Scanning molecular sieve chromatography of interacting protein systems. II. Determination of large zone transport parameters by the difference profile method at low solute concentration

The experimental determination of difference profiles for the study of large zone transport processes by scanning molecular sieve chromatography is described. Using the difference profile method, the progesterone-induced purple glycoprotein of the porcine uterus was found to exist as monomeric units in high ionic environment, with a partition coefficient of 0.269, partition cross-section of 0.488, partition radius of 25 A and a molecular weight of 33500 g mole . The technique was further applied in examining the association-dissociation properties of oxyhemoglobin. In a high tonic environment, the partition coefficient was found to be 0.365 for dimer and the partition cross-section, 0.419; for the tetramer in low ionic strength solution, the partition coefficient was 0.275 and the partition cross-section 0.377, with a dissociation constant of 1.03 x 10(-6) mole/1. This new technique should prove applicable in (1) readily locating the centroid positions of transport boundary profiles at the lowest practicable protein concentration limits, (2) demonstrating the characteristic boundary shape and concentration-dependent centroid position for an interacting solute, (3) determining the axial dispersion coefficient characteristic of solute turbulence within the gel matrix, and (4) distinguishing the boundary between low and high ionic strength solvent phases in the gel column.     

Identification of some plasma metabolites of 6, 7-3-H-estrone glucosiduronate in male dogs.

Following the constant infusion of 6, 7-3-H-estrone glucosiduronate in male dogs for a period of 120 minutes, the radioactive metabolites present in the plasma were separated by solvent partition, DEAE-Sephapadex, Celite partition and thin layer chromatography. The identities of the individual estrogens and estrogen conjugates were confirmed by specific activity determinations after chromatography in several different solvent systems, enzyme hydrolysis and steroids and their derivatives. Most of the radioactivity in the plasma was identified as estrone glucosiduronate. The major metabolite present was estradiol-17-beta-3-glucosiduronate. Small amounts of estradiol-17-alpha-3-glucosiduronate and free estrone were also identified. Three other minor conjugates were separated, but positive identification could not be made.     

Separation of estrogen conjugates by high pressure liquid chromatography.

High pressure liquid chromatography using a prepacked commercial strong anion exchanger column (mu Partisil 10 SAX, 25 cm x 4.6 mm) was used to separate a mixture of eight estrogen conjugates. Chromatographic conditions using a 0.01 M potassium phosphate or 0.1 M NaCl as solvent in the isocratic mode are described for the separation of estrone glucosiduronate, 17beta-estradiol-3-glucosiduronate, 17beta-estradiol-17-glucosiduronate, estriol-3-glucosiduronate, estriol-16alpha-glucosiduronate, estriol-17-glucosiduronate, estrone sulfate and 17beta-estradiol-3-sulfate. This system gives high resolution of the estrogen conjugates in small eluent volumes in less than 30 min. The advantages of this high pressure liquid chromatographic system over other methods of separation are discussed.     

Purification of gangliosides by liquid-liquid partition chromatography

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.     

Isolation and identification of antiplasmodial N-alkylamides from Spilanthes acmella flowers using centrifugal partition chromatography and ESI-IT-TOF-MS

The development of new antiplasmodial drugs is of primary importance due to the growing problem of multi-drug resistance of malaria parasites. Spilanthes acmella, a plant traditionally used for the treatment of toothache, was targeted as a lead for its potential antiplasmodial activity. A systematic approach for investigating a suitable centrifugal partition chromatography (CPC) solvent system for N-alkylamides separation was reported. The partition behavior of three N-alkylamides has been studied using several biphasic solvent mixtures in search of an adequate CPC solvent system for this class of compounds. Major N-alkylamides in S. acmella were isolated from a methanolic crude extract of flowers by CPC with the solvent system heptanes-ethyl acetate-methanol-water (3:2:3:2, v/v/v/v). Four N-alkylamides were purified and the structures were illustrated by electrospray ionization-ion trap-time of flight-mass spectrometry (ESI-IT-TOF-MS), 1H nuclear magnetic resonance (1H NMR) and 13C nuclear magnetic resonance (13C NMR). The CPC fractions, which contained natural mixtures of phytochemicals, demonstrated significantly higher antiplasmodial activity compared to corresponding purified N-alkylamides, thus suggesting that interactions between these N-alkylamides may potentiate antiplasmodial bioactivity.     

