What is chromatography: Principle, method & types

Chromatography is a non-destructive procedure for resolving a complex mixture into its individual fractions or compounds. This is a separation procedure and the separated entities are identified by other analytical techniques like UV-visible, Infrared, NMR (nuclear magnetic resonance), Mass spectrometry etc. In applications for quantitative analysis the measurement of the area under the curve in chromatogram is done.

Chromatography derives its name from two words as chromo= colour and graphy= writing. I.e colour bands are formed in the procedure which are measured or analysed. This colour bands are due to separation of individual compounds at different lengths on the column as seen in column chromatography and on paper in paper chromatography.

But in the most modern chromatography methods used like HPLC, Gas chromatography etc.no colour bands can be seen.

Chromatography is defined as the process of separation of the individual components of a mixture based on their relative affinities towards stationary and mobile phases.

Chromatography Principle: The samples are subjected to flow by mobile liquid phase onto or through the stable stationary phase. As in the definition the principle involved is separation of fractions of mixture based on their relative affinity towards the two phases during their travel.

The fraction with greater affinity to stationary phase travels slower and shorter while that with less affinity travels faster and longer.

Based on the mode or method employed in separation chromatography is broadly classified as

1. Adsorption chromatography: Here the stationary phase is a solid while the mobile phase is liquid. The compounds travel on the stationary phase under the influence of mobile phase based on their relative adsorption to the solid stationary phase.

2. Partition mode: In this mode both the stationary and mobile phase are liquids. So the compounds have affinity based on their partition into the individual liquid phases. The one with greater partition to stationary phase has higher affinity to stationary phase and vice versa.

Based on the nature of stationary phase it is of two types:

a) Normal phase chromatography: Here the stationary phase is polar in nature and hence the compounds with higher polarity elute out last while non polars come out first.

b) Reverse phase chromatography: Here the stationary phase is non-polar in nature and hence the compounds with lower polarity elute out last and vice-versa.

In most HPLC analysis, the mode used is reverse phase chromatography as many of the biological, phytochemical compounds and even drugs are polar in nature.

For more details read types of chromatography

Chromatography Methods

There are many developments which have occurred in chromatography over years based on the requirements and also technology employed in evaluation of mixtures.

Chromatography methods can be broadly divided into planar chromatography and columnar chromatographic methods.

In planar chromatography the stationary phase is a plane surface i.e (two dimension surface where only length and breadth are taken as area) on which chromatograms are formed.

This method is adopted in techniques like

1. Paper chromatography.

2. Thin layer chromatography.

3.High performance thin layer chromatography (HPTLC).

In Columnar chromatography, there is use of a column on whose walls lies a stationary phase and the mobile phase is flushed through the column.

The techniques which employ this method are

1. Column chromatography.

2. Gas chromatography.

3. High Pressure liquid chromatography (HPLC).

4. Size exclusion chromatography.

5. Ion exchange chromatography. etc.

Both techniques have their Pros and cons.

Planar methods have the advantages like faster separation, visualization of formed chromatograms or spots and are less expensive techniques. But are not useful for preparative chromatography.

In Columnar chromatography, the advantages like better or effective separations even for complex mixtures, possibility of preparative chromatography i.e to yield large amount of compounds by separation of mixtures etc.

But they have disadvantage of being expensive, time consuming and cumbersome techniques.