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Inspired by the interpretation of two-dimensional (2D) nuclear magnetic resonance spectra, an efficient strategy was proposed for pinpointing bioactive components from complex natural products. An off-line comprehensive countercurrent chromatography (CCC) × high-performance liquid chromatography (HPLC) was employed to achieve a 2D chemical chromatogram, and 2D bioassay profilings were obtained from bioassays of the eluent of the first dimension (

Analytical enantioseparations of five N-alkyl drugs, fluoxetine hydrochloride, labetalol, venlafaxine hydrochloride, trans-paroxol, and atropine sulfate, were investigated by reverse phase high-performance liquid chromatography with sulfobutylether-β-cyclodextrin as chiral mobile phase additive. Effects of various factors such as composition of mobile phase, concentration of cyclodextrins, and column temperature on retention and enantioselectivity were studied. Apparent formation constant between methanol, acetonitrile, and sulfobutylether-β-cyclodextrin were determined to be 2.90 × 10

Cassiae Semen (CS) is consumed as fried tea or medicinal food in Asian areas. Its two commercial forms, namely raw and fried CS, exert different clinical applications, in which fried CS is commonly applied as a functional tea for losing weight. To prevent confusion in the use of the two forms of CS, a comprehensive strategy by combining plant metabolomics and spectrum-effect relationship analyses was developed for the fast and efficient discrimination of raw and fried CS, and further for the discovery of the potential hypoglycemic metabolites of CS to control its quality. First, the plant metabolic profiling of raw and processed samples was performed by UHPLC-QTOF-MS/MS. A total of 1111 differential metabolites were found to well distinguish the raw and fried CS after analyzing by MPP software. Subsequently, α-glucosidase inhibition of raw and fried CS was investigated. As a result, fried CS demonstrated much stronger α-glucosidase inhibition activity than the raw sample. By analyzing the spectrum-effect relationship with GRA, BCA, and PLSR, 14 potential hypoglycemic-related compounds were discovered. As anticipated, they were also found as the differential metabolites of the raw and fried samples with a potential hypoglycemic effect, which might be beneficial for the quality control of CS tea. Additionally, molecular docking analysis was conducted to reveal the underlying inhibition mechanisms of the four most critical constituents, including physcion, chrysoobtusin, aurantio-obtusin, and obtusifolin. This study provides a powerful tool for the discrimination of processed samples and fast screening of the active constituents in complex natural products on a high-throughput basis.

Carboxymethyl-β-cyclodextrins (CM-β-CDs) with five kinds of degrees of substitution were synthesized and characterized. Analytical enantioseparation of six basic drugs containing N-alkyl groups, including pheniramine, chlorpheniramine, labetalol, propranolol, venlafaxine, and trans-paroxol, was achieved by reversed-phase high-performance liquid chromatography (RP-HPLC) using the synthesized CM-β-CD as chiral mobile phase additives. Key influence factors were optimized, including organic modifier, pH value, CM-β-CD with different degrees of substitution, and concentration of CM-β-CD. The mobile phase was composed of methanol and 10 mmol L

Screening and analysis of bioactive components from natural products is a fundamental part of new drug development and innovation. Two-dimensional (2D) chromatography has been demonstrated to be an effective method for screening and preparation of specific bioactive components from complex natural products.To collect details of application of 2D chromatography in screening of natural product bioactive components and to outline the research progress of different separation mechanisms and strategies.Three screening strategies based on 2D chromatography are reviewed, including traditional separation-based screening, bioactivity-guided screening and affinity chromatography-based screening. Meanwhile, in order to cover these aspects, selections of different separation mechanisms and modes are also presented.Compared with traditional one-dimensional (1D) chromatography, 2D chromatography has unique advantages in terms of peak capacity and resolution, and it is more effective for screening and identifying bioactive components of complex natural products.Screening of natural bioactive components using 2D chromatography helps separation and analysis of complex samples with greater targeting and relevance, which is very important for development of innovative drug leads.

A method for quantitative determination of the main active ingredient in traditional Chinese medicine was developed and validated, using liquid-liquid chromatography combined with high performance ...

