大豆异黄酮的分离与纯化

天然大豆异黄酮作为健康食品在防治骨质疏松和癌症方面具有一定功效。由于化合物结构相似,异黄酮甙元特别是高纯度黄豆黄素（glycitein）的获得有一定难度,文献报道大多是通过盐酸水解异黄酮甙的方法获得,而这种方法对环境污染和工厂生产设备腐蚀较大。本文报道了用醇溶剂进行固相提取以及硅胶柱色谱方对含有三种大豆异黄酮甙元的混合物产品进行分离。通过低成本和无环境污染的固相提取方法得到纯度为97％的黄豆黄素和纯度超过95％的大豆黄素（daidzein）;95％纯度的另一种大豆甙元金雀异黄素（genistein）则通过硅胶柱色谱分离得到。应用硅胶柱色谱,一次性分离了一种含有两个异黄酮甙：大豆甙（daidzin）和黄豆甙（glycitin）的产品。     

大孔吸附树脂结合硅胶柱层析纯化环孢菌素A

采用大孔吸附树脂层析结合硅胶柱层析,对环孢菌素A的分离纯化进行研究,确定了最佳层析条件,建立了工业化制备环孢菌素A的工艺。大孔吸附树脂层析选用D101树脂作为吸附介质,提取液丙酮含量控制在50%,最大吸附量为35 mg/g湿树脂,洗脱剂选用丙酮;硅胶柱层析选用42~64μm硅胶作为层析介质,最优层析条件为柱床高径比10∶1,流动相配比V(石油醚)∶V(丙酮)=70∶30,流速80 mL/m in,环孢菌素A上样质量浓度100 g/L,硅胶层析平均收率为84.2%,环孢菌素A纯度可达到97%以上,整个工艺总收率为65%~70%。     

Determination of deoxynivalenol in cereals by immunoaffinity clean-up and ultra-high performance liquid chromatography tandem mass spectrometry

An immunoaffinity column (IAC) was prepared with a new deoxynivalenol (DON) monoclonal antibody and used as a clean-up tool before ultra-high performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) analysis of DON in cereals. The developed IAC clean-up method showed high recoveries for DON. They ranged from 61% to 103% in wheat, rice, and millet with intra-day and inter-day variations below 19% and 17%, respectively. The column capacity was 2.86μg DON per mL of gel, and it maintained above 0.68μg/mL of gel after 10 cycles of usage at 2 days intervals. The limit of detection (LOD) and limit of quantification (LOQ) were 0.3 and 0.8μg/kg, respectively. Twenty-one out of 40 analyzed commercial cereal samples were positive at DON concentrations from 7 to 534μg/kg.     

铜藻岩藻黄素提取及纯化工艺研究

为了对岩藻黄素的提取、纯化进行系统研究,进而为高纯度岩藻黄素的工业化生产提供研究基础,筛选了适用于提取铜藻（Sargassum horneri）鲜藻中岩藻黄素的有机溶剂,并通过单因素实验和正交实验确定了最佳的提取溶剂浓度、提取温度、提取时间、料液比等工艺参数。随后采用硅胶柱层析法进行纯化,并通过单因素实验确定了最佳的硅胶柱床高度、上样量和洗脱流速。最后采用制备液相法对经层析纯化的岩藻黄素进一步纯化。结果表明,有机溶剂萃取的最佳工艺条件为：甲醇浓度90%,提取温度50 ℃,提取时间1 h,料液比1∶10,此条件下岩藻黄素提取率达到(0.258 9±0.003 6) mg·g-1鲜重（FW）［(1.078 8±0.015 0) mg·g-1干重（DW）］。硅胶柱层析的最佳工艺条件为：硅胶柱床高度10 cm,上样量6 g,洗脱流速10 mL·min-1,此条件下岩藻黄素得率为0.176 5 mg·g-1FW（0.735 3 mg·g-1 DW）,纯度为87.01%±0.88%。经制备液相进一步纯化后,岩藻黄素得率为0.127 1 mg·g-1 FW（0.529 4 mg·g-1 DW）,纯度为99.27%±0.22%。研究所用工艺简单,岩藻黄素得率高,为高纯度岩藻黄素的制备提供了实验基础。     

