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1.
高速逆流双水相色谱法纯化卵白蛋白 总被引:7,自引:0,他引:7
生物大分子的液_固色谱纯化过程中固相载体会产生产物吸附、变性等不良影响。高速逆流色谱无需固相载体 ,且具有高分便率和高回收率的优点 ,其中有机相 水相体系在分离天然产物中应用广泛 ,而应用双水相体系分离生物大分子尚处于研究阶段。双水相高速逆流色谱体系的建立与仪器设备及操作工艺条件密切相关 ,因此利用多分离柱高速逆流色谱仪 ,研究了PEG1000-无机盐双水相体系对标准蛋白质混合物以及卵白蛋白的分离。pH值和PEG浓度对不同种类蛋白质的分配系数影响不同 ,实验发现在pH9.2的150% (W/W)PEG1000 170% (W/W)磷酸钾盐体系中 ,细胞色素C、溶菌酶和肌红蛋白的分配系数差异较大 ,且分布合理 ,因而采用该体系在 0 8mL min流速 ,85 0r min转速的条件下 ,成功分离了细胞色素C、溶菌酶和肌红蛋白的混合物。实验也发现在pH9 2的 16 0 % (W/W)PEG10 0 0 17 0 % (W/W)磷酸钾盐体系中 ,鸡蛋清样品中的主要蛋白质成分:卵转铁蛋白、卵白蛋白和溶菌酶的分配系数差异最大 ,因而采用该体系在 1 8mL min流速、85 0r mi转速的条件下,200min内从鸡蛋清样品中成功分离卵白蛋白,其电泳纯度为100%,收率为95%. 相似文献
2.
【目的】应用高速逆流色谱法(High-speed counter-current chromatography,HSCCC)实现对小孢拟盘多毛孢Pestalotiopsis microspora PM-1菌株代谢产物中除草活性物质的首次分离。【方法】以正己烷:乙酸乙酯:甲醇:水(4:5:4:5,V/V/V/V)为最佳的两相溶剂体系,上相(水相)为固定相,下相(有机相)为流动相,正相洗脱。【结果】在流速2 mL/min、转速900 r/min、检测波长254 nm的条件下进行分离,得到4个馏分,其中馏分Ⅱ对马唐的活性较强。馏分Ⅱ经高效液相色谱法(High performance liquid chromatography,HPLC)以乙腈:水=75:25(V/V)为流动相经过C18柱进一步分离检测,所得馏分A对马唐种子萌发有较强的抑制作用。其保留时间为7.954 min,该峰经二极管阵列光谱检测为单一组分。【结论】利用最佳的高速逆流色谱条件和高效液相色谱条件对小孢拟盘多毛孢Pestalotiopsismicrospora PM-1菌株代谢产物进行分离,可获得除草活性化合物——馏分A。 相似文献
3.
pH-区带精制逆流色谱分离高F值寡肽新技术研究 总被引:1,自引:0,他引:1
高F值寡肽是一种由2~8个氨基酸组成的生理活性肽.本研究采用胃蛋白酶和α-胰凝乳蛋白酶对草鱼蛋白进行了两步酶解,高效液相色谱对酶解液进行了肽谱分析,以制备改善肝性脑病的高F值寡肽.结果表明,草鱼蛋白酶解液中含有高支低芳氨基酸的寡肽组分,开发一种pH-区带精制逆流色谱分离新技术,所用体系为甲基叔丁基醚:正丁醇:乙腈:水(2:2:1:5,、V/V/V/V),上相中加入20 mM三乙胺和20%磷酸二(2-乙基己基)酯(DEHPA)作为固定相,下相分两部分,分别加入10 mM HCl和20 mM HCl作为流动相,进行梯度洗脱.成功地对酶解液中的混合组分进行了分离,得到了5个高F值(F>20)的分离组分,估测其F值(F=OD214nm/DD280nm)高达24.7~36.4.该结果为利用低值鱼蛋白酶解制备功能性活性肽提供了新的制备方法. 相似文献
4.
