is based upon adsorption or partition chromatography. It was first used by Stahl in 1958. It is an
important tool for analysis of natural compound as well as synthetic compound in pharmaceutical
industry or forensics. The adsorbent or stationary phase is applied by coating up to a thickness of 0.3
mm on a clean glass plate or aluminum foil. The plates are than activated at 105’C for 30 minutes. The
spots are located by either UV light (254nm or 366nm) or by using chemical reagent.

Different compounds have different Rf Values

𝐷𝑖𝑠𝑡𝑎𝑛𝑐𝑒 𝑡𝑟𝑎𝑣𝑒𝑙𝑙𝑑 𝑏𝑦 𝑠𝑜𝑙𝑢𝑡𝑒𝑠
𝑅𝑓 =
𝐷𝑖𝑠𝑡𝑎𝑛𝑐𝑒 𝑡𝑟𝑎𝑣𝑒𝑙𝑙𝑒𝑑 𝑏𝑦 𝑠𝑜𝑙𝑣𝑒𝑛𝑡
ℎ𝑅𝑓 = 𝑅𝑓 × 100

Paper Chromatography
Paper chromatography is a type of partition or liquid-liquid chromatography. It is economical. Filtered
paper is used for this purpose. Separation of molecules occurs between water molecules trapped with
cellulose and mobile phase

Rf calculation is like TLC.

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HPLC- High Performance Liquid Chromatography

In HPLC mostly adsorption and partition chromatography is performed (other can also be performed). In
HPLC separation take place under high pressure (to increase flow). HPLC is most versatile dependable
faster and sensitive type of chromatography. It is frequently used in quality control of drugs.

Gas- Liquid Chromatography

Used by Jane and Martin in 1952. This technique is used for separating volatile oil or volatile substance.
The basis of separation is partitioning of sample between gas and liquid. Gas is the mobile phase and
liquid (stationary phase)

Nitrogen and helium are most commonly used gases. High temperature are used to produce
fragmentation of less or non-volatile compounds.

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