Electrokinetic Capillary Chromatography Research Articles

Capillary zone electrophoresis and micellar electrokinetic chromatography of solution of polyaniline particles

The behavior of two different fractions of the electrically conducting polymer polyaniline (PANI) was studied in order to characterize them using fast, simple, and cheap methods of capillary zone electrophoresis. In contradistinction to the expectations that the particles will bear a positive charge on their surface, in the milieu of electrolytes used for electrophoresis (0.01-0.05 M buffers of pH 4-8.5), the surface was demonstrated to be slightly negatively charged, which led to a slow migration to the anode. An evident charge was given to the particles after their interaction with micelles formed from surface active components, cetyltrimethylammonium bromide (CTAB) and sodium dodecyl sulfate (SDS). An active electrophoretic migration and full separation of the PANI fractions was achieved by micellar electrokinetic chromatography (MEKC) with negatively charged SDS micelles. With this method, a reliable qualitative as well as quantitative analysis was performed in the concentration range of 0.1-1.6 mg/mL with a reproducibility of migration times better than 1.5%.

Capillary electrokinetic chromatography with charged linear polymers as a nonmicellar pseudostationary phase: determination of capacity factors and characterization by solvation parameters.

A permanent polycation, polydiallyldimethylammonium (PDADMA), is applied as a linear, polymeric, replaceable, and nonmicellar pseudostationary phase for the separation of neutral analytes by capillary electrokinetic chromatography. It is shown that this polymer used in the background electrolyte is able to separate the analytes even if it does not form micelles under the given conditions. The most favorable aspect for practical use lays in the simple replacement of the separation media after each run, thus generating highly reproducible conditions. To determine the capacity factors of the analytes, a new method, based on an isotachophoretic regime, has been introduced for the measurement of the electrophoretic mobility of the polymeric pseudo-stationary phase. The capacity factors in the separation system, derived from the mobilities of the polymer, the electroosmotic flow, and the mobilities of 15 individual aromatic analytes, range between 0.3 and 1.2 for the given separation media (aqueous solution of acetate buffer, pH 5.2, with 4% w/w PDADMA). The type of interaction in the pseudochromatographic system was clarified from solvation parameters based on the linear free energy relationship model. It was found that pi and n electron interactions and hydrogen-bond basicity of the polymer, as compared with the aqueous bulk phase, are the main cause of retention of the analytes.

Capillary Electrochromatography: A Powerful Tool for the Resolution of Complex Mixtures

Abstract Methods such as capillary electrophoresis (CE) and capillary liquid chromatography (LC) are particularly useful in the analysis of complex mixtures because of their high resolving power. Continued refinement of these techniques has led to a novel separation method known as capillary electrochromatography (CEC), also referred to as capillary electrokinetic chromatography, which is a combination of capillary LC and conventional CE. As in LC, mobile phase is transported through a column where partitioning of solutes occurs. In CEC, mobile phase flow is generated by electroosmosis, which arises when an electric potential is applied across the long axis of the capillary column. The favorable flow dynamics of electroosmosis result in higher chromatographic efficiencies in CEC relative to LC. If the species of interest are charged, a second separation mechanism is afforded by electrophoresis. CEC combines the high efficiency of capillary electrophoretic separations with the broad range of selectivity obtainable in LC. As such, CEC provides numerous advantages over other separation methods. This study introduces the theory, principles, instrumentation, and applications of this technique.

Chiral separations using polymeric dipeptide surfactants: effect of number of chiral centers and steric factors

Two polymeric dipeptide chiral surfactants (PDCSs), poly sodium N-undecanoyl isoleucyl-valinate (SUILV) with three chiral centers and poly sodium N-undecanoyl leucyl-valinate (SULV) with two chiral centers, have been evaluated and compared as chiral pseudo-stationary phases in electrokinetic capillary chromatography. The performance of these surfactants, in terms of enantioselectivity was examined using anionic, cationic and neutral analytes. Analyses of the data suggest that the enantiomeric resolutions of the analytes with these two PDCSs are dependent upon steric factors rather than number of stereogenic centers.

