Limonene in Arizona liquid systems used in countercurrent chromatography. I Physicochemical properties

Limonene is a biorenewable cycloterpene solvent derived from orange peel waste. Its potential as a “green” solvent to replace heptane was recently evaluated. Countercurrent chromatography (CCC) is a preparative separation technique using biphasic liquid systems. One liquid phase is the mobile phase; the other liquid phase is the stationary phase held in place by centrifugal fields. A particular range of special proportions of the heptane/ethyl acetate/methanol/water system is called the Arizona (AZ) liquid system when the heptane/ethyl acetate ratio is exactly the same as the methanol/water ratio. A continuous polarity decrease is obtained between the most polar A composition (ethyl acetate/water or 0/1/0/1 v/v) and the least polar Z composition (heptane/methanol or 1/0/1/0 v/v), replacing heptane by limonene and methanol by ethanol produce biphasic liquid systems much more environmentallyfriendly than the original AZ compositions. The chemical compositions of the two liquid phases of 12 AZ limonene/ethyl acetate/ethanol/water proportions were fully determined by Karl-Fisher titration of water and by gas chromatography for the organic solvents. The results were compared with the compositions of the corresponding AZ mixtures containing heptane and methanol. Significant differences in ethyl acetate and ethanol distribution between phases of the two systems with identical volume proportions were established. The ratio of the upper phase over the lower phase volumes and the phase density difference are important in CCC, there are also significant differences between the classic and “green” AZ systems that are discussed.     

Alkane effect in the Arizona liquid systems used in countercurrent chromatography

Countercurrent chromatography (CCC) is a separation technique that uses a biphasic liquid system; one liquid phase is the mobile phase, the other liquid phase is the stationary phase. Selection of the appropriate liquid system can be a problem in CCC, since it is necessary to select both the “column” and the mobile phase at the same time as the first is completely dependent on the second. A range of systems with various proportions of solvents were developed to ease this choice; 23 variations of the heptane/ethyl acetate/methanol/water biphasic liquid system were labeled A to Z. This range proved to be extremely useful and became the popular Arizona (AZ) liquid system. However, authors often replace the heptane with hexane. In this work, the chemical compositions of the upper phases and the lower phases of 55 Arizona systems made with various alkanes (pentane, hexane, heptane, isooctane and cyclohexane) were determined by gas chromatography and Karl Fischer titration. The test mixture separated consisted of five steroid compounds. The lower phases were found to have similar compositions when different alkanes were used, but the upper phases were found to change. Exchanging heptane for hexane or isooctane produced minimal changes in the CCC chromatogram, while changing the proportions of the solvents resulted in an exponential change in the retention volumes. The high density of cyclohexane made liquid stationary phase retention difficult. All Arizona systems equilibrated within 30 min, but were not stable: water slowly hydrolyzed the ethyl acetate (as shown by a continuous decrease in the pH of the lower aqueous phase), especially in the water-rich systems (early alphabet letters).     

Limonene in Arizona liquid systems used in countercurrent chromatography. II Polarity and stationary-phase retention

The previous article in this series described the physico-chemical properties and chemical compositions of the two phases of the limonene–ethyl acetate–ethanol–water biphasic liquid system. This system was designed to be a “green” version of the so-called Arizona (AZ) scale of heptane–ethyl acetate–methanol compositions in which the heptane–ethyl acetate volume ratio is exactly the same as the methanol–water ratio. The first major difference between the standard and “green” AZ systems is the difference in upper and lower phase densities. The higher density of limonene compared with heptane greatly reduces the density difference of the “green” system: half the compositions have a density difference lower than 0.06 g mL^?1, precluding their use in hydrodynamic CCC columns. The other major difference is the phase polarity. The better distribution of ethanol between the upper organic and lower aqueous phases of the “green” AZ scale renders them more polar than their counterparts in standard heptane-based compositions. The test solutes aspirin and coumarin have higher distribution constants in the “green” AZ compositions. It is revealed that a hydrostatic column is suitable for use with all “green” compositions, with very good phase retention and limited driving pressure at high flow rates. A hydrodynamic column only functioned at limited flow rates with polar compositions of sufficient phase-density difference. The CCC chromatograms obtained with different compositions and columns are shown, and their peak position and sharpness discussed.     

