Ion exchange chromatography, amino acids

Briddon, A., Total plasma homocysteine as part of the routine aminogram by ion-exchange chromatography, Amino Acids, 15, 235, 1998. 

Separation can also be done by ion-exchange chromatography employing an anion- or a cation-exchange resin. An amino acid analyzer is an instmment that automates ion-exchange chromatography. Amino acids can be synthesized by a Hell-VoUiard-Zelinski reaction (followed by reaction with excess NH3), a Strecker synthesis, reductive amination, a A-phthalimidomalonic ester synthesis, or an acetamidomalonic ester synthesis. 

Incomplete oxidation can be a problem. Higher recoveries of cysteine and cystine have been achieved by reduction of those amino acids with 2-mercaptoethanol followed by incubation with 4-vinylpyridine. This converts cysteine and cystine to S-(4-pyridylethyl)-L-cysteine, a derivative that can be separated by ion-exchange chromatography. Performic acid oxidation of methionine in the presence of phenol is a suitable method for analysis of cysteine. 

Conventional extraction procedures and evaporation techniques are applied. Depending on the nature of the compounds, different clean-up procedures are recommended ion exchange for amino acids and organic acids, pyridine solutions for sugars. Because the final volume of the solution must be small, evaporations must be carried out in vacuo (Rinco evaporators) or by lyophilization. Sometimes inorganic ions (Na, Mg ) interfere in chromatography, and desalting may have to be carried out. Usually, commercial electrolytic desalters are used for this purpose. 

Ion-exchange chromatography of amino acids on acid resins is a very efficient analytical procedure but is difficult to adapt to preparative separations. On the other hand, ion-exchange chromatography of acidic amino acids and peptides on basic resins is an efficient technique for preparative separations and is used mainly for that purpose. Thus, nearly all isolations of acid y-glutamyl derivatives from natural material have employed this technique. 

These are the definitions of the two characteristic dissociation constants normally expressed in terms of p K. When three dissociating groups are present in a molecule there are three piC values, ie, pfC, P 3- knowledge of these piC values is important in the separation or isolation of each amino acid by ion-exchange chromatography. 

The automated amino acid analy2er depends on ion-exchange chromatography (117) and is now a routine tool for the analysis of amino acid mixtures (118). This most advanced machine can detect as Htde as 10 pmol in ninhydrin reaction analysis. One-half to two hours are required for each analysis. An analysis chart is shown in Figure 2. 

Fig. 2. Amino acid analysis by automated ion-exchange chromatography. Standard column, 4.6 mm ID x 60 mm Ninhydrin developer. Computer print out indicates retention time (RT), height and area of peaks, and the ratio of the height of an amino acid in the sample to the height of a standard amino acid.

Amino acids have high melting or decomposition points and are best examined for purity by paper or thin layer chromatography. The spots are developed with ninhydrin. Customary methods for the purification of small quantities of amino acids obtained from natural sources (i.e. l-5g) are ion-exchange chromatography (see Chapter 1). For general treatment of amino acids see Greenstein and Winitz [The Amino Acids, Vols 1-3, J.Wiley Sons, New York 1961] and individual amino acids in Chapters 4 and 6. 

The analysis of amino acids involves chromatographic issues similar to those encountered in analysis of simple amines. Underivatized amino acids have, with a few exceptions, weak UV absorbance and a strong tendency to interact with stationary phases in undesirable ways. Underivatized amino acids are normally separated with ion exchange chromatography, then visualized post-column by reaction with ninhydrin, o-phthaladehyde (OPA), or other agents. Underivatized tryptophan and the metabolites kynurenine, 3-hydroxykynurenine, kynurenic acid, and 3-hydroxyanthranilic acid, were separated on a Partisphere 5-p ODS column with fluorescent detection.121... 

Hamilton, P. B., Ion exchange chromatography of amino acids. A single column, high resolving, fully automatic procedure, Anal. Chem., 35, 2055, 1963. 

Houpert, Y., Tarallo, P., and Siest, G., Amino acid analysis by ion-exchange chromatography using a lithium elution gradient. Influence of methanol concentration and sample pH, /. Chromatogr., 115, 33, 1975. 

Chin, C. C. Q., Ion-exchange chromatography of some amino acid derivatives found in proteins, Meth. Enzymol., 106, 17, 1984. 

Murren, C., Stelling, D., and Felstead, G., An improved buffer system for use in single-column gradient-elution ion-exchange chromatography of amino acids, /. Chromatogr., 115, 236, 1975. 

Le Boucher, J., Charret, C., Coudray-Lucas, C., Giboudeau, J., and Cynober, L., Amino acid determination in biological fluids by automated ion-exchange chromatography Performance of Hitachi L-8500A, Clin. Chem., 43,1421,1997. 

Albin, D. M., Wubben, J. E., and Gabert, V. M., Effect of hydrolysis time on the determination of amino acids in samples of soybean products with ion-exchange chromatography or precolumn derivatization with phenyl isothiocyanate,. Agr. Food Chem., 48, 1684, 2000. 

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