Conventional Capillary Zone Electrophoresis Research Articles

Gradient Elution Moving Boundary Electrophoresis with Channel Current Detection

Gradient elution moving boundary electrophoresis (GEMBE) is a recently described technique for electrophoretic separations in short (1-3 cm) capillaries or microchannels. With GEMBE, the electrophoretic migration of analytes is opposed by a bulk counterflow of separation buffer through the separation channel. The counterflow velocity is varied over the course of a separation so that analytes with different electrophoretic mobilities enter the separation channel at different times and are detected as moving boundary, stepwise increases in the detector response. The resolution of a GEMBE separation is thus dependent on the rate at which the counterflow velocity is varied (rather than the length of the separation channel), and relatively high resolution separations can be performed with short microfluidic channels or capillaries. In this paper we describe an implementation of the GEMBE technique in which a very short (2.5-3.5 mm) capillary or microchannel is used as both the separation channel and a conductivity detection cell. Because the channel is so short, only a single moving boundary "step" is present in the channel at any given time, and the measured current through the channel can therefore be used to give a signal comparable to what is normally generated by more complicated detector arrangements. A theoretical description of the new technique is given along with simulation and experimental data relevant to the optimization of the method parameters such as channel length, counterflow acceleration, and applied field strength. A key theoretical prediction is that although this technique is expected to be a factor of 10 or 20 slower than conventional capillary zone electrophoresis, separation times of the order 1 s or less can still be achieved, making it applicable for ultrahigh-throughput analyses when implemented in a multiplexed format.

Micellar electrokinetic biofluid analysis of biogenic amines using on-line sample concentration and UV laser-induced native fluorescence detection

A simple and sensitive sweeping micellar electrokinetic chromatography method coupled with UV laser-induced native fluorescence detection has been developed for quantitative analysis of biogenic amines in biofluids. The background electrolyte comprised 30 mmol L −1 phosphoric acid and 20 mmol L −1 sodium dodecyl sulfate. The concentration limits of detection of analytes using sweeping-micellar electrokinetic chromatography (sweeping-MEKC) were in the range 7–100 nmol L −1, which were 250–3600-fold improvement for dopamine, DOPA and epinephrine compared with conventional capillary zone electrophoresis. An improvement of approximately 20-fold was observed for all analytes compared with typical micellar electrokinetic chromatography conditions. Baseline separation was achieved for the all analytes within 12 min and migration-time and peak-area repeatability were better than RSD 0.35% and 5.68%, respectively. The developed method was applied to measure the biogenic amines in biofluids extracted from wheat phloem sap, human plasma and human urine.

Determination of Phenolic Acids in Herba Epilobi by ITP–CE in the Column-Coupling Configuration

The combination of capillary isotachophoresis (ITP) and capillary zone electrophoresis (CZE) in the column-coupling configuration has been optimized in a mode in which the background electrolyte employed in the CZE step was different from the leading and terminating electrolytes of the ITP step. The optimum composition of the electrolyte system was 0.01 M HCl, 0.02 M IMI, 0.2% HEC, pH 7.2 (leading electrolyte), 0.01 M HEPES, pH 8.2 (terminating electrolyte), and 25 mM MES, 50 mM TRIS, 30 mM boric acid, 0.2% HEC, pH 8.3 (background electrolyte). All solutions contained 20% methanol. The timing of the transfer of isotachophoretically stacked analyte zones into the CZE column was also optimized. An ITP–CZE method with UV detection at 270 nm was developed for separation of nine phenolic acids (protocatechuic, syringic, vanillic, cinnamic, ferulic, caffeic, ρ-coumaric, chlorogenic, and gentisic acids) in a model mixture and used for assay of some of these acids in a methanolic extract of herba epilobi. Application of ITP–CZE resulted in 100-fold better sensitivity than conventional CZE; limits of detection ranged between 10 and 60 ng mL−1. When MES–TRIS–borate-based buffer, pH 8.3, was used in the CZE separation step the linearity of the ITP–CZE response was satisfactory (correlation coefficients were from 0.9937 to 0.9777). Repeatability was also satisfactory (RSD values ranged between 0.77% and 1.28% for migration times and between 1.65% and 13.69% for peak area).

