Influence of Mobile Phase Composition on the Enantioseparation of Methoxyl Flavanones with Self-prepared CDMPC Column and Chiral Recognition Mechanism

Chiral stationary phases (CSPs) based on the cellulose derivatives have proven to be one type of the most useful CSPs in high-performance liquid chromatography because of their versatility, durability and loadability1, 2. Flavanone was often used as a standard chiral compound for evaluating cellulose derivative for chiral columns1, 3. Krause and Galensa4 reported the enantioseparation of flavanone and its seven derivatives on six kinds of commercial chiral column. 4-Methoxyl flavanone, 5-…     

Chiral separation of diastereomeric flavanone-7-O-glycosides in citrus by capillary electrophoresis

The 2S- and 2R-diastereomers of major flavanone-7-O-glycosides found in sweet orange (Citrus sinensis), mandarine (Citrus deliciosa), grapefruit (Citrus paradisi), lemon (Citrus limon), and sour or bitter orange juice (Citrus aurantium) were separated for the first time by chiral capillary electrophoresis (CE) employing various buffers with combined chiral selectors. Native cyclodextrins (CDs), neutral and charged CD derivatives were examined as chiral additives to the background electrolyte (BGE). Separation efficiency has not proved satisfactory with one single CD as chiral selector in the buffer, a full and simultaneous separation could often be achieved only by using combined buffer with two different CDs. Chiral separation of major flavanones in sweet orange, mandarine and grapefruit juices raised more difficulties than in lemon and sour orange juices as narirutin will not readily build complexes with most CDs. Diastereomeric flavanones of mature and immature grapefruits were compared and some differences were found: naringin showed different diastereomeric ratio and 2S-prunin appeared only in immature grapefruit. Marmalade was also examined by chiral CE. Its major flavanones corresponded to flavanone pattern of mixed sour and sweet oranges.     

Enantiomer separation of drugs by micellar electrokinetic chromatography using chiral surfactants

A review surveying enantiomer separations by micellar electrokinetic chromatography (MEKC) using chiral surfactants is described. MEKC is one of the most popular techniques in capillary electrophoresis, where neutral compounds can be analyzed as well as charged ones, and the use of chiral micelles enable one to achieve the enantioseparation. The chiral MEKC systems are briefly reviewed according to the types of chiral surfactants along with typical applications. As chiral micelles or pseudostationary phases in MEKC, various natural and synthetic chiral surfactants are used, including several low-molecular-mass surfactants and polymerized surfactants or high-molecular-mass surfactants. Cyclodextrin modified MEKC using chiral micelles is also considered.     

Enantioselectivity of alcohol-modified polymeric surfactants in micellar electrokinetic chromatography

A novel method of modifying sodium undecanoyl-L-leucinate (SUL) micelles employed in chiral separation of analytes in micellar electrokinetic chromatography (MEKC) to enhance selectivity toward specific analytes is discussed. The current study aimed at modifying the SUL micelles by introducing different alcohols into the mono-SUL micelles. The micellar solutions were then polymerized in the presence of alcohols followed by postpolymerization extraction of the alcohols to yield alcohol-free polymeric surfactants (poly-L-SUL). The effects of hexanol (C(6)OH) and undecylenyl alcohol (C(11)OH) on micellar properties of this surfactant were investigated by use of surface tensiometry, fluorescence spectroscopy, pulsed field gradient-nuclear magnetic resonance (PFG-NMR), and MEKC. The surface tension and PFG-NMR studies indicated an increase in the critical micelle concentration (cmc) and micellar size upon increasing the alcohol concentration. Fluorescence measurements suggested that alcohols induce closely packed micellar structures. Coumarinic and benzoin derivatives, as well as (+/-)-1, 1'-binaphthyl-2,2'-dihydrogen phosphate (BNP) were used as test analytes for MEKC experiments. Examination of MEKC data showed remarkable resolutions and capacity factors of coumarinic derivatives obtained with modified poly-L-SUL as compared to the unmodified poly-L-SUL. Evaluation of fluorescence, PFG-NMR, and MEKC data suggest a strong correlation between the polarity and hydrodynamic radii of alcohol-modified micelles and the resolution of the test analytes.     