高速逆流色谱用于天然产物分离和指纹图谱构建

利用国产高速逆流色谱分离纯化雪莲黄酮类成分和丹参醌类成分。雪莲分离选用氯仿 甲醇 水 (10∶7∶3)体系 ,固定相保留率 72 % ,仪器参数 80 0r min 2mL min ,采用一步洗脱法 ,7h内得到 14个组分 ;丹参分离选用正己烷 乙醇 水 (10∶5 5∶4 5 )体系 ,固定相保留率达到 78 8% ,采用分步洗脱 ,3个产地丹参在 13h内各分离得到 12个洗脱组分。HSCCC洗脱图谱可以表现出不同产地丹参的差别 ,并且各对应洗脱峰保留时间的相对标准偏差 <3% ,因此提出将HSCCC作为构建中药指纹图谱的方法之一 ,其可行性需要通过与常规的指纹图谱构建方法比较之后做出评价。     

Isolation and structure of somatostatin from porcine hypothalami.

The isolation and structure of somatostatin (GH-RIH) from pig hypothalami are described. This hormone was purified by preparative gel filtration, solvent extraction, countercurrent distribution in two solvent systems, ion-exchange and partition chromatography, and analytical gel filtration. The somatostatin activity was followed by in vitro bioassays and a radioimmunoassay. The isolated product was homogeneous chromatographically and had biological and immunological properties similar to synthetic somatostatin corresponding to the ovine hormone. The primary structure of porcine somatostatin was shown to be H-Ala-Gly-cyclo-(Cys-Lys-Asn-Phe-Phe-Trp-Lys-Thr-Phe-Thr-Ser-Cys)-OH. Other immunologically and biologically active form(s) of somatostatin were also detected.     

刺果紫玉盘的化学成分研究

采用95%乙醇提取,溶剂萃取,硅胶柱色谱分离等方法,从刺果紫玉盘根的乙醇提取物中分离得到了6个化合物,经NMR、MS等光谱学方法分别鉴定为:(-)1,6-desoxypipoxide(1)、piperenol A(2)、zeylena(3)、grandi-floracin(4)、顺式桂皮酸(5)和苯甲酸(6)。所有这些化合物都为从该植物中首次分离鉴定。     

Preparation of radiolabeled GM2 and GA2 gangliosides.

GM2 and GA2 gangliosides from the brain of a patient who died of Sandhoff's disease were purified by solvent partition, silicic acid and silica gel column chromatography, and silica gel preparative thin-layer chromatography. They were tritiated in the terminal N-acetylgalactosamine residue using galactose oxidase and sodium [3H]borohydride with the inclusion of catalase and peroxidase into the oxidation reaction. The specific activities were 4.62 X 10(8) dpm/mumol of GM2 ganglioside and 5.54 X 40(7) dpm/mumol of GA2 ganglioside. The addition of catalase and peroxidase to the tritiation procedure is recommended.     

New metabolites of riboflavin appear in human urine

By surveying compounds having isoalloxazine derived from flavins on a high performance liquid chromatogram with fluorescence detection, two new flavin derivatives were found in human urine. These two compounds were purified by partition chromatography on a cellulose column and by paper chromatography with several solvent systems, and their structures were determined to be 7 alpha-hydroxyriboflavin and 8 alpha-hydroxyriboflavin. The relative distributions, measured by high performance liquid chromatography, of 7 alpha- and 8 alpha-hydroxyriboflavin, riboflavin, and hydroxyethylflavin and its derivative were calculated to be 31.1, 5.0, 25.6, 4.9, and 21.9%, respectively, to total flavins in normal human urine obtained in early morning. The excretion of 7 alpha- and 8 alpha-hydroxyriboflavin in human urine indicates the occurrence of a metabolic pathway of the isoalloxazine ring of flavin at its 7 alpha and 8 alpha positions.     

Three New Flavone Glycosides from Drymaria diandra B1.