两种水溶性抗菌活性物质的分离提取

对水溶性、不解离的极性物质分离时,一般采用吸附层析和凝胶层析等途径。实验通过硅胶柱层析、葡聚糖凝胶柱层析以及硅胶ＧＦ２５４制备型薄板层析,从发酵液样品中分离出两种有抗菌活性的纯物质。薄层层析的展开剂为二氯甲烷－四氢呋喃－甲醇－水（２５：３０：２）,分离出的组分中Ｒｆ＝０．７和Ｒｆ＝０．８两种物质有抗菌活性。硅胶柱层析洗脱过程为梯度洗脱,先用１５０ｍｌ上述展开剂洗脱,再用二氯甲烷－甲醇（２０：８０）     

General method for purification of α-amino acid-n-carboxyanhydrides using flash chromatography

We describe the application of flash column chromatography on silica gel as a rapid and general method to obtain pure α-amino acid-N-carboxyanhydride (NCA) monomers, the widely used precursors for the synthesis of polypeptides, without the need for recrystallization. This technique was effective at removing all common impurities from NCAs and was found to work for a variety of NCAs, including those synthesized using different routes, as well as those bearing either hydrophilic or hydrophobic side chains. All chromatographed NCAs required no further purification and could be used directly to form high molecular weight polypeptides. This procedure is especially useful for the preparation of highly functional and low melting NCAs that are difficult to crystallize and, consequently, to polymerize. This method solves many long-standing problems in NCA purification and provides rapid access to NCAs that were previously inaccessible in satisfactory quality for controlled polymerization. This method is also practical in that it requires less time than recrystallization and often gives NCAs in improved yields.     

An improved enzymatic cycle for nicotinamide-adenine dinucleotide phosphate.

We describe the use of column chromatography on the nonpolar adsorbent. Amberlite XAD-2, and on silanized silica gel in the desalting and partial purification of cobalamins. These techniques are both simpler and more versatile than phenol extraction, without sacrificing efficiency. In addition, a solvent system for thin-layer chromatography on silanized silica gel is described which rapidly separates naturally occurring cobalamins.     

Nicotinamide as a Plant Growth Regulator Isolated from Rice Hulls

Aqueous 70% methanol extract of rice (Oryza sativa, var. Hadsaduri) hulls was found to contain an effective plant growth regulator, which was located at Rf 0.7 on a histogram on silica gel (Kieselgel GF254) TLC (iso-PrOH: 14% NH₄OH, 10:2). After preliminary purification by ion exchange, the crude extract was repeatedly purified by column chromatography. The active substance thus obtained as colorless crystals was identical with authentic nicotinamide. The growth promotive effect of nicotinamide in co-operation with ammonium ion on the intact plant at low concentration was observed.     

Purification of gramicidin A.

A simple chromatographic purification of the naturally occurring ion channel-forming pentadecapeptide gramicidin A (gA) is presented. This procedure allows gA to be isolated in gram quantities from the commercially available mixture of isomers after chromatography on silica gel. The gramicidin A obtained in this manner is greater than 95% pure as determined by 1HNMR, HPLC, and amino acid analysis.     

Determination of deoxynivalenol (DON) in blood, bile, urine and excrement samples from swine using immunoaffinity chromatography and LC-UV-detection

A work up procedure is described by which DON concentrations in blood, bile, urine and excrements from swine can be quantified by HPLC and UV- detection at λ = 220 nm. The central step thereby is the purification and concentration of DON by means of an immunoaffinity column. While, in our experiments, the quantification of DON in blood and urine was straightforward an additional purification step by a preparative HPLC run prior to immunoaffinity chromatography was needed when bile and excrements were investigated. However, when low DON concentrations in blood and urine are expected, a preparative HPLC run prior to immunoaffinity chromatography is recommended as well, because larger amounts of sample materials should be analyzed and more impurities interfere with the column proteins. In our study, using spiked samples, recoveries ranged from 75—90% and limits of detection were 0.01 to 0.02 μg/ml.     

