Organic Stationary Phase Research Articles

Identification, separation by spiral high-speed counter-current chromatography, and quantification of 7-chloro-5-methyl-2H-1,4-benzothiazin-3(4H)-one, an impurity in the thioindigoid color additive D&C Red No. 30 and its lakes

An impurity in the color additives D&C Red No. 30 (R30) and D&C Red No. 30 lakes (R30L) was newly identified and characterized as 7-chloro-5-methyl-2H-1,4-benzothiazin-3(4H)-one (BTZ), and its extent and level in certified batches of these color additives was determined. BTZ was extracted from the dye with ethanol, resulting in a crude extract enriched to a concentration of over 60%. BTZ was then separated from a portion of the enriched extract by high-speed counter-current chromatography using a spiral-tube assembly column with intermittently pressed tubing of 60 ml capacity. It was the first reported use of such a column to separate a small, moderately hydrophobic compound. The two-phase solvent system was also moderately hydrophobic, consisting of hexane-ethyl acetate-methanol-water (5:2:5:2), and the retention of the organic stationary phase measured after the separation was 83.3%. The separation yielded BTZ of two purity grades, the higher of which (~95.5%) was used as a standard to quantify the impurity in 37 batches of R30 and R30L using an HPLC method developed and validated for that purpose. Analyses revealed a wide range of BTZ levels across batches, <0.05 - 0.84%, and suggested that BTZ contamination could be reduced by appropriate adjustments in the manufacturing process. An explanation of the likely source of BTZ – as a side-reaction product in a particular step of the manufacturing process – was also presented.

Preparative separation of high-purity trans- and cis-ferulic acid from wheat bran by pH-zone-refining counter-current chromatography

Ferulic acid stereoisomers are the most abundant phenolic acids in cereal bran. However, it is challenging to separate them because of the similar structures and properties. In this study, a preparative separation method of ferulic acid stereoisomers from the crude extract of wheat bran was successfully developed. The method contained a two-step separation, the traditional counter-current chromatography (CCC, hexane: ethyl acetate: methanol: water = 2:5:2:4) was followed with a pH-zone-refining CCC (hexane: ethyl acetate: acetonitrile: water = 2:5:2:2, 10 mmol L−1 trifluoroacetic acid in organic stationary phase and 10 mmol L−1 ammonia in aqueous mobile phase). Trans-ferulic acid and cis-ferulic acid with HPLC high purity over than 99% and 98% can be yielded in large-scale separation. Moreover, it is found that different proton affinity, deprotonation ability and interaction site of hydrogen bond result in distinct partition behavior of stereoisomers, which is illustrated by quantitative analysis of molecular surface. This contributes to our in-depth understanding of the separation mechanism toward pH-zone refining CCC. The developed method can be applied in the exploitation of ferulic acids and related phenolic acids from other resources.

Aporphine Alkaloids from Triclisia dictyophylla Diels by pH-Zone Refining Countercurrent Chromatography

A new dimeric aporphine alkaloid, biscocsarmine, 2, together with lirioferine, 1, and cocsarmine, 3, were isolated from the ethanol extract of the stems of Triclisia dictyophylla Diels (Menispermaceae) using a preparative pH-zone refining CCC in a single step. The first solvent system family tested for this fractionation was hexane–ethyl acetate–methanol–water (HEMWat) at a series of solvent ratios, and hydrochloric acid and triethylamine at 5 mM as eluter/retainer. None of these HEMWat ratios, however, gave suitable K for the target alkaloids. Ethyl acetate–n-butanol–water (EBuWat) was tested instead and the selected solvent system was composed of ethyl acetate–n-butanol–water 5:5:10 (v/v), where triethylamine (60 mM) was added to the upper organic stationary phase as a retainer and hydrochloric acid (5 mM) to the aqueous mobile phase as an eluter. The chemical structures of the isolated compounds were elucidated based on comprehensive spectroscopic and spectrometric techniques. This is the first report of aporphine alkaloids in this plant species.

