Capillary Zone Electrophoresis Column Research Articles

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).

Assay of phenolic compounds in red wine by on-line combination of capillary isotachophoresis with capillary zone electrophoresis

The on-line combination of capillary zone electrophoresis (CZE) with capillary isotachophoresis (ITP) increases significantly the separation capability and sensitivity of capillary electrophoresis. This technique was used for separation and quantification of fourteen selected natural constituents in red wine belonging to flavonoids and phenolic acids. The leading electrolyte (LE) in the ITP pre-separation step was 10 mM HCl of pH * 7.2 with Tris as counterion, the terminating electrolyte (TE) was 50 mM boric acid of pH * 8.2 (adjusted with barium hydroxide). The background electrolyte in the electrophoretic step contained 25 mM β-hydroxy-4-morpholinopropanesulfonic acid (MOPSO), 50 mM Tris, 15 mM boric acid and 5 mM β-cyclodextrin of pH * 8.5. The content of methanol in all electrolytes was 20% (v/v). For exact timing of the transfer of isotachophoretically stacked analyte zones into the CZE column and for the control of the residual amount of leading and terminating ITP electrolytes picric acid was used as coloured marker. The R.S.D. values ( n=6) ranged between ∼0.1% (for 0.25 μg ml −1 rutin) and ∼11% (for 0.25 μg ml −1 of quercitrin). Detection limits were 30 ng ml −1 for phenolic acids, quercitrin and rutin, 100 ng ml −1 for quercetin, kaempferol and epicatechin and 250 ng ml −1 for catechin. A single analysis took 45 min.

On-line coupling of capillary isotachophoresis and zone electrophoresis for the assay of phenolic compounds in plant extracts.

The combination of capillary isotachophoresis (ITP) and capillary zone electrophoresis (CZE) in the column coupling configuration was optimized in a mode where the electrolyte for the CZE step is different from the leading and terminating ITP electrolytes. Two colored markers, picric acid and 1-nitroso-2-naphthol, were used for exact timing of the transfer of isotachophoretically stacked analyte zones into the CZE column and for the control of the residual amount of the leading and terminating ITP electrolytes entering the CZE capillary together with the analytes, thus controlling the duration of transient ITP migration in the CZE capillary and ensuring good separation of the analytes and reproducibility of the migration times (relative standard deviations 1%). ITP-CZE was applied to the simultaneous assay of several cinnamic acid derivatives and flavonoids in methanolic extracts of Sambucus flowers and Crataegus leaves and flowers. The preconcentrating and cleansing effect of the ITP step allowed injection of relatively large sample volumes (30 microL). The limits of detection were approximately 20-50 ng x mL(-1) and 100 ng x mL(-1) for the acids and flavonoids, respectively ( thick similar 200-times lower compared to conventional CE) with spectrophotometric detection at 254 nm. The ITP-CZE exhibited satisfactory linearity and precision when using CZE buffer of pseudo "pH" 9.0; 1-nitroso-2-naphthol was employed as the internal standard. The separation took approximately 35 min. The ITP-CZE results for rutin, hyperoside, and vitexin-2-O"-rhamnoside were in good accordance with those obtained previously by high-performance liquid chromatography.

Large-volume sample stacking for on-capillary sample enrichment in the determination of naphthalene- and benzenesulfonates in real water samples by capillary zone electrophoresis.

We investigated the on-line preconcentration of a test mixture of 15 substituted and unsubstituted naphthalene(NSs) and benzenesulfonates (BZSs) by large-volume sample stacking (LVSS). Analyses were carried out by capillary zone electrophoresis (CZE) with on-column UV detection. In particular, we focused on how experimental variables such as the inside diameter of the capillary, the volume of sample introduced and polarity switching influenced the enrichment procedure. The best results were obtained when 300 nl were injected and stacked using a bubble cell capillary. Under these conditions, LVSS increased the detector response of conventional hydrodynamic injection by a factor of 40. The limits of detection of the method were between 5 and 10 microg l(-1). Determinations were reproducible, in terms of peak area and migration time, under such conditions. The performance of the method was examined by determining NS and BZS in real samples, such as tap, river and surface waters and inflow/outflow waters from a water treatment plant. Real samples were injected directly into the CZE column with little or no preparation.

