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.     

Three‐phase solvent systems for the comprehensive separation of a wide variety of compounds from Dicranostigma leptopodum by high‐speed counter‐current chromatography

A three‐phase solvent system was efficiently applied for high‐speed counter‐current chromatography to separate secondary metabolites with a wide range of hydrophobicity in Dicranostigma leptopodum. The three‐phase solvent system of n‐hexane/methyl tert‐butyl ether/acetonitrile/0.5% triethylamine (2:2:3:2, v/v/v/v) was selected for high‐speed counter‐current chromatography separation. The separation was initiated by filling the column with a mixture of intermediate phase and lower phase as a stationary phase followed by elution with upper phase to separate the hydrophobic compounds. Then the mobile phase was switched to the intermediate phase to elute the moderately hydrophobic compounds, and finally the polar compounds still retained in the column were fractionated by eluting the column with the lower phase. In this research, 12 peaks were eluted out in one‐step operation within 110 min, among them, eight compounds with acceptable purity were obtained and identified. The purities of β‐sitosterol, protopine, allocryptopine, isocorydione, isocorydine, coptisine, berberrubine, and berberine were 94.7, 96.5, 97.9, 86.6, 98.9, 97.6, 95.7, and 92.8%, respectively.     

Application of accelerated solvent extraction coupled with high-performance counter-current chromatography to extraction and online isolation of chemical constituents from Hypericum perforatum L

Accelerated solvent extraction (ASE) coupled with high-performance counter-current chromatography (HPCCC) was successfully used for the extraction and online isolation of five chemical constituents from the plant Hypericum perforatum L. The upper phase of the solvent system of ethyl acetate-methanol-water (5:2:5, v:v:v) was used as both the ASE solvent and the HPCCC stationary phase. Two hydrophobic compounds including 28.4 mg of hyperforin with a HPLC purity of 97.28% and 32.7 mg of adhyperforin with a HPLC purity of 97.81% were isolated. The lower phase of ethyl acetate-methanol-n-butanol-water (5:2:2.5:12, v:v:v:v) was used as both the ASE solvent and CCC stationary phase. Three hydrophilic compounds of 12.7 mg of 3,4,5-O-tricaffeoylquinic acid with a HPLC purity of 98.82%, 15.2 mg of 1,3,5-O-tricaffeoylquinic acid with a HPLC purity of 99.46% and 42.5mg of 3-O-caffeoylquinic acid with a HPLC purity of 96.90%, were obtained in a one-step extraction-separation process with less than 3h from 10.02 g of raw material of H. perforatum. The targeted compounds isolated, collected and purified by HPCCC were analyzed by high performance liquid chromatography (HPLC), the chemical structures of all five compounds above mentioned were identified by UV, MS and NMR.     

Isolation of all-trans lycopene by high-speed counter-current chromatography using a temperature-controlled solvent system

The effect of solvent system, partition coefficient, retention of stationary phase, column, revolution speed, and flow rate of mobile phase are well known parameters to effect HSCCC (high-speed counter-current chromatography) separations. Temperature effects on chromatographic techniques like HPLC and GC are well studied, but the influence of temperature on CCC solvent systems is hardly investigated. This paper presents the influence of temperature on several key parameters (partition coefficient, settling time, volume ratios) in the hydrophobic HSCCC solvent system hexane:dichloromethane:acetonitrile (30:11:18, v/v/v) used for the isolation of lycopene from tomato paste at 10, 15, 20 and 25 degrees C.     

A Three-Phase Solvent System in High-Speed Counter-Current Chromatographic for the Separation and Purification of Bioactive Constituents from Acanthus ilicifolius

A new three-phase solvent system, n-hexane–acetonitrile–dichloromethane–water–ethyl acetate (5:5:1:5:1.5, v/v/v/v/v) was developed for the high-speed counter-current chromatographic (HSCCC) separation and purification of five bioactive constituents, syringic acid (1), vomifoliol (2), vanillic acid (3), 6-hydroxy-2-benzoxazolinone (4), and 2-benzoxazolinone (5), from Acanthus ilicifolius.