The relationship between polymer structures on silica particles and the separation characteristics of the corresponding columns for hydrophilic interaction chromatography

Silica particles with various pore sizes were modified with poly(acrylamide) via surface-initiated atom-transfer radical polymerization (SI-ATRP) under different reaction conditions. Twenty different columns were prepared and characterized according to a test method for hydrophilic interaction chromatography (HILIC) columns. Hydrophilic retention by the SI-ATRP columns was much higher than that of poly(acrylamide) columns prepared via free-radical polymerization and many commercially available HILIC columns. The SI-ATRP columns displayed greater selectivity for –OH groups than any of the HILIC columns based on their α(U/2dU) values. SI-ATRP functionalization was used to increase the polymer chain density on the silica particles, which suggested a brush-type morphology, and improved hydrophilic selectivity. This indicated that hydrophilic retention and selectivity could be controlled by adjusting the morphology of the organic stationary phase. This stationary phase design strategy was validated experimentally by the effective separation of highly hydrophilic analytes. The findings of this study will greatly contribute to the creation of better separation media.

Isolation and purification of six aristolochic acids with similar structures from Aristolochia manshuriensis Kom stems by pH-zone-refining counter-current chromatography

Aristolochic acids (AAs), the major components in Aristolochia manshuriensis Kom stems (AMK), may cause Chinese herb nephropathy during clinical application. Therefore, it is necessary to distinguish AMK from other herbs and Chinese medicines using AAs with high purity as standards. So, an efficient method for separation and purification of AAs is required because of their similar structures. In this study, six AAs with purities of >98% were obtained by pH-zone-refining counter-current chromatography (PZRCCC) in a single run. The optimum two-phase solvent system was petroleum ether–ethyl acetate–methanol–water (3:7:3:7, v/v). Triethylamine (10 mM) was added to the aqueous mobile phase and trifluoroacetic acid (10 mM) to the organic stationary phase. As a result, 9.7 mg aristolochic acid IIIa, 12.0 mg aristolochic acid IVa, 32.2 mg aristolochic acid II, 103.7 mg aristolochic acid I, 24.6 mg aristolic acid II, and 26.1 mg aristolic acid I were obtained from 800 mg AAs crude extract. The elution order of AAs during PZRCCC separation corresponded with the pKa values and hydrophobicities of the target compounds. PZRCCC is an efficient method for isolation of AAs with similar structures.

Rapid isolation of acidic cannabinoids from Cannabis sativa L. using pH-zone-refining centrifugal partition chromatography

This work introduces an effective methodology for the isolation of acidic cannabinoids from fiber-type Cannabis sativa L. Supercritical fluid extraction (SFE) was initially employed to obtain an enriched extract of acidic cannabinoids. Subsequently, fractionation was performed by using centrifugal partition chromatography (CPC) with the pH-zone-refining method. The biphasic solvent system that was selected consisted of n-hexane/ethyl acetate/ethanol/water 8:2:5:5 (v/v/v/v). Trifluoroacetic acid was added as retainer in the organic stationary phase, while triethylamine was used as eluter in the aqueous mobile phase. The most promising CPC fractions containing cannabidiolic acid (CBDA) and cannabidivarinic acid (CBDVA) were further purified by liquid-liquid extraction. Following this procedure, 1.86 g of CBDA (>85%) were recovered from 9 g of extract, with 1.08 g thereof having a purity of more than 95%, as determined by HPLC-PDA analysis. Moreover, 91 mg of CBDVA with greater than 85% purity were obtained. This methodology can be efficiently used for the large-scale purification of CBDA and after minor modifications could be readily adaptable for the isolation of other acidic cannabinoids, based on their ionizable character.