表面活性剂在高效毛细管电泳中的作用

表面活性剂作为缓冲液添加剂已广泛用于高效毛细管电泳中,综述了阴离子、阳离子、两性离子、非离子及手性等多种表面活性剂在离子、中性分子、手性化合物、多肽和蛋白质分离等方面的作用,介绍了其作用机理与改善高效毛细管电泳分离的原理。     

Separation of O- and C-allyl glycoside anomeric mixtures by capillary electrophoresis and high-performance liquid chromatography

Rapid and reliable methods for the analysis of O- and C-allyl galactopyranosides and glucopyranosides are presented, based on capillary zone electrophoresis (CZE) and micellar electrokinetic capillary chromatography (MEKC). In MEKC, the formation of chromophoric and charged complexes between the saccharides and borate as well as the hydrophobic interactions with micelles jointly contributed to the selective separation and sensitive detection of all the investigated anomeric couples. Some non-purified synthesis mixtures of C-allyl glycosides were successfully characterised without pre-treatment. MEKC buffer conditions for which glycosides separation was successfully achieved were then exported and applied to reverse-phase liquid chromatography (RP-HPLC), for the quantitative isolation of each allyl glycoside anomer. Identification of the obtained anomeric products was performed by electrospray mass spectrometry and (13)C NMR spectroscopy. Glycoside-solvent interactions driving the selective anomeric separation were shortly addressed and discussed on the basis of sugar derivatives structural differences.     

Separation and selectivity of benzophenones in micellar electrokinetic chromatography using sodium dodecyl sulfate micelles or sodium cholate modified mixed micelles

The separation and selectivity of nine benzophenones in micellar electrokinetic chromatography (MEKC) using sodium dodecyl sulfate (SDS) micelles or sodium cholate (SC) modified mixed micelles were investigated in the pH range 6.5-8.0. The results indicate that the combined effects of buffer pH and SC concentration can greatly affect the separation and selectivity of benzophenones, particularly for benzophenones possessing a hydroxyl substituent at the 4-position of the aromatic ring with respect to the carbonyl moiety when using SDS-SC mixed micelles. Better separability can be obtained with SDS-SC mixed micelles than with SDS micelles. Complete separation of nine benzophenones in MEKC can be achieved with an appropriate choice of buffer pH and the concentration of SDS micelles or SC modified mixed micelles. The dependence of the migration order of those benzophenones based on their structures and solute-micelle interactions is discussed.     

Analysis of flavanone-7-O-glycosides in citrus juices by short-end capillary electrochromatography

The separation of the major flavanone-7-O-glycoside constituents of Citrus was carried out by isocratic reversed phase capillary electrochromatography using a 75 microm i.d. silica fused column packed with 5 microm ODS silica gel. In comparison to HPLC mode, capillary electrochromatography resolution of flavanone glycosides was obtained with a high selectivity factor. Optimum separation conditions were found using a mixture of ammonium formate (pH 2.5)--acetonitrile (8:2, v/v) as the mobile phase by the short-end injection mode. Under these conditions all the investigated flavanones were baseline-resolved within short analysis time (i.e. between 5 and 10 min). A study, evaluating the intra- and inter-day repeatability as well as limit of detection and method linearity, was developed in accordance with the analytical procedures for method validation. The developed method was applied for the quantitative analysis of flavanone glycosides in commercial fruit juices (sweet orange, lemon and grapefruit).     