Quasi-isoelectric buffers for protein analysis in a fast alternative to conventional capillary zone electrophoresis☆

Two different approaches are here reported for obtaining ultra-narrow pI cuts from 2-pH unit wide carrier ampholyte ranges, as commercially available, for use as quasi-isoelectric buffers in capillary electrophoresis separations of proteins. One of them uses multicompartment electrolyzers endowed with isoelectric membranes (Immobiline technology); the other employs the Rotofor equipment. Although the first approach results in more precise pI cuts, the latter technique is much faster, easier to handle and permits the immediate collection of 20 fractions in a single run. This results in ultra-narrow, ca. 0.1-pH unit intervals, uniformly spaced apart along the original wider gradient utilized for the fractionation. It is here shown that such quasi-isoelectric buffers, especially those in the pH 8-9 interval, have the unique property of coating the silica wall, thus preventing interaction of the proteins with the silica surface, that would otherwise totally disrupt the separation. On the contrary, such a shielding is not obtained in control, non isoelectric buffers (such as phosphate), that give very poor separations in uncoated capillaries. It is hypothesized that such a unique shielding effect is due to the oligo-amino backbone of the carrier ampholytes, typically composed (in the Vesterberg's synthetic approach) of 4-6 nitrogens spaced apart by ethylene moieties. Although such oligoprotic buffers should bear, in the isoelectric state, just one positive and one negative charge, they might be transiently ionized upon contact with the silanols, thus inducing a cooperative binding to the silica wall.

Analysis of metal ions by sweeping via dynamic complexation and cation-selective exhaustive injection in capillary electrophoresis.

To improve the detection sensitivity of metal ions in capillary zone electrophoresis (CZE), a novel method that combines complex formation and on-line sample preconcentration by sweeping was developed. Sweeping is defined as the picking and accumulating of analytes by a carrier in the background solution, with which they have considerable affinity. In this sweeping method, using ethylenediaminetetraacetic acid as carrier, dynamic complexation to form a UV-absorbing chelate and on-line preconcentration occur simultaneously during a run. The technique was validated in terms of the limit of detection, reproducibility, and sensitivity enhancement. Detection responses of some divalent metal ions, in terms of peak heights, were improved from 60- to 180-fold, relative to conventional CZE which employed precapillary complexation. The limits of detection were in the range of (1.8-23.4) x 10(-8) M. This method was applied to the analysis of trace metal ions in factory wastewater. Furthermore, sweeping in conjunction with sample stacking accompanying electrokinetic injection, cation-selective exhaustive injection (CSEI-sweeping), was also examined. Up to 140 000-fold improvement in detector responses for some divalent and trivalent metal ions was realized by CSEI-sweeping. The limits of detection were in the range (2.4-25.2) x 10(-11) M.

Salt effects in capillary zone electrophoresis: V. Adsorption and retention of electrically neutral analytes

Two electrically neutral analytes previously observed to be separated from the neutral marker in capillary zone electrophoresis (CZE) experiments [sulphanilamide (SAA) and sulphaguanidine (SGW)] have been examined to determine the basis for separation. The degree of separation increases markedly with buffer concentration and improves with increasing field strength. On the basis of the apparent electrophoretic mobilities in conventional CZE, migration times in a zero EOF environment were calculated for SAA, SGW and six other sulphonamides that were known to be ionized. These six markers were used to test the legitimacy of our predictions and to correct for small discrepancies between the predicted and observed migration times. It was concluded that SAA and SGW have negligible electrophoretic mobilities and that they are retained in the electrical double layer close to the capillary wall. A mechanism for adsorption is proposed and the generality of the phenomenon is discussed.

