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.     

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.     

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%.     

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.     

Use of the ionic liquid 1-butyl-3-methylimidazolium hexafluorophosphate in countercurrent chromatography

Room temperature ionic liquids (RTIL) are molten salts that are liquids at room temperature. Their liquid state makes them possible candidates as solvents in countercurrent chromatography (CCC), which uses solvents as both the mobile and stationary phases. The study focuses on 1-butyl-3-methylimidazolium hexafluorophosphate (BMIM PF₆), an easy to synthesize and purify RTIL whose melting point is –8°C. It is shown that BMIM PF₆ behaves like a solvent of significant polarity (comparable with that of ethanol). The ternary phase diagram water–acetonitrile–BMIM PF₆ is given, because it was necessary to add acetonitrile to reduce the ionic liquid viscosity. The 40:20:40% w/w water–acetonitrile–BMIM PF₆ biphasic liquid system was found to be appropriate as a biphasic liquid system for CCC. Different aromatic solutes, including bases, acids, and neutral compounds, were injected into the CCC column to estimate their distribution constants between the ionic liquid-rich phase and the aqueous phase. The resulting K_il/w constants were compared with the corresponding literature octanol–water partition coefficients, K_o/w. The important drawbacks in the use of RTIL in CCC are clearly pointed out: high viscosity producing pressure build-up, UV absorbance limiting the use of the convenient UV detector, and non-volatility precluding the use of the evaporative light-scattering detector for continuous detection.     

Extractant Effect on the Retention of the Stationary Phase in a Rotating Coiled Column

The effects of physicochemical properties of two-phase liquid systems (interfacial tension and differences in density and viscosity) on the retention of the stationary phase in the column were examined. These effects mainly determine the separation parameters of compounds. Extractant/decane–aqueous phase systems were used; their physicochemical properties changed both as a result of adding an extractant (di-(2-ethylhexyl) phosphoric acid, trioctylamine, or tributyl phosphate) to the organic solvent and because of a change in the composition of the aqueous phase. Aqueous ammonium sulfate of varying concentration was used as a mobile phase. It was shown that interfacial tension substantially affects the behavior of the systems under consideration. An increase in the ammonium sulfate concentration only slightly affects the retention factor of the stationary phase in the column. With a proper choice of the stationary phase, countercurrent chromatography can be used for the extraction of components from salt solutions of various concentrations.     

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.     

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.     

Studies on the retention behaviour of some thiazolylazo derivatives in reversed phase liquid chromatography

The retention behaviour of thiazolylazo derivatives, 4-(2-thiazolylazo) resorcinol (TAR), 4-(2-thiazolylazo)-orcinol (TAO), 2-(2-thiazolylazo)-4-methylphenol (TAC) and 1-(2-thiazolylazo)-2-naphtol (TAN) was studied by reversed phase liquid chromatography. The optimum conditions for the separation of four thiazolylazo derivatives were examined with respect to column, flow rate, mobile phase composition and pH of mobile phase. These derivatives were separated simultaneously on Symmetry C₈ column using composition of acetonitrile/water (60/40, v/v) as mobile phase. The capacity factor (k′) has been decreased at higher pH than pK_a of solute which may due to the increasing concentration of the ionized species as increase the pH of mobile phase. The dependence of log k′ on the volume faction of water in the binary mobile phase and k′ on the liquid–liquid extraction distribution ratio (D_c) in acetonitrile–water (60/40, v/v)/n-octane extraction system for thiazolylazo derivatives were obtained good linear relationship. The results showed that the retention behaviour of these derivatives was mainly affected by the hydrophobic interaction between thiazolylazo as solute and mobile phase.     

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.     

Silver ion coordination countercurrent chromatography: Separation of β‐elemene from the volatile oil of Curcumae Rhizoma

In this work, the antitumor 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 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%.     

