Separation of isomers of dienoic acids by electromigration techniques

Different capillary electromigration techniques were employed to resolve geometrical isomers of sorbic acid, decadienoic acid, and ethyl sorbate. Since these substances differ in their polarity, shape, and size, various electromigration approaches were investigated to separate the four geometrical isomers of each compound. With capillary electrophoresis (CE) modified with a cyclodextrin (β‐CD) the four isomers of sorbic acid were separated using a buffer that consists of 60 mM tetraborate and 8 mg/mL β‐CD. The separation of decadienoic acid geometrical isomers was not possible, even at elevated tetraborate and cyclodextrin concentrations. The four isomers of decadienoic acid were successfully separated using micellar electrokinetic chromatography (MEKC) with a buffer consisting of 30 mM tetraborate and 100 mM SDS and microemulsion electrokinetic chromatography (MEEKC). Ethyl sorbate is the least polar of all the studied substances and its isomers could not be separated by MEKC or MEEKC. The resolution was improved and isomers were fully separated using capillary electrochromatography (CEC) with ODS stationary phase and a mobile phase consisting of 10 mM boric acid in 50% acetonitrile. Minor differences in the polarity and the shape of isomers and high resolving power of the applied techniques were sufficient for separation of very similar compounds. We have shown that versatile electromigration techniques can be applied for separation of geometrical isomers of dienoic acids and their esters.     

Determination of food colorants by microemulsion electrokinetic chromatography

A microemulsion electrokinetic chromatography (MEEKC) method was developed to analyze and detect eight food colorants (tartrazine, fast green FCF, brilliant blue FCF, allura red AC, indigo carmine, sunset yellow FCF, new coccine, and carminic acid), which are commonly used as food additives in various food products. The effects of sodium dodecyl sulfate (SDS) surfactant, organic modifier, cosurfactant, and oil were examined in order to optimize the separation. The amount of organic modifier (acetonitrile) and SDS surfactant were determined as apparent influences on the separation resolution while the type of oil and cosurfactant rarely affected the separation selectivity of the eight colorants. A highly efficient MEEKC separation method, where the eight colorants were separated with baseline resolution within 14 min, was achieved by using a microemulsion solution of pH 2.0 containing 3.31% SDS, 0.81% octane, 6.61% 1-butanol, and 10% acetonitrile. This optimal MEEKC method has a higher separation efficiency and similar detection limit when compared to conventional capillary electrophoresis (CE) method. Furthermore, a sample pretreatment is rarely needed when this MEEKC technique is used to analyze colorants in food products, whereas a suitable sample pretreatment (for example solid-phase extraction) has to be employed prior to CE separation in order to eliminate matrix interferences resulting from the constituents of the food sample.     

Separation of binaphthyl enantiomers by capillary zone electrophoresis and electrokinetic chromatography

The capillary electrophoretic (CE) separation of the enantiomers of three binaphthyl compounds is investigated. Several CE modes such as cyclodextrin (CD) modified capillary zone electrophoresis (CZE) (CD-CZE), micellar electrokinetic chromatography (MEKC), cyclodextrin electrokinetic chromatography (CD-EKC), etc. are employed for the simultaneous enantiomer separation of the three solutes. The successful separation was achieved by combining two modes, in other words by using more than two chiral selectors. A development of the CE enantiomer separation is demonstrated for the binaphthyl compounds. The enantioselectivity of binaphthyl compounds is alo briefly discussed.     

Capillary electrophoresis in pharmaceutical analysis: a survey on recent applications

Capillary electrophoresis (CE) has a significant role in drug discovery and manufacturing processes and has a potential to grow further, due to new developments that can provide highly sensitive and high throughput analysis. This review illustrates recent applications of CE in pharmaceutical analysis (2005-present). The history, principles, instruments, and conventional modes of CE are briefly described. Applications for drug analysis by various techniques of CE are presented in six tables: capillary zone electrophoresis (CZE) (Table I), micellar electrokinetic chromatography (MEKC) and microemulsion electrokinetic chromatography (MEEKC) (Table II), non-aqueous CE (NACE) (Table III), chiral CE (Table IV), CE-mass spectrometry (MS) microchip CE (Table V), and multiplexed CE (MCE) (Table VI).     

