Neuroendocrine Peptides - Analysis by Reversed Phase High Performance Liquid Chromatography

Abstract

A high performance liquid chromatographic (HPLC) method is described for determining the biologically active neuroendocrine peptides thyrotropin releasing hormone (TRH), leucine (leu) and methionine (met) enkephalin, angiotensin II, delta sleep inducing peptide (DSIP), luteinizing hormone releasing hormone (LHRH), substance P and growth hormone release inhibiting factor (somatostatin). The selection of mobile phases was limited to these systems that do not exhibit strong absorbtion at 215 nm and 254 nm. Under isocratic conditions at room temperatures with the appropriate selection of mobile phase it was possible by reversed phase chromatography to resolve all of the peptides investigated. We can resolve with the systems employed peptides differing by only one amino acid in chain length as well as peptides differing by only one amino acid in the chain sequence. The method is rapid, does not require derivitization, can be used with aqueous matrixes and is sensitive in the nanomolar range.

Our research has shown that most synthetic peptides lack purity and that all of the peptides except LHRH lack stability when stored in aqueous sterile solution at 8°C for four weeks. The implications of this latter finding are under investigation.     

Single Step Purification of a Synthetic Peptide Fragment of Neurofilaments by Preparative High Performance Liquid Chromatography

Abstract

It was recently shown that fragment Lys-Ser-Pro-Val (1,2) is the major site in vivo neurofilament phosforilation. A suitable method for the preparation of this peptide was therefore studied paying particular attention to the purification step. Even thought solubility of this peptide in methyl t-butil ether does not allow the standard purification. It is possible achieve a good separation in a single step by HPLC, after a simple extraction with acidic water.     

Preparative Purification of Peptides by Countercurrent Chromatography on the Ito Coil Planet Centrifuges

Abstract

For the purification of up to 1000 mg of synthetic peptides by countercurrent chromatography, the coil planet centrifuges have proven useful in the research laboratory. Besides the earlier described horizontal flow-through coil planet centrifuge which chromatographs substances in all solvent systems at room temperature, the multi-layer coil planet centrifuge affords more rapid chromatography. However, separations using n-butanol, especially suited for peptides, have to be conducted at elevated temperatures. A new machine that has characteristics of both of the foregoing instruments is the compact horizontal flow-through coil planet centrifuge which promises rapid chromatography with full retention of the stationary phase.     

多肽和蛋白质的反相高效液相色谱研究--色谱行为的多样性

以Ｃ８柱为固定相,研究了微管结合蛋白２中两段重要肽段（肽１,肽２）和猪胰岛素在反相色谱上的保留行为。当以甲醇溶液为流动相时,猪胰岛素保留因子的对数值（ｌｎＫ’）随流动相有机溶剂体积分数（ψ）的变化呈现很好的线性关系。当以乙腈为流动相测定肽１的ｌｎＫ’随ψ变化关系时,所得结果显示ｉｎＫ’与ψ呈现良好的二次曲线关系。以乙腈为流动相测定肽１和肽２和Ｖａｎ’ｔ Ｈｏｆｆ曲线（ｌｎｋ’与１／Ｔ的关系图）时,     

Liquid Adsorption Chromatography on Preparative Scale of Protected Synthetic Peptides

Abstract

This paper reports the purification of synthetic protected peptides on a preparative scale by means of adsorption chromatography on silica gel 60 columns.

The protected peptides described were precursors of a free asymmetrical cystine peptide corresponding to the insulin sequence A^18–21-B^19–26 and were obtained in analytically pure form. Selection of solvent systems for isocratic or stepwise elution depended largely on R_F data obtained from thin-layer chromatograms which were used for monitoring and optimizing synthetic reactions.     

High Performance Liquid Chromatography and Proteolytic Enzyme Characterization of Peptides in Tooth Pulp Extracts

Abstract

Metabolic profiles are obtained for peptides contained in tooth pulp extracts. To determine which high performance liquid chromatographic peaks are due to peptides, a series of proteolytic enzymes (chymotrypsin, trypsin, and carboxypeptidase A) are utilized. Results from treatment of extracts with immobilized enzymes demonstrate that virtually all peaks in this reverse phase system are due to peptides. This current study is a necessary component in a larger research program focusing on quantification of enkephalin-and endorphin-related peptides in biologic extracts including brain and tooth pulp tissue.     

