The development of cation-exchange chromatography in free amino acids analysis

Although it was not called a liquid chromatograph, the first such instrument was introduced in late 1959 by Beckman Instruments and was designated the M.S. Amino Acid Analyzer in honour of Moore and Stein. Inject valves had yet to be invented, the buffer switching valves were glass stopcocks and the column temperature was controlled by circulating water baths. Two positive displacement pumps eluted two glass columns (acids and neutrals basics). The eluants were a set of four buffers plus a fifth solution of the appropriate hydroxide called the column regenerant. Low temperature (~30ºC) favoured the threonine/serine resolution on the acids/neutrals column while high temperature (~60ºC) favoured the tyrosine phenylalanine on the basics column. Amino acids were detected with a spectrophotometer after derivatisation with ninhydrin reagent. Analysis time for physiological fluids was 72 hours.

Approximately 10 years later, Waters Corporation introduced the first instrument called a ‘liquid chromatograph’. The three most significant differences between this liquid chromatograph and the amino acid analysers of the time were:

• its ability to form continuous gradientsbetween two eluants;
• a single column temperature; and
• silica- or alumina-based stationary phases.

As instrument and column technology improved, liquid chromatography (LC) became increasingly popular as a method of separating and detecting a wide variety of organic compounds. In the early 1980s, Pickering Laboratories introduced the first isothermal, two-eluant gradient amino acid analysis based on the then available high-performance liquid chromatography (HPLC) technology of ternary gradient pumps and single temperature elution. Adding a specially designed post-column derivatisation system made it possible to analyse amino acids using contemporary HPLC equipment. Total analysis time was shortened. A little later, most HPLC manufacturers offered one of several pre-column derivatisation methods for amino acid analysis by reversed-phase chromatography.

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