毛细管微乳电动色谱应用的研究进展

近年来毛细管微乳电动色谱(MEEKC)研究的范围不断扩大,其分离分析的化合物类型也不断增多。该文综述了2002年以来MEEKC应用的研究进展,指出了目前MEEKC研究中存在的一些问题,对今后的研究发展方向进行了展望。     

Application of microemulsion electrokinetic chromatography to the analysis of a wide range of pharmaceuticals and excipients

Microemulsion electrokinetic chromatography (MEEKC) is a capillary electrophoresis (CE) technique in which solutes partition with moving oil droplets present in a microemulsion buffer. Ionised species will also separate by electrophoresis. In this paper MEEKC is shown to give highly efficient and relatively rapid separations for a wide range of pharmaceuticals, vitamins and excipients. A single set of operating conditions was used to resolve both water-soluble and insoluble compounds. The method was also used to separate both ionic and neutral compounds. The method was especially useful in the analysis of water-insoluble neutral compounds such as steroids and lecithin, which are difficult to analyse by CE. The method was found to be both quantitative and highly repeatable. The quality of the separation was found to be dependent upon the sample diluent used if large injection volumes are employed. The use of MEEKC for the determination of complex mixtures such as multi-ingredient formulations and drug-related impurities was successfully demonstrated. MEEKC offers significant advantages over many forms of CE and capillary electrochromatography (CEC) and should be considered as an extremely useful option in pharmaceutical analysis.     

Determination of Low-Molecular-Mass Phenolic and Non-Phenolic Lignin Degradation Compounds in Wood Digestion Solutions by Capillary Electrophoresis

 Different capillary electrophoretic techniques were investigated and compared for the separation of low-molecular-mass phenolic and neutral lignin degradation compounds. Simple capillary zone electrophoresis (CZE) was not suited for this problem. Switching to micellar electrokinetic capillary chromatography (MEKC) by adding micelle-forming reagents to the carrier electrolyte enhanced the separation performance considerably. Alternatively, microemulsion electrokinetic chromatography (MEEKC) was investigated. This is a CE technique in which analytes interact with moving oil droplets present in a microemulsion buffer. Using the optimized carrier electrolyte systems and a 60 cm×50 μm I.D. bubble cell capillary it was possible to analyse solutions of different wood digestion procedures and to investigate differences in lignin degradation products during use of different kinds of wood (eucalyptus, beech, scotch pine and acacia). Received August 25, 1999. Revision April 13, 2000     

Investigation of solvent effects in capillary electrophoresis for the separation of biological porphyrin methyl esters

The effects of organic solvents on the capillary electrophoresis (CE) separation of a number of important biological porphyrin methyl esters - six weakly basic, hydrophobic cyclic tetrapyrroles possessing two and four to eight methyl ester groups around the periphery of the porphyrin ring - were investigated in the mode of micellar electrokinetic chromatography (MEKC), microemulsion electrokinetic chromatography (MEEKC), and nonaqueous CE. In aqueous MEKC, partial separation of the six neutral porphyrin methyl esters was obtained with an organic modifier (acetonitrile) in the concentration range between 20 and 40%, in which sodium dodecyl sulfate (SDS) molecules might be present in the form of SDS micelles and/or SDS micelle-like aggregates. Relatively stable SDS micelles can be formed in nonaqueous MEKC using formamide as the separation medium, but the separation of the target analytes remained unsatisfactory. Improved resolution of all six porphyrin methyl esters was obtained using MEEKC with the running buffer consisting of 0.8% w/w n-heptane (oil phase), 2.25% w/w SDS and 1.0% w/w Brij 35 (mixed surfactant), 6.6% w/w 1-butanol (cosurfactant), and 30% v/v 2-propanol (second cosurfactant), but reproducibility in terms of peak areas for certain porphyrins (especially uroporphyrin I octamethyl ester) was found to be very poor. Best separation performances were achieved with nonaqueous CE separations in which the weakly basic porphyrin methyl esters were protonated under strongly acidic conditions (e.g., using 10 mM perchloric acid) in mixed organic solvents. For example, using a 50:50 mixture of methanol and acetonitrile as the separation medium, baseline separation of all six (positively charged) porphyrin methyl esters can be obtained within 3 min and the average precision (RSD, N = 13) in terms of migration time and peak area were 0.55 and 2.16%, respectively.     

