Electroosmotic flow control of fluids on a capillary electrophoresis microdevice using an applied external voltage

Independent control of electroosmosis is important for separation science techniques such as capillary zone electrophoresis and for the movement of fluids on microdevices. A capillary electrophoresis microdevice is demonstrated which provides more efficient control of electroosmosis with an applied external voltage field. The device is fabricated in a glass substrate where a 5.0 cm separation channel (30 microm wide) is paralleled with two embedded electrodes positioned 50 microm away in the substrate. With this structure, greatly reduced applied external potentials (< or = 120 V compared to tens of kilovolts) still effectively altered electroosmosis. The efficiency for the control of electroosmosis by the applied external field is improved by approximately 40 times over published values.     

Electroosmotic capillary flow with nonuniform zeta potential

The present work is an analytical and experimental study of electroosmotic flow (EOF) in cylindrical capillaries with nonuniform wall surface charge (zeta-potential) distributions. In particular, this study investigates perturbations of electroosmotic flow in open capillaries that are due to induced pressure gradients resulting from axial variations in the wall zeta-potential. The experimental inquiry focuses on electroosmotic flow under a uniform applied field in capillaries with an EOF-suppressing polymer adsorbed onto various fractions of the total capillary length. This fractional EOF suppression was achieved by coupling capillaries with substantially different zeta-potentials. The resulting flow fields were imaged with a nonintrusive, caged-fluorescence imaging technique. Simple analytical models for the velocity field and rate of sample dispersion in capillaries with axial zeta-potential variations are presented. The resulting induced pressure gradients and the associated band-broadening effects are of particular importance to the performance of chemical and biochemical analysis systems such as capillary electrokinetic chromatography and capillary zone electrophoresis.     

Protocol for resolving protein mixtures in capillary zone electrophoresis.

The separation of protein mixtures by capillary zone electrophoresis can be plagued by wall adsorption of the protein components, causing peak broadening and distortion. A method is presented for overcoming this problem by adding ethylene glycol to the protein sample and by choosing the running buffer and protein sample to be at different pH values and molarities. This protocol appears to work for a wide class of proteins having different molecular weights and pI values. The method has been applied to the analysis of proteins in human serum. Compared to the traditional method of agarose gel electrophoresis, the present method is more rapid and offers better resolution, suggesting its potential as a clinical diagnostic of certain disease states.     

Multiplexed capillary zone electrophoresis and micellar electrokinetic chromatography with internal standardization.

Although liquid chromatography and gas chromatography are the main workhorses in the analytical laboratory, samples can only be analyzed consecutively in an instrument. In this study, capillary zone electrophoresis and micellar electrokinetic chromatography separations are performed in a 96-capillary array system with laser-induced fluorescence detection. Migration times of four kinds of fluoresceins and six polyaromatic hydrocarbons (PAHs) are normalized to one of the capillaries using two internal standards. The relative standard deviations after normalization are 0.6-1.4% for the fluoresceins and 0.1-1.5% for the PAHs. Quantitative calibration of the separations based on peak areas is also performed, again with substantial improvement over the raw data. This opens up the possibility of performing massively parallel separations for high-throughput chemical analysis for process monitoring, combinatorial synthesis, and clinical diagnosis.     

On-column sample gating for high-speed capillary zone electrophoresis

High-speed zone electrophoresis in a fused-silica capillary is described. Elevated electric fields and short capillary lengths allow a mixture of fluorescein isothiocyanate (FITC) labeled amino acids to be separated in times as short as 1.5 s. Formation of the analyte zone at the head of the capillary is controlled by laser-induced photolysis of a tagging reagent. This gating procedure allows rapid and automated introduction of sample into the capillary. Ultimately, Joule heating of the buffer limits the speed and efficiency of the separation.     

High-speed capillary zone electrophoresis with online photolytic optical injection

We report an online, optical injection interface for capillary zone electrophoresis (CZE) based upon photophysical activation of a caged, fluorogenic label covalently attached to the target analyte. This injection interface allows online analysis of biomolecular systems with high temporal resolution and high sensitivity. Samples are injected onto the separation capillary by photolysis of a caged-fluorescein label using the 351-364 nm irradiation of an Ar+ laser. Following injection, the sample is separated and detected via laser-induced fluorescence detection at 488 nm. Detection limits for online analysis of arginine, glutamic acid, and aspartic acid were less than 1 nM with separation times less than 5 s and separation efficiencies exceeding 1,000,000 plates/m. Rapid injection of proteins was demonstrated with migration times less than 500 ms and 0.5 nM detection limits. Online monitoring was performed with response times less than 20 s, suggesting the feasibility of this approach for online, in vivo analysis for a range of biologically relevant analytes.     

