Chromatographic techniques for Bio-medical researchers

Meaning of chromatography

Chromatography is the science of separation techniques . The technique is used to fractionate mixture of gases, liquids  or dissolved solids. The name chromatography (Greek: chroma means color and graphein means writing) literally means writing in color. In other words writing out the ‘signature ‘ of a mixture in color.

Definition of chromatography

Chromatography is defined as  a physical separation technique used to separate macromolecules  based on differences in their structure and / or composition i.e.their size, shape or charge (Heftmann 1992 ). Chromatography is a dynamic separation system which partitions chemical substances between two phases (Biphasic system)-a stationary phase (SP) and a mobile phase (MP). Chromatographic – like separation processes occur in nature

–for e.g. migration of water through soil  results in purification of water. Chromatography is a science which studies the separation of molecules

Discovery of chromatography

The Russian botanist Mikhail S. Tswett (1872-1919) found that pigment composition became separated when plant pigment (chlorophyll) together with petroleum ether went through calcium carbonate layer. Chromatography is a method  in which the components of a mixture are separated on an adsorbent column in  a flowing system (Tswett,1906)

Biphasic systems of chromatography 

 Stationary phase consists of small solid particles with micro porous surface. Mobile phase can  be a gas or a liquid which carries the components of a mixture. The rate of movement of a given component of a mixture depends on the degree of solubility  in the solvent system. More soluble substances travel more slowly down the column than the less soluble.

Rf   value : relative front 

The relation of the distance traveled by compound to that of the solvent front is called Rf value. Parameters influencing Rf value includes temperature, solvent system, direction of flow and type of paper.
Rf value   = Distance   traveled  by the solute

                        --------------------------------------

                   Distance traveled by the solvent   

Kinds of chromatographic techniques            

Column chromatography
A mixture of components dissolved in a solvent is poured over a column of solid adsorbent . The column is eluted with the same or a different solvent. The stationary phase is solid. The mobile phase (the eluent) is liquid.

Paper chromatography

        The paper adsorbs water from the atmosphere of the developing chromatogram. The water is the stationary phase. The eluting solvent is the mobile phase.

Chromatographic  principles   of   separation

1- Elution development - The components of the mixture are separated into zones by the passage of one or more solvents through the column. This technique is most widely used in GC, GLC, LLC and LSC.
2. Gradient elution - A gradual change in composition of the eluting solvent is used to achieve separation of compounds of widely varying affinities for the stationary phase. The solvent composition gradient may be linear with increasing or decreasing concentration, pH, polarity or ionic strength.
3. Frontal analysis - No solvent is used for irrigation. The solution itself is added continuously.
4. Displacement analysis - The components in the mixture are adsorbed on the column. The irrigation of the column is carried out with the solution of another substance, having a higher preferential adsorption on the column than that of the components of the mixture sought.

Research  applications  of chromatography

There are two kinds of research applications .i.e. analytical and preparative applications. Analytical application is to determine the chemical composition of a biological sample. Preparative application is to  purify and collect one or more components of a biological sample.

Significance of chromatographic methods

•         They serve to resolve and  identify the separated components of a mixture.

•         Very small quantities of substances could be analyzed qualitatively and quantitatively.

•         The equipment is very simple except HPLC

•         No special skill is required for performing the method

•         The results are remarkably reproducible.