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A new protease was isolated from the intestine of small red scorpionfish. After ammonium sulfate precipitation, the enzyme was purified to homogeneity by a two-step chromatography with 6.69% recovery and fivefold increase in specific activity. The molecular weight of the protease was about 24 kDa as estimated by size exclusion chromatography and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The enzyme optimum pH and temperature were pH 10.0 and 40°C, respectively. The protease was stable at temperatures below 40°C and over a broad pH range (8.0-11.0). The Km and Kcat values were 0.36 mmol L-1 and 1.61 s-1, respectively. Interestingly, the enzyme presented specificity for casein, egg albumin, bovine serum albumin, and gelatin with obtained degrees of hydrolysis ranging from 3.14% to 6.58%, after 2 h of hydrolysis. Moreover, gluten hydrolysate analysis confirmed protein hydrolysis and solubilization. These results suggested the potential use of the protease in the preparation of food protein hydrolysates with interesting properties. [ABSTRACT FROM AUTHOR]

Magnetic poly(2-hydroxyethyl methacrylate-co-N-methacryloyl–L-phenyl alanine), p(HEMA-co-MAPA) nanoparticles were produced by emulsion polymerization technique. The nanoparticles were characterized by zetasizer, atomic force microscopy (AFM), transmission electron microscopy (TEM), electron spin resonance spectroscopy (ESR), and Fourier transformed infrared spectroscopy (FTIR) techniques. Experimental conditions were optimized for DNA adsorption in aqueous media. The effects of several factors (i.e., temperature, pH, equilibrium concentration of DNA, salt type, and ionic strength) on adsorption capacity were examined in batch studies. The maximum DNA adsorption capacity was 211.3 mg/g for nanoparticles consisting of magnetite. The adsorption isotherms and kinetics were also examined. The Langmuir model and pseudo second-order kinetics are the best fitted to the data. Following the ten adsorption-desorption cycles, the decrease in adsorption capacity was only 12%. Finally, plasmid DNA was purified fromE.colilysate by magnetite nanoparticles consisting of MAPA. The purity and molecular mass of purified plasmid DNA were determined by the use of agarose gel electrophoresis resulting in 7000 base pairs for molecular mass and OC pDNA form following elution. [ABSTRACT FROM AUTHOR]

Flavonoids are the main chemical constituents ofSmilax chinaL. and thought to be responsible for the anti-inflammatory activity ofSmilax chinaL. In this study, a suitable HSCCC method was developed to separate the three main flavonoids in a one-separation from the anti-chronic pelvic inflammation disease (CPID) effective fraction ofSmilax chinaL. with a two-phase solvent system composed of n-hexane-ethyl acetate-methanol-water (1:10:1:10, v/v/v/v). 42 milligrams of astilbin, 10 mg neoisoastilbin, and 6 mg quercetin-3-O-α-L-rhamnoside were separated from 200 mg of the anti-CPID effective fraction ofSmilax chinaL. with purity of 98.3%, 98.7%, 98.5%, respectively, by HPLC analysis. Orthogonal test L9(34) was also applied to investigate the optimum extracting conditions of the three flavonoids. HSCCC was successfully used for the isolation and purification of the three flavonoids from the anti-CPID effective fraction ofSmilax chinaL. and the neoisoastilbin was first separated fromSmilax chinaL. [ABSTRACT FROM AUTHOR]

Forsythoside A and suspensaside A were isolated and purified fromForsythia suspensaby one step of high-speed counter-current chromatography for the first time using a solvent system of ethyl acetate-n-butanol-methanol-water (4:0.5:0.5:5,v/v). The purities of the two compounds were 98.19% and 98.05%, respectively, as determined by HPLC. Their structures were identified by MS, UV, and NMR analysis. Such a simple and effective method was fairly useful to prepare pure compounds as reference substances for related study onForsythia suspensa. [ABSTRACT FROM PUBLISHER]