     

高速逆流色谱法分离制备母丁香和公丁香中的5种非挥发性化合物

应用高速逆流色谱法从母丁香和公丁香中快速分离了3种已知非挥发性化合物,并利用相同方法从公丁香中分离出2种色原酮类化合物。两相溶剂系统A为正己烷-乙酸乙酯-甲醇-水(5:8:6:13, v/v/v/v),系统B为正己烷-乙酸乙酯-甲醇-水(5:8:9:10, v/v/v/v),以系统A的上相为固定相,系统A和B的下相为流动相,利用梯度洗脱方式,在主机转速为880 r/min、流速1.2 mL/min条件下,成功地从70 mg母丁香粗提物中分离得到12.3 mg鞣花酸、9.6 mg鼠李素、17.2 mg槲皮素,从50 mg公丁香粗提物中分离得到5,7-二甲氧基-2-甲基色原酮10.2 mg、5,7-二甲氧基-2,6-二甲基色原酮8.6 mg,纯度均在96%以上。各化合物的结构均由质谱和核磁共振氢谱、碳谱鉴定。利用该方法可以对丁香不同药用部位中的非挥发性化合物进行有效的分离和纯化。     

Using the liquid nature of the stationary phase in countercurrent chromatography. IV. The cocurrent CCC method

The retention volumes of solutes in countercurrent chromatography (CCC) are directly proportional to their distribution coefficients, K(D) in the biphasic liquid system used as mobile and stationary phase in the CCC column. The cocurrent CCC method consists in putting the liquid "stationary" phase in slow motion in the same direction as the mobile phase. A mixture of five steroid compounds of widely differing polarities was used as a test mixture to evaluate the capabilities of the method with the biphasic liquid system made of water/methanol/ethyl acetate/heptane 6/5/6/5 (v/v) and a 53 mL CCC column of the coil planet centrifuge type. It is shown that the chromatographic resolution obtained in cocurrent CCC is very good because the solute band broadening is minimized as long as the solute is located inside the "stationary" phase. Pushing the method at its limits, it is demonstrated that the five steroids can still be (partly) separated when the flow rate of the two liquid phases is the same (2 mL/min). This is due to the higher volume of upper phase (72% of the column volume) contained inside the CCC column producing a lower linear speed compared to the aqueous lower phase linear speed. The capabilities of the cocurrent CCC method compare well with those of the gradient elution method in HPLC. Continuous detection is a problem due to the fact that two immiscible liquid phases elute from the column. It was partly solved using an evaporative light scattering detector.     

Preparation of the monomers of gingerols and 6-shogaol by flash high speed counter-current chromatography

The flash high speed counter-current chromatographic (FHSCCC) separation of gingerols and 6-shogaol was performed on a HSCCC instrument equipped with a 1200-ml column (5 mm tubing i.d.) at a flow rate of 25 ml/min. The performance met the FHSCCC feature that the flow rate of mobile phase (ml) is equal to or greater than the square of the diameter of the column tubing (mm). The separation employed the upper phase of stationary phase of the n-hexane-ethyl acetate-methanol-water (3:2:2:3, v/v) as the stationary phase. A stepwise elution was performed by eluting with the lower phase of n-hexane-ethyl acetate-methanol-water (3:2:2:3, v/v) for first 90 min and the lower phase of the n-hexane-ethyl acetate-methanol-water (3:2:6:5, v/v) for the second 90 min. In each separation 5 g of the ethyl acetate extract of rhizomes of ginger was loaded, yielding 1.96 g of 6-gingerol (98.3%), 0.33 g of 8-gingerol (97.8%), 0.64 g of 6-shogaol (98.8%) and 0.57 g of 10-gingerol (98.2%). The separation can be expected to scale up to industrial separation.     

Application of supercritical fluid extraction coupled with counter–current chromatography for extraction and online isolation of unstable chemical components from Rosa damascena

Supercritical fluid extraction (SFE) coupled with high‐speed counter‐current chromatography (HSCCC) was successfully used for the extraction and online isolation of the unstable compounds from Rosa damascene in a single extraction and separation operation in two stages. The solvent systems of SFE/HSCCC were optimized with the help of multiexponential function model. At the first stage, the upper phase of the solvent system of n‐butanol–tert‐butyl methyl ether–acetonitrile–0.1% aqueous TFA (1.7:1.0:0.8:4.0, v/v/v/v) was used as both the SFE entrainer and the HSCCC stationary phase, and the target compounds were eluted with the corresponding lower phase to separate the hydrophobic compounds. At the second stage, the upper phase of the solvent system of n‐hexane–ethyl acetate–methanol–water (3.2:1.0:2.8:2.6, v/v/v/v) was used as both the SFE entrainer and the HSCCC stationary phase, followed by elution with the corresponding lower phase to separate the moderate hydrophobic compounds. Six compounds including formononetin, delphinidin, cyaniding, 5,6,4′‐trihydroxy‐7,8‐dimethoxy flavone, 5,3′‐dihydroxy‐7,8‐dimethoxy flavone, and 5‐hydroxy‐6,7,8,3′,4′‐pentamethoxy flavone were successfully separated in one extraction–separation operation within 300 min. The targeted compounds were identified by MS and NMR spectroscopy. This research has opened up great prospects for industrial application of SFE/HSCCC to the extraction and separation of unstable compounds.     

