柱前衍生化HPLC法分离苏氨酸对映体的研究
首发时间:2012-07-25
摘要:以邻苯二甲醛/ N-异丁酰基-L-半胱氨酸(OPA/IBLC)为手性衍生化试剂,建立苏氨酸手性分离和光学纯度检查的高效液相色谱法。采用Hedera ODS-2色谱柱(250 mm × 4.6 mm, 5 μm),以0.1% 醋酸铵-甲醇-乙腈(70 : 27 : 3)为流动相,检测波长为338 nm。考察了溶液pH值、试剂用量、衍生化时间及温度对衍生化反应的影响。结果表明,在优化的色谱条件下,苏氨酸的对映体分离度为1.6,L-苏氨酸最低检测限约为0.05 μg/mL。该方法灵敏度高,重现性好,可用于L-苏氨酸光学纯度的检查控制。
关键词: 苏氨酸 邻苯二甲醛/ N-异丁酰基-L-半胱氨酸 柱前衍生化 高效液相色谱法 手性分离 纯度检查
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Separation of Threonine Enantiomer by Pre-column Derivatization HPLC
Abstract:The chiral separation and optical purity test of threonine were investigated by high performance liquid chromatography using ortho-phthalaldehyde/N-isobutyryl-L-cysteine (OPA/IBLC) as the pre-column derivatizing reagent. The diastereoisomers produced were separated on a Hedera ODS-2 column (250 mm×4.6 mm, 5 μm) with a mobile phase consisting of 0.1% acetate-methanol-acetonitrile (70 : 27 : 3) at the detection wavelength of 338 nm. The effects of solution pH, the amount of OPA/IBLC, the derivatizing time and the derivatizing temperature were discussed. The results show that under the optimized chromatographic condition, resolution of threonine is 1.6 and the lowest detectable limit of L-threonine is 0.05 μg/mL. The method is sensitive and highly reproducible and it can be used in the purity test of L-threonine.
Keywords: threonine ortho-phthalaldehyde/N-isobutyryl-L-cysteine pre-column derivatization HPLC chiral separation optical purity
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