Separation of butyltin and phenyltin species by ion-exchange chromatography with complexing mobile phases

Summary Butyltin and phenyltin species have been separated by ion-exchange chromatography using silica-based and polymer-based columns. Mobile phases consisted of methanol-water mixtures containing polyfunctional carboxylic acids, which can act as complexing agents for organotin species. The best results were achieved with a system based on a methanol mobile phase containing malic and oxalic acids and a polymer-based column, which allowed the separation of tri- and diorganotin compounds and some resolution between monobutyltin and monophenyltin.     

Characterisation of Tokaj wines based on free amino acids and biogenic amines using ion-exchange chromatography

Summary Tokaj wines (Szamorodni and Aszu wines) of Hungarian origin were investigated on the basis of free amino acids and biogenic amines. The separation and determination of these compounds was performed by an amino acid analyser equipped with an ion-exchange resin column. The total amount of free amino acids and biogenic amines was higher in Aszu wines than in Szamorodni wines. The main amino acids were proline and arginine, while the major biogenic amines were tyramine and putrescine. The free amino acid and biogenic amine content of Aszu wines depended on the vineyards the wines originated from. Presented at Balaton Symposium '01 on High-Performance Separation Methods, Siófok, Hungary, September 2–4, 2001     

Dynamic ion-exchange chromatography with post-column reaction detection for the determination of the rare earth elements in monazites

Summary Separation and determination of lanthanum, cerium, praseodymium, neodymium and samarium in monazites have been achieved by dynamic ion-exchange chromatography. The ore samples are decomposed by sulfuric acid and the rare earths are separated in a group as oxalates. The rare earth elements are then separated from each other on a column of bonded phase silica by gradient elution with 0.05 to 0.5 M lactic acid (pH 3.5) in the presence of 0.01 M sodium 1-octanesulfonate. Post-column reaction with Arsenazo III is used for detection and quantification of the individual rare earth elements. Results are quoted for lanthanum, cerium, praseodymium, neodymium and samarium in monazites. Detection limit is 1 μg ml⁻¹ with a S/N ratio of 3. The separation is complete within 27 min valley to valley resolution. Precision of better than 1% can usually be obtained.     

Isolation of some quaternary alkaloids from the extract of roots ofChelidonium Majus L. by column and thin-layer chromatography

Summary A chromatographic system comprising untreated silica gel and an aqueous buffer—methanol eluent was investigated for the micropreparative separation of the alkaloids ofChelidonium majus L. The relationship between the logarithm of the retention factorsk of the alkaloids and volume fraction of methanol at pH^* 6 was linear for volume fractions in the range of 0.1 to 0.4. From this relationship it is possible to estimate the value of the retention factor for a given alkaloid in pure buffer and then the maximum volume of alkaloid extract that can be sampled on micropreparative column filled with silica gel.     

Separation of hordein proteins from european barley by high-performance liquid chromatography: Its application to the identification of barley cultivars

Summary High-performance liquid chromatography using either reversed phase or anion-exchange techniques was used for the fractionation of hordein proteins extracted from European barley. A reversed phase method is presented which utilises an Ultrapore column packed with a wide pore (30 nm) C-3 alkylbonded silica support. Using this method up to 20 components may be separated in 54 min. Elution profiles were found to be reproducible. A further method using rapid anion-exchange chromatography indicated that up to 13 components may be separated, a number which is comparable to that found with electrophoresis. The separation of proteins extracted from different barley cultivars indicated that on the basis of elution profiles high-performance liquid chromatography using either reversed phase or anion-exchange offers considerable potential as a method for barley cultivar identification.     

Reverse-phase liquid chromatography ofp-tert-butylcalixarenes

Summary Thep-tert-butylcalix[n]arenes (n=4,6,7,8) andp-tert-butyldihomooxacalix[4]arene have been separated by reverse phase liquid chromatography. Optimum conditions have been obtained on a Spherisorb ODS1, 5 μm C18 column by isocratic ambient elution with acetonitrile-methyltert-butyl ether. Calibration plots have been obtained from purified calixarenes and the reliability of the method is confirmed from test mixtures of calixarenes of known composition.     

