Buffer Modifier Research Articles

Semipermanent capillary coatings in mixed organic–water solvents for CE

This report describes the creation of semipermanent capillary coatings that are compatible with organic-water solvent systems in CE. The coatings are created by simply rinsing the fused-silica capillary with long double-chain cationic surfactants, such as dimethyl-ditetradecyl ammonium bromide (2C(14)DAB), dihexadecyldimethyl ammonium bromide (2C(16)DAB), and dimethyldioctadecyl ammonium bromide (2C(18)DAB). These surfactants generate semipermanent bilayer coatings on the capillary surface, which display a high degree of stability in buffers containing up to 60% v/v of organic solvents, such as methanol and ACN. The coating stability increases with increasing hydrophobicity of the surfactant, i.e., with increasing chain length. For instance, the EOF changes by only 1.2% in a 2C(18)DAB-coated capillary after 130 capillary volumes of rinsing with 60% v/v methanol containing buffer. The bilayer coatings allow separations to be performed without the need to regenerate the coating between runs or to maintain the EOF modifier in the run buffer. Rapid separations (<2 min) of anions and basic drugs with migration time reproducibility of less than 0.5% RSD and efficiencies of 0.4-0.6 million plates/m are obtained. In addition, selectivity changes for small anions and cationic drugs are also observed when the organic solvent content is adjusted.

Quantification in capillary electrophoresis-mass spectrometry: Long- and short-term variance components and their compensation using internal standards

Different approaches were chosen to examine ionization reproducibility of analytes after separation by capillary electrophoresis-mass spectrometry (CE-MS) in a commercially available sheath-flow electrospray interface. For this task three different standard samples were examined. Sample 1 contained neostigmine bromide (cationic), paracetamol (PCM) (neutral) and nicotinic acid (anionic component). Results were evaluated using internal standard (IS) calculations. Sample 2 represented an isotopically labelled IS of the quantified substance (PCM/D4-PCM), while sample 3 (neostigmine bromide/scopolamine hydrobromide) provided an IS closely migrating to the tested substance. Furthermore, short-time variations inside the interface were examined by multiple injections of the same substance. For sample 1, the relative standard deviations (RSD%s) were between 8 and 25% (n at least 58) for the peak area ratios. Multiple injected samples gave 5.5-19.4% (n = 25) for peak area RSD%. Using a closely migrating IS, sample 3, RSD%s between 6.5 and 10% (n at least 63) were achieved. With isotopically labelled IS, sample 2, an RSD% of 3-4% was achieved for peak area ratios over long periods (n = 25), for shorter periods (n = 9) even 1-2% RSD% was obtained. Keeping the instrument settings constant, the influence on the ionization efficiency and reproducibility was tested, varying the buffer pH, the organic buffer modifier and the sample concentration. Repeatabilities of migration time and peak area were measured and compared. Two 10 mM ammonium acetate buffers with pH 4.0 and 8.5 were investigated. No influence of buffer pH on peak area reproducibility was found. Isopropanol as organic buffer modifier significantly improved the ionisation leading to larger peak areas, but reduced reproducibility. The basic buffer produced slightly better RSD%s for migration times (2.5-4.0%) (n = 180) and faster analysis for the different test analytes of sample 1, while with the acetic buffer, RSD%s from 3.9 to 6.0% were obtained (n at least 163). The positioning of the capillary turned out to be the crucial parameter to ensure reproducible results. Thus, a procedure was established to ensure a defined ion-intensity level after capillary changes. The investigation of the different sample concentrations gave negligible differences in RSD%, showing that the signal-to-noise ratio was not the crucial parameter for reproducibility here, in contrast to CE-UV detection.