In order to find new structural and biologically active compounds, the constituents from the whole plant of Drymaria diandra B1. （Caryophyllaceae） were investigated and three new flavone glycosides, named drymariatins B （1）, C （2）, and D （3）, were isolated by solvent partition, Si gel, sephadex LH-20, and Rp- 18 column chromatography. Using spectroscopic methods, including two-dimensional nuclear magnetic resonance analysis, the structures of these compounds were elucidated as 6-C-（2-deoxy-β-D-fucopyranosyl）- 5,7,4＇-trihydroxyl-flavone, 6-C-（2-deoxy-β-D-fucopyranosyl）-7-O-（β-D-glucopyranosyl）-5,4＇-dibydroxyl- flavone, and 6-C-（3-keto-β-digitoxopyranosyl）-7-O-（β-D-glucopyranosyl）-5,4＇-dihydroxyl-flavone.     

Separation of individual sulfated bile acid conjugates as calcium complexes using reversed-phase partition thin-layer chromatography.

A method for separating individual monosulfated primary bile acid conjugates by reversed-phase partition thin-layer chromatography on octadecyl-bonded silica gel is described. The solvent system is acetonitrile containing calcium, probably as calcium carbamate. Excellent resolution of the 3- and 7-monosulfated glycine conjugates, as well as 3- and 7-monosulfated taurine conjugates of cholic and chenodeoxycholic acids is reported. A convenient class separation of sulfated from nonsulfated primary bile acid conjugates by adsorption thin-layer chromatography on low-polarity silica gel is also described.     

Purification of betulinic acid from Eugenia florida (Myrtaceae) by high-speed counter-current chromatography

A high yield of betulinic acid (up to 17% from the ethanolic extract) was found in the leaves of Eugenia florida collected in south-eastern Brazil, making this species a potential commercial source of the title compound. Extracts of E. florida were subjected to solvent partition, and rapid high-speed counter-current chromatography (HSCCC) was applied to the semi-crude extracts to afford betulinic acid in high purity. The mobile and stationary phases were derived from the two-phase solvent system composed of n-hexane-ethyl acetate-methanol-water (10:5:2.5:1). The developing solvent system (stationary and mobile phases) for optimum HSCCC separation was chosen by dissolving the fraction to be chromatographed in the proposed solvent mixture and determining the amount of betulinic acid in each phase by densitometric TLC. Purified betulinic acid was characterized by 13C-NMR, GC-MS and co-injection of its methyl ester with standards in GC-FID. The HSCCC technique is commonly employed to isolate triterpene glycosides, but is applied in this study to an aglycone.     

Liquid-gel partition chromatography of vitamin A compounds; formation of retinoic acid from retinyl acetate in vivo

A clear separation of retinol, retinal, and retinoic acid has been achieved by liquid-gel partition chromatography on Sephadex LH-20 with solvent mixtures of chloroform, Skellysolve B, and methanol. A mixture of retinyl esters, retinol, retinal, and retinoic acid has been resolved on hydroxyalkoxypropyl Sephadex using Skellysolve B and acetone. There is no decomposition of any of the vitamin A compounds during chromatography, and recovery is complete. The combination of mildness and potential for resolution makes liquid-gel partition chromatography a superior tool for the separation of vitamin A compounds. This method has been applied to the study of vitamin A metabolism at physiological levels in the vitamin A-deficient rat. Retinyl palmitate, an ester of retinoic acid, retinal, retinol, retinoic acid, and a polar metabolite have been demonstrated in various tissues of the rat 12 hr after a dose of 2 micro g of [11-(14)C]retinyl acetate.     

The separation of peptide hormone diastereoisomers by reverse phase high pressure liquid chromatography. Factors affecting separation of oxytocin and its diastereoisomers--structural implications.

Experimental conditions and parameters involved in high performance liquid chromatography (HPLC) separations of the peptide hormone oxytocin and seven of its diastereoisomers, namely [1-hemi-D-cystine]-, [2-D-tyrosine]-, [4-D-glutamine]-, [5-D-asparagine]-, [6-hemi-D-cystine-], [7-D-proline]-, and [8-D-leucine]-oxytocin, on reverse phase columns were investigated. The effects of solvent, pH, and salt concentration were studied. Using the solvent systems 10% tetrahydrofuran-ammonium acetate buffer or 18% acetonitrile-ammonium acetate buffer and the muBondapak C18 support, oxytocin was separated from each of its diastereoisomers under all conditions studied, but the order of elution of diastereoisomers was highly dependent on solvent and to a lesser extent on pH. Separations of the hormone and its diastereoisomers on reverse phase HPLC and on classical partition chromatography on Sephadex G-25 were compared. The results are discussed in terms of the interactions of the solute with the reverse phase column and the solvent system. Implications of these findings in terms of the different solution conformations of the peptides are discussed.     