Separation of the potential G-quadruplex ligands from the butanol extract of Zanthoxylum ailanthoides Sieb. & Zucc. by countercurrent chromatography and preparative high performance liquid chromatography

G-quadruplex DNA structure is considered to be a very attractive target for antitumor drug design due to its unique role in maintaining telomerase activities. Therefore, discovering ligands with high stability of G-quadruplex structure is of great interest. In this paper, pH-zone refining counter current chromatography (CCC) and preparative high performance liquid chromatography (HPLC) were employed for the separation of potent G-quadruplex ligands from the n-butanol fraction of the crude extract of Zanthoxylum ailanthoides, which is a traditional Chinese medicine recently found to display high inhibitory activity against several human cancer cells. The 75% aqueous ethanol extract of the stem bark of Z. ailanthoides and its fractions with petroleum ether, ethyl acetate and n-butanol displayed almost the same G-quadruplex stabilization ability. Here, pH-zone refining CCC was used for the separation of the alkaloids from the n-butanol fraction by a seldom used solvent system composed of dichloromethane-methanol-water (4:1:2.5) with 10mM TEA in the organic stationary phase as retainer and 10mM HCl in the aqueous mobile phase as eluter. Compounds I, II and III were obtained, with purity greater than 95%, in the quantities of 31.2, 94.0, and 26.4mg respectively from 300mg of lipophilic fraction within 80min, which were identified as three tetrahydroprotoberberines isolated for the first time in this plant. In addition, a phenylpropanoid glycoside compound IV (Syringin), an isoquinoline (Magnoflorine, V), and two lignin isomers (+)-lyoniresiol-3α-O-β-d-glucopyranoside (VI) and (−)-lyoniresinol −3α-O-β-D −glucopyranoside (VII) were isolated by traditional CCC together with preparative HPLC. Compounds IV, V, VI and VII were obtained, with purity greater than 95%, in the quantities of 4.0, 13.2, 6.7, and 6.5mg respectively from 960mg of hydrophilic fraction. Among the seven isolated compounds, tetrahydroprotoberberine I, II and III were found to display remarkable stabilization effects on G-quadruplex by increasing G-quadruplex's Tm approximately 10°C, which may be the most potent G-quadruplex ligands in Z. ailanthoides.

A general ionic liquid pH-zone-refining countercurrent chromatography method for separation of alkaloids from Nelumbo nucifera Gaertn

The alkaloids from lotus (Nelumbo nucifera Gaertn) are effective in lowering hyperlipemia and level of cholesterol. However, there is not a general method for their separation. In this work, a general ionic liquid pH-zone-refining countercurrent chromatography method for isolation and purification of six alkaloids from the whole lotus plant was successfully established by using ionic liquids as the modifier of the two-phase solvent system. The conditions of ionic liquid pH-zone-refining countercurrent chromatography, involving solvent systems, concentration of retainer and eluter, types of ionic liquids, the content of ionic liquids as well as ionic liquids posttreatment, were optimized to improve extraction efficiency. Finally, the separation of these six alkaloids was performed with a two-phase solvent system composed of n-hexane-ethyl acetate-methanol-water-[C4mim][PF6] at a volume ratio of 5:2:2:8:0.1, where 10mM TEA was added to the organic stationary phase as a retainer and 3mM HCl was added to the aqueous mobile phase as an eluter. As a result, six alkaloids including N-nornuciferine, liensinine, nuciferine, isoliensinine, roemerine and neferine were successfully separated with the purities of 97.0%, 90.2%, 94.7%, 92.8%, 90.4% and 95.9%, respectively. The established general method has been respectively applied to the crude samples of lotus leaves and lotus plumules. A total of 37.3mg of liensinine, 57.7mg of isoliensinine and 179.9mg of neferine were successfully purified in one run from 1.00g crude extract of lotus plumule with the purities of 93.2%, 96.5% and 98.8%, respectively. Amount of 45.6mg N-nornuciferine, 21.6mg nuciferine and 11.7mg roemerine was obtained in one step separation from 1.05g crude extract of lotus leaves with the purity of 96.9%, 95.6% and 91.33%, respectively.

