Isolation of quercetin-3-O-L-rhamnoside from Acer truncatum Bunge by high-speed counter-current chromatography

Preparative high-speed counter-current chromatography (HSCCC) was successfully used for isolation and purification of quercetin-3-O-L-rhamnoside from the ethyl acetate extract of the leaves of Acer truncatum Bunge using a two-phase-system composed of ethyl acetate-ethanol-water at a volume ratio of 5:1:5 (v/v/v). In a single operation, 41.9mg of quercetin-3-O-L-rhamnoside was obtained from 366mg of the crude extract. High-performance liquid chromatography (HPLC) analyses of the CCC fraction revealed that the purity of quercetin-3-O-L-rhamnoside was over 96%. Its structure was identified by MS, 1H NMR and 13C NMR. Quercetin-3-O-L-rhamnoside was obtained from this plant for the first time.     

Application of preparative high-speed counter-current chromatography for separation of chlorogenic acid from Flos Lonicerae

Chlorogenic acid, an ester formed between caffeic acid and quinic acid, is a major phenolic compound in the traditional Chinese medicinal herb Flos Lonicerae. The separation and purification of chlorogenic acid from the crude extract of Flos Lonicerae was achieved by high-speed counter-current chromatography (HSCCC). A high acid, highly polar two-phase solvent system containing n-butanol-acetic acid-water (4:1:5) was run on a preparative scale. The upper phase was used as the mobile phase in the head to tail elution mode. A 300-mg quantity of the crude extract containing 5.97% chlorogenic acid was loaded on a 342-ml HSCCC column. Double separations were performed with the same solvent system yielding 16.9 mg chlorogenic acid at 94.8% purity with approximately 90% recovery.     

Application of preparative high-speed counter-current chromatography for separation and purification of arctiin from Fructus Arctii

Following an initial clean-up step on the AB-8 resin (polystyrene resin, 0.3-1.25 mm: NanKai Chemical Factory, Tianjin, China), high-speed counter-current chromatography (HSCCC) was used to purify an arctiin from an extract of the fruits of the Arctium lappa L. Arctiin is a major lignan compound in the traditional Chinese medicinal herb A. lappa L. The two-phase solvent system used was composed of ethyl acetate-n-butanol-ethanol-water at an optimized volume ratio of 5:0.5:1:5 (v/v/v/v). The upper phase was used as the mobile phase in the head to tail elution mode. A total amount of 159 mg of arctiin at 98% purity was obtained from 350 mg of the crude extract (containing 49% arctiin) with 91% recovery. The preparative isolation and purification of arctiin by HSCCC was completed in 5 h in a separation. Identification of the target compound was performed by LC-electrospray ionization MS and 13C-NMR. The structure of the product was further confirmed by comparison with authentic sample (National Institute of the Control of Pharmaceutical and Biological Products, Beijing, China).     

Application of analytical and preparative high-speed counter-current chromatography for separation of lycopene from crude extract of tomato paste

Lycopene was isolated from 100 mg of crude extract of tomato paste containing about 9% of lycopene. Analytical high-speed counter-current chromatography (HSCCC) was first used for the systematic selection of the two-phase solvent system. Then preparative HSCCC separation was performed with a nonaqueous solvent system composed of n-hexane-dichloromethane-acetonitrile at an optimum volume ratio of 10:3.5:6.5. This yielded 8.6 mg of lycopene at over 98.5% purity as determined by HPLC analysis.     

Application of preparative high-speed counter-current chromatography for isolation and separation of schizandrin and gomisin A from Schisandra chinensis

Following an initial cleaning-up step on the D101 macroporous resin, a preparative high-speed counter-current chromatography (HSCCC) with a two-phase solvent system composed of n-hexane-ethyl acetate-methanol-water (1:0.9:0.9:1, v/v) was used to isolate and separate schizandrin and gomisin A from Schisandra chinensis. A total of 107 mg schizandrin and 36 mg gomisin A with purities of 99.5% and 99.1% were obtained from 400 mg crude extract in one-step elution and less than 3 h, and the structure identification was performed by UV, IR, MS, 1H NMR and 13C NMR.     

