以羟丙基-β-环糊精为手性添加剂拆分2-取代芳基丙酸:取代基对手性识别的影响（英文）

以羟丙基-β-环糊精为手性添加剂,采用反相高效液相色谱法对2-取代芳基丙酸类物质进行了手性拆分。考察了流动相的组成,包括缓冲溶液、有机改性剂以及添加剂的浓度等。缓冲溶液的pH值、有机改性剂的种类与浓度,以及添加剂的浓度对色谱峰的保留时间和分离度均有较大的影响。以YMC ODS-C_(18)(150 mm×4.6 mm,5μm)为色谱柱,乙腈-0.10 mol/L磷酸盐缓冲液(pH 3.3,含25 mmol/L添加剂)为流动相,测定了各2-取代芳基丙酸与羟丙基-β-环糊精的包结常数,考察了羟丙基-β-环糊精对各物质的包结形式。实验结果表明,羟丙基-β-环糊精与各对映体均以1∶1的形式包结,同时发现推电子取代基更有利于羟丙基-β-环糊精的包结行为,为羟丙基-β-环糊精对手性拆分的影响提供了一个有利的参考因素。     

Chiral separation of brompheniramine enantiomers by recycling high‐speed countercurrent chromatography using carboxymethyl‐β‐cyclodextrin as a chiral selector

A recycling high‐speed countercurrent chromatography protocol was proposed for the enantioseparation of brompheniramine by employing β‐cyclodextrin derivatives as a chiral selector. The two‐phase solvent system of n‐hexane/isobutyl acetate/0.10 mol/L phosphate buffer solution with a volume ratio of 2:4:6 was selected by a series of extraction experiments. Factors that affected the distribution of the enantiomers over the two‐phase system (e.g., the type and concentration of β‐cyclodextrin derivatives = pH value of the aqueous solution, and the separation temperature) were also investigated. In addition, the theory of thermodynamics is applied to verify the feasibility of the enantioseparation process and the corresponding results demonstrate that this separation process is feasible. The optimized conditions include carboxymethyl‐β‐cyclodextrin concentration of 0.010 mol/L, pH of 7.5, and temperature of 5°C. Under the optimal conditions, the purities of both monomer molecules were over 99%, and the recovery yields were 88% for (+)‐brompheniramine and 85% for (–)‐brompheniramine, respectively.     

Enantioseparation of aldols by high‐performance liquid chromatography on polysaccharide‐based chiral stationary phases that bear chlorinated substituents

Two families of aldols, obtained from the condensation of aromatic aldehydes with cyclohexanone or acetone (ten examples in each group), were analyzed by high‐performance liquid chromatography in normal phase elution mode on three polysaccharide‐based chiral stationary phases of the Lux series, namely, Lux Cellulose‐2, Lux Cellulose‐4 and Lux Amylose‐2, which share the common feature of chlorinated substituents in the chiral selectors. Following simple optimization steps, the enantioseparation of all aldols derived from cyclohexanone was achieved and the highest values of separation factor (α, 1.32 < α < 2.20) and resolution (R_s, 4.5 < R_s <17.2) were observed on Lux Cellulose‐2, with the only exception of the 4‐nitro‐substituted derivative that was better resolved on Lux Cellulose‐4. On the contrary, Lux Amylose‐2 was the best choice for aldols derived from acetone and only specific analytes in this group were resolved on the cellulose‐based supports. A variable‐temperature study of selected compounds allowed us to determine thermodynamic parameters of the enantioseparation process, which was enthalpy‐controlled in all the cases except one.     

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%.     

Preparation of chiral oxazolinyl‐functionalized β‐cyclodextrin‐bonded stationary phases and their enantioseparation performance in high‐performance liquid chromatography

A simple procedure for the synthesis of three new oxazolinyl‐substituted β‐cyclodextrins (6‐deoxy‐6‐R‐(–)‐4‐phenyl‐4,5‐dihydrooxazolinyl‐β‐cyclodextrin, 6‐deoxy‐6‐S‐(–)‐4‐phenyl‐4,5‐dihydrooxazolinyl‐β‐cyclodextrin, and 6‐deoxy‐6‐S‐(–)‐(4‐pyridin‐1‐ium‐4‐methyl‐benzenesulphonate)‐4,5‐dihy‐drooxazolinyl‐β‐cyclodextrin) and their covalent bonding to silica are reported. The ability of these chiral stationary phase columns for separating compounds is also presented and discussed. Twenty‐eight compounds were examined in the polar‐organic mobile phase mode, and 11 β‐nitroethanols were tested in the reversed‐phase mode. Excellent enantioseparations were achieved for most of the analytes, even for several challenging compounds. The rigid and flexible structures of mono‐substituted chiral groups and the fragments around the rim of the β‐cyclodextrin cavity played an important role in the separation process. Factors such as π–π stacking, dipole–dipole interactions, ion‐pairing, and steric hindrance effects were found to affect the chromatographic performance. Moreover, the buffer composition, and percentages of organic modifiers in the mobile phase, were investigated and compared. The mechanisms involved in the separation were postulated based on the chromatographic data.     