Comprehensive isotachophoresis–capillary zone electrophoresis using directly inserted columns having different diameters with a periodic counterflow and dual ultraviolet detectors

AbstractIsotachophoresis (ITP) coupled to capillary zone electrophoresis (CE) in a comprehensive manner was used to separate mixture components in both insufficient and sufficient quantities without heart‐cutting or splitting. Examples of comprehensive ITP‐CE involving multiple CE injections of preconcentrated ITP zones are demonstrated. In the comprehensive arrangement, all of the sample in the first dimension (ITP) is subjected to analysis in the second dimension (CE), without significant sample loss or decrease in sample detectability resulting from removal of a portion of the sample. This is especially important for analytes at low concentrations which may form a single mixed zone instead of individual ITP zones. Direct on‐line coupling of ITP to CE in this comprehensive arrangement involved the use of columns having different diameters with one directly inserted inside of the other. A computer‐controlled counterflow was applied when the isotachophoretic sample stack reached the bifurcation point. Large volume (10 μL) injections were made using an electrically insulated commercial polymeric rotary valve injector for increased reproducibility compared to previous comprehensive ITP‐CE studies, with ITP and CE retention time RSD's ranging from 2 to 5%. An ultraviolet (UV) detector positioned at the bifurcation point was used to determine the beginning of CE injection. Application of a splitting voltage at the bifurcation point showed no affect on analyte transfer into the CE column. In using multiple injections of the ITP band(s), CE column overloading was not observed. © 2001 John Wiley & Sons, Inc. J Micro Sep 13: 361–370, 2001

Capillary zone electrophoresis with on-line isotachophoretic sample pretreatment: Sample clean-up aspects

A theoretical model intended for characterizations of capillary zone electrophoresis (CZE) separations of samples of unknown matrix compositions with on-line isotachophoresis (ITP) sample pretreatment was developed. Assuming the use of the column-coupling capillary electrophoresis (CE) separation system, this model explicitly defines (i) potential sample interferents for a given CZE analyte in both stages of the ITP–CZE combination and (ii) residual sample interferents in the CZE stage, i.e., interfering sample constituents which cannot be removed from the sample by the ITP pretreatment using a particular combination of the ITP and CZE electrolyte systems. Relationships applicable to estimations of sizes of these groups of sample interferents are provided by the model. A calculation procedure based on these relationships was developed to test the model. The results from these tests obtained for a group of CZE analytes of different acid–base properties (p-sulfanilic acid, hippuric acid, 2,6-dinitrophenol, and o-nitrophenol) show that an effective use of the ITP–CZE combination in the column-coupling separation system requires (i) that the ITP and CZE electrolyte systems introduce different separation mechanisms for the analyte and (ii) that the size of the sample fraction in which the analyte is transferred into the CZE column after the ITP pretreatment is kept at a minimum. Numerical data obtained in these tests for p-sulfanilic acid were in reasonable agreement with the ones provided by computer simulations of the CZE and ITP–CZE separations with the sample matrix consisting of 264 anionically migrating constituents of known migration properties. ©1999 John Wiley & Sons, Inc. J Micro Sep 11: 141–153, 1999

Comprehensive on-line RPLC-CZE-MS of peptides

Peptide standards and tryptic digests of ribonuclease B are separated by comprehensive two-dimensional reversed phase liquid chromatography (RPLC) and capillary zone electrophoresis (CZE) and detected on-line by electrospray mass spectrometry. The RPLC column is coupled to the CZE column by a transverse flow gating interface. A new rugged microelectrospray needle is described that combines high ionization efficiency, low flow rates, and a sheath flow. The result is a system combining the separation capabilities of both RPLC and CZE with on-line mass spectrometric detection, all in about 15 min.

