The separation of o-phthalaldehyde derivatives of amino acids by reversed-phase chromatography on octylsilica columns

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Amino acids were reacted with o-phthalaldehyde and 2-mercaptoethanol and were separated using a simple linear gradient from 10 to 65% methanol over 15 min on an octyl silica (C8) column by reversed-phase chromatography. The separation obtained was found to be sensitive to the pH, ionic strength, and tetrahydrofuran concentration of aqueous solvent A [THF: sodium acetate (45 m m), pH 5.7, (4:96)]. These effects were characterized and used to design a rapid (17 min) separation of the amino acids commonly found in acid hydrolysates of proteins. A more involved procedure was used to separate the more complex mixture of amino acids that are found in enzymatic hydrolysates of proteins or in physiological fluids. The simplicity of the methods allows their use on different chromatographic systems with little or no alteration.