Electroosmotic flow in composite microchannels and implications in microcapillary electrophoresis systems

The electroosmotic flow in laminated excimer laser-ablated microchannels has been studied as a function of the depth of the rectangular channels, and particular emphasis has been given to the difference in the zeta-potentials between the lamination layer and the ablated substrate. Experimental electroosmotic flow follows the tendency predicted by a recently published model. The zeta-potentials of lamination and ablated surfaces were determined for poly(ethylene terephthalate) and poly(carbonate) substrates by fitting the experimental data with a numerical implementation of this model. In the experimentally investigated range of channel cross sections, a linear fit to the data gives a good approximation of the zeta-potentials for both materials. Moreover, a flow injection analysis of fluorescein dye has been performed to show the severe loss in numbers of theoretical plates, caused by Taylor dispersion, when such microchannels, dedicated to microcapillary electrophoresis, are used.     

On-line concentration of proteins and peptides in capillary zone electrophoresis with an etched porous joint

A novel approach for on-line concentration of proteins and peptides in capillary electrophoresis (CE) is presented. A short section (approximately 0.5-1 cm) along the capillary was etched with HF. The etched section became a porous membrane that allowed electrical conductivity but prevented passage of the analyte ions. The capillary was isolated into two parts by the etched section. Thus, we were able to use three buffer vials to perform CE experiments in the capillary by applying high voltages independently. Concentration and separation were performed at the two respective regions. When high voltage was applied to the concentration capillary (between the inlet end and the etched section), proteins and peptides were concentrated at the etched portion, because the porous capillary wall allowed only small buffer ions to pass through and there was no electric field gradient beyond that point. After focusing, the narrow sample zone was introduced into the separation capillary (between the etched section and the outlet end) by hydrodynamic flow or by electroosmotic flow. Finally, conventional CE was carried out for separation of the analytes. Several different concentration schemes for proteins and peptides were successfully demonstrated by using this new approach.     

The principle cause for lower plate numbers in capillary zone electrophoresis with most organic solvents.

With longitudinal diffusion as an unavoidable source of peak broadening, the peak efficiency (expressed by the plate number, N) in capillary zone electrophoresis depends on the ratio of electrophoretic mobility, mu, and tracer- or self-diffusion coefficient, D. Both parameters are functions of the ionic strength of the electrolyte solution. According to theory, the mobility is decreased with increasing ionic strength by the relaxation effect (depending on the relative permittivity) and the electrophoretic effect (depending on the relative permittivity and the viscosity of the solvent), whereas the diffusion coefficient is decreased only by the relaxation effect. This allows the theoretical predictions that the plate number, which is proportional to the ratio mu/D, decreases with increasing ionic strength and that the magnitude of this reduction depends on the solvent. Taking the values for relative permittivity and viscosity allows forecasting that, in general, water as a solvent exhibits the smallest lowering of the plate number, as compared to organic solvents. The theoretical predictions are confirmed by the data for the ratio calculated from measured mobilities and diffusion coefficients for iodide as the analyte ion in water, methanol, and acetonitrile with ionic strength of the background electrolyte varying between 0.005 and 0.080 mol L(-1). Whereas the experimentally observed plate number per volt is reduced from its "ultimate value" of about 20 (analyte charge number z = 1, zero ionic strength) in water by only 10%, the decrease at the same ionic strength in methanol and acetonitrile reaches 25 to 30%. Thus, the maximum plate number should read Nmax approximately equals 13 zU (with U being the effective voltage) for these solvents with ionic strengths normally applied in capillary electrophoresis. This reduction is not stemming from inappropriate experimental conditions, but has fundamental physicochemical causes.     

Suppression of electroosmotic flow and prevention of wall adsorption in capillary zone electrophoresis using zwitterionic surfactants

Addition of zwitterionic surfactants such as dodecyldimethyl(3-sulfopropyl)ammonium hydroxide, hexadecyldimethyl(3-sulfopropyl)ammonium hydroxide, and coco (amidopropyl)hydroxyldimethylsulfobetaine (Rewoteric AM CAS U) to an electrophoretic buffer suppress the electroosmotic flow by 50-90%. Onset of suppression occurs at approximately the critical micelle concentration of the surfactant. CAS U effectively suppresses the electroosmotic flow over the pH range 3-12. Addition of 2 mM CAS U to the electrophoretic buffer prevents adsorption of cationic proteins lysozyme, α-chymotrypsinogen A, cytochrome c, and ribonuclease A. Migration time reproducibility for these proteins is ～1% RSD within 1 day and 2-5% from day to day. Efficiencies in excess of 750?000 plates/m and recoveries of >80% were observed for protein injections from distilled water. Alternatively if 2 mM CAS U is added to samples, recoveries were quantitative, although efficiencies decreased to 325?000-600?000 plates/m. The natural electroosmotic flow of the capillaries is regenerated simply by rinsing with sodium hydroxide.     

