Introduction to

Chromatography
ORIGIN OF TERM:
The word Chromatography has been derived from Greek word “chroma” mean "color" and
“graphein” mean "to write“.
DEFINITION:
Chromatography can be defined as
“A non-destructive procedure for resolving a complex mixture into its individual
fractions or compounds”.
OR
“A physical method of separation that distributes components to separate between two phases, one stationary
(stationary phase), the other (the mobile phase) moving in a definite direction”.

HISTORY:
Chromatography was first employed in Russia by the Italian-born scientist Mikhail Tsvet in 1906 during his research
on plant pigments. He used liquid-adsorption column chromatography with calcium carbonate as adsorbent and
petrol ether/ethanol mixtures as eluent to separate chlorophylls and carotenoids. The method was described on 30
December 1901. He continued to work with chromatography in the first decade of the 20th century, primarily 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.
New types of chromatography developed during the 1930s and 1940s made the technique useful for many separation
processes.
Chromatography technique developed substantially as a result of the work of Archer John Porter Martin and Richard
Laurence Millington Synge during the 1940s and 1950s. They established the principles and basic techniques of partition
chromatography, and their work encouraged the rapid development of several chromatographic methods designated as
paper chromatography, gas chromatography, and high performance liquid chromatography. Since then, the technology has
advanced rapidly. Researchers found that the main principles of Tsvet's chromatography could be applied in many different
ways, resulting in the different varieties of chromatography. Advances are continually improving the technical performance
of chromatography, allowing the separation of increasingly similar molecules.
COMPONENTS OF CHROMATOGRAPHY:

Mobile phase: A solvent that flows in a different direction through the supporting medium to elute the mixture
components. It may be a liquid (LC and Capillary Electro chromatography (CEC)), a gas (GC), or a supercritical
fluid (supercritical-fluid chromatography, SFC). The mobile phase consists of the sample being separated /analyzed
and the solvent that moves the sample through the column. In the case of HPLC the mobile phase consists of a non-
polar solvent(s) such as hexane in normal phase or polar solvents in reverse phase chromatography and the sample
being separated. The mobile phase moves through the chromatography column (the stationary phase) where the
sample interacts with the stationary phase and is separated.

Stationary phase: A layer of solid, gel or liquid film coating immobilized on the supporting medium that interacts
with the analyte to be separated. Examples include the silica layer in thin layer chromatography.

Supporting medium: A solid surface on which the stationary phase is bound or coated.

Eluate: The eluate is the mobile phase leaving the column.

Eluent: The eluent is the solvent that carries the analyte.

Eluotropic series: An eluotropic series is a list of solvents ranked according to their eluting power.
 PRINCIPLE:
Chromatography is introduced as a technique for separating and/or identifying the components in a mixture. The basic
principle is that components in a mixture have different tendencies to adsorb onto a surface or dissolve in a solvent. All
chromatographic methods require one static part (the stationary phase) and one moving part (the mobile phase).
The separation is based on differential partitioning between the mobile and stationary phases. Subtle differences in a
compound's partition coefficient result in differential retention on the stationary phase and thus changing the separation.
A.Adsorption
Adsorption chromatography was developed first. It has a solid stationary phase and a liquid or gaseous mobile phase.
(Plant pigments were separated at the turn of the 20th century by using a calcium carbonate stationary phase and a liquid
hydrocarbon mobile phase. The different solutes travelled different distances through the solid, carried along by the
solvent.) Each solute has its own equilibrium between adsorption onto the surface of the solid and solubility in the solvent,
the least soluble or best
Adsorbed ones travel more slowly. The result is a separation into bands containing different solutes. Liquid
chromatography using a column containing silica gel or alumina is an example of adsorption chromatography (Fig. 1).

