高效液相色谱法测定非天然氨基酸的光学纯度

 以（1- 氟-2,4-二硝基苯基）-5-L-缬氨 酰胺为手性试剂、反相高效液相色谱法测定非天然氨基酸的光学 纯度。梯度洗脱，流动相A：含体积分数为0.1％的三氟乙酸的乙腈，流动相B：体积分 数为0.1％的三氟乙酸水溶液。L-和D-对映体得到良好分离。准确测定了25种非天然 氨基酸L-和D-对映体的光学纯度。

Determination of Chiral Purity of Synthetic Amino Acids by High Performance Liquid Chromatography

A series of synthetic beta-heterocyclic and beta-naphthyl alanine and aromatic substituted phenylalanine were separated by reversed-phase HPLC as diastereomeric derivatives obtained after derivatization with (1-fluoro-2,4-dinitrophenyl)-5-L-valinamide (FDNP-Val-NH2). The separation was performed with gradient elution. The mobile phase A was acetonitrile containing 0.1% trifluoroacetic acid and mobile phase B was 0.1% trifluoroacetic acid. According to the hydrophobicity of the side chain of the amino acids, 6 different linear gradient programs were selected and all gradients completed in 20 minutes. Good resolution was obtained for neutral amino acids. All L-L diastereomer of neutral amino acids (the first letter refers to the configuration of amino acid) were eluted faster than the D-L diastereomer with retention times less than 16 minutes. In the same gradient eluting system, the hydrophobic parameter (pi value) and the position of the substituent in benzene ring of phenylalanine also influence the retention time of diastereomers. Usually the retention time of the phenylalanine with a large pi value was longer than that with a small one. Among the three phenylalanines containing the same substituent, compounds with the substituent in 2-postion gave the shortest retention time. In the case of basic amino acids, the D-L diastereomers eluted prior to the L-L diastereomers and showed rather poor resolution. The chiral purity (ee) of the optical active amino acids (synthetic DL-amino acids after resolution by different enzymes) were calculated with the equation as follows: ee = A(L- or D-) - A(D- or L-)]/A(L-) + A(D-)]] x 100% (L- or D-), A: peak area; L-, D-: amino acid diastereomer.