Ondansetron, a highly selective 5-hydroxytryptamine 3 receptor antagonist, was successfully enantioseparated by recycling countercurrent chromatography using sulfobutyl ether-β-cyclodextrin as chiral selector. Important factors for the enantioseparation were optimized, including different organic solvent, type of substituted β-cyclodextrin, pH of aqueous phase, concentration of chiral selector and separation temperature. A biphasic solvent system composed of n-hexane: n-butyl acetate: 0.1 mol L-1 phosphate buffer solution pH 9.2 with 50 mmol L-1 of sulfobutyl ether-β-cyclodextrin (2.5:7.5:10, v/v/v) was selected. Under optimized separation conditions, 5 mg of ondansetron was enantioseparated using recycling countercurrent chromatography, yielding 1.2 mg and 1.5 mg of ondansetron enantiomers with 97.5% and 95.8% purity and the recovery reached 48-60%. This article is protected by copyright. All rights reserved.

Nine types of hydroxypropyl-β-cyclodextrin (HP-β-CD) with different degrees and distributions of substitution were synthesised, and nine racemates were selected to investigate the effect of different degrees and distributions of substitution of HP-β-CD on the enantioseparation factor.

Two anionic β-cyclodextrins as chiral selectors were successfully applied in the enantioseparation of N-methyl duloxetine, duloxetine, and fluoxetine by countercurrent chromatography. Sulfobutyl ether-β-cyclodextrin and carboxymethyl-β-cyclodextrin showed opposite enantioselectivity for both duloxetine and N-methyl duloxetine enantiomers. Two biphasic solvent systems, n-hexane: 0.1 mol/L phosphate buffer pH 7.6 with 50 mmol/L of sulfobutyl ether-β-cyclodextrin (1:1, v/v) and n-hexane: 0.1 mol/L phosphate buffer pH 7.2 with 50 mmol/L of carboxymethyl-β-cyclodextrin (1:1, v/v), were selected for N-methyl duloxetine. Enantioseparation of duloxetine was achieved by recycling countercurrent chromatography using a solvent system composed of n-butyl acetate: 0.1 mol/L phosphate buffer pH 7.2 with 20 mmol/L of sulfobutyl ether-β-cyclodextrin or carboxymethyl-β-cyclodextrin (1:1, v/v). A solvent system composed of n-hexane: n-butyl acetate: 0.1 mol/L phosphate buffer pH 7.6 containing 20 mmol/L of sulfobutyl ether-β-cyclodextrin (6:4:10, v/v) was selected for enantioseparation of fluoxetine.

2-(4-Chlorophenyl)succinic acid was successfully enantioseparated by countercurrent chromatography using hydroxypropyl-β-cyclodextrin as chiral selector. A two-phase solvent system composed of n-hexane-ethyl acetate-0.1 mol/L phosphate buffer with pH 2.65 (5:5:10, v/v) was selected. Enantioselective liquid-liquid extraction was used to optimize the enantioseparation conditions. Meanwhile, the influence of injection volume on resolution in countercurrent chromatography was investigated and a linear relationship between the inflection point of injection volume and sample loading was tentatively obtained. The peak resolution will decrease significantly when the injection volume over the inflection point was used. In addition, it could be found that the smaller amount of sample loading, the larger impact of injection volume on resolution could be observed, which might serve as a good reference for the selection of sample volume in enantioseparations by countercurrent chromatography. Under optimized conditions, 20 mg of 2-(4-chlorophenyl)succinic acid racemate dissolved in 10 mL of aqueous phase was successfully enantioseparated by countercurrent chromatography. The recovery for both of the enantiomer of (±)-2-(4-chlorophenyl)succinic acid reached within 70-75% with a purity of 99.0%.

As a liquid-liquid partition chromatography, counter-current chromatography has advantages in large sample loading capacity without irreversible adsorption, which has been widely applied in separation and purification fields. The main factors, including partition coefficient, two-phase solvent systems, apparatus, and operating parameters greatly affect the separation process of counter-current chromatography. To promote the applications of counter-current chromatography, it is essential to develop theoretical research to master the principles of counter-current chromatographic separations so as to achieve predictions before laborious trials. In this article, recent progress about separation prediction methods are reviewed from a point of the steady and unsteady state of the mass transfer process of counter-current chromatography and its mass transfer characteristics, and then it is divided into three aspects: prediction of partition coefficient, modeling the thermodynamic process of counter-current chromatography, and modeling the dynamic process of counter-current chromatography.

