Developments in high‐speed countercurrent chromatography and its applications in the separation of terpenoids and saponins

High‐speed countercurrent chromatography is a liquid–liquid separation chromatographic technique, which has the unique feature of eliminating irreversible adsorption using liquid support medium, and is widely used in research and development of traditional Chinese medicine, biochemistry, food, environment analysis, and so on. In this review, some new developments of countercurrent chromatography, for instance cross‐axis countercurrent chromatography, dual countercurrent chromatography, foam countercurrent chromatography, and pH‐zone‐refining countercurrent chromatography are presented. Furthermore, the research and progress in high‐speed countercurrent chromatography techniques and its application in the separation and purification of terpenoids and saponins are reviewed.     

Off‐line comprehensive two‐dimensional reversed‐phase countercurrent chromatography with high‐performance liquid chromatography: Orthogonality in separation of Polygonum cuspidatum Sieb. et Zucc

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.     

Elution–extrusion countercurrent chromatography separation of six pairs of isomeric iridoids from Cornus officinalis Sieb. et Zucc. guided by ion current extraction in mass spectrometry

A mass spectrometry–guided elution–extrusion countercurrent chromatography protocol was developed to separate chemical components from Cornus officinalis Sieb. et Zucc. In this study, ion current extraction, a mass spectrometry–based data postacquisition method, was utilized to boost the separation power and scope of countercurrent chromatography technique. As a peak repicking and denoising tool, ion current extraction was carried out to process the liquid chromatography with mass spectrometry and the countercurrent chromatography with mass spectrometry data. So the target components were reacquired in the created extracted ion current patterns with enhanced responses and diminished background noise, which facilitated the distribution constant determination (by liquid chromatography with extracted ion current) and the targets fractionation (by countercurrent chromatography with extracted ion current). Under the guidance of the extracted ion currents of the target components and with the support of elution–extrusion mode in the countercurrent chromatography separation, six pairs of minor iridoid isomers were obtained in shortened experimental duration. Besides, a reciprocal shifted symmetry plot was established to represent the elution–extrusion countercurrent chromatography chromatogram. The results demonstrated the capability of the ion current extraction–guided elution–extrusion countercurrent chromatography protocol in discovery, analysis, and fractionation of low‐concentration and structurally similar chemicals from a complicated sample.     

A practicable strategy for enrichment and separation of four minor flavonoids including two isomers from barley seedlings by macroporous resin column chromatography,medium‐pressure LC,and high‐speed countercurrent chromatography

Separation of minor compounds especially with similar polarities and structures from complex samples is a challenging work. In the present study, an efficient method was successfully established by macroporous resin column chromatography, medium‐pressure liquid chromatography, and high‐speed countercurrent chromatography for separation of four minor flavonoids from barley seedlings. Macroporous resin column chromatography and medium‐pressure liquid chromatography were used for enrichment of these four flavonoids. High‐pressure liquid chromatography analysis showed the total content of these four flavonoids increased from 2.2% in the crude extract to 95.3% in the medium‐pressure liquid chromatography fraction. It was indicated that the combination of macroporous resin column chromatography and medium‐pressure liquid chromatography could be a practicable strategy for enrichment of minor compounds from complex sample. Then, high‐speed countercurrent chromatography was employed for separation of these four flavonoids using ethyl acetate/n‐butanol/water (0.1% glacial acetic acid) (4:1:5, v/v/v) as solvent system. As a result, four flavonoids including two isomers with purities higher than 98% were obtained. Interestingly, two flavonoids existing in one high‐pressure liquid chromatography peak were also successfully separated. All these indicated high‐speed countercurrent chromatography had great potential for separation of compounds with similar structures and polarities. This study provides a reference for efficient enrichment and separation of minor compounds from complex sample.     