利用高速逆流色谱法从雷公藤植物粗提物分离得到一个化合物.两相溶剂体系为正己烷/乙酸乙酯/甲醇/水(2∶3∶3∶2,V/V/V/V),水相作流动相,有机相作固定相.经单晶X-衍射分析确定该化合物为雷酚内酯异构体.晶体参数为:晶体为正交晶系,空间群为P2(1)2(1)2(1);晶胞参数为:a=0.71913(10) nm,... 相似文献
5.
采用紫外-可见分光光度计法进行了高速逆流色谱技术分离芦荟多糖的溶剂系统研究,得出了高速逆流色谱分离芦荟多糖的溶剂系统为w(PEG600)∶w(KH2PO4)∶w(K2HPO4)∶w(H2O)=5∶15∶15∶65,加入NaCl的质量分数为2%。在水浴温度30℃,转速600 r/min,下相流速为2 mL/min的条件下,采用高速逆流色谱技术成功分离出芦荟多糖粗品,得到APS-1和APS-2两个组分,经Sephadex G-100凝胶柱层析技术和高效凝胶渗透色谱技术初步分析:APS-1和APS-2均为单一组分。 相似文献
6.
糖的高效液相色谱分析研究——Ⅰ.中性糖、氨基糖和双糖的分析 总被引:1,自引:0,他引:1
本文报道常见的中性糖(包括醛糖、酮糖和脱氧糖)、氨基糖和双糖在氨基结合键固定相色谱柱上,以不同比例的乙腈-水和乙腈-水-甲醇体系作流动相时的高效液相色谱行为,测定了它们的保留时间和_R 和容量因子K'以及它们的定量方法,并对这些结果进行了讨论。 相似文献
7.
本文报道常见的中性糖(包括醛糖、酮糖和脱氧糖)、氨基糖和双糖在氨基结合键固定相色谱柱上,以不同比例的乙腈-水和乙腈-水-甲醇体系作流动相时的高效液相色谱行为,测定了它们的保留时间t_R和容量因子K′以及它们的定量方法,并对这些结果进行了讨论。 相似文献
8.
白坚木皮醇是天然橡胶制胶乳液中存在的一种具有极高药用价值的天然化合物,多存在于制胶废水中。由于其极性高且溶于水,在常用的反相液相色谱柱中保留值较低,分析和分离都较困难。本研究选用SHI-SEIDO PC HILIC色谱柱建立了亲水色谱法(HILIC)测定白坚木皮醇的方法。以乙腈-水(V/V,60/40)为流动相,二极管阵列(DAD)为检测器时,检测波长为190 nm,白坚木皮醇的保留因子为1.07,线性范围为0.05~4mg/mL,检测限0.01 mg/mL。 相似文献
9.
《天然产物研究与开发》2016,(7)
为筛选黄连中α-葡萄糖苷酶抑制剂,本研究采用高效液相色谱-电喷雾质谱联用技术(HPLC-DAD-MS)对黄连提取物中的化学成分进行分析鉴定,并采用高速逆流色谱分离其中的活性成分。选用反相C18色谱柱,以0.02%醋酸溶液(A)和甲醇(B)为流动相,进行梯度洗脱;利用电喷雾质谱(ESI-MS)正离子模式在线检测化学成分;以α-葡萄糖苷酶作为生物靶分子,以超滤质谱技术筛选酶抑制剂。再经高速逆流色谱分离纯化,以乙酸乙酯-正丁醇-乙醇-水(3.0∶1.7∶0.5∶6.0,v/v/v/v)为两相溶剂系统,所得分离收集液经高效液相色谱法检测。实验通过HPLC-DAD-MS共鉴定出5个化学成分,分别为药根碱、表小檗碱、黄连碱、巴马亭和小檗碱。通过HSCCC分离得到两种α-葡萄糖苷酶抑制剂巴马亭和小檗碱。利用液相色谱-超滤-质谱-高速逆流色谱联用技术可以快速分离鉴定黄连中的化合物。此方法对于筛选有效成分具有快速和灵敏等优势。 相似文献
10.