Preparative separation of bioactive constitutes from Zanthoxylum planispinum using linear gradient counter‐current chromatography

Counter‐current chromatography is a chromatographic technique with a support‐free liquid stationary phase. In the present study, a successful application of linear gradient counter‐current chromatographic method for preparative isolation of bioactive components from the crude ethanol extract of Zanthoxylum planispinum was presented. The application of n‐hexane/ethyl acetate/methanol/water quaternary solvents, in terms of “HEMWat” or “Arizona” solvent families, in gradient elution mode was evaluated. Results indicated that slightly proportional changes of biphasic liquid systems provided the possibility of gradient elution in counter‐current chromatography, maintaining stationary phase retention in the column. With the selected quaternary solvent systems composed of n‐hexane/ethyl acetate/methanol/water (2:1:2:1 and 3:2:3:2, v/v), and optimized gradient programs, in total seven fractions were separated in 4.5 h. Most of the purified compounds could be obtained at the milligram level with over 80% purity. The present study indicated that the linear gradient counter‐current chromatographic approach possessed unique advantages in terms of separation efficiency, exhibiting great potential for the comprehensive separation of complex natural extracts.     

Using the liquid nature of the stationary phase in countercurrent chromatography. IV. The cocurrent CCC method

The retention volumes of solutes in countercurrent chromatography (CCC) are directly proportional to their distribution coefficients, K(D) in the biphasic liquid system used as mobile and stationary phase in the CCC column. The cocurrent CCC method consists in putting the liquid "stationary" phase in slow motion in the same direction as the mobile phase. A mixture of five steroid compounds of widely differing polarities was used as a test mixture to evaluate the capabilities of the method with the biphasic liquid system made of water/methanol/ethyl acetate/heptane 6/5/6/5 (v/v) and a 53 mL CCC column of the coil planet centrifuge type. It is shown that the chromatographic resolution obtained in cocurrent CCC is very good because the solute band broadening is minimized as long as the solute is located inside the "stationary" phase. Pushing the method at its limits, it is demonstrated that the five steroids can still be (partly) separated when the flow rate of the two liquid phases is the same (2 mL/min). This is due to the higher volume of upper phase (72% of the column volume) contained inside the CCC column producing a lower linear speed compared to the aqueous lower phase linear speed. The capabilities of the cocurrent CCC method compare well with those of the gradient elution method in HPLC. Continuous detection is a problem due to the fact that two immiscible liquid phases elute from the column. It was partly solved using an evaporative light scattering detector.     

Direct purification of tanshinones from Salvia miltiorrhiza Bunge by high‐speed counter‐current chromatography without presaturation of the two‐phase solvent mixture

Tanshen, the rhizome of Salvia miltiorrhiza Bunge, is a famous Traditional Chinese Medicine for multiple therapeutic remedies. This work presents the isolation and purification of tanshinone I and tanshinone IIA from the extract of the rhizome of S. miltiorrhiza by using high‐speed counter‐current chromatography (CCC) without presaturation of the two‐phase solvent mixture. The CCC method combines the results of CCC solvent system selection and components analyses of solvent mixture by GC, and thus it is possible to add accurately each individual solvent to prepare single saturated solvent phase without presaturation. The optimum CCC solvent system is a system of hexane–ethyl acetate–ethanol–water (8:2:7:3, v/v), which has been determined by usual solvent system selection and CCC runs. As a result, over 98% pure tanshinone IIA and over 94% pure tanshinone I have been obtained by using less solvent volume. Their structures have been identified by ESI‐MS, NMR spectra.     