In vitro screening and isolation of human aromatase inhibitors from Cicer arietinum by a novel continuous online method combining chromatographic techniques

Ultrafiltration liquid chromatography with mass spectrometry can efficiently and rapidly screen and identify ligands from the seeds of Cicer arietinum for human aromatase. Using this method, we identified 11 major compounds, including organic acids, organic acid glycosides, flavone glycosides, isoflavones, and isoflavone glycosides, as potent human aromatase inhibitors. A continuous online method, including pressurized liquid extraction, countercurrent chromatography, and preparative liquid chromatography, was developed for scaling up the production of these compounds with high purity and efficiency. The bioactivity of the separated compounds was assessed by an in vitro enzyme inhibition assay. This novel approach using a combination of ultrafiltration liquid chromatography with mass spectrometry and pressurized liquid extraction with countercurrent chromatography and preparative liquid chromatography as well as an in vitro enzyme inhibition assay could be applied to efficiently screen and isolate human aromatase inhibitors from complex samples and to the large‐scale production of functional food and nutraceutical ingredients.     

Efficient separation of tocopherol homologues in vegetable oil by ionic‐liquid‐based countercurrent chromatography using a non‐aqueous biphasic system

Tocopherol homologues are important fat‐soluble bioactive compounds with high nutritional value. However, it is of great challenge to separate these homologues because of their high structural similarities. In this work, ionic‐liquid‐based countercurrent chromatography was used for the separation and purification of tocopherol homologues. Conventional countercurrent chromatography and ionic‐liquid‐based countercurrent chromatography solvent systems were evaluated in respect of partition coefficient, separation factor, and stationary phase retention factor to separate these targets. Kind of ionic liquids, amount of ionic liquid, and sample amount were systematically optimized. A novel countercurrent chromatography non‐aqueous biphasic system composed of n‐hexane‐methanol‐1‐butyl‐3‐methylimidazolium chloride was established. The baseline separation of tocopherol mixtures was obtained in one cycle process. The ionic liquid played a key role in the countercurrent chromatography separation, which resulted in difference of partition behavior of individual tocopherol in the whole system through different hydrogen‐bonding affinity. Finally, n‐hexane‐methanol‐1‐butyl‐3‐methylimidazolium chloride (5:5:3, v/v) water‐free biphasic system was successfully applied to separate tocopherol homologues from vegetable oil that was not achieved beforehand. This method can be widely employed to separate many similar molecules such as tocotrienols, tocomonoenols, and marine‐derived tocopherol in food samples.     

Preparative isolation and analysis of alcohol dehydrogenase inhibitors from Glycyrrhiza uralensis root using ultrafiltration combined with high‐performance liquid chromatography and high‐speed countercurrent chromatography

A simple, rapid, and effective assay based on ultrafiltration combined with high‐performance liquid chromatography and high‐speed countercurrent chromatography was developed for screening and purifying alcohol dehydrogenase inhibitors from Glycyrrhiza uralensis root extract. Experiments were carried out to optimize binding conditions including alcohol dehydrogenase concentration, incubation time, temperature, and pH. By comparing the chromatograms, three compounds were found possessing alcohol dehydrogenase binding activity in Glycyrrhiza uralensis root. Under the target‐guidance of ultrafiltration combined with the high‐performance liquid chromatography experiment, liquiritin ( 1 ), isoliquiritin ( 2 ), and liquiritigenin ( 3 ) were separated by high‐speed countercurrent chromatography using ethyl acetate/methanol/water (5:1:4) as the solvent system. The alcohol dehydrogenase inhibitory activities of these three isolated compounds were assessed; compound 2 showed strongest inhibitory activity with an IC₅₀ of 8.95 μM. The results of the present study indicated that the combinative method using ultrafiltration, high‐performance liquid chromatography and high‐speed countercurrent chromatography could be widely applied for the rapid screening and isolation of enzyme inhibitors from complex mixtures.     