Sample capacity in preparative high-speed counter-current chromatography

High-speed counter-current chromatography (HSCCC) is a versatile technique in preparative separation and purification of pure compounds from complex matrices. As a preparative chromatography, there is a need to maximize the column production. Based on the plate theory of Van Deemter, the effect of the sample load on the separation was investigated in a preparative HSCCC with a 1000 ml column capacity. The test samples of hydroquinone, pyrocatechol and phenol were separated using a two-phase solvent system of n-hexane-ethyl acetate-ethanol-water (1:1:1:1, v/v/v/v) at different sample loads. The results showed that for the case of HSCCC, the agreement of the effect of sample load on peak height and peak width between the Van Deemter's theory and the experiments is excellent. Furthermore, the factors limiting the mass load, including the resolution between the peaks, the partition isotherm and the solute solubility were also discussed.     

Completed preparative separation of alkaloids from Cephaltaxus fortunine by step-pH-gradient high-speed counter-current chromatography

Cephalotaxine-type alkaloids are the anti-cancer components in twigs, leaves, roots and seeds of Cephalotaxus fortunine. It is very important to use the limited resource by finding an efficient purification technology of the alkaloids. Separation of cephalotaxine-type alkaloids in Cephalotaxus fortunine by step-pH-gradient high-speed counter-current chromatography (step-pH-gradient HSCCC) was studied in this paper. The step-pH-gradient HSCCC was performed on a HSCCC instrument equipped with a 400-mL column, using the upper phase of ethyl acetate–n-hexane–water, with added 0.01% trifluoroacetic acid (TFA) as stationary phase, and the lower phase of ethyl acetate–n-hexane–water, with added 2% NH₄OH, 0.2% NH₄OH and 0.05% TFA as mobile phase. For each separation, 800 mg of extract of cephalotaxine-type alkaloids was separated to yield 9.3 mg of drupacine, 15.9 mg of wilsonine, 130.4 mg of cephalotaxine, 64.8 mg of epi-wilsonine, 12.8 mg of fortunine and 35.6 mg of acetylcephalotaxine with purities 81.2%, 85.7%, 95.3%, 97.5%, 89.1% and 96.2%, respectively. The recovery of each alkaloid was more than 90%. The structures of the six alkaloids were identified by electrospray ionization mass spectrum (ESI-MS) and ¹H and ¹³C NMR.     

Application of preparative high-speed counter-current chromatography for the separation of flavonoids from the leaves of Byrsonima crassa Niedenzu (IK)

The methanolic extract of the leaves of the medicinal plant Byrsonima crassa (Malpighiaceae) contain flavonoids with antioxidant activity. They were separated in a preparative scale using high-speed counter-current chromatography. The optimum solvent system used was composed of a mixture of ethyl acetate-n-propanol-water (140:8:80 (v/v/v)) and led to a successful separation between monoglucosilated flavonoids (quercetin-3-O-alpha-L-arabinoside, quercetin-3-O-beta-D-galactoside) and the biflavonoid amentoflavone in only 3.5 h. The purities of quercetin-3-O-alpha-L-arabinoside (95 mg), quercetin-3-O-beta-D-galactoside (16 mg) and the biflavonoid amentoflavone (114 mg) were all isolated at purity over 95%. Identification was performed by 1H NMR, 13C NMR and UV analyses.     

Application of preparative high-speed counter-current chromatography for the separation of two alkaloids from the roots of Tabernaemontana catharinensis (Apocynaceae)

The methanolic extract of Tabernaemontana catharinensis (Apocynaceae) roots, which contains alkaloids with several biological activities, was separated on a preparative scale using high-speed counter-current chromatography. The optimum solvent system was found to be a mixture of CHCl(3)-MeOH-H(2)O [5:10:6 (v/v/v)] and led to a successful separation of two monoterpenic indole alkaloids, voachalotine (1) and 12-methoxy-N(b)-methylvoachalotine (2) in approximately 4.0 hours. The alkaloids were all isolated at purities over 95%, and their structures were established on the basis of spectroscopic methods, including 1D and 2D NMR and EI/MS.     

