Ion-Exchange Separation of Amino Acids with Postcolumn Orthophthalaldehyde Detection

Abstract

Moore's and Stein's classical ion-exchange separation of amino acids remains the standard by which all methods are judged. The adaptation of liquid chromatography (LC) equipment to amino acid analysis was inevitable because microprocessor control of gradients allowed almost infinite variation in gradient shape, producing superior resolution with only 2 buffers. The versatility of LC equipment allowed the instruments to be used for other assays. Adaptation of orthophthalaldehyde (OPA) to amino acid analysis increased detection sensitivity to the picomole range. A method for essential amino acids analysis in mechanically separated red meat and poultry products has been adapted to liquid chromatography using postcolumn hypochlorite oxidation, OPA derivatization, and fluorescence detection. Separation is achieved with 2 sequential concave exponential gradients combining ionic strength and pH increases with halide-containing buffers. Hydroxyproline and proline are detected with increasing sensitivity through the use of 3-mercaptopropionic acid in a stabilized OPA reagent. Sample preparation is a critical part of the method. A defatting procedure removes fat and other nonprotein nitrogenous substances. The hydrolysis procedure is designed to protect tryptophan which can be routinely assayed in hydrolysates with a modified flow program. Corrosion damage to the equipment by halide buffers has brought about a search for alternative methodology.