Stereoisomer separation of flavanones and flavanone‐7‐O‐glycosides by means of nanoliquid chromatography employing derivatized β‐cyclodextrins as mobile‐phase additive

A nanoliquid chromatographic method for the stereoisomer separation of some flavanone aglycones and 7‐O‐glycosides has been proposed employing a C18 capillary column and a chiral mobile‐phase additive such as cyclodextrin. The chiral separation of eriodictyol, naringenin, and hesperitin was obtained by addition of carboxymethyl‐β‐cyclodextrin to the mobile phase, whereas eriocitrin, naringin, narirutin, and hesperidin diastereoisomers were resolved by using sulfobutyl ether‐β‐cyclodextrin. The influence of the composition of the mobile phase, the length of the capillary column, and the flow rate on the chiral recognition were investigated. At optimum conditions, baseline separation for the selected aglycones and glycosylated forms were achieved with a mobile phase consisting of 50 mM sodium acetate buffer pH 3 and 30% methanol containing 20 mM of carboxymethyl‐β‐cyclodextrin and 10 mM of sulfobutyl ether‐β‐cyclodextrin, respectively. Precision, linearity, and sensitivity of the method were tested. Limits of detection and quantification for the studied flavanone glycosides were in the range 1.3‐2.5 and 7.5‐12.5 µg/mL, respectively. The method was used for the determination of the diastereomeric composition of the flavanone‐7‐O‐glycosides in Citrus juices after solid‐phase extraction procedure.     

Achiral and chiral separations using MEKC,polyelectrolyte multilayer coatings,and mixed mode separation techniques with molecular micelles

Mixed mode (MM) separation using a combination of MEKC and polyelectrolyte multilayer (PEM) coatings is herein reported for the separation of achiral and chiral analytes. Many analytes are difficult to separate by MEKC and PEM coatings alone. Therefore, the implementation of a MM separation provides several advantages for overcoming the limitations of these well‐established methods. In this study, it was observed that achiral separations using MEKC and PEM coatings individually resulted in partial resolution of eight very similar aryl ketones when the molecular micelle (sodium poly(N‐undecanoyl‐L ‐glycinate)) concentration was varied from 0.25 to 1.00% w/v and the bilayer number varied from 2 to 4. However, when MM separation was introduced, baseline resolution was achieved for all eight analytes. In the case of chiral separations, temazepam, aminoglutethimide, benzoin, benzoin methyl ether, and coumachlor were separated using the three separation techniques. For chiral separations, the chiral molecular micelle, sodium poly(N‐undecanoyl‐L ‐leucylvalinate), was employed at concentrations of 0.25–1.50% w/v for both MEKC and PEM coatings. Overall, the results revealed partial separation with MEKC and PEM coatings individually. However, MM separation enabled baseline separation of each chiral mixture. The separation of achiral and chiral compounds from different compound classes demonstrates the versatility of this MM approach.     

Use of multivariate analysis for optimization of separation parameters and prediction of migration time, resolution, and resolution per unit time in micellar electrokinetic chromatography

The optimization of separation parameters in chromatography for better separation and resolution of analytes continues to be a labor intensive procedure usually performed by a trial and error method. A multivariate analysis in the form of multilinear regression (MLR) is used to optimize separation parameters and predict the migration behavior, resolution, and resolution per unit time of achiral (4-chlorophenol, pentachlorophenol, clonazepam, and diazepam) and chiral (1,1'-binaphthyl 2,2'-dihydrogen phosphate (BNP), and 1,1'-bi-2-naphthol (BOH)) compounds in MEKC. Separations of achiral and chiral analytes were performed using an achiral (poly(sodium N-undecylenic sulfate)) molecular micelle and chiral (poly(sodium N-undecanoyl-L-leucylvalinate) or poly(sodium N-undecanoyl-L-isoleucylvalinate)) molecular micelle, respectively, at various operating temperatures, applied voltages, pH values, and molecular micelle concentrations in the BGE. The separation parameters were subsequently used as input variables for MLR models. The models were validated with independent samples. The root-mean-square percent relative error (RMS%RE) is used as a figure of merit for characterizing the performance of the migration time, resolution, and resolution per unit time models. The RMS%RE obtained for predicted migrated times, resolutions, and resolution per unit time of 4-chlorophenol, pentachlorophenol, clonazepam, diazepam, BNP, and BOH ranged between 8 and 19%. The same experimental procedure was used to optimize the separation parameters of six other chiral analytes of different compound class. The predicted migration times, resolutions, and resolution per unit time of the chiral as well as the achiral analytes compare favorably with the experimental migration times and resolutions, indicating versatility and wide applicability of the technique in MEKC.     