Counter‐current chromatographic method for preparative scale isolation of picrosides from traditional Chinese medicine Picrorhiza scrophulariiflora

Apocynin, androsin, together with picroside I, II and III from crude extracts of Picrorhiza scrophulariiflora were isolated by means of high‐speed counter‐current chromatography (CCC) combining elution‐extrusion (EE) and cycling‐elution (CE) approach. The EECCC took full advantages of the liquid nature of the stationary phase for a complete sample recovery and extended the solute hydrophobicity window, while CECCC showed its unique advantage in achieving effective separation of special compounds through preventing stationary phase loss. In the present work, the biphasic liquid system composed of n‐hexane/ethyl acetate/methanol/water (1:2:1:2, v/v/v/v) was used for separation of apocynin and androsin, ethyl acetate/n‐butanol/water/formic acid (4:1:5:0.005, v/v/v/v) for picroside I, II and III. However, due to the extremely similar K values (K₁/K₂≈1.2), picroside I and III were always eluted together by several biphasic solvent systems. In this case, the CECCC exhibited great superiority and baseline separated in the sixth cycle using ethyl acetate/water (1:1, v/v) as biphasic liquid system. Each fraction was analyzed by UPLC‐UV and ESI‐MS analysis, and identified by comparing with the data of reference substances. Compared with classical elution, the combination of EE and CE approach exhibits strong separation efficiency and great potential to be a high‐throughput separation technique in the case of complex samples.     

Screening of antioxidant phenolic compounds in Chinese Rhubarb combining fast counter‐current chromatography fractionation and liquid chromatography/mass spectrometry analysis

In this paper, an effective method combing fast elution‐extrusion counter‐current chromatography (CCC) and LC/MS for rapid screening of antioxidative phenolic compounds in Chinese Rhubarb is presented. An integrated three‐coil CCC column (40 mL each coil) was used to accomplish the optimization of biphasic liquid system. In a single run (approximately 40 min), the solvent system composed of n‐hexane/ethyl acetate/methanol/water (1:1:1:1, v/v) was selected as optimum CCC liquid system for fast fractionation of the crude ethanol extract. With a 140 mL‐capacity CCC instrument, 100 mg Chinese Rhubarb extract was separated under the optimized conditions, producing six fractions in only 100 min. The quantities of each fraction were ～15 mg. In addition, each fraction was subjected to antioxidant activity assay and characterized by LC/MS analysis. Fifty compounds, including phenolic acids, phenolic glucosides and hydroxyanthraquinones, were detected by LC/MS/MS analysis. As a result, gallic acid together with Fr I showed excellent antioxidant activity, which was well consistent with previous studies and exhibited great potential for natural drug discovery program of the present method.     

Preparative separation of anti‐oxidative constituents from Rubia cordifolia by column‐switching counter‐current chromatography

An effective column‐switching counter‐current chromatography (CCC) protocol combining stepwise elution mode was successfully developed for simultaneous and preparative separation of anti‐oxidative components from ethyl acetate extract of traditional Chinese herbal medicine Rubia cordifolia. The column‐switching CCC system was interfaced by a commercial low‐pressure six‐port switching valve equipped with a sample loop, allowing large volume introduction from the first dimension (1^st‐D) to the second dimension (2^nd‐D). Moreover, to extend the polarity window, three biphasic liquid systems composed of n‐hexane/ethyl acetate/methanol/water (1:2:1:2, 2:3:2:3, 5:6:5:6 v/v) were employed using stepwise elution mode in the 1^st‐D. By valve switching technique the whole interested region of 1^st‐D could be introduced to second dimension for further separation with the solvent system 5:5:4:6 v/v. Using the present column‐switching CCC protocol, 500 mg of crude R. cordifolia extract were separated, producing milligram‐amounts of four anti‐oxidative components over 90% pure. Structures of purified compounds were identified by ¹H and ¹³C NMR.     

