Practical Protein Chemistry - A. Darbre 1989

Enantiomeric analysis of amino acid mixtures by high-performance liquid chromatography
Reagents
HPLC Solutions

Buffer I. Glacial acetic acid is added to a 0.2 M pyridine solution until pH 3.4 is reached.

Buffer II. Glacial acetic acid is added to a 0.4 M pyridine solution until pH 5.3 is reached.

OPA Reagent. Dissolve 30 g of boric acid in 1 L of Water, add KOH to adjust the pH to 9.5–10, and then add 2.5 g of EDTA. Dissolve 1.0 g of o-phthaldialdehyde and 0.5 mL of 2-mercaptoethanol in 10 mL of methanol, and slowly pour this solution into the first one while stirring. Filter the resulting mixture through a Millipore filter (0.22 µm).

Chiral Mobile phase. Dissolve 8 mM (1.384 g) of N,N-di-n-propyl-L-Alanine and 4 mM (0.8 g) of copper acetate in 1 L of water (pH 5.3–5.5), filter the solution through the same filter, and degas under vacuum.

Class="center">Table 9.1. HPLC Separation of Amino Acids. Column: Spherisorb LC-18, 5 µm, 150X4.6 mm; chiral additive: Cu(N,N-di-n-propyl-L-alanine)₂

^a Retention time was determined individually for each amino acid starting from The Emergence of the column void volume V₀.

^b Void time V₀ is 5 min, determined by the appearance of the solvent peak on the chromatogram.

^c Void time V₀ is 3.7 min.

^d Ala and Pro were individually separated on a Supelco LC-18 column (150X4.6 mm); detection was performed via the ninhydrin reaction, measuring optical density at 570 nm (Ala) and 440 nm (Pro). Void time V₀ is 5 min.