Chiral separation of dansyl-DL-amino acids with micellar systems containing copper (II) ion and N-n-dodecyl-L-proline in electrokinetic capillary chromatography.

Enantiomers of dansylated DL-amino acids were resolved by chiral copper (II)-N-n-dodecyl-L-proline (1) complexes incorporated in micelles of sodium dodecyl sulfate (SDS) in electrokinetic capillary chromatography (EKC). This resolution is caused by formation of diastereomeric ternary complexes consisting of chiral ligand 1, central copper (II) ion and enantiomeric amino acid derivatives in micellar phase. However, the resolution was not observed when SDS with an anionic polar head grop was replaced with dodecyl trimethylammonium brode (DTMAB) with a cationic polar head group. The ratio between copper (II) ion and 1 in the complex in either SDS or DTMAB was measured by UV-visible spectra, which respond to the d-d transition of copper (II). Mechanism of separation should be discussed in terms of effect of surfactant structures on constitution of copper (II) ion and 1 in the micellar phase and that of arene substituent structures linked to sulfonamide units in amino acid derivatives to be separated.

Capillary electrokinetic chromatography with polyethyleneimine as replaceable cationic pseudostationary phase: Influence of methanol and acetonitrile on separation selectivity

The effect of methanol and acetonitrile, respectively, on the separation of neutral compounds (benzyl alcohol, phenols) is investigated in electrokinetic chromatographic (EKC) systems consisting of polyethyleneimine (PEI) as charged, polymeric, replaceable pseudostationary phase. The separation systems consist of a buffer solution (2-morpholinoethanesulfonic acid, pH 7.0, 20 mM) containing 0.3–0.9% (w/v) PEI as additive and a varying percentage of methanol (0–50%, v/v) or acetonitrile (0–30%, v/v). EKC is carried out in fused-silica capillaries [47.0 cm (effective length 40.3 cm)×100 μm I.D.]. They are dynamically coated with PEI, resulting in an electroosmotic flow directed towards the anode. The neutral analytes are migrating with the electroosmotic flow, and are retarded by the electrically driven counterflow of PEI. Separation of the analytes follows in the sequence benzyl alcohol, phenol, resorcinol, pyrogallol, reflecting the increasing hydrogen bond acidity and polarity (polarizibility) of the solutes. However, addition of methanol or acetonitrile causes a drastic loss of resolution, whereby the relative retention of the separands (related to benzyl alcohol) indicates a decrease of retardation upon addition of the organic solvents.

Chiral separations by open tubular capillary electrokinetic chromatography

The inner walls of 50 μm fused-silica capillaries are etched by ammonium hydrogendifluoride and then modified by the silanization/hydrosilation method with a chiral selector. Three different types of selectors were evaluated: lactone, β-cyclodextrin and naphthylethylamine. Each of the bonded chiral stationary phases provided at least partial separation for one type of racemic solute. These results confirm that bonded organic moieties on the etched inner wall of a capillary can provided sufficient solute–bonded phase interactions to influence the retention of molecules driven through a capillary by electroosmosis or a combination of electroosmosis and electrophoretic mobility.

Recent trends in enantioseparations using capillary electromigration techniques

Capillary electrophoresis (CE) is increasingly used for analytical-scale enantioseparations. Alongside more traditional methods, such as capillary zone electrophoresis and capillary electrokinetic chromatography, several special techniques (partial filling of the CE capillary, non-aqueous separations) and capillary electrochromatography are currently being developed. The introduction of new types of chiral selectors, the combination of different chiral selectors, and mechanistic studies involving other instrumental techniques are the most recent developments in this field. The on-line coupling of chiral CE with mass spectrometer may be very useful for bioanalytical problem solving.

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.