Two-dimensional counter-current chromatography: 1st traditional counter-current chromatography, 2nd acid-base elution counter-current chromatography

An on-line column-switching counter-current chromatography (CCC) with solid-phase trapping interphase is presented in this paper. The large volume injection is avoided using solid-phase trapping interphase. Thereby, totally different chemical composition biphasic solvent system can be utilized to enhance system orthogonality. In the present work, a 140 mL-capacity CCC instrument was used in the first dimension, and a parallel three-coil CCC centrifuge (40 mL each coil) was used in the second dimensional separation allowing three injections at the same time. With biphasic solvent system composed of n-hexane: ethyl acetate: methanol: water (1:1:1:1, v/v), five well-separated fractions were obtained in the first dimension. Two fractions were online concentrated and further separated in the second dimension with solvent system composed of methyl tert-butyl ether: acetonitrile: water (2:2:3, v/v), where trifluoroacetic acid (10 mM) was added to the upper organic phase as a retainer and triethylamine (10mM) to the aqueous mobile phase as an eluter. Four hydroxyanthraquinones were successfully separated and purified from Chinese medicinal plant Rheum officinale in only one step.     

Centrifugal partition chromatography directly interfaced with mass spectrometry for the fast screening and fractionation of major xanthones in Garcina mangostana

Xanthones are well known for their interesting phytochemical properties, which make them attractive to the pharmaceutical and medicinal industry. We have therefore developed a method to analyse the major xanthones in Garcina mangostana. The xanthones were extracted by pressurized liquid extraction with ethanol and separated at the semi-preparative scale by centrifugal partition chromatography (CPC) with a biphasic solvent system consisting of heptane/ethyl acetate/methanol/water (2:1:2:1, v/v/v/v). A CPC-electrospray ionisation MS coupling was performed and used to simultaneously separate and identify the compounds. Thanks to a variable flow splitter and an additional stream of ethanol/1 mol L⁻¹ ammonium acetate (95:5, v/v), all the compounds were ionised, detected and monitored whatever the solvents used in mobile phase for the CPC separation. The dual mode or elution–extrusion which are less solvent-consuming and faster than the elution mode were used without loss of ionisation and detection.     

Separation of caffeoylquinic acids and flavonoids from Asteris souliei by high‐performance counter‐current chromatography and their anti‐inflammatory activity in vitro

Eleven compounds were successfully separated from Asteris souliei by using a two‐step high‐performance counter‐current chromatography method. The first step involved a reversed phase isocratic counter‐current chromatography separation using hexane/ethyl acetate/methanol/water (1:0.8:1:1 v/v/v/v), which produced three fractions, the first two of which were mixtures. The second step used step‐gradient reversed‐phase counter‐current chromatography with hexane/butanol/ethyl acetate/methanol/water (1:0.5:3.5:1:4 v/v/v/v/v) initially followed by hexane/ethyl acetate/methanol/water (1:2:1:2 v/v/v/v) to separate Fraction 1 into seven compounds; and hexane/ethyl acetate/methanol/water (1:1:1:1.2 v/v/v/v) to separate Fraction 2 into three further compounds. The chemical structures of the separated compounds were identified by ESI‐MS and NMR spectroscopy (¹H and ¹³C). Baicalin ( 5 ), eriodictyol ( 7 ), apigenin‐7‐glycoside ( 8 ), quercetin ( 9 ), luteolin ( 10 ), and apigenin ( 11 ) showed obvious inhibitory effects on lipopolysaccharide‐induced nitric oxide production in RAW264.7 cells at a concentration of 10 μg/mL.     

