Improved cross-axis synchronous flow-through coil planet centrifuge for performing counter-current chromatography. II. Studies on retention of stationary phase in short coils and preparative separations in multilayer coils

Performance of the apparatus was evaluated in terms of stationary phase retention, partition efficiency and sample loading capacity. Preliminary studies with short coils revealed high retention of the stationary phase under a proper combination of the head-tail elution and planetary motion. Preparative capability of the apparatus was successfully demonstrated on efficient multigram separations of 2,4-dinitrophenyl amino acids, indole auxins, and bacitracin in a pair of large multilayer coils with a total capacity of 1.5 l.     

pH-gradient counter-current chromatography isolation of natural antioxidant chlorogenic acid from Lonicera japonica Thumb. using an upright coil planet centrifuge with three multi-layer coils connected in series

A new pH-gradient counter-current chromatography method for the isolation of chlorogenic acid from flowers and buds of Lonicera japonica Thumb. has been successfully established using a novel upright coil planet centrifuge with three multi-layer coils connected in series with 600 mL capacity. The crude extracts were first prepared by direct extraction with hot water and following concentration to remove the solution. Then the two-phase solvent system composed of ethyl acetate-n-butanol-water (2:1:3, v/v) was applied to the separation. Its neutral upper phase was used as stationary phase, whereas both its neutral lower phase and base lower phase with 10mM NH(3) were employed as mobile phase with gradient elution in the head to tail mode. As a result, 330 mg quantity of crude extract was purified in one-step separation for 180 min, yielding 20.5mg chlorogenic acid with over 98% purity. Structure of the compound is further identified by electrospray ionization tandem mass spectrometry (ESI-MS/MS) and nuclear magnetic resonance (NMR).     

Preparative counter-current chromatography isolation of liensinine and its analogues from embryo of the seed of Nelumbo nucifera GAERTN. using upright coil planet centrifuge with four multilayer coils connected in series

Preparative counter-current chromatography (CCC) isolation of liensinine and its analogues, isoliensinine and neferine from embryo of the seed of Nelumbo nucifera GAERTN. has been successfully performed for the first time using upright coil planet centrifuge with four multilayer coils connected in series with 1600 mL capacity. Two kinds of two-phase solvent systems were applied to preparative CCC isolation. The first was the system composed of light petroleum (b.p. 60-90 degrees C)-ethyl acetate-tetrachloromethane-chloroform-methanol-water (1:1:4:4:6:2, v/v) which was very suitable for fast and small-scale CCC isolation. The second was the system composed of ethyl acetate-tetrachloromethane-methanol-water (1:6:4:1, v/v), which was the optimum for large-scale CCC isolation. Using the first system, 1102 mg of the crude alkaloid was purified in one-step separation of 150 min, yielding 350 mg neferine, 100 mg isoliensinine and 95 mg liensinine with over 95% purity. While using the second solvent system, 5850 mg of the crude alkaloid was purified in one-step separation of 9 h, yielding 2545 mg neferine, 698 mg isoliensinine and 650 mg liensinine with over 97% purity. Structures of the compounds were identified by electrospray ionization multiple mass spectrometry, one- and two-dimensional NMR.     

高速逆流色谱与硅胶柱层析联用分离岩黄连中的2种原小檗碱型季胺生物碱

建立了分离岩黄连2种生物碱的方法。岩黄连醇提物经溶剂萃取后的水相提取物,经过硅胶柱层析粗分离,对其中一个流分采用高速逆流色谱法,以正己烷-乙酸乙酯-正丁醇-乙醇-水-氨水(体积比为0.4:1.5:4:0.4:5:0.11)为两相溶剂系统,下相为固定相,上相为流动相,通过一次高速逆流色谱,即可从64.81 mg样品中分离得到9.28 mg纯度为97.0%的去氢碎叶紫堇碱(dehydrocheilanthifoline)和4.38 mg纯度为90.7%的脱氢异阿朴卡维汀(dehydroisoapocavidine),并通过与对照品HPLC保留时间的比较,以及ESI-MS测定分子量,确认了化合物的结构。该方法降低了反复的柱层析导致的样品损失,也为上述两种化合物的药理研究提供了物质基础。     