Theoretical description of the influence of external radial fields on the electroosmotic flow in capillary electrophoresis

The effect of applying a radial voltage on the electroosmotic flow in capillary electrophoresis has been studied from a theoretical point of view. Based on Stern's model for the electric double layer on the surface of a fused silica capillary and on the Gouy-Chapman theory for the diffuse layer, equations describing the relation between the electroosmotic mobility and the radial electric field were derived. The thickness of the stagnant solution layer on the surface of the capillary, an important parameter in the calculations, was estimated from the electroosmotic mobility found in high-pH solutions. The theory developed predicts the experimental findings that the effect of the radial field levels off at high applied voltages and that it is smaller when the electroosmotic mobility without radial field is already high. The theoretical results were compared with experimental data taken from the literature. A good quantitative agreement was found.     

Dynamic coating using polyelectrolyte multilayers for chemical control of electroosmotic flow in capillary electrophoresis microchips

Poly(dimethylsiloxane) (PDMS) capillary electrophoresis (CE) microchips were modified by a dynamic coating method that provided stable electroosmotic flow (EOF) with respect to pH. The separation channel was coated with a polymer bilayer consisting of a cationic layer of Polybrene (PB) and an anionic layer of dextran sulfate (DS). According to the difference in charge, PB- and PB/ DS-coated channels supported EOF in different directions; however, both methods of channel coating exhibited a pH-independent EOF in the pH range of 5-10 due to chemical control of the effective zeta-potential. The endurance of the PB-coated layer was determined to be 50 runs at pH 3.0, while PB/DS-coated chips had a stable EOF for more than 100 runs. The effect of substrate composition and chip-sealing methodology was also evaluated. All tested chips showed the same EOF on the PB/DS-coated channels, as compared to uncoated chips, which varied significantly. No significant variation for separation and electrochemical detection of dopamine and hydroquinone between coated and uncoated channels was observed.     

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.     

Determination and modeling of peptide pKa by capillary zone electrophoresis

In this work, pKa values of polyglycines, poly(L-alanines), and poly(L-valines) with a number of residues up to 10 were determined in different conditions of ionic strength (10 and 100 mM) and temperature (from 15 to 60 degrees C) by capillary electrophoresis. For each peptide family, the pKa values were modeled as a function of the number of residues, the temperature, and the ionic strength. Next, using this set of experimental data, a semiempirical model was developed in order to predict pKa values for any oligopeptide having neutral lateral chains. This model only needs, as input parameters, the number of residues and the pKa of terminal amino acids in their free form. It can predict the peptide pKa values at a given ionic strength and temperature. Comparisons with experimental data from the literature demonstrated that the prediction was possible with a standard deviation of approximately 0.1 pH unit.     

Phosphopeptide isomer separation using capillary zone electrophoresis for the study of protein kinases and phosphatases.

Methods for the rapid separation of phosphopeptide isomers (peptides with the same sequence but with phosphates on different residues) were developed using capillary zone electrophoresis with ultraviolet (CZE-UV) detection. Uncoated, cationic and neutral capillaries were used with both acidic and basic peptides. These methods enabled the assay of several protein kinases (mitogen activated protein kinase, protein kinase A, GST-tyrosine kinase) and phosphatases (acid, alkaline, and protein tyrosine phosphatase) and the determination of the sites of phosphorylation and dephosphorylation. Incubations of nonphosphorylated or phosphorylated peptide with kinases or phosphatases took place directly in the instrument's autosampler and were monitored over several hours using CZE-UV.     

Attomole amino acid determination by capillary zone electrophoresis with thermooptical absorbance detection

Attomole quantities of 4-(dimethylamino)azobenzene-4'-sulfonyl chloride derivatized amino acids are separated by using capillary zone electrophoresis in a mixed acetonitrile/aqueous buffer system. Detection is performed with an on-column thermooptical absorbance detection technique based on a 130-mW argon ion pump laser. Detection limits for the concentration of analyte injected onto the column range from 5 x 10(-8) M for methionine to 5 x 10(-7) M for aspartic acid. Only 37 amol of methionine and 450 amol of aspartic acid are contained within the subnanoliter injection volume. It is interesting to note that these limits are a factor of 4 superior to the best fluorescence detection limit reported for chromatographic separation of amino acids. A subnanoliter sample of derivatized human urine was analyzed with this technique; quantities of amino acids contained within the sample are 3 orders of magnitude greater than the detection limit.