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PubMed2006

Band broadening inside the chromatographic column: the interest of a liquid stationary phase.

Band broadening inside chromatographic columns was studied by Giddings 40 years ago. This theory is revisited pointing out that the band width depends only on the band position, x, inside the column and the height equivalent to a theoretical plate, H, and not on the solute affinity for the stationary phase. The band standard deviation, sigma, inside the column is simply sigma = square root [xH]. This property can be used in countercurrent chromatography (CCC), a chromatographic technique that works with a liquid stationary phase. Two possibilities are presented: 1-extrusion of the liquid stationary phase called elution-extrusion method, and 2-slow motion of the stationary phase in the same direction as the mobile phase, called cocurrent CCC method. A mixture of five steroids, prednisone, prednisolone acetate, testosterone, estrone and cholesterol, with partition coefficient varying from 0.1 to 40, is used with a 53 mL CCC column to show the method capabilities. The elution-extrusion method is discontinuous; however, it allows saving dramatic amounts of solvent and time. Cholesterol could be fully resolved in 2h and 120 mL instead of 7 h and 1.2 L using the classical elution way. The cocurrent CCC method is continuous and was able to resolve cholesterol at baseline in 40 min using 110 mL. Detection is difficult due to the fact that two immiscible liquid phases enter the detector.

Acetates↗
PubMed2006

Bioassay-guided purification and identification of antimicrobial components in Chinese green tea extract.

The Chinese green tea extract was found to strongly inhibit the growth of major food-borne pathogens, Escherichia coli O157:H7, Salmonella Typhimurium DT104, Listeria monocytogenes, Staphylococcus aureus, and a diarrhoea food-poisoning pathogen Bacillus cereus, by 44-100% with the highest activity found against S. aureus and lowest against E. coli O157:H7. A bioassay-guided fractionation technique was used for identifying the principal active component. A simple and efficient reversed-phase high-speed counter-current chromatography (HSCCC) method was developed for the separation and purification of four bioactive polyphenol compounds, epicatechin gallate (ECG), epigallocatechin gallate (EGCG), epicatechin (EC), and caffeine (CN). The structures of these polyphenols were confirmed with mass spectrometry. Among the four compounds, ECG and EGCG were the most active, particularly EGCG against S. aureus. EGCG had the lowest MIC90 values against S. aureus (MSSA) (58 mg/L) and its methicilin-resistant S. aureus (MRSA) (37 mg/L). Scanning electron microscopy (SEM) studies showed that these two compounds altered bacterial cell morphology, which might have resulted from disturbed cell division. This study demonstrated a direct link between the antimicrobial activity of tea and its specific polyphenolic compositions. The activity of tea polyphenols, particularly EGCG on antibiotics-resistant strains of S. aureus, suggests that these compounds are potential natural alternatives for the control of bovine mastitis and food poisoning caused by S. aureus.

Anti-Bacterial Agents↗
PubMed2006

Multiple dual-mode centrifugal partition chromatography, a semi-continuous development mode for routine laboratory-scale purifications.

Nowadays, centrifugal partition chromatography (CPC) separations can be routinely achieved at the laboratory scale. The solvent system selection has been made easy, as generic sets of solvent systems are described in publications and books. This approach, however, generally reduces the scope of optimization strategies for two important parameters: selectivity and sample solubility. This can be very limiting for the preparative separation of structurally similar compounds. Multiple dual-mode (MDM) CPC has been developed to provide an easy-to-use alternative technique to circumvent this problem. A MDM separation consists of a succession of dual-mode runs (i.e. multiple inversion of stationary and mobile phase) that can only be achieved because both chromatographic phases are liquids. This original elution mode is thus a semi-continuous process with a classical sample injection and which only requires a single CPC column. Underlying mechanisms of MDM were studied using a model mixture of acenaphthylene and naphthalene. A mixture of two synthetic pairs of diastereomers was then successfully submitted to MDM CPC, in the framework of the synthesis of biologically active compounds.

Acenaphthenes↗
PubMed2006

Purification of coenzyme Q10 from fermentation extract: high-speed counter-current chromatography versus silica gel column chromatography.