Gold Nanoparticle-Modified Etched Capillaries for Open-Tubular Capillary Electrochromatography

The use of gold nanoparticles in conjunction with etched capillary-based open-tubular capillary electrochromatography (OTCEC) to improve the efficiency of separation and the selectivity between selected solutes is described. The fused-silica capillaries (50-microm i.d.) were etched with ammonium hydrogen difluoride, followed by prederivatization of the new surface with (3-mercaptopropyl)trimethoxysilane (MPTMS) for the immobilization of dodecanethiol gold nanoparticles, for OTCEC. The electrochromatography of a "reversed-phase" test mixture and of selected polycylic aromatic hydrocarbons was investigated, and efficient separations and high theoretical plate numbers per meter were obtained. The electroosmotic flow characteristics of the etched gold nanoparticle capillary, unetched gold nanoparticle capillary, bare capillary, and etched bare capillary were studied by varying the percentage of organic modifier in buffer, buffer pH, and separation voltage. Optical microscopy and scanning electron microscopy were used to examine the process of etching and modification and the surface features of the etched gold nanoparticle capillary. The results confirm that dodecanethiol gold nanoparticles bonded on the etched inner wall of the fused-silica capillary can provide sufficient solute-bonded phase interactions to obtain OTCEC separations with reproducible retention, as well as characteristic reversed-phase behavior, even with the inner diameter of the capillary of 50 microm.

Application of dodecyldimethyl (2-hydroxy-3-sulfopropyl) ammonium in wall modification for capillary electrophoresis separation of proteins

A zwitterionic surfactant, dodecyldimethyl (2-hydroxy-3-sulfopropyl) ammonium (C12H25N+(CH3)2CH2CHOHCH2SO3-), named dodecyl sulfobetaine (DSB), was used as a novel modifier to coat dynamically capillary walls for capillary electrophoresis separation of basic proteins. The DSB coating suppressed the electroosmotic flow (EOF) in the pH range of 3-12. At high DSB concentration, the EOF was suppressed by more than 8.8 times. The DSB coating also prevented successfully the adsorption of cationic proteins on the capillary wall. Anions, such as Cl-, Br-, I-, SO4(2-), CO3(2-), and ClO4-, could be used as running buffer modifiers to adjust the EOF for better separation of analytes. Using this dynamically coated capillary, a mixture of eight inorganic anions achieved complete separation within 4.2 min with the efficiencies from 24,000 to 1,310,000 plates/m. In the presence of ClO4- as EOF adjustor, the separation of a mixture containing four basic proteins (lysozyme, cytochrome c, alpha-chymotrypsinogen A, and myoglobin) yielded efficiencies of 204,000-896,000 plates/m and recoveries of 88%-98%. Migration time reproducibility of these proteins was less than 0.5% relative standard deviation (RSD) from run to run and less than 3.1% RSD from day to day, showing promising application of this novel modifier in protein separation.

Separation of purine and its derivatives by capillary zone electrophoresis

The separation of a group of 17 purine and its derivatives by capillary zone electrophoresis is presented. A systematic approach was used to study the effect of pH, buffer type, organic modifiers, applied potential, sodium dodecyl sulfate (SDS) and cyclodextrins on the separation of these purine derivatives. An ideal condition was found for their separation, which was 30 mmol/L sodium borate buffer (pH 9–9.5), 10% (V/V) methanol buffer modifier and 20 kV. Under this condition, the 17 purine derivatives were baseline separated and the linear correlation coefficient for adenine, uric acid and 2-thioxanthine was 0. 99 over two orders of magnitude. The variation of peak areas was less than 4. 6% (n=5) and that of migration times was in the range of 0%–3%, while the samples were injected hydrodynamically at a height of 15 cm and an injection time of 8–10 s. In addition, alcohol, 1-propanol, 1-butanol and acetonitrile were also effective additives in the separation. However, SDS and various β-cyclodextrin (β-CDs) were found to do no good to their separation.

Cyclodextrin-modified micellar electrokinetic chromatography for the analysis of Esterom, a topical product consisting of hydrolyzed benzoylecgonine in propylene glycol.

Esterom, a new drug currently in human clinical trials, is a mixture of compounds in a propylene glycol vehicle. It is being evaluated as a topical treatment to aid in the relief of muscle pain and to increase range of motion. Benzoylecgonine is the major component of Esterom and there are at least nine other minor constituents, including four hydroxypropyl esters that have multiple diasteriomers. The aim of the study was to develop a capillary electrophoresis method for the simultaneous separation of the main components in Esterom, including the multiple proposed diastereomers of the esters. Due to the complex sample composition, the use of micelles and cyclodextrins as buffer modifiers was evaluated. A cyclodextrin-modified micellar electrokinetic chromatography method was able to determine 7 of the 8 UV-active Esterom components, with baseline separation of 7 of the 10 diastereomers of the hydroxypropyl esters.