Isolation and purification of alkaloids from lotus leaves by ionic‐liquid‐modified high‐speed countercurrent chromatography

An effective high‐speed countercurrent chromatography method was successfully established by using ionic liquids as the modifier of the two‐phase solvent system. Adding a small amount of ionic liquids significantly shortens the separation time and improves the separation efficiency. The conditions of ionic‐liquid‐modified high‐speed countercurrent chromatography including solvent systems, types and content of added ionic liquids, and ionic liquids posttreatment were investigated. The established method was successfully applied to separate alkaloids from lotus leaves using a two‐phase solvent system composed of petroleum ether/ethyl acetate/methanol/water/[C₄mim][BF₄] (1:5:1:5:0.15, v/v/v/v/v). Four alkaloids pronuciferine (1.7 mg), N‐nornuciferine (4.3 mg), nuciferine (3.1 mg), and roemerine (2.1 mg) were obtained with the purities of 90.53, 92.25, 99.86, and 98.63%, respectively, from 100 mg crude extract of lotus leaves. The results indicated that the ionic‐liquid‐modified high‐speed countercurrent chromatography method was suitable for alkaloid separation from lotus leaves and would be a promising method for the separation of alkaloids from other natural products.     

PREPARATIVE ISOLATION AND PURIFICATION OF CHEMICAL CONSTITUENTS OF BELAMCANDA BY MPLC, HSCCC AND PREP-HPLC

Combined with medium-pressure liquid chromatography (MPLC) and preparative high-pressure liquid chromatography (Prep-HPLC), high-speed countercurrent chromatography (HSCCC) was successfully applied for separation and purification of isoflavonoids from the extract of belamcanda. HSCCC separation was performed on a two-phase solvent system composed of methyl tert-butyl ether -ethyl acetate - n-butyl alcohol - acetonitrile -0.1% aqueous trifluoroacetic acid at a volume radio of 1:2:1:1:5. Semi-purified peak fractions from HSCCC separation were further purified by Prep-HPLC. Nine well-separated fractions were analyzed by HPLC-UV absorption spectrometry to determine their purities and characterized with ESI-MS(n). Except for peaksland VII (unknown) seven compounds were identified as apocynin (peak II), mangiferin (peak III), 7-O-methylmangiferin (peak IV), hispidulin (peak V), 3'-hydroxyltectoridin (peak VI), iristectorin B (peak VII), isoiridin (peak IX).     

Separation of six xanthones from Swertia franchetiana by high‐speed countercurrent chromatography

In our present study, two groups of xanthones isomers (1‐hydroxy‐3,5,8‐trimethoxyxanthone and 1‐hydroxy‐3,7,8‐trimethoxyxanthone; 1,8‐dihydroxy‐3,7‐dimethoxyxanthone and 1,8‐dihydroxy‐3,5‐dimethanolxanthone) and other two xanthones (3‐methoxy‐1,5,8‐trihydroxyxanthone and 3,5‐dimethoxy‐1‐hydroxyxanthone) were separated from Swertia franchetiana . First, a solvent system composed of petroleum ether/methanol/water (2:1:0.6, v/v) was developed for the liquid–liquid extraction of these xanthones from the crude extract. Then, an efficient method was established for the one‐step separation of these six xanthones by high‐speed countercurrent chromatography using n‐hexane/ethyl acetate/methanol/ethanol/water (HEMEW; 6:4:4:2:4, v/v) as the solvent system. The results showed that liquid–liquid extraction could be well developed for efficient enrichment of target compounds. Additionally, high‐speed countercurrent chromatography could be a powerful technology for separation xanthones isomers. It was found ethanol could be a good methanol substitute when the HEMEW system could not provide good separation factors.     

Isolation and characterization of methoxylated flavones in the flowers of Primula veris by liquid chromatography and mass spectrometry

Characterization of six flavones, which were named substances G1, G2, G3, G4, G5 and G6 according to their R_F values in normal-phase thin-layer chromatography, is reported. The pure flavones were purified after maceration with methanol by normal-phase solid-phase extraction, normal-phase medium-pressure liquid chromatography, normal-phase preparative thin-layer chromatography and preparative reversed-phase high-performance liquid chromatography (RP-HPLC). The collected fractions of several isolation steps were analyzed by normal-phase (NP) and RP-HPLC. Detection and identification of the substances G was accomplished by UV detection at 213–216 nm, diode array UV detection, or fluorescence detection (λ_ex=330 nm; λ_em=440 nm). The molecular mass, the elementary composition, and the structure of the six components was determined by electron-impact high-resolution mass spectrometry (EI-HRMS). Substance G4 was identified as 3′,4′,5′-trimethoxyflavone. The substances G1–G6 were shown to be mono-, di- tri- and pentamethoxyflavones. HPLC–electrospray ionization tandem mass spectrometry (ESI-MS–MS) of the flavones was carried out employing a 150×2 mm I.D. column packed with a 3 μm/100 Å octadecylsilica stationary phase and a mobile phase comprising 1.0% acetic acid in water–acetonitrile (50:50). Comparative RP-HPLC–ESI-MS of the raw methanol extract and the isolated substances G1–G6 proved that the isolated compounds were pure and were not artifacts. Finally, RP-HPLC–ESI-MS–MS was used to identify substances G1–G6 in phytopharmaceutical drugs.     

