Mathematical model of computer-programmed intermittent dual countercurrent chromatography applied to hydrostatic and hydrodynamic equilibrium systems

Dual high-speed countercurrent chromatography (dual CCC) literally permits countercurrent flow of two immiscible solvent phases continuously through the coiled column for separation of solutes according to their partition coefficients. Application of this technique has been successfully demonstrated by separation of analytes by gas–liquid and liquid–liquid two-phase systems. However, the method cannot be directly applied to the system with a set of coiled columns connected in series, since the countercurrent process is interrupted at the junction between the columns. However, this problem can be solved by intermittent dual CCC by eluting each phase alternately through the opposite ends of the separation column. This mode of application has an advantage over the conventional dual CCC in that the method can be applied to all types of CCC systems including hydrostatic equilibrium systems such as toroidal coil CCC and centrifugal partition chromatography. Recently, the application of this method to high-speed CCC (hydrodynamic system) has been demonstrated for separation of natural products by Hewitson et al. using a set of conventional multilayer coil separation columns connected in series. Here, we have developed a mathematical model for this intermittent dual CCC system to predict retention time of the analytes, and using a simplified model system the validity of the model is justified by a series of basic studies on both hydrodynamic and hydrostatic CCC systems with a computer-programmed single sliding valve. The present method has been successfully applied to spiral tube assembly high-speed CCC (hydrodynamic system) and toroidal coil CCC (hydrostatic system) for separation of DNP-amino acid samples with two biphasic solvent systems composed of hexane–ethyl acetate–methanol–0.1 M hydrochloric acid (1:1:1:1 and 4:5:4:5, v/v).     

Allan J. Bard and Cynthia G. Zoski (Eds): Electroanalytical Chemistry

Coupled with evaporative light scattering detection, a high-speed counter-current chromatography (HSCCC) method was applied to the separation and purification of three tauro-conjugated cholic acids of taurochenodeoxycholic acid (TCDCA), taurohyodeoxycholic acid (THDCA) and taurohyocholic acid (THCA) from Pulvis Fellis Suis (Pig gallbladder bile) for the first time. The two-phase solvent system composed of chloroform-methanol-water-acetic acid (4:4:2:0.3, v/v/v/v) was selected for the one-step separation where the lower phase was used as the mobile phase in the head to tail elution mode. The revolution speed of the separation column, flow rate of the mobile phase and separation temperature were 800 rpm, 1.5 ml/min and 25°C respectively. From 100 mg of the crude extract, 10.2 mg of TCDCA, 11.8 mg of THDCA and 5.3 mg of THCA were obtained with the purity of 94.6%, 96.5% and 95.4%, respectively. in one step separation The HSCCC fractions were analyzed by high-performance liquid chromatography (HPLC) and the structures of the three tauro-conjugated cholic acids were identified by ESI-MS, (1)H NMR and (13)C NMR.     

High-Speed Countercurrent Chromatography of Dipeptides on a Polar Two-Phase Solvent System

Abstract

The performance of the high-speed countercurrent chomatograph was evaluated by separating dipeptide samples on a polar biphasic solvent composed of n-butanol-acetic acid-water (4:1:5, v/v/v). Best results were obtained with a set of multilayer coils of small helical diameter by eluting with the upper nonaqueous phase in a head to tail direction at a flow rate of 1 or 2 ml/min. Four components were completely resolved in 5 to 11 hours. Other types of coiled columns such as a multilayer coil with large helical diameter (in a commercial model) and eccentric dual-layer coil assemblies mounted on a horizontal coil planet centrifuge also yielded satisfactory separations.     

Studies on the performance of different coiled column configurations for compact type-I countercurrent chromatography

Three types of novel coiled column configurations, i.e. a triangular coiled column and elliptical coiled columns I and II, were designed for type-I countercurrent chromatography and their performances were evaluated with two solvent systems each with suitable test samples. Three dinitrophenyl (DNP) amino acids (DNP-DL-glu, DNP-β-ala and DNP-L-ala) were separated with a moderately hydrophobic two-phase solvent system composed of hexane-ethyl acetate-metanol-0.1 M hydrochloric acid (1:1:1:1, v/v), while two dipeptides (tryptophyl-tyrosine and valyl-tyrosine) were separated with a polar solvent system composed of 1-butanol-acetic acid-water (4.75:0.25:5, v/v). The overall results indicated that the performance of compact type-I countercurrent chromatography was improved by elliptical coiled column I which was mounted with its maximum coil diameter perpendicular to the surface of the column holder. Hydrodynamic effects involved in these separations were discussed.     