Separation and selectivity in micellar electrokinetic chromatography using sodium dodecyl sulfate micelles or Tween 20-modified mixed micelles

The separation and selectivity of eight aromatic compounds ranging from hydrophilic to hydrophobic properties in micellar electrokinetic chromatography (MEKC) using sodium dodecyl sulfate (SDS) micelles or Tween 20-modified mixed micelles were investigated. The effect of different operation conditions such as SDS and Tween 20 modifier surfactant concentration, buffer pH, and applied voltage was studied. The resolution and selectivity of analytes could be markedly affected by changing the SDS micelle concentration or Tween 20 content in the mixed micelles. Applied voltage and pH of running buffers were used mainly to shorten the separation time. Complete separation of eight analytes could be achieved with an appropriate choice of the concentration of SDS micelles or Tween 20-modified mixed micelles. Quicker elution and better precision could be obtained with SDS-Tween 20 mixed micelles than with SDS micelles. The mechanisms that migration order of those analytes was mainly based on their structures and solute-micelle interactions, including hydrophobic, electrostatic, and hydrogen bonding interactions, were discussed.     

聚(苯乙烯-co-甲基丙烯酸)自组装胶束假固定相电动色谱的特性

研究了两亲性无规共聚物聚(苯乙烯-co-甲基丙烯酸)(P(St-co-MAA))(单体摩尔比分别为6:4和7:3)自组装胶束的物理化学性质,及其作为假固定相(PSP)的胶束电动色谱性能。测定了聚合物胶束的临界胶束浓度(CMC),对胶束内核微环境的极性、表面电荷密度和流体力学直径等微结构参数进行了表征,对时间窗口、亚甲基选择性等电动色谱参数进行了测定,并与聚(甲基丙烯酸甲酯-co-甲基丙烯酸)(P(MMA-co-MAA))胶束、十二烷基硫酸钠(SDS)胶束体系进行了比较;利用线性溶剂化能关系(LSER)研究了聚合物PSP的选择性差异。结果表明:P(St-co-MAA)体系具有最小的CMC、最宽的时间窗口和最好的亚甲基选择性;LSER表明,疏水作用是决定聚合物PSP选择性的最主要因素,氢键酸度其次,特别是P(St-co-MAA)(单体摩尔比7:3)体系具有最高的作用参数,显示了该PSP具有较高的分离选择性。     

A comparative study of micellar and microemulsion EKC for the analysis of benzoylurea insecticides and their analogs

An investigation of the basic factors which govern the microemulsion EKC (MEEKC) and MEKC for the separation of four benzoylurea (BU) insecticides and their four analogs was carried out. In MEEKC, the separation of eight BU compounds was optimized by changing the microemulsion composition, such as concentration of SDS, octane, n-butanol, and isopropanol percentages, as well as capillary temperature. Separation optimization was also carried out for MEKC, showing that ACN and a high level of another additive gamma-CD were needed to achieve effective separation of these analytes. Although separation with baseline resolution was achieved by either MEEKC or MEKC methods, the separation selectivity resulting from the proposed MEEKC method was completely different from that of MEKC. In addition, analytical time in MEEKC was longer than that in MEKC, but in view of theoretical plate numbers, detection limits, and reproducibility, both methods were effective for the analysis of BU insecticides and their analogs.     