The inclusion interaction between hydroxypropyl-β-cyclodextrin (HP-β-CD) and 21 2-aryl carboxylic acids was investigated by UV (ultraviolet) spectrophotometer. The inclusion constant of each 2-aryl carboxylic acids with HP-β-CD was determined by Benesi-Hildebrand's equation. According to our previous work, it was found that a high inclusion constant for inclusion complex formed by a racemate and cyclodextrin was always observed with the fact that a high enantioseparation factor was achieved for the racemate in enantioseparation by liquid-liquid chromatography, which suggested that high binding combination between racemate and cyclodextrin is very important for a successful enantioseparation in enantioselective liquid-liquid extraction. Among all the studied subjects, mandelic acid enantiomer, 2,3-diphenylpropionic acid enantiomer, and naproxen enantiomer were selected for the further study. The inclusion constants of enantiomers of these three subjects were determined by UV spectra, which indicated that a necessary difference in inclusion constants between enantiomer and cyclodextrin was also essential. It was found in UV spectra that the absorbance of the analytes with the addition of cyclodextrin would increase or decrease, which was determined by the type of electron excitation. The conformation changes of small molecules can lead to the changes of chromophore valence electron clouds distribution, causing the HOMO-LUMO energy difference decreased. Thus, a red shift of the wavelength of the maximum absorption was produced indicating that the possibility of the molecular interaction of enantiomers with HP-β-CD exists.

The elution behavior of four sesquiterpenoids in volatile oil of Curcumae Rhizoma on reverse-phase high-performance liquid chromatography with methyl-β-cyclodextrin as mobile phase additive was stu...

In this work, relationships between solvent strength of organic phase (ψ) for two biphasic solvent systems in high speed countercurrent chromatography, hexane-ethyl-acetate-methanol-water (HEMWat) and ethyl acetate-n-butanol-water (EBuWat), and partition coefficient (K) were investigated using four retention models, including Jandera's model (ABM), Neue-Kuss model (NK), linear-solvent-strength model (LSS) and quadratic-solvent-strength model (QSS). Experimental results showed that ABM model had the best fitting results for HEMWat system while NK model and QSS model had good fitting results in EBuWat system. Thus, a mathematical relationship between partition coefficient (K) and solvent strength of organic phase (ψ) could be obtained by measurement of partition coefficients of the target compounds with three different volume ratios of organic phase. At the same time, a functional map was proposed to construct to get a maneuverable region so that an optimal two-phase solvent system for separation of a target compound could be selected easily, which saved a lot of manpower for high speed countercurrent chromatographic separation. The application of this new method was declared by successful separation of two components, apigenin-6-C-β-D-xylopyranosyl-8-C-α-L-arabinopyranoside and vicenin-3, from dried leaves of Dendrobium officinale Kimura et Migo using high speed countercurrent chromatography.

In the present study, high-performance liquid chromatography micro-fraction bioactive evaluation and high speed countercurrent chromatography were performed on screening, identification and isolation of antioxidants from Citrus peel. Three compounds were screened as antioxidants and tyrosinase inhibitors using 2,2'-azino-bis (3-ethyl-benzothiazoline-6-sulfonic acid) radical cation scavenging assay and tyrosinase activity test, then they were identified as eriocitrin, narirutin and hesperidin. Moreover, the solvent system ethyl acetate-n-butanol-water (6:4:10, v/v/v) was used for separation of ethyl acetate extract of Citrus peel by high speed countercurrent chromatography. In total, 0.45 mg of eriocitrin with 87.10% purity, 2.04 mg of narirutin with 95.19% purity and 1.35 mg of hesperidin with 95.19% purity were obtained from 20 mg of ethyl acetate extract of Citrus peel in a single run and then each component was subjected to 2,2'-azino-bis (3-ethyl-benzothiazoline-6-sulfonic acid) radical cation scavenging assay and tyrosinase inhibition assay. Eriocitrin showed great antioxidant activity (the half-maximum concentration: 3.65 µM) and tyrosinase inhibition activity (the half-maximum concentration: 115.67 µM), while narirutin and hesperidin exhibited moderate activity. Tyrosinase inhibition activity for eriocitrin in vitro was reported for the first time. Furthermore, molecular docking between eriocitrin and mushroom tyrosinase was also studied.

A comprehensive two-dimensional (2D) countercurrent chromatography (CCC) × gas chromatography (GC) was investigated for characterization of chemical constituents of Artemisia argyi essential oil, and orthogonality for the 2D chromatographic system was evaluated. A solvent system composed of n-hexane/acetonitrile/methanol (2:2:1, v/v/v) was selected for first dimensional separation of Artemisia argyi essential oil. Then all CCC fractions were analyzed by GC, which provided a wealth of information regarding the composition of the essential oil. Visualization of chemical compositions obtained from the comprehensive 2D CCC × GC separation was achieved by creation of a 2D contour plot map. Total peak capacity was evaluated and approximately 1392 peaks were obtained through a comprehensive 2D CCC × GC separation. A high spatial coverage and a low linear correlation coefficient were achieved. Meanwhile, all compounds were identified by GC-MS. The obtained 2D contour plot could be divided into six zones to show the characteristic chemical compositions. Six zones could be divided into different component groups, including monoterpenes, sesquiterpenes, monoterpene alcohols, phenols, aldehydes, ketones and esters, which could be used to identify compounds that have not been reported, and to predict the structure of unknown compounds in Artemisia argyi essential oil and comprehensively characterize fingerprint peak.

