Comparison of oil-in-water and water-in-oil microemulsion electrokinetic chromatography as methods for the analysis of eight phenolic acids and five diterpenoids

Oil-in-water (O/W) and water-in-oil (W/O) MEEKC were compared for their abilities to separate and detect eight phenolic acids and five diterpenoids in Radix et Rhizoma Salviae Miltiorrhizae (RRSM). The effects of oil type and concentration, organic modifier, SDS, and buffer concentration on separation were examined in order to optimize the two methods. Oil contents and organic modifier were found to markedly influence the separation selectivity for both O/W and W/O systems. SDS concentration rarely affected separation resolution for O/W MEEKC, and separation of eight phenolic acids and five diterpenoids could be improved by changing the buffer concentration for W/O MEEKC. A highly efficient O/W MEEKC separation method, where the 13 compounds were separated with baseline resolution, was achieved by using a microemulsion solution of pH 8.0 containing 0.6% cyclohexane, 3.0% SDS, 6.0% 1-butanol, and 3.0% ACN. The W/O MEEKC was unable to resolve all the components. In addition, the analytic time in O/W MEEKC was shorter than that in W/O MEEKC. Finally, the developed O/W MEEKC method was successfully applied to determine analytic compounds in RRSM samples.     

Screening of octanol-water partition coefficients for pharmaceuticals by pressure-assisted microemulsion electrokinetic chromatography

A rapid screening assay for the determination of octanol-water partition coefficients (log P(OW)) of pharmaceuticals was developed by using pressure-assisted microemulsion electrokinetic chromatography (MEEKC). The microemulsion system contains 50 mM sodium dodecyl sulfate, 0.87 M l-butanol, 82 mM heptane, and 50 mM borate-phosphate (2:3) at pH 10. Ten standard compounds with known log P(OW) values from -0.26 to 4.88 were used for constructing the calibration curve of log P(OW) against the MEEKC retention factor, log k. The log P(OW) values of the compounds were calculated based on the log k values measured by MEEKC and the slope and intercept of the calibration curve. For 13 literature and 32 Roche compounds, about 90% of the log P(OW) values measured by MEEKC are within 0.5 log units of the values from the literature and potentiometric titration. The throughput is about 2 samples/h using +20 kV voltage plus 5 mbar air pressure for separation. This MEEKC method is applicable for log P(OW) screening of weakly basic, weakly acidic, and neutral pharmaceuticals with log P(OW) = 0-5 and pKa < or = 10.     

Migration mechanism of bases and nucleosides in oil-in-water microemulsion capillary electrophoresis.

The electrophoretic behaviors of five bases and corresponding nucleosides in the oil in water (o/w) microemulsion capillary electrophoresis, microemulsion electrokinetic chromatography (MEEKC), were examined in comparison with those in normal capillary zone electrophoresis (CZE). The microemulsion systems were composed of heptane, sodium dodecyl sulfate (SDS), 1-butanol and 10 mM phosphate buffer (pH 7.0) or toluene, SDS, 1-butanol and 5 mM carbonate buffer (pH 10.0). CZE was carried out in the range of pH 9.7-10.9, and the dissociation constants, pKa, of the bases and nucleosides and the electrophoretic mobilities of the anionic forms were determined. The electrophoretic behaviors of the solutes in the microemulsion systems were analyzed from their pKa, the electrophoretic mobilities of the anions determined by CZE, and the distribution constants, K(D), of the neutral forms between the microemulsion droplets and the outer aqueous phase. The importance of adsorption mechanism in MEEKC system was suggested from the correlation between log K(D) and log P.     