Two-dimensional counter-current chromatography for the preparative separation of prenylflavonoids from Artocarpus altilis

A two-dimensional counter-current chromatographic system (2D-CCC) for preparative isolation and purification of three prenylflavonoids from Artocarpus altilis is presented. An upright CCC instrument (CCC1, total capacity: 1600 ml) was used as the first dimension. Effluent of interest from CCC1 was collected on-line into a 30 ml sample loop by a laboratory-prepared column-switching interface and introduced into a high-speed CCC instrument (CCC2, total capacity: 210 ml) for the second dimension separation. With this 2D-CCC system and a pair of two-phase solvent systems composed of n-hexane-ethyl acetate-methanol-water (5:5:7:3 and 5:5:6.5:3.5, v/v/v/v), which had been selected by high-speed CCC, about a 500 mg amount of the crude extract was separated, yielding 9 mg of compound 1, 28 mg of compound 2 and 78 mg of compound 3. The purities of the three prenylflavonoids were 98.7 (1), 98.3 (2) and 97.2% (3), respectively, as determined by HPLC analysis. Their chemical structures were identified by electrospray ionization MS, (1)H NMR and (13)C NMR.     

Application of high-speed counter-current chromatography to the preparative separation and purification of baicalin from the Chinese medicinal plant Scutellaria baicalensis

Baicalin was separated and purified for the first time from the traditional Chinese medicinal plant Scutellaria baicalensis Georgi by high-speed counter-current chromatography. Crude baicalin was obtained by extraction with methanol-water (70:30, v/v) from S. baicalensis Georgi. The separation was performed in two steps with a two-phase solvent system composed of n-butanol-water (1:1, v/v), in which the lower phase was used as the mobile phase at a flow-rate of 1.0 ml min(-1) in the head-to-tail elution mode. A total of 37.0 mg of baicalin at 96.5% purity was yielded from 200 mg of the crude baicalin (containing 21.6% baicalin) with 86.0% recovery as determined by HPLC analysis.     

Separation of tanshinones from Salvia miltiorrhiza Bunge by high-speed counter-current chromatography using stepwise elution

High-speed counter-current chromatography (HSCCC) was successfully used for isolation and purification of tanshinones from the roots of Salvia miltiorrhiza Bunge by stepwise elution. A set of three solvent systems and other experimental conditions were determined by analytical HSCCC. Using the optimized conditions, the preparative HSCCC separation was performed on 50 mg of crude light petroleum extract yielding pure tanshinones of tanshinone HA (7 mg), tanshinone I (3 mg) and cryptotanshinone (4 mg) all at purities of over 95% in a single run.     

Rapid separation of flavonoids by analytical high-speed counter-current chromatography

A commercial model of the analytical high-speed counter-current chromatography instrument was used for separation of flavonoids from a crude ethanol extract of dried fruits of sea buckthorn (Hippophae rhamnoides). Using a two-phase solvent system of chloroform-methanol-water (4:3:2), a five-fold increase in flow-rate of the mobile phase from 60 to 300 ml/h resulted in a rapid separation of five components in less than 15 min without significant loss in peak resolution. Major flavonoid component, isorhamnetin, was identified in its pure state by mass spectrometric analysis.     

高速逆流色谱分离制备椭圆叶花锚中的2种口山酮苷元

椭圆叶花锚的主要活性成分为口山酮类化合物,这类化合物具有利胆、抗炎、抗菌及抗病毒活性。应用高速逆流色谱法建立了2种高纯度口山酮苷元的分离制备方法。对椭圆叶花锚氯仿萃取部位运用高速逆流色谱分离纯化,以正己烷-乙酸乙酯-甲醇-水(5:5:7:5, v/v/v/v)为两相溶剂系统,上相为固定相,下相为流动相。在主机转速800 r/min,流动相流速1.5 mL/min,检测波长254 nm条件下进行分离制备。所得产物经高效液相色谱分析检测,其化学结构由核磁共振氢谱(1H NMR)和核磁共振碳谱(13C NMR)鉴定。在此条件下,从100 mg粗样品中一步分离得到18 mg 1-羟基-2,3,5-三甲氧基口山酮,14 mg 1-羟基-2,3,4,5-四甲氧基口山酮。经高效液相色谱分析,其纯度均达98%以上。该方法简便、快速,所得产物纯度高,适合于椭圆叶花锚口山酮苷元的制备分离。     

Preparative separation of minor saponins from Platycodi Radix by high-speed counter-current chromatography