Optimized separation of pharmacologically active xanthones from Securidaca inappendiculata by micellar electrokinetic chromatography and microemulsion electrokinetic chromatography

Micellar electrokinetic chromatography (MEKC) has been used for the separation of nine xanthones from Securidaca inappendiculata by using sodium dodecyl sulfate (SDS) micellar system, and the effects of different buffer modifiers including sodium cholate (SC), organic solvent and cyclodextrins (CDs) on the separation selectivity have been extensively studied, based on the systematically optimization of such parameters as pH, concentration of running buffer and SDS concentration. The results indicated that the additions of modifiers could greatly affect the separation selectivity of xanthones, and the migration order of those xanthones depends on their structures and solute–micelle-modifier interactions. As a special variant of electrokinetic chromatography, microemulsion electrokinetic chromatography (MEEKC) has also been utilized for this separation for the comparison with MEKC, showing that MEEKC has better selectivity.

Enantioseparation of amino acid derivatives by capillary zone electrophoresis using vancomycin as chiral selector.

The separation of racemic derivatized amino acids (N-acetyl) into their enantiomers was achieved using capillary zone electrophoresis employing vancomycin as a chiral selector. Due to the strong absorption properties of the chiral selector at the low wavelengths used, the partial-filling countercurrent method was adopted in order to improve method sensitivity. In the separation system studied, the chiral selector filled only a part of the capillary and, due to the appropriate selection of the pH, was moving in the opposite direction of the analytes keeping the detector free from absorbing compounds. The effect of several experimental parameters on the enantioresolution of analytes was studied, e.g., vancomycin concentration (0-5 mM), pH of the background electrolyte (pH 4-7), capillary temperature (15-35 degrees C), and the presence of an organic modifier in the run buffer (methanol or ethanol or n-propanol). N-Acetyl glutamic acid, serine, cystine, tyrosine, and proline were all baseline-resolved into their enantiomers and the enantioresolution factor (R(s)) was increased by raising the vancomycin concentration. pH 4 allowed the baseline resolution of the five studied analytes in the presence of 2.5 mM of chiral selector and an increase in pH caused a decrease of R(s).

Calculation of the electrophoretic mobility of amines in methanol-aqueous electrolyte systems.

An equation for calculating the electrophoretic mobility of an analyte with respect to the concentration of organic modifier in mixed aqueous-organic modifier running buffer was derived from mixture response surface methodology. In order to assess the accuracy and predictability of the proposed model, the electrophoretic mobilities of four aliphatic amines and imidazole (background absorber) were measured in mixed water-methanol running buffer containing 0-100% v/v methanol by capillary zone electrophoresis with indirect UV detection. The accuracy of the proposed model was examined by fitting all the experimental data points. The predictability of the model was then evaluated by employing six training data points to compute the model constants, and the mobility at the other data points was predicted by the trained model. The proposed model gave accurate results for the correlation and prediction, with mean percentage errors for the amines studied of 0.6% to 0.5% and 1.5% to 1.1%, respectively.

Capillary electrophoretic separation, immunochemical recognition and analysis of the diastereomers quinine and quinidine and two quinidine metabolites in body fluids.

The capillary electrophoretic separation and immunochemical recognition of the two naturally fluorescing, cationic diastereomers quinine (QN) and quinidine (QD), their hydroderivatives and two major QD metabolites (3-hydroxyquinidine and quinidine-N-oxide) was investigated. Plain aqueous phosphate buffers and an alkaline buffer containing dodecyl sulfate micelles are shown to be incapable of resolving the two diastereomers. However, incorporation of an additional chemical equilibrium (with beta-cyclodextrin) in the case of capillary zone electrophoresis (CZE) and the presence of a small amount of an organic solvent as buffer modifier (2-propanol) in dodecyl sulfate based micellar electrokinetic capillary chromatography (MECC), were found to provide separation media which lead to complete resolution of QN, QD and the other compounds of interest. Furthermore, for MECC- and CZE-based immunoassay formats, a commercially available antibody against QD was found to be a perfect discriminator between QD and QN. It was determined to recognize QD and the two QD metabolites (cross reactivity of 20--30%) but not QN. MECC and CZE with laser induced fluorescence (LIF) detection are shown to be suitable to determine QD and metabolites in urine and plasma (quinidine-N-oxide only) collected after single dose intake of 50 mg QD sulfate and of QN in urine, saliva and serum samples that were collected after self-administration of 0.5 l of quinine water (25 mg of QN). With direct injection of a body fluid, MECC with LIF was found to provide 10 ng/ml detection limits for QD and QN. This ppb sensitivity is comparable to that obtained in HPLC assays that are based upon drug extraction. Furthermore, MECC and CZE assays with UV detection are shown to provide the ppm sensitivity required for therapeutic drug monitoring and clinical toxicology of QD and QN.

