A New Spherical Hydroxyapatite for High Performance Liquid Chromatography of Proteins

Abstract

Basic properties of a newly developed hydroxyapatite column and results of its application to the separation of proteins are described. The hydroxyapatite was completely spherical and porous beads in appearance by scanning electron microscopy, and showed superior properties to other types of hydroxyapatite column. The column was mechanically strong enough to show the pressure limit of 140–150 kg/cm². The hydroxyapatite column showed excellent mechanical and chemical stability, and was applicable to high speed and high resolution separation of proteins. Proteins are recovered in high yield after the chromatography.     

4′-甲基-7-(2-萘磺酰基)大豆素的细胞吸收及代谢研究

对新型大豆素萘磺酸酯衍生物DD的细胞吸收和代谢变化进行研究,并根据实验结果预测其可能存在的体外代谢途径。利用高效液相色谱法对DD在人主动脉血管平滑肌细胞(HAVSMCs)内的吸收和利用率进行研究,并利用液相色谱-质谱联用技术考察其细胞代谢变化。HAVSMCs对DD的吸收率达到了74.3%,远远高于大豆素的细胞吸收率。细胞代谢研究表明,DD中4′-和7-位上的取代基影响其代谢途径和代谢产物。与4′-位的甲基相比,DD的7-位的萘磺酰基更容易水解。对药物分子进行适当的结构修饰和改造,可以增强药物分子的脂溶性,进而提高药物分子在细胞中的吸收利用率,说明药物的吸收利用率与其理化性质密切相关。     

Recent Developments in the Use of Group-Specific Ligands for Affinity Bioseparations

Abstract

One of the major obstacles encountered in the commercialization of biochemical products is their separation and purification since it can represent up to 60% of the total production costs^l. Using conventional methods such as filtration, centrifugation, extraction, precipitation, and chromatography usually requires many separation steps in order to isolate biomolecules with similar physicochemical properties. Conventional techniques suffer from a lack of selectivity for a specific biomolecule in a complex mixture, especially when the biomolecules in the mixture have similar properties. A dramatic increase in selectivity is obtained when the method of recovery is based on affinity interactions between the desired macromolecule and its complementary ligand.     

Cell Separations on the Countercurrent Chromatograph

Abstract

Separation of cells differing only subtly has been achieved by partitioning between the two phases formed by solution of dextran and polyethylene glycol in water. Cell populations which have related, but not identical, surface properties seldom exhibit sufficiently different partition behavior to be separated in a single extraction. In such cases, repeated partitions are carried out via countercurrent distribution or countercurrent chromatography to effect the separation. Potential advantages of countercurrent chromatography are its ease and rapidity of operation. In the present work we describe our approach to determining and possibly improving the efficiency of polymer phase partitioning by countercurrent chromatography.     

REE bound polysaccharides in leaves of Dicranopteris dichotoma by MAA

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Preservation of Biological LH and FSH Activity after Application on HPLC. Comparison between Cation-Exchange and Reverse-Phase High Performance Liquid Chromatography

Abstract

A cation-exchange high-performance liquid chromatography procedure is described for the separation of bioactive lutropin from follitropin in a human urinary gonadotropin standard preparation (1st IRP 70/45). The results clearly demonstrate that recovered protein retained most of its biological activity following the chromatography. Due to a lack of efficient and quick methods for purifying pituitary or urinary gonadotropins, this method could prove to be valuable for this purpose. This method has also been shown to be quick (less than 20 min) and efficient for separation of impurities or degradation products from bovine follitropin with 92% preservation of its biological activity. Our results suggest that ion exchange HPLC procedure will play a powerful role in the isolation of gonadotropins and other biologically active compounds.     

High Performance Liquid Chromatography of Proteins on a Hydroxyapatite Column

Abstract

High performance liquid chromatography on a spherical ceramic type hydroxyapatite has been applied successfully for the separation of various kinds of proteins. Twenty-one proteins of various origin, having an isoelectric point of 3.3–11.0 and a molecular weight of 11,000–190,000 daltons, were loaded on the column and eluted by linear gradient of sodium phosphate at pH 6.8. The chromatography showed good resolution and high recovery for the proteins. The analysis of the retention behavior, relation between capacity ratio and physicochemical properties of proteins, showed a tendency that the capacity ratio of protein increased with the pI value of the protein.     

Chromatography Liquide a Haute Pression DE Colorants Alimentaires Synthetiques

Abstract

The synthetic food dyes separation is described using the paired-ion chromatography. The method used allows the separation of the most permitted food colours and the orange GGN (CI 15980). Differentiation of this dye from its isomer, the sunset yellow (CI 15985), using the usual chromatographic methods still now remains unsuccessful.     