Enantioseparation of pheniramine enantiomers by high‐speed countercurrent chromatography using β‐cyclodextrin derivatives as a chiral selector

The enantioselective separation of pheniramine was studied by a high‐speed countercurrent chromatography method using β‐cyclodextrin derivatives as a chiral selector. Several key variables, for instance, type of organic solvent and chiral selector, concentration of chiral selector, pH value of aqueous phase, and temperature on the enantioselectivity, were investigated systematically by liquid–liquid extraction experiments. Combining the results of extraction experiments and high‐speed countercurrent chromatography, the most suitable conditions for separation of pheniramine enantiomers were obtained with the two‐phase system that consisted of isobutyl acetate/aqueous phase, containing 0.02 mol/L carboxymethyl‐β‐cyclodextrin, pH 8.50 at 278.15 K. Under the optimal conditions, pheniramine enantiomer was successfully resolved after four cycles of high‐speed countercurrent chromatography. By using high‐performance liquid chromatography to analyze the fractions, the purities of both (+)‐pheniramine and (–)‐pheniramine were over 99% and the recovery of this method was up to 85–90%.     

Preparative isolation of diterpenoids from Salvia bowleyana Dunn roots by high-speed countercurrent chromatography combined with high-performance liquid chromatography

The root of Salvia bowleyana Dunn (Lamiaceae) is used as a traditional Chinese medicine that has multiple therapeutic effects. In this study, an efficient strategy was developed to separate diterpenoid compounds, which are the main active ingredients in Salvia bowleyana Dunn roots, from complex crude extracts by high-speed countercurrent chromatography combined with preparative high-performance liquid chromatography. A two-phase solvent system comprising n-hexane–ethyl acetate–methanol–water (7:3:7:3, v/v/v/v) was selected for high-speed countercurrent chromatographic separation. Three major diterpenoids, 6α-hydroxysugiol ( 7 ), sugiol ( 8 ), and 6, 12-dihydroxyabieta-5,8,11,13-tetraen-7-one ( 9 ) were obtained at purities of 98.9, 95.4, and 96.2%, respectively, and minor diterpenoids were enriched via one-step separation. The enriched minor diterpenoids were further purified by continuous preparative high-performance liquid chromatography to yield two new norabietanoids ( 1 , 6 ) and four known compounds ( 2 – 5 ). The structures of these new compounds were determined using NMR spectroscopy, high-resolution electrospray ionization mass spectrometry, and electronic circular dichroism spectroscopy. The results suggest that high-speed countercurrent chromatography combined with preparative high-performance liquid chromatography efficiently isolates diterpenoids, including minor components, from complex natural products.     

Separation of epimeric aromatic acid (−)‐menthol esters by countercurrent chromatography using hydroxypropyl‐β‐cyclodextrin as an additive

The separation of ten epimeric aromatic acid (−)‐menthol esters by countercurrent chromatography with hydroxypropyl‐β‐cyclodextrin as the mobile phase additive was investigated, and methods for the analysis of all the epimeric esters by reversed‐phase high‐performance liquid chromatography were established. A biphasic solvent system composed of n‐hexane/20–70% methanol containing 50 mmol/L of hydroxypropyl‐β‐cyclodextrin (1:1, v/v) was selected, which provided high separation factors for five of the epimeric esters, and successful separations by countercurrent chromatography were achieved. The complete separation of five pairs of epimeric ester was obtained with the purity being over 98% for each peak fractions, as determined by high‐performance liquid chromatography. The recovery of each analyte from the eluted fractions reached around 80–88%.     

Separation and characterisation of five polar herbicides using countercurrent chromatography with detection by negative ion electrospray ionisation mass spectrometry.

Five polar herbicides were separated and characterised using high-speed analytical countercurrent chromatography (HSACCC) in conjunction with online electrospray mass spectrometry (ESI-MS). The countercurrent chromatography used a standard isocratic biphasic solvent system of hexane/ethyl acetate/methanol/water in reverse phase to effect the separation of these five environmentally important compounds. The chromatograph was coupled to a triple quadrupole mass spectrometer via a standard electrospray liquid chromatography interface that was able to give mass spectra in negative ion mode of each compound. Limits of detection are reported for this series of compounds along with representative negative ion ESI-MS data and calibrations for the separation.     