Amperometric Ion Detection in Capillary Zone Electrophoresis by Ion Transfer across a Liquid−Liquid Microinterface

Ion transfer across the electrically charged interface of two immiscible electrolytes has been employed for the amperometric detection of ions in capillary zone electrophoresis (CE). The method is inherently selective to hydrophobic ions, which are characterized by a low standard Gibbs energy of partition between the aqueous CE buffer solution and 2-nitrophenyl n-octyl ether employed as organic phase. The applicability of this detection scheme for nonredox ions is demonstrated by means of the cations choline and acetylcholine and by some alkyl and aryl sulfate and sulfonate anions. The response of the detector is sufficiently fast; no additional peak broadening has been observed. Plate numbers of 100 000(170 000 m -1 ) were determined for the quickly migrating choline cation. It is shown that the use of buffer modifiers such as organic solvent additives, cyclodextrins, and polyelectrolytes is compatible with the detector within certain boundaries. The limit of detection was ∼ 1 μM for the ions tested.

Correlation of the capacity factor in vesicular electrokinetic chromatography with the octanol:water partition coefficient for charged and neutral analytes.

The aim of this study was to develop a method based upon electrokinetic chromatography (EKC) using oppositely charged surfactant vesicles as a buffer modifier to estimate hydrophobicity (log P) for a range of neutral and charged compounds. Vesicles were formed from cetyltrimethylammonium bromide (CTAB) and sodium n-octyl sulfate (SOS). The size and polydispersity of the vesicles were characterized by electron microscopy, dynamic light scattering, and pulsed-field gradient NMR (PFG-NMR). PFG-NMR was also used to determine if ion-pairing between cationic analytes and free SOS monomer occurred. The CTAB/SOS vesicles were used as a buffer modifier in capillary electrophoresis (CE). The capacity factor (log k') was calculated by determining the mobility of the analytes both in the presence and absence of vesicles. Log k' was determined for 29 neutral and charged analytes. RESULTS; There was a linear relationship between the log of capacity factor (log k') and octanol/water partition coefficient (log P) for both neutral and basic species at pH 6.0, 7.3, and 10.2. This indicated that interaction between the cation and vesicle was dominated by hydrophobic forces. At pH 4.3, the log k' values for the least hydrophobic basic analytes were higher than expected, indicating that electrostatic attraction as well as hydrophobic forces contributed to the overall interaction between the cation and vesicle. Anionic compounds could not be evaluated using this system. Vesicular electrokinetic chromatography (VEKC) using surfactant vesicles as buffer modifiers is a promising method for the estimation of hydrophobicity.

CHIRAL SEPARATION OF SEVERAL THIOBARBITURATES ON A CELLULOSE TRIS(4-METHYLBENZOATE) PHASE UNDER REVERSED-PHASE CONDITIONS

The chiral separation of several thiobarbiturates on a tris(4-methylbenzoate) phase (Chiralcel OJ-R, 15 × 0.46 cm and Chiralcel OJ-R 15 × 0.20 cm) under reversed-phase conditions is described. The separation properties of a normal sized and a newly developed narrow-bore column were compared. The influence of buffer and organic modifiers on enantioselectvity was investigated. The three mobile phases used were water/acetonitrile (70:30,v/v), water/acetonitrile/methanol (50:25:25, v/v), and water/acetonitrile/methanol (55:15:30, v/v). Of seven thiobarbiturates investigated, five have been resolved. The values of α (1.11–1.85) and R s (0.71–5.75) have been reported.

