东北林蛙皮肤活性肽的反相高效液相色谱纯化体系建立

Reversed Phase High Performance Liquid Chromatography Analysis of Antimicrobial Peptides from the Skin of Rana dybowskii

为建立东北林蛙皮肤抗菌肽反相高效液相纯化体系,将东北林蛙(Rana dybowskii)皮肤分泌物冻干粉用0.1%TFA水溶液溶解,并对分析样品进行全波长扫描,确定检测波长,通过SunfireTM C18(5μm,4.6mm×150mm)色谱分析柱对分离条件进行了优化。结果表明:分泌物在216nm和280nm处均有明显的吸收峰;乙腈能使样品较早被洗脱,而甲醇则能延迟样品的洗脱;流速与样品保留时间、分离度成反相关。经试验证实,最佳液相色谱检测条件为:检测波长216nm和280nm;流动相为V(乙腈)∶V(甲醇)∶V(水/TFA)=9∶1∶90;流速0.5mL/min;进样量20μL。

An experiment was conducted to establish the RP-HPLC purification system of antimicrobial peptides from the skin of Rana dybowskii. The lyophilized skin secretion of Rana dybowskii was dissolved in 0. 1 percent TFA and identified by ultraviolet full wavelength scanner, and C18 column SunfireTM（5 μm 4.6 × 150 mm） was used to optimize the mobile phase composition in HPLC by RP-HPLC. Result showed that UV detection wavelength was performed at 216 nm and 280 nm, and the sample was cluted early by acetonitrile and later by methanol. The flow rate was in an inverse correlation to retention time and resolution. The optimal conditions were obtained as the detection wavelength 216 nm and 280 nm, mobile phase （acetonitrile, methanol and water） at a volume ratio of 9： 1： 90, flow rate 0.5 mL·min^-1 , and sample size 20 μL each time.