In the present work, influence of solvent strength of aqueous phase for two frequently-used biphasic solvent system in partition coefficient (K) of selected solutes were mainly studied, and a new method for selection of biphasic solvent system was proposed for high-speed countercurrent chromatographic separations. Solvent strength was referred to the typical theory that was deeply investigated in conventional reversed-phase liquid chromatography. Experimental results showed that a linear relationship between log(K) of solutes and apparent content of methanol in biphasic solvent system was found for the biphasic solvent system hexane-ethyl acetate-methanol-water (HEMWat), which was consistent with the relationship between real content and apparent content of methanol in this system. Meanwhile, a quadratic relationship was found between log(K) of solutes and apparent content of methanol in biphasic solvent system chloroform-methanol-water (ChMWat), in which it was found that the relationship between real content and apparent content of methanol in this system was also quadratic. In addition, a visual and simple method was proposed to select a suitable biphasic solvent system for separation of target compounds by high-speed countercurrent chromatography with isocratic elution, which saves a lot of manpower and material resources in order to find a suitable two-solvent system. An optimal biphasic solvent system for isolation of several tested compounds by high-speed countercurrent chromatography was easily obtained using our proposed method.

The combined use of pH-zone-refining counter-current chromatography (CCC) and conventional CCC was successfully employed for the preparative separation and purification of baicalin and wogonoside from the traditional Chinese medicinal herb Scutellaria Baicalensis Georgi. A more environmentally friendly biphasic solvent system composed of n-butanol-ethyl acetate-water (2:3:5, v/v) was used for pH-zone-refining CCC, in which trifluoroacetic acid was added to a final concentration of 10 mmol L−1 in the organic phase and ammonia was added to a final concentration of 10 mmol L−1 in the aqueous phase while a two-phase solvent system composed of ethyl acetate-ethanol-3 mmol L−1 hydrochloric acid (10:1:10, v/v) was used for the conventional CCC. Two elution modes including reverse displacement mode and normal displacement mode were investigated for pH-zone-refining CCC. The first run provided 186.7 mg of baicalin with a purity of 95.3% and 143.4 mg of a mixture of baicalin and wogonoside from 500 mg of the crude extracts of Scutellaria Baicalensis Georgi by pH-zone-refining CCC with reverse displacement elution mode. The mixture was further separated by means of a second run in conventional CCC, yielding 64.3 mg of baicalin with a purity of 98.2% and 46.1 mg of wogonoside with a purity of 98.9%.

Four stereoisomeric components were produced during the synthesis of the antidepressant drug (1S, 4S)-sertraline hydrochloride due to the two chiral carbon centers in its chemical structure, including (1S, 4S), (1R, 4R), (1S, 4R), and (1R, 4S)-isomer. Stereoselective separation of the target isomer (1S, 4S)-sertraline from the medicinal reaction mixtures by countercurrent chromatography using hydroxypropyl-β-cyclodextrin as the stereoselective selector was investigated. A biphasic solvent system composed of n-hexane/0.20 mol/L phosphate buffer solution with pH 7.6 containing 0.10 mol/L of hydroxypropyl-β-cyclodextrin (1:1, v/v) was selected for separation of cis-sertraline and trans-sertraline using reverse phase elution mode and (1S, 4S)-sertraline was separated with (1R, 4R)-sertraline using recycling elution mode. A fabricated in-house analytical countercurrent chromatographic apparatus was used for optimization of the separation conditions. Stationary phase retention and peak resolution were investigated for separation of cis-sertraline and trans-sertraline by the analytical apparatus.