本文首次采用高速逆流色谱结合高效液相色谱的方法对桂枝正丁醇相进行分离纯化。首先,以石油醚-乙酸乙酯-甲醇-水(8∶2∶6∶4,v/v)为高速逆流色谱溶剂系统,将桂枝正丁醇萃取相分为两个馏分,然后结合制备高效液相,共分离得到4个高纯度化合物。通过核磁共振波谱鉴定其化学结构,分别为香豆素(1)、反式-邻甲氧基桂皮酸(2)、桂皮酸(3)、反式-桂皮醛(4),这四种化合物纯度经高效液相检测均大于95%。该方法简便、快速、节省溶剂,可以对桂枝正丁醇相进行快速有效的分离纯化,具有较好的实用价值,为桂枝资源的进一步开发应用提供了技术和物质支持。 相似文献
11.
Laabe于1987年提出了生物催化剂工程(Biocatalyst engineering)和介质工程(Medium engineering)的概念[1]。有机相生物催化中溶剂的选择也是介质工程的内容之一。纯酶在有机相中的催化作用已有大量报道[2],但对完整细胞研究甚步。本文以甲基单胞菌(Methylomonas Z201)完整细胞为生物催化剂.丙烯环氧化为指标反应.研究有机溶剂对活性的影响并对催化活性——溶剂疏水性进行了相关性分析。研究了水一十六烷两相体系中十六烷含量和搅拌速度对丙烯环氧化速度的影响和细胞的操作稳定性。 相似文献
12.
Use of a Novel Sub‐2 µm Silica Hydride Vancomycin Stationary Phase in Nano‐Liquid Chromatography. II. Separation of Derivatized Amino Acid Enantiomers
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A novel vancomycin silica hydride stationary phase was synthesized and the particles of 1.8 µm were packed into fused silica capillaries of 75 µm internal diameter (I.D.). The chiral stationary phase (CSP) was tested for the separation of some derivatized amino acid enantiomers by using nano‐liquid chromatography (nano‐LC). Some experimental parameters such as the type and the content of organic modifier, the pH, and the concentration of the buffer added to the mobile phase were modified and the effect on enantioselectivity, retention time, and enantioresolution factor was studied. The separation of selected dansyl amino acids (Dns‐AAs), e.g., Asp, Glu, Leu, and Phe in their enantiomers was initially achieved utilizing a mobile phase containing 85% (v/v) methanol (MeOH) and formate buffer measuring the enantioresolution factor and enantioselectivity in the range 1.74–4.17 and 1.39–1.59, respectively. Better results were obtained employing a more polar organic solvent as acetonitrile (ACN) in the mobile phase. Optimum results (Rs 1.41–6.09 and α 1.28–2.36) were obtained using a mobile phase containing formate buffer pH 2.5/water/MeOH/ACN 6:19:12.5:62.5 (v/v/v/v) in isocratic elution mode at flow rate of 130 nL/min. Chirality 27:767–772, 2015. © 2015 Wiley Periodicals, Inc. 相似文献
13.
MOTIVATION: Liquid chromatography-tandem mass spectrometry (LC-MS/MS) is a powerful tool in proteomics studies, but when peptide retention information is used for identification purposes, it remains challenging to compare multiple LC-MS/MS runs or to match observed and predicted retention times, because small changes of LC conditions unavoidably lead to variability in retention times. In addition, non-contiguous retention data obtained with different LC-MS instruments or in different laboratories must be aligned to confirm and utilize rapidly accumulating published proteomics data. RESULTS: We have developed a new alignment method for peptide retention times based on linear solvent strength (LSS) theory. We found that log k(0) (logarithm of retention factor for a given organic solvent) in the LSS theory can be utilized as a 'universal' retention index of peptides (RIP) that is independent of LC gradients, and depends solely on the constituents of the mobile phase and the stationary phases. We introduced a machine learning-based scheme to optimize the conversion function of gradient retention times (t(g)) to log k(0). Using the optimized function, t(g) values obtained with different LC-MS systems can be directly compared with each other on the RIP scale. In an examination of Arabidopsis proteomic data, the vast majority of retention time variability was removed, and five datasets obtained with various LC-MS systems were successfully aligned on the RIP scale. 相似文献
14.
