Determination of bioactive peptides using capillary zone electrophoresis/mass spectrometry.

Mixtures of bioactive peptides have been analyzed by capillary zone electrophoresis/mass spectrometry (CZE/MS) using an on-line coaxial continuous-flow fast atom bombardment interface. High separation efficiencies (up to 410,000 theoretical plates) were obtained from low femtomole levels of peptides. The analysis of basic peptides was accomplished by using aminopropyl-silylated CZE columns to minimize zone broadening due to adsorption effects. CZE/MS/MS data were acquired from femtomole levels of peptides in electrophoretic real time.     

Stacking ionizable analytes in a sample matrix with high salt by a transient moving chemical reaction boundary method in capillary zone electrophoresis

The paper presents a novel on-line transient moving chemical reaction boundary method (tMCRBM) for simply but efficiently stacking ionizable analytes in high-salt matrix in capillary zone electrophoresis (CZE). The powerful function and stability of the tMCRBM are elucidated with the ionizable test analytes of L-phenylalanine (Phe) and L-tryptophan (Trp) in the matrix with 85.6-165.6 mM sodium ion and further compared with the normal CZE of Phe and Trp samples dissolved in running buffer. The results verify that (1) the on-line tMCRBM mode can evidently increase separation efficiency, peak height, and resolution, (2) with the mode, the analytes in a 28-cm high-salt matrix plug can be stacked successfully and further separated well, (3) the values of relative standard deviation of peak height, peak area, and migrating time range from 3.9% to 6.1%; the results indicate the high stability of the technique of tMCRBM-CZE. The techniques implies obvious potential significance for those ionizable analytes, e.g., protein, peptide, and weak alkaline or acidic compound, in such matrixes as serum, urine, seawater, and wastewater, with high salt, which has a deleterious effect on isotachophoresis (ITP) and especially on electrostacking and field-amplified sample injection (FASI). The mechanism of stacking of zwitterionic analytes in a high-salt matrix by the tMCRBM relies on non-steady-state isoelectric focusing (IEF) but not on transient ITP, electrostacking, and FASI.     

Capillary electrochromatography for separation of peptides driven with electrophoretic mobility on monolithic column.

A mode of capillary electrochromatography for separation of ionic compounds driven by electrophoretic mobility on a neutrally hydrophobic monolithic column was developed. The monolithic column was prepared from the in situ copolymerization of lauryl methacrylate and ethylene dimethacrylate to form a C12 hydrophobic stationary phase. It was found that EOF in this hydrophobic monolithic column was very poor, even the pH value of mobile phase at 8.0. The peptides at acidic buffer were separated on the basis of their differences in electrophoretic mobility and hydrophobic interaction with the stationary phase; therefore, different separation selectivity can be obtained in CEC from that in capillary zone electrophoresis (CZE). Separation of peptides has been realized with high column efficiency (up to 150,000 plates/meter) and good reproducibility (migration time with RSD <0.5%), and all of the peptides, including some basic peptides, showed good peak symmetry. Effects of the mobile phase compositions on the retention of peptides at low pH have been investigated in a hydrophobic capillary monolithic column. The significant difference in selectivity of peptides in CZE and CEC has been observed. Some peptide isomers that cannot be separated by CZE have been successfully separated on the capillary monolithic column in this mode with the same buffer used.     

Strategy for on-line preconcentration in chromatographic separations

In chromatographic separations, the heights of peaks are proportional to the concentrations of sample components present in an injected mixture. In general, an increase in the peak height cannot be achieved by simply increasing the injection time or the sample plug length. An exception occurs if some form of on-line preconcentration is possible. We present a new strategy for achieving on-line preconcentration by the use of a porous chromatographic material that acts as a solid-phase extractor as well as a stationary-phase separator. We are able to realize significant on-line preconcentration using capillary columns filled with a photopolymerized sol-gel (PSG). More than 2-cm plugs of sample solution can be loaded into the capillary and concentrated using a running buffer that is the same as the injection buffer (to avoid solvent gradient effects). As a demonstration, mixtures of three different polycyclic aromatic hydrocarbons, eight different alkyl phenyl ketones, and five different peptides in solutions of aqueous acetonitrile have been injected onto the PSG column and separated by capillary electrochromatography. The preconcentration is marked in terms of peak heights, with up to 100-fold increase for the PAH mixture, 30-fold for the alkyl phenyl ketone mixture, and 20-fold for the peptide mixture. Preconcentration takes place because of the high mass-transfer rates possible in the highly porous structure, and the extent of preconcentration follows the retention factor k for a given analyte.     