Separation of five flavone glycosides including two groups with similar polarities from Dracocephalum tanguticum by a combination of three high-speed counter-current chromatography modes

The separation of compounds with similar polarities is challenging. In the present study, five flavone glycosides, including two groups with similar polarities, were obtained from Dracocephalum tanguticum by three high-speed counter-current chromatography modes, including flow rate conversion mode, recycling mode, and heart-cut mode. With flow rate conversion mode, compounds 3 and 4 with similar polarities and compound 5 were separated by high-speed counter-current chromatography with ethyl acetate/methanol/water (5.0% acetic acid) (8:2:10, v/v) system. The flow rate was controlled as: 1.8 mL/min for 0–160 min, 2.2 mL/min for 160–200 min, and 2.5 mL/min for 200–400 min. However, compounds 1 and 2 with similar polarities were not separated due to the similar distributive properties. Then, a recycling and heart-cut mode were introduced to improve the separation efficiency. The heart-cut mode was introduced in the second and third cycles, and compounds 1 and 2 were well separated in the fourth cycle. Consequently, five flavone glycosides, including two groups with similar polarities were obtained and identified as cosmosiin (1), pedaliin (2), quercetin-3-O-rutinoside (3), pedaliin-6''-acetate (4), and sorbifolin-6-O-β-glucopyranoside (5). The current strategy provides a reference for separating compounds with similar polarities from a crude sample.     

Separation of polar antioxidants from Rhizoma Polygonatum Odorati by high-speed counter-current chromatography with a hydrophilic solvent system

Some highly polar compounds are quality control makers for medicinal herbs. However, investigation of them has been hampered because the existing fractionation steps are difficult and laborious to purify them. Similar situations happen to Rhizoma Polygonatum Odorati, a widely used food supplement and medicinal herb with strong antioxidant activity, and up to date, only ethyl acetate fraction of Rhizoma Polygonatum Odorati has been comprehensively investigated. Here, HSCCC using a hydrophilic solvent system composed of n-butanol–ethanol–2 M ammonium sulfate (1:1:2, v/v/v) was performed to isolate highly polar antioxidants in n–butanol fraction of Rhizoma Polygonatum Odorati, guided by DPPH–HPLC experiment. Afterward, Sephadex LH-20 column chromatography eluted by methanol was selected to eliminate ammonium sulfate and purify co-eluted compounds in HSCCC collected fractions. Finally, nine compounds, including four nucleosides, cytidine (1), uridine (4), guanosine (5), and adenosine (8); two nucleobases, guanine (3), and adenine (6); and three amino acids, tyrosine (2), phenylalanine (7), and tryptophan (9) with purities over 98% were achieved and identified by UV, MS, and ¹H NMR data. Notably, compounds 1–9 were first reported in genus Polygonatum. The results indicated that the proposed method was an efficient approach to isolate and purify highly polar compounds from complex extracts.     

Isolation of three glucaric acids from Leonurus japonicus Houtt. by using high-speed countercurrent chromatography combined with semi-preparative high-performance liquid chromatography

The isomerism of glucaric acids and the complexity of the composition of Leonurus japonicus Houtt. increased the difficulty of the separation of glucaric acids from the herb. In the present study, three glucaric acids were isolated from Leonurus japonicus Houtt. by using high-speed countercurrent chromatography combined with semi-preparative high-performance liquid chromatography. Cation exchange resin chromatography was applied to remove the alkaloids and enrich the glucaric acid fractions. Preliminary separation of the glucaric acid extract by high-speed countercurrent chromatography was carried out at 45℃ by using an optimized solvent system of ethyl acetate/n-butanol/formic acid/water (1:1:0.01:2, v/v/v/v) with satisfied stationary phase retention and separation factor. The semi-preparative high-performance liquid chromatography was used for further separation and purification of the target fractions, and three monomeric compounds were obtained with purities of 90.0, 91.0, and 95.3%. UV spectroscopy, NMR spectroscopy, and mass spectrometry were employed to identify their structures, which were assigned as 2-syringyl glucaric acid, 2,4-disyringyl glucaric acid, and 3,4-disyringyl glucaric acid, respectively, and 2,4-disyringyl glucaric acid was reported for the first time.     