Systematic investigations of different capillary electrophoretic techniques for separation of methylquinolines

The migration behaviour of isoquinoline, quinoline, and methyl derivatives of quinoline in different capillary electrophoretic modes has been systematically investigated. Optimised separation conditions were established by varying the key parameters (solvent, pH, temperature, surfactant concentration, core phase) for aqueous and non‐aqueous capillary zone electrophoresis (NACE), micellar electrokinetic chromatography (MEKC) with anionic or non‐ionic micelles (SDS, Brij 35), and microemulsion electrokinetic chromatography (MEEKC) with charged or uncharged microemulsion droplets. A separation of all quinolines could be achieved by MEEKC with charged droplets, by MEKC or by formamide‐based NACE. Comparing the separations with respect to separation selectivity, substantial changes in migration order could be observed between the different techniques. Regarding separation efficiency, the number of theoretical plates and limits of detection (LOD) have been compared. The best LODs were achieved using SDS as surfactant in MEKC, followed by MEEKC.     

Orthogonal separations of nicotine and nicotine-related alkaloids by various capillary electrophoretic modes

The migration behaviour of nicotine and related tobacco alkaloids was investigated using three different capillary electrophoretic (CE) modes. Novel separations were achieved both using microemulsion electrokinetic chromatography (MEEKC) and nonaqueous CE (NACE). Improved resolution compared to previous studies was obtained using free-solution CE (FSCE). Each technique resulted in different, orthogonal separation selectivity. The suitability of each method for application to the analysis of nicotine lozenges is discussed. The FSCE method was applied to the analysis of nicotine lozenges due to its compatibility with an established lozenge extraction solvent. The method used gave good injection precision and linearity. Good agreement of CE and high-performance liquid chromatography (HPLC) results was obtained. The CE method is therefore considered suitable for the quantitative determination of nicotine in nicotine lozenges.     

Anion-selective exhaustive injection-sweeping microemulsion electrokinetic chromatography

In this study, anion-selective exhaustive injection-sweeping (ASEI-sweeping) technique, which is a selective on-line sample concentration technique, was first proposed in microemulsion electrokinetic chromatography (MEEKC) for analyses of eight acidic phenolic compounds. In contrast to a capillary that is typically filled with nonmicellar background solution in conventional ASEI-sweeping MEKC method, in the proposed ASEI-sweeping MEEKC method, a capillary is filled with a low pH microemulsion solution (pH 2.0), and then with a short acid plug (pH 2.0, 1.9 cm) before field-amplified sample injection. This proposed design has two functions. First, the microemulsion solution that is present at the front of capillary column is able to avoid phase separation of microemulsion solution during MEEKC separation. Second, the presence of the short acid plug would effectively limit the partition behavior of acid analytes with the oil droplets in the microemulsion during field-amplified sample injection; otherwise, the stacking effect of acid analytes would be markedly reduced. This optimal ASEI-sweeping MEEKC method afforded about 96,000-fold to 238,000-fold increases in detection sensitivity in terms of peak areas without any separation efficiency loss when compared to normal MEEKC separation. Furthermore, trace levels (about 3 ng/g) of gallic acid and catechin in foods were also detected successfully by the proposed ASEI-sweeping MEEKC technique.     

Pharmaceutical applications of micelles in chromatography and electrophoresis

This review surveys the use of micelles as separation media in chromatography and electrophoresis. Applications to pharmaceuticals whose molecular masses are relatively small are focused on in this review. In high-performance liquid chromatography (HPLC), chromatography using micelles and reversed-phase stationary phases such as octadecylsilylized silica gel (ODS) columns is known as micellar liquid chromatography (MLC). The main application of MLC to pharmaceutical analysis is the same as in ion-pair chromatography using alkylsulfonate or tetraalkylammonium. In most cases, selectivity is much improved compared with other short alkyl chain ion-pairing agents such as pentanesulfonate or octanesulfonate. Direct plasma/serum injection can be successful in MLC. Separation of small ions is also successful by using gel filtration columns and micellar solutions. In electrophoresis, especially capillary electrophoresis (CE), micelles are used as pseudo-stationary phases in capillary zone electrophoresis (CZE). This mode is called micellar electrokinetic chromatography (MEKC). Most of the drug analysis can be performed by using the MEKC mode because of its wide applicability. Enantiomer separation, separation of amino acids and closely related peptides, separation of very complex mixtures, determination of drugs in biological samples etc. as well as separation of electrically neutral drugs can be successfully achieved by MEKC. Microemulsion electrokinetic chromatography (MEEKC), in which surfactants are also used in forming the microemulsion, is successful for the separation of electrically neutral drugs as in MEKC. This review mainly describes the typical applications of MLC and MEKC for the analysis of pharmaceuticals.     