New small-scale cross-axis coil planet centrifuge. Partition efficiency and application to purification of bullfrog ribonuclease

The new small-scale cross-axis coil planet centrifuge (X-axis CPC) previously designed and fabricated in our laboratory has a distinctive feature such that four separation columns of similar weight are mounted symmetrically around the rotary frame to achieve stable balancing of the centrifuge under a high revolution speed. In this column layout, neighboring columns must be rotated in the opposite direction if viewed from the center of the centrifuge to avoid twisting the interconnecting flow tubes. The effect of rotational direction of the columns on the partition efficiency was evaluated with separation of a set of test samples such as cytochrome c, myoglobin, and lysozyme using an aqueous-aqueous polymer phase system composed of 12.5% (w/w) polyethylene glycol (PEG) 1000 and 12.5% (w/w) dibasic potassium phosphate under 1000 rpm of column revolution. A series of experiments was performed using a set of two diagonally located columns (connected in series) each consisting of five coiled layers of 1 mm I.D. with a total capacity of 27.0 mL. Both right- and left-handed coils were tested each under the optimized conditions for choice of mobile phase and direction of the column rotation so that the satisfactory volume of the mobile phase was retained in the column by the aid of Archimedean screw effect. The results of these studies showed that one particular combination of handedness of the coil and direction of the rotation yielded the best peak resolution for each mobile phase. In order to demonstrate the capability of the apparatus, the purification of ribonuclease (RNase) from the extract of bullfrog egg, sialic acid binding lectin (cSBL), was carried out using both organic-aqueous and aqueous-aqueous polymer phase systems. When using the 16.0% (w/w) PEG 1000-6.3% (w/w) dibasic potassium phosphate-6.3% (w/w) monobasic potassium phosphate system, cSBL was successfully separated from other proteins present in the extract while commercial RNase A was eluted at near the solvent front by the lower phase mobile. The cSBL retained its native RNase activity. The overall results demonstrated that the present new small-scale X-axis CPC is useful for the purification of bioactive compounds without loss of their native activities.     

Isolation and purification of inflacoumarin A and licochalcone A from licorice by high-speed counter-current chromatography

High-speed counter-current chromatography (HSCCC) technique in semi-preparative scale has been applied to isolate and purify bioactive flavone compounds from the ethanol extract of Glycyrrhiza inflata Bat., a particular plant species of licorice. HSCCC separation was performed with a two-phase solvent system composed of n-hexane-chloroform-methanol-water (5:6:3:2, v/v) by eluting the lower mobile phase at a flow rate of 1.8 ml/min and a revolution speed of 800 rpm. Purification was performed with a two-phase solvent system composed of n-hexane-chloroform-methanol-water (1.5:6:3:2, v/v) by eluting the lower mobile phase at a flow-rate of 1.5 ml/min and a revolution speed of 800 rpm. Two major flavone peaks: inflacoumarin A and licochalcone A were collected and the respective yields of the peaks amount to 6 mg (8.6%, w/w) and 8 mg (11.4%, w/w) from 70 mg of the crude extract sample. The purities of inflacoumarin A and licochalcone A reached 99.6% and 99.1%, respectively, after a sequential purification run. The structures of inflacoumarin A and licochalcone A were positively confirmed by 1H NMR and 13C NMR, 1H-13C-COSY, UV, FT-IR and electron ionization MS analyses.     

Preparative separation of high-purity cordycepin from Cordyceps militaris(L.) Link by high-speed countercurrent chromatography

A high-speed counter-current chromatography (HSCCC) technique in a preparative scale has been applied to separate and purify cordycepin from the extract of Cordyceps militaris(L.) Link by a one-step separation. A high efficiency of HSCCC separation was achieved on a two-phase solvent system of n-hexane-n-butanol-methanol-water (23:80:30:155, v/v/v/v) by eluting the lower mobile phase at a flow rate of 2 ml/min under a revolution speed of 850 rpm. HSCCC separation of 216.2 mg crude sample (contained cordycepin at 44.7% purity after 732 cation-exchange resin clean-up) yielded 64.8 mg cordycepin with purity of 98.9% and 91.7% recovery. Identification of the target compound was performed by UV, IR, MS, (1)H NMR and (13)C NMR.     

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.     