Separation and determination of biphenyl nitrile compounds by microemulsion electrokinetic chromatography with mixed surfactants

A mixture of six biphenyl nitrile compounds and three related substances with high hydrophobicity and similar structures was successfully separated by microemulsion electrokinetic chromatography (MEEKC) within 30 min. The microemulsion system contained 100 mM sodium dodecyl sulfate (SDS), 80 mM sodium cholate (SC), 0.81% v/v heptane, 7.5% v/v n-butanol, 10% v/v acetonitrile, and 10 mM borate. The addition of SC, organic modifiers, sample preparation, and temperature all showed remarkable effects on the separation. The capacity factor (k) was calculated by using dodecyl benzene as the marker for microemulsion, and the calculated partition coefficient log P(o/w) of the solutes was in the range of 3.35-7.38. The log k values matched well with the log P(o/w) with a correlation coefficient of 0.96. In addition, the linear correlation coefficients of each compound between peak area and concentration were from 0.996 to 0.998 with the repeatability RSD value < 1.2% for migration time and < 4.8% for peak area, and the highest theoretic plate number was > 586000. MEEKC was compared with micellar electrokinetic chromatography (MEKC) indicating that the former method is more suitable for this separation and can be used for the quality control of biphenyl nitrile compounds in the synthesis of liquid crystals.     

Analysis of anticancer platinum(II)-complexes by microemulsion electrokinetic chromatography: separation of diastereomers and estimation of octanol-water partition coefficients

Microemulsion electrokinetic chromatography (MEEKC) was applied for the separation and lipophilicity estimation of oxaliplatin and eight novel anticancer oxaliplatin derivatives. Solubility and permeability have to be balanced in modern drug development, and the octanol-water partition coefficient (log P) still represents one of the most useful quantifiable parameters providing a reasonable estimation of a drug's lipophilicity. Therefore, the capacity factors from MEEKC were correlated to log P values derived by the traditional shake flask method. The MEEKC method was accomplished using a microemulsion of heptane/sodium dodecyl sulfate (SDS)/butanol in phosphate buffer at pH 7.4 and 37 degrees C with all analytes being in a neutral state during the run. This experimental setup allowed a baseline separation of all platinum complexes within 11 min. Remarkably, beside the very good resolution and precision of the measurements, separation of diastereomers of the complexes and quantification of the diastereomeric ratios could be achieved. Correlating the capacity factors with the corresponding log P values resulted in a linear dependency with a correlation factor of r = 0.9935. Consequently, the applied MEEKC method was found to be a highly valuable technique not only for the separation of platinum complexes but as well for the estimation of the octanol-water partition coefficient with many advantages in comparison to other methods.     

Determination of octanol-water partition coefficients for carbonate esters and other small organic molecules by microemulsion electrokinetic chromatography

Microemulsion electrokinetic chromatography (MEEKC) was assessed as a tool for determination of octanol-water partition coefficients using 34 solutes encompassing 8 carbonate esters. It was confirmed that microemulsions containing 1.44-2.88% w/w SDS, 6.49% w/w 1-butanol, and 0.82% w/w n-heptane constitute a good model of octanol-water partitioning in the pH range of 1.4-7.4. Use of the migration index concept led to improved repeatability of the MEEKC method compared to the use of retention factors. Using a dynamical coating, a high electroosmotic flow at pH 1.4 and 4.75 was achieved expanding the practical pH working range of the MEEKC system. The correlation obtained between the migration index and log P was unaffected by pH indicating that the properties of the microemulsion droplets and, thus, partitioning are independent of pH. No evidence for congeneric behavior was found for the sample set comprising solutes with different hydrogen bonding properties suggesting that simple reference compounds can be used as calibrators. Lipophilicity estimates for the series of carbonate esters were obtained. The increase in lipophilicity with chain length was smaller than expected from the Hansch substituent constant, pi.     

Selectivity in microemulsion electrokinetic chromatography

Microemulsion electrokinetic chromatography (MEEKC) is a most promising separation technique providing good selectivity and high separation efficiency of anionic, cationic as well as neutral solutes. In MEEKC lipophilic organic solvents dispersed as tiny droplets in an aqueous buffer by the use of surfactants provide a pseudo-stationary phase to which the solutes may have an affinity either to the surface or they may even partition into the droplets. When the droplets are charged, typically negatively, they will migrate opposite to the electroosmotic flow and hence separation of neutral solutes may take place. In the present paper focus has been set on how to change selectivity in MEEKC. Changes in the nature of surfactant as well as in pH have been shown to be powerful tools in changing the selectivity. The type of lipophilic organic phase is of less importance for the separation of fairly lipophilic solutes. Also changes in the temperature surrounding the capillary may alter the selectivity.     