Several methods have been demonstrated in the recovery and purification of cutinase; however, hydrophobic interaction membrane chromatography (HIMC) has yet to be implemented for this purpose. In this study, two factors, that is, buffer pH and ammonium sulphate concentration for HIMC operation, were optimized using response surface methodology (RSM), aiming to obtain the highest cutinase purification factor and recovery possible. The experimental design used was a central composite design (CCD) comprised of 13 experimental runs. The analysis of variance (ANOVA) indicated that a quadratic model best represented the system. Based on theR2values, it can be concluded that around 90% of the variability in the system can be explained by the fitted models. The optimized HIMC conditions were buffer pH 6.0 and 1.3 M ammonium sulphate, which was expected to provide 27-fold cutinase purification factor and 85% recovery. However, the actual values deviated from the predicted values with 13% and 18% error, respectively. The statistical analysis showed that only the ammonium sulphate concentration had a great influence on cutinase purification factor. Meanwhile, for cutinase recovery, both parameters were important in determining the optimal condition for cutinase adsorption and desorption to the chromatographic media. [ABSTRACT FROM AUTHOR]

An effective high–speed counter–current chromatography (HSCCC) method was established for semi-preparative isolation and purification of bioactive chemical constituents from the Tibetan medicinal plantPedicularis longifloravar.tubiformis(Klotzsch) P. C. Tsoong. With a two-phase solvent system composed of chloroform–methanol–water (8:4:5, v/v), 40 mg of extract was separated to yield luteolin (12.5 mg), apigenin (9.6 mg), and chrysoeriol (4.8 mg), with purities of 99.3, 98.2, and 98.6%, respectively, as determined by high-performance liquid chromatography (HPLC). The chemical structures of these three components were identified by1H NMR and13C NMR. [ABSTRACT FROM PUBLISHER]

The detrimental effect of heterogeneity on the efficiency of particle packed columns has been a long standing issue in chromatographic separations. This heterogeneity arises from the difference in packing density between the wall and central regions of the chromatographic bed and imparts radial variance in mobile phase velocity through the column. In conjunction with the uneven distribution of the sample band through frits and distributors at the head of the column, the resultant effect is a decrease in efficiency due to the formation of a parabolic-“like” sample band. We introduce the concept of “Active Flow Management” to counteract the effects of column heterogeneity via the separation of the flow eluting from the center of the column from that of the flow eluting near the peripheral wall region. The flow streams from the two aforementioned regions are separated by the use of a specialized column outlet fitting. By varying the ratio of flow eluting from the center to the peripheral region it was possible to increase the efficiency of a 100 × 21 mm column by up to 57% compared to a column operated in the conventional manner. [ABSTRACT FROM AUTHOR]

A successful method of preparative high-speed counter-current chromatography (HSCCC) was established for direct isolation and purification of hydrolysis products ofEpimedium koreanumNakai to obtain the secondary glycosides and aglycons of flavonoids inE. koreanum. The two-phase solvent systems of HSCCC composed n-hexane-ethyl acetate-methanol-water at the ratios of 6:3.5:6:5 (v/v) and 6:3.5:8:5 (v/v) were used in a stepwise elution. Totally, 60.1 mg of FractionIthat would be isolated and purified by preparative HPLC, 9.4 mg of icartin (4), 3.5 mg of desmethylicartin (5), 4.7 mg of 3′′-ethyoxyl-icartin (6), 7.3 mg of anhydroicaritin (7), and 10.1 mg of β-anhydroicaritin (8) were obtained in one-step separation from 100 mg of the crude materials with purities of 97.7%, 96.2%, 99.2%, 99.5%, and 91.0%, respectively. And FractionIof HSCCC was purified by the preparative HPLC to obtain 12.7 mg of pletypetaloside (I-1), 6.8 mg of icariside I (I-2) and 16.3 mg of 3′′-ethyoxyl-pletypetaloside (I-3) with purities of 90.8%, 93.4%, and 99.2%. All of the chemical structures were identified on the basis of widely spectral technology including 2D-NMR. Among them, compoundsI-3and6are novel. [ABSTRACT FROM AUTHOR]