Separation of betacyanins from purple flowers of Gomphrena globosa L. by ion-pair high-speed counter-current chromatography

Betacyanins, known as antioxidants and chemopreventive natural compounds with colourful properties, were extracted from purple flowers of Gomphrena globosa L. belonging to the Amaranthaceae family and separated for the first time by ion-pair high-speed counter-current chromatography (HSCCC). The pigments were detected by LC-DAD-ESI-MS/MS technique. Separation of betacyanins (300mg) by HSCCC was accomplished in four solvent systems: tert-butyl methyl ether – butanol – acetonitrile – water (0.7% and 1.0% HFBA – heptafluorobutyric acid – system I and III) and tert-butyl methyl ether – butanol – methanol – water (0.7% and 1.0% HFBA – system II and IV) (2:2:1:5, v/v/v/v) in the head-to-tail mode. The mobile phase (aqueous phase) was run at 2.0ml/min and the column rotation speed was 860rpm.The applied systems enabled to study the influence of HFBA concentration as well as systems polarity on betacyanins separation. Comparison of the systems containing 0.7% HFBA (systems I–II) demonstrates that the replacement of acetonitrile by methanol increases the resolution (Rs) between all betacyanins and does not influence the retention of the stationary phase (Sf=76%). Higher concentration of the acid in systems III–IV slightly decreases Sf to 71% in the systems with 1.0% HFBA. Comparison of the resolution values for betacyanins in the systems with 0.7% and 1.0% HFBA demonstrates that higher concentration of the acid improves the separation effectiveness for all betacyanins as a result of increasing of the chemical affinity of the pigments to the organic stationary phase in HSCCC. The systems III–IV with 1% HFBA are the most effective for the separation of all the studied betacyanins.

One-step preparative isolation of aristolochic acids by strong ion-exchange centrifugal partition chromatography

Aristolochic acids are biologically active and highly toxic secondary metabolites that recently became an important pharmacological tool for in vivo models of acute kidney disorders. Only aristolochic acid I and a mixture of aristolochic acids I and II are currently commercially available. A method based on the strong-ion exchange displacement mode in centrifugal partition chromatography was developed to extract and purify arictolochic acids, naturally present in Aristolochia bracteolata Lam. (Aristolochiaceae). The extraction and purification of aristolochic acids were performed at a flow rate of 2 mL min−1 in the descending mode by using the biphasic solvent system methyl-ter-butylether, acetone, methanol and water (3:1:1:3, v/v/v/v). Trioctylmethylammonium chloride (Aliquat 336®) was used as the anion exchanger in the organic stationary phase and sodium iodide as the displacer in the aqueous mobile phase. From 5 g of crude extract of A. bracteolata, 45.5 mg, 77.2 mg and 35.1 mg of aristolochic acids I, II and IIIa were obtained respectively with purities greater than 95%.

Industrial case study on alkaloids purification by pH-zone refining centrifugal partition chromatography

The industrial potential of pH-zone refining centrifugal partition chromatography has been evaluated by studying the purification of pharmaceutical ingredients at the pilot scale. For the first time, a scale up methodology based on both column capacity and mass transfer efficiency as invariants was developed. The purification of catharanthine and vindoline from an industrial crude extract of aerial parts of Catharanthus roseus, was used as a case of study. Toluene/CH3CN/water (4/1/5, v/v/v) was selected as biphasic solvent system, triethylamine as retainer in the organic stationary phase and sulphuric acid as displacer in the aqueous mobile phase. The separation intensification was performed on a 36mL CPC column equipped with 832 partition twin-cells. The combined effects of four parameters (displacer and retainer concentrations for intensive parameters, flow rate and rotational speed for extensive parameters) were studied by design of experiment in order to maximize both recoveries and productivities. Then, scale change was done on two larger columns (305mL and 1950mL of capacity) equipped with only 231 and 238 partition cells. For this step, it has been shown that the global mass transfer coefficient k0a (the efficiency of a column design) and the stationary phase retention (the capacity of the column) were relevant and useful scale up invariants.A CPC model based on acid-base equilibriums and interfacial mass transfer in continuously stirred tank reactors in series was used to predict fully separations on larger CPC column at the optimized operating conditions and to guide the CPC user in its scale-up strategy. The experimental validation on pilot CPC column, by injecting up to 150g of Catharanthus roseus crude extract on the 1950mL column highlighted the preservation of the separation quality, the non-linear character of the scale up in centrifugal partition chromatography and that a productivity of about 4kg of processed crude extract per day can be reached by implementing developed methodology.