Preparation of cyclodextrin‐modified monolithic hybrid columns for the fast enantioseparation of hydroxy acids in capillary liquid chromatography

Cyclodextrins and their derivatives are one of the most common and successful chiral selectors. However, there have been few publications about the use of cyclodextrin‐modified monoliths. In this study, organic hybrid monoliths were prepared by the immobilization of derivatized β‐cyclodextrin alone or with l‐ 2‐allylglycine hydrochloride to the polyhedral oligomeric silsesquioxane methacryl substituted monolith. The main topic of this study is a combined system with dual chiral selectors (l‐ 2‐allylglycine hydrochloride and β‐cyclodextrin) as monolithic chiral stationary phase. The effect of l‐ 2‐allylglycine hydrochloride concentration on enantioseparation was investigated. The enantioseparation of the four acidic compounds with resolutions up to 2.87 was achieved within 2.5 min on the prepared chiral monolithic column in capillary liquid chromatography. Moreover, the possible mechanism of enantioseparation was discussed.     

Separation of isorhamnetin 3‐sulphate and astragalin from Flaveria bidentis (L.) Kuntze using macroporous resin and followed by high‐speed countercurrent chromatography

D4020 resin offered the best dynamic adsorption and desorption capacity for total flavonoids based on the research results from ten kinds of macroporous resin. A column packed with D4020 resin was used to optimize the separation of total flavonoids from Flaveria bidentis (L.) Kuntze extracts. The content of flavonoids in the product was increased from 4.3 to 30.1% with a recovery yield of 90%. After the treatment with gradient elution on D4020 resin, the contents of isorhamnetin 3‐sulfate and astragalin were increased from 0.49 to 8.70% with a recovery yield of 74.1% and 1.16 to 30.8%, with a recovery yield of 92.2%, respectively. Further purification was carried out by one‐run high‐speed countercurrent chromatography yielding 4.5 mg of isorhamnetin 3‐sulfate at a high purity of 96.48% and yielding 24.4 mg of astragalin at a high purity of over 98.46%.     

Characterization and purification of polyunsaturated fatty acids from microalgae by gas chromatography-mass spectrometry and countercurrent chromatography

Summary The present study was undertaken in order to characterize then to purify fatty acids from marine phytoplankton. From a crude mixture of fatty acid methyl esters it was possible to isolate by countercurrent chromatography a mixture of four polyunsaturated fatty acid methyl ester identified as being hexadecatrienoic acid methyl ester, octadecatetraenoic acid methyl ester, eicosapentaenoic acid methyl ester and docosahexaenoic acid methyl ester by gas chromatography coupled with mass spectrometry in electron impact and in positive-ion chemical ionization mode. The four polyunsaturated fatty acids are in different ratios in mixtures from the two microorganisms:Skeletonema costatum andIsochrysis galbana.     

Enantioseparation of hydroxyeicosatetraenoic acids by hydroxypropyl‐γ‐cyclodextrin‐modified micellar electrokinetic chromatography

Complete resolution of hydroxyeicosatetraenoic acid (HETE) enantiomers was achieved using hydroxypropyl‐γ‐cyclodextrin (HP‐γ‐CD)‐modified MEKC. The optimum running conditions were determined to be utilizing a 30 mM phosphate–15 mM borate buffer (pH 9.0) containing 30 mM HP‐γ‐CD and 75 mM SDS as the BGE, application of +30 kV as the effective voltage, and carrying out the experiment at 15°C. The eluents were detected at 235 nm. The method was used successfully for the simultaneous separations of (S)‐ and (R)‐enantiomers of regioisomeric 8‐, 11‐, 12‐, and 15‐HETEs. Subsequently, the optimized method was applied to evaluate the stereochemistry of 8‐ and 12‐HETEs from the marine red algae, Gracilaria vermiculophylla and Gracilaria arcuata, respectively. The 8‐HETE was found to be a mixture of 98% (R)‐enantiomer and 2% (S)‐enantiomer, while the 12‐HETE was a mixture of 98% (S)‐enantiomer and 2% (R)‐enantiomer. The present study demonstrates that the HP‐γ‐CD‐modified MEKC method is simple and sensitive and provides unambiguous information on the configuration of natural and synthetic HETEs.     