Separation of mixtures of solvents with wide bore capillary columns connected in series

The gas chromatographic separation of the components of a complex mixture of industrial solvents, not possible on a single stationary phase owing to coelution of some of the compounds, has been achieved on two serially connected wide bore capillary columns of different polarity. The analysis of a mixture of twenty five compounds was optimized using the serial gas chromatography theory of Purnell and Williams. The capacity factors of sample components measured with the two columns coupled in series were found to agree with those predicted by theory, regardless of the order in which they were connected.     

Separation of WAP-8294A components, a novel anti-methicillin-resistant staphylococcus aureus antibiotic, using high-speed counter-current chromatography.

The WAP-8294A complex was isolated from the fermentation broth of Lysobacter sp. WAP-8294, whose major component, WAP-8294A2, showed a strong activity against Gram-positive bacteria including methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant Enterococci in vitro, and also exhibited a potent activity against MRSA in vivo. The previous separation procedure using the conventional chromatographic methods was laborious and time-consuming, and the recovery of the desired compound was often unsatisfactory. In the present study, high-speed counter-current chromatography (HSCCC) was applied to the separation of the main components of the WAP-8294A complex. Due to the high polarity of the target compounds, we selected a hydrophilic two-phase solvent system composed of n-butanol-ethyl acetate-aqueous 0.005 M trifluoroacetic acid (1.25:3.75:5, v/v/v) which provided a suitable range of partition coefficient values for these compounds. Although the settling time of this solvent system was much longer than the optimum range, suggesting a low retention of the stationary phase under the standard experimental conditions, the separation was successfully performed at the low flow-rate of 0.5 ml/min. A sample size of 25 mg yielded pure fractions of three components (1-6 mg). The identification of each component was carried out by HPLC and fast atom bombardment mass spectrometry. The method will contribute to the clinical development of WAP-8294A2 as an anti-MRSA agent.     

Scaling up of high-speed countercurrent chromatographic apparatus with three columns connected in series for rapid preparation of (−)-epicatechin

In the present study, compact high-speed countercurrent chromatographic apparatus was constructed with three columns connected in series. Two sets of columns were prepared from 10 mm and 12 mm I.D. tubing to form 12 L and 15 L capacities, respectively. Performance of these columns was compared for the separation of (−)-epicatechin gallate (ECG) from a tea extract by flash countercurrent chromatography (FCCC). In each separation, 200 g of the tea extract in 1600 mL of mobile phase was loaded onto the column. The 12 L column gave 7.5 L (35 g of ECG) and the 15 L column gave 9 L (40 g of ECG) of ECG solution without impurities. The ECG solution was directly hydrolyzed by tannase into (−)-epicatechin. The hydrolysate was purified by flash chromatography on AB-8 macroporous resin to give 52 g of EC (purity 99.1%). This scaled up apparatus could be used for the industrial separation of natural products.     

