乙醛酸的高效液相色谱法分析条件探讨

Research on Glyoxalic Acid Analysis Based on HPLC Method

乙醛酸可与2,4-二硝基苯肼(DNPH)反应生成乙醛酸腙,该腙类物质对紫外-可见光有较强的特征吸收。本研究对乙醛酸采用高效液相色谱法(HPLC)测定,探究乙醛酸与DNPH反应,衍生成腙过程中的影响因素。实验发现,乙醛酸与DNPH衍生过程中会产生顺式和反式两种异构体。该顺反异构体的生成及生成条件从未被报道过。两种异构体的生成比例与衍生时间、衍生温度、pH值以及DNPH用量等因素有关。其中,衍生时间对乙醛酸腙顺反异构体生成比例的影响最为显著。长时间衍生有利于反式异构体向顺式异构体的转化。同时还发现,当乙醛酸的衍生条件:温度为40℃,pH值为3,DNPH与醛基的物质的量比为30,反应时间60min时,可完全满足乙醛酸的定量要求。但是使用乙醛酸腙顺式或反式单一异构体面积积分值(365nm)与乙醛酸质量浓度做标准曲线不能实现对乙醛酸的准确定量。使用两种异构体色谱峰面积的算术加和与乙醛酸质量浓度作标准曲线定量,则可实现乙醛酸的外标法准确定量。在上述衍生体系下,乙醛酸质量浓度在7～140 mg/kg范围内,乙醛酸腙的峰面积与乙醛酸的质量浓度之间呈良好的线性关系,线形相关系数为0.9999。七次重复测定相对标准偏差为1.06%,结果重复性好。加标回收率在98.9%～102.3%之间。

Glyoxalic acid reacts with DNPH （2,4 - Dinitrophenylhydrazine） and produces glyoxalic acid hydrazone （ GAH）, which shows strong absorption to ultraviolet light at 365nm. In this paper, a method to quantify-glyoxalie acid were developed, and the four key factors were exanfined using high performance liquid chromatography （HPLC）. The results shown that both eis and trans GAH isomers were observed. Also the ratio of eis to trans GAH were affected by four factors including the amount of DNPH in derivazation process, the reaetionfime, temperature and pH. However, derivazation time significantly infflueneed the ratio of eis to trans GAH. It demonstrated that the longer derivazation time, the higher amount of trans GAHwe get, compared to eis GAH. The proper derivazation conditions were set as follows ： the reaction temperature of 40℃,the pH of the system of 3 ,the molar ratio of DNPH to glyoxalic acid of 30, and the reaction time of 60 nfin. Compared to peak area integration of single isomer, the sum of eis and tran GAH showed better linear correlation to the concentration glyoxalic aeidwith a linearity of coefficient value of 0. 9999. Relative standard deviation （RSD） of seven parallel tests is 1.06% and recovery ranged from 98.9% to 102.3%.