Optimization of separation and migration behavior of chloropyridines in micellar electrokinetic chromatography

The separation and migration behavior of pyridine and eight chloropyridines, including three monochloropyridines, four dichloropyridines, and 2,3,5-trichloropyridine were investigated by micellar electrokinetic chromatography using either sodium dodecyl sulfate (SDS) as an anionic surfactant or SDS-Brij 35 mixed micelles. Various parameters such as buffer pH, SDS concentration, Brij 35 concentration and methanol content that affect the separation were optimized. Complete separation of these chloropyridines was optimally achieved with a phosphate buffer containing SDS (30 mM) and methanol (10%, v/v) at pH 7.0. The resolution and selectivity of analytes could be considerably affected by the addition of methanol and/or Brij 35 to the background electrolyte. The migration order of these chloropyridines depends primarily on their hydrophobicity. However, electrostatic interactions may also play a significant role in the determination of the migration order of the positional isomers of chloropyridines.     

Methodology for predicting the separation of proteins by hydrophobic interaction chromatography and its application to a cell extract

Ligand-exchange micellar electrokinetic capillary chromatography was used for the chiral resolution of underivatized and dansyl amino acid enantiomers simultaneously. The separation was achieved by chiral copper(II)-L-valine complexes incorporated in micelles of sodium dodecyl sulfate (SDS). The enantioresolution was strongly affected by SDS and a concentration of 20 mM SDS was shown to be necessary for the separation. Other impacting factors were investigated including pH, the molar ratio of copper(II) to L-valine and the total concentration of complex. Using the proposed method, 11 different dansyl amino acids and two underivatized amino acids were separated successfully with a running electrolyte of 20 mM NH4OAc, 4 mM CuSO4, 8 mM L-valine and 20 mM SDS at pH 9.0 in less than 25 min. Experiments were also performed with other amino acid ligands in order to vary the stability and the sterical arrangement of the copper(II) complexes and the possible chiral recognition mechanism was also discussed briefly.     

Enantiomeric separation of dihydroxyphenylalanine (DOPA), methyldihydroxyphenylalanine (MDOPA) and hydrazinomethyldihydroxyphenylalanine (CDOPA) by using capillary electrophoresis with sulfobutyl ether-beta-cyclodextrin as a chiral selector

Capillary electrophoresis (CE) was successfully applied to the enantiomer resolution of racemic structurally related compounds, namely dihydroxyphenylalanine (DOPA), methyldihydroxyphenylalanine (MDOPA) and hydrazinomethyldihydroxyphenylalanine (CDOPA). The chiral resolution was performed in an untreated fused-silica capillary by using a phosphate buffer at pH 2.5 or 3.0 supplemented with sulfobutylated beta-cyclodextrin (SBE-CD). Resolution was strongly influenced by the concentration of the chiral selector added to the background electrolyte. In fact, 2-5 mM of SBE-CD enabled the resolution of DOPA and MDOPA enantiomers, while CDOPA optical isomers were resolved by using either 0.5 mM or 6-20 mM of SBE-CD. The latter separation conditions (reversed polarity mode) made it possible to obtain inversion of migration order.     

Optimization of the separation of phenolic compounds by micellar electrokinetic capillary chromatography

A group of phenolic compounds including phenolic aldehydes, acids and flavonoids are separated by micellar electrokinetic chromatography (MECC). The influence of buffer (concentration and pH), concentration of sodium dodecylsulphate (SDS) and applied voltage were studied. To increase the selectivity of the separation and the resolution of the solutes organic solvents are added to the separation buffer, the best results were obtained when methanol was used at lower percentages. An optimized buffer (150 mM boric acid (pH 8.5)-50 mM SDS-5% methanol) provides the optimum separation with regard to resolution and migration time. This method was applied to the determination of these compounds in wine samples with good results.     

