Further Reading

Gehrke CW, Roach D, Zumwalt RW et al. (eds) (1968) Quantitative Gas-liquid Chromatography of Amino Acids in Proteins and Biological Substances: Macro, Semimicro and Micro Methods. Columbia, MO: Analytical Biochemical Laboratories.

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The first approach to the automatic liquid chromatography (LC) of amino acids (AAs) - known today as ion exchange chromatography (IEC) - was published by Spackman et al. in 1958. In over 40 years later, it now takes less than 5 min (Figure 1) to separate and quantitate the essential protein AAs instead of 2 days. Early separations were carried out by post-column derivatization.

Over the last 20 years LC has offered unlimited possibilities in both the preparative and analytical scale. The wide choice and sophisticated columns, detectors, derivatization procedures, development of modern instrumentation and data-handling systems reduce time and costs, and give versatility and automation in Good Laboratory Practice (GLP)- controlled conditions for selectivity, sensitivity and repro-ducibility. It is the responsibility of the researcher to choose the most appropriate method for the given task. The most popular LC method for analysis of both free AAs (present in many natural matrices, biological fluids and tissues, feed and foodstuffs) and of those constituents of protein hydrolysates is now reversed-phase (RP) chromatography after pre-col-umn derivatization of the AAs.

Numerous methods for derivatization are available in the literature. This article will discuss the advantages and drawbacks of the commonly used derivatives.

Current trends in AA analysis identify the best conditions for enantiomer separation and the development of LC-mass spectrometry (LC-MS).

Husek P and Macek K (1975) Gas chromatography of amino acids. Journal of Chromatography 113: 139.

Konig WA (1987) The Practice of Enantiomer Separation by Capillary Gas Chromatography. Heidelberg: HUthig.

MacKenzie SL (1981) Recent developments in amino acid analysis by gas-liquid chromatography. In: Glick D (ed.) Methods of Biochemical Analysis, vol. 27, p. 1. New York: Interscience.

Weinstein B (1966) Separation and determination of amino acids and peptides by gas liquid chromatography. In: Glick D (ed.) Methods of Biochemical Analysis, vol. 14, p. 203. New York: Interscience.

Zumwalt RW, Kuo KCT and Gehrke CW (eds) (1987) Amino Acid Analysis by Gas Chromatography. Boca Raton, FL: CRC Press.

To attain one of the main advantages of LC - separating the 'classical 20' as underivatized AAs - has appealed to chromatographers. In spite of a number of efforts, the simultaneous LC of underivatized AAs has remained of secondary importance. Determination of a few selected AAs, such as tryptophan or sulfur-containing AAs, has proved to be fruitful for special tasks.

The aim of various investigations was to render unnecessary the time-consuming derivatization techniques. However, the characteristics of the free AAs are considerably different from each other and their various structural properties did not permit their easy resolution. Thus, in attempting to achieve better separation of free AAs, further means of discrimination were needed. For this purpose special techniques have been introduced, such as the use of various phase systems, ion pair and ligand exchange chromatography, column-switching techniques or anion exchange chromatography with electrochemical detection.

The solvent-generated ion exchange phase system ensured the gradient elution of 19 AAs (Figure 2A): some, but not all, are baseline-separated. A simple isocratic method using aqueous, copper acetate/alkyl-sulfonate additives containing acetate buffer (pH 5.6) as mobile phase, a conventional RP column and UV detection (230-240 nm) at different temperatures and varying the concentrations of additives was unable to separate the classical 20 protein amino acids. Significant improvement in the separation can be obtained by column switching (Figure 2B), as well as by using an anion exchange column, a quaternary

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