Analysis of Group Retention Contributions for Peptides Separated by Reversed Phase High Performance Liquid Chromatography

Abstract

Within the framework provided by solvophobic theory, selectivities for unprotected peptides separated on fully porous, microparticulate, chemically bonded alkylsilicas can be ascribed to differences between the effective hydrophobic contact areas of the solutes. Furthermore, this theoretical treatment predicts that retention behaviour differences can be evaluated from topological parameters which accomodate the influence of amino acid side chain and end group contributions in the retention process. With data obtained for 57 peptides, including a variety of peptide hormones, eluted under the same conditions from a μBondapak C₁₈ column, these predictions have been rigorously tested using two methods of numerical analysis. The results provide further evidence that the hydrophobic group retention contributions of the amino acid residues in small peptides have an essentially additive effect on peptide retention with alkylsilicas. Divergences in retention behaviour are interpreted in terms of specific silanophilic and solvation interactions.     

高效液相色谱整体柱技术的进展

高效液相色谱整体柱（又名连续床）具有制备相对简单、原料易得以及聚合组分在一定范围内可调节的优点,是近年来得到迅速发展的新型色谱柱。本文综述了目前高效液相色谱（HPLC）制备整体柱的典型高聚物体系、制备各种整体柱时反应条件的影响,并简要介绍了它的表征方法和应用。     

Rapid Purification of Synthetic Bombesin by Countercurrent Chromatography on the Multi-Layer Coil Planet Centrifuge

Abstract

A temperature-controlled multi-layer coil planet centrifuge rapidly yields highly efficient preparative separations of polar compounds. Capability of the method was demonstrated on a one-step purification of crude synthetic bombesin with a two-phase solvent system composed of n-butanol/dichloroacetic acid/water (100:1:100). Under an elevated temperature at 45°C to 50°C, the bombesin peak was eluted within two hours. Reversed phase HPLC analysis of the bombesin fractions showed over 98% purity. The method may be applicable to many other peptides and polar compounds.     

多联吡啶衍生物的RP-HPLC分析

多联吡啶在分子元件、生物模拟、药理病理研究、分析检测等领域中应用前景[1 ] 广阔。我们曾合成了新型多联吡啶衍生物— 6,6″ -二甲基 - 4′-苯基 - 2 ,2′,6′,2″ -三联吡啶 (ＴＰＹ) [2 ] 并研究了它与过渡金属离子形成络合物的性能 ,但其产率低 ,限制了它的实际应用。本文探讨合成ＴＰＹ缩合反应中各物质的色谱行为及各物质ＲＰ -ＨＰＬＣ分离的适宜条件。1　实验部分1 1　仪器与药品Ｗａｔｅｒｓ 60 0高效液相色谱仪 (美国Ｗａｔｅｒｓ公司 ) ,2 4 87紫外 -可见分光光度检测器 ,Ｍ32数据采集和处理系统 ,Ｒｈｅｏｄｙｎｅ 772 5ｉ手…     

The Purification of Xanthene Dyes by Reverse Phase High Performance Liquid Chromatography

Abstract

A reliable method for the separation of fluorescein dyes from their impurities was developed using high performance liquid chromatography and involved a μBondapak C₁₈ reverse phase column and mixtures of methanol and ammonium acetate buffer. This technique was used to verify the purity of commercial products as well as to aid in the development of an empirical theory related to retention of halogenated fluorescein dyes by reverse phase columns.     

用反相高效液相色谱法纯化白细胞介素－2条件初探

用反相高效液相色谱法纯化白细胞介素－２条件初探胡明（沈阳军区军事医学研究所沈阳１１００３１）关键词反相高效液相色谱法，白细胞介素－２１前言高效液相色谱法（ＨＰＬＣ）是一项分辨率很高的液相色谱技术，具有速度快，灵敏度高、样品可回收等优点。除被广泛地应用...     

反相高效液相色谱分离纯化天然除虫菊酯

天然除虫菊酯含有6种有效成分,但结构相似,分离困难。本研究以除虫菊酯精油为原料,通过优化反相高效液相色谱分离条件,分离纯化了除虫菊酯6种有效成分,纯度均达到99%。然后用气相色谱-质谱定性分析确证6种成分。本研究所建立的天然除虫菊酯6种有效成分的反相高效液相色谱分离纯化方法,为天然除虫菊酯除虫菊酯残留检测提供了准确的判断标准,为阐明天然除虫菊酯6种单一有效成分的杀虫机理研究奠定了基础,也为除虫菊酯6种单一有效成分纯品的生产提供了重要的方法参考。     

Purification of Human Platelet Monoamine Oxidase B by High Performance Liquid Chromatography