On-line concentration of acidic compounds by anion-selective exhaustive injection-sweeping-micellar electrokinetic chromatography

An easy, simple, and highly efficient on-line preconcentration method for acidic compounds in capillary electrophoresis was investigated. It combined two on-line concentration techniques, field-amplified sample injection (FASI) and sweeping. A low-pH (2.5) background electrolyte was used to suppress the electroosmotic flow (EOF), obviating the need of a coated capillary, as well as to neutralize the weakly acidic analytes. After injection of a plug of water inside the separation capillary, negative voltage was applied to initialize FASI for a much longer time than usual. The anions experienced a high electric field and moved quickly to the boundary of the water and the low-pH nonmicellar electrolyte. When the anions encountered the low-pH electrolyte, they were neutralized and a focused sample zone was formed. Then both inlet and outlet vials were changed to those containing the low-pH micellar background electrolyte. As negative voltage was applied, the anionic micelles moved into the capillary, and sweeping and separation began. The novelty in the present procedure is that a low-pH buffer is used to suppress the EOF and also the ionization of the analytes, without need of any other additives or use of a coated capillary. This method afforded 100,000-fold improvement in peak heights for some phenoxy acidic herbicides. The detection limits for these compounds could be low as 100 pg/mL     

Integration of electrokinetic-based multidimensional separation/concentration platform with electrospray ionization-Fourier transform ion cyclotron resonance-mass spectrometry for proteome analysis of Shewanella oneidensis

This work focuses on the development of a multidimensional electrokinetic-based separation/concentration platform coupled with electrospray ionization-Fourier transform ion cyclotron resonance mass spectrometry (ESI-FTICR-MS) for achieving the high resolution and ultrasensitive analysis of complex protein/peptide mixtures. A microdialysis junction is employed as the interface for on-line combination of capillary isoelectric focusing (CIEF) with transient capillary isotachophoresis/zone electrophoresis (CITP/CZE) in an integrated platform. Besides the excellent resolving power afforded by both CIEF and CZE separations, the electrokinetic focusing/stacking effects of CIEF and CITP greatly enhance the dynamic range and detection sensitivity of MS for protein identification. The constructed multidimensional separation/concentration platform is demonstrated for the analysis of Shewanella oneidensis proteome, which has considerable implications toward the bioremediation of environmental pollutants. The electrokinetic-based platform offers the overall peak capacity comparable to those obtained using multidimensional chromatography systems, but with a much shorter run time and no need for column regeneration. Most importantly, a total of 1174 unique proteins, corresponding to 26.5% proteome coverage, are identified from the cytosolic fraction of S. oneidensis, while requiring <500 ng of proteolytic digest loaded in the CIEF capillary. The ultrasensitive capabilities of electrokinetic-based proteome approach are attributed to the concentration effect in CIEF, the electrokinetic stacking of CITP, the nanoscale peak volume in CZE, the "accurate mass tag" strategy for protein/peptide identification, and the high-sensitivity, high-resolution, and high-mass measurement accuracy of FTICR-MS.     

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.     

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.     

On-line concentration and separation of proteins by capillary electrophoresis using polymer solutions

Proteins were separated in 0.6% poly(ethylene oxide) (PEO) solutions using a capillary filled with buffers prior to analysis and were detected by laser-induced native fluorescence using a pulsed Nd:YAG laser. PEO solutions entered the capillary by electroosmotic flow (EOF) during the separation. The composition and concentration of the buffer affected the adsorption of PEO molecules on the capillary surface and, consequently caused changes in the EOF. Short separation times (< 7 min) were achieved on a sample solution of five proteins in a 0.6% PEO solution containing 5 microg/mL ethidium bromide using a capillary pre-filled with 100 mM TRIS-borate (TB) buffers (pH 10,0). We also extended this method for on-line concentration and separation of proteins. Proteins dissolved in low-conductivity media stacked in both TB buffers and in PEO solutions. The peak height was proportional to the injection volume up to 2.1 microL using an 80-cm capillary filled with 400 mM TB buffers. Using large injection volumes (2.1 microL), we achieved a limit of detection (S/N = 3) of 31 pM for carbonic anhydrase, which was a 1696-fold sensitivity enhancement compared to a conventional injection method (1 kV for 10 s). In high-conductivity media (urine matrix), stacking occurred at the boundary between the sample zone and PEO solutions. A urine sample without any pretreatment was analyzed, and after stacking, several peaks were detected. Spiking the urine sample with human serum albumin (HSA) affected the fluorescent intensity of some analytes as a result of interaction with HSA.     

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.     

