CHROMATOGRAPHY

Definition: “Chromatography is a physical method of separation in which the components to be

separated are distributed between two immiscible phases, one of which is stationary (stationary

phase) while the other moves in a definite direction (mobile phase).”

Chromatography may be preparative or analytical. The purpose of preparative

chromatography is to separate the components of a mixture for further use (and is thus a form

of purification). Analytical chromatography is done normally with smaller amounts of material

and is for measuring the relative proportions of analytes in a mixture. The two are not mutually

exclusive.

Chromatography is one of the versatile separation techniques and it is used to separate, analyze,

identify, purify and quantify the components present in the mixture.

The term chromatography was first introduced in 1906 by a Russian botanist, “Mikhail Tswett.”

The word chromatography is derived from two Greek words “chromatus” and “graphein”,

meaning “colour” and “to write”. He worked on the separation of chlorophylls and other
pigments including xanthophylls and carotenoids in a plant extract. Tswett discovered that by

washing the compounds through a column packed with an adsorbent medium (calcium

carbonate), the least adsorbed pigments were washed through the column quickly, while the

strongly adsorbed pigments immobilized by their attraction to the column packing. Adsorption in

this column process is directly related to the affinity of the solute for either the adsorbent

(stationary phase) or the flowing solvent (mobile phase) and the process is generally referred to

as column chromatography. So, chromatography is a technique for separating a sample into its

constituent components and then measuring or identifying the components in some way. The

components to be separated are distributed between two naturally immiscible phases.

The heart of any chromatography is the stationary phase, which is sometimes a solid and

sometimes a liquid. The liquid stationary phase is attached to a support, a solid inert material.

The sample, often in vapour form or dissolved in a solvent, is moved across or through the

stationary phase. It is pushed along by liquid or gas-the mobile phase. As the mobile phase

moves through the stationary phase, the sample components undergo a large number of

exchanges (partitions) between the two phases.

The difference in the chemical and physical properties of the components in the sample is used to

bring about the separation and govern the rate of movement (called migration) of the individual

components.
Basic Principle of Chromatography:

In Chromatography, the components to be separated are distributed between two phases;

stationary phase and mobile phase. These components are separated due to their relative

affinities for the stationary and mobile phase. Separation of the sample components is based on

the difference in rates of migration among the components. The mixture components are

distributed between two phases according to the distribution or partition coefficient (Kc).

Kc= [Xs] / [Xm]

⸫ Xs = Concentration of Solute Components in the Stationary Phase.

Xm = Concentration of Solute Components in the Mobile Phase.

STATIONARY PHASE:

The stationary phase is one of the two phases forming a chromatographic system. It may be a

solid, a gel or a liquid. If a liquid, it may be supported on a solid. This solid may or may not
contribute to the separation process. This liquid may also be chemically bonded to the solid

(bonded phase) or immobilized onto it (immobilized phase).

• Bonded phase: A stationary phase which is covalently bonded to the support particles or

to the inside wall of column tubing.

• Immobilized phase: A stationary phase which is immobilized on the support particles or

on the inner wall of the column tubing.

MOBILE PHASE:

A fluid which percolates through or along the stationary bed, in a definite direction is called

mobile phase. It may be a liquid or a gas. In gas chromatography the expression carrier gas may

be used for the mobile phase.

Terms that are often used in Chromatography:

Effluent: “The mobile phase leaving the column is called effluent.”

Sample: “The mixture consisting of a number of components, the separation of which is

attempted on the chromatographic bed as they are carried or eluted by the mobile phase.”

Sample components: “These are chemically pure constituents of the sample. They may be un-

retained (i.e. not delayed) by the stationary phase, partially retained (i.e. eluted at different times)

or retained permanently.”

The term Elute or Analyte are also acceptable for a sample component.

Zone: “A region in the chromatographic bed where one or more components of the sample are

located is called zone. The term Band may also be used for it.”
Chromatogram: “A chromatogram is the visual output of the chromatography. In the case of an

optimal separation, different peaks or patterns on the chromatogram correspond to different

components of the separated mixture.”

Figure: Showing chromatogram

CLASSIFICATION OF CHROMATOGRAPHY:

CLASSIFICATION ACORDING TO THE PHYSICAL STATE OF THE MOBILE

PHASE:

1. Gas chromatography (GC):

Gas chromatography (GC) is a separation technique in which the mobile phase is a gas. The

stationary phase can be a solid or liquid. If stationary phase is solid then it is termed as “Gas-

Solid Chromatography (GSC)” and if stationary phase is liquid then it is termed as “Gas-Liquid

chromatography (GLC)”.

Gas chromatography is always carried out in a column, which is typically "packed" or

"capillary". Gas chromatography (GC) is based on a partition equilibrium of analyte between a

solid stationary phase (often a liquid silicone-based material) and a mobile gas (most often
Helium). The stationary phase is adhered to the inside of a small-diameter glass tube (a capillary

column) or a solid matrix inside a larger metal tube (a packed column). It is widely used in

analytical chemistry and well suited for use in the petrochemicals, environmental monitoring and

remediation, and industrial chemical fields. It is also used extensively in chemical research.