Matrix-assisted laser desorption mass spectrometry of proteins isolated by capillary zone electrophoresis.

A simple method for the off-line coupling of laser desorption mass spectrometry (LDMS) and capillary zone electrophoresis (CZE) is described. Representative mass spectra of subpicomole quantities of proteins isolated from CZE are presented and discussed. The current detection limit for bovine alpha-lactalbumin is 100 fmols injected onto the CZE column. Horse heart myoglobin was demonstrated to be stable in CHES/KCl, a CZE buffer, for at least 1 month, suggesting that some isolates can be safely stored for long time periods prior to LDMS analysis. Protein stability in 0.1% aqueous trifluoroacetic acid (TFA), a common solvent for LDMS, must also be considered. In the special case of porcine pepsinogen, significant (greater than 50%) degradation was observed within 5 min in TFA. In favorable cases, mass measurement accuracies of +/- 0.02% were obtained for protein isolates. Factors limiting mass measurement accuracy are presented. Finally, the possibility of identifying protein isolates, by combining N-terminal sequencing, molecular mass measurements, and selective peptide "mapping" procedures, is discussed.

Optimization of capillary zone electrophoresis/electrospray ionization parameters for the mass spectrometry and tandem mass spectrometry analysis of peptides

The solution chemistry conditions necessary for optimum analysis of peptides by capillary zone electrophoresis (CZE)/electrospray ionization mass spectrometry and CZE electrospray ionization tandem mass spectrometry have been studied. To maximize the signal-to-noise ratio of the spectra it was found necessary to use acidic CZE buffers of low ionic strength. This not only increases the total ion current, but it also serves to fully protonate the peptides, minimizing the distribution of ion current across the ensemble of possible charge states. The use of acidic buffers protonates the peptides, which is advantageous for mass spectrometry and tandem mass spectrometry analysis, but is problematic with CZE when bare fused silica CZE columns are used. These conditions produce positively charged peptides, and negatively charged silanol moieties on the column wall, inducing adsorption of the positively charged peptides, thus causing zone broadening and a loss in separation efficiency. This problem was circumvented by the preparation of chemically modified CZE columns, which, when used with acidic CZE buffers, will have a positively charged inner column wall. The electrostatic repulsion between the positively charged peptides and the positively charged CZE column wall minimizes adsorption problems and facilitates high efficiency separations. Full-scan mass spectra were acquired from injections of as little as 160 fmols of test peptides, with CZE separation efficiencies of up to 250,000 theoretical plates.

Variance contributions to band spread in capillary zone electrophoresis

A peak variance method is described and used to determine contributions to band spread in capillary zone electrophoresis (CZE) for model systems consisting of amino acids, peptides and proteins. A theoretical and experimental approach is proposed for isolating time-independent contributions to band spread from the time-dependent contributions to band spread in CZE. The significant time-independent contributions to CZE band spread include injection and detection, while important time-dependent contributions to band spread in CZE are due to molecular diffusion, Joule heating and deviation from ideal electroosmotic plug flow. The contribution of diffusion to band spread in CZE is experimentally determined by using a new approach involving multiple passes of the sample band past the detector, allowing measurement of the total variance of a sample band at periodically increasing residence times in the CZE column. Molecular diffusion is confirmed to be the major contributor to band spread under optimized CZE conditions. The experimental values for diffusion coefficients obtained in studies (without an externally applied electric field) are subsequently used to isolate the more subtle contributions to band spread which include Joule heating and the nature of electroosmotic ( i.e., flow deviation from plug). Important time-independent contributions, i.e., injection, detection and voltage “on-off” switching are isolated for several analytes and compared to their total experimentally determined time-independent variance.