Development of capillary electrophoresis methods for the analysis of fluoroquinolones and application to the study of the influence of humic substances on their photodegradation in aqueous phase

Analytical techniques in capillary zone electrophoresis (CZE) and capillary electrokinetic chromatography (MEKC) were developed for the analysis of fluoroquinolone carboxylic acids (norfloxacin, ciprofloxacin, ofloxacin, enrofloxacin, danofloxacin) and their major degradation products. The theoretical determination of the charge densities of the studied compounds allowed the rapid development of the separation buffer conditions. These rapid estimations can be used as an effective screening tool in capillary electrophoresis (CE) method development. The two CE methods were applied to follow the photostability of enrofloxacin with and without humic substances under natural sunlight conditions. Enrofloxacin showed an average half-life of 2.0 h under summer sunlight conditions and the photolysis kinetic decreased in the presence of humic acids. The presence of humic substances in irradiated solution caused changes in the measured photodegradation product profile. Studies in affinity capillary electrophoresis (ACE) of enrofloxacin and its degradation products with the dissolved humic acids showed a lower adsorption potential of enrofloxacin to the humic phase than the degradation products. The adsorption of some photodegradation products to the dissolved humic matrix may explain the differences in the measured photodegradation products concentration in irradiated solutions. ACE turned out to be a rapid screening tool for the comparison of the adsorption potential of active ingredients and their degradation products to dissolved organic phases using very small amounts of sample.

Amino acid order in polymeric dipeptide surfactants: effect on physical properties and enantioselectivity.

The effect of amino acid order on chiral selectivity in polymeric dipeptide surfactants, as well as the physical properties of the surfactants, is investigated. An understanding of enantioselectivity of such dipeptide surfactants is crucial to the design of more efficient polymeric surfactants and has implications in other areas of research such as enantioselective interactions of amino acid based compounds (i.e., enzymes, hemoglobin, antibodies, etc.). It should be noted that such polymeric surfactants are not easily crystallized. Therefore, in a manner similar to the study of proteins, fluorescence spectroscopy is a powerful tool used to study the structure-function relationship of these polymeric surfactants. The microenvironments inside the core of 18 polymeric surfactants were characterized using the environmentally sensitive probes pyrene and 6-propionyl-2-(dimethylamino)naphthalene (Prodan). The surfactants examined in this study include all possible dipeptide combinations of the L-form of alanine, valine, and leucine and the achiral amino acid glycine (except glycine-glycine) as well as the single amino acid surfactants of alanine, valine, and leucine. The results of the fluorescent probe studies led to a proposed structure of the polymeric dipeptide surfactants in solution. The implications of the proposed structure for chiral selectivity were tested with two model atropisomers, (+/-)1,1'-bi-2-naphthol and (+/-)1,1'-bi-2-naphthyl-2,2'-diyl hydrogen phosphate, using capillary electrokinetic chromatography.

Electrokinetic chromatography of twelve monomethylbenz[a]anthracene isomers using a polymerized anionic surfactant.

A method for the separation of twelve monomethyl-substituted benz[a]anthracene isomers using poly-(sodium undecylenic sulfate) (poly-SUS) surfactant by means of electrokinetic capillary chromatography (EKC) is described. Several parameters such as concentration of acetonitrile (ACN), pH, as well as applied voltage were studied to optimize the EKC separation. ACN at a concentration of 35% v/v, 12.5 mM phosphate-borate buffer, 30 kV with 0.5% w/v poly-SUS at a pH of 9.5 provided a resolution of a mixture of nine out of twelve methylbenz[a]anthracene (MBA) isomers in 50 min. The results of this study suggest that molecular length of MBA rather than length-to-breath ratio plays an important role in the elution order of some isomers.