Rational development of a selection model for solvent gradients in single-step separation of ginsenosides from Panax ginseng using high-speed counter-current chromatography

A new model of solvent gradients selection was rationally developed for the preparative separation of target compounds. The solvent gradients were selected based on a three-stage screening process where stationary phase retention was ensured by introducing a new parameter termed as the phase ratio. The phase ratio was calculated after mixing the upper phase of a solvent system with the lower phase of a different solvent system (1:1, v/v). The developed model was applied to the one-step separation of eight ginsenosides from Panax ginseng. Three gradients were selected on the basis of new model and eight ginsenosides, Rb(1), Rb(2), Rc, Rd, Re, Rg(1), Rf, and Rh(1), were efficiently separated by high-speed counter-current chromatography coupled with evaporative light scattering detector. The structures of all compounds were characterized by electrospray-ionization mass spectrometry and nuclear magnetic resonance spectroscopy.     

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.     

Combination of supercritical fluid extraction with counter‐current chromatography to isolate anthocyanidins from the petals of Chaenomeles sinensis based on mathematical calculations

Supercritical fluid extraction (SFE) coupled with high‐speed counter‐current chromatography (HSCCC) was successfully used for the extraction and on‐line isolation of the anthocyanidins from the petals of Chaenomeles sinensis in two stages. The SFE parameters were optimized by an orthogonal test, and the solvent systems of SFE and HSCCC were calculated and optimized with the help of a multiexponential function model. In the first stage, the lower phase of the solvent system of n‐butanol/tert‐butyl methyl ether/acetonitrile/0.1% aqueous TFA (0.715:1.0:0.134:1.592, v/v/v/v) was used as both the SFE modifier and the HSCCC stationary phase, after extraction, the extractants were pumped into HSCCC column, and then eluted with the corresponding upper phase to isolate the moderately hydrophobic compounds. In the second stage, the upper phase of the solvent system of n‐butanol/ethyl acetate/acetonitrile/0.1% aqueous TFA (1.348:1.0:0.605:2.156, v/v/v/v) was used as both the SFE modifier and the HSCCC stationary phase, followed by elution with the corresponding lower phase to separate the hydrophobic compounds. With the help of two‐stage SFE/HSCCC, six compounds including delphinidin‐3‐O‐glucoside (Dp3G), cyanidin‐3‐O‐glucoside (Cy3G), peonidin‐3‐O‐glucoside (Pn3G), delphinidin (Dp), peonidin (Pn), and malvidin (Mv) were successfully separated within 300 min. The targeted compounds were identified by UV spectrophotometry, MS, and NMR spectroscopy. This research has opened up great prospects for the industrial application of SFE–HSCCC for the automatic extraction and separation of unstable compounds.     

高速逆流色谱法分离制备中药诃子中的没食子酸

利用高速逆流色谱法从100 mg诃子醇提物中一次性分离制备得到8.6 mg没食子酸。通过分析型高速逆流色谱对5种溶剂系统进行筛选,确定以正己烷-乙酸乙酯-甲醇-水(体积比为1:5:1:5)为两相溶剂体系并放大到制备型上,以上相为固定相,下相为流动相,在主机转速850 r/min、流动相流速2 mL/min、检测波长254 nm的条件下进行分离制备,获得4个分离峰(组分Ⅰ、Ⅱ、Ⅲ、Ⅳ)。经高效液相色谱检测,按照面积归一法计算,其中组分Ⅲ的纯度达96.40%。经电喷雾电离质谱分析,并结合与没食子酸标准品的高效液相色谱测定结果的对比,确定组分Ⅲ为没食子酸。该方法简便、快速、重复性好,适合于诃子中没食子酸的分离制备。     

Studies on the effect of column angle in centrifugal helix counter-current chromatography

