Abstract
Publisher Summary This chapter examines the important liquid chromatography (LC) methodologies available for amino acid analysis, considering two major goals: Extending the limits of detection and reducing the time required for achieving high-quality separations. The chapter reviews the post-column derivatization method using a ninhydrin reagent, which reacts with the eluates from a cation-exchange column. This method has long been employed in LC for amino acid analysis of both protein hydrolysates and free amino acids, such as may occur in physiological fluids. Recent methods, which utilize o-phthalaldehyde (OPA) as the reagent in post-column derivatization from an ion-exchange column and in pre-column derivatization with a reversed-phase column, are also discussed. Post-column derivatization, using ninhydrin takes advantage of the fact that a primary amino acid molecule reacts with two molecules of ninhydrin to form Ruhemann's Purple, which has a characteristic wavelength of ca. 570 nm. Ninhydrin reagent is prepared by dissolving ninhydrin and a reductant in a mixture of a buffer solution and an organic solvent. Methyl cellosolve, in which ninhydrin is highly soluble, is often used as the organic solvent.
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