Separation, quantification and identification

Abstract

Numerous texts have been published on separation methods of use in biochemistry and clinical chemistry. Almost all such methods are based on some form of chromatography, both for the separation and identification of the compounds of interest and for their subsequent quantification, and may be considered as complementary to one another, with newer techniques superseding the older, and being replaced in turn by others or by extensions and improvements of the basic analytical techniques. It has been said that: ‘Every scientific advance is an advance in method. The invention of a new specialized laboratory procedure brings about rapid conquests in new fields of science and technology: finally it exhausts itself and is replaced by a still more practical method’ (Zechmeister and Von Cholnoky, 1943) and this is particularly true for work on organic acids. Major advances in the knowledge of organic acids in physiological fluids and of their significance in human disease were made by the use of column and paper chromatographic methods. Much of this technology has been replaced by thin-layer chromatography (TLC) which is still routinely employed today in work on organic acids, although TLC has itself been largely superseded by gas chromatography (GC). The latter, in combination with mass spectrometry (MS) as an aid to absolute identification of separated compounds, gave added impetus to research on organic acids in man, and it is fair to say that most of the major advances and discoveries in the field have been made with the use of GC-MS.