Preparative separation of five flavones from flowers of Polygonum cuspidatum by high‐speed countercurrent chromatography

A preparative high‐speed countercurrent chromatography method was successfully used for the isolation of five minor flavones from Polygonum cuspidatum flowers. Among them, three compounds were obtained from P. cuspidatum for the first time. A twin two‐phase solvent system composed of n‐hexane/ethyl acetate/ethanol/water (1:6:3:6, v/v/v/v) and petroleum ether/ethyl acetate/methanol/water (2:4:3:3, v/v/v/v) was developed. Compounds were obtained from the fraction B and fraction C prepurified by silica gel column chromatography. Five minor compositions, 6.8 mg of hesperidin, 11.2 mg of phloridzin, 4.9 mg of luteolin, 5.3 mg of hyperin, and 3.7 mg of luteoloside were obtained from 140 mg of the fraction B and 110 mg of fraction C with a purity of 95.3, 96.4, 98.0, 96.8, and 95.3%, respectively, as determined by high‐performance liquid chromatography. The structures of these compounds were identified by ¹H and ¹³C NMR spectroscopy.     

Rapid screening of bioactive components from Zingiber cassumunar using elution-extrusion counter-current chromatography

Elution-extrusion counter-current chromatography (EECCC) takes full advantages of the liquid nature of the stationary phase. It effectively extends the solute hydrophobicity window that can be studied and renders the CCC technique particularly suitable for rapid analysis of complex samples. In this paper, EECCC was used to screen the crude ethanol extract of Zingiber cassumunar and to isolate milligram-amounts of bioactive components. The two column volume (2V(C)) EECCC method was applied to rapidly optimize the composition of the biphasic liquid system in both reversed- and normal-phase separation mode. With the n-hexane/ethyl acetate/methanol/water 1/1/1/1 (v/v) system, 100mg of crude Z. cassumunar extract were fractionated on a 140 mL-capacity semi-preparative hydrodynamic CCC column and 0.5 g on a 1600 mL column for large-scale preparation. Satisfactory separation efficiency was achieved in both cases, producing milligram-amounts of four phenylbutenoids over 90% pure and of a mixture of diastereoisomers (phenylbutenoid dimers). However, the global throughputs of the two columns were 8 and 11 mg/h, not very different. This is due to the fact that the 1600 mL column could not retain the liquid stationary phase as well as the smaller 140 mL column. It was necessary to work at much lower flow rate than calculated. Methanol was added as a post-column clarifying reagent for stable continuous UV detection. A lipophilic biphasic liquid system composed of n-hexane/acetonitrile/water (5/3/2, v/v) allowed to resolve the pair of diastereoisomers with the larger preparative instrument producing 35 mg of the (+/-)-trans form 99.1% pure and 28 mg of the (+/-)-cis isomer 98.1% pure. Compared with classical elution, the EECCC approach exhibits strong separation efficiency and great potential to be a high-throughput separation technique in the case of complex samples.     

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.     

Activity coefficient at infinite dilution, azeotropic data, excess enthalpies and solid–liquid-equilibria for binary systems of alkanes and aromatics with esters

Binary azeotropic data have been measured at different pressures for ethyl acetate + heptane, methyl acetate + heptane, isopropyl acetate + hexane and isopropyl acetate + heptane by means of a wire band column. Additionally activity coefficients at infinite dilution have been determined for ethyl acetate and isopropyl acetate in decane and dodecane in the temperature range between 303.15 and 333.15 K with the help of the dilutor technique. Furthermore excess enthalpies for the binary systems methyl acetate + hexane, methyl acetate + decane, ethyl acetate + hexane and ethyl acetate + decane at 363.15 and 413.15 K have been studied with the help of isothermal flow calorimetry. Finally solid–liquid equilibria for the systems ethyl myristate + benzene and ethyl myristate + p-xylene have been investigated by a visual technique. All these data have been used for the revision and extension of the group interaction parameters of the group contribution method modified UNIFAC (Dortmund) and the group contribution equation of state VTPR. The experimental data was compared with the results predicted using the group contribution method modified UNIFAC (Dortmund) and the group contribution equation of state VTPR.     

