Chromatography definition, principle, techniques & uses

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Chromatography is derived from Greek terms chromo= color & gram=bands. Hence as the name indicates, in chromatography there is the formation of colored bands. These bands are indicative of different components in the sample. In the Initial stages, it was developed as column chromatography. Where in into a long vertical column, samples with mobile phase was allowed to percolate through solid stationary phase like sand. So there were bands of separated compounds at different heights or lengths of column appearing as colored bands. Further passage of mobile phase through the column leads to leakage of the components from column bottom into different beakers kept for each band.

Chromatography definition:

Chromatography is a qualitative and quantitative analytical technique where in a sample mixture under test is subjected to preferential separation into different components under the influence of a moving phase (mobile phase) over a stationary phase. These separated components are later identified for their quantity.

Chromatography principle:

Chromatography is based on the principle of separation of compounds into different bands (color graphs) and then identification of those bands.

The preferential separation is done due to differential affinities of compounds towards stationary and mobile phase. After separation of the compounds they are identified by suitable detection methods.

The differences in affinities arises due to relative adsorption or partition coefficient in between components towards the both phases.

Adsorption varies due to polarity of components towards stationary phase. If both stationary phase and components in sample are polar, the rate of travel for polar component is slow and hence get separated from rest of sample is passed out of the column last. Similarly if both stationary phase and component in sample is non-polar, then non- polar component comes out last due to slow rate of travel in the column under the influence of mobile phase. So the stationary phase and mobile phase are always opposite. I.e if stationary phase is polar, the mobile phase use is non-polar and vice-verse.

Ex: In a sample with mixture of lipids and proteins. Lipids are non-polar and proteins are polar in nature. Suppose here Sand which is polar in nature is used as stationary phase  and non-aqueous solvents like Hexane or Acetone which are non-polar in nature are used as mobile phase. During separation, lipids come out  of the column first while proteins come out latter due to their higher adsorption to sand stationary phase.

Partition varies due to solubility of components into different liquids. So here both mobile phase and stationary phase must be liquids. The stationary phase liquid is in the form of a thin layer of film on a hard back ground in the column. So when a sample mixture having components of differences in solubility in different liquids, of same nature i.e either polar or non-polar, they get separated into two liquid phases i.e mobile phase and stationary phase based on their partition coefficient in between two liquids.

Chromatography definition

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Ex: Iodine in between water and trichloromethane. Though iodine is soluble in both, it is non-polar in nature and hence is more soluble in triclorormethane than that in water. So most of the iodine separates into it and very small amount stays in water.

Chromatography techniques:

They vary based on different Types of Chromatography. But as a brief description general to all types the techniques are

1. Stationary phase: The stationary phase used can be solid or liquid. The solid used in stationary phase should have particles of uniform size and shape. More over their shape should be preferably spherical. If liquid is used as stationary phase, the liquid is spread as a uniform layer on the solid background.

2. The mobile phase: The mobile phase used is a liquid or gas and it should be free of particles, contamination and impurities..Moreover mobile phase should be opposite in polarity to the stationary phase. I.e. if stationary phase is polar in nature, mobile phase has to be non-polar and vice-verse.

3. Flow rate: the flow rate of mobile phase over stationary phase is always kept constant for uniform and reliable results.

4. Temperature: The temperature of the experimental chamber or chromatography lab is kept uniform. Alteration in temperature can alter the flow rate, the state of mobile phase and also the detector efficiency.

5. Treatment of sample before or after the process of separation in chromatography. This is termed as pre-column or post column derivation. This is especially followed in gas chromatography for some solvents.

6. Some other techniques like ascending, descending and radial mode of development of chromatograms is followed.

7. The test sample or standard sample used in the analysis is moistened with the solvent preferably the mobile phase and it should be soft, powder form or homogeneous or pulpy but not hard.

8. Preparative or analytic technique is employed. For preparative hplc, the sample quantity is more and is aimed to get pure compound. While in analytic technique sample s just analysed for identification of sample and also determination of its quantity..

Chromatography Uses :

Chromatography is widely used as analytic technique for clinical diagnosis, in scientific research, in quality control of drugs, food manufacture industries.

It is specifically used to identify and measure the molecules having similar chemistry,  optical absorbance etc.

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