Modelling alterations in the partition coefficient in in vitro biological systems using headspace gas chromatography

Headspace gas chromatography was used to determine the physiological media–air partition coefficient (K) of four volatile organic solvents of industrial importance. The experimental conditions were those likely to be used in in vitro metabolic and toxicological studies on volatile compounds. The addition of solvent to the liquid phase from a stock solution in ethanol, or the presence of organic material at concentrations normally seen in in vitro studies, did not significantly alter the K value. Binary solvent addition resulted in a dose-dependent decrease in K for each solvent that was also influenced by the solvent solubility and the constituents of the liquid matrix. The aromatic solvents exerted the greatest effect and showed the greatest change in K value.     

Macrocyclic trichothecene toxins produced by a strain of Stachybotrys atra from Hungary.

A strain of Stachybotrys atra isolated from a field case of stachybotryotoxicosis in Hungary was cultured in Hungary. All of the compounds toxic to brine shrimp were separated from the culture extract by solvent partition, column chromatography, and preparative thin-layer chromatography. Two of the toxic compounds were identified as verrucarin J and satratoxin H by comparison with pure standards resolved by high-pressure liquid chromatography and characterized by mass spectrometry. Two other toxic components were identified as roriden E and satratoxin G on the basis of their mass spectra. The fifth toxic compound was identified as a macrocyclic trichothecene based on the following findings: a positive 4-(p-nitrobenzyl)pyridine color reaction, hydrolysis resulting in verrucarol verified by combined gas chromatography-mass spectrometry, and a characteristic trichothecene proton-nuclear magnetic resonance spectrum. This macrocyclic trichothecene has a molecular ion (528) identical to satratoxin H, and its mass spectrum is similar; however, its Rf value on Silica Gel G differs.     

Isolation of secondary metabolites from roots of Plumbago auriculata Lam. by countercurrent chromatography

Centrifugal liquid-liquid partition chromatography presents significant advantages for the separation and purification of plant metabolites owing to the short operational time of the process and the elimination of possible irreversible adsorption of compounds. The crude chloroform extract from roots of Plumbago auriculata was analysed by countercurrent chromatography using hexane:ethyl acetate:methanol:water (40:10:10:2, v/v) as solvent system. The isolation of the naphthoquinones plumbagin and epi-isoshinanolone, the steroids sitosterol and 3-O-glucosylsitosterol, plumbagic and palmitic acids was easily achieved. Naphthoquinones are typical components of Plumbago species and they show interesting biological activities.     

Scanning molecular sieve chromatography of interacting protein systems. II. Determination of large zone transport parameters by the difference profile method at low solute concentration.

The experimental determination of difference profiles for the study of large zone transport processes by scanning molecular sieve chromatography is described. Using the difference profile method, the progesterone-induced purple glycoprotein of the porcine uterus was found to exist as monomeric units in high ionic environment, with a partition coefficient of 0.269, partition cross-section of 0.488, partition radius of 25 A and a molecular weight of 33,500 g/mole. The technique was further applied in examining the association-dissociation properties of oxyhemoglobin. In a high tonic environment, the partition coefficient was found to be 0.365 for dimer and the partition cross-section, 0.419; for the tetramer in low ionic strength solution, the partition coefficient was 0.275 and the partition cross-section 0.377, with a dissociation constant of 1.03 x 10(-6) mole/l. This new technique should prove applicable in (1) readily locating the centroid positions of transport boundary profiles at the lowest practible protein concentration limits, (2) demonstrating the characteristic boundary shape and concentration-dependent centroid position for an interacting solute, (3) determining the axial dispersion coefficient characteristic of solute turbulence within the gel matrix, and (4) distinguishing the boundary between low and high ionic strength solvent phases in the gel column.     

Preparative isolation and purification of geniposide from gardenia fruits by centrifugal partition chromatography

The iridoid glycoside, geniposide was purified by centrifugal partition chromatography (CPC) with a two-phase solvent system composed of ethyl acetate:isopropanol:water (3:2:5, v/v) from an 80% methanolic extract of fruits of Gardenia jasminoides. Preparative CPC yielded 56.2 mg of geniposide in a one-step separation of 500 mg of extract, with a purity of 95% as determined by HPLC. Isolated geniposide was identified from its 1H-NMR, 13C-NMR and MS spectra.