High-speed counter-current chromatography (HSCCC) is applied to the purification of coenzyme Q(10) (CoQ(10)) for the first time. CoQ(10) was obtained from a fermentation broth extract. A non-aqueous two-phase solvent system composed of heptane-acetonitrile-dichloromethane (12:7:3.5, v/v/v) was selected by analytical HSCCC and used for purification of CoQ(10) from 500 mg of the crude extract. The separation yielded 130 mg of CoQ(10) at an HPLC purity of over 99%. The overall results of the present studies show the advantages of HSCCC over an alternative of silica gel chromatography followed by recrystallization. These advantages extend to higher purity (97.8% versus 93.3%), recovery (88% versus 74.3%) and yield (26.4% versus 23.4%). An effort to avoid the toxic, expensive solvent CH(2)Cl(2) was unsuccessful, but at least its percentage is low in the solvent system.

Agrobacterium tumefaciens↗
PubMed2006

Isolation of hydroxycinnamoyltartaric acids from grape pomace by high-speed counter-current chromatography.

A method for the isolation of caftaric, coutaric and fertaric acids from grape pomace by high-speed counter-current chromatography (HSCCC) was developed. Using a system of hexane/ethyl acetate/methanol/water 3:7:3:7 (v/v/v/v) and 0.5% trifluoroacetic acid (TFA) in the head-to-tail elution mode, the target compounds were separated from co-extracted polyphenolics and subsequently isolated in a second run (tert-butyl-methyl ether/acetonitrile/n-butanol/water, 2:2:1:5 (v/v/v/v) and 0.5% TFA; tail-to-head elution mode). The concomitant flavonoid quercetin 3-glucuronide was also isolated with the present method. The compounds were characterized by 1H NMR spectroscopy, by LC/electrospray ionization (ESI)-MS/MS in the negative ionization mode, and by UV spectroscopy. A purity of 97.0% (2.0% Z-isomer) for caftaric acid, 97.2% (4.8% Z-isomer) for coutaric acid, and 90.4% (13% Z-isomer) for fertaric acid was obtained from 10 g of grape pomace with yields of 62, 48 and 23%, respectively. Caftaric and coutaric acids may be used for in vitro and in vivo studies and as reference substances for analytical purposes.

Countercurrent Distribution↗
PubMed2006

Preparative separation of gambogic acid and its C-2 epimer using recycling high-speed counter-current chromatography.

A recycling counter-current chromatographic system was first set up with a high-speed counter-current chromatography instrument coupled with a column switching valve. This system was first successfully applied to the preparative separation of epimers, gambogic acid and epigambogic acid from Garcinia hanburyi using n-hexane-methanol-water (5:4:1, v/v/v) as the two-phase solvent system. As a result, 28.2 mg gambogic acid and 18.4 mg epigambogic acid were separated from 50 mg of mixture. Their purities were both above 97% as determined by HPLC. The chemical structures were then identified by their (1)H NMR and (13)C NMR spectra.

Countercurrent Distribution↗
PubMed2006

Three-phase solvent systems for comprehensive separation of a wide variety of compounds by high-speed counter-current chromatography.

Three-phase solvent systems were efficiently utilized for high-speed counter-current chromatography (HSCCC) to separate multiple components with a wide range of hydrophobicity. The compositions of three-phase systems were optimized according to their physical parameters such as volume ratio, viscosity and specific gravity of upper (UP), middle (MP) and lower (LP) phases. The three-phase systems composed of n-hexane-methyl acetate-acetonitrile-water (4:4:3:4, v/v/v/v) was selected for HSCCC separation of a mixture of 15 standard compounds with a wide range in hydrophobicity from beta-carotene to tryptophan. The separation was initiated by filling the column with a mixture of MP and LP both as a stationary phase followed by elution with UP to separate the hydrophobic compounds. Then the mobile phase was switched to MP to elute the moderately hydrophobic compounds, and finally the polar compounds still retained in the column were fractionated by eluting the column with LP. The system successfully resolved all 15 compounds in one-step operation in 70 min.

Chromatography, Liquid↗
PubMed2006

Orthogonal test design for optimization of supercritical fluid extraction of daphnoretin, 7-methoxy-daphnoretin and 1,5-diphenyl-1-pentanone from Stellera chamaejasme L. and subsequent isolation by high-speed counter-current chromatography.