In this study; sheep carbonic anhydrase-II (SCA-II) (E.C: 4.2.1.1) was purified from sheep liver and in vitro effects of heavy metals on the enzyme was examined. SCA-II isozyme was purified with about 203 purification fold, a specific activity of 2320 EU/mg-protein and a yield of 72 by using Sepharose-4B-L tyrosine-sulfanilamide affinity gel chromatography. Purity of the SCA-II enzyme was verified by SDS-PAGE technique and subunit molecular mass of the enzyme was found as 29 kDa. In addition to this, inhibitory effects of some metal ions on the enzyme were examined. In this study, sheep liver tissue was chosen; because the liver is an organ in which metal wastes of air, water and food are collected and it is easy to obtain the liver tissue. Because of the very important duties of CA enzyme on living beings, the effect of metals on the CA enzyme was investigated. [ABSTRACT FROM AUTHOR]

Prenylated isoflavonoids such as 6,8-diprenylorobol, 6,8-diprenylgenistein and 4′-O-methylalpinumisoflavone were purified from n-hexane extract of C. tricuspidata fruits using centrifugal partition chromatography (CPC) with a n-hexane-ethyl acetate-methanol-water (7:3:6:4, v/v, ascending mode) solvent system in a one-step within 200 min. One-step preparative CPC yielded 22.4 mg of 6,8-diprenylorobol and 26.7 mg of 6,8-diprenylgenistein with 95% and 85% purity, respectively, from 350 mg of crude hexane extract. Further purification by crystallization in methanol yielded 4′-O-methylalpinumisoflavone (9.3 mg) at over 95% purity based on the HPLC analysis. The chemical structures of purified compounds were elucidated by 1H, 13C NMR, and ESI-MS. [ABSTRACT FROM AUTHOR]

The article presents a study of the monoprotection of triamines using alkyl phenyl carbonates. It discusses the investigations carried out by Sayre and coworkers and Bruice and Willis regarding the acylation of 1,2-diaminoethane with different acylating reagents and the acylation of one of the amino groups in α,ω-diamine respectively. It prepares a series of monoprotected triamines from the triamines and alkyl phenyl carbonates. It develops a simple method for monoprotection of triamines in methanol with alkyl phenyl carbonates avoiding chromatographic purification.

A facile synthesis of 1,3,4-thiadiazine-5-yl-pyran-2-one derivatives is achieved via a three-component reaction involving 3-(2-bromoacetyl)-4-hydroxy-6-methyl-2H-pyran-2-one, thiocarbohydrazide with various carbonyl compounds in one-pot under stirring. The main advantage of this procedure is the short reaction time, high yields, simple workup, and purification of products by non-chromatographic methods, i.e. by simple recrystallization from ethanol. The structures of newly prepared compounds have been established by elemental analysis and spectral data. [image omitted] [ABSTRACT FROM AUTHOR]

Three flavonoid glycosides including quercetin-3-O-[2''-O-(6'''-O-E-sinapoyl)-β-D-glucopyranosyl]-β-D-glucopyranoside(I), quercetin-3-O-[2''-O-(6'''-O-E-feruloyl)-β-D-glucopyranosyl]-β-D-glucopyranoside(II) and quercetin-3-O-[2''-O-(6'''-O-E-feruloyl)-β-D-glucopyranosyl]-β-D-galactopyranoside(III) were isolated and purified from Hedyotis diffusa Willd by high-speed counter-current chromatography (HSCCC). This run was carried out with a two-phase solvent system composed of n-hexane-ethyl acetate-n-butanol-methanol-1.0% acetic acid (1:1:3.5:1:4.5, v/v) by eluting the lower phase as the mobile phase with a flow-rate at 2.0 ml/min. Consequently, 29.6 mg of I, 35.1 mg of II, 41.3 mg of III with purities of over 95% were obtained from 200 mg of the crude extracts in a single run in less than 130 min. The structure of the isolated compounds was confirmed by MS, 1H NMR, and 13C NMR analysis. [ABSTRACT FROM AUTHOR]