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.     

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.     

Enantioseparation of acetyltropic acid by countercurrent chromatography with sulfobutyl ether‐β‐cyclodextrin as chiral selector

Acetyltropic acid is an important synthetic intermediate for preparation of tropane alkaloid derivatives, which can be used as anticholinergic drugs, deliriants, and stimulants. In the present work, acetyltropic acid was successfully enantioseparated by countercurrent chromatography using sulfobutyl ether‐β‐cyclodextrin as chiral selector. A biphasic solvent system composed of n‐butyl acetate/n‐hexane/0.1 mol/L citrate buffer at pH = 2.2 containing 0.1 mol/L of sulfobutyl ether‐β‐cyclodextrin (7:3:10, v/v) was selected, which produced a suitable distribution ratio D_S = 1.14, D_R = 2.31 and a high enantioseparation factor α = 2.03. Baseline separation was achieved for preparative enantioseparation of 50 mg of racemic acetyltropic acid. A method for chiral analysis of acetyltropic acid by conventional reverse phase liquid chromatography with hydroxylpropyl‐β‐cyclodextrin as mobile phase additive was established, and formation constants of inclusion complex were determined. It was found that different substituted β‐cyclodextrin should be selected for enantioseparation of acetyltropic acid by countercurrent chromatography and reverse phase liquid chromatography.     

Determination of chlorophenols in red wine using ionic liquid countercurrent chromatography as a new pretreatment method followed by high‐performance liquid chromatography

A countercurrent chromatography method for the enrichment and cleanup of chlorophenols from food samples was successfully established by using an ionic‐liquid‐modified two‐phase solvent system composed of dichloromethane containing 2% 1‐butyl‐3‐methylimidazolium bis(trifluoromethanesulfonyl)imide and water. The column was firstly filled with the organic stationary phase, and then a large volume of sample was pumped into the column after it was equilibrated with pure water at the rotation speed. Finally, the trace amounts of chlorophenols extracted and enriched in the stationary phase were eluted out by an alkaline mobile phase and determined by high‐performance liquid chromatography. Under optimized conditions, the enrichment and cleanup of the chlorophenols can be fulfilled online with enrichment factors (34–65) and high recoveries (84.69–95.23%). The method has been applied to the determination of chlorophenols in real red wine samples with the limits of detection in the range of 1.89–4.21 μg/L. The present method is highly suitable for the pretreatment of large volume of aqueous sample for the determination of trace amounts of contaminants in food and environmental samples.     

Separation and purification of 9,10‐dihydrophenanthrenes and bibenzyls from Pholidota chinensis by high‐speed countercurrent chromatography

Stilbenoids are the main components of leaves and stems of Pholidota chinensis. In the present investigation, high‐speed counter‐current chromatography was used for the separation and purification of two classes of stilbenoids, namely, bibenzyls and 9,10‐dihydrophenanthrenes, on a preparative scale from whole plants of P. chinensis with different solvent systems after silica gel column chromatography fractionation. n‐Hexane/ethyl acetate/methanol/water (1.2:1:1:0.8, v/v/v/v) was selected as the optimum solvent system to purify 1‐(3,4,5‐trimethoxyphenyl)‐1′,2′‐ethanediol ( 1 ), coelonin ( 2 ), 3,4′‐dihydroxy‐5,5′‐dimethoxybibenzyl ( 3 ), and 2,?7‐?dihydroxy‐?3,?4,?6‐?trimethoxy‐?9,?10‐?dihydrophenanthrene ( 4 ). While 2,7‐dihydroxy‐3,4,6‐trimethoxy‐?9,?10‐?dihydrophenanthrene ( 5 ), batatasin III ( 6 ), orchinol ( 7 ), and 3′‐O‐methylbatatasin III ( 8 ) were purified by n‐hexane/ethyl acetate/methanol/water (1.6:0.8:1.2:0.4, v/v/v/v). After the high‐speed counter‐current chromatography isolation procedure, the purity of all compounds was over 94% assayed by ultra high performance liquid chromatography. The chemical structure identification of all compounds was carried out by mass spectrometry and ¹H and ¹³C NMR spectroscopy. To the best of our knowledge, the current investigation is the first study for the separation and purification of bibenzyls and 9,10‐dihydrophenanthrenes by high‐speed counter‐current chromatography from natural resources.     