Supercritical fluid extraction (SFE) of daphnoretin, 7-methoxy-daphnoretin and 1,5-diphenyl-1- pentanone from Stellera chamaejasme L. was performed. An orthogonal L9 (3)4 test design was applied to select the optimum extraction parameters including pressure, temperature, modifier and sample particle size on yield using an analytical-scale SFE system. The process was then scaled up by 100 times using a preparative SFE system under the optimized conditions of 25 MPa of pressure, 45 degrees C of temperature, 40-60 mesh of sample particle size and modified CO2 with 20% methanol. The yield of the crude extract from preparative SFE was 2.65%, which contained daphnoretin 25.2%, 7-methoxy-daphnoretin 22.8% and 1,5-diphenyl-1-pentanone 21.1%, respectively. Then the crude extract was successfully isolated and separated by preparative high-speed counter-current chromatography (HSCCC) with a two-phase solvent system composed of n-hexane-ethyl acetate-methanol-water (10:13:13:10, v/v) by increasing the flow-rate of the mobile phase stepwise from 1.0 to 2.0 ml/min after 90 min. The target compounds isolated and purified by HSCCC were analyzed by high-performance liquid chromatography (HPLC). The separation produced total of 69.2mg of daphnoretin at 99.2% purity, 63.4 mg of 7-methoxy-daphnoretin at 98.7% purity and 58.3 mg of 1,5-diphenyl-1-pentanone at 98.1% purity from 300 mg of the crude extract in one-step separation. The recoveries of daphnoretin, 7-methoxy-daphnoretin and 1,5-diphenyl-1-pentanone were 90.8, 91.5 and 90.4%, respectively, in HSCCC isolation step and the chemical structure identification was carried out by MS, 1H NMR and 13C NMR.

Chromatography, High Pressure Liquid↗
PubMed2007

Rapid purification and scale-up of honokiol and magnolol using high-capacity high-speed counter-current chromatography.

In this paper, a rapid separation approach has been developed using high-capacity high-speed counter-current chromatography (high-capacity HSCCC) to isolate and purify honokiol and magnolol, which are the main bioactive constituents from Houpu. The optimization of the solvent selection process, sample loading volume and flow rate is systematically studied using analytical high-capacity HSCCC. The optimized parameters obtained rapidly at analytical scale were used for a 1000 x scale-up preparative run using pilot scale high-capacity HSCCC in a MAXI-DE centrifuge. A crude sample of 43 g was successfully separated and the fractions were analysed by high-performance liquid chromatography (HPLC). This large scale preparative single step run yielded 16.9 and 19.4 g of honokiol and magnolol with purities of 98.6 and 99.9%, in only 20 min. This is the first time that high-performance counter-current chromatography has been used to purify multiple gram grade bioactive compounds in less than 1h and at such high concentrations of final products (10.8 g/l for magnolol and 7.0 g/l for honokiol).

Biphenyl Compounds↗
PubMed2006

Preparative isolation and purification of chemical constituents from the root of Adenophora tetraphlla by high-speed counter-current chromatography with evaporative light scattering detection.

Preparative high-speed counter-current chromatography (HSCCC), as a continuous liquid-liquid partition chromatography with no solid support matrix, combined with evaporative light scattering detection (ELSD) was employed for systematic separation and purification of non-chromophoric chemical components from Chinese medicinal herb Adenophora tetraphlla (Thunb.), Fisch. Nine compounds, including alpha-spinasterol, beta-sitosterol, nonacosan-10-ol, 24-methylene cycloartanol, lupenone, 3-O-palmitoyl-beta-sitosterol, 3-O-beta-d-glucose-beta-sitosterol, eicosanoic acid and an unknown compound, were obtained. The compounds were all above 95% determined by high-performance liquid chromatography (HPLC)-ELSD, and their structures were identified by (1)H NMR and chemical ionization mass spectroscopy (CI-MS). The results demonstrate that HSCCC coupled with ELSD is a feasible and efficient technique for systematic isolation of non-chromophoric components from traditional medicinal herbs.

Campanulaceae↗
PubMed2006

One-step separation and purification of 3,4-dihydroxyphenyllactic acid, salvianolic acid B and protocatechualdehyde from Salvia miltiorrhiza Bunge by high-speed counter-current chromatography.