Purification of growth-promoting peptides and proteins, and of histones, by high pressure silica gel chromatography.

A rapid method for the purification of histones and a variety of growth-promoting proteins and peptides by chromatography on silica gel has been developed. The isolation of the growth-promoting components of serum has been hampered by excessive losses associated with the use of water-based purification mens in acidic methanol-H2O solutions (eg. insulin, albumin, the somatomedins) provides a basis for purification on high-pressure silica gel columns, while peptides and histones can be purified in similar solvents. After column chromatography, the solvent is removed by flash-evaporation, or the protein may be precipitated directly from the solvent by neutralization of the pH and the addition of ethanol. The retention of biological activity (eg. somatomedin-C binding to insulin receptors and cell-growth stimulation) and recovery are excellent.     

The Azure Dyes their Purification and Physicochemical properties. II. Purification of Azure B

A method is described for the purification of the dye azure B in quantities sufficient for biological staining experiments on a larger scale. The method is based on the use of column chromatography. Two columns are employed. In column A with silica gel as adsorbent the azure B fraction is isolated from a suitable substrate ('technical' azure B gained by a modification of Bernthsen's synthesis of methylene blue, or plychrome methylene blue) using an acetate-formate mixture as eluent. In column B, on an Amberlite polyineric adsorbent (XAD-2) the acetate-formate anions are exchanged for chloride. Regeneration of both columns is possible: KMnO₄, Na₂S₂O₄ and water are run through column A, 5% NaOH, methanol and water through column B. Purification of azure B on economic terms is thus attained. The opinion is expressed that this method is also applicable to the purification of other cationic dyes.     

The azure dyes: their purification and physicochemical properties. II. Purification of azure B.

A method is described for the purification of the dye azure B in quantities sufficient for biological staining experiments on a larger scale. The method is based on the use of column chromatography. Two columns are employed. In column A with silica gel as adsorbent the azure B fraction is isolated from a suitable substrate ('technical' azure B gained by a modification of Bernthsen's synthesis of methylene blue, or polychrome methylene blue) using an acetate-formate mixture as eluent. In column B, on an Amberlite polymeric adsorbent (XAD-2) the acetate-formate anions are exchanged in chloride. Regeneration of both columns is possible: KMnO4, Na2S2O4 and water are run through column A; 5% NaOH, methanol and water through column B. Purification of azure B on economic terms is thus attained. The opinion is expressed that this method is also applicable to the purification of other cationic dyes.     

离心液相色谱法快速分离纯化神经节苷脂

神经节苷脂在脊椎动物的神经组织细胞膜上含量丰富,除有受体的功能外,还有许多其它功能。采用离心液相色谱法,以国产硅胶Ｇ－６０为填料,分离纯化神经节苷脂,取得了满意的效果。经分离后产物的脂结合唾液酸（ＬＢＳＡ）含量,由１４．３％提高到２４．１％。以蛋白质含量为代表的含氮化合物由２２．０％降至９．６％。回收率为９０．２５％。特点是操作简便、周期短、成本低、效果好。优于ｌａｔｒｏｂｅａｄｓ柱层析法。     

中试规模纯化海洋芽孢杆菌源脂肽类化合物

本次研究旨在建立经济可行的海洋芽孢杆菌源脂肽类化合物的中试规模纯化工艺。对包括酸化沉淀、甲醇浸提、溶剂沉淀、盐析、萃取、硅胶柱层析和HZ806大孔树脂吸附工艺在内的可放大的成熟单元工艺进行反复试验,考察脂肽类化合物表面活性对单元工艺的影响。严格遵循以高收率为前提循序渐进逐步减少杂质的原则,组合上述单元工艺对目标产物进行提取和纯化,并最终获得高纯度脂肽样品。新工艺可从1 t海洋芽孢杆菌Bacillus marinus B-9987的发酵液中,以百克量级的规模制备87.51%–100%纯度的脂肽类化合物样品,收率81.73%。本研究首次实现了高纯度的海洋芽孢杆菌源脂肽类化合物的百克量级制备;允许发酵生产阶段使用天然培养基,缓解了脂肽中游发酵生产和下游大规模纯化之间的矛盾;且各单元工艺规避了脂肽类化合物水溶液的乳化起泡和不经济的大体积水溶液蒸发浓缩。新工艺实用可行,经济合理。     

A preparative method for purification of riboflavin 5'-monophosphate.

