高压液相色谱技术分离制备杆菌肽各组分的研究
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摘要
目的开发制备型高压液相色谱分离杆菌肽各组分的方法。方法根据杆菌肽各组分化学结构性质,采用纳微Unisil C_(18)柱,通过改变流动相pH和流动相配比,以及不同的上样量,探索杆菌肽各组分分离的方法。收集分离得到的不同组分,经脱盐-冷冻干燥,得到相应杆菌肽组分冻干粉。结果纳微Unisil C_(18)柱可以用于杆菌肽各组分的分离,流动相配比为甲醇(A)∶[1.54‰乙酸铵水溶液(乙酸调pH至5.0)-乙腈(9∶1,V/V)](B)=9.0∶11.0(V/V),总流速40 m L·min~(-1),上样量200mg,检测波长为254 nm。制备得到的杆菌肽各组分纯度均大于95%。结论该方法上样量大,稳定性好,收集到的各组分纯度高,为杆菌肽各组分的分离制备提供了借鉴。
Objective To develop the separation and preparation method of bacitracin components by preparative HPLC.Methods The appropriate separation method was determined through the p H of mobile phase,the ratio of mobile phase,and the loading quantity of sample by using nanomicro Unisil C_(18).Freeze-dried powders of bacitracin components were obtained by desalination and freeze drying of the collected solution.Results Nanomicro Unisil C_(18) chromatographic column could be used for separation.The ratio of mobile phase was A∶ B = 9.0∶ 11.0(V/V) [A was methanol and B consisted of 1.54‰ ammonium acetate solution(p H adjusted to 5.0)-acetonitrile(9∶ 1,V/V) ].The flow rate was 40 m L·min-1 and the sample load was 200 mg.The wavelength was 254 nm.The purities of bacitracin components were all above 95%.Conclusions The method is of great loading quantity of sample,good stability,and high purity of the prepared components.It provides reference for the separation and preparation of bacitracin components.
Objective To develop the separation and preparation method of bacitracin components by preparative HPLC.Methods The appropriate separation method was determined through the p H of mobile phase,the ratio of mobile phase,and the loading quantity of sample by using nanomicro Unisil C_(18).Freeze-dried powders of bacitracin components were obtained by desalination and freeze drying of the collected solution.Results Nanomicro Unisil C_(18) chromatographic column could be used for separation.The ratio of mobile phase was A∶ B = 9.0∶ 11.0(V/V) [A was methanol and B consisted of 1.54‰ ammonium acetate solution(p H adjusted to 5.0)-acetonitrile(9∶ 1,V/V) ].The flow rate was 40 m L·min-1 and the sample load was 200 mg.The wavelength was 254 nm.The purities of bacitracin components were all above 95%.Conclusions The method is of great loading quantity of sample,good stability,and high purity of the prepared components.It provides reference for the separation and preparation of bacitracin components.
引文
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[9]王茉莉,宋更申,常俊山.HPLC法同时测定杆菌肽原料药中主成分的含量及有关物质[J].中国药房,2015,26(6):834-837.
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[11]李伟,欧阳藩.制备色谱操作条件的优化[J].生物技术通报,2000(2):32-35.
[12]宁德生,梁小燕,方宏,等.半制备高压液相色谱法制备罗汉果苷V标准品[J].食品科学,2010,31(12):137-140.
[13]GOVAERTS C,LI C,ORWA J,et al.Sequencing of bacitracin A and related minor components by liquid chromatography/electrospray ionization ion trap tandem mass spectrometry[J].Rapid Commun Mass Spectrom,2003,17(12):1366-1379.
[2]徐加兵,魏晓东,那可,等.杆菌肽产生菌的诱变育种及培养基优化[J].中国医药工业杂志,2013,44(5):446-448,452.
[3]徐超,吕凤霞,别小妹,等.地衣芽孢杆菌Bacitracin A高产工业培养基优化[J].食品工业科技,2016,37(18):197-201,207.
[4]胡立勇,宋友礼.杆菌肽的研究[J].国外医药:抗生素分册,1988,9(1):55-64.
[5]RAUSSENS V,NARAYANASWAN V,GOORMAGHTIGH E,et al.Hydrogen/deuterium exchange kinetics of apolipophorin-Ⅲin lipid-free and phospholipid-bund states.An analysis by Fourier transform infrared spectroscopy[J].J Biol Chem,1999,271(38):89.
[6]谢瑞,李力,张明亮,等.杆菌肽高产菌株的诱变选育[J].食品工业科技,2015,36(11):188-192,196.
[7]杨宏,李军,孙立文,等.一种提取分离杆菌肽的方法:中国,CN102153633A[P].2011-08-17.
[8]宋如,彭敬梅,张健,等.高效液相色谱法测定杆菌肽的含量[J].河北化工,2009,32(9):70-71.
[9]王茉莉,宋更申,常俊山.HPLC法同时测定杆菌肽原料药中主成分的含量及有关物质[J].中国药房,2015,26(6):834-837.
[10]PAVLI V,KMETEC V.Pathways of chemical degradation of polypeptide antibiotic bacitracin[J].Biol Pharm Bull,2006,29(11):2160-2167.
[11]李伟,欧阳藩.制备色谱操作条件的优化[J].生物技术通报,2000(2):32-35.
[12]宁德生,梁小燕,方宏,等.半制备高压液相色谱法制备罗汉果苷V标准品[J].食品科学,2010,31(12):137-140.
[13]GOVAERTS C,LI C,ORWA J,et al.Sequencing of bacitracin A and related minor components by liquid chromatography/electrospray ionization ion trap tandem mass spectrometry[J].Rapid Commun Mass Spectrom,2003,17(12):1366-1379.