Gradient elution isotachophoresis for enrichment and separation of biomolecules

A novel format for performing capillary isotachophoresis (ITP) is described -- gradient elution ITP (GEITP). GEITP merges the recently described electrophoretic separation technique of gradient elution moving boundary electrophoresis (GEMBE) with an ITP enrichment step. GEMBE utilizes a combination of continuous sample injection with a pressure-controlled counterflow; as the counterflow is reduced, analytes are sequentially eluted onto the separation column and detected as boundary interfaces. By incorporating leading electrolytes into the counterflow and terminating electrolytes into the sample matrix, an ionic interface can be formed near the capillary inlet. The discontinuous buffer system forms highly enriched analyte zones outside of the capillary, which are then eluted onto the separation capillary as the counterflow is reduced. Separation of fluorescent analytes was achieved either through discrete electrolyte spacers added to the sample or by using ampholyte mixtures to form a continuum of spacers. As the ITP process occurs off-column, extremely short length separations can be achieved, as demonstrated by a separation in 30 microm. The effects of various parameters on the GEITP enrichment process are investigated, including initial counterflow rates, electric field, leading electrolyte concentration, and counterflow acceleration, which is an adjustable parameter allowing for highly flexible separations. Typical enhancements in limits of detection and sensitivity were greater than 10,000-fold and were achieved in less than 2 min, yielding low-picomolar detection limits using arc lamp illumination and low-cost CCD detection. An optimized system afforded greater than 100,000-fold improvement in detection of carboxyfluorescein in 8 min. Specific examples of enrichment and separation demonstrated include the following: small dye molecules, DNA, amino acid mixtures, and protein mixtures.     

Method for internal standard introduction for quantitative analysis using on-line solid-phase extraction LC-MS/MS

A novel approach for on-line introduction of internal standard (IS) for quantitative analysis using LC-MS/MS has been developed. In this approach, analyte and IS are introduced into the sample injection loop in different steps. Analyte is introduced into the injection loop using a conventional autosampler (injector) needle pickup from a sample vial. IS is introduced into the sample injection loop on-line from a microreservoir containing the IS solution using the autosampler. As a result, both analyte and IS are contained in the sample loop prior to the injection into the column. Methodology allowed to reliably introduce IS and demonstrated injection accuracy and precision comparable to those obtained using off-line IS introduction (i.e., IS and analyte are premixed before injection) while maintaining chromatographic parameters (i.e., analyte and IS elution time and peak width). This new technique was applied for direct analysis of model compounds in rat plasma using on-line solid-phase extraction (SPE) LC-MS/MS quantification. In combination with on-line SPE, IS serves as a surrogate IS and compensates for signal variations attributed to sample preparation and instrumentation factors including signal suppression. The assays yielded accuracy (85-119%), precision (2-16%), and analyte recovery comparable to those obtained using off-line IS introduction. Furthermore, on-line IS introduction allows for nonvolumetric sample (plasma) collection and direct analysis without the need of measuring and aliquoting a fixed sample volume prior to the on-line SPE LC-MS/MS analysis. Therefore, this methodology enables direct sample (plasma) analysis without any sample manipulation and preparation.     

Sweeping with electrokinetic injection and analyte focusing by micelle collapse in two-dimensional separation via integration of micellar electrokinetic chromatography with capillary zone electrophoresis

A novel integrated concentration/separation approach involving online combination of sweeping with electrokinetic injection and analyte focusing by micelle collapse (AFMC) with heart-cutting two-dimensional (2D) capillary electrophoresis (CE) in a single capillary was developed for analysis of Herba Leonuri and mouse blood samples. First, a new sweeping with an electrokinetic injection preconcentration method was developed to inject a large volume sample solution and significantly enhance detection sensitivity. Then, the preconcentration scheme was integrated to the 2D-CE to provide significant analyte concentration and extremely high resolving power. The sample was preconcentrated by sweeping with electrokinetic injection and separated in first dimension micellar electrokinetic chromatography (MEKC). Then, only a desirable fraction of the first dimension separation was transferred into the second dimension of the capillary by pressure and further analyzed by capillary zone electrophoresis (CZE) acting as the second dimension. As the key to successful integration of MEKC and CZE, an AFMC step was integrated between the two dimensions to release analytes from the micelle interior to a liquid zone and to overcome the sample zone diffusion caused by mobilization pressure. The injected sample plug lengths for flavonoids under 15 kV for 60 min were experimentally estimated as 546 cm. The dual concentration methods resulted in the increased detection factors of 6000-fold relative to the traditional pressure injection method. The relative standard deviation (RSD) values of peak height, peak area, and migration time were 2.7-4.5%, 1.9-4.3%, and 4.7-6.8% (n = 10), respectively. The limits of detection (S/N = 3) were in the range of 7.3-36.4 ng/L, and the theoretical plate numbers (N) were in the range of 1.7-4.3 × 10(4) plates/m. This method has been successfully applied to determine flavonoids in Herba Leonuri and postdosing mouse blood samples. The pharmacokinetic study also demonstrated that the proposed concentration/separation method was convenient and sensitive and would become an attractively alternative method for online sample concentration and separation in complex samples.     