Molecularly imprinted CEC sorbents: investigations into polymer preparation and electrolyte composition

The influence of the sorbent preparation protocol and separation parameters on the selectivity and chromatographic efficiency of super-porous molecularly imprinted polymer (MIP) monoliths in capillary electrochromatography (CEC) was studied. Chiral templates were employed and enantiomer separation and resolution were used as measures of imprint selectivity and column efficiency, respectively; the latter was in addition studied by chromatography of non-related aromatic structures. The polymer preparation was varied with respect to monomer composition in the pre-polymerisation mixture and also the use of single versus multiple template(s). The separation parameters investigated were type and content of organic solvent and surfactant modifier in the electrolyte. It was found that acetone and acetonitrile in buffer mixtures provided enantiomer separation of enantiomers of the template and also structural analogues; however, the degree of separation was greatly influenced by the content in the electrolyte. Three surfactants, sodium dodecylsulfate (SDS), cetyltrimethylammonium bromide (CTAB) and polyoxyethylene sorbitanmonolaurate (Tween 20), were examined as electrolyte modifiers. It was found that addition of SDS decreased and CTAB and Tween 20 increased the enantiomer separation. SDS and CTAB could be used up to 1 mM concentration whereas Tween could be used up to 90 mM concentration without causing baseline disturbances. The effects found and demonstrated strongly suggest that these parameters are to be considered during optimisation of an MIP-CEC system.     

Determination of lignans in Schisandra chinensis using micellar electrokinetic capillary chromatography

Micellar electrokinetic capillary chromatography (MEKC) has been developed as a promising method for the determination of lignans in plant samples. The separation conditions have been optimized with respect to the different parameters including sodium dodecyl sulfate (SDS) and acetonitrile concentration, pH of the background electrolyte, separation voltage, and capillary temperature. The background electrolyte consisting of 40 mM SDS and 35% acetonitrile in 10 mM tetraborate buffer (pH 9.3) was found to be the most suitable electrolyte for this analysis. The applied voltage of 28 kV (positive polarity) and the capillary temperature 25 degrees C gave the best separation of lignans. The interday reproducibility of the peak areas and the migration times was below 2.0%. The results of MEKC analyses were compared with those obtained by capillary electrochromatography (CEC) and reversed-phase high-performance liquid chromatography (RP-HPLC). The possibilities of using this method for the determination of lignans in drug and in serum samples were also tested.     

Capillary electrophoretic chiral separation of Cinchona alkaloids using a cyclodextrin selector

A new capillary electrophoretic method for the chiral separation of four major Cinchona alkaloids (quinine/quinidine and cinchonine/cinchonidine) was developed using heptakis-(2,6-di-O-methyl)-beta-cyclodextrin as the chiral selector. The inner walls of the separation capillary were modified with a thin polyacrylamide layer, which substantially reduced the electroosmotic flow and improved the chiral resolution and the reproducibility of the migration time of the analytes. Various operation parameters were optimised, including the pH, the capillary temperature, the concentration of the background electrolyte, and the concentration of the chiral selector. Baseline separation of the two diastereomer pairs was achieved in 12 minutes in ammonium acetate background electrolyte pH 5.0 with addition of cyclodextrin in a concentration of 3 mM or higher.     

Separation and determination of lignans from seeds of Schisandra species by micellar electrokinetic capillary chromatography using ionic liquid as modifier

In this paper, a micellar electrokinetic chromatographic (MEKC) method using ionic liquid as modifier for the quantification of the active components of lignans found in the medicinal herbs Schisandra species was developed for the first time. Preliminary investigations employing sodium dodecyl sulfate (SDS) as surfactant did not lead to the necessary resolution of the studied compounds, the addition of ionic liquid 1-butyl-3-methylimidazolium tetrafluoroborate (BMIM-BF4) to the SDS micellar system resulted in the complete separation of all the compounds. The effects on the separation by several parameters such as BMIM-BF4 and SDS concentration, applied voltage, background electrolyte pH and concentration, were evaluated. Under the optimal conditions (5 mM borate-5 mM phosphate buffer in the presence of 20 mM SDS and 10 mM BMIM-BF4, pH 9.2, applied voltage 25 kV and detection at 254 nm), the method successfully applied to the determination of lignans in extracts of Schisandra chinensis (Turcz.) Baill. and Schisandra henryi C.B. Clarke in less than 13 min. The separation mechanism was also discussed.     

