Xueli Cao

Separation and purification of isoflavones from Pueraria lobata by high-speed counter-current chromatography

High-speed counter-current chromatography (HSCCC) was applied to the semipreparative separation and purification of puerarin and related isoflavones from a crude extract of Pueraria lobata. Analytical HSCCC was used for the preliminary selection of a suitable solvent system composed of ethyl acetate-n-butanol-water (2:1:3, v/v/v). Using the above solvent system the preparative HSCCC was successfully performed yielding six relatively pure isoflavones including puerarin from 80 mg of the crude extract in one-step separation.

Determination of triclosan, triclocarban and methyl-triclosan in aqueous samples by dispersive liquid–liquid microextraction combined with rapid liquid chromatography

In this study, dispersive liquid-liquid microextraction (DLLME) combined with ultra-high-pressure liquid chromatography (UHPLC)-tunable ultraviolet detection (TUV), has been developed for pre-concentration and determination of triclosan (TCS), triclocarban (TCC) and methyl-triclosan (M-TCS) in aqueous samples. The key factors, including the kind and volume of extraction solvent and dispersive solvent, extraction time, salt effect and pH, which probably affect the extraction efficiencies were examined and optimized. Under the optimum conditions, linearity of the method was observed in the range of 0.0500-100 microg L(-1) for TCS, 0.0250-50.0 microg L(-1) for TCC, and 0.500-100 microgL(-1) for M-TCS, respectively, with correlation coefficients (r(2))>0.9945. The limits of detection (LODs) ranged from 45.1 to 236 ng L(-1). TCS in domestic waters was detected with the concentration of 2.08 microg L(-1). The spiked recoveries of three target compounds in river water, irrigating water, reclaimed water and domestic water samples were achieved in the range of 96.4-121%, 64.3-84.9%, 77.2-115% and 75.5-106%, respectively. As a result, this method can be successfully applied for the rapid and convenient determination of TCS, TCC and M-TCS in real water samples.

Separation and identification of polyphenols in apple pomace by high-speed counter-current chromatography and high-performance liquid chromatography coupled with mass spectrometry.

Apple pomace, a by-product in the processing of apple juice, was investigated as a potential source of polyphenols. Two methods of separation and purification of polyphenols from apple pomace extract were established by combination of gel chromatography with high-speed counter-current chromatography (HSCCC) and solvent extraction with HSCCC, respectively. The optimal separation was performed on a Sephadex LH-20 column using gradient aqueous ethanol as eluting solvent from 0% to 100% in increments of 10%. HPLC analysis indicated that main polyphenols existed in fractions eluted between 40% and 50% aqueous ethanol. The fractions of interest from column were separated by HSCCC with the solvent system hexane-ethyl acetate-1% aqueous acetic acid (0.5:9.5:10, v/v/v). Ethyl acetate fractionation of the apple pomace extract followed by direct HSCCC separation by the same solvent system in the volume ratio of 1:9:10 also produced a good separation of the main polyphenols of interest. Six high-purity polyphenols were achieved tentatively and identified by HPLC/MS: chlorogenic acid (1, m/z 354), quercetin-3-glucoside/quercetin-3-glacaside (2, m/z 464), quercetin-3-xyloside (3, m/z 434), phloridzin (4, m/z 436), quercetin-3-arabinoside (5, m/z 434), and quercetin-3-rhamnoside (6, m/z 448). These results provided a preliminary foundation for further development and exploration of apple pomace.

Separation of Dammarane‐Saponins from Notoginseng, Root of Panax notoginseng (Burk.) F. H. Chen, by HSCCC Coupled with Evaporative Light Scattering Detector

Abstract High‐speed countercurrent chromatography (HSCCC) coupled with evaporative light scattering detection, was applied to the separation of saponins from notoginseng, the main root of Panax notoginseng (Burk.) F. H. Chen. Five individual dammarane saponins have been isolated with the solvent systems composed of CHCl3/MeOH/2‐BuOH/H2O (5:6:1:4, v/v/v/v) and EtOAc/1‐BuOH/H2O (1:1:2, v/v/v), successively. They were identified as ginsenoside‐Rg1, ginsenoside‐Rd, notoginsenoside‐R1, ginsenoside‐Re, and ginsenoside‐Rb1 by FAB‐MS and 13C NMR, together with HPLC and TLC analysis.

Separation of salidroside from Rhodiola crenulata by high-speed counter-current chromatography

High-speed counter-current chromatography (HSCCC) was used to purify salidroside from an extract of Rhodiola crenulata with two steps using a two-phase solvent system composed of ethyl acetate-n-butanol-water (1:4:5, v/v) in the first run and chloroform-methanol-isopropanol-water (5:6:1:4) in the second run. The method yielded 21.9 mg of salidroside from 1.216 g of the crude sample at 98% purity determined by HPLC analyses. Identification was performed by 1H NMR, 13C NMR, and MS.

Supercritical fluid extraction of catechins from Cratoxylum prunifolium Dyer and subsequent purification by high-speed counter-current chromatography.

Supercritical fluid extraction of tea catechins including epigallocatechin-3-O-gallate (EGCG) and epicatechin-3-O-gallate (ECG) from Cratoxylum prunifolium Dyer was performed. The optimization of parameters was carried out using an analytical-scale supercritical fluid extraction (SFE) system designed in our laboratory. Then the extraction was scaled up by 100 times using a preparative SFE system under a set of optimized conditions of 40 degrees C, 25 MPa and modified CO2 with 80% ethanol aqueous solution. The combined yield of EGCG and ECG reached about 1 mg per 1 g of tea leaves where the solubility was near 1.4 x 10(-4) mass fraction of CO2 fluid. EGCG and ECG of high purity (>98%) were obtained from the crude preparative extract by high-speed counter-current chromatography.