Separation and preconcentration of trace manganese from various samples with Amberlyst 36 column and determination by flame atomic absorption spectrometry

This work assesses the potential of a new adsorptive material, Amberlyst 36, for the separation and preconcentration of trace manganese(II) from various media. It is based on the sorption of manganese(II) ions onto a column filled with Amberlyst 36 cation exchange resin, followed by the elution with 5 mL of 3 mol/L nitric acid and determination by flame atomic absorption spectrometry (FAAS) without interference of the matrix. Different factors including pH of sample solution, sample volume, amount of resin, flow rate of sample solution, volume and concentration of eluent, and matrix effects for preconcentration were investigated. Good relative standard deviation (3%) and high recovery (>95%) at 100 μg/L and high enrichment factor (200) and low analytical detection limit (0.245 μg/L) were obtained. The adsorption equilibrium was described well by the Langmuir isotherm model with maximum adsorption capacity of 88 mg/g of manganese on the resin. The method was applied for the manganese determination by FAAS in tap water, commercial natural drinking water, commercial treated drinking water and commercial tea bag sample. The accuracy of the method is confirmed by analyzing the certified reference material (tea leaves GBW 07605). The results demonstrated good agreement with the certified values.     

Preparative isolation and purification of flavone compounds from sophora japonica L. by high-speed counter-current chromatography combined with macroporous resin column separation

High-speed counter-current chromatography combined with macroporous resin column separation was applied to the isolation and purification of genistein-7,4'-di-O-beta-D-glucoside (I), genistein-7-O-beta-D-glucopyranoside-4'-O-[(alpha-L-rhamnopyransoyl)-(1-2)-beta-D-glucopyranoside] (II), kaempferol-3-O-beta-D-sophoroside(III), quercetin-3-O-beta-L-ramnopyranosyl-(1 - 6)-beta-D-glucopyranoside (IV), genistein-4'-beta-L-rhamnopyransoyl-(1 - 2)-alpha-D-glucopyranoside (V), and kaempferol-3-O-beta-L-ramnopyranosyl-(1 - 6)-beta-D-glucopyranoside (VI) from the Chinese medicinal herb Sophora japonica L. The crude extracts from the pericarps of Sophora japonica L. were pre-separated on a D-101 macroporous resin column and divided into two parts as sample 1 and sample 2. An 80-mg portion of sample 1 was separated by using n-butanol-acetic acid (1%) (5:5, v/v) as the two-phase solvent system and yielded 30.1 mg of compound I, 23.3 mg of compound II. A 120 mg portion of sample 2 was separated by using ethyl acetate-n-butanol-acetic acid (1%) (5:0.8:5, v/v) as the two-phase solvent system and yielded 5.5 mg of compound III, 31.7 mg of compound IV, 37.4 mg of compound V, and 6.2 mg of compound VI. The purities of compounds I, II, III, IV, V, and VI were 98.7, 98.2, 97.8, 98.5, 99.3, and 98.9%, respectively, as determined by HPLC. The chemical structures of these components were identified by 1H-NMR and 13C-NMR.     

Separation and purification of furanocoumarins from Toddalia asiatica (L.) Lam. using microwave-assisted extraction coupled with high-speed counter-current chromatography

A method of microwave-assisted extraction coupled with high-speed counter-current chromatography was established for separation and purification of isopimpinellin, pimpinellin and phellopterin from Toddalia asiatica (L.) Lam. The conditions of MAE including the extraction solvent, size of sample, solid/liquid ratio, extraction temperature and extraction time were optimized by a mono-factor test. That is, 2.0 g dried powder of T. asiatica (L.) Lam of 0.30-0.15 mm size was extracted with 20 mL (solid/liquid ratio of 1:10, g/mL) methanol under 50 °C for 1 min. The crude extract was separated and purified by high-speed counter-current chromatography with hexane-ethyl acetate-methanol-water (5:5:5.5:4.5, v/v/v/v) solvent system. 0.85 mg/g of isopimpinellin, 2.55 mg/g of pimpinellin and 0.95 mg/g of phellopterin were obtained from original sample in one-step within 240 min, the purity determined by high performance liquid chromatography was 95.0%, 99.1% and 96.4%, respectively. Their chemical structures were further identified by mass spectroscopy and nuclear magnetic resonance spectroscopy. The results demonstrated that microwave-assisted extraction coupled with high-speed counter-current chromatography was a feasible, economical and efficient technique for rapid extraction, separation and purification of effective compounds from natural products.     