Combinative application of pH-zone-refining and conventional high-speed counter-current chromatography for preparative separation of caged polyprenylated xanthones from gamboge.

An efficient method for the preparative separation of four structurally similar caged xanthones from the crude extracts of gamboge was established, which involves the combination of pH-zone-refining counter-current chromatography and conventional high-speed counter-current chromatography for the first time. pH-zone-refining counter-current chromatography was performed with the solvent system composed of n-hexane/ethyl acetate/methanol/water (7:3:8:2, v/v/v/v), where 0.1% trifluoroacetic acid was added to the upper organic stationary phase as a retainer and 0.03% triethylamine was added to the aqueous mobile phase as an eluter. From 3.157 g of the crude extract, 1.134 g of gambogic acid, 180.5 mg of gambogenic acid and 572.9 mg of a mixture of two other caged polyprenylated xanthones were obtained. The mixture was further separated by conventional high-speed counter-current chromatography with a solvent system composed of n-hexane/ethyl acetate/methanol/water (5:5:10:5, v/v/v/v) and n-hexane/methyl tert-butyl ether/acetonitrile/water (8:2:6:4,v/v/v/v), yielding 11.6 mg of isogambogenic acid and 10.4 mg of β-morellic acid from 218.0 mg of the mixture, respectively. The purities of all four of the compounds were over 95%, as determined by high-performance liquid chromatography, and the chemical structures of the four compounds were confirmed by electrospray ionization mass spectrometry and NMR spectroscopy. The combinative application of pH-zone-refining counter-current chromatography and conventional high-speed counter-current chromatography shows great advantages in isolating and enriching the caged polyprenylated xanthones.

Optimisation and establishment of separation conditions of organic acids from Usnea longissima Ach. by pH-zone-refining counter-current chromatography: Discussion of the eluotropic sequence

The major bioactive constituents of Usnea longissima Ach. are organic acids. However, few recent literatures involve the preparative separation of these organic acids. In the present study, pH zone-refining counter-current chromatography is used to separate organic acids from crude sample of U. longissima Ach. The crude extract was separated with the two-phase solvent system Pet–EtAc–MeOH–H2O (5:5:3:7, v/v) with 10mM TFA in organic stationary phase and different concentration of the eluter in aqueous mobile phase for the screening of the most suitable separation conditions. From the crude extract (1.2g), 74.0mg of orsellinic acid at 92.7% purity, 55.5mg of 4-O-methylorsellinic acid at 97.7% purity, 353.5mg of evernic acid at 93.8% purity, 102.0mg of barbatic acid at 94.8% purity, 19.4mg of diffractaic acid at 92.2% purity, and 44.9mg of usnic acid at 95.7% purity were obtained using the selected conditions in which the concentration of TFA in stationary phase was 10mM and the concentration of NaOH in mobile phase was 10–20mM. The purities of the separated organic acids were measured by HPLC. And the data of electrospray ionization-liquid chromatography/mass spectrometry (ESI–LC/MS), 1H NMR, and 13C NMR were used for confirming chemical structures.

One-step preparative isolation of aristolochic acids by strong ion-exchange centrifugal partition chromatography

Aristolochic acids are biologically active and highly toxic secondary metabolites that recently became an important pharmacological tool for in vivo models of acute kidney disorders. Only aristolochic acid I and a mixture of aristolochic acids I and II are currently commercially available. A method based on the strong-ion exchange displacement mode in centrifugal partition chromatography was developed to extract and purify arictolochic acids, naturally present in Aristolochia bracteolata Lam. (Aristolochiaceae). The extraction and purification of aristolochic acids were performed at a flow rate of 2 mL min−1 in the descending mode by using the biphasic solvent system methyl-ter-butylether, acetone, methanol and water (3:1:1:3, v/v/v/v). Trioctylmethylammonium chloride (Aliquat 336®) was used as the anion exchanger in the organic stationary phase and sodium iodide as the displacer in the aqueous mobile phase. From 5 g of crude extract of A. bracteolata, 45.5 mg, 77.2 mg and 35.1 mg of aristolochic acids I, II and IIIa were obtained respectively with purities greater than 95%.