Stationary phase retention and preliminary application of a spiral disk assembly designed for high-speed counter-current chromatography

A spiral disk assembly composed of five single-channel units was designed for high-speed counter-current chromatography (HSCCC). The retention of different solvent systems ranging from moderately polar to polar organic-aqueous systems to aqueous two-phase systems (ATPs) was investigated under different elution modes. The results indicated that the spiral disk assembly can produce excellent retention of stationary phase for moderately polar organic-aqueous solvent systems, such as chloroform-methanol-water (4:3:2) and hexane-ethyl acetate-methanol-water (1:1:1:1) by pumping lower mobile phase from head (H) to tail (T), and upper mobile phase from tail (T) to head (H) even at a high flow-rate (8 mL/min, Sf>70%), regardless of whether the inlet is at the inner or outer terminal of the channel. This makes it possible for fast analysis of some small molecular compounds. This has been proved in the separation of mixtures of three flavones, including isorhamnetin, kaempferol, and quercetin. The spiral disk assembly can also provide satisfactory retention for polar to ATPS such as 1-butanol-acetic acid-water (4:1:5) (<3 mL/min, Sf>70%), 12.5% poly(ethylene glycol) (PEG) 1000-12.5% K2HPO4-75% water (< or =1 mL/min, Sf>70%) and 4% PEG 8000-5% Dextran T500-91% water (< or =0.5 mL/min, Sf>50%) by pumping lower mobile phase from inner terminal (I) to outer terminal (O), and upper mobile phase from outer terminal (O) to inner terminal (I) at a low flow-rate, while this is not possible with the multilayer coil column. Acceptable resolutions were achieved when it was used for the separation of peptides such as Leu-Tyr and Val-Tyr, and proteins including cytochrome c and myoglobin, lysozyme and myoglobin, and fresh chicken egg-white proteins.

Separation of Aloins A and B from Aloe Vera Exudates by High Speed Countercurrent Chromatography

Abstract Aloin is the main anthraquinone in aloe leaf, which occurs naturally as a mixture of two diastereoisomers, aloin A and aloin B, and currently served as one of the important control constituents in most of the commercial aloe products. High speed countercurrent chromatography (HSCCC) has been employed for the preparative separation of individual isomers combined with preseparation on silica gel chromatography. Three solvent systems composed of chloroform‐methanol‐water (4:2:3), ethyl acetate‐methanol‐water (5:1:5), and butanol‐ethyl acetate‐water (1:3:4) have been used in HSCCC. 6 g of extract of dry aloe leaf exudates yielded 202 mg of aloin A (>98%) and 140 mg aloin B (>96%). Their structures have been confirmed by FAB‐MS and 1HNMR through gradient enhanced nuclear overhauser effect spectroscopy (Goesy).

Separation and purification of 10-deacetylbaccatin III by high-speed counter-current chromatography.

High-speed counter-current chromatography (HSCCC) was used for the separation and purification of 10-deacetylbaccatin III from the needle extract of Chinese yew (Taxus chinensis). The crude needle extract (500 mg/5 ml) was first separated with a two-phase solvent system composed of n-hexane-ethyl acetate-ethanol-water (2:5:2:5, v/v) and the partially purified fraction was again purified with a different solvent system composed of n-hexane-chloroform-methanol-water (5:25:34:20, v/v). HPLC analysis of the final fraction showed that the purity of 10-deacetylbaccatin (20 mg) was over 98%. The chemical structure was confirmed by fast atom bombardment MS and 1H NMR.

Determination of Alternaria mycotoxins in wine and juice using ionic liquid modified countercurrent chromatography as a pretreatment method followed by high-performance liquid chromatography.

Alternariol (AOH), alternariol monomethyl ether (AME), and tenuazonic acid (TeA) are some of the main Alternaria mycotoxins that can be found as contaminants in food materials. The objective of this study was to develop a pretreatment method with countercurrent chromatography (CCC) for enrichment and cleanup of trace Alternaria mycotoxins in food samples prior to high-performance liquid chromatography (HPLC) analysis. An Analytical CCC instrument with a column volume 22.5mL was used, and a two-phase solvent system composed of ethyl acetate and water modified with 6% [HOOMIM][Cl] in mass to volume ratio was selected. Under the optimized CCC operation conditions, trace amounts of AOH, AME, and TeA in large volume of liquid sample were efficiently extracted and enriched in the stationary phase, and then eluted out just by reversing the stationary phase as mobile phase in the opposite flowing direction tail-to-head. The enrichment and elution strategies are unique and can be fulfilled online with high enrichment factors (87-114) and high recoveries (81.14-110.94%). The method has been successively applied to the determination of Alternaria mycotoxins in real apple juice and wine samples with the limits of detection (LOD) in the range of 0.03-0.14μgL(-1). Totally 12 wine samples and 15 apple juice samples from the local market were analyzed. The detection rate of AOH and AME in both kinds of the samples were more than 50%, while TeA was found in relatively high level of 1.75-49.61μgL(-1) in some of the apple juice samples. The proposed method is simple, rapid, and sensitive and could also be used for the analysis and monitoring of Alternaria mycotoxin in other food samples.