Separation of lidocaine and its metabolites by capillary electrophoresis using volatile aqueous and nonaqueous electrolyte systems

The separation of the basic drug lidocaine and six of its metabolites has been investigated both by using volatile aqueous electrolyte system, at low pH and by employing non-aqueous electrolyte systems. In aqueous systems, the best separation of the compounds under the investigated conditions was achieved by using the electrolyte 60 mM trifluoroacetic acid (TFA)/triethylamine (TEA) at pH 2.5 containing 15% methanol. With this electrolyte, all seven compounds were well separated with high efficiency and migration time repeatability. The separations with bare fused-silica capillaries and polyacrylamide-coated capillaries were compared with higher separation efficiency with the latter. On the other hand, near baseline separation of all the seven compounds was also obtained by employing the non-aqueous electrolyte, 40 mM ammonium acetate in methanol and TFA (99:1, v/v), with comparable migration time repeatability but lower separation efficiency relative to the aqueous system.     

Separation of cold medicine ingredients by capillary electrophoresis

This study demonstrates the separation of cold medicine ingredients (e.g., phenylpropanolamine, dextromethorphan, chlorpheniramine maleate, and paracetamol) by capillary zone electrophoresis and micellar electrokinetic chromatography. Factors affecting their separations were the buffer pH and the concentrations of buffer, surfactant and organic modifiers. Optimum results were obtained with a 10 mM sodium dihydrogen-phosphate-sodium tetraborate buffer containing 50 mM sodium dodecyl sulfate (SDS) and 5% methanol (MeOH), pH 9.0. The carrier electrolyte gave a baseline separation of phenylpropanolamine, dextromethorphan, chlorpheniramine maleate, and paracetamol with a resolution of 1.2, and the total migration time was 11.38 min.     

Chiral separation of raltitrexed by cyclodextrin-modified micellar electrokinetic chromatography

A rapid and effective method was developed for the chiral separation of raltitrexed (RD) enantiomers by carboxymethyl-beta-cyclodextrin (CM-β-CD)-modified micellar electrokinetic chromatography (MEKC). Optimization of conditions including the type and concentration of the chiral selector, concentration of sodium dodecyl sulfate (SDS), pH and concentration of the background electrolyte (BGE), capillary temperature, and applied voltage was investigated. The enantiomers of raltitrexed could be separated with satisfactory resolution and linear response by using 75 mM Tris-phosphate at pH 8.0 containing 30 mM SDS and 8 mM CM-β-CD as buffer system. Furthermore, the usefulness of this method was demonstrated in a purity test of a real synthetic drug sample. Figure Chiral separation of raltitrexed by CM-β-CD MEKC was optimized and applied to test the purity of a synthetic drug sample     

Micellar electrokinetic chromatography as a fast screening method for the determination of 1,4-dihydropyridine calcium antagonists

A micellar electrokinetic chromatographic method (MEKC) was optimized for the separation of six calcium antagonists. The effects of the buffer (concentration and pH), concentration of sodium dodecyl sulphate (SDS), the organic modifier, the injection time, and the voltage applied were studied. A final appropriate electrolyte of 50 mM borate buffer, pH 8.2, containing 20 mM SDS and 15% (v/v) acetonitrile was found to provide the optimum separation with respect to resolution and migration time. The samples were introduced hydrostatically for 4 s at 50 mbar injection pressure and the applied voltage was +25 kV. The screening of the six compounds was achieved in less than 15 min: nifedipine (migration time, tm = 6.9 min), nimodipine (tm = 10.1 min), felodipine (tm = 12.2 min), nicardipine hydrochloride (tm = 12.7 min), lacidipine (tm = 13.5 min) and amlodipine besylate (tm = 14.1 min, tm = 8 min). The method developed showed to be linear at least up to 70 micrograms/ml with a detection limit of about 5 micrograms/ml for each compound. The within-day and inter-day area reproducibility (R.S.D.) were, respectively, lower than 4.8 and 8.6% for six replicate samples.     