2. Liquid chromatography: (LC)

Liquid chromatography (LC) is a separation technique in which the mobile phase is a liquid.

Stationary phase can be a Solid or a liquid supported on solid. If stationary phase is solid, then it

is termed as “Liquid-Solid Chromatography (LSC)” and if the stationary phase is liquid then it is

termed as “Liquid-Liquid Chromatography (LLC)”.

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 referred to as high performance liquid chromatography (HPLC).

In the HPLC technique, the sample is forced through a column that is packed with a stationary

phase composed of irregularly or spherically shaped particles, a porous monolithic layer, or a

porous membrane by a liquid (mobile phase) at high pressure. HPLC is historically divided into

two different sub-classes based on the polarity of the mobile and stationary phases. Methods in

which the stationary phase is more polar than the mobile phase (e.g. toluene as the mobile phase,

silica as the stationary phase) are termed normal phase liquid chromatography (NPLC) and the

opposite (e.g. water-methanol mixture as the mobile phase and C18 silica = octadecylsilyl as the

stationary phase) is termed reversed phase liquid chromatography (RPLC). Ironically the

"normal phase" has fewer applications and RPLC is therefore used considerably more.
CLASSIFICATION ACCORDING TO THE MECHANISM OF SEPARATION:

1. Adsorption chromatography:

A separation technique in which separation is based mainly on the difference between the

adsorption affinities of the sample components for the surface of an active solid.

It is one of the oldest types of chromatography.

2. Partition chromatography:

A separation technique in which separation is based mainly on differences between the

solubilities of the sample components in the stationary phase (gas chromatography) or on

differences between the solubilities of the components in the mobile and stationary phase

(liquid chromatography).
3. Ion-exchange chromatography:

A separation technique in which separation is based mainly on differences in the ion

exchange affinities of the sample components. Ion-exchange chromatography (or ion

chromatography) is a process that separates ions and polar molecules based on their

affinity to the ion-exchanger.

Principle:

Ion exchange chromatography retains analyte molecules based on ionic interactions. The

stationary phase surface displays ionic functional groups (R-X) that interact with the

analyte ions of opposite charge.

Stationary Phase used in ion-exchange chromatography is “ion-exchange resins bonded to

solid support” and “buffer solution” are used as Mobile Phase.

i. Cation Exchange Chromatography:

“Cation exchange chromatography retains positively charged cations because the

stationary phase surface displays negatively charged functional groups.”

ii. Anion Exchange Chromatography:

“Anion exchange chromatography retains anions using positively charged

functional groups on the surface of stationary phase.”

Exclusion chromatography is a separation technique in which separation is based mainly

on exclusion effects, such as differences in molecular size and/or in charge. The term

“Size-exclusion chromatography” may also be used when separation is based on

molecular size. It separates molecules according to their size, shape & molecular weight.

It is usually applied to large molecules or macromolecular complexes such as proteins

and industrial polymers. Porous gels or porous beads are used as Stationary phase,

however aqueous or organic solvent is used as mobile phase.

Size exclusion chromatography was first invented by Grant Henry Lathe and Colin R

Ruthven.

Principle:

Size-exclusion chromatography works by trapping smaller molecules in the pores of the

adsorbent material (porous beads) which acts as stationary phase. The larger molecules

simply pass by the pores because those molecules are too large to enter the pores.

Therefore the larger molecules are excluded and flow through the column more quickly

than smaller molecules and are separated first. In short, the smaller the molecule, the

longer the retention time.

This expression characterizes the particular variant of chromatography in which the

unique biological specificity of the analyte and ligand interaction is utilized for the

separation.

It is the most selective type of chromatography and is a method generally used for

separating bio-molecules from a mixture, based on highly specific binding interaction

between one kind of solute molecule and a second molecule that immobilized on the

stationary phase such as the interaction between antigen and antibody or enzyme and

substrate or receptor and ligand.

CLASSIFICATION ACCORDING TO THE SHAPE OR GEOMETRY OF

CHROMATOGRAPHIC BED:

1. Column chromatography:

“It is a separation technique in which the stationary phase is present within the column or

a tube”. Two types of columns are used in chromatography:

a) Packed column: The particles of the solid stationary phase or the support coated with

a liquid stationary phase are fully packed inside the column and may fill the whole

inside volume of the tube.

They are usually constructed of glass or metal (stainless steel, aluminium, and

copper).

b) Capillary column: The particles of the solid stationary phase or the liquid stationary

phase supported on solid are coated on the inner surface of column or tube. They are
 also called “Open Tubular columns” because the middle part of the tube has an open

and unrestricted path for the mobile phase.

They are usually constructed of fused silica with a coating of polymer on it.

2. Planar chromatography:

“It is a separation technique in which the stationary phase is present as or on a planar

surface.”

That planar surface can be a paper as in case of paper chromatography (PC) or it can be a

layer of solid particles spread on a support e.g. a glass plate coated with thin layer of

stationary phase as in case of thin layer chromatography (TLC). Sometimes planar

chromatography is also termed as open bed chromatography.