Free solution capillary electrophoresis and micellar electrokinetic resolution of amino acid enantiomers and peptide isomers with L- and D-Marfey's reagents

Separation of amino acid enantiomers and peptide isomers has been made possible through the use of Marfey's reagent and high-performance capillary electrophoresis (HPCE). Samples of amino acids and peptides were first derivatized with Marfey's reagent and subsequently analyzed by HPCE. Different modes of separation were investigated including free solution and micellar electrokinetic chromatography. The use of micellar electrokinetic chromatography in combination with L- and D-Marfey's reagent offered unequivocal means to confirm the presence of D-amino acid in an unknown sample. This method is also particularly useful for the analysis of peptide isomers.     

Separation of ritalin racemate and its by-product racemates by capillary electrophoresis

Ritalin, [(+)-threo]methylphenidate hydrochloride, is a chiral drug substance with two chiral centers. The drug substance may contain three pairs of enantiomers, [(+)-threo], [(-)-threo], [(+)-erythro] and [(-)-erythro] isomers, and its degradation products, threoritalinic acid racemate. Determination of the optical purity of ritalin drug substance and the amount of its by-product isomers is a critical step in the single-isomer drug development. In order to efficiently recognize the three pairs of enantiomers by one method, capillary electrophoresis (CE) was employed for the separation. The three pairs of enantiomers in CE showed different enantioselectivities with eight different types of CDs. Only 2,6-di-o-methyl-beta-cyclodextrin (DM-beta-CD) and carboxymethyl-beta-cyclodextrin (CM-beta-CD) showed enantioselectivity to all these pairs of enantiomers. With respect to separation resolution and efficiency, DM-beta-CD was chosen as the chiral selector. For optimization of the separation conditions, the concentration of DM-beta-CD, pH of the buffer solution, and temperature of the capillary were further studied.     

毛细管电泳在手性氨基酸拆分中的研究进展

毛细管电泳(capillary electrophoresis,CE)作为一种强有力的手性分离技术,由于操作简单、试剂消耗少及柱效高等优点,受到广泛关注,是近年来手性分离领域的研究热点.氨基酸是组成蛋白质的基本单元,且大多数氨基酸具有手性中心,手性氨基酸是生命体系的一个重要特征.具有手性中心的氨基酸,其对映体间的生物活性往往存在着较大的差异,因此,氨基酸的手性拆分对了解人体及动物生命活动起着举足轻重的作用.主要总结了近5年来毛细管电泳的3种分离模式(毛细管区带电泳、胶束电动毛细管色谱、毛细管电色谱)在氨基酸手性拆分中的发展和应用.     

Twenty‐one years of microemulsion electrokinetic chromatography (1991–2012): A powerful analytical tool

Microemulsion electrokinetic chromatography (MEEKC) is a CE separation technique, which utilizes buffered microemulsions as the separation media. In the past two decades, MEEKC has blossomed into a powerful separation technique for the analysis of a wide range of compounds. Pseudostationary phase composition is so critical to successful resolution in EKC, and several variables could be optimized including surfactant/co‐surfactant/oil type and concentration, buffer content, and pH value. Additionally, MEEKC coupled with online sample preconcentration approaches could significantly improve the detection sensitivity. This review comprehensively describes the development of MEEKC from the period 1991 to 2012. Areas covered include basic theory, microemulsion composition, improving resolution and enhancing sensitivity methods, detection techniques, and applications of MEEKC.     

Separation of linoleic acid oxidation products by micellar electrokinetic capillary chromatography and nonaqueous capillary electrophoresis

In this work the suitability of micellar electrokinetic capillary chromatography (MEKC) and nonaqueous capillary electrophoresis (CE) to the analysis of the primary oxidation products of linoleic acid was studied with uncoated fused-silica capillaries. The primary autoxidation products of linoleic acid are the four hydroperoxide isomers 13-hydroperoxy-cis-9, trans-11-octadecadienoic acid, 13-hydroperoxy-trans-9, trans-11-octadecadienoic acid, 9-hydroperoxy-trans-10,cis-12-octadecadienoic acid, 9-hydroperoxy-trans-10, trans-12-octadecadienoic acid. Addition of a surfactant such as sodium dodecyl sulfate (SDS) or sodium cholate (SC) into the running buffer (20-30 mM 3-(cyclohexylamino)-1-propanesulfonic acid (CAPS) or ammonium acetate, pH 9.5-11) was required to enhance the water solubility of the sample and selectivity of the separation. MEKC proved to be a promising new technique for the separation of the primary oxidation products of lipids giving results comparable to high performance liquid chromatography (HPLC). Partial separation of hydroperoxide isomers was also achieved using nonaqueous CE with methanol-acetonitrile-sodium cholate as running buffer.     