Migration behavior and separation of benzenediamines, aminophenols and benzenediols by capillary zone electrophoresis

The migration behavior and separation of five benzendiamines, five aminophenols and three benzenediols were investigated in capillary zone electrophoresis. The results indicate that benzendiamines and aminophenols are optimally separated with a phosphate buffer at pH 5, whereas benzenediol isomers are best separated at pH about 12. The addition of surfactant monomers of tetradecyltrimethylammonium bromide to a phosphate buffer at pH 5 under the conditions of reversed electroosmotic flow is effective for separating these dye intermediates, except for the separation of 1,2-benzenediol from 1,3-benzenediol. The addition of sodium tetraborate as an electrolyte modifier is effective in the separation of 1,2-benzenediol from 1,3-benzenediol, but the latter comigrates with the 1,4-benzenediol isomer at pH 5.0. The electrophoretic mobility of ionized analytes can be described with Offord's equation, and the migration order depends on their ratios of charge to mass. In addition, the pKa values of these analytes in 50 mM phosphate buffer are reported.     

Microchip separations of transition metal ions via LED absorbance detection of their PAR complexes

Micellar electrokinetic chromatography was utilized in the electrophoretic separation of seven transition metal ions, colorimetrically complexed by 4-(2-pyridylazo)resorcinol (PAR) on a glass capillary electrophoresis microchip. Detection of the PAR metal chelates was demonstrated using a green light emitting diode (540 nm) and a miniature photomultiplier tube. Parameters investigated included the effect of buffer type, pH and surfactant concentration (sodium dodecyl sulfate, SDS) on the separation efficiency. The optimally determined background electrolyte contained 10 mM ammonium phosphate buffer (pH 7.5), 1 mM PAR to prevent kinetic lability problems and 75 mM SDS for enhanced resolution. The separation of seven transition metal ions, Co2+, V3+, Ni2+, Cu2+, Fe2+, Mn2+ and Cd2+, was achievable in under 65 s, with the resolution of each metal ion in excess of 1.60. Detection limits obtained ranged from 400 ppb for Ni2+ to 1.2 ppm for Mn2+.     

Optimization of the separation of flavonoids by micellar electrokinetic capillary chromatography: Effect of organic solvents

Summary A group of flavonoids of special interest to wine quality were separated by micellar electrokinetic chromatography (MEKC) with diode array detection. Their separation was optimized as a function of the buffer concentration and pH, the concentration of sodium dodecyl sulfate (SDS) and the applied voltage. Selectivity and resolution of the solutes were increased by adding organic solvents to the separation buffer, the best results being obtained at lower concentrations. An optimized buffer with 5% methanol provided optimum separation with regard to efficiency, resolution and migration time. The optimized method was applied to the determination of these compounds in wine samples.     

Separation of natural pyrethrum extracts using micellar electrokinetic chromatography

The separation of the six pyrethrin esters in a technical pyrethrum extract (Riedel-de-Ha?n, Cresent Chemical Co. Inc. Hauppauge, NY, USA) by micellar electrokinetic chromatography (MEKC) using both sodium dodecyl sulfate (SDS) and a polymerized surfactant as pseudo-stationary phases has been investigated and optimized. Parameters such as pH, SDS and polymerized sodium N-undecyl sulfate (poly-SUS) concentration, type and concentration of background electrolyte and organic modifier, as well as the acetonitrile/water ratio in the sample were studied to optimize the resolution, efficiency, and analysis time. An optimized separation of the six pyrethrin esters was achieved in 25 min with 25 mM Tris, buffered at pH 9, containing 30 mM SDS, 25% (v/v) acetonitrile, and an equal volume ratio of acetonitrile/water sample matrix at a voltage of 25 kV. The use of 0.5% (w/v) poly-SUS enhanced resolution of the pyrethrin esters and shortened the total analysis time from 25 to 20 min, compared to the SDS mediated separation. The optimized MEKC results are compared to the HPLC separation of these esters and show an improvement in efficiency and total analysis time.     

Chiral separation with ligand-exchange micellar electrokinetic chromatography using a D-penicillamine-copper(II) ternary complex as chiral selector

D-Penicillamine is demonstrated for the first time as a chiral ligand for the enantioseparation of dansyl amino acids based on ligand-exchange micellar electrokinetic chromatography (LE-MEKC). Copper(II) was used as the central ion in the ternary complex. The effect of surfactant on the resolution was significant. A concentration of 20 mM sodium dodecyl sulfate (SDS) was shown to be necessary for the separation. Other important parameters, such as the concentration ratio of D-penicillamine (D-PEN) to Cu2+, the kind of metal central ion, the type and pH value of buffer, were also investigated. N-Acetyl-D-penicillamine and L-valine (Val), with similar structure to D-penicillamine, were applied as their copper(II) complexes as chiral selector and the chiral recognition mechanism is briefly discussed. Under optimum experimental conditions, i.e., 20 mM NH4OAc, pH 6.5, a 2:1 concentration ratio of D-penicillamine to Cu(II), 4 mM CuSO4 and 8 mM D-penicillamine, the chiral separation of eight pairs of different dansyl amino acid enantiomers was accomplished with resolution ranging from 1.1 to 5.9. When L-PEN was used instead of D-PEN, reversal of the migration order was observed.     