The performance of the coiled column of centrifugal counter-current chromatography was investigated by changing the angle between column axis and centrifugal force in the separation of dipeptides or DNP-amino acids each with suitable two-phase solvent systems. In general, retention of the stationary phase (Sf) decreased, and peak resolution (Rs) increased as the column angle was increased. The first series of experiments was performed using a polar two-phase solvent system composed of 1-butanol–acetic acid–water (4:1:5, v/v/v) to separate two dipeptide samples, Trp-Tyr and Val-Tyr, at a flow rate of 1 ml/min at 1000 rpm. When the column angle was changed from 0° to 90°, Rs increased from 1.05 (Sf = 60.1%) to 1.17 (Sf = 38.7%) with the lower phase mobile and from 1.02 (Sf = 67.8%) to 1.14 (Sf = 47.4%) with the upper phase mobile, respectively. The second series of experiments was similarly performed with a more hydrophobic two-phase solvent system composed of hexane–ethyl acetate–methanol–0.1 M hydrochloric acid (1:1:1:1, v/v/v/v) to separate three DNP-amino acids, DNP-glu, DNP-β-ala and DNP-ala, at a flow rate of 1 ml/min at 1000 rpm. When the column angle was changed from 0° to 90°, Rs increased from 1.38 (1st peak/2nd peak) and 1.20 (2nd peak/3rd peak) (Sf = 61.1%) to 1.66 and 1.45 (Sf = 34.4%) with the lower phase mobile and from 1.14 and 0.63 (Sf = 72.2%) to 1.53 and 0.87 (Sf = 51.1%) with the upper phase mobile, respectively. The overall results of our studies indicate that increasing the column angle against the radially acting centrifugal force enhances the mixing of two phases in the column to improve the peak while decreasing the stationary phase retention by interrupting the laminar flow of the mobile phase.     

A Simple and Rapid Reversed Phase High Performance Liquid Chromatographic Method for Quantification of Alprazolam and α-Hydroxyalprazolam in Plasma

Abstract

A simple and rapid reversed-phase liquid chromatographic method for the determination of alprazolam and a-hydroxyalprazolam in plasma is described. Flunictrazepam was used as internal standard. Plasma samples were buffered with sodium borate and extracted with dichloromethane /n-pentane 4:6 v/v for 60 sec on a vortex apparatus. Extraction solvent was evaporated to dryness and extraction residues were reconstituted in the mobile phase. Samples were chromatographed on a 5μ Lichrospher RP-18 column (25cm × 4mm i. d) using acetonitrile/water 40:60 v/v as the mobile phase. The column effluent was monitored at 230nm. The lower limit of detection was 1ng/ml for alprazolam and a-hydroxyalprazolam while the lower limit of quantification was 2ng/ml for both compounds. Peak height and plasma     

Comprehensive separation of secondary metabolites in natural products by high-speed counter-current chromatography using a three-phase solvent system

High-speed counter-current chromatography (HSCCC) using the three-phase solvent system n-hexane-methyl acetate-acetonitrile-water at a volume ratio of 4:4:3:4 was applied to the comprehensive separation of secondary metabolites in several natural product extracts. A wide variety of secondary metabolites in each natural product was effectively extracted with the three-phase solvent system, and the filtered extract was directly submitted to the HSCCC separation using the same three-phase system. In the HSCCC profiles of crude natural drugs listed in the Japanese Pharmacopoeia, several physiologically active compounds were clearly separated from other components in the extracts. The HSCCC profiles of several tea products, each manufactured by a different process, clearly showed their compositional difference in main compounds such as catechins, caffeine, and pigments. These HSCCC profiles also provide useful information about hydrophobic diversity of whole components present in each natural product.     

Performance comparison of three types of high-speed counter-current chromatographs for the separation of components of hydrophilic and hydrophobic color additives

The performance of three types of high-speed counter-current chromatography (HSCCC) instruments was assessed for their use in separating components in hydrophilic and hydrophobic dye mixtures. The HSCCC instruments compared were: (i) a J-type coil planet centrifuge (CPC) system with a conventional multilayer-coil column, (ii) a J-type CPC system with a spiral-tube assembly-coil column, and (iii) a cross-axis CPC system with a multilayer-coil column. The hydrophilic dye mixture consisted of a sample of FD&C Blue No. 2 that contained mainly two isomeric components, 5,5'- and 5,7'-disulfonated indigo, in the ratio of ～7:1. The hydrophobic dye mixture consisted of a sample of D&C Red No. 17 (mainly Sudan III) and Sudan II in the ratio of ～4:1. The two-phase solvent systems used for these separations were 1-butanol/1.3M HCl and hexane/acetonitrile. Each of the three instruments was used in two experiments for the hydrophilic dye mixture and two for the hydrophobic dye mixture, for a total of 12 experiments. In one set of experiments, the lower phase was used as the mobile phase, and in the second set of experiments, the upper phase was used as the mobile phase. The results suggest that: (a) use of a J-type instrument with either a multilayer-coil column or a spiral-tube assembly column, applying the lower phase as the mobile phase, is preferable for separating the hydrophilic components of FD&C Blue No. 2; and (b) use of a J-type instrument with multilayer-coil column, while applying either the upper phase or the lower phase as the mobile phase, is preferable for separating the hydrophobic dye mixture of D&C Red No. 17 and Sudan II.     