Determination of naturally occurring hydroxynaphthoquinone polymers by size-exclusion chromatography

Summary Polymerization of alkannin, shikonin, and their derivatives, potent pharmaceutical substances, crucially affects their use in pharmaceuticals, cosmetics, and as food colorants, because it leads to loss of their antimicrobial activity, reduction of the lustre of their red coloration, and a decrease in their solubility. In this study size-exclusion chromatography (SEC) has been used for the first time for qualitative and quantitative analysis of monomeric and polymeric hydroxynaphthoquinone alkannin and shikonin derivatives. The purity and degree of polymerization has been determined to evaluate severalAlkanna tinctoria root samples from different geographical sources, and commercial samples of alkannin and shikonin, as pharmaceutical raw materials. Conditions for extraction of hydroxynaphthoquinones fromAlkanna tinctoria roots with olive oil were optimized in terms of polimerization, aiming to improve the biological activity of the final pharmaceutical product, Helixderm.     

Reversed-phase liquid chromatography coupled on-line with capillary gas chromatography use of an anion-exchange membrane to remove an ion-pair reagent from the eluent

Summary In order to enable the coupling of reversed-phase liquid chromatography (RPLC) with capillary gas chromatography (GC), the performance of an anion-exchange micromembrane device has been studied to remove the ion-pair reagent methanesulphonic acid from an acetonitrile/water LC eluent. The regenerant in the membrane was tetrabutylammonium hydroxide dissolved in acetonitrile/water, which effects an anion-exchange of methanesulphonate ions for regenerant hydroxide ions. The efficiency of the exchange process was found to be 99.9%. This enabled the direct introduction of the LC eluent, free of ions and with the proper acetonitrile/water ratio, into the GC. The applicability of the on-line LC-micromembrane-GC system has been illustrated for the potential drug eltoprazine, which is quantitatively recovered with a coefficient of variation for standard solutions of 3% at the 150 g/ml analyte level.     

The study for isolation and purification of R-phycoerythrin from a red alga

An effective procedure for the rapid extraction and purification of the biliprotein R-phycoerythrin from a red alga,Ceramium isogonum, was developed. The purified R-phycoerythrin ofC. isogonum consisted of three components with mol wt 180,000 (6β subunits), 70,000 (6α subunits), and 30,400 (γ subunit), respectively. The phycoerythrin is suitable for use as a natural food coloring and can also be used as a fluorescent label.     

Optimization of the mobile phase conditions for the purification ofTaql endonuclease

Summary Taql endonuclease was purified by high performance ion exchange liquid chromatography with linear gradient elution. Of the chromatographic media tested as mobile phases for the HPLC purification ofTaql endonuclease, sodium acetate (pH 5.0) and phosphate (pH 7.0) buffers were appropriate for use with cation exchange columns, and L-histidine (pH 6.0) and Tris (pH 8.0) buffers with anion exchange columns.     

Optimization of monoclonal antibody purification by ion-exchange chromatography. Application of simple methods with linear gradient elution experimental data

Simple methods for the optimization of ion-exchange chromatography of proteins in our previous papers were applied to cation-exchange chromatography purification of monoclonal antibodies (Mab). We carried out linear gradient elution experiments, and obtained the data for the peak salt concentration and the peak width. From these data, the distribution coefficient as a function of salt concentration, and the height equivalent to a theoretical plate (HETP) as a function of mobile phase velocity were calculated. The optimized linear gradient elution conditions were determined based on the relationship between buffer consumption and separation time. The optimal stepwise elution conditions were determined based on the relationship between the distribution coefficient and the salt concentration.     

Large-Scale Isolation and Purification of Scoparone from Herba artemisiae scopariae by High-Speed Counter-Current Chromatography

A preparative high-speed counter-current chromatography with a two-phase solvent system composed of n-hexane-ethyl acetate-methanol- water (1:1:0.45:1.55, v/v/v/v) was successfully performed to isolate scoparone (6,7-dimethoxycoumarin, 6,7-dimethylesculetin, 6,7-DME) from the plant of Herba artemisiae scopariae, a traditional Chinese medicine. 233.5 mg Scoparone with the purity of 96.8% (determined by HPLC) was obtained in one-step elution from 800 mg crude extract. The recovery of scoparone was 91.8%, and the chemical structure of this compound was identified by IR, MS, ¹H NMR and ¹³C NMR spectrum.     