Comparison of migration modeling in micellar electrokinetic chromatography by linear regression and by use of an artificial neural network

The concentrations of modifier (methanol or acetonitrile) and surfactant (sodium dodecyl sulfate, SDS) in the running buffer are important factors influencing the mobility of analytes in micellar electrokinetic chromatography (MEKC). Response surfaces of the effective mobility can be used to predict mobility as a function of the buffer composition. This paper describes the modeling, by multiple linear regression and by use of a back-propagation neural network, of the response surfaces of the effective mobility of cocaine and related compounds, of fluvoxamine and its possible impurities, and of a mixture of alkylphenones. Special attention has been paid to selection of the proper order of the regression models, to selection of the proper network configuration, and to the predictive quality of the models. The regression models were selected by backward elimination of non-significant terms starting with a full second-order polynomial model. The proper network configuration was selected by finding the optimum number of hidden neurons by lateral inhibition and subsequent determination of the optimum number of training cycles by a cross-validation method. On the basis of the multiple determination coefficient,R 2, the descriptive performances of the neural network and the regression models are almost the same. However, the predictive performance of the regression models and the neural network models are approximately equal for migration in MEKC. The relative prediction error of the effective mobility is ca 2.5% for cocaine and related compounds, 1 – 7% for the alkylphenones, and 1.5 – 2.2% for fluvoxamine and its possible impurities; this is approximately the same order of magnitude, or slightly higher, than the relative standard deviation of the replicate measurements.

Capillary electrophoresis of seed 2S albumins from Lupinus species

Two modes of capillary electrophoresis (CE) — free-solution capillary zone electrophoresis (CZE) and sodium dodecyl sulfate capillary electrophoresis (SDS-CE) using a non-gel sieving matrix — have been developed for comparative analysis of low-molecular-mass 2S albumin isoforms from lupins. The albumin fraction and 2S albumins were separated in uncoated fused-silica capillary by CZE with 0.02 M phosphate buffer, pH 7.3, containing the sodium salt of phytic acid. The use of phytic acid (0.025 M) as buffer modifier and ion-pairing agent improved migration reproducibility, peak shape and separation efficiency. The reduced 2S albumins were separated by SDS-CE using a high concentration (0.3–0.5 M) mixture of tris(hydroxymethyl)aminomethane and borate buffers in uncoated fused-silica capillary. Of the various polymers used as non-gel sieving matrix, SDS-CE with a 10% dextran solution was found to be suitable for separation of 2S albumin polypeptides with molecular masses of 4000–7000 and 8000–11 000. The addition of glycerol or ethylene glycol to the SDS separating buffer improved the resolution of polypeptides. The examined Lupinus species showed species-specific CZE and SDS-CE migration profiles of the 2S albumins.

Applications of a sulfonated-polymer wall-modified open-tubular fused-silica capillary in capillary zone electrophoretic separations

A fused-silica capillary that is wall-modified via chemically bonding a sulfonated polymer to the capillary wall has a uniform negative charge density on its surface and produces an electroosmotic flow (EOF) greater than 4·10 −4 cm 2 V −1 s −1. The EOF is nearly independent of buffer pH over the pH range of 2 to 10 and is lower than the EOF obtained for the bare fused-silica capillary at the more basic pH but is higher at the more acidic buffer pH. Optimization of buffer pH can be based on analyte p K a values to improve the overall quality of the capillary zone electrophoresis (CZE) separation of complex mixtures of weak acid and base analytes. Because of the high EOF in an acidic buffer, the capillary is useful for the separation of weak organic bases which are in their cation forms in the acidic buffer. EOF for the sulfonic acid bonded phase capillary can be adjusted via buffer additives such as organic solvent, tetraalkylammonium salts, multivalent cations and alkylsulfonic acids. The advantages of utilizing buffer pH and the EOF buffer modifiers to enhance migration time, selectivity, and resolution in CZE separations with this capillary are illustrated using a series of test analyte mixtures of inorganic anions, carboxylic acids, alkylsulfonic acids, benzenesulfonic acids, sulfas, pyridines, anilines or small-chain peptides.

Experimental designs and response surface modeling applied for the optimization of metal-cyanide complexes analysis by capillary electrophoresis.

