Practical Protein Chemistry - A. Darbre 1989

Enantiomeric analysis of amino acid mixtures by high-performance liquid chromatography
HPLC
Group separation

Amino Acids are divided into 4 groups:

1) acidic: Cya, Asp, Thr, Ser, Glu;

2) neutral: Gly, Ala, Val, Met, Leu, Ile;

3) aromatic: Phe, Tyr, Trp;

4) basic: Lys, His, Arg.

Apply 20 µL of The amino acid solution to a stainless steel Column (50x4.6 mm) packed with a cation-exchange resin (5 µm) of the polystyrene-divinylbenzene sulfonic acid type, equilibrated with buffer I. The first three groups of amino acids, with the exception of Trp, are eluted with the same buffer. Fig. 9.1 shows the chromatogram and Separation conditions. Buffer II is used to elute Trp, Lys, His, and Arg. The column eluate is mixed with OPA reagent delivered at a flow rate of 0.6 mL/min, enters a coiled reaction Cell (the transit time through the coil is sufficient for the reaction to complete), and then passes into a fluorescence detector (Waters, 420 AC). The initial run establishes the exact retention times of the amino acids. The Procedure is then repeated under identical conditions, but without treating the components with the reagent or detecting the resulting derivatives. Amino acids are collected directly at the column outlet at specific time intervals, for example, acidic amino acids between 1 and 9 min, neutral ones at 10–15 min, and Other Amino Acids individually to avoid large buffer volumes (Phe 20–24 min; Tyr 34–38 min; Trp 62–66 min, Lys 75–81 min, His 84–89 min, Arg 175–188 min). The Trp, Tyr, and Phe fractions are pooled, as are the His, Lys, and Arg fractions.

To reduce separation time, the initial amino acid mixture can be separated on a column (50x2.0 mm) using buffer II exclusively (starting immediately). This results in a faster elution of Trp, Lys, His, and Arg. All other amino acids elute together and can subsequently be separated using buffer I.

The eluate fractions grouped by amino acid Class are evaporated under a stream of nitrogen and then vacuum-dried for 20 min. The residue is dissolved in 200 µL of the chiral Mobile phase for the next separation step.