On-line back-extraction field-amplified sample injection method for directly analyzing cocaine and thebaine in the extractants by solvent microextraction

This paper describes a novel method that applies on-line back-extraction field-amplified sample injection (OLBE-FASI) to the extractants by solvent microextraction (SME) in capillary zone electrophoresis (CZE). To our knowledge, it provides the first report that the water-immiscible solvent samples were directly analyzed by CZE. The water-immiscible solvent sample, sealed with a water plug in the sample vial, was used for direct electroinjection. The water plug with a moderate content of organic solvent, low-conductivity, and the presence of a small amount of H+ provided the highest sensitivity for analyzing positively chargeable compounds, such as cocaine and thebaine. The linear range was at least 2 orders of magnitude, with the square of the correlation coefficient (r2) > 0.9999, and a separate calibration over the range 0.016-10 microg/mL showed the linear range to be approaching 3 orders of magnitude. Detection limits were in the range of 2-10 ng/mL. Because the need to perform solvent exchange (from organic to aqueous solution) was eliminated, the OLBE-FASI method could be conveniently coupled with SME. In the present work, SME-OLBE-FASI-CE was validated for quantitative purposes, and applications to human urine were demonstrated.     

Head-Column Field-Amplified Sample Stacking in Binary System Capillary Electrophoresis: A Robust Approach Providing over 1000-Fold Sensitivity Enhancement

Effective electrokinetic field-amplified sample injection occurring at the capillary inlet from a sample volume equivalent exceeding that of the capillary up to 10-fold is described and demonstrated to provide over 1000-fold sensitivity enhancement. Successful application of this head-column field-amplified sample stacking approach to the analysis of positively chargeable, hydrophobic compounds in binary system capillary electrophoresis is shown to require an initially introduced low-conductivity zone (water plug) of >1 mm length, a sample injection voltage <20 kV, and an injection time interval <60 s. Following these conditions for more than 1500 runs with capillaries of 50 μm i.d. and about 20 cm effective length, damaging heat production during electroinjection within the low-conductivity zone at the column inlet (boiling of solvent and possible deposition of solutes or fusing of capillary walls) could be prevented. The solute amount injected by head-column field-amplified sample stacking is further shown to be dependent on the organic fraction and the buffer in the sample solution. High content of organic solvent, low conductivity, and the presence of a small amount of H(+) (50-100 μM) provides the highest sensitivity for analysis of positively chargeable model substances, including amiodarone and desethylamiodarone. Solutes present at the nanomolar level can thereby be accumulated from a sample volume equivalent of about 4 μL (with injection of about 20 nL of sample solvent into the capillary) and measured by UV absorption detection. To prevent disturbances caused by electrolysis, sample vials should be employed only once. The data obtained further show that quantitation can be reliably performed using internal calibration based on peak height (RSDs for inter- and intraday determinations are on the 2% level). However, due to variation of the roughness of the capillary walls and cuts, the time interval between operational steps, and trace adsorption onto the capillary walls, the length of the water zone drawn by capillary action on the inlet side is not constant, and external calibration therefore cannot be employed for quantitation.     

On-line coupling of capillary electrochromatography, capillary electrophoresis, and capillary HPLC with nuclear magnetic resonance spectroscopy

A novel capillary NMR coupling configuration, which offers the possibility of combining capillary zone electrophoresis (CZE), capillary HPLC (CHPLC), and for the first time capillary electrochromatography (CEC) with nuclear magnetic resonance (NMR), has been developed. The hyphenated technique has a great potential for the analysis of chemical, pharmaceutical, biological, and environmental samples. The versatile system allows facile changes between these three different separation methods. A special NMR capillary containing an enlarged detection cell suitable for on-line NMR detection and measurements under high voltage has been designed. The acquisition of 1D and 2D NMR spectra in stopped-flow experiments is also possible. CHPLC NMR has been performed with samples of hop bitter acids. The identification and structure elucidation of humulones and isohumulones by on-line and stopped-flow spectra has been demonstrated. The suitability of the configuration for electrophoretic methods has been investigated by the application of CZE and CEC NMR to model systems.     

