氨基酸的2,3-二甲醛基喹喔啉荧光衍生及高效液相色谱分离分析

将荧光试剂2,3-二甲醛基喹喔啉首次应用到氨基酸的高效液相色谱分离分析中。考察了该荧光试剂与伯胺氨基酸衍生反应的pH值、比例、反应时间等影响因素,确定衍生反应的最佳条件为:100 mmol/L硼砂缓冲液(pH 9.5),二巯基乙醇、衍生试剂与氨基酸的比例为9∶3∶1,室温下反应5 min。采用C18色谱柱,经二元梯度洗脱,荧光检测,17种伯胺蛋白氨基酸得到较好分离。氨基酸的浓度在0.01～1.00 mmol/L范围内与其衍生物峰面积呈较好的线性关系,氨基酸衍生物的检出限为0.02～0.07μg/L。应用该方法对麦冬中游离氨基酸的含量进行测定,结果满意。此方法快速,高效,灵敏度高,精密度好。

Separation of Amino Acids by High Performance Liquid Chromatography with Quinoxaline-2,3-dicarboxaldehyde as Fluorescent Derivation Reagent

A fluorescence reagent quinoxaline-2,3-dicarboxaldehyde was firstly applied in the separation of amino acids by high performance liquid chromatography(HPLC).The reaction of primary amine amino acids with fluorescence reagent was studied in detail.The best conditions for the reaction:buffer:pH 9.5 100 mmol/L borate,a mole ratio of 2-mercapto ethanol,fluorescence reagent and amino acid:9 ∶ 3 ∶ 1,reaction time:5 min at room temperature.Under the optimal conditions,seventeen derivatives were detected with fluorescence detector after separation on a C18 column by gradient elution.The calibration curves were linear over the range of 0.01-1.00 mmol/L for all derivatives,with detection limits of 0.02-0.07 μg/L.This method was simple,rapid and sensitive,and was used for the measurement of the concentration of amino acid in ophiopogon of the Chinese traditional medicines with satisfactory results.