Micellar electrokinetic chromatography systems for the separation of mixtures of charged and uncharged compounds

In this study, the migration behavior of charged and uncharged analytes was investigated under different conditions. Effective mobilities - electrophoretic mobilities under the influence of micelles - of cations, anions, and neutrals were measured at neutral, basic, and acidic pH (7.5, 11, and 2.2) using background electrolytes containing different sodium dodecyl sulfate (SDS) concentrations (0-90 mM) and acetonitrile (ACN) proportions (0-75%). SDS concentration and ACN proportion were found to have a tremendous effect on the effective mobilities and migration order of the model compounds. Although the SDS micelles preferably interact with neutrals and cations, hydrophobic bonds can also occur with anions. Cations, anions, and neutrals having rather different migration behaviors, it is possible to considerably enhance the selectivity of the method by adjusting properly the SDS concentration and the ACN proportion. These observations confirm the interest of using micellar electrokinetic chromatography not only for the separation of neutral substances but also to analyze charged compounds.     

Simultaneous determination of nine acetylcholinesterase inhibitors using micellar electrokinetic chromatography

MEKC was used for the separation of nine acetylcholinesterase inhibitors (AChEIs). AChEIs are an important group of drug compounds that are used medicinally to treat Alzheimer's disease and Myasthenia Gravis. At the time of the experiment, this is the first time that nine AChEIs are used simultaneously in a study. Several chromatographic parameters, such as buffer concentration, pH, surfactants and their concentration, background electrolyte composition, etc., were evaluated to optimize the separation. The optimum separation of the nine AChEIs was achieved in less than 15 min by using 12.5 mM Na(2)HPO(4), 12.5 mM Na(2)B(4)O(7) and 20 mM SDS at pH 10, an applied voltage of 30 kV and a temperature of 25 °C. The reproducibility of the method was also evaluated by computing the RSDs of the migration times and the areas of the nine analyte-peaks, and the migration time and the area of the peak that corresponds to rivastigmine added in the blood sample. The RSD values of the migration times and the peak areas were less than 2% and 6%, respectively, in most cases. The limits of detection and quantification were 0.5 μg/mL and 1.7 μg/mL, respectively. The MEKC method developed was applied to a real blood sample that was obtained from a patient who was not under any of this medication. The sample was spiked with rivastigmine in order to establish the ability of the method to separate the drug from other components that might exist in the blood sample.     

Combined use of ionic liquid and sulfobutylether‐β‐cyclodextrin as electrolyte additives for separation and determination of camptothecin alkaloids by CZE

This work reported that ionic liquid (IL) ([Bmim] [PF₆]) and sulfobutylether‐β‐CD (SBE‐β‐CD) were used as electrolyte additives for the separation and determination of camptothecin (CPT) alkaloids by CZE. Separation parameters such as the buffer type, pH, and concentration of the running buffer, the concentration of SBE‐β‐CD and IL, temperature, and separation voltage were all investigated in order to achieve the maximum possible resolution. The four analytes were baseline separated within 10 min in capillary at the separation voltage of 15 kV with a running buffer consisting of 20 mM borate buffer, 20 mM IL, and 100 mM SBE‐β‐CD at pH 9.0. Under such conditions, good linearity about two orders of magnitudes of peak areas was achieved for the investigated CPT alkaloids with the correlation coefficients ranging from 0.9946 to 0.9985. For all analytes, detection limits (S/N = 3) and quantitation limits (S/N = 10) range from 0.05 to 0.92 μg/mL and 0.17 to 3.06 μg/mL, respectively. The proposed method has not only been successfully applied to the separation and determination of CPT alkaloids but also showed that IL seemed to be a promising additive in CZE separation.