Platycosides (PSs), the saponins found in the root of Platycodon grandiflorum (Jacq.) A. DC. (Platycodi Radix), are typically composed of oleanene backbones with two side chains; one is a 3-O-glucose linked by a glycosidic bond, and the other is a 28-O-arabinose-rhamnose-xylose-apiose linked by an ester bond. Minor saponins, acetylated isomers of the major saponin on either the 2' or 3' position of rhamnose, were isolated from Platycodi Radix using a multi-step process including high-speed counter-current chromatography (HSCCC) and preparative reversed-phase high-performance liquid chromatography (RP-HPLC). After the separation of the major components, the enriched minor saponin fraction was used for this study. A two-phase solvent system consisting of chloroform-methanol-isopropanol-water (3:2:2:3, v/v) was used for HSCCC. HSCCC separation of the enriched minor saponin fraction yielded 2'-O-acetylplatycodin D, 3'-O-acetylpolygalacin D, 2'-O-acetylpolygalacin and a mixture of 3'-O-acetylplatycodin D and polygalacin D. The mixture fraction from HSCCC separation was further purified by preparative RP-HPLC, giving 3'-O-acetylplatycodin D and polygalacin D at a purity of over 98.9%. The developed method provides the preparative and rapid separation of minor saponins in the crude extract of Platycodi Radix. To the best of our knowledge, this is the first on the separation of acetylated PSs by HSCCC.     

Protein separation using toroidal columns by type-J synchronous counter-current chromatography towards preparative separation

Separation of large bioactive molecules such as proteins, DNAs and RNAs using aqueous two-phase systems (ATPSs) and liquid–liquid partition-based counter-current chromatography (CCC) can avoid risks of sample loss and denaturation, and greatly reduce processing time. We have constructed toroidal columns (length 26–140 m, column volume 51–280 ml, bore size 1.6 mm) suitable for mounting onto a commercially available preparative CCC apparatus. With the use of an ATPS containing 12.5% (w/w) PEG1000 and 12.5% (w/w) K₂HPO₄ and at a rotational speed of 800 rpm for the rotor of the CCC device, the lower phase (i.e. the phosphate-enriched phase) has been used as the mobile phase and a pair of proteins, myoglobin and lysozyme, as model proteins for demonstrating the separation capability of the CCC system. For a toroidal column with a length of 53.5m and a column volume of 107.5 ml, and operated for the Coriolis force parallel flow mode at 0.62 ml/min, protein sample loading (containing 2.2 mg/ml myoglobin and lysozyme, respectively) at 1.7% and 7.4% to the column volume led to peak resolution (with theoretical plate number TP and stationary phase retention S_f shown in the parenthesis) of R_s = 1.5 (N = 211 and N = 113 TP for myoglobin and lysozyme, respectively, and S_f = 45.0%), and R_s = 1.4 (218 and 152 TP, and S_f = 34.0%). However, further increase of the loading to 13% failed to separate the two proteins. Although proteins eluted at positions predictable from the distribution coefficients, they showed broader peaks when compared with small dipeptides under identical CCC operating conditions. This confirms that the molecular weight of the partitioned species is an important factor causing peak broadening on CCC chromatograms. These results paved the way for further scaling-up toroidal CCC columns for processing larger quantities of samples containing large biomolecules.     

Versatile two-phase solvent system for alkaloid separation by high-speed counter-current chromatography

In order to find a versatile high speed counter-current chromatography solvent system that can be used as a general prefractionation system for most alkaloids, the crude extracts of five Chinese traditional medicinal herbs, Cortex phellodendri, Semen strychni, green tea, Sophora flavescens ait, and Datura mete L. were resolved. All separations were performed only with a two-phase system composed of CHCl3-CH3OH-water (4:3:2). The water had different acidities controlled by adding NaH2PO4 or HCl to each sample. The fractionated components were identified by thin-layer chromatography, which confirmed this solvent system was versatile and very useful for the separation of alkaloids.     

Preparative separation of rhein from Chinese traditional herb by repeated high-speed counter-current chromatography

High-speed counter-current chromatography (HSCCC) was repeatedly used for isolation and purification of rhein from Rheum officinale Baill (Dahuang) with a two-phase solvent system composed of n-hexane-ethyl acetate-methanol-water (3:7:5:5, v/v), which had been selected by analytical (HSCCC). Using two preparative units of the HSCCC centrifuge, about a 500 mg amount of the crude extract was separated, yielding 6.7 mg of rhein at a high purity of over 97%.     

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.