ANALYSIS OF AMPICILLIN AND ITS DEGRADATION PRODUCTS BY CAPILLARY ELECTROPHORESIS

Capillary zone electrophoresis (CZE) and micellar electrokinetic capillary chromatography (MECC) have been employed for the separation of ampicillin and its degradation products, especially the oligomers of ampicillin which are readily formed during storage and in aqueous solution. Both CZE and MECC are suitable for the separation of the oligomers of ampicillin. MECC is, however, more suitable for the separation of other degradation products. Electrophoresis conditions used for the analysis of ampicillin and its degradation products were as follows: length of fused (uncoated) silica capillary 44 cm (36 cm effective length), 50 μm i.d.; voltage, 15 kV; temperature, 25°C; detection wavelength, 215 nm; separation buffer, 40 mM sodium dihydrogen phosphate, and 100 mM sodium dodecyl sulfate (SDS) adjusted to pH 7.5. The influence of various parameters on the separation such as SDS concentration and the pH of the buffer were investigated. The method shows good repeatability, linearity and sensitivity. The robustness of this method was examined by applying a full factorial design to test the influence of the pH of the buffer, the SDS concentration and the buffer concentration.

Separation of neutral compounds by capillary electrokinetic chromatography using polyethyleneimine as replaceable cationic pseudostationary phase.

Polyethyleneimine (PEI, molecular weight 6 x 10(5) - 1 x 10(6)) is applied as a positively charged pseudostationary phase for electrokinetic chromatography (EKC) of uncharged mono- and oligophenols. EKC is carried out in PEI-coated fused-silica capillaries (with electroosmotic flow directed towards the anode) in 2-(N-morpholino)ethanesulfonic acid (MES) buffer (pH 7.0, 20 mM) with PEI added to the solution in concentrations up to 0.70% w/v. The pseudostationary phase leads to a retardation of the solutes mainly according to the number (and the position) of the OH-groups of the separands, and is not influenced significantly by methyl groups. For 0.70% w/v PEI solution, for instance, the relative retention, rho, has values between 0.33 and 0.53. For the systems with the highest resolution of the separands (0.25-0.30% PEI) 190,000 plates per meter are observed. The results indicate that the separation selectivity is mainly caused by ion-dipole interactions between the OH-groups of the solutes and the pseudostationary phase.

Separation of neutral compounds by capillary electrokinetic chromatography with a replaceable charged linear polymer as pseudo-stationary phase

Neutral substances are separated by capillary electrokinetic chromatography using a negatively charged linear polymer (partially hydrolyzed polyacrylamide), dissolved in the buffer (pH 7, at a concentration of 2%), as pseudo-stationary phase. The solutes are transported by the electroosmotic flow towards the cathode, whereas the charged polymer forms an electrically driven counterflow; the non-charged parts of the polymer interact with the solutes, thus leading to retardation and separation of the analytes (dimethyl sulfoxide, phenol, pyrogallol). Plate numbers are 20 000 to 24 000, and are not much lower than theoretically reachable, taking longitudinal diffusion as the only peak dispersion process into account.

Use of a New Diaminobutane Dendrimer in Electrokinetic Capillary Chromatography

A modified polyamidoamine-starburst dendrimer (PAMAM-SBD), 16-cascade:(1,4-diaminobutane)[4-1,4]:(1,6-diaza-7-oxo-nonyl idene)2:propanoic acid methyl ester (DABD), was synthesized and used as a carrier in electrokinetic capillary chromatography. The utility of DABD is demonstrated with the separation of a mixture of naphthalene derivatives and five neutral aromatic molecules without the need for an organic solvent as a modifier. Separation of a mixture of five neutral aromatic molecules were optimized through dendrimer concentration, pH, and organic modifier. The modified polyamidoamine-starburst dendrimer offered selectivity for polynuclear aromatic hydrocarbons as well as naphthalene based derivatives. However, DABD did not provide as much selectivity for neutral aromatic compounds in the presence of organic modifier.

