Chromatography

Principle,Types,and Application

CHROMATOGRAPHY Chromatography is a physical method of separation in which the components to be separated are distributed between two phases. It is a analytical process used to separate and purify organic and inorganic substances and macromolecules.

Chromatography
It is equally important for the fraction of Complex mixtures. Seperation of closely related compounds. Isomers. Unstable compounds .

History
Mikhail Tswett, Russian, 1872-1919 (Botanist)
In 1906 Tswett used to chromatography to separate plant pigments He called the new technique chromatography because the result of the analysis was 'written in color' along the length of the adsorbent column
Chroma means color and graphein means to write

Principle
Based on adsorption and differential migration of components of a mixture. This requires 2 alternative phases, mobile phase consisting of moving solvent and stationary/immobile phase called matrix. The mobile phase moves through the stationary phase. The materials forming stationary phase contain sites to which molecules from mobile phase can bind.

 As individual molecules from mobile phase interact with materials of stationary phase, their progress through the immobile phase is retarded this is called impedence.
Greater the affinity of a particular type of molecules for the stationary phase , slower the movement . Since different components of a mixture have different affinity for the stationary phase, so they are retarded to different degrees. The molecules with least affinity for the stationary phase are retarded the least and appear in the first fraction in the stationary phase. Thus , Chromatography works on propelling of molecules through chromatographic column and their selective impedence by matrix (stationary phase).

Classification
a. b.

According To Mobile Phase:

Liquid chromatography: mobile phase is a liquid. (LLC, LSC). Gas chromatography : mobile phase is a gas. (GSC, GLC).

Classification
According To The Packing Of The Stationary Phase:

1- Thin layer chromatography (TLC): the stationary phase is a thin layer supported on glass, plastic or aluminium plates. 2- Paper chromatography (PC): the stationary phase is a thin film of liquid supported on an inert support.
3- Column chromatography (CC): stationary phase is packed in a glass column

Classification
According To The Force Of Separation:

1- Adsorption chromatography.

2- Partition chromatography.
3- Ion exchange chromatography. 4- Gel filtration chromatography.

5- Affinity chromatography

Paper Chromatography
A method of partition chromatography using filter paper strips as carrier or inert support. The factor governing separation of mixtures of solutes on filter paper is the partition between two immiscible phases. One is usually water adsorbed on cellulose fibres in the paper (stationary phase). The second is the organic solvent flows past the sample on the paper (mobile phase).

Paper chromatography

Application
It is widely used because of simplicity,rapidity, and high resolving power. Used in separating the components of blood and urine. Seperation of antibodies. Study of new amino acids in bacteria. Study of various steps of photosynthesis.

Column Chromatography
It is also called adsorption chromatography. It was developed by Tswett in 1903. In this the solution or cell extract is passed through a column of solid inert material packed in a glass tube. The column act as adsorbent.

Application
Used to separate fats. Carotenoids(plant pigments). Steroids. Chlorophyll and their derivatives. ADSORBENT MEDIUM Silica Charcoal Alumina

Starch and sucrose

Thin layer chromatography

Modified version of column chromatography. In TLC,adsorbent is spread on a glass sheet to form a thin layer. The adsorbent used in TLC may be cellulose,alumina,silica,cellulose ion exchange resin . TLC is a useful technique because it is relatively quick and requires small quantities of material. It is a method for identifying substances and testing the purity of compounds.

Thin layer chromatography

Ion exchange chromatography

Molecules are seperated by the difference in their electric charge. The ion exchange resin used as adsorbent consist of poly styrene beads to which ionisable groups added chemically. The most used ion exchange resins are DEAE cellulose and CM cellulose

DEAE cellulose is +vely charged,and is anion exchanger. it acts by binding vely charged molecules.
CM cellulose cation exchanger

Gel filtration Chromatography

Seperation of protein,nucleic acid depends on their size,shape,molecular weight. Depends on the ability of molecules to enter the pores within the beads of beaded gel. The adsorbent material consist of tiny beads that are packed into a column in a glass tube and act as molecular sieve. The solution of proteins slowly percolates through the beads.

Application
Purification of protein Enzymes Anti bodies Hormone Nucleic acid Also used to determine the weight of protein and enzymes.

Affinity chromatography
Based on the unique property of protein ,that interact with specific compounds such as enzymes with substrate, receptors with ligands and antibodies with antigen. Each of these type of protein can be removed from solution mixture of these protein passes through a column conain immobilized ligand. The protein of interest binds with ligand and all other protein pass through. Non specifically bound proteins removed by washing the column with buffer. Commonly employed combinations of immobilized ligand and protein used in affinity chromatography are an antibody to purify antigen, a hormone for a receptors, an inhibitor for an enzyme, and lectin to purify a glycoprotein. E.g. an impure preparation of insulin receptors.

Partition Chromatography
Based on the partitioning of a substance between two liquid phase.
Substance which are more soluble in mobile phase will pass rapidly through system while those which favors the stationary phase will be retarded. Two types, normal phase partition chromatography. and reverse phase partition chromatography.

Application
Used to separate broad range of non polar, polar ionic biomolecules, like peptides,proteins,oligosaccharides,and vitamins.

Hydrophobic interaction chromatography.

Based on the difference in the hydrophobicity of biomolecules.
Mainly to purify proteins. The hydrophic residues are scattered over the surface of different protein in different manner.

The hydrophobic region are covered with an ordered film of water molecule that effectively mask the hydrophobic groups.
It can be exposed by the presence of high salt concentration facilitating protein-protein interaction

The presence of hydrophic groups attached to a suitable matrix, facilitates protein matrix interaction.
Stationary phase: alkyl or phenyl groups attached to an agarose matrix.

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