HIGH PERFORMANCE LIQUID CHROMATOGRAPHY (HPLC)
The excellent and the most wonderful technique of ‘high performance liquid chromatography’ (HPLC) is nothing but an outcome of the various theories and instrumentation that were originally advocated for liquid chromatography (LC) and gas chromatography (GC). By the late 1960’s, analysts invariably used to have the bliss of excellent experience of achieving the goal of superb separations of complex mixtures in seconds rather than in minutes, with the aid of electronic integrators to get an exact access of areas under elution bands, and above all the ‘computer-printouts’ of the complete analysis with the flick of a finger. In many favourable instances the smallest possible quantities ranging from nanogram to picogram* could be detected with utmost ease and convenience.
In reality, some of the serious limitations too often encountered in GC ultimately brought about the development of HPLC, for instance :
(i) In GC the mixture of components are usually screened in the vapour phase. Hence, either a stable vapour from the mixture is obtained directly or indirectly converting the substance in it to such derivatives that are thermally stable. One 20% of chemical compounds usually come across in analysis are suitable for GC directly i.e., without making their corresponding appropriate deriva-tives,
(ii) The remainder 80% of the chemical compounds are either thermally unstable or involatile in nature, and
(iii) Compounds essentially having highly polar or ionizable function groups are very prone to ‘tailing’ by GC-analysis.
Therefore, HPLC has been evolved as a dire confluence of need, technological supremacy, the emer-gence of newer theoretical concepts and ideas towards development along rational lines, and above all-‘the human desire to minimise work’. HPLC offers numerous advantages as stated below :
· Capable of handling ‘macromolecules’,
· Suitable for pharmaceutical compounds,
· Efficient analysis of ‘labile natural products’,
· Reliable handling of inorganic or other ionic species, and
· Dependable analysis of biochemicals.
Interestingly, in HPLC the stationary phase and the mobile-phase is able to interact with the sample selectively. Besides, such interactions as hydrogen bonding or complexation which are absolutely not possible in the GC-mobile phase may be accomplished with much ease in the HPLC-mobile phase. Furthermore, the spectrum of these selective interactions may also be enhanced by an appropriate chemical modification of the silica surface i.e., the stationary phase. Therefore, HPLC is regarded as a more versatile technique than GC and capable of achieving more difficult separations.