Determination of cyanine-labeled amino acid enantiomers by cyclodextrin-modified capillary gel electrophoresis combined with diode laser fluorescence detection

Enantiomeric amino acids were labeled with a cyanine derivative (Cy5) for selective determination by capillary electrophoresis combined with diode laser-induced fluorometry. Cyanine-labeled glutamic acid has a high negative charge so that it was difficult to detect by conventional capillary zone electrophoresis due to its electrophoretic mobility being greater than the electroosmotic mobility. Thus, cyclodextrin-modified capillary gel electrophoresis using replaceable poly(vinylpyrrolidone) (PVP), which effectively suppresses the electroosmotic flow, was employed to separate Cy5-labeled amino acid enantiomers. The separation of six amino acid enantiomers could be achieved by using 1% PVP solution containing 70 m M γ-cyclodextrin. It was also shown that PVP does not affect the selectivity of optical isomers.

Vacancy Capillary Zone Electrophoresis and Differential Capillary Zone Electrophoresis: Variations on a Well-Known Theme

Two new variants on capillary zone electrophoresis (CZE) are described and experimentally investigated. In vacancy CZE the sample, diluted with a background electrolyte, fills the electrode vessels and the separation capillary. When pure background electrolyte is injected, the resulting electropherogram represents the composition of the sample. The electropherogram is almost identical with the result of a conventional CZE experiment. In differential CZE the sample again fills the electrode vessels and the separation compartment. After injection of a slightly different sample, a differential electropherogram is obtained that represents the differences between the two samples. The retention times of both new variants are comparable with conventional CZE, the separation efficiency, in terms of theoretical plates, is marginally lower, and both show good quantitative characteristics. The concept of vacancy electrophoresis explains the origin of unexpected system peaks in conventional CZE when multiple co-ions in the background electrolyte are used.

Advanced possibilities on multi-element separation and detection of metal ions by capillary zone electrophoresis using precapillary complexation I. Separation aspects

Many metal ions can be seperated by capillary zone electrophoresis (CE) using cyclohexane-1,2-diaminetetraacetic acid (CDTA) as a complexing reagent for precapillary derivatization of analytes. Adding an organic solvent, such as ethylene glycol, permits a wide selection in separation resolution. The effect of this electrolyte additive, as well as that of tetraalkylammonium salts, anionic and cationic surfactants, varying borate buffer concentration and some operational variables (applied voltage, sample volume, etc.), was studied in detail with regard to electrophoretic and electroosmotic mobility. The gain in resolution is due to the enhancing effect of electrolyte changes on the migration range and peak efficiency. An impact of modest electrophoretic velocity changes is believed to occur in a much lesser degree. Under optimized conditions, excellent separations of 23 metal-CDTA complexes within 10 min were obtained. Aluminium(III), antimony(III), bismuth(III), chromium(III), mercury(II), palladium(II), silver(I), tallium(I), tin(IV), uranium(VI), vanadium(IV,V) and zirconium(IV), which are scarcely subject to conventional CE procedures, were separated, along with several other metal ions, using a 20 m M sodium borate buffer (pH 9.0) containing 5% ethylene glycol and 1 m M CDTA. The problem of improving the detectability for complexed metal ions was also addressed.

On-line partial filling micelllar electrokinetic chromatography-electrospray ionization mass spectrometry

On-line combination of partial filling micellar electrokinetic chromatography (PF-MEKC) and electrospray ionization mass spectrometry (ESI-MS) is demonstrated for the analysis of triazine herbicides including atrazine, propazine, ametryne and prometryne. In comparison with conventional micellar electrokinetic chromatography (MEKC), PF-MEKC involves filling a small portion of the capillary with a sodium dodecyl sulfate (SDS) micellar solution for achieving the separation. In PF-MEKC, the triazine analytes first migrate into the micellar plug where the separation occurs and then into the electrophoresis buffer which is free of surfactant. Consequently, the electroosmotic transfer of neutral triazine herbicides to ESI-MS at the end of PF-MEKC capillary is comparable to conventional capillary zone electrophoresis ESI-MS. Therefore, PF-MEKC-ESI-MS provides a mechanism for the separation and mass detection of neutral molecules without the interference of surfactant.