Liquid countercurrent chromatography: A new approach to creating a concentration gradient in the stationary phase

The capability of dynamic phase mixing in a two-phase liquid system in a rotating coiled column, which is used to separate substances in liquid countercurrent chromatography, was studied. The effects of the hydrodynamic conditions of the chromatographic process and the physicochemical properties of the two-phase liquid systems now in use on the efficiency of phase mixing in a rotating coiled column were found. A new approach to creating a concentration gradient in the stationary phase was proposed. The following three modes of mixing stationary-phase regions within a column were found: the formation of a stable reagent concentration gradient within the rotating coiled column, the slow mixing of zones, and the complete mixing of zones.__________Translated from Zhurnal Analiticheskoi Khimii, Vol. 60, No. 4, 2005, pp. 404–411.Original Russian Text Copyright © 2005 by Maryutina, Rakcheev.     

Simulation of the Mechanism of Liquid Stationary Phase Retention in a Rotating Coil Column: Hydrophobic Liquid Systems

A mathematical model was developed which describes the behavior of various liquid systems in a rotating coil column used for liquid countercurrent chromatography. The conditions of retaining the liquid stationary phase were described in detail for hydrophobic (rapidly settling) systems. Various forces that act on liquids in the column were considered. An equation was derived for the steady-state retention process. Mathematical expressions were obtained, which make it possible to estimate the stationary-phase volume retained in a column with consideration for the physicochemical properties of the liquid system in use, the rate of column rotation, and the main design parameters of the planet centrifuge. This volume directly affects the column capacity and separation efficiency and it is crucial for choosing liquid systems and experimental conditions.     

Preparative enantioseparation of loxoprofen precursor by recycling countercurrent chromatography with hydroxypropyl‐β‐cyclodextrin as a chiral selector

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.     

Simulation of the Mechanism of Liquid Stationary Phase Retention in a Rotating Coiled Column: Hydrophilic Liquid Systems

A mathematical model was developed which describes the behavior of various liquid systems in a rotating coiled column used in countercurrent chromatography. The conditions of retaining the liquid stationary phase were described in detail for hydrophilic (slowly settling) systems. Various forces that act on liquids in the column were considered. Equations were derived for the steady-state retention process. Mathematical expressions were obtained which make it possible to estimate the stationary-phase volume retained in a column with consideration for the physicochemical properties of the liquid system in use, the speed of column rotation, and the main design parameters of the planet centrifuge. This volume directly affects the column capacity and separation efficiency, and it is crucial for choosing liquid systems and experimental conditions.     

Analysis of C60 and C70 fullerenes using high-performance liquid chromatography–Fourier transform infrared spectroscopy

The performance of Fourier transform infrared spectroscopy (FT-IR) detection coupled to high-performance liquid chromatography for the analysis of C₆₀ and C₇₀ fullerenes was investigated. The isocratic separation method involved an octadecylsilane (ODS) column and an acetonitrile–toluene (1:1) mobile phase. The hyphenated system was designed with a split valve to control eluent volume leading to the FT-IR detector; this allowed for additional coupling of the liquid chromatograph to ultraviolet–visible detection. On-line FT-IR spectra of C₆₀ and C₇₀ were matched with standard off-line FT-IR spectra from the literature. In addition, with band chromatograms individual fullerenes can be identified using FT-IR active modes known specifically for each fullerene. Few changes to a pre-existing HPLC–UV method were necessary for the HPLC–FT-IR method, and there was no need for fraction collection to identify the fullerenes C₆₀ and C₇₀.