Highly efficient and selective separations of a wide range of analytes obtained by an optimised microemulsion electrokinetic chromatography method

Summary Microemulsion Electrokinetic Chromatography (MEEKC) has previously been reported to be useful for the separation of a range of hydrophobic solutes. The previous reports had tended to show relatively long separation times. In this report the separation and operation conditions were optimized and a method was successfully employed in a range of novel applications. High efficiencies, rapid separations and high repeatability were demonstrated. The applications developed included a range of both neutral and charged compounds. Both water-soluble and water-insoluble compounds were also well separated. The novel applications developed included analysis of basic drugs, various aromatic acids, a range of neutral aromatics, profiling of ink components, direct injection of urine for profiling purposes and the analysis of a range of amino acids and their derivatives. The method could also be used for hydrophobicity measurements of the resolved solutes based upon their migration times. This report clearly shows that MEEKC can be widely applied to a range of compounds and is especially useful for complex mixtures containing solutes with varying charge and hydrophobicity.     

磷脂微乳电动色谱的定量结构保留关系研究

以大豆磷脂为主要的表面活性剂,制备适合毛细管电动色谱使用的不同构成比的微乳体系, 应用溶剂化参数模型研究了中性溶质在其中的定量结构保留关系.使用动态涂层毛细管, 以二甲基亚砜和十二烷基苯分别作为电渗流和微乳液滴迁移的标记物, 测定了26个具有不同结构小分子中性化合物在17种微乳电动色谱体系下的保留因子, 建立了线性溶剂化能量关系(LSER)方程.通过比较两体系的LSER方程系数比较体系相似性.结果表明, 本研究建立的磷脂微乳电动色谱体系在线性溶剂化特征上和其它构成的微乳电动色谱体系相似.对溶质保留贡献较大的是溶质体积和有效氢键碱度, 油相种类及浓度对溶质的保留选择性无明显影响.     

On-line concentration sample stacking coupled with water-in-oil microemulsion electrokinetic chromatography

This study describes for the first time, the ability of a normal stacking mode (NSM) on-line concentration step coupled with water-in-oil (W/O) microemulsion electrokinetic chromatography (MEEKC), using six common penicillin antibiotics (oxacillin, penicillin V, penicillin G, nafcillin, ampicillin, and amoxicillin) as test analytes. Optimization of penicillin separation in the conventional W/O MEEKC system demonstrated that change in the type and concentration of the oil phase (1-butanol) and column temperature had a pronounced effect on the separation. With the subsequent development of the NSM coupled with W/O MEEKC, improved separation and detection sensitivities were observed when an organic solvent plug (1-propanol; 1.04 cm) was placed between the W/O microemulsion and the sample solutions. This could be attributed to the solution viscosity difference between the aqueous sample zone and the organic solvent plug causing the penicillin to be stacked in this 1-propanol plug. The optimal NSM W/O MEEKC provided about 12-fold increase in detection sensitivity compared with conventional sample injection (50 mbar, 3 s). Finally, this proposed method was successfully applied in the analyses of several food samples (porcine organs) spiked with penicillin.     

Microemulsion electrokinetic chromatography as a suitable tool for lipophilicity determination of acidic,neutral, and basic compounds

In the present work, several MEEKC systems are studied to assess their suitability for lipophilicity determination of acidic, neutral, and basic compounds. Thus, several microemulsion compositions over a wide range of pH values (from 2.0 to 12.0), containing heptane, 1?butanol and different types and amounts of surfactant (SDS or sodium cholate: from 1.3 to 3.3%) are characterized using Abraham's solvation model. The addition of acetonitrile (up to 10%) is also studied, since it increases the resolution of the technique for the most lipophilic compounds. The system coefficients obtained are very similar to those of the 1?octanol/water, used as the reference lipophilicity index, allowing simple and linear correlations between the 1?octanol/water partition values (log P_o/w) and MEEKC mass distribution ratios (log k_MEEKC). Variations in the microemulsion composition (aqueous buffer, surfactant, concentration of ACN) did not significantly affect the similarity of the MEEKC systems to log P_o/w partition.     