A linear solvation energy relationship study for the reactivity of 2‐(4‐substituted phenyl)‐cyclohex‐1‐enecarboxylic, 2‐(4‐substituted phenyl)‐benzoic,and 2‐(4‐substituted phenyl)‐acrylic acids with diazodiphenylmethane in various solvents

The reactivities of 2‐(4‐substituted phenyl)‐cyclohex‐1‐enecarboxylic acids, 2‐(4‐substituted phenyl)‐benzoic acids, and 2‐(4‐substituted phenyl)‐acrylic acids with diazodiphenylmethane in various solvents were investigated. To explain the kinetic results through solvent effects, the second‐order rate constants of the examined acids were correlated using the Kamlet–Taft solvatochromic equation. The correlations of the kinetic data were carried out by means of multiple linear regression analysis, and the solvent effects on the reaction rates were analyzed in terms of initial and transition state contributions. The signs of the equation coefficients support the proposed reaction mechanism. The solvation models for all investigated carboxylic acids are suggested. The quantitative relationship between the molecular structure and the chemical reactivity is discussed, as well as the effect of geometry on the reactivity of the examined molecules. © 2010 Wiley Periodicals, Inc. Int J Chem Kinet 42: 430–439, 2010     

Continuous separation of maslinic and oleanolic acids from olive pulp by high‐speed countercurrent chromatography with elution‐extrusion mode

In this work, a continuous high‐speed countercurrent chromatography method has been developed on the basis of elution‐extrusion mode and this method was successfully applied to the separation of maslinic and oleanolic acid from the extract of olive pulp. In the process of ‘elution’, the sample solution was continuously loaded into the column and the maslinic acid was steadily eluted out in this step while highly retained oleanlic acid always stayed in the column. In the process of ‘extrusion’, the oleanlic acid was pushed out of the column with the stationary phase. In this way, we achieved a large sample loading. A total of 120 mL sample solution (about 89.55% of the column volume) which contains 600 mg olive pulp extract was pumped in the apparatus by a constant‐flow pump and the maslinic and oleanolic acids were largely separated within 120 min. Both of these two compounds presented high yields and high purities (271.6 mg for maslinic acid with 86.7% and 83.9 mg oleanolic acids with 83.4%).     

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.     

Enzymatic transformation of platycosides and one‐step separation of platycodin D by high‐speed countercurrent chromatography

Platycosides, the saponins found in the roots of Platycodon grandiflorum (Platycodi Radix), are typically composed of oleanane triterpenes with two side chains. In platycosides, platycodin D, a glucose unit at C‐3, is a major component, which has several pharmacological activities. Because of the high demand for this compound, we attempted to enzymatically convert platycodin D₃ and platycoside E, having two and three glucose units at C‐3, respectively, into platycodin D. In this study, we tested the ability of several glycosidases to transform platycosides, or more specifically, the ability to transform platycoside E and platycodin D₃ into platycodin D. To obtain pure platycodin D on a preparative scale, high‐speed countercurrent chromatography with a solvent system of ethyl acetate/n‐butanol/water (1.2:1:2, v/v/v) was used for the separation of the enzymatically transformed product. Approximately 39.4 mg of platycodin D (99.8% purity) was obtained from 200 mg of the product in a one‐step separation. The results strongly support the advantage of enzymatic transformation of the platycosides for the efficient enrichment of platycodin D in the complicated extract of the medicinal plant.     

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.     

The rôle of countercurrent chromatography in the fractionation of complex mixtures

The biologically active pricipals in nature are frequently present as only a few parts per million of complex mixtures of non-volatile components and often have limited stability. Their isolation often requires the application of all available techniques, such as adsorption chromatography, ion exchange procedures, size exclusion techniques, and solvent partition methods consistent with their physical properties and stability. The process of countercurret chromatography is essentially liquid-liquid chromatography in which the stationary liquid bed is retained in the column by a force field rather than by a solid supporting matrix. Adsorption effects are thereby eliminated. The technique is particularly advantageous in the preparative separation of milligram to gram quantities of polar and labile organic compounds and bio-particulate materials such as cells and cell fragments. Virtually any twophase solvent system, either aqueous or non-aqueous may be employed. Countercurrent chromatography (CCC) provides a convenlent alternative to adsorption chromatography for fractionation of natural products or other complex mixtures. In some cases, this high resolution method offers advantages with regard to the avoidance of contamination from solid adsorbents, versatility, and relatively inexpensive operation. The article covers some of the applications, selection of solvents, and advantages of CCC.