A preparative column chromatography method was developed for preparation of pure riboflavin 5′-monophosphate. A crude preparation of riboflavin phosphate(s) was chromatographed on DEAE-cellulose to provide a mixture of riboflavin 4′- and 5′-monophosphates. The 5′-isomer was isolated by chromatography on a column of silica gel with an ethanol:1 m triethylammonium bicarbonate, pH 7.5 (85:15) solvent system.     

Solvent systems for improved isolation and separation of territrems A and B.

Territrems A and B, tremorgenic mycotoxins in the rice culture of Aspergillus terreus, were extracted with hot chloroform. The toxins were cleaned on a silica gel column by direct adsorption-concentration of the extracts followed by desorption with chloroform-acetone (9:1, vol/vol). Crude toxin mixtures were separated by silica gel column chromatography and eluted with benzene-ethyl acetate (65:35, vol/vol). The method gave 112 mg of territrem A, 379 mg of territrem B, and 80 mg of their mixture from 4 kg of rice per batch. The criteria of extraction, cleanup, and separation are provided.     

A rapid method for the purification of RNA polymerase holoenzyme from Escherichia coli

A method is described for the rapid purification of RNA polymerase holoenzyme from small amounts of Escherichia coli cells. Chromatography of a crude extract on a single-stranded DNA agarose column followed by gel filtration chromatography gave 95% pure holoenzyme. The enzyme had kinetic characteristics on T7 DNA identical to those of RNA polymerase purified by other more laborious procedures.     

Purification of Growth-Promoting Peptides and Proteins,and of Histones,by High Pressure Silica gel Chromatography

A rapid method for the purification of histones and a variety of growth-promoting proteins and peptides by chromatography on silica gel has been developed. The isolation of the growth-promoting components of serum has been hampered by excessive losses associated with the use of water-based purification methods. The solubility of many growth-promoting serum proteins in acidic methanol-H₂O solutions (eg. insulin, albumin, the somatomedins) provides a basis for purification on high-pressure silica gel columns, while peptides and histones can be purified in similar solvents. After column chromatography, the solvent is removed by flash-evaporation, or the protein may be precipitated directly from the solvent by neutralization of the pH and the addition of ethanol. The retention of biological activity (eg. somatomedin-C binding to insulin receptors and cell-growth stimulation) and recovery are excellent.     

A chemical method for the quantitative determination of 2-hydroxyestrone in human urine

A highly specific chemical procedure for the quantitative determination of 2-hydroxyestrone in the urine of pregnant women is described. The assay consists of the following steps: 1) Hot acid hydrolysis of 20 ml of urine, 2) purification of 2-hydroxyestrone by “reducing chromatography” on paper and silica gel column, 3) conversion of 2-hydroxyestrone to the phenazine compound, 4) purification of the phenazine derivative by alumina column chromatography, and 5) spectroscopic quantitation of the phenazine. For internal yield correction [4-¹⁴C]2-hydroxyestrone is added after urine hydrolysis. High specificity of the method is especially guaranteed by the specific transformation of 2-hydroxyestrone to a stable phenazine derivative and by rigorous chromatographic purification of the estrogen as well as of the phenazine. The method can be used for the determination of amounts of less than 1 μg of 2-hydroxyestrone/20 ml of urine. From the data obtained the coefficient of variation is calculated to be ±3.7%. The urinary excretion of 2-hydroxyestrone in late pregnancy was found to vary within a wide range of 30–800 μg of 2-hydroxyestrone/24 hours.It seems possible to extend this method to the determination of other 2-substituted estrogens present in urine.