Yang Y Gu D Aisa HA Ito Y 《Journal of chromatography. B, Analytical technologies in the biomedical and life sciences》2011,879(32):3802-3805
The performance of protein separation using the figure-8 column configuration in centrifugal counter-current chromatography was investigated under various flow rates and revolution speeds. The separation was performed with a two-phase solvent system composed of polyethylene glycol 1000/potassium phosphate each at 12.5% (w/w) in water and with lysozyme and myoglobin as test samples. In order to improve tracing of the elution curve, a hollow fiber membrane dialyzer was inserted at the inlet of the UV detector. The results showed that the retention of stationary phase (Sf) and resolution (Rs) increased with decreased flow rate and increased revolution speed. The highest Rs of approximately 1 was obtained at a flow rate of 0.01 mL/min under a revolution speed of 1200 rpm with a 3.4 mL capacity column. 相似文献
15.
A liquid-liquid partition chromatographic technique was applied to separate amphiphilic glycolipids. A two-phase solvent system composed of n-butanol-t-butyl methyl ether-acetonitrile-water at a volume ratio of 3:1:1:5 was found to be suitable for separating the gangliosides from total lipids extracted from rat brain by liquid-liquid partition chromatographic systems, namely centrifugal partition chromatography (CPC) and high-speed counter-current chromatography. GM1 could be separated rapidly by using the upper phase as stationary phase for both systems. Moreover, various kinds of gangliosides (GM1, GD1a, GD1b, GT1b) could be separated individually by using the lower phase as stationary phase by CPC. The sample can be recovered without loss by these systems. 相似文献
16.
A convenient method for the separation of molecular species of sphingomyelin by reversed-phase high-performance liquid chromatography (HPLC) is described. Sphingomyelin species from bovine brain and sheep and pig erythrocytes were resolved into 10-12 separate peaks on a micro -BondaPak C(18) or Nucleosil-5-C(18) reversedphase column with methanol-5 mM potassium phosphate buffer, pH 7.4, 9:1 (v/v) as the solvent. Detection was at 203-205 nm. The sphingomyelin species were primarily resolved due to specific hydrophobic interaction of their fatty acid and sphingoid chains with the alkyl ligand of the stationary phase. The retention time of the sphingomyelin species increased progressively as the number of carbon atoms in the hydrophobic chains increased in the homologous series. The presence of one double bond in the molecule reduced the retention time significantly. Introduction of a second double bond in the fatty acid side chain did not reduce the retention time to the same extent as the first double bond. The presence of a trans double bond in the sphingoid moiety increased the retention time of sphingomyelin more than did a cis double bond in the fatty acid side chain. The differential hydrophobic interaction observed between the ligand of the stationary phase and different alkyl chains of the sphingomyelin species illustrates that reversed-phase HPLC technique can be conveniently used to study the extent of relative hydrophobicity of different types of alkyl chains.-Jungalwala, F. B., V. Hayssen, J. M. Pasquini, and R. H. McCluer. Separation of molecular species of sphingomyelin by reversed-phase high-performance liquid chromatography. 相似文献
17.