Protein separation by nonsynchronous coil planet centrifuge with aqueous-aqueous polymer phase systems

Counter-current chromatographic separation of proteins was performed using a rotary-seal-free nonsynchronous coil planet centrifuge (CPC) fabricated in our laboratory. This apparatus has a unique feature that allows a freely adjustable rotational rate of the coiled separation column at a given revolution speed. The separation was performed using a set of stable proteins including cytochrome c, myoglobin and lysozyme with two different types of aqueous-aqueous polymer phase systems, i.e., PEG (polyethylene glycol) 1000-dibasic potassium phosphate, and PEG 8000-dextran T500 in 5 mM potassium phosphate buffer. Using a set of multilayer coiled columns prepared from 0.8 mm I.D. PTFE tubing with different volumes (11, 24, 39 ml), the effect of the column capacity on the partition efficiency was investigated under a given set of experimental conditions. Among these experiments, the best separation of proteins was attained using the 39 ml capacity column with a 12.5% (w/w) PEG 1000-12.5% (w/w) dibasic potassium phosphate system at 10 rpm of coil rotation under 800 rpm. With lower phase mobile at 0.2 ml/min in the head-to-tail elution, the resolution between cytochrome c and myoglobin was 1.6 and that between myoglobin and lysozyme, 1.9. With upper phase mobile in the head-to-tail elution, the resolution between lysozyme and myoglobin peaks was 1.5. In these two separations, the stationary phase retention was 35.0 and 33.3%, respectively. Further studies were carried out using a pair of eccentric coil assemblies with 0.8 mm I.D. PTFE tubing at a total capacity of 20 ml. A comparable resolution was obtained using both lower and upper phases as a mobile phase in a head-to-tail elution. The results of our studies demonstrate that the nonsynchronous CPC is useful for protein separation with aqueous-aqueous polymer phase systems.     

Large-Scale Preparative Counter-Current Chromatography with a Coil Planet Centrifuge

Abstract

Development of the large-scale preparative countercurrent chromatographic schemes has been continued by increasing the diameter of the separation column. A 0.55 cm i.d. FEP tube was coaxially coiled around the holder (7.5 cm, 10 cm or 15 cm in diameter) of a horizontal flow-through coil planet centrifuge (15 cm revolutional radius). Performance of each column was evaluated on the separation of dinitrophenyl amino acid samples with a two-phase solvent system composed of chloroform, acetic acid, and 0.1N hydrochloric acid (2:2:1) by using both aqueous and nonaqueous phases as the mobile phase. Experiments with the short preliminary columns (114 ml capacity) revealed that the hydrodynamic distribution of the two solvent phases was sensitively affected by the helical diameter of the column. However, by choosing the proper elution mode of the mobile phase, satisfactory results were obtained with the helical diameters of 7.5 cm and 15 cm at a high flow rate of 500 ml/h under a moderate revolutional speed of 300 rpm. With the long coiled columns (750 ml capacity), the preparative capability of the present scheme was successfully demonstrated on separations of the 1g-quantity sample mixture under optimized operational conditions. Overall results indicated that the sample-loading capacity of the present scheme can be further increased by the use of longer and/or larger-diameter columns.     

Separation and identification of water-soluble salvianolic acids from Salvia miltiorrhiza Bunge by high-speed counter-current chromatography and ESI-MS analysis

High-speed counter-current chromatography (HSCCC) technique in semi-preparative scale has been applied to separate and purify salvianolic acids from the water extract of Danshen, Salvia miltiorrhiza Bunge. High efficiency HSCCC separation was achieved on a two-phase solvent system composed of a mixture of n-hexane-ethyl acetate-water-methanol (1.5:5:5:1.5, v/v) by eluting the lower mobile phase at a flow-rate of 1.7 ml/min and a revolution of 850 rpm. A total of five well separated peaks were obtained in the HSCCC chromatogram, and their purities determined by HPLC-UV absorption. These peaks were characterized by UV-vis spectra and ESI-MS, and the data compared with the reference standards. Salvianolic acid B was positively identified as one of the major peaks. Three of the remaining four peaks were also tentatively identified as rosmarinic acid, lithospermic acid, and salvianolic acid E, an isomer of salvianolic acid B, all are members of the salvianolic acids group. In a typical run, tens of milligrams of samples can be separated with high efficiency to yield tens of milligrams of purified materials with over 98% purity. HSCCC thus provides a cost-effective alternative to preparative scale HPLC for the semi-preparative scale separation and purification of salvianolic acids in Danshen. With appropriate modifications, the technique should also be applicable to other herbs in general.     