Determination of chlorophenols in red wine using ionic liquid countercurrent chromatography as a new pretreatment method followed by high-performance liquid chromatography.

A countercurrent chromatography method for the enrichment and cleanup of chlorophenols from food samples was successfully established by using an ionic-liquid-modified two-phase solvent system composed of dichloromethane containing 2% 1-butyl-3-methylimidazolium bis(trifluoromethanesulfonyl)imide and water. The column was firstly filled with the organic stationary phase, and then a large volume of sample was pumped into the column after it was equilibrated with pure water at the rotation speed. Finally, the trace amounts of chlorophenols extracted and enriched in the stationary phase were eluted out by an alkaline mobile phase and determined by high-performance liquid chromatography. Under optimized conditions, the enrichment and cleanup of the chlorophenols can be fulfilled online with enrichment factors (34-65) and high recoveries (84.69-95.23%). The method has been applied to the determination of chlorophenols in real red wine samples with the limits of detection in the range of 1.89-4.21 μg/L. The present method is highly suitable for the pretreatment of large volume of aqueous sample for the determination of trace amounts of contaminants in food and environmental samples.

A general separation method of phenolic acids using pH-zone-refining counter-current chromatography and its application to oat bran

pH-zone-refining counter-current chromatography technique for the separation of natural and synthetic mixtures has been widely used, especially for organic acids and alkaloids. Phenolic acids are very important compounds due to the potential treatment for a wide variety of diseases. However, there is not a general method for their separation. In this work, the conditions of pH-zone-refining counter-current chromatography, involving solvent systems, concentration of retainer and eluter, flow rate of mobile phase as well as sample pretreatment, were optimized to improve extraction efficiency and reduce separation time. Finally a general separation method for seven common phenolic acids has been established using pH-zone-refining counter-current chromatography. The separation of these phenolic acids was performed with a two-phase solvent system composed of methyl tert-butyl ether/acetonitrile/water at a volume ratio of 4.75: 0.25: 5, where 3mM trifluoroacetic acid was added to the organic stationary phase as a retainer and 3mM NH4OH was added to the aqueous mobile phase as an eluter. As a result, seven phenolic acids, including syringic acid, 4-hydroxyphenylacetic acid, vanillic acid, caffeic acid, p-hydroxybenzoic acid, ferulic acid and p-coumaric acid were successfully separated with the purities of 95.9%, 67.3%, 96.9%, 82.4%, 97.0%, 91.0%, and 97.2%, respectively. The established general method has been applied to the crude sample of oat bran pretreated with AB-8 resin. A total of 49.5mg of syringic acid, 109.2mg of p-coumaric acid and 184.5mg of ferulic acid were successfully purified in one run from 1.22g crude extract with the purities of 95.2%, 93.0%, and 91.8%, respectively.

Separation of amlodipine besilate enantiomers by biphasic recognition recycling high-speed counter-current chromatography

Biphasic recognition recycling high-speed counter-current chromatography (HSCCC) was applied for the separation of amlodipine besilate (ADB) enantiomers. The biphasic recognition system was established by adding hydrophilic sulfobutyl ether-β-cyclodextrin (SBE-β-CD) in the aqueous mobile phase and lipophilic d-iso-butyl tartrate (d-IBTA) in the organic stationary phase, which is capable of preferentially recognizing (+)-enantiomer and (−)-enantiomer respectively. The two-phase solvent system, which was composed of ethyl acetate–aqueous solution (1:1, v/v) plus the above chiral selectors, was selected as per the distribution ratios and separation factor of ADB enantiomers. Impacts of the type and concentration of chiral selectors, pH value of the aqueous solution as well as temperature on the separation efficiency were investigated. By means of preparative HSCCC, pure enantiomers were obtained through the separation of 30mg racemates with a purity greater than 97.5% and a recovery rate between 80% and 84%. Experimental results revealed that biphasic recognition recycling high-speed counter-current chromatography offers a promising means to separate racemates with a low separation factor in a highly efficient manner.