Separation of Thiamin,Thiamin Antagonists and Their Phosphate Esters by High-Performance Liquid Chromatography

Abstract

Methods are presented for the separation of the phosphate esters of thiamin, oxythiamin or pyrithiamin and the separation of thiamin and oxythiamin by high-performance liquid chromatography. Detection by means of a UV monitor and fluorometer with a post-column oxidation system is described which allows for selective detection of thiochrome, pyrichrome and their respective phosphate esters. Anion exchange and ion-pair reverse phase chromatography are the chromatographic modes of separation.     

Chromatographic and adsorption properties of the mixed stationary phase poly(1-trimethylsilyl-1-propyne)/poly(1-phenyl-1-propyne)

Adsorption and chromatographic properties of the mixed stationary phase poly-(1-trimethylsilyl-1-propyne)/poly(1-phenyl-1-propyne) (PTMSP/PPP) composed as 97: 3 by weight have been investigated by methods of low-temperature nitrogen adsorption and gas chromatography on packed columns. The resultant phase has uniform mesoporous structure. The chromatographic properties of the mixed phase are significantly different from the properties of the original porous polymers PTMSP and PPP. The adsorbent obtained by modifying Chromosorb P NAW with a mixture of polymers provides the selective separation of chlorosubstituted, saturated, and aromatic hydrocarbons.     

Separation of Organic Acids in Plant Tissue by HPLC with a Twin Phase,Ion Exchange and Reverse Phase,Column

Abstract

No single isocratic chromatographic technique allows the complete separation of common organic aliphatic and alicyclic acids of plants. In order to obtain a better isocratic separation with a single HPLC method we combined in one chromatography assay the Ion Exchange and Reverse Phase technics in building a twin phase column. The first attempts are promising. This double chromatography based on the polarity of molecule (Reverse Phase) and on its acidic characteristics (Ion Exchange) has the advantages of both methods and allows good separations of the acids.     

Application of Thin-Layer Chromatography to High-Performance Liquid Chromatographic Separation of Steroidal Hormones and Cephalosporin Antibiotics

Abstract

High-performance liquid chromatographic (HPLC) separation of steroidal hormones and cephalosporin antibiotics was investigated by adsorption chromatography and reversed-phase chromatography, respectively.

Prior to the HPLC separation of these pharmaceuticals, silica gel thin-layer adsorption chromatography of steroidal hormones and reversed-phase thin-layer partition chromatography of cephalosporin antibiotics with chemically bonded dimethylsilyl silica gel were performed in order to obtain suitable HPLC separation systems.

In the separation of steroidal hormones, the same binary mobile phase ratios of TLC did not give satisfactory results in HPLC. For the sharp separation in HPLC, solvent strength in the binary solvent mixture used for TLC had to be decreased.

The difference in solvent strength for efficient separation between TLC and HPLC might be attributed to the fact that in HPLC the solvent elution power acts in an isocratic manner while in TLC it acts in a gradient manner.

On the other hand, a correlation of mobility between TLC and HPLC separation for cephalosporin antibiotics was obtained, and the possibility of direct transfer of chromatographic systems from TLC to HPLC for separation of these antibiotics was confirmed.     

基于巯基-烯点击化学法的β-环糊精固定相多模式色谱保留行为研究

基于巯基硅胶与单取代-6A-烯丙氨基-β-环糊精的巯基-烯点击化学反应,制备了β-环糊精(Click TE-CD)共价键合固定相。元素分析结果表明β-环糊精被成功键合到硅胶表面。以黄酮苷类化合物为模型,考察了Click TE-CD固定相在亲水、反相和超临界流体色谱等分离模式下的色谱保留行为。黄酮苷类化合物保留时间随流动相中乙腈含量的变化呈现典型的U型曲线,表明Click TE-CD固定相具有亲水/反相的双重保留特性。应用几何学方法测得Click TE-CD固定相在反相/亲水、亲水/超临界、反相/超临界混合模式下的正交性分别为69.8%、50.8%、50.8%。对比复杂中药样品降香提取物在反相、亲水、超临界等模式下的分离情况,结果表明Click TE-CD固定相在分离中药复杂样品方面具有极大潜力,可以在一根色谱柱上通过分离模式的改变,实现二维液相色谱的分离。Click TE-CD固定相不同分离模式的分离性能和较好的正交性表明该固定相具有在液相色谱方法发展和二维液相色谱分离方面应用的潜力。     

Computer Enhanced Spectroscopic Separation of Coeluted Components in Supercritical Fluid Chromatography Coupled with an Ultraviolet Multichannel Detector

Abstract

Computer enhanced spectroscopic separation technique has been applied to identification of coeluted components in supercritical fluid chromatography by using an ultraviolet photodiode array multichannel detector. The powerful function such as peak deconvolution procedure has successfully offered thespectroscopic complete separation for even partially or completely coeluted components in super critical fluid chromatography.     

