微流控芯片毛细管电泳柱后衍生激光诱导荧光检测氨基酸

Analysis of amino acids by microfluidic chip capillary electrophoresis with post column derivation and laser induced fluorescenc

建立了微流控芯片毛细管柱后扩散衍生激光诱导荧光检测氨基酸的方法。利用微流控芯片的二维平面结构特征,在分离通道末端增加支通道,通过扩散法引入柱后衍生试剂,避免了电压引入法对分离通道流型的影响,因而提高了分离效率。考察了支通道长度、衍生试剂液面高度、检测点位置对衍生结果的最优条件,考察了衍生试剂引入方法、催化剂种类、缓冲溶液种类对检测结果的影响。用20 mmol/L硼砂-NaOH（pH=10）溶液作为电泳缓冲溶液,与柱后衍生试剂1.0 mmol/L NDA＋8.0 mmol/L 2-ME＋35 mmol/L硼砂（pH 10.0）的30%（V/V）甲醇溶液反应,精氨酸、苯丙氨酸、天冬酰胺、脯氨酸、丙氨酸、甘氨酸在不加任何添加剂的情况下可达到基线分离。本法用于板蓝根药材中主要游离氨基酸的分离检测,相对标准偏差小于4.4%（n=5）,回收率为92.3%～98.6%。所测板蓝根药材中精氨酸和脯氨酸含量分别为14.97,8.02 mg/g。

A new method for the determination of amino acids by microfluidic chip capillary electrophoresis with post-column derivation and laser induced fluorescence detection was developed.Based on the two dimentional character of a planar microchip,a side channel was constructed in the terminal of separation channel.The derivation reagent,naphthalene-2,3-dicarboxaldehyde（NDA）,was introduced into the separation channel by dispersion under a gravity from a side channel,then the changing of fluid shape in separation channel caused by voltage introduction method was avoided,thereby the separation efficiency was increased.Influence of side channel length,derivation reagent volume,detection position,derivation reagent introducing method,catalyst type,and buffer solution on separation performance were explored.20 mmol./L Na2BO4-NaOH buffer（pH 10.0） was used as electrophoresis electrolyte,1.0 mmol/L naphthalene-2,3-dicarboxaldehyde,8.0mmol/L 2-mere-aptoethanol and 35 mmol/L soldium tetraborate buffer（pH 10.0） in 30%（V/V） methanol were used as derivation reagents,and arginine,asparagine,proline,phenylalanine,alanine,and glycine got a baseline separation under the optimized conditions without any additives.The method has been successfully employed to analyze the main free amino acids in root of Isatis indigotica Fort.The linear regressions of the standard curves were determined for Arg and Pro.The method was carried out over the concentration range of 3.484 ～ 10.452 mg/L for Arg and 2.302 ～ 6.906 mg/L for Pro.The recoveries were in ranges of 92.3% ～98.6% and 94.6% ～ 98.3% for Arg and Pro respectively with the relative standard deviations below than 4.4%（n =5）.The contents of Arg and Pro in Isatis indigotica Fort were determined as 14.97 mg/g and 8.02 mg/g respectively.