Preparative separation of quinolone alkaloids in Evodia rutaecarpa (Juss.) Benth was conducted by high performance counter-current chromatography (HPCCC) with a pair of two solvent systems consisting of n-hexane-methanol-water-acetic acid (2:1:1:0.2, v/v) and (5:4:2:0.1, v/v). Consequently, 31.78 mg 1-methyl-2-nonyl-4 (1H)-quinolone (I), 59.25 mg 1-methyl-2-(6-undecenyl)-4 (1H)-quinolone (II), 333.27 mg evocarpine (III), 101.13 mg 1-methyl-2-(6,9-pentadecadienyl)-4(1H)-quinolone (IV), 132.17 mg dihydroevocarpine (V), and 86.99 mg 1-methyl-2-(10-pentadecenyl)-4(1H)-quinolone (VI) were obtained from 1.3 g of the crude extract. The structures of these compounds were identified by mass spectrometer (MS), nuclear magnetic resonance (1H NMR and 13C NMR). [ABSTRACT FROM AUTHOR]

Centrifugal partition chromatography (CPC) is a type of counter current separation technique and was first invented by Sanki Eng. Ltd. in Kyoto. Since the year of 1982, CPC has been used to isolate or fractionate bioactive materials and has shown its efficiency in the purification process of natural products from plants and microorganisms. This review focuses on the CPC applications for the purification of over 250 compounds from 60 plant materials and microorganisms between 1994 to September 2009. [ABSTRACT FROM AUTHOR]

α-diketones are important precursors for the synthesis of a wide variety of compounds. Many methods for the synthesis of 1,2-diketones using a variety of reagents have been reported. Almost all the methods result in the formation of by-products in small or major amounts. In this paper, the development of a column chromatographic method for the separation of a diketone from keto-ester is reported. The column conditions standardized for the purification of the diketone from keto-ester may also find application in similar cases involving the presence of more polar by-products formed along with the diketone during the course of the reaction. [ABSTRACT FROM AUTHOR]

Black currant is an important material in the food industry, but little research has been reported on the isolation of phenolic acids because of their low content. In the present study, high-speed countercurrent chromatography (HSCCC) has been successfully used for the preparative isolation of the minor phenolic compounds from the ethyl acetate extracts of black currant fruit. The HSCCC separation was performed with a two phase solvent system composed of n-hexane/EtOAc/MeOH/H2O (5:15:4:7 v/v) at a flow rate of 1.5 mL/min. From a 500 mg crude sample 0.8 mg of protocatechuic acid, 1.0 mg of caffeic acid, 0.5 mg of 4-hydroxybenzoic acid, and 2.5 mg of myricetin were purified by a one step HSCCC operation,. Their chemical structures were confirmed by MS and NMR. [ABSTRACT FROM AUTHOR]

The application of high speed countercurrent chromatography to the preparative purification of crude single walled carbon nanotubes is described. The purification was accomplished on the basis of an experiment that dispersed the single walled carbon nanotubes with aniline, the result mixture was separated by high speed countercurrent chromatography using the two phase system composed of chloroform/methanol/water = 4/4/2. The sizes of SWNTs separated were observed by scanning electron microscopy. The experimental results demonstrated that the high speed countercurrent chromatography possessed good efficiency for purification of single walled carbon nanotubes. [ABSTRACT FROM AUTHOR]

The determination of organotin compounds in bottom sediments is a complex process that requires a number of analytical steps, i.e. sample collection, transport and storage; extraction of analytes from sediment; derivatization; extract purification; enrichment; and the final chromatographic measurement. The whole process is time and labour consuming, and subject to securing sample representativeness. In this review the most frequently encountered problems and the examples of possible analytical solutions are presented, which encompass the specific steps of speciation analysis of these toxic compounds. [ABSTRACT FROM AUTHOR]