William S. Powell 《Analytical biochemistry》1981,115(2):267-277
Prostaglandins and monohydroxy fatty acids derived from 8,11,14-icosatrienoic acid and arachidonic acid have been separated by high-pressure liquid chromatography using a cation-exchange column loaded with silver ions. The retention times in a variety of solvent systems have been determined for prostaglandin E1(PGE1), PGF1α, PGD2, PGE2, PGF2α, 6-oxoPGF1α, 15-hydroxy-8,11,13-icosatrienoic acid, 5-hydroxy-6,8,11,14-icosatetraenoic acid, 8-hydroxy-5,9,11,14,-icosatetraenoic acid, 9-hydroxy-5,7,11,14-icosatetraenoic acid, 11-hydroxy-5,8,12,14-icosatetraenoic acid, 12-hydroxy-5,8,10,14-icosatetraenoic acid, 15-hydroxy-5,8,11,13-icosatetraenoic acid, 8,11,14,-icosatrienoic acid, and arachidonic acid. The mechanisms involved in the interaction of solutes with the stationary phase have been investigated. Retention times on silver ion columns appear to be determined by a combination of interactions between (a) the silver ions of the stationary phase and double bonds of the solute and (b) polar groups of the stationary phase and polar groups of the solute. The relative contributions of these two types of interactions to the retention of solutes can be varied over a wide range by altering the composition of the solvent. In this way the selectivity of the stationary phase can be controlled in order to optimize the separation of any given group of solutes. The maximum separation of solutes on the basis of the number of double bonds they possess is obtained by using polar solvents containing low concentrations of acetonitrile. As the polarity of the mobile phase is reduced or the concentration of acetonitrile increased, the selectivity of the stationary phase tends to resemble that of normal-phase chromatography on silicic acid. 相似文献
18.
D Kuang 《Nucleic acids symposium series》1980,(7):89-98
A solvent extraction method for separating synthetic protected oligodeoxyribonucleotides was used in our laboratory based on the lipophilic property of the protecting group of 5'-OH of the oligomers. The extraction of synthetic products protected with MMTr is complete by ether or ether-chloroform (6:1 V/V) for mononucleosides, by chloroform for dinucleoside monophosphates, by dichloromethane:n-butanol (4:1 V/V) for trimer or tetramer, and is nearly complete by dichloromethane:n-butanol (2:1 V/V) for hexamer. The 5'-end phosphorylated nucleotides, oligonucleotides and their symmetrical pyrophosphates remain in water phase. The following synthetic products of protected oligodeoxyribonucleotides have been isolated with this method, all above 85% in purity: (Formula: see text). 相似文献
19.
The macrocyclic antibiotics represent a relatively new class of chiral selectors in CE, HPLC, and TLC. We have examined the use of the macrocyclic antibiotic vancomycin as a chiral selector in HPLC for the separation of 1,4-dihydropyridines (DHPs) calcium antagonists (CAs). Chromatographic data of six 1,4-dihydropyridine calcium channel blockers obtained on the vancomycin chiral stationary phase (Chirobiotic V) were compared with those obtained on an alpha(1)-acid glycoprotein (AGP) HPLC stationary phase. Optimization of pH and organic modifier was carried out in order to modulate the retention properties of each system. All chiral neutral DHPs were resolved on the AGP column, whereas on Chirobiotic V only basic DHPs showed a split peak. The analytical chromatographic procedure on Chirobiotic V proved suitable for semipreparative separation, since the separation factor on the analytical column was high enough to obtain pure enantiomers with high yields. 相似文献
20.
采用高速逆流色谱(HSCCC)技术从蛹虫草子实体粗提物中分离制备高纯度虫草素和N6-(2-羟乙基)-腺苷。利用高效液相色谱(HPLC)测定目标产物在溶剂体系中的分配系数,优化HSCCC分离虫草素和N6-(2-羟乙基)-腺苷的溶剂体系,确定了以乙酸乙酯-正丁醇-1.5%氨水(1:4:5,V/V/V)为HSCCC的两相溶剂体系,并运用此溶剂体系,上相为固定相,下相为流动相,主机转速850r/min,流动相流速为1.5mL/min,检测波长为254nm条件下进行分离制备,在250min内从200mg蛹虫草子实体粗提物中一步分离得到10.8mg纯度99%的虫草素和6.1mg 纯度98%的N6-(2-羟乙基)-腺苷。该方法简便、快速,为虫草素和N6-(2-羟乙基)-腺苷的大量制备建立了基础。 相似文献