The development of capillary zone electrophoresis (CZE) methods for the determination of metal cyanide complexes in real samples showed some problems, such as the low detection signal of Au (CN)2- and the low resolution between Ni(II) and Fe(II) cyanides in gold processing solutions, and the lack of separation of Pt(CN)4(2-) and Pd(CN)4(2-) in the leachates from automobile catalytic converters. To optimize some analytical parameters, the present study thus focused on the application of experimental designs and multiregression models. The following factors were examined by a two-level factorial design: applied voltage, injection time, detection wavelength, buffer ion, ionic strength and buffer modifiers. For optimization of the CZE method, subsequent response-surface experiments with the important factors were made with the two kinds of leaching solutions. Optimal analytical conditions were obtained in each case, giving good detection signals and resolution for the components of the studied leachates.

Electrophoretic separation and characterisation of gliadin fractions from isolates and nanoparticulate drug delivery systems

Gliadin is a group of polymorphic proteins that are commonly used for the preparation of many pharmaceutical and cosmetic products. The aim of this study was to develop an analytical HPCE procedure for the identification and quantification of gliadin samples. For this purpose, the HPCE buffer composition were optimised to improve the resolution of separations. There-fore, several buffer modifiers were tested for use with 50 mM phosphate buffer in HPCE separations. Twenty percent acetonitrile and 0.05% hydroxypropylmethyl-cellulose provided optimal resolution while maintaining excellent reproducibility. In conclusion, this method can be successfully applied to the elucidation of the different gliadin fractions in protein isolates and nanoparticulate dosage forms.

Separation of resin acids using cyclodextrin-modified capillary electrophoresis.

A cyclodextrin-modified capillary electrophoretic method has been developed for the analysis of eleven common resin acids using a pH 4.5, 20 mM sodium acetate buffer containing 10% acetonitrile, 20 mM methyl-beta-cyclodextrin (MECD) and 30 mM sulfobutylether-beta-cyclodextrin (SBCD) as buffer modifiers. At pH below their pKa (< 5.7-6.4) the resin acids were virtually unionized and insoluble; however, they formed water-soluble inclusion complexes with MECD (20 mM) or SBCD (30 mM) even at pH 4.5. The analytes were separated in 25 min and, with the exception of two pairs, 12- or 14-chlorodehydroabietic/12,14-dichlorodehydroabietic acid and dehydroabietic/palustric acid, the remaining resin acids were baseline-separated. Analysis time was significantly shortened (< 12 min) at pH 9.25 using 30 mM SBCD and 20 mM MECD in 20 mM sodium borate. Resin acids were baseline-separated with the exception of two pairs, pimaric/sandaracopimaric acid and 12- or 14-chlorodehydroabietic/abietic acid. The addition of 7.5% methanol to the running buffer resolved the abietic acid peak. Both HPLC and micellar capillary electrokinetic chromatography using 20 mM deoxycholic acid, 10% acetonitrile in 20 mM sodium borate, pH 9.25, failed to resolve the resin acids. The simple capillary electrophoretic method developed would be useful for the rapid separation and characterization of several important resin acids in pulp mill effluents and other contaminated samples.

Reverse Polarity Capillary Zone Electrophoretic Analysis of Nitrate and Nitrite in Natural Water Samples

This paper describes the application of reverse polarity capillary zone electrophoresis (CZE) for rapid and accurate determination of nitrate and nitrite in natural water samples. Using hexamethonium bromide (HMB) as an electroosmotic flow modifier in a borate buffer at pH 9.2, the resolution of nitrate and nitrite was accomplished in less than 3 min. CZE was compared with ion chromatographic (IC) and cadmium reduction flow injection analysis (Cd-FIA) methods which are the two most commonly used standard methods for the analysis of natural water samples for nitrate and nitrite. When compared with the IC method for the determination of nitrate and nitrite, CZE reduced analysis time, decreased detection limits by a factor of 10, cut laboratory wastes by more than 2 orders of magnitude, and eliminated interferences commonly associated with IC. When compared with the cadmium reduction method, CZE had the advantage of simultaneous determination of nitrate and nitrite, of being able to be used in the presence of various metallic ions that normally interfere in cadmium reduction, and of decreased detection limits by a factor of 10.