The solvent that is put into a column is called the eluent, and the liquid that flows out of the end of the column is called the
eluate.
B.Partition
In partition chromatography the stationary phase is a non-volatile liquid which is held as a thin layer (or film) on the surface
of an inert solid. The mixture to be separated is carried by a gas or a liquid as the
mobile phase. The solutes distribute themselves between the moving and the stationary phases, with the more soluble
component in the mobile phase reaching the end of the chromatography column first (Fig. 2).
Paper chromatography is an example of partition chromatography.
C. Ion exchange
Ion exchange chromatography is similar to partition chromatography in that it has a coated solid as the stationary phase. The
coating is referred to as a resin (agarose, cellulose), and has ions (either cations or anions, depending on the resin) covalently
bonded to it and ions of the opposite charge are electrostatically bound to the surface. When the mobile phase (always a
liquid) is eluted through the resin the electrostatically bound ions are released as other ions are bonded preferentially (Fig. 3).
Ion exchange retains analyte molecules on the column based on coulombic (ionic) interactions. The stationary phase surface
displays ionic functional groups (R-X) that interact with analyte ions of opposite charge.ion exchange is further
subcategorized as:
1. Cation-exchange
Cation exchange retains positively charged cations because the stationary phase displays negatively charged functional
groups.
2. Anion-exchange
Anion exchange retains negatively charged anions because the stationary phase displays positively charged functional
groups.
Domestic water softeners work on this principle.
D. Molecular exclusion
Molecular exclusion differs from other types of chromatography in that no equilibrium state is established between the
solute and the stationary phase. Instead, the mixture passes as a gas or a liquid through a porous gel. The pore size is
designed to allow the large solute particles to pass through uninhibited. The small particles, however, permeate the gel and
are slowed down so the smaller the particles, the longer it takes for them to get through the column. Thus separation is
according to particle size (Fig. 4).
CHROMATOGRAPHIC METHODS CLASSIFICATION
Classification of chromatography according to the phase involved:
(a) The primary division of chromatographic techniques is based on the type of mobile phase used in the system:
Type of Chromatography Type of Mobile Phase
Gas chromatography (GC) gas
Liquid chromatography (LC) liquid

(b)Further divisions can be made based on the type of stationary phase used in the system:

Gas Chromatography

Name of GC Method Type of Stationary Phase

Gas-solid chromatography solid, underivatized support
Gas-liquid chromatography liquid-coated support
Bonded-phase gas chromatography chemically-derivatized support

Liquid Chromatography

Name of LC Method Type of Stationary Phase

Adsorption chromatography solid, underivatized support
Partition chromatography liquid-coated or derivatized support
Ion-exchange chromatography support containing fixed charges
Size exclusion chromatography porous support
Affinity chromatography support with immobilized ligand
(2) Classification according to the packing of the stationary phase:

(Methods of holding the Stationary Phase):

In column chromatography the stationary phase is contained in a tube called the column.
Planar chromatography. In this geometry the stationary phase is configured as a thin two-dimensional sheet .
(i) In paper chromatography a sheet or a narrow strip of paper serves as the stationary phase.
(ii) In thin-layer chromatography a thin film of a stationary phase of solid particles bound together for mechanical
strength with a binder, such as calcium sulfate, is coated on a glass plate or plastic or metal sheet.
(3)Classification according to the force of separation:
THEORY OF CHROMATOGRAPHY

CHROMATOGRAM:
A chromatogram is the visual output of the chromatograph. Different peaks or patterns on the chromatogram correspond
to different components of the separated mixture.
Plotted on the x-axis is the retention time and plotted on the y-axis a signal corresponding to the response created by the
analyte exiting the system. The signal is proportional to the concentration of the specific analyte separated.

CHROMATOGRAPH:
A chromatograph is equipment that enables a sophisticated separation, e.g. gas chromatographic or liquid
chromatographic separation.
RETENTION TIME:
The retention time is the characteristic time takes for a particular analyte to pass through the system (from the column
inlet to the detector) under set conditions.

Where:
tR = retention time
tM = void time
Wb = baseline width of the peak in time units
Wh = half-height width of the peak in time units
Retardation factor
The retardation factor (Rf) is defined as the ratio of the distance traveled by the center of a spot to the distance traveled
by the solvent front.
Rf can be mathematically described by the following ratio:
Rƒ = Distance travelled by a Solute
Distance travelled by a Solvent
OR
Distance traveled by component from application point
Rf =---------------------------------------------------------------------------
Distance traveled by solvent from application point

If Rƒ value of a solution is zero, the solute remains in the stationary phase and thus it is immobile.
If Rƒ value = 1 then the solute has no affinity for the stationary phase and travels with the solvent front.

Rf is a function of partition co-efficient and is a constant or a given substance, provided the conditions of chromatographic
system are kept constant.
FACTORS AFFECTING RF VALUE
 The temperature
 The purity of the solvents used
 The quality of the paper, adsorbents & impurities present n the adsorbents
 Chamber saturation techniques, method of drying & development
 The distance travelled by the solute & solvent
 Chemical reaction between the substances being partitioned.
 pH of the solution