高速逆流色谱分离纯化防风中升麻素苷和5-O-甲基维斯阿米醇苷

建立了高速逆流色谱分离制备防风中有效成分升麻素苷和5-O-甲基维斯阿米醇苷的方法.防风根的粉末经甲醇浸泡提取和减压蒸馏,得粗提浸膏.以V(乙酸乙酯):V(正丁醇):V(水)=2:7:9为溶剂,上相为固定相,下相为流动相,流速2.0 mL/min.从316 mg防风粗提物中一步分离得到13.9 mg升麻素苷和25 mg 5-O-甲基维斯阿米醇苷,纯度分别为98.1%和99 2%.采用ESI-MS, 1H NMR 和13C NMR对目标化合物的结构进行了鉴定.     

高速逆流色谱法分离纯化黄芪中的芒柄花素和毛蕊异黄酮

采用高速逆流色谱法(HSCCC),以正己烷-氯仿-甲醇-水组成二相系统作为固定相与流动相,对黄芪的乙酸乙酯粗提 物进行了分离纯化。 结果发现:以正己烷-氯仿-甲醇-水(体积比为1.5∶3∶3∶2)组成的系统可以从黄芪的乙酸乙酯粗 提物中分离出毛蕊异黄酮,纯度可达95％以上,并可以初步纯化芒柄花素;接着用正己烷-氯仿-甲醇-水(体积比为4∶4∶5 ∶4)组成的系统进一步纯化芒柄花素,其纯度达95％以上。利用该方法,可以对中药黄芪中的异黄酮进行快速的分离和纯 化。     

Studies on the effect of column angle in figure-8 centrifugal counter-current chromatography

The performance of the figure-8 column configuration in centrifugal counter-current chromatography was investigated by changing the angle between the column axis (a line through the central post and the peripheral post on which the figure-8 coil is wound) and the centrifugal force. The first series of experiments was performed using a polar two-phase solvent system composed of 1-butanol-acetic acid-water (4:1:5, v/v) to separate two dipeptide samples, Trp-Tyr and Val-Tyr, at a flow rate of 0.05 ml/min at 1000 rpm. When the column angle was changed from 0° (column axis parallel to the centrifugal force) to 45° and 45° to 90° (column axis perpendicular to the centrifugal force), peak resolution (Rs) changed from 1.93 (Sf=37.8%) to 1.54 (Sf=30.6%), then to 1.31 (Sf=40.5%) with the lower mobile phase and from 1.21 (Sf=38.8%) to 1.10 (Sf=34.4%), then to 0.99 (Sf=42.2%) with the upper mobile phase, respectively, where the stationary phase retention, Sf, is given in parentheses. The second series of experiments was similarly performed with a more hydrophobic two-phase solvent system composed of hexane-ethyl acetate-methanol-0.1M hydrochloric acid (1:1:1:1, v/v) to separate three DNP-amino acids, DNP-glu, DNP-β-ala and DNP-ala, at a flow rate of 0.05 ml/min at 1000 rpm. When the column angle was altered from 0° to 45° and 45° to 90°, Rs changed from 1.77 (1st peak/2nd peak) and 1.52 (2nd peak/3rd peak) (Sf=27.3%) to 1.24 and 1.02 (Sf=35.4%), then to 1.69 and 1.49 (Sf=42.1%) with the lower mobile phase, and from 1.73 and 0.84 (SF=41.2%) to 1.44 and 0.73 (Sf=45.6%), then to 1.21 and 0.63 (Sf=55.6%) with the upper mobile phase, respectively. The performance of figure-8 column at 0° and 90° was also compared at different flow rates. The results show that Rs was increased with decreased flow rate yielding the highest value at the 0° column angle with lower mobile phase. The overall results of our studies indicated that a 0° column angle for the figure-8 column enhances the mixing of two phases in the column to improve peak resolution while decreasing the stationary phase retention by interrupting the laminar flow of the mobile phase.