Three kinds of polyphenols of Salvia miltiorrhiza Bunge, 3,4-dihydroxyphenyllactic acid, salvianolic acid B and protocatechualdehyde, were separated and purified in one step with solvent system n-hexane-ethyl acetate-methanol-acetic acid-water (1:6:1.5:1.5:8) by high-speed counter-current chromatography. Acetic acid was successfully used to increase the partition of high polar target compounds in organic phase to modify partition coefficient value. 3,4-Dihydroxyphenyllactic acid, salvianolic acid B and protocatechualdehyde were purified from 100mg water extracted crude sample of Salvia miltiorrhiza Bunge at purity of 97.6%, 94.2% and 98.2% and at yield of 98.6%, 73.6% and 90.2%. High-speed counter-current chromatography together with organic/aqueous solvent system supplied an efficient method to purify water-soluble compounds directly from crude samples of traditional Chinese medicines.

Benzaldehydes↗
PubMed2006

Isolation of chlorophylls a and b from spinach by counter-current chromatography.

A method for the isolation of chlorophylls from spinach by counter-current chromatography was developed. An initial extraction protocol was devised to avoid the notorious sensitivity of chlorophylls to degradation by light, heat, oxygen, acids and bases. Further purification and separation of chlorophylls a and b were achieved using counter-current chromatography. Chlorophyll structures and purities were established by HPLC, fast atom bombardment mass spectrometry and nuclear magnetic resonance. Purity was estimated to be >95% (100% by HPLC). Typical yields from 30g of freeze-dried spinach were 300mg of chlorophyll a and 100mg of chlorophyll b.

Chlorophyll↗
PubMed2006

Preparative isolation of huperzines A and B from Huperzia serrata by displacement centrifugal partition chromatography.

The pH-zone refining centrifugal partition chromatography technique was used to separate the two acetylcholinesterase inhibitors huperzines A and B from a crude alkaloid extract of the club moss Huperzia serrata. Complete co-elution of huperzines A and B was initially observed with the well-known methyl tert-butyl ether-acetonitrile-water (4:1:5, v/v/v) solvent system with triethylamine (8mM) as the displacer and methane sulfonic acid (6mM) as the retainer. An efficient biphasic system was designed on the basis of solvent association that provided selectivity in the elution mode: n-heptane/ethyl acetate/n-propanol/water (5:15:35:45, v/v/v/v). Lowering the bridge solvent content (n-propanol) of this system increased the polarity difference between the two phases thus adapting it to the pH-zone refining mode. Thus, the purification of these compounds was achieved using the biphasic system n-heptane/ethyl acetate/n-propanol/water (10:30:15:45, v/v/v/v) with triethylamine (8mM) as the displacer and methane sulfonic acid (6mM) as the retainer.

Alkaloids↗
PubMed2006

Solvent systems for countercurrent chromatography: an aqueous two phase liquid system based on a room temperature ionic liquid.

A new aqueous two phase liquid system (ATPS) based on the ionic liquid 1-butyl-3-methyl imidazolium chloride (BMIM Cl), potassium dibasic phosphate (K(2)HPO(4)) and water was recently proposed in the literature. The full phase diagram of this ATPS was prepared and some tie lines were fully determined. It was compared to classical ATPSs based on polyethylene glycol with an average molecular mass of 1000 (PEG 1000) and 10,000 (PEG 10000) and K(2)HPO(4). Two countercurrent chromatography (CCC) columns, a hydrostatic Sanki and a J type hydrodynamic CCC columns were used to test the liquid phase retention of these ATPSs in all possible configurations. It was found that the BMIM Cl ATPS liquid phases were much easier to retain in the two CCC columns than the PEG 1000 ATPS phases. Using protein and alcohol solutes, it was established that the BMIM Cl ATPS has a polarity completely different from that of the PEG 1000 ATPS. For example, ovalbumin partitions equally between the two phases of the PEG 1000 ATPS (K(D)=1.4) when it is completely located in the BMIM Cl upper phase of the ionic liquid ATPS (K(D)=180). The discrimination factor of the ionic liquid system and its intrinsic hydrophobicity were respectively found three times higher and ten times lower than the respective values of the PEG 1000 ATPS.

