Kochhar S, Christen P
Biochemisches Institut der Universität Zürich, Switzerland.
Anal Biochem. 1989 Apr;178(1):17-21. doi: 10.1016/0003-2697(89)90348-5.
Amino acids are quantitatively determined by precolumn derivatization with 1-fluoro-2,4-dinitrophenyl-5-L-alanine amide and reversed-phase high-performance liquid chromatography with photometric detection at 340 nm. Excellent chromatographic resolution of a mixture of the derivatives of 20 amino acids including proline and cystine is achieved within 110 min by linear gradient elution with acetonitrile in 13 mM trifluoroacetate plus 4% (by vol) tetrahydrofuran. The limit of detection is 50 pmol. Amino acid analyses of acid hydrolysates of the several proteins gave results equivalent to those obtained by conventional ion-exchange-based amino acid analysis. The simplicity of the procedure allows its use on any multipurpose high-performance liquid chromatographic system.
氨基酸通过使用1-氟-2,4-二硝基苯基-5-L-丙氨酸酰胺进行柱前衍生化，并结合在340nm处进行光度检测的反相高效液相色谱法定量测定。通过在13mM三氟乙酸加4%（体积）四氢呋喃中用乙腈进行线性梯度洗脱，在110分钟内可实现包括脯氨酸和胱氨酸在内的20种氨基酸衍生物混合物的出色色谱分离。检测限为50皮摩尔。对几种蛋白质的酸水解产物进行氨基酸分析，结果与通过传统的基于离子交换的氨基酸分析获得的结果相当。该方法的简便性使其可用于任何多用途高效液相色谱系统。