Evaluation of the effect of column orientation in type-I high-speed counter-current chromatography

The performance of type-I high-speed counter-current chromatography was evaluated by changing the column inclination against the rotating centrifugal force field. The separations were performed with two different solvent systems composed of 1-butanol–acetic acid–water (4.75:0.25:5, v/v) (BAW) and hexane–ethyl acetate–methanol–0.1 M HCl (1:1:1:1, v/v) (HEMW) using dipeptides and DNP-amino acid as test samples, respectively. A set of short coiled columns connected in series is mounted around the holder hub in two different ways: in the parallel orientation, all column units are arranged in parallel to each other and mounted on the holder at various angles against the horizontal plane. In the zigzag configuration, the neighboring units of the same column are mounted symmetrically forming various angles apart. In the parallel configuration, for both the BAW and HEMW systems, Sf (the retention of stationary phase) first increased as the column angle decreased from 90° to 60° and then decreased, as the column angle further decreased from 60° to 0°, while Rs (peak resolution) continually declined over the entire column angle range from 90° to 0°. But, for both solvent systems, with the zigzag configuration, retention of stationary phase and resolution both decreased as the column angle decreased from 90° to 0°. In general, Sf and Rs for separation of dipeptides in the BAW system, from 90° to 15°, is better for the parallel orientation than for the zigzag configuration. However, at 0°, Sf and Rs are better for the zigzag orientation. In the DNP-amino acid separation with the HEMW system, retention of the stationary phase and Rs for the parallel orientation is better than that for the zigzag orientation from 90° to 30°, whereas from 30° to 0° the results are opposite. Over all results of our studies revealed that the formally used column orientation [5] at 90° inclination yields the highest peak resolution in both solvent systems.     

Isolation and Purification of Lipids by Centrifugal Partition Chromatography

Abstract

Methodologies for the separation of various lipid classes including free fatty acids (FA) and fatty acid ethyl esters (FAEE), monoglycerides (MG), diglycerides (DG), triglycerides (TG), phosphoglycerides (PG), and glycosphingolipids (GSL) by Centrifugal Partition Chromatography are presented. The fatty acid ethyl esters, Hexadecanoate, Octadecanoate, cis-9-Octadecenoate; and cis,cis-9,12-Octadecadienoate, and all-cis-9,12,15-Octadecatrienoate, were separated at 800 rpm; flow rate of 2.0 ml/min; using n-hexane/acetonitrile (1:1, v/v) as the solvent system and normal ascending and reversed descending elution modes respectively. The fatty acid ethyl esters, all-cis-5,8,11,14,17-Icosapentaenoate and all-cis-4,7,10,13,16,19-Docosahexaenoate, were separated at 600 rpm; flow rate of 3.3 ml/min; using n-hexane/methanol/water (1/0.9/0.1, v/v/v) as the solvent system and normal ascending mode. FA and MG; and DG and TG were separated at 600 rpm; 10     

Development and evaluation of a spiral tube column for counter-current chromatography

An improved type-J counter-current chromatography (CCC) planet centrifuge with two spiral tube columns (volume 2×15 mL, β value 0.3-0.7, tubing 0.8 mm id) was developed and evaluated for its retention ability of four typical different solvent systems including heptane-methanol (1:1, v/v) (A), hexane-ethyl acetate-methanol-water (1:1:1:1, v/v) (B), n-butanol-acetic acid-water (4:1:5, v/v) (C), PEG1000-K(2)HPO(4)-water (12.5:12.5:75, w/w) (D) under eight different operation modes. The results indicated that the spiral tube column could significantly increase the retention of four typical solvent systems compared with a traditional multilayer coil column with similar parameters (volume 35 mL, β value 0.3-0.7, tubing 0.8 mm id). The retention of stationary phase (S(f)) for the less polar system (A) and moderately polar solvent system (B) can be increased by about 10%, and for the polar system (C) and aqueous two-phase system (ATPS) (D) by 30-40%. The preliminary applications of this spiral tube column to the separation of small molecular compounds such as moderately polar theaflavins, polar anthocyanins and dipeptides were successful. Acceptable resolution can be obtained between cytochrome c and myoglobin, lysozyme and myoglobin when it was applied on protein separation; however, it still needs to be improved with regard to its column efficiency.     

Size Exclusion Chromatography of Nonionic and Anionic Copolymers of Vinylpyrrolidone