Countercurrent Distribution↗
PubMed2007

Separation of solanesol in tobacco leaves extract by slow rotary counter-current chromatography using a novel non-aqueous two-phase solvent system.

Non-aqueous solvent system composed of sunflower oil-ethanol was found to be suitable for the separation of lower polarity components. Employing this solvent system, 3 g of tobacco leaves extract containing 15% solanesol was separated using a slow rotary counter-current chromatograph equipped with an 1100 mL column made of 5.7 mm I.D. convoluted PTFE tube. The separation yielded 1.5 g of yellow oily product in which solanesol occupied 26.8% determined by HPLC. The tobacco tar and other main impurities in the tobacco leaves extract were removed after slow rotary counter-current chromatographic separation. The separation processing may be developed as a technique for producing a solanesol product with food grade.

Chromatography, Thin Layer↗
PubMed2004

Purification of alcohol dehydrogenase from bovine liver crude extract by dye-ligand affinity counter-current chromatography.

Alcohol dehydrogenase (ADH) was extracted from a crude bovine liver homogenate by dye-ligand affinity counter-current chromatography (CCC) using a cross-axis coil planet centrifuge (x-axis CPC). The purification was performed using two types of polymer phase systems composed of 4.4% polyethylene glycol (PEG) 8000-7.0% dextran T500-0.1 M potassium phosphate buffers and 16% PEG 1000-12.5% potassium phosphate buffers, both containing a procion red dye as an affinity ligand at various pH values. The best purification was achieved using the PEG 1000-potassium phosphate system at pH 7.3 containing 0.05% procion red as a ligand. The upper PEG-rich phase containing procion red was used as the stationary phase and a crude bovine liver homogenate was eluted with the potassium phosphate-rich lower phase at 0.5 ml/min. After elution of bovine liver proteins in the homogenate, ADH still retained in the stationary phase was collected from the column by eluting with the PEG 1000-rich upper phase. Collected fractions were analyzed by ADH enzymatic activity and by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) to detect contaminant proteins in the ADH fractions. The ADH was purified directly from crude bovine liver extract within 6h with minimum loss of its enzymatic activity.

Alcohol Dehydrogenase↗
PubMed2004

Purification of glucosyltransferase from cell-lysate of Streptococcus mutans by counter-current chromatography using aqueous polymer two-phase system.

Counter-current chromatography (CCC) using a cross-axis coil planet centrifuge (X-axis CPC) was applied to the purification of glucosyltransferase (GTF) from a cell-lysate of cariogenic bacteria. The purification was performed using an aqueous polymer two-phase system composed of 4.4% (w/w) polyethylene glycol (PEG) 8000-6% (w/w) dextran T500 containing 10mM phosphate buffer at pH 9.2 by eluting the upper phase (UP) at 1.0ml/min. The bacterial GTF in the cell-lysate of Streptococcus mutans was selectively retained in the dextran-rich lower stationary phase. The column contents were diluted and subjected to hydroxyapatite (HA) chromatography to remove the polymers from the GTF. Fractions eluted with 500mM potassium phosphate buffer were analyzed by GTF enzymatic activity as well as sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The GTF purity in the final product was increased about 87 times as that in the cell-lysate with a good recovery rate of about 79% through this purification process.

Chromatography, Liquid↗
PubMed2006

Two-step purification of scutellarin from Erigeron breviscapus (vant.) Hand. Mazz. by high-speed counter-current chromatography.

Scutellarin, a flavone glycoside, popularly applied for the treatment of cardiopathy, has been purified in two-step purification by high-speed counter-current chromatography (HSCCC) from Erigeron breviscapus (vant.) Hand. Mazz. (Deng-zhan-hua in Chinese), a well-known traditional Chinese medicinal plant for heart disease. Two solvent systems, n-hexane-ethyl acetate-methanol-acetic acid-water (1:6:1.5:1:4, v/v/v/v/v) and ethyl acetate-n-butanol-acetonitrile-0.1% HCl (5:2:5:10, v/v/v/v) were used for the two-step purification. The purity of the collected fraction of scutellarin was 95.6%. This study supplies a new alternative method for purification of scutellarin.

Apigenin↗