In this work, pH-zone-refining countercurrent chromatography was investigated in the preparative separation of two bioactive components, oleanolic acid and ursolic acid, from three different natural products, Aralia chinensis, apple peels and Eriobotrya japonica Thunb. Oleanolic acid and ursolic acid are structurally isomeric pentacyclic triterpene acids that are widely distributed in many natural products. However, it was difficult to separate these components with high purity by conventional methods. A biphasic solvent system composed of n-hexane–dichloromethane–methanol–water (7 : 3 : 2 : 8, v/v) was selected, in which an optimized concentration of 10 mmol L−1 trifluoroacetic acid was added in the upper phase as the retainer and 10 mmol L−1 ammonia (with 25–28% NH3) was added in the aqueous phase as the eluter. Consequently, 38.56 mg of oleanolic acid with 99.01% purity was separated from 100 mg of the crude extract of Aralia chinensis, while 65.6 mg of a mixture of ursolic acid (90.98%) and oleanolic acid (6.51%) and 46.6 mg of a mixture of ursolic acid (74.35%) and oleanolic acid (23.61%) were separated from 100 mg of the crude extract of apple peels and 100 mg of the crude extract of Eriobotrya japonica Thunb., respectively, by pH-zone-refining countercurrent chromatography using the above selected biphasic solvent system. The results showed that pH-zone-refining countercurrent chromatography is an efficient method for the preparative separation of pentacyclic triterpene acids from natural products.

The minor ginsenosides with less polarity from Panax notoginseng leaves may have more potent biological activities. Four minor saponins, i.e. gypenoside XVII, ginsenoside Rd2, notoginsenoside Fe and notoginsenoside Fd, were successfully separated from Panax notoginseng leaves after biotransformation by one-step counter-current chromatography using the biphasic solvent system consisting of n-butanol-ethyl acetate-water (1:4:5, v/v/v). 30 mg of the refined extract of Panax notoginseng leaves produced 1 mg of gypenoside XVII, 4 mg of notoginsenoside Fe, 2.5 mg of ginsenoside Rd2, and 8.4 mg of notoginsenoside Fd, with purity of 74.9%, 95.2%, 87.3% and 97.6%, respectively. Besides, orthogonality evaluation for the separation of the four saponins using counter-current chromatography and liquid chromatography was briefly discussed in this paper. Four minor saponins were successfully separated from each other on preparative scale by counter-current chromatography from Panax notoginseng leaves, which will facilitate to provide an ample of these minor saponins for further pharmacological studies. This article is protected by copyright. All rights reserved.

To avoid stationary phase irreversibly adsorption and tedious and time-consuming separation steps, high-speed counter-current chromatography was employed for the preparative separation of anti-tumor compound antroquinonol from solid fermentation culture of Antrodia camphorata for the first time. A Box-Behnken experimental design, based on three parameters including liquid-to-solid ratio, extraction time and extraction temperature, was applied to optimize the ultrasonic extraction procedure. The optimal extraction condition was set as below: liquid-to-solid ratio: 49.57:1; extraction time: 55.76 min; extraction temperature was arranged as 44.21 °C. Meanwhile, an optimized solvent system containing petroleum ether, ethyl acetate, methanol and water (4:1:4:1, v/v/v/v) was selected for the preparative separation of antroquinonol at a flow rate of 2.0 mL/min. The yield of isolated antroquinonol was determined to be 6.0 mg from 0.67 g of ethyl acetate extracts. The isolated antroquinonol was elucidated by ultra high performance liquid chromatography tandem mass spectrometry, and NMR spectroscopy, and by comparison with literature data. The purity of isolated antroquinonol was determined to be 97.12%. This study confirmed high-speed counter-current chromatography was powerful and cost-effective for the preparative separation of the high-potently anti-tumor compound antroquinonol from solid fermentation culture of Antrodia camphorata. This article is protected by copyright. All rights reserved.

Maslinic acid and corosolic acid with high purity were successfully separated from Eriobotrya japonica (Thunb.) leaves by two-step countercurrent chromatographic separation. Two biphasic solvent systems composed of petroleum ether-ethyl acetate-ethanol-water (6:4:5:5, v/v) and petroleum ether-ethyl acetate-ethanol-0.10 mol/L of hydroxypropyl-β-cyclodextrin with pH 7.0 (8:2:3.5:6.5, v/v) were selected according to the partition performance of the main structural isomeric pentacyclic triterpenes. The influences of pH value and concentration of hydroxypropyl-β-cyclodextrin in separation of two isomers were investigated. In first step countercurrent chromatographic separation, a mixture of two target structural isomers (14.12 mg of sample I) was separated from 40.00 mg of a partially purified sample. In second step countercurrent chromatographic separation, maslinic acid and corosolic acid were completely isolated from 12.00 mg of sample I with hydroxypropyl-β-cyclodextrin as aqueous phase additive. The recoveries of the two isomers were over 90%, yielding 5.18 mg of maslinic acid and 5.47 mg of corosolic acid, respectively.