Dimensional Separation Of Low Molecular Weight Compounds

Abstract

The current status of separation of low molecular weight compounds by size exclusion chromatography and the hybrid modes, size partition and size adsorption chromatography is reviewed.     

High-Performance Liquid Chromato- Graphic Assay for Kinetic and Inhibition Properites of Monoamine Oxidase form A

Abstract

A rapid and convinient method to study the kinetic and inhibition properties of rat brain monoamine oxidase form A by high-performance liquid chromatography is described. The procedure consists of homogenization of the brain tissue, enzymatic reaction with or without inhibitors using 5-hydroxytryptamine as the substrate, termination of the reaction with perchloric acid, removal of protein by centrifugation and detection of reaction products at 280 nm after HPLC separation. The method was applied to determine K_m and V_max values of 5-HT for MAO-A and to study the inhibition properties of MAO-A using clorgyline and 1-deprenyl as inhibitors.     

Use of Centrifugal Partition Chromatography and Proteins in The Preparative Separation of Amino Acid Enantiomers

Abstract

The growth of analytical methodologies for the separation of enantiomers has been impressive. Attention is now turning to the large scale separation of enantiomers. Often scaling-up sensitive analytical separations is ineffective and inefficient. Centrifugal partition chromatography (CPC) may be a viable alternative for the preparative separation of racemic mixtures in some cases. The use of proteins as chiral selectors in CPC is examined. Attention was focused on proteins that previously were used as bonded phases in analytical LC columns. The enzymatic properties of α-chymotrypsin allowed it to be used as a bioreactor in conjunction with CPC. When proteins are used as components of the stationary or mobile phase there can be problems with denaturation. However, when used in external incubation processes or as column bioreactors coupled with CPC, effective gram-scale separations can be performed. Tryptophan methyl ester was used as a model compound to evaluate this approach.     

Processing impact on tocopherols and triglycerides composition of soybean oil and its deodorizer distillate evaluated by high-performance liquid chromatography

In the present study, high-performance liquid chromatography (HPLC) was used for the separation of tocopherols and triglycerides of processed soybean oil and deodorizer distillate (DD). The results of normal and reversed-phase modes of HPLC revealed that concentrations of tocopherols and triglycerides content were decreased during the neutralization, bleaching, and deodorization processes. The loss of individual tocopherols ranged between 55.16% and 63.25%. During processing, triglycerides containing stearic-oleic-linoleic (SOL) moieties and palmitic-palmitic-linoleic (PPL) fragments showed greater reduction up to 38.14% and 37.69%, respectively. Among tocopherols and triglycerides; γ-tocopherol and oleic-oleic-oleic (OOO) were found to be in greater concentrations 5.53% and 19.78%, respectively in DD as compared to their counterparts. A maximum reduction of tocopherols was observed in the deodorization step. DD was found to be a rich source of bioactive components; therefore, it could be used for many industrial applications including pharmaceutical formulations, cosmetics, and food industries.     

Analysis of a Mixture of Polychlorinated Biphenyls,DDT and its Analogues by High-Performance Liquid Chromatography

Abstract

A simple and rapid method for separation and quantitative analysis of polychlorinated biphenyis (PCBs) and chlorinated pesticides (DDT and its analogues DDE and DDD in their o, o'-and p, p'-isomers) is described. The procedure consists of two steps: a) transformation of DDT and its analogues in o, p'-and p, p'-dichlorobenzophenone (DCBP); b)determination of the amount of PCBs and ∑DDT as DCBP by HPLC. Results obtained confirm that HPLC can be considered as an alternative or a supplementary methodology to conventional methods such as gas chromatography. The method is applied to marine organisms.     

Preparative Liquid Chromatographic Method for the Characterization of Minor Constituents of Lubricating Base Oils

Abstract

In an effort to isolate, identify, and measure the properties of the active ingredients in a lubricating base oil, a high performance liquid chromatography (HPLC) separation scheme has been developed. The preparative mode of production is necessary to yield sufficient amounts of minor constituents for property measurements in terms of friction, wear, and oxidation characteristics.

In friction and wear control, the polarity of the molecular species is more important than the function groups in the species. Therefore the design of the separation scheme is based on the relative polarity of various functional groupings. Because the effort is directed towards identifying key components rather than analysis of the major compositions, mass recovery requirement is critical.