A universal concept for stacking neutral analytes in micellar capillary electrophoresis

Unlike recent studies that have depended on manipulation of separation buffer parameters to facilitate stacking of neutral analytes in micellar capillary electrophoresis (MCE) mode, we have developed a method of stacking based simply on manipulation of the sample matrix. Many solutions for sample stacking in MCE are based on strict control of pH, micelle type, electroosmotic flow (EOF) rate, and separation-mode polarity. However, a universal solution to sample stacking in MCE should allow for free manipulation of separation buffer parameters without substantially affecting separation of analytes. Analogous to sample stacking in capillary zone electrophoresis by invoking field amplification of charged analytes in a low-conductivity sample matrix, the proposed method utilizes a high-conductivity sample matrix to transfer field amplification from the sample zone to the separation buffer. This causes the micellar carrier in the separation buffer to stack before it enters the sample zone. Neutral analytes moving out of the sample zone with EOF are efficiently concentrated at the micelle front. Micelle stacking is induced by simply adding salt to the sample matrix to increase the conductivity 2-3-fold higher than the separation buffer. This solution allows free optimization of separation buffer parameters such as micelle concentration, organic modifiers, and pH, providing a method that may complement virtually any existing MCE protocol without restricting the separation method.     

Simultaneous screen for microsomal stability and metabolite profile by direct injection turbulent-laminar flow LC-LC and automated tandem mass spectrometry

A LC-LC/MS/MS method has been developed that significantly increases the throughput in metabolism screening of drug candidates during lead optimization in discovery. This was accomplished by the reduction of sample preparation time through an on-line extraction of a drug and its metabolites from microsomal proteins using turbulent flow chromatography. Following its injection onto a column at turbulent flow, the drug and its metabolites are backwashed onto a reverse-phase column via on-line column switching and resolved chromatographically at a laminar flow of 2 mL/min. This tandem turbulent-laminar flow chromatographic system in a total cycle time of 8 min can achieve adequate separation of isomeric metabolites of venlafaxine, haloperidol, or adatanserin. Further improvement in throughput can be achieved by multiplexing both microsomal stability assessment and metabolite profiling into a single analysis. This is made possible by the ability of the ion-trap mass spectrometer to perform simultaneously multiple-reaction monitoring for microsomal stability and data-dependent multiple-stage mass spectrometric analysis for metabolite profiling within a single LC analysis. Such a LC-LC/MS/MS approach can dramatically shorten the time for providing metabolism feedback to the drug discovery process.     

Automated instrumentation for comprehensive isotachophoresis-capillary zone electrophoresis

An automated comprehensive isotachophoresis-capillary zone electrophoresis (ITP-CZE) system is described. The sample is focused in the first capillary by ITP and injected repeatedly into the second smaller diameter capillary for more rapid CZE separation. Since only small portions of the concentrated zones are sequentially injected for CZE separation, overloading was not observed. Moreover, the sensitivity is enhanced because all of the concentrated zones are analyzed and the results are summed. A single detector (only for the CZE dimension) is required, and accurate timing for CZE injection is not necessary. The system was evaluated using a mixture of angiotensins. The effect of addition of leading electrolyte at the junction of the ITP and CZE capillaries before each CZE run on comprehensive ITP-CZE peak area was studied, and leading electrolyte volumes between 7 and 11 microL led to the best sensitivity. Under optimized conditions, a detection limit of approximately 5 nM could be achieved by injecting 10 microL of angiotensin solution.     

Mechanistic study on analyte focusing by dynamic pH junction in capillary electrophoresis using computer simulation

Dynamic pH junction is an on-line preconcentration method in capillary electrophoresis (CE) based on electrokinetic focusing of weakly ionic analytes with in large sample volumes in a multisection electrolyte system. In this report, experiments and computer simulations were performed to gain a better insight of the analyte focusing mechanism when a dynamic pH junction was used. A computer program, SIMUL, was used to simulate the band-narrowing process of a group for phenol derivatives under optimized buffer conditions, which were compared with experimental results. Computer simulations revealed the formation of a sharp moving pH boundary within the sample zone causing efficient focusing of long plugs of weakly acidic analytes based on their pKa. These studies offered useful information for understanding the band-narrowing process by control of the depth and lifetime of the moving pH boundary as a function of analyte pKa, sample pH, and injection length. The change in pH of the sample within the capillary was also estimated by measuring the absorbances of an analyte at two different wave-lengths. Optimization of analyte focusing resulted in enhanced detection responses of about 60-450-fold in terms of peak heights for some phenol derivatives' relation to conventional injections. Dynamic pH junction represents a novel approach to control band dispersion (peak width) and selectivity (mobility) of specific analytes for high-resolution CE separations.