Off-line two-dimensional countercurrent chromatography has been widely applied to the isolation of complex samples, but little research on the investigation of orthogonality in the selection of biphasic solvent systems is available. In the present work, the orthogonality in the selection of a biphasic solvent system for liquid-liquid chromatographic separation of aqueous extract and ether extract from the traditional Chinese medicinal plant Polygonum cuspidatum Sieb. et Zucc was evaluated by the correlation coefficient and space occupancy rate. In total, 25 different biphasic solvent systems were tested, and 313 system combinations were analysed. A convex hull methodology was used to determine the separation space and to optimize separation conditions. The correlation coefficient matrix was transformed into dendrograms and a colour map to visualize the dissimilarity between, and orthogonality for, all solvent systems. The aqueous extracts from Polygonum cuspidatum were separated using selected biphasic solvent systems with high orthogonality: ethyl acetate-ethanol-water (70:1:70, v/v) and petroleum ether-ethyl acetate-water (1:5:5, v/v). The ether extracts from Polygonum cuspidatum were also separated using selected biphasic solvent systems with high orthogonality: petroleum-ethyl acetate-methanol-aqueous 0.25 M NH3•H2O (5:5:5:5, v/v) and petroleum-ethyl acetate-methanol-water (5:5:5:5, v/v). Thirteen compounds were successfully obtained. The experimental results demonstrated that the evaluation of orthogonality provided an alternative strategy to select an applicable solvent system for the separation of complex samples using off-line two-dimensional countercurrent chromatography.

An efficient and target-oriented pH-peak-focusing countercurrent chromatographic method was established for large-scale separation of baicalin and wogonoside from the crude exact of traditional Chinese medicinal herb Scutellaria baicalensis Georgi. An optimized two-phase solvent system composed of n-butanol-ethyl acetate-methanol-water (1:4:0.5:5, v/v) was selected. Trifluoroacetic acid (10 mmol/L) was added to the upper organic phase, used as the stationary phase. One liter of the aqueous lower phase was used as the mobile phase for 0-350 min, and then 10 mmol/L ammonia was added to remaining 1 L of the aqueous lower phase and used as the mobile phase for 350-600 min. In total, 493.2 mg of baicalin with 98.6% purity and 88.6 mg of wogonoside with 98.9% purity were obtained from 1.0 g of crude exact of S. baicalensis by countercurrent chromatography in a single run. The acid dissociation constant (pKa) and oil-water partition coefficient values of two components were measured to better understand the mechanism of separation. Results showed that pH-peak-focusing countercurrent chromatography with a polar solvent system added with trifluoroacetic acid could be an efficient method for large-scale isolation of organic acids, which are difficult to separate with conventional countercurrent chromatography due to their poor solubility in non-polar solvents.

The analysis of anionic compounds via the sensitive FLM-EKS method remains challenging due to their inverse migration behaviors to EOF in the capillary. A highly sensitive ionic liquid coating-based online preconcentration methodology via electrokinetic supercharging (EKS) coupled to a free liquid membrane (FLM) was first proposed and successfully used to determine vanillic acid (VA), salvianic acid A sodium (SAAS) and ferulic acid (FA) in a complex sample matrix. EKS was recognized as the first step preconcentration, and its separation conditions were optimized. Then, the second preconcentration step, namely FLM, which was performed by simple injection of a short water-immiscible solvent plug as a membrane interface prior to electrokinetic injection (EKI) of the sample, was further investigated to increase the stacking efficiency. The influences of multifarious parameters on FLM-EKS performance were examined, and the optimal FLM-EKS conditions were identified: a buffer electrolyte of 20 mM Tris-HOAc (pH=6) with 5% (v/v) methanol; hydrodynamic injection (HI) of a leading electrolyte (50 mM NH4Cl, 30 s, 0.5 psi) followed by the HI of 2-nitrophenyl octyl ether (NPOE) as the FLM (5 s, 0.5 psi); EKI of the sample (120 s, -10 kV) and there with the HI of terminator (20 mM N-cyclohexyltaurine, CHES, 40 s, 0.5 psi). The newly developed method yielded analytes with 2006-2862-fold enhancement compared to the normal injection, there by producing detection limits of 1.5-5.0 ng mL-1 with satisfactory repeatability below 2.92%, which provides 1.5-2.0 times higher stacking efficiency than that obtained by EKS only. Feasibility in real sample analysis was demonstrated by using Naoxintong capsules and rat feces, which suggests that the presented methodology has a high potential for the reliable and sensitive determination of trace anionic components in complex sample matrices.