Abstract

Size exclusion chromatography (SEC) of poly(vinylpyrrolidoneco-vinylacetate), PVPVA, and poly(vinylpyrrolidone-co-dimethylamincethylmethacrylate-co-vinylcaprolactan),PVPDMMAEMAVC, is evaluated in terms of resolution between polymer and solvent peaks using aqueous and nonaqueous mobile phases. A 1:1 (v/v) water/methanol, 0.1M LiNO₂ mobile phase is used with four Waters Ultrahydrogel columns with pore sizes of 120Å, 500Å, 1000Å, and 2000Å. DMF, 0.1M LiNO₃ mobile phase is used with three different two column sets. Two of the column sets are comprised of a mixed bed packing of poly(styrene-co-divinyl benzene), obtained from Shodex (KD80M), followed by either an Ultrahydrogel 120Å or a PLge1 100Å. The third two column set is a PLge1 10⁴Å followed by a PLgel 500Å. Any of the second columns in these sets improve separation between the trailing end of the polymer peak and the leading end of the solvent peak but the column set using an Ultrahydrogel 120Å yields a separation of these peaks whose valley is closer to the baseline. The aqueous mobile phase with the four column set yields a separation between the trailing end of the polymer peak and the leading end of the solvent peak whose valley is closest to the baseline. Recovery of PVPVA and PVPDMAEMAVC from the columns evaluated is 100%. Vinylpyrrolidone compositions of PVPVA ranging from 30 to 70 mole % were studied using both mobile phases. SEC of     

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.     

Semimicro Size Exclusion Chromatography For Oligomers. I. Column Packing Procedure

Abstract

Polystyrene gels of a particle diameter 10 ± 2 μm for the use in oligomer separation were packed into 1.5 mm i.d. × 25 cm length columns by the balanced density slurry-packing technique under a constant flow rate of 500 μL/min. The slurry solvent was a mixture of toluene and chloroform (50.5/49.5, v/v). The example of the number of theoretical plates (N) of these columns was 8600 plates/25 cm (HETP = 29.1 μm) at flow rate of 40 μL/min by injecting 1 μL of 0.5% benzene solution. Sixteen columns were connected and the overall value of N was 103000 plates/4 m. A typical example of oligomer separation was demonstrated. A constant-flow technique is preferable to a constant-pressure technique. When two or three column blanks were packed together, the columns located at the outlet of the packer-column assembly had higher values of N. Optimum flow rate of the slurry solvent when three column blanks were packed together lay between 400 and 500 μL/min. The packing efficiency, that is, the probability of getting valid columns was about 60%. Viscous slurry solvents were not effective to get efficient columns. To pack gels in the less swollen state gave sometimes efficient columns. Pressure monitoring in progress of packing was very effective to foresee the column efficiency.     

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).     

Dynamic extraction in rotating coiled columns, a new approach to direct recovery of polycyclic aromatic hydrocarbons from soils

A new approach to the direct recovery of polycyclic aromatic hydrocarbons (PAHs) from environmental solid samples has been proposed. It has been shown that rotating coiled columns (RCCs) earlier used mainly in countercurrent chromatography can be successfully applied to the fast continuous-flow extraction of PAHs from soils. A particulate solid sample was retained in the rotating column as the stationary phase under the action of centrifugal forces while a mixture of organic solvents (acetone-cyclohexane, 1:1, v/v) was continuously pumped through. The separation procedure requires less than half an hour, complete automation being possible. No clean-up step is needed before the subsequent HPLC- analysis of extracts. Besides, the dynamic multistage extraction performed in the rotating column at room temperature and normal pressure may have nearly the same efficiency as accelerated batch solvent extraction repeated three times at 150 degrees C and 14 MPa. Contents of PAHs in extracts obtained by using both methods are in good agreement with the certified data on the PAHs concentrations in the soil samples. The use of appropriate "mild" solvents for the dynamic extraction in rotating columns may be very perspective for the simulation of naturally occurring processes and determination of environmentally-relevant forms of PAHs and other pollutants in environmental solids. A particular emphasis could be placed on time-resolved (kinetic) studies of the mobilization of toxicants in soil systems.     

Separation and purification of four compounds from Desmodium styracifolium using off‐line two‐dimensional high‐speed counter‐current chromatography

An off‐line 2D high‐speed counter‐current chromatography technique in preparative scale has been successfully applied to separate and purify the main compounds from the ethyl acetate extract of Desmodium styracifolium. A two‐phase solvent system composed of n‐hexane/ethyl acetate/methanol/water at an optimized volume ratio of 1:2:1:2 v/v/v/v was used. Conventional high‐speed counter‐current chromatography was used as the first dimension, and the upper phase of the solvent system was used as the stationary phase in the head‐to‐tail elution mode at a flow rate of 2.0 mL/min and a rotation speed of 900 rpm. Recycling high‐speed counter‐current chromatography served as the second dimension to separate an impure fraction of the first dimension. A total of four well‐separated substances including vanillic acid ( 1 ), β‐sitosterol ( 2 ), formononetin ( 3 ), and aromadendrin ( 4 ) were obtained, and their purities and structures were identified by HPLC–MS and ¹H NMR spectroscopy. The results illustrated that off‐line 2D high‐speed counter‐current chromatography is an effective way to isolate compounds in complex samples.