Separation of radioactive metabolites in cultured tea cells fed with [14C]phenylalanine using high-speed counter-current chromatography

Separation of radioactive metabolites in cultured tea cells fed with [14C]phenylalanine was conducted using high-speed counter-current chromatography. Among seven components obtained our studies focused two metabolites, i.e. (-)-epicatechin and D,L-catechin. The specific radioactivity of (-)-epicatechin was 212.01 KBq/mg, amounting to 8.5% of the total radioactivity of ethyl acetate extract while that of D,L-catechin was 1.0006 MBq/mg or 5.4% of the total.     

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.     

Selective recognition of Triamterene in biological samples by molecularly imprinted monolithic column with a pseudo template employed

Melamine (MAM) was employed as a pseudo template to prepare a molecularly imprinted polymer monolithic column which presents the ability of selective recognition to Triamterene (TAT), whose structure was similar to that of MAM. Methacrylic acid and ethylene glycol dimethacrylate were applied as functional monomer and cross‐linker, respectively, during the in situ polymerization process. Chromatographic behaviors were evaluated, the results indicated that the molecularly imprinted polymer monolithic column possessed excellent affinity and selectivity for TAT, and the imprinting factor was high up to 3.99 when 7:3 of ACN/water v/v was used as mobile phase. In addition, the dissociation constant and the binding sites were also determined by frontal chromatography as 134.31 μmol/L and 132.28 μmol/g, respectively, which demonstrated that the obtained molecularly imprinted polymer monolith had a high binding capacity and strong affinity ability to TAT. Furthermore, biological samples could be directly injected into the column and TAT was enriched with the optimized mobile phase. These assays gave recovery values higher than 91.60% with RSD values that were always less than 3.5%. The molecularly imprinted monolithic column greatly simplified experiment procedure and can be applied to preconcentration, purification, and analysis of TAT in biological samples.     

Introduction of large volumes of water-containing samples into a gas chromatograph. Improved retention of volatile solutes through the swing system

The swing system is designed for introducing large volumes of water-containing samples into a gas chromatograph. Sample evaporation and solvent-solute separation are performed in separate compartments. This widens the application range to compounds of higher volatility. Sample evaporation takes place in a hot chamber packed with Carbofrit. Solvent-solute separation is performed in a cascade of increasing powers of retention. While high boiling solutes are retained in an oven-thermostatted retaining precolumn, the more volatile components are retained by a packed bed of sorbents of increasing powers of retention situated in a programmed temperature vaporiser. For elution, the gas flow is reversed and the solutes are discharged from the heated packed bed through the retaining precolumn into the separation column.     

Fast and quantitative analysis of branched-chain amino acids in biological samples using a pillar array column

In this study, a fast and quantitative determination method for branched-chain amino acids (BCAAs), namely leucine, isoleucine, and valine, was developed using a pillar array column. A pillar array column with low-dispersion turns was fabricated on a 20?×?20-mm² microchip using multistep ultraviolet photolithography and deep reactive ion etching. The BCAAs were fluorescently labeled with 4-fluoro-7-nitro-2,1,3-benzoxadiazole (NBD-F), followed by reversed-phase separation on the pillar array column. The NBD derivatives of the three BCAAs and an internal standard (6-aminocaproic acid) were separated in 100 s. The calibration curves for the NBD-BCAAs had good linearity in the range of 0.4–20 μM, using an internal standard. The intra- and interday precisions were found to be in the ranges of 1.42–3.80 and 2.74–6.97 %, respectively. The accuracies for the NBD-BCAA were from 90.2 to 99.1 %. The method was used for the analysis of sports drink and human plasma samples. The concentrations of BCAAs determined by the developed method showed good agreements with those determined using a conventional high-performance liquid chromatography system. As BCAAs are important biomarkers of some diseases, these results showed that the developed method could be a potential diagnostic tool in clinical research.