Enantioseparation of phenothiazines in cyclodextrin-modified micellar electrokinetic chromatography

In this study, enantioseparations of five phenothiazines in cyclodextrin (CD)-modified micellar electrokinetic chromatography (MEKC) were investigated using a citrate buffer containing tetradecyltrimethylammonium bromide (TTAB) as a cationic surfactant at low pH. Beta-cyclodextrin (beta-CD) and hydroxylpropyl-beta-CD (HP-beta-CD) were selected as chiral selectors. The results indicate that the separation window is greatly enlarged by beta-CD concentration and that the separability and selectivity of phenothiazines are remarkably influenced by the concentrations of both beta-CD and TTAB, as well as buffer pH. The interaction of thioridazine with beta-CDs is considerably reduced in the presence of TTAB micelles due to competitive complexation of thioridazine with TTAB micelles, which is pH-dependent. As a result, effective enantioseparation of thioridazine is simultaneously achievable with that of trimeprazine and promethazine or ethopropazine in MEKC with addition of either beta-CD or HP-beta-CD, respectively, to a micellar citrate buffer containing TTAB at pH 3.5. Better enantioresolution of thioridazine in MEKC than in capillary zone electrophoresis can be obtained.     

On-chip chiral and achiral separation of amphetamine and related compounds labeled with 4-fluoro-7-nitrobenzofurazane

Amphetamine and analogous compounds have been labeled with 4-fluoro-7-nitrobenzofurazane and analyzed on a microfabricated chip. Separation of norephedrine, ephedrine, cathinone, pseudoephedrine, methcathinone, amphetamine and methamphetamine is demonstrated using micellar electrokinetic capillary chromatography (MEKC) and laser-induced fluorescence (LIF) detection. Chiral separations of individual drugs were studied using neutral and negatively charged cyclodextrins (CDs) with and without the addition of an organic modifier and/or sodium dodecyl sulfate (SDS). The best results were obtained using a highly sulfated gamma-CD (HS-gamm-CD) in combination with a low concentration of SDS. To obtain complete separation of a mixture of (+/-)-norephedrine, (+/-)ephedrine, (+/-)-pseudoephedrine, (+/-)-methcathinone, (+/-)-amphetamine and (+/-)-methamphetamine it was necessary to add a small amount (1.5 mM) of SDS to the separation buffer. Optimized chiral separation was achieved within 7 min using an S-folded separation channel, a separation voltage of 8 kV and a buffer consisting of 50 mM phosphate (pH 7.35), 10 mM HS-gamma-CD and 1.5 mM SDS.     

Separation mechanism and determination of flavanones with capillary electrophoresis and high-performance liquid chromatography

To probe separation mechanism and determination with capillary zone electrophoresis (CZE) and liquid chromatography (LC), nine compounds with identical flavanone skeleton were studied. Optimum separation of LC was attained with gradient of acetonitrile and 5mM phosphate buffer (pH 6.9). For CE, electrolyte buffer was 4.5mM SDS in 32mM sodium tetraborate buffer (pH 9.2). The distinguishing feature in this work was successful separation of monohydroxyl stereoisomers by CZE. Polarity is generally increased with hydroxyl groups. In a separation mechanism study, polarity would be reduced by intramolecular hydrogen bond between hydroxyl of C5 and carbonyl group of C4. Comparison of the retention results among monohydroxyl flavanones shows polarity with hydroxyl at C6 the least, and that at C4' and C7 nearly equal. Also, elution order of flavones and flavanones would be adverse due to the hydroxyl at C3 in LC. From the numerical value pK(a) of flavanone, the C7-OH is the smallest, and two hydroxyl groups in an adjacent position is always less than the unique one caused by forming a stable 5-membered ring. Investigation of separation mechanism yield only the effect of constituent but also reasonable explanation for contradictory results between Wulf and our laboratory, this due to the hydroxyl at C3.     

