反相高效液相色谱柱前衍生法测定发酵液中的5氨基乙酰丙酸
5氨基乙酰丙酸,反相高效液相色谱法,梯度洗脱,柱前衍生,发酵液,,5氨基乙酰丙酸，反相高效液相色谱法，梯度洗脱，柱前衍生，发酵液,摘要
摘 要 建立了同时检测发酵液中5氨基乙酰丙酸和甘氨酸的柱前衍生反相高效液相色谱法。使用ZORBAX SBAQ型色谱柱(250 mm×4.6 mm i.d.，5 μm)，以甲醇50 mmol/L醋酸钠缓冲溶液(pH 6.2)作为流动相梯度洗脱。流速为1 mL/min，柱温25℃，254 nm波长下检测30 min。在此条件下，5氨基乙酰丙酸和甘氨酸在浓度为0.8~23 mmol/L和1.3~66 mmol/L时与色谱峰面积值之间线性关系良好，回归系数均在0.9997以上。发酵液产物的加标回收率在94.9%~104.7%。将检测结果与分光光度法相比，符合较好。测试数据表明，此方法简单、可靠，对于发酵产物的分离效果好。关键词 5氨基乙酰丙酸，反相高效液相色谱法，梯度洗脱，柱前衍生，发酵液
Determination of 5Aminolevulinic Acid in E.coli Fermentation Broth by Reversed Phase High Performance Liquid Chromatography with Precolumn Derivatization
Fan Li, Lin Jianping*, Liu Xiaoxia, Qin Gang, Cen Peilin
(Institute of Bioengineering, Zhejiang University, Hangzhou 310027)
Abstract A reversed phase high performance liquid chromatography with precolumn derivatization was developed for the simultaneous determination of 5aminolevulinic acid (ALA) and glycine (Gly) in E. coli fermentation broth. A ZORBAX SBAQ column (250 mm×4.6 mm i.d.，5 μm)was used for separation, and the elution was performed with linear gradient elution of methanol and 50 mmol/L sodium acetate solution (adjustment of pH to 6.2 with acetic acid ). The flow rate was 1 mL/min, the temperature of column was 25℃, and the detection wavelength was at 254 nm with the detection time of 30 minutes. The dynamic linear range is 0.8-23 mmol/L and 1.3-66 mmol/L for ALA and Gly respectively, and the correlation coefficients are both above 0.9997. The average recoveries of ALA in 3 samples of fermentation broth ranged from 94.9% to 1047%. The results agreed well with those of spectrophotometry. So the developed method is simple and reliable. ......
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