Development of an efficient method for the preparative isolation and purification of chlorophyll a from a marine dinoflagellate Amphidinium carterae by high-speed counter-current chromatography coupled with reversed-phase high-performance liquid chromatography

In our research into chlorophylls of marine dinoflagellates, chlorophyll a was separated rapidly from the hexane extract of Amphidinium carterae in three steps. The first step was silica gel column chromatography, where elution was performed with 0–50% ethyl acetate in n-hexane. The second was high-speed counter-current chromatography using a two-phase solvent system consisting of n-hexane–ethyl acetate–methanol–water (5:5:5:1, v/v), and the third step was preparative reversed-phase high-performance liquid chromatography using a solvent system of acetone–water (89:11, v/v). HPLC analysis showed that the purity of chlorophyll a from the second step was over 83%, and after the third it was over 99%. Thirty milligrams of chlorophyll a was isolated from a crude sample of 250 mg of chlorophylls, and its structure was identified by analyzing its MS, ¹H NMR and ¹³C NMR spectra.     

Analysis of terpenes fromGinkgo biloba L. extracts by reversed phase high-performance liquid chromatography

Summary The main terpenes ofGinkgo biloba L. extracts (bilobalide, ginkgolide A, ginkgolide B and ginkgolide C) have been separated by isocratic elution on a 3 μm C₁₈ Spherical column using 2-propanol:water (10∶90) as eluent.     

Determination of caffeoylquinic acids and flavonoids inCynara scolymus L. by high performance liquid chromatography

Summary The main phenolic compounds in dried extracts fromCynara scolymus (artichoke)—monocaffeoylquinic acids, dicaffeoylquinic acid, and flavonoids–have been separated by high-performance liquid chromatography. By use of a narrow bore C₁₈ column and an acidic mobile phase this HPLC method enabled improved separation within 31 min with significantly reduced solvent consumption compared with other methods. The method was validated to demonstrate its linearity, precision, accuracy, and robustness. Twelve commercial samples were analyzed. Monocaffeoylquinic acids were the most abundant phenolic compounds; the amounts present ranged from 0.48 to 4.24%. The amounts of dicaffeoylquinic acids and flavonoids were smaller—from 0.03 to 0.52%. The method is a good combination of efficiency and economy and should be especially useful for commercial applications.     

Large scale purification of synthesized oligodeoxyribonucleotides by reversed-phase chromatography

Summary A reversed-phase chromatographic column suitable for the purification of chemically synthesized large oligodeoxyribonucleotides (oligo-DNA) was prepared. The specifications of this column are; the selected silica (Toyo Soda silica) with large pore size (at least 150 ) and small particle diameter (5 m desired) should be grafted only with monochloro alkylating reagent of long alkyl chain (sufficiently C₁₈) so that the carbon content of the resultant packing material is 15–16%. Using this column, we could isolate the targeted large oligo-DNA (up to 50mer) in a large scale (75 g per one cycle) from the impurities in the reaction mixture formed during the automated synthesis by the phosphite method.     

Isolation and purification of digalactosyldiacylglycerols

Summary A convenient method for the isolation of digalactosyldiacylglycerols from plant material has been developed using solid phase extraction. Commerical cartridges were utilized to prepare 100 mg fractions of galactolipids containing more than 97% of digalactosyldiacylglycerol. The purity of the fractions was monitored by an isocratic normal phase analytical HPLC procedure, which was optimized using factorial design. The optimized system was successfully scaled up to a semi-preparative HPLC system with a capacity of about 10 mg/injection which was used to further purify the digalactosyldiacylglycerol fraction.     

Enzymatic hydrolysis of pectic substances by gel-permeation chromatography

Summary A rapid liquid chromatographic (HPLC) method for the determination of pectinolytic activity of enzymes produced by Aspergillus Niger and Rhizopus species is reported.Compared with more conventional methods, HPLC is more reliable and has a much improved maximum sensitivity. The limit of quantitation of galacturonic acid is 0.1g.