Quick method for separating target compounds from the bark of Maqian (Zanthoxylum myriacanthum var. pubescens) by high‐performance countercurrent chromatography

Choosing a suitable solvent system for a countercurrent chromatography separation presents a challenge for many researchers. In this study, we introduce a quick method of separating a target compound from the bark of Zanthoxylum myriacanthum var. pubescens by countercurrent chromatography. This method relies on the thin‐layer chromatography based generally useful estimation of solvent systems. This paper will present how to quickly choose a suitable solvent system with a thin‐layer chromatography based generally useful estimation of solvent systems working chart. O‐Methyltembamide ( 1 ) was enriched by countercurrent chromatography using n‐hexane/ethyl acetate/methanol/water (6:4:6:4) as the solvent system. Further purification was achieved by high‐performance liquid chromatography with purities of 98.2% from Z. myriacanthum var. pubescens bark.     

Countercurrent Chromatography for Isolation of Flavonol Glycosides From Ginkgo Biloba Leaves

Abstract

A procedure which combined countercurrent chromatography with gradient elution and preparative high-performance liquid chromatography was developed for the isolation and the purification of the seven predominant flavonol glycosides from Ginkgo biloba leaves.     

Enantioseparation of 3‐phenyllactic acid by chiral ligand exchange countercurrent chromatography

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.     

Separation and purification of two minor typical diarrhetic shellfish poisoning toxins from harmful marine microalgae via combined liquid chromatography with mass spectrometric detection

A novel method was developed for the purification of two typical diarrhetic shellfish poisoning toxins from toxin‐producing marine microalgae using macroporous resin, high‐speed countercurrent chromatography–mass spectrometry, and semipreparative high‐performance liquid chromatography–mass spectrometry. Analytical high‐performance liquid chromatography–mass spectrometry was used for identification and purity analysis of okadaic acid and dinophysistoxin‐1 because they exhibit no visible or ultraviolet absorption. First, four kinds of macroporous resins were investigated, and HP‐20 macroporous resin was selected for the preenrichment and cleanup of the two target toxins. Second, the resin‐purified sample was further purified using high‐speed countercurrent chromatography coupled with a mass spectrometer. The purities of the obtained okadaic acid and dinophysistoxin‐1 were 89.0 and 83.0%, respectively, as determined through analytical high‐performance liquid chromatography–mass spectrometry. Finally, further purification was carried out using semipreparative high‐performance liquid chromatography with mass spectrometry, and the purities of the final okadaic acid and dinophysistoxin‐1 products were both over 98.0% based on the analytical high‐performance liquid chromatography–mass spectrometry chromatograms and fraction spectra. This work demonstrates that the proposed purification process is a powerful method for the preparation of high‐purity okadaic acid and dinophysistoxin‐1 from toxin‐producing marine microalgae. Moreover, it is particularly important for the purification and preparation of minor toxins that exhibit no visible or ultraviolet absorption from harmful marine algae.     

pH‐zone‐refining elution–extrusion countercurrent chromatography: Separation of hydroxyanthraquinones from Cassiae semen