A sensitive capillary electrophoresis strategy was developed by combining salting-out assisted liquid-liquid extraction and online three-step stacking to detect trace quantities of antitumour indole alkaloids in complex biological samples. The proposed strategy fully exploits these two technologies such that extraction, online stacking and separation are combined in a fast and efficient manner. First, salting-out assisted liquid-liquid extraction was used to extract three indole alkaloids (vinblastine sulfate, catharanthine sulfate and vindoline) from complex biomasses. An appropriate volume of acetonitrile (ACN) was mixed with a faecal aqueous solution to precipitate proteins. The mixed solution was vortexed, followed by the addition of ammonium sulfate ((NH

Retention behavior of two structural isomeric pentacyclic triterpenic acids, maslinic acid and corosolic acid, was investigated by reverse phase high performance liquid chromatography (HPLC) with hydroxypropyl-β-cyclodextrin (HP-β-CD) as mobile phase additive. Inclusion complexation of maslinic acid, corosolic acid with hydroxypropyl-β-cyclodextrin was evaluated under different concentration of hydroxypropyl-β-cyclodextrin. Apparent formation constant (Km) between methanol and hydroxypropyl-β-cyclodextrin was determined to be 13.82 L mol−1 under 25 °C using UV-spectrophotometry. Two retention models were employed individually for evaluation of inclusion complexation between the two pentacyclic triterpenic acids and hydroxypropyl-β-cyclodextrin. It was found that a higher apparent formation constant (Kf) for corosolic acid and hydroxypropyl-β-cyclodextrin was obtained, 19115 L mol−1, indicating that a greater affinity of hydroxypropyl-β-cyclodextrin with corosolic acid was produced compared with that of maslinic acid, 11775 L mol−1, in the selected mobile phase, and stoichiometric ratio for both of inclusion complex was found to be 1:1. Thermodynamic analysis showed that a negative standard enthalpy change (ΔH) and an entropy change (ΔS*) for analyte transfer were obtained, where ΔH of maslinic acid and corosolic acid was found to be −10.188 kJ mol−1 and −10.650 kJ mol−1, ΔS* of two compounds was −2.092 and −2.180, respectively, indicating that transfer of structural isomers from mobile phase to stationary phase was enthalpically driven. Meanwhile, positive values were obtained for standard enthalpy change and standard entropy change, 136 kJ mol−1 and 274 kJ mol−1 and 536 J mol−1 K−1and 1004 J mol−1 K−1, for inclusion complexation between maslinic acid, corosolic acid and hydroxypropyl-β-cyclodextrin, while negative values were obtained for Gibbs free energy during formation of inclusion complex, −160 kJ mol−1 and −299 kJ mol−1, indicating a spontaneous inclusion reaction happened.

Three key intermediates in the preparation of the nonsteroidal anti-inflammatory drug naproxen were successfully separated and purified with high purity from synthetic mixtures by countercurrent chromatography with a selected biphasic solvent system. The biphasic solvent system composed of n-hexane/ethyl acetate/methanol/water (9:1:9:1, v/v/v/v) was selected according to partition performance of the three components using thin-layer chromatography. Fifty milligrams of the synthetic mixture after the three-step reaction was injected into a preparative countercurrent chromatography separation column and yielded 3.5, 14.0, and 8.0 mg of three key intermediates with 95.0, 99.0, and 98.0% purity, and the recovery of each component was 65.2, 71.2, and 69.6%, respectively. The results indicated that countercurrent chromatography is an efficient alternative and economical method for the separation and purification of intermediate components from synthetic mixtures.

Recycling countercurrent chromatography was successfully applied to the resolution of 2-(4-bromomethylphenyl)propionic acid, a key synthetic intermediate for synthesis of nonsteroidal anti-inflammatory drug loxoprofen, using hydroxypropyl-β-cyclodextrin as chiral selector. The two-phase solvent system composed of n-hexane/n-butyl acetate/0.1 mol/L citrate buffer solution with pH 2.4 (8:2:10, v/v/v) was selected. Influence factors for the enantioseparation were optimized, including type of substituted β-cyclodextrin, concentration of hydroxypropyl-β-cyclodextrin, separation temperature, and pH of aqueous phase. Under optimized separation conditions, 50 mg of 2-(4-bromomethylphenyl)propionic acid was enantioseparated using preparative recycling countercurrent chromatography. Technical details for recycling elution mode were discussed. The purities of both the S and R enantiomers were over 99.0% as determined by high-performance liquid chromatography. The enantiomeric excess of the S and R enantiomers reached 98.0%. The recovery of the enantiomers from eluted fractions was 40.8-65.6%, yielding 16.4 mg of the S enantiomer and 10.2 mg of the R enantiomer. At the same time, we attempted to enantioseparate the anti-inflammatory drug loxoprofen by countercurrent chromatography and high-performance liquid chromatography using a chiral mobile phase additive. However, no successful enantioseparation was achieved so far.