Use of poly(sodium oleyl-L-leucylvalinate) surfactant for the separation of chiral compounds in micellar electrokinetic chromatography

A chiral amino acid-based monomeric and polymeric surfactant, sodium oleyl-L-leucylvalinate) (L-SOLV) and poly(sodium oleyl-L-leucylvalinate) (poly-L-SOLV) were synthesized and used for chiral separations in micellar electrokinetic chromatography (MEKC). Poly-L-SOLV was used successfully in the separation of various enantiomers of neutral, acidic, and basic analytes such as 1,1'-bi-2-napthol, 1,1'-binaphthyl-2,2'-diamine, benzoin, hydrobenzoin, benzoin methylether, warfarin, and coumachlor obtaining well-resolved peaks but with only partial separation of temazepam. In addition, the atropisomer 1,1'-binaphthyl-2, 2'-dihydrogen phosphate was chosen to study the applicability of the polymeric surfactant over a wide range of parameters such as concentration, temperature, voltage, and pH. The most striking characteristic of this new surfactant is its high hydrophobicity. It is favorable to interactions with hydrophobic chiral analytes, and thus may provide better chiral recognition for the compounds.     

Evaluation of novel amylose and cellulose-based chiral stationary phases for the stereoisomer separation of flavanones by means of nano-liquid chromatography

Three polysaccharide-based chiral stationary phases, Sepapak^® 1, Sepapak^® 2 and Sepapak^® 3 have been evaluated in the present work for the stereoisomer separation of a group of 12 flavonoids including flavanones (flavanone, 4′-methoxyflavanone, 6-methoxyflavanone, 7-methoxyflavanone, 2′-hydroxyflavanone, 4′-hydroxyflavanone, 6-hydroxyflavanone, 7-hydroxyflavanone, hesperetin, naringenin) and flavanone glycosides (hesperidin, naringin) by nano-liquid chromatography (nano-LC). The behaviour of these chiral stationary phases (CSPs) towards the selected compounds was studied in capillary columns (100 μm internal diameter (i.d.)) packed with the above mentioned CSPs using polar organic, reversed and normal elution modes. The influence of nature and composition of the mobile phase in terms of concentration and type of organic modifier, buffer type and water content (reversed phase elution mode) on the enantioresolution (R_s), retention factor (k) and enantioselectivity (α) was evaluated. Sepapak^® 3 showed the best chromatographic results in terms of enantioresolution, enantioselectivity and short analysis time, employing a polar organic phase mode. A mixture of methanol/isopropanol (20/80, v/v) as mobile phase enabled the chiral separation of eight flavanones with enantioresolution factor (R_s) in the range 1.15–4.18. The same analytes were also resolved employing reversed and normal phase modes with mixtures of methanol/water and hexane/ethanol at different ratios as mobile phases, respectively. Loss in resolution for some compounds, broaden peaks and longer analysis times were observed with these last two chromatographic elution modes.     

Control of separation selectivity in micellar electrokinetic chromatography by modification of the micellar phase with solubilized organic compounds

On the basis of the data on the distribution of various neutral solutes between sodium dodecyl sulfate (SDS) micelles and water, the control of separation selectivity in micellar electrokinetic chromatography (MEKC) by modification of the micellar phase with organic additives has been proposed and applied to the separation of simple model compounds. It was found that the distribution constants between the micelles and water (K_d,mc), which were determined by means of MEKC, of the solutes possessing hydrophilic functional groups are much larger than those between heptane and water (K_d,hep), whereas the K_d,mc values of the solutes possessing no hydropholic groups are comparable to their K_d,hep values. This indicates that the former solutes are preferentially solubilized in the Stern layer of the micelles and that the latter are located in the hydrocarbon core. In MEKC separations of aromatic compounds and metal acetylacetonates, considerable changes in separation selectivity were caused by the addition of compounds possessing both hydrophilic functional groups such as alcohols, phenol and ketones to the SDS micellar solution. The variations of the retention factors of the analytes could be explained in terms of saturation of the solubilization sites in the Stern layer with the modifiers, specific interaction of the modifiers with the analytes via hydrogen bonding in the micelles, and expansion of the core volume with the hydrocarbon parts of the modifiers. Such effects of the micellar modification could improve the resolution as well as the selectivity of MEKC separations.