Characterization of continuous electrophoretic separations in narrow channels coupled to small bore capillaries

Continuous electrophoretic separations in narrow channels have been carried out by coupling conventional capillary zone electrophoresis to open channel structures with rectangular cross sections. Presented here is an examination of the results of continuous separations conducted under various experimental conditions. The characterization is conducted using analysis of both continuous and plug injections of material into the channel, and the parameters used to analyze the separation are lateral dispersion, longitudinal dispersion, and signal intensity. Longitudinal dispersion indicates the amount of band broadening in the direction parallel to channel flow and, therefore, determines the spatial resolution of the separation. Lateral dispersion corresponds to band broadening perpendicular to channel flow and affects the accuracy with which the time of sample introduction into the channel can be determined. The results from these experiments indicate that (a) a cooled channel system provides better results when longer analyses are conducted, (b) a capillary step rate yielding a band introduction angle of 45 degrees relative to the direction of flow in the channel provides the best compromise between longitudinal and lateral dispersions (this value typically equals 0.9 s/25-μm step for the system used in this research), (c) a capillary to channel linear velocity ratio of 3 to 5 yields improved signals relative to lower ratios due to more sample introduced (velocity ratios above this may lead to loss of spatial resolution), and (4) fast channel flows yield more symmetrically shaped bands than do slower flows due to more efficient transfer of material from the capillary-channel junction into the channel. Detection limits of 41 μM are obtained with a linear range from 0.12 mM to 1.9 mM for laser induced fluorescence detection.

Ion-association capillary electrophoresis: New separation mode for equally and highly charged metal chelates

A new separation method for highly charged metal chelates based on ion association in capillary zone electrophoresis (CZE) has been developed. Metal chelates of AI(III), Co(III), CR(III) and FE(III) ions with 2,2′-dihydroxyazobenzene-5,5′-disulphonate could not be separated by a conventional CZE system (electrophoretic buffer: [NaH 2PO 4] T = 0.02 M, pH 7.0, V = 21.7 kV, i = 20 μgA, l = 50 cm), because they have fixed and identical (−5) charges. However, when 25 m M tetrabutylammonium bromide was added to the same electrophoretic buffer, each of the four chelates was well resolved and migrated in the order Fe(III), Co(III), Cr(III) and Al(III) with theoretical plate numbers of 210 000–250 000 per 50 cm within 11 min ( V= 21.3 kV, I = 40 μA). The detection limits ( S/N = 3) as determined by spectrophotometric detection (λ = 494 nm, I.D. = 50 μm) were 1.00, 1.89 and 3.11 fmol for Al, Co and Fe, respectively, in 6.0 · 10 −9 dm 3 of sample solution injected. The effect on the separation of the sizes, types and the concentrations of quaternary ammonium ions added to the electrophoretic buffer was also investigated. As a result, some evidence was obtained that the ion-association reaction between the chelates and ammonium ions probably took the major role in the separation mechanism.

Application of sample stacking to gravity injection in capillary electrophoresis

AbstractSample stacking can be used to increase the amount of analyte loaded onto a capillary column without loss in resolution. After the sample is loaded onto the column, the analytes are then separated by conventional capillary zone electrophoresis. When using gravity injection, over an order of magnitude improvement in detection limit is achieved. There is an optimal point for the amount of material which can be injected onto the column based on the resolution desired for the separation.

Effect of Surfactant Structures on the Separation of Cold Medicine Ingredients by Micellar Electrokinetic Chromatography

Using micellar electrokinetic chromatography (micellar EKC or MEKC) the retention behavior of twelve active ingredients used in cold medicines was investigated. The role of five different anionic surfactants was investigated by MEKC and the results were compared with those obtained by conventional capillary zone electrophoresis (CZE). The relative retention order of the 12 ingredients was significantly different among the five surfactants; the different elution orders were ascribed to the differences in the hydrophilic groups of the surfactants. The effects of surfactant concentration and buffer pH were also investigated. The ingredients were successfully separated within 30min by micellar EKC with high theoretical plate numbers (~200 000), and selectivity was much improved in comparison with CZE. Micellar EKC was applied by the internal standard method to the quantitation of some active ingredients combined in commercial preparations.