In this work, the anti-tumor constituent β-elemene was selectively separated from the volatile oil of the Curcumae Rhizoma by countercurrent chromatography with silver nitrate as selective reagent based on the formation of coordination complexes. A biphasic solvent system composed of n-hexane/methanol/water (2:1.5:0.5, v/v/v) was selected, in which 0.15 mol L−1 of silver nitrate was added to the aqueous phase. The aqueous phase was used as the stationary phase for separation of β-elemene by countercurrent chromatography after it was partially purified from the volatile oil by silica gel column chromatography. An enriched β-elemene fraction was obtained by silica gel column chromatography to improve the percentage of β-elemene from 16.5 to 46.1%. Subsequently, β-elemene was further purified from 445 mg of the partially purified sample of volatile oil by countercurrent chromatography with silver nitrate as a selective reagent, yielding 145 mg of β-elemene with greater than 99% purity, as determined by gas chromatography-mass spectrometry. The recovery of β-elemene from the crude volatile oil through two steps was around 63.6%. This article is protected by copyright. All rights reserved

3-Phenyllactic acid is an antimicrobial compound with broad-spectrum activity against various bacteria and fungus. The observed difference in pharmacological activity between optical isomeric 3-phenyllactic acid necessitates a method for enantioseparation. Chiral ligand exchange countercurrent chromatography was investigated for the enantioseparation of 3-phenyllactic acid with a synthesized chiral ligand. A two-phase solvent system was composed of n-butanol/hexane/water (0.4:0.6:1, v/v/v) to which N-n-dodecyl-l-hydroxyproline was added to the organic phase as chiral ligand and cupric acetate was added in the aqueous phase as a transitional metal ion. The influence factors were optimized by enantioselective liquid-liquid extraction. Baseline enantioseparation of racemic 3-phenyllactic acid by analytical high-speed countercurrent chromatography was achieved. The optical purities of enantiomeric 3-phenyllactic acid reached 99.0%, as determined by chiral high-performance liquid chromatography.

Two structural isomeric pentacyclic triterpenes, oleanolic acid and ursolic acid, were considered as the models for the quality control of many traditional Chinese herbal medicines and they have been proved to own important pharmacological activities. In the present work, liquid chromatographic and liquid-liquid chromatographic separation with high peak resolution of structural isomeric oleanolic acid and ursolic acid using hydroxypropyl-β-cyclodextrin as mobile phase additive was successfully achieved, respectively. A high peak resolution, RS=8.143, was achieved for the two structural isomeric compounds by conventional reverse phase high performance liquid chromatography, which was greatly improved compared with the published values. Meanwhile, a biphasic solvent system composed of n-hexane-ethyl acetate-0.1 mol/L hydroxypropyl-β-cyclodextrin (9:1:10, v/v) was selected for liquid-liquid chromatography, which provided a high peak resolution, RS = 6.573, for analytical apparatus and Rs = 8.500 for semi-preparative apparatus after optimization by liquid-liquid extractions. Two elution modes including reverse phase mode and normal phase mode were investigated for preparative separation of two acids from crude exact of Eriobotrya japonica Thunb. Furthermore, the inclusion complex between each of the two structural isomers and hydroxypropyl-β-cyclodextrin were also investigated for high performance liquid chromatography and liquid-liquid chromatography, respectively, in which formation constants were determined for oleanolic acid and ursolic acid.

Off-line comprehensive two-dimensional reversed-phase countercurrent chromatography with high-performance liquid chromatography was investigated in separation of crude ethanol extract from traditional Chinese medicinal herb Polygonum cuspidatum Sieb. et Zucc. Two-dimensional contour plots for countercurrent chromatography with high-performance liquid chromatography was obtained after comprehensive separation was completed. Total peak capacity was evaluated and approximately 810 peaks were obtained through a comprehensive two-dimensional separation. A highly orthogonality of 52.23% and a large separation space occupancy of 88.86% were achieved. Meanwhile, it was found that several components could be well separated by countercurrent chromatography while they could not be separated by high-performance liquid chromatography, and vice versa, which further indicated the orthogonality of the two separation methods. The off-line comprehensive two-dimensional countercurrent chromatography with high-performance liquid chromatography provided a promising and powerful method for separation of complex natural products.