Separation constitutes an essential pre-requisite for analysis of complex matrices before its constituents can be identified and quantified. Classical techniques for separation in involved processes such as :
- Solvent extraction
Chromatography added a new dimension to separation sciences providing fast identification and estimation of sample constituents.
The present article will introduce you to chromatographic techniques which have large-scale applications in areas such as pharmaceuticals, foods, environmental studies, petroleum refining and forensic investigations. The areas covered are Planar Chromatography, Gas Chromatography and High Performance Liquid Chromatography.
Planar chromatography involves separation of mixture constituents on a plane surface and includes paper chromatography, thin-layer chromatography and electrochromatography. Presently thin-layer chromatography and its advanced version HPTLC are favoured techniques. Separation of complex mixtures on a thin layer of adsorbent applied on metal or glass plate. The mobile phase comprising of a single or a mixture of solvents moves the components through the stationary phase by capillary action.The separated components are detected using:
- Inherent colours of separated spots
- Use of fluorescence or derivatization reactions
- Radioactive measurement using radio tracers
- Viewing chambers using UV light
TLC and HPTLC offer low cost and maintenance free option in comparison to more advanced techniques and it is possible to preserve separation plates or photographs for longer periods. However, the speed of analysis is slow in comparison to the later techniques
Gas Chromatography (GC)
Gas chromatography is the natural choice for separation of gases and low molecular weight volatile components of mixtures. The basis of separation is distribution of solutes between the solid adsorbent (Gas- solid chromatography) or a liquid layer adsorbed on the inert solid support (Gas -liquid chromatography). The packings inside the column can be raised to temperatures around 400°C in isothermal or programmed manner which adds to the versatility of the range of separations.
Choice of wide range of detectors such as FID, TCD, ECD, NPD, FTD, etc extend the separations to different chemical categories of compounds to include pharmaceutical residual solvents, food flavours, pesticide residues and petroleum fraction analysis.
High Performance Liquid Chromatography (HPLC)
High performance liquid chromatography is based on same operational principle as GC but the main difference is that a liquid is used as a mobile phase instead of gas. It has found applications and analysis of thermally labile compounds having high molecular weight ranges. A wide choice of column packings, detectors and mobile phases has resulted in sub-classification of HPLC technique as
- Normal phase chromatography
- Reverse phase chromatography
- Ion-exchange chromatography
- Size exclusion chromatography
Subsequent articles will deal with hyphenated techniques such as LC – MS and GC – MS which have moved separation science to limits not thought of before in terms of sensitivity and speed of analysis. Today HPLC is making inroads into more and more unexplored areas of biochemistry, life sciences and clinical research.