Seven hydroxyanthraquinones were successfully separated from the traditional Chinese medicinal herb Cassiae semen by conventional and pH‐zone‐refining countercurrent chromatography with an environmentally friendly biphasic solvent system, in which elution–extrusion mode was investigated for pH‐zone‐refining countercurrent chromatography for the first time. A two‐phase solvent system composed of n‐hexane/ethyl acetate/ethanol/water (5:3:4:4, v/v/v/v) was used for the conventional countercurrent chromatography while the same system with a different volume ratio n‐hexane/ethyl acetate/ethanol/water (3:5:2:6, v/v/v/v) was used for pH‐zone‐refining countercurrent chromatography, in which 20 mmol/L of trifluoroacetic acid was added in the organic phase as a retainer and 15 mmol/L of ammonia was added to the aqueous phase as an eluter. A 400 mg crude sample could be well separated by pH‐zone‐refining countercurrent chromatography, yielding 53 mg of aurantio‐obtusin, 40 mg of chryso‐obtusin, 18 mg of obtusin, 24 mg of obtusifolin, 10 mg of emodin, and 105 mg of the mixture of chrysophanol and physcion with a purity of over 95.8, 95.7, 96.9, 93.5, 97.4, 77.1, and 19.8%, as determined by high‐performance liquid chromatography. Furthermore, the difference in elution sequence between conventional and pH‐zone‐refining mode was observed and discussed.     

Combined application of chromatographic techniques for the separation of phenolic compounds from Stenoloma chusanum Ching

High‐speed countercurrent chromatography combined with preparative high‐performance liquid chromatography was successfully used to separate seven phenolic compounds from Stenoloma chusanum Ching. A biphasic solvent system composed of hexane/ethyl acetate/methanol/water (1:2:1:2, v/v) was used for the first step high‐speed countercurrent chromatography separation in elution–extrusion mode. A mobile phase composed of acetonitrile (18%) and pure water (82%) was used for further preparative high‐performance liquid chromatography purification. In total, the combined separation yielded seven compounds, including 3,4‐dihydroxy benzoic acid, 3,4‐dihydroxy benzaldehyde, esculetin, caffeic acid, syringic acid, luteolin, and apigenin, at a purity of over 90%. Esculetin was separated from Stenoloma chusanum Ching for the first time. The results suggest that the proposed combination method is a useful strategy for separating compounds from complex samples.     

Separation and purification of two taxanes and one xylosyl‐containing taxane from Taxus wallichiana Zucc.: A comparison between high‐speed countercurrent chromatography and reversed‐phase flash chromatography

10‐Deacetylbaccatin III, an important semisynthetic precursor of paclitaxel and docetaxel, can be extracted from Taxus wallichiana Zucc. A process for the isolation and purification of 10‐deacetylbaccatin III ( 1 ), baccatin III ( 2 ), and 7β‐xylosyl‐10‐deacetyltaxol ( 3 ) from the leaves and branches of Taxus wallichiana Zucc. via macroporous resin column chromatography combined with high‐speed countercurrent chromatography or reversed‐phase flash chromatography was developed in this study. After fractionation by macroporous resin column chromatography, 80% methanol fraction was selected based on high‐performance liquid chromatography and liquid chromatography with mass spectrometry qualitative analysis. A solvent system composed of n‐hexane, ethyl acetate, methanol, and water (1.6:2.5:1.6:2.5, v/v/v/v) was used for the high‐speed countercurrent chromatography separation at a flow rate of 2.5 mL/min. The reversed‐phase flash chromatography separation was performed using methanol/water as the mobile phase at a flow rate of 3 mL/min. The high‐speed countercurrent chromatography separation produced compounds 1 (10.2 mg, 94.4%), 2 (2.1 mg, 98.0%), and 3 (4.6 mg, 98.8%) from 100 mg of sample within 110 min, while the reversed‐phase flash chromatography separation purified compounds 1 (9.8 mg, 95.6%) and 3 (4.9 mg, 97.9%) from 100 mg of sample within 120 min.     

HPLC Purification of Solid-Phase Generated Synthetic Bombesin

Abstract

A scheme based on ion-exchange and reverse-phase high pressure liquid chromatography has been utilized for the semi-preparative and preparative purification of the solid phase generated model peptide bombesin. The final product showed a purity ≥ 99% in analytical reverse-phase high pressure liquid chromatography and was identical to authentic bombesin as demonstrated by different physico-chemical and biological criteria. The results are discussed and compared to those obtained using countercurrent chromatography.     

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

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