An in-tandem study comprising two affinity chromatographic columns, Blue Avidgel P (for the selective removal of albumin) and an aza-arenophilic gel (Gel A) containing a nitrogen capping nucleophile (for the selective removal of IgG), was conducted to obtain high purity IgG from serum. The results demonstrated that highly pure IgG (>97%) can be obtained from the phenethylamine capped aza-arenophilic gel column after the serum was passed through the Blue Avidgel P column in contrast to the 38% IgG purity obtained when the serum was passed through a Gel A column alone under similar conditions. Further studies show that IgG selectivity is not dependent on the nature of the support material used in the synthesis of these aza-arenophilic gels since the synthesis of aza-arenophilic gels using either Sepharose or Fractogel yield similar results. [ABSTRACT FROM AUTHOR]

Screening and confirmatory methods for the determination of Salbutamol (SAL) and Clenbuterol (CL) in swine urine were established. The polyclonal antibody against SAL was prepared, which was used to develop the indirect competitive enzyme-linked immunosorbent assay (ELISA) with the limit of detection of 0.5 ng/ml, and to prepare the immunoaffinity chromatography column. The immunoaffinity column could extract and purify SAL and CL simultaneously, with a binding capacity of 400 ng for SAL and 416 ng for CL. After purification, the swine urine samples were screened by ELISA for the presence of SAL and/or CL, and the positive samples were further confirmed and quantified by gas chromatography-mass spectrometry (GC-MS). The results showed that 95% of the positive samples were confirmed by GC-MS with various levels of SAL (1.1-4.6 ng/ml) and/or CL (1.9-229.1 ng/ml) residues in the incurred samples, and all the negative samples were confirmed with no SAL and/or CL residues. [ABSTRACT FROM AUTHOR]

The first report of the use of task-specific ionic liquid as soluble support for the Gewald synthesis of 2-aminothiophenes is reported in this article. This synthetic method is simple and efficient, and the products are obtained in good to excellent yields with high purities, without the need for chromatographic purification. [ABSTRACT FROM AUTHOR]

Significant amounts of vitamin B 12 (about 116.3∼⃒556.3 µg/100 g) were determined in a Japanese salted and fermented salmon kidney “Mefun.” A corrinoid compound was purified to homogeneity from Mefun and partially characterized. TLC and HPLC patterns of the purified corrinoid compound were identical to those of authentic vitamin B 12 , but not to inactive corrinoids. The vitamin B 12 found in Mefun was not derived from concomitant vitamin B 12 ‐synthesizing bacteria, but had accumulated in the salmon kidney. Gel filtration experiments demonstrated that most of the vitamin B 12 found in Mefun was recovered in the free vitamin B 12 fractions. These results indicate that Mefun would be an excellent vitamin B 12 (free form) source for elderly persons with food‐bound vitamin B 12 malabsorption. [ABSTRACT FROM AUTHOR]

Ion‐Exchange centrifugal partition chromatography using benzalkonium chloride as a strong exchanger (SIXCPC) was successfully used to purify rosmarinic acid from a crude extract that was produced by callus culture. The purification process was carried out on a gram scale using the ternary biphasic system CHCl 3 : n ‐BuOH∶water 4.5:1:4.5 v/v/v in the ascending mode (mobile aqueous phase and stationary organic phase). Two particular points are discussed: the influence of benzalkonium chloride on ternary solvent system stability and the advantage of injecting the analytes as sodium salts rather than molecular acids. [ABSTRACT FROM AUTHOR]

A facile, preparative low-pressure chromatographic process was developed for purification of paclitaxel from crude extract of Taxus spp. The process consisted of normal-phase and a reversed-phase chromatography without utilization of any liquid-liquid partition step. The normal phase chromatography was designed lo separate paclitaxel from a majority of unwanted compounds to obtain an initial purification. Silica gel and Al2O3, were compared as packing materials for this step. The result demonstrated that basic Al2O3 was better than silica gel to give a higher throughput. A further advantage of the Al2O3 was its ability of removing l0-deacetyl-7-epipaclitaxel. a compound difficult to be separated from the paclitaxel in subsequent purification. The chromatographic fraction of A2O3 was purified by a subsequent reversed-phase column chromatography, employing a novel uniform porous microsphere, named PST, as the packing material. Combination of the two steps of column chromatography and one step of crystallization was able to purify paclitaxel, from 0.46% to more than 98% with the total recovery- of 71%. This integrated chromatographic procedure was reproducible, efficient, and simple to use. [ABSTRACT FROM AUTHOR]

