Analysis of Free Amino Acids During Fermentation of Iturin A by High Performance Liquid Chromatography

doi:10.13560/j.cnki.biotech.bull.1985.2018-0012

Abstract

Abstract: A fast and efficient HPLC with phenylisothiocyanate（PITC）as the pre-column derivatization to detect the concentrations of free amino acids was established and utilized to analyze the dynamic change patterns of free amino acids in the fermentation process of iturin A. The method using PITC as pre-column derivatizing reagent for HPLC analysis,liquid chromatography conditions were optimized. Amino acids were separated on a Vensuil-AA amino acid analysis column（4.6 mm × 250 mm,5 μm）,with optimal conditions：0.1 mol/L sodium acetate buffer（pH 6.4±0.1）-acetonitrile（66∶5）as mobile phase A and acetonitrile -water（4∶1）as mobile phase B by gradient elution,the flow rate was 1.0 mL/min,the UV detection wavelength was set at 254 nm,and chromatographic column temperature was 40℃. The results showed that gradient elution program,pH of the mobile phases and chromatographic column temperature had important effects on the analysis time of amino acids,peak separation and peak shapes. Sixteen amino acids were separated drastically in 35 min under the optimal chromatographic conditions. In addition,each amino acid was separated with fine linearity（R²> 0.9986）in a range of the test concentration,and the average recoveries were 83.84% to 108.02% with RSD less than 2.77%. The method was accurate and reliable with favorable precision and stability. Based on this method,the dynamic changes of free amino acids during the fermentation of iturin A was studied,by which three different changing trends of various amino acids were revealed.

Key words: phenylisothiocyanate, high performance liquid chromatography, free Amino acids, iturin A

ZHAO Jie, YUE Hua, GOU Xue-lei, ZHOU Jin-yan, TAN Hong. Analysis of Free Amino Acids During Fermentation of Iturin A by High Performance Liquid Chromatography[J]. Biotechnology Bulletin, 2018, 34(8): 151-158.

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