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.     

Improvement of microemulsion electrokinetic chromatography for measuring octanol-water partition coefficients

Microemulsion electrokinetic chromatography (MEEKC) has been used to indirectly measure octanol-water partition coefficients (log P(ow)) of compounds. In order to obtain an accurate log P(ow) value, the electrophoretic mobilities of the microemulsion phase (mu(me)) and the analyte (mu(eff)) in MEEKC must be accurately required. However, in conventional MEEKC, the shortage of obtaining mu(me) with a tracing method was discovered, and the influences of concentration, injection volume of analyte, and high electric field on measuring mu(eff) were also found. In this paper, a novel method called improved MEEKC (I-MEEKC) was developed to avoid the problems mentioned above. In I-MEEKC, a nonlinearity fitting program was used to obtain mu(me) to avoid the error from tracing mu(me); the extrapolating method was used to eliminate the effects of concentrations and injection volumes of analytes on mu(eff) measurement, and an enough stable microemulsion was selected to eliminate the effect of high electric field on mu(eff )measurement. Then the novel method was applied to estimate log P(ow) of uncharged compounds and charged pharmaceuticals compared to the conventional MEEKC. The log P(ow) of all analytes obtained by I-MEEKC agreed with those obtained by classical shake flask or literature values, the errors between them were within 0.1 logarithm units, better than the ones by conventional MEEKC.     

具有油水分配系数测算功能的中药指纹图谱

建立了一种能同时测算中药中主要成分的油水分配系数Pow的功能化中药指纹图谱。采用微乳液电动色谱,以中药女贞子为例,对运行缓冲液浓度、pH值、十二烷基硫酸钠浓度等参数进行了优化。利用SPSS程序对磺胺等从女贞子中提取的6种标准化合物组分的迁移时间tm与其log Pow文献值进行非线性拟合,得到女贞子提取组分的标准方程线性关系良好（r=0.9880）。在相同的实验条件下测得化合物的log Pow与文献报道值较好的符合,证明该方法可靠。获得了能代表组分疏水化学特征的中药指纹图谱,测得女贞子的主要有效成分齐墩果酸的log Pow为3.63。该指纹图谱较为可靠地提供了女贞子中各种成分的log Pow,直观地反映出各组分油水分配性质的分布,对中药有效成分提取溶剂的选择、剂型的设计及制剂工艺的改进具有指导意义。     

The use of novel ionic liquid-in-water microemulsion without the addition of organic solvents in a capillary electrophoretic system

In this work, a new ionic liquid-in-water (IL/W) microemulsion without requiring toxic organic solvents was investigated as a pseudostationary phase (PSP) in CE. As observed during the IL/W microemulsion system, a fast and an efficient separation of eight phenolic acids was achieved using 1-butyl-3-methylimidazolivmhexa fluorophosphate (bmimPF(6)) as oil drops, Tween 20 as the surfactant, and borate as the BGE. The effects of oil phase, surfactant, buffer and pH on the separation were explored in detail to evaluate the novel PSP. In contrast, the detection efficiency of these same analytes was markedly decreased using oil-in-water (O/W) MEEKC. We have also validated the practicality of the IL/W microemulsion method by quantitative determination of acidic compounds in pharmaceutical injection. The results obtained indicated that an additional association between the IL cations and analytes tested seemed to play a prominent role in the separation mechanism exhibited by this novel PSP compared with the conventional O/W MEEKC.     