Acetyl esterase (acetic-ester acetylhydrolase, EC 3.1.1.6) from citrus peel, whose natural role is not well known, catalyses, in vitro, the hydrolysis of acetyl groups from a wide range of substrates. This enzyme was extracted from Mediterranean orange peel, largely available in Italy, and purified 190-fold by a single chromatographic step on Sepabeads FP-HG. SDS polyacrylamide gel electrophoresis of the purified enzyme showed a major protein band, corresponding to a molecular mass of 45 kDa. Both free and immobilised enzyme were used in biotransformations. The enzyme removed the acetyl group in the 3 position of β-lactamic antibiotics, such as cephalosporin C and the intermediate 7-aminocephalosporanic acid with ≥98% conversion and 91-93% product yield. [ABSTRACT FROM AUTHOR]

Epigallocatechin (EGC) was prepared by the degallation of Epigallocatechin-O-3-gallate (EGCG). EGCG was completely converted to EGC and gallic acid by adding 4 mg tannase/100 mg EGCG at the concentration of 2 mg/mL under pH 6.0, at 35°C for 30 min. EGC was then separated from the reaction mixture by HSCCC, using a two-phase solvent system composed of hexane–ethyl acetate–water (1/9/10, v/v/v). Finally, 1.3 g of EGC at 97% purity was prepared from about 2.3 g of EGCG at 85% purity, indicating that EGC was almost completely recovered by HSCCC. [ABSTRACT FROM AUTHOR]

Peroxidase from turnip roots was purified using metal affinity chromatography up to a specific activity of 337 units/mg protein with 3.02 RZ and 63.5% recovery. After purification, the enzyme showed 2–3 bands on sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE). The molecular weight of the purified enzyme was found to be 37–39 kD with matrix assisted laser desorption ionization mass spectrometer (MALDI-MS). The enzyme showed maximum activity in phosphate buffer, pH 6.0, and lowest activity in borate buffer at the same pH. The Km of the enzyme was found to be 7.07×10-4 mM. Turnip peroxidase also contains an iron moiety which is found to be about 0.28%. The enzyme showed 50% inhibition of its specific activity with ethylene diamine tetraacetic acid (EDTA). [ABSTRACT FROM AUTHOR]

Preparative scale purification of PEG-protein conjugates by centrifugal precipitation chromatography is discussed. By utilizing the transport of ammonium sulfate across a membrane and the centrifugal force, an ammonium sulfate gradient is formed and used for differential precipitation of PEG-protein conjugates. Native proteins can be separated from PEG-protein conjugates by using this technique. [ABSTRACT FROM AUTHOR]

Centrifugal partition chromatography linked with evaporative light scattering detection has been investigated to achieve the large scale-up isolation of oraposide which is a phenylpropanoid glycoside having a radical-scavenging activity. A two-phase solvent system has been studied to improve the yield of pure product and to decrease the cost of purification. A comparison with preparative HPLC has been made. [ABSTRACT FROM AUTHOR]

Column chromatography on aminopropyl-derivatized silica and precipitation of a complex with perchloric acid have been evaluated as methods for the purification of di-tert-butylcyclohexano-18-crown-6 (DtBuCH18C6), a compound frequently employed for the selective extraction of strontium from acidic nitrate media. Both methods are shown to provide a simple and effective means of eliminating inactive sample components (i.e., impurities or stereoisomers incapable of extracting strontium) from the crown ether and enriching the material in 4(z),4′(z) cis-syn-cis DtBuCH18C6, a stereoisomer capable of highly efficient strontium extraction. [ABSTRACT FROM AUTHOR]