Elvacite 2669 as pseudostationary phase: Electrokinetic capillary chromatography and light scattering study

The applicability of a polyelectrolyte, Elvacite 2669, as a pseudostationary phase in capillary electrokinetic chromatography for the separation of some neutral hydrophobic compounds was investigated. In addition, its behavior in buffer solutions for capillary electrophoretic separations was studied by dynamic light scattering. With the polymer concentration held constant and the methanol–water ratio varied, study was made of the viscosity, the migration times of some neutral hydrophobic compounds, and the light scattering properties of the buffers. The effect of varying the polymer concentration on the separation of the compounds was also investigated. All studies were made under basic conditions. Data on the aggregation behavior, including the size and aggregation number of the polyelectrolyte under different conditions, are given and the capillary electrophoretic results are interpreted in terms of the dynamic light scattering data. In addition, the interaction of Elvacite 2669 with the fused silica wall was investigated, and the results showed that polymer adsorption onto the capillary wall occurred at polymer concentrations at and above 0.5 wt%. Both the capillary electrophoretic and dynamic light scattering results showed the behavior of Elvacite 2669 to be highly dependent on the methanol–water ratio in the buffer. © 1998 John Wiley & Sons, Inc. J Micro Sep 10: 557–565, 1998

Bonded polymethacrylate stationary phases for open tubular liquid chromatographic and electrochromatographic separations

Using a new procedure developed in our laboratory for the preparation of thick polymethacrylate films bonded in fused silica capillaries, crosslinked polymeric stationary phases were synthesized in situ in 25-μm-i.d. capillaries. The effect of monomer and crosslinker concentrations on the resulting polymer film was studied by open tubular capillary electrokinetic chromatography (OTCEC) using p-hydroxy benzoates (parabens) as test solutes. Retention of analytes showed interesting trends versus monomer and crosslinker concentrations, exhibiting maxima at certain combinations rather than a continuous increase in capacity factor with increasing concentration. Because the electroosmotic flow can arise from both exposed silica and polymer surfaces, the flow velocity did not exhibit an obvious trend versus monomer and/or crosslinker concentration. Open tubular liquid chromatographic (OTLC) separations were also achieved using head pressure as the driving force for bulk flow, without employing a high-voltage power supply or micro LC pump. © 1998 John Wiley & Sons, Inc. J Micro Sep 10: 99–105, 1998

Comparative study of capillary zone electrophoresis and micellar electrokinetic chromatography for the separation of twelve aromatic sulphonate compounds

Two modes of capillary electrophoresis (CE), capillary zone electrophoresis (CZE) and micellar electrokinetic chromatography (MEKC), were investigated for the separation of 12 aromatic sulphonate compounds. In CZE, although the voltage applied, the buffer concentration and the pH were optimized for effective separation of the compounds studied, under the best conditions four of the five amino compounds coeluted, as did naphthalene-1-sulphonic acid and naphthalene-2-sulphonic acid. In MEKC, sodium dodecyl sulphate (SDS) and Brij 35 were chosen as the anionic and nonionic surfactants and the effect of the concentration of micelles was examined. The effect of adding methanol as the organic modifier was also investigated with each of these micellar systems. All the analytes, including the isomers, were completely separated by use of MEKC with Brij 35 but when SDS was used only 11 compounds were separated because two amino compounds coeluted.

Enantiomeric Separations by Use of Calixarene Electrokinetic Chromatography

Chiral acylcalix[4]arene amino acid derivatives have been synthesized and used as pseudostationary phases in capillary electrophoresis (CE). Use of these calix[4]arene (CX4) derivatives with electrokinetic capillary chromatography (4-EKC) resulted in enantioseparation of three binaphthyl derivatives at pH values above 7. The structures of the CX4 derivatives, i.e., (N-l-alaninoacyl)calix[4]arene (CX4-l-Ala) and (N-l-valinoacyl)calix[4]arene (CX4-l-Val), and the buffer pH were found to be important factors in chiral separation. When CX4-l-Ala was used as a pseudostationary phase in the CE buffer, baseline resolution was achieved for 1,1‘-bi-2-naphthol (BINOL) and 1,1‘-binaphthyl-2,2‘-diamine (BNA). The addition of sodium dodecyl sulfate (SDS) to the CX4-l-Ala solution resulted in baseline resolution of the enantiomers of 1,1‘-binaphthyldiyl hydrogen phosphate (BNHP). In contrast, chiral separations of BNHP, BINOL, and BNA were successfully achieved in a single run with use of CX4-l-Val as a chiral selector...