Recent advances in the development and application of microemulsion EKC

Microemulsion EKC (MEEKC) is an electrodriven separation technique. Separations are typically achieved using oil-in-water microemulsions, which are composed of nanometre-sized oil droplets suspended in an aqueous buffer. The droplets are stabilised by a surfactant and a cosurfactant. The novel use of water-in-oil microemulsions has also been investigated. This review summarises the advances in the development of MEEKC separations and also the different areas of application including determination of log P values, pharmaceutical applications, chiral analysis, natural products and bioanalytical separations and the use of new methods such as multiplexed MEEKC and high speed MEEKC. Recent applications (2004-2006) are tabulated for each area with microemulsion composition details.     

Determination of lipophilicity by reversed-phase high-performance liquid chromatography. Influence of 1-octanol in the mobile phase

Lipophilicity was evaluated using a novel RP-HPLC stationary phase (Discovery-RP-Amide-C16) with and without 1-octanol added to the mobile phase. A set of 46 drugs and flavonoids characterized by a broad structural diversity and a wide log Poct range (-0.69 to 5.70) was selected for this study. This set consists of neutral solutes and solutes with acidic or ampholytic functionalities which were maintained neutral at pH 2.5 or 4. In our conditions, the addition of 1-octanol in the mobile phase proved a key factor to derive a lipophilicity index log k(w) highly correlated with log Poct for all investigated solutes. 1-Octanol improved the correlation between log Poct and log k(w) mainly by influencing the retention behavior of the solutes with log Poct values below +3. This study brings additional evidence that under proper experimental conditions of stationary and mobile phases, RP-HPLC is a very useful method to obtain log Poct values.     

Recent applications of microemulsion electrokinetic chromatography

Compared to MEKC, the presence of a water-immiscible oil phase in the microemulsion droplets of microemulsion EKC (MEEKC) gives rise to some special properties, such as enhanced solubilization capacity and enlarged migration window, which could allow for the improved separation of various hydrophobic and hydrophilic compounds, with reduced sample pretreatment steps, unique selectivities and/or higher efficiencies. Typically, stable and optically clear oil-in-water microemulsions containing a surfactant (SDS), oil (octane or heptane), and cosurfactant (1-butanol) in phosphate buffer are employed as separation media in conventional MEEKC. However, in recent years, the applicability of reverse MEEKC (water-in-oil microemulsions) has also been demonstrated, such as for the enhanced separation of highly hydrophobic substances. Also, during the past few years, the development and application of MEEKC for the separation of chiral molecules has been expanded, based on the use of enantioselective microemulsions that contained a chiral surfactant or chiral alcohol. On the other hand, the application of MEEKC for the characterization of the lipophilicity of chemical substances remains an active and important area of research, such as the use of multiplex MEEKC for the high-throughput determination of partition coefficients (log P values) of pharmaceutical compounds. In this review, recent applications of MEEKC (covering the period from 2003 to 2005) are reported. Emphases are placed on the discussion of MEEKC in the separation of chiral molecules and highly hydrophobic substances, as well as in the determination of partition coefficients, followed by a survey of recent applications of MEEKC in the analysis of pharmaceuticals, cosmetics and health-care products, biological and environmental compounds, plant materials, and foods.     

Microemulsion electrokinetic capillary chromatography of sulfated disaccharides derived from glycosaminoglycans.

Microemulsion electrokinetic capillary chromatography (MEEKC) is a capillary electrophoresis technique in which neutral and ionized species can be resolved according to their partitioning into moving oil droplets present in the operating buffer. In this report, we present for the first time the application of MEEKC in the analysis of glycosaminoglycans. An efficient method for the separation of the variously sulfated delta-disaccharides obtained following digestion of chondroitin and dermatan sulfates with chondro/ dermato lyases and derivatization with 2-aminoacridone is described. Nonsulfated, mono-, di-, and trisulfated delta-disaccharides were completely separated using the microemulsion octane/butan-1-ol/Sodium dodecyl sulfate (SDS) in 10 mM borate buffer, pH 9.3, at 25 kV. Agreement of the obtained disaccharide composition with literature values showed that MEEKC can be used for the analysis of glycosaminoglycans.