反向高效液相色谱测定发酵液中的聚苹果酸

Determination of Poly(malic acid) in Fermentation Broth by RP-HPLC

摘要:

采用高效液相色谱柱AlltimaTM C18 (4.6 mm×150 mm, 4 μm)上定量测定发酵生产的聚苹果酸，流动相为乙腈和0.025 mol/L磷酸二氢钾混合溶液（磷酸调pH 2.5）（V:V=5:95），流速1.0 mL/min，检测波长210 nm，柱温25 ℃，进样量5 μL。发酵液经离心除菌体、纯沉除多糖、水解处理后进样，可以将其中的L-苹果酸完全分离定量。采用液相方法的回收率为98.70%~100.65%，RSD为0.4~1.11%。采用试剂盒的方法的回收率为96.7~97.23%，RSD为0.57~1.19%。可见，液相的精确度和试剂盒相差不大。但液相的价格相对比试剂盒要便宜很多，说明该法是测定发酵液中聚苹果酸的一种很有效的方法。

Abstract:

For the detection of poly (malic acid) fermentation broth, a reversed phase high performance liquid chromatography method was developed. The samples was analyzed on a column of AlltimaTM C18 (4.6 mm×150 mm, 4 μm) with a mixture of acetonitrile and 0.025 mol/L KH2PO4 buffer (pH 2.5) (5:95, v:v) used as mobile phase at the flow rate of 1.0mL/min. The detection wavelength was 210 nm; the column temperature was 25 ℃ and the volum of injection was 5 μL. Before sampling, the fermentation broth was centrifuged to remove fungis, added with organic solvent to remove polysaccharide and hydrolysed. L-malic acid were completely separated and determined in 3 minutes. The recovery and relative standard deviation (RSD, n=3) of L-malic acid by RP-HPLC method were 98.70%~100.65%, and 0.40%~1.11%, respectively. The recovery and relative standard deviation (RSD, n=3) of L-malic acid by L-malic acid kit were 96.70%~97.23% and 0.57%-1.19%, respectively. The method was proved to be a effective way to determine the poly (malic acid) in fermentation broth.