Preparative separation of alkaloids from Picrasma quassioides (D. Don) Benn. by conventional and pH-zone-refining countercurrent chromatography.

Two high-speed countercurrent chromatography (HSCCC) modes were compared by separation of major alkaloids from crude extract of Picrasma quassioides. The conventional HSCCC separation was performed with a two-phase solvent system composed of petroleum ether–ethyl acetate–methanol–water (5:5:4.5:5.5, v/v/v/v) with 200 mg loading. pH-Zone-refining CCC was performed with two-phase solvent system composed of petroleum ether–ethyl acetate–n-butanol–water (3:2:7:9, v/v/v/v) where triethylamine (10 mM) was added to the upper organic stationary phase and hydrochloric acid (5 mM) was added to the lower aqueous phase with 2 g loading. From 2 g of crude extract, 87 mg of 5-methoxycanthin-6-one (a), 38 mg of 1-methoxy-β-carboline (b), 134 mg of 1-ethyl-4,8-dimethoxy-β-carboline (c), 74 mg of 1-ethoxycarbonyl-β-carboline (d), 56 mg of 1-vinyl-4,8-dimethoxy-β-carboline (e) and 26 mg of 1-vinyl-4-dimethoxy-β-carboline (f) were obtained with purities of over 97.0%. The results indicated that pH-zone-refining CCC is an excellent separations tool at the multigram level.

Separation of phenylsuccinic acid enantiomers using biphasic chiral recognition high-speed countercurrent chromatography.

High-speed countercurrent chromatography (HSCCC) combined with biphasic chiral recognition was successfully applied to the resolution of phenylsuccinic acid enantiomers. D-Isobutyl tartrate and hydroxypropyl-β-cyclodextrin were employed as lipophilic and hydrophilic selectors dissolved in the organic stationary phase and aqueous mobile phase, respectively. The two-phase solvent system was made up of n-hexane/methyl tert-butyl ether/water (0.5:1.5:2, v/v/v). Impacts of the type and concentration of chiral selectors, the pH value of the aqueous phase solution as well as the temperature on the separation efficiency were investigated. By means of preparative HSCCC, pure enantiomer was obtained by separating 810 mg of racemate with a purity >99.5% and a recovery rate between 82 and 85%. The experimental results indicate that biphasic recognition HSCCC provide a promising means for efficient separation of racemates.

Preparative Separation of Six Rhynchophylla Alkaloids from Uncaria macrophylla Wall by pH-Zone Refining Counter-Current Chromatography

pH-Zone refining counter-current chromatography was successfully applied to the preparative isolation and purification of six alkaloids from the ethanol extracts of Uncaria macrophylla Wall. Because of the low content of alkaloids (about 0.2%, w/w) in U. macrophylla Wall, the target compounds were enriched by pH-zone refining counter-current chromatography using a two-phase solvent system composed of petroleum ether–ethyl acetate–isopropanol–water (2:6:3:9, v/v), adding 10 mM triethylamine in organic stationary phase and 5 mM hydrochloric acid in aqueous mobile phase. Then pH-zone refining counter-current chromatography using the other two-phase solvent system was used for final purification. Six target compounds were finally isolated and purified by following two-phase solvent system composed of methyl tert-butyl ether (MTBE)–acetonitrile–water (4:0.5:5, v/v), adding triethylamine (TEA) (10 mM) to the organic phase and HCl (5 mM) to aqueous mobile phase. The separation of 2.8 g enriched total alkaloids yielded 36 mg hirsutine, 48 mg hirsuteine, 82 mg uncarine C, 73 mg uncarine E, 163 mg rhynchophylline, and 149 mg corynoxeine, all with purities above 96% as verified by HPLC Their structures were identified by electrospray ionization-mass spectrometry (ESI-MS) and 1H-NMR spectroscopy.