Microemulsion and micellar electrokinetic chromatography of Hematoporphyrin D: a starting material of hematoporphyrin derivative

An investigation of the basic factors which govern the microemulsion electrokinetic chromatography (MEEKC) and micellar electrokinetic chromatography (MEKC) separation of Hematoporphyrin D and its base hydrolysis product, hematoporphyrin derivative (HpD), was performed. These model compounds contain a complex mixture of porphyrin monomers, dimers and/or oligomers, and were utilized to gain insights into the MEEKC/micellar electrokinetic chromatography (MEKC) separation of samples containing highly lipophilic substances. For example, the organic modifier/cosurfactant (1-butanol) and/or oil phase (e.g., 1-octanol in comparison to ethyl acetate) were found to have an apparent influence on the separation selectivity of Hematoporphyrin D, the extent of which was dependent on the chemical nature of the surfactant employed (e.g., sodium dodecyl sulfate vs. sodium cholate). An interesting and important finding was that the presence of an organic modifier (methanol or acetonitrile at a concentration of 20% or higher) in the sample matrix as well as in the run buffer was essential for the optimal MEEKC or MEKC separation of a number of porphyrin monomers (including hematoporphyrin IX and its acetates, most likely hydroxyacetate, diacetate, and vinyl acetate, as well as its dehydration products, hydroxyethylvinyldeuteroporphyrin and protoporphyrin) contained in Hematoporphyrin D. On the other hand, the use of these optimized conditions for the MEEKC or MEKC separation of various oligomeric porphyrin species in HpD were unsatisfactory. As HpD is a well-known and effective photosensitizing agent in photodynamic therapy (a new approach for cancer treatment), the improved separation and characterization of various monomeric and oligomeric porphyrin species in HpD and its starting material, such as Hematoporphyrin D, is a challenging and important task.     

Stacking and separation of urinary porphyrins in capillary electrophoresis: optimization of concentration efficiency and resolution

We demonstrated that anionic porphyrins could be stacked and separated in micellar electrokinetic chromatography (MEKC) and microemulsion electrokinetic chromatography (MEEKC) by applying acetonitrile and high salt content in human urine sample matrix. The introduction of sample containing acetonitrile and sodium chloride into the CE capillary at more than 10% of the total capillary volume resulted in the improvement of peak resolution and the enhancement of detection sensitivity. The achieved acetonitrile stacking enrichment factors of six porphyrins ranged from 12 to 32 in MEKC and from 28 to 33 in MEEKC, respectively. The stacking technique was successfully applied for analyzing porphyrins present in urine samples that were deproteinized with acetonitrile. For the analysis of coproporphyrin isomers, addition of the sodium cholate (SC) into micelle and microemulsion solutions provided adequate resolution. Calibration curves obtained for the determination of coproporphyrin isomers were found linear between 30 and 400 nmol L⁻¹, and the limit of detection (LOD) was 20 nmol L⁻¹ in MEEKC. Intra- and interday precisions (n = 11) in the microemulsion separation system for the isomers at spiked concentrations of 40-400 nmol L⁻¹ in urine were in the range of 0.1-0.4% and 0.7-7.6% for migration time and peak area, respectively. Coproporphyrin III, coproporphyrin I and uroporphyrin were detected at levels of 80.7 nmol L⁻¹, 32.3 nmol L⁻¹ and 19.8 nmol L⁻¹, respectively, in the urine samples collected from healthy individuals. Different porphyrin profiles, however, were observed in urine samples from porphyria cutanea tarda (PCT) patients.     

蛋白质的微乳液毛细管电动色谱分离研究

考察了用微乳液毛细管电动色谱(MEEKC)分离蛋白质时微乳液组成等不同因素对分离的影响,并与胶束电动色谱进行对比,探讨了其分离机理,为蛋白质的分离鉴定提供了一种有力的工具.