Abstract

Human platelet monoamine oxidase (MAO B), a membrane bound enzyme was purified to homogeneity by DEAE-Sephacel column chromatography, chromatofocusing, and high performance liquid chromatography (HPLC). The crucial purification step was HPLC on a anion exchange column (SynChropak AX 300). The HPLC column was eluted initially with potassium phosphate buffer (100 mM, pH 7.4) for 10 min at a flow rate of 1.0 ml/min, followed by a gradient (0–1%) of octyl-β-D-glucopyranoside (octylglucoside) in the same buffer for 10 min, and finally with buffered octylglucoside (1%) for 40 min. The elution of pargyline-bound or active MAO was established by determining either radioactivity in each fraction when MAO B had previously been covalently labeled with [³H]-pargyline [³H(G)] or catalytic activity using [¹⁴C-methylene]-benzylamine as substrate. [³H]-pargyline-bound and active MAO B eluted from the column at approximately 34 min. The extent of homogeneity and the subunit M_r (approximately 59,000) of MAO B were determined by sodium-dodecyl sulfate polyacrylamide gel electrophoresis followed by silver staining for proteins.     

High Performance Liquid Chromatography of Dithiolthiones

Abstract

Sixteen dithiolthiones including oltipraz (5-(2-Pyrazinyl)-4-methyl-1-1, 2 dithiol-3-thione) and anethol dithiolthione (P-methoxyphenyl-1,2-dithiol-3-thione) have been studied using an isocratic high performance liquid chromatographic system. The retention characteristics of these compounds were determined using a conventional C 18 reverse phase column and a μ Bondapack phenyl column. The mobile phases used consisted of several concentrations of methanol and water. All compounds studied were adequately detected in the nanogram range (30–100 ng on column) using ultra violet detection (UV) set at 300 nm. Several analogs and isomers were separated. The retention characteristics of the 16 compounds studied are reported for both columns using 4 mobile phases and chromatograms of reference standards are presented.     

高效液相色谱法同时测定调味料中的8种合成着色剂

建立一种同时测定调味料中8种合成着色剂的高效液相色谱分析方法。样品经30％甲醇水溶液提取,采用Diamonsil—C18柱（250mm×4．6mm）,以甲醇-1．55g／L乙酸铵为流动相,在最佳梯度洗脱条件下对8种合成着色剂进行分离,二极管阵列检测器检测,外标法定量。8种合成着色剂的线性范围为0．5～10．0μg／mL,回收率为95．0％～101％。对样品进行6次重复测定,峰面积测定结果的相对标准偏差为0．84％-3．51％（n=6）。     

离子对高效液相色谱法测定果汁中的合成色素

采用聚酰胺吸附前处理果汁饮料,用离子对反相液相色谱法测定柠檬黄、日落黄、苋菜红、胭脂红四种合成色素。以含对离子试剂四丁基溴化铵0．005mol／L的甲醇：水=45：55(V／V)为流动相,15min内可完成四种合成色素的分离,回收率为89．8％～102．2％,变异系数为1．81％～2．22％。     

Liquid Crystals as Stationary Phases for High Performance Liquid Chromatography

Abstract

Liquid crystals have not yet been used as stationary phases in High Performance Liquid Chromatography. This is surprising since Gas Chromatography has demonstrated some remarkable separations, many of which are not possible with normal stationary phases, that have been achieved where liquid crystals have been employed as the stationary phase. The objective of the work reported here was to evaluate the chromatographic properties of several cholesteric liquid crystals as stationary phases in HPLC. Included in this study was an investigation of the feasibility of bonding a cholesteric moiety to a solid support for use in HPLC. The columns showed a dramatic increase in capacity factor for steroid molecules as the temperature of the column was increased.     

Preparation of High Efficiency Columns for High Performance Liquid Chromatography

Abstract

This method employs a specially constructed slurry reservoir, which features an internal taper and a quick-disconnect pump fitting. A major innovation is the use of a three-solvent system to maintain a slurry of packing material as a ‘slug’ within the reservoir. A measured amount of carbon tetrachloride is poured into the empty reservoir-column assembly. The column packing material is introduced as an isopropanol slurry and the reservoir is filled with isooctane. The assembly is then connected to a Haskel DSTV 122C pump, and the column is packed with isooctane at 10,000 psi pressure.

This procedure provides HPLC columns with efficiencies of 40,000 plates per meter for 10-micron silica, 30,000 plates per meter for 10-micron C₁₈, and 70,000 plates per meter for 5-micron C₁₈ packing materials. We have used this method for preparation of over 35 HPLC columns during the past two years.     

Non-Porous Silica Supports for High Performance Liquid Chromatography

Abstract

The physical and chromatographic properties of two types of non-porous HPLC support materials were examined. Loading capacities of non-porous supports were about 10 fold less than of wide-pore gels, while their physical strength was much greater, suggesting that non-porous supports could be useful for polymer separations. Such materials appeared to be less suitable for low-molecular-weight solutes, except for fundamental retention studies, where the absence of pore structure offers a less ambiguous support matrix.