A simple and efficient protocol for large‐scale preparation of three flavonoids from the flower of Daphne genkwa by combination of macroporous resin and counter‐current chromatography

In this paper, macroporous resin column chromatography and counter‐current chromatography (CCC) were applied for large‐scale preparative separation of three flavonoids from the flower of Daphne genkwa, a famous Chinese medicinal herb. Nine kinds of resins were investigated by adsorption and desorption tests and D101 macroporous resin was selected for the first cleaning‐up, in which 40% aqueous ethanol was used to remove the undesired constituents and 90% aqueous ethanol was used to elute the targets. The crude extract after the first step was directly subjected to the preparative CCC purification using the solvent system composed of n‐hexane–ethyl acetate–methanol–water (4:5:4:5, v/v). The compounds apigemin (823 mg), 3‐hydroxyl‐genkwanin (842 mg) and genkwanin (998 mg) with the purities of 98.79, 97.71 and 93.53%, respectively, determined by HPLC were produced from 3‐g crude extract only in one CCC run. Their chemical structures were identified by MS, UV and the standards.     

Distribution ratio, distribution constant and partition coefficient. Countercurrent chromatography retention of benzoic acid

There is some confusion in chromatography between terms such as solute distribution ratio, distribution constant and partition coefficient. These terms are very precisely defined in the field of liquid-liquid systems and liquid-liquid extraction as well as in the field of chromatography with sometimes conflicting definitions. Countercurrent chromatography (CCC) is a chromatographic technique in which the stationary phase is a support-free liquid. Since the mobile phase is also liquid, biphasic liquid systems are used. This work focuses on the exact meaning of the terms since there are consequences on experimental results. The retention volumes of solutes in CCC are linearly related to their distribution ratios. The partition coefficient that should be termed (IUPAC recommendation) distribution constant is linked to a single definite species. Using benzoic acid that can dimerize in heptane and ionize in aqueous phase and an 18 mL hydrodynamic CCC column, the role and relationships between parameters and the consequences on experimental peak position and shape are discussed. If the heptane/water distribution constant (marginally accepted to be called partition coefficient) of benzoic acid is 0.2 at 20 °C and can be tabulated in books, its CCC measured distribution ratio or distribution coefficient can change between zero (basic aqueous mobile phase) and more than 25 (acidic aqueous mobile phase and elevated concentration). Benzoic acid distribution ratio and partition coefficient coincide only when both dimerization and ionization are quenched, i.e. at very low concentration and pH 2. It is possible to quench dimerization adding butanol in the heptane/water system. However, butanol additions also affect the partition coefficient of benzoic acid greatly by increasing it.     

Purification of the seven tetranortriterpenoids in neem (Azadirachta indica) seed by counter-current chromatography sequentially followed by isocratic preparative reversed-phase high-performance liquid chromatography

Counter-current chromatography (CCC) sequentially followed by isocratic preparative reversed-phase high-performance liquid chromatography was used to isolate the seven bio-actives (azadirachtin A, azadirachtin B, azadirachtin H, desacetylnimbin, desacetylsalannin, nimbin and salannin) from the seed concentrate (NSC) of the neem tree (Azadirachta indica A. Juss). Reproducible, narrow polarity range, high purity fractions were obtained from repeated injections of the NSC (700 mg loadings/injection), on to a relatively small volume CCC coil (116 mL). The CCC biphasic solvent system chosen was hexane:butanol:methanol:water (1:0.9:1:0.9, v/v). A mass balance of injected material showed that 95+% were recovered.     

Scale-up of counter-current chromatography: Demonstration of predictable isocratic and quasi-continuous operating modes from the test tube to pilot/process scale

Predictable scale-up from test tube derived distribution ratios and analytical-scale sample loading optimisation is demonstrated using a model sample system of benzyl alcohol and p-cresol in a heptane:ethyl acetate:methanol:water phase system with the new 18 L Maxi counter-current chromatography centrifuge. The versatility of having a liquid stationary phase with its high loading capacity and flexible operating modes is demonstrated at two different scales by separating and concentrating target compounds using a mixture of caffeine, vanillin, naringenin and carvone using a quasi-continuous technique called intermittent counter-current extraction.