HPLC-PAD法优化乌骨鸡活性肽分离纯化

Optimization of isolation conditions for active peptides from Black-Bone Silky Fowl (Gallus gallus domesticus Brisson) by HPLC-PAD

乌骨鸡是一种具有药用价值的鸡种,乌骨鸡的鸡肉蛋白通过酶促水解后可获得多肽产物乌骨鸡活性肽,营养价值高,分离纯化活性肽是研究乌骨鸡应用价值的基础。利用装置C18反相色谱柱或TSKgel凝胶色谱柱的高效液相色谱仪进行乌骨鸡活性肽的分离优化,针对流动相、流速进行优化的同时,参考波长、进样量,以出峰数为主,结合分离度等考察分离效果。结果表明:C18柱的出峰数随甲醇含量及流速的降低而增加;三氟乙酸或磷酸盐作缓冲液时出峰数基本一致,但分离度不同;综合因素得pH 6.0磷酸盐/6%甲醇/水体系的流动相、0.2 mL/min的流速为该实验下的最佳分离条件。TSKgel柱的乙腈分离效果优于甲醇,梯度洗脱效果较好;综合因素得出0.1%三氟乙酸/(10%~25%)乙腈/水体系的流动相、0.5 mL/min的流速为该实验下的最佳分离条件。

Black-Bone Silky Fowl is a kind of chicken with medicinal value,the active peptides with high nutritional value can be obtained by enzymatic hydrolysis of the muscle protein.The separation and purification of active peptides are the basis of the application value of Black-Bone Silky Fowl.Two common methods,C18 reversed-phase chromatography and TSKgel filtration chromatography,were used to optimize isolation conditions for active peptides from Black-Bone Silky Fowl based on HPLC-PDA.The effects of these aspects on the isolation were studied,such as a raw material mixture ratio of mobile phase,flow rate,injection amount and wavelength.Isolation effects were based on the peak numbers in combination with peak resolution.When isolated by C18 reversed-phase chromatography,the number of peaks was increased by the decrease of methanol concentration and flow rate.Moreover,peak numbers were almost the same but not peak resolution.The optimizing isolation conditions were pH 6.0 sodium phosphate/6%methanol/distilled water and 0.2 mL/min flow rate.When isolated by TSKgel filtration chromatography,isolation effect of acetonitrile was better than methanol,and gradient elution effect of acetonitrile was better.The optimizing isolation conditions were 0.1%trifluoroacetic acid/(10%-25%)acetonitrile/distilled water and 0.5 mL/min flow rate.