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Because electric field distribution is determined by emitter size and shape, sprayer tip geometry determines the optimum liquid flow rate that can be processed by the electrospray ionization interface. Electric field is the highest at the sharpest edge of an electrode; therefore, for a beveled tip, the field is highest at the very tip, and for a conventional symmetrically tapered tip, the field is the highest around the rim of the electrode. Electrospray performance as a function of flow rate was investigated using both continuous infusion and peak-based analysis. The sharpest symmetrical emitter gave the most stable electrospray ionization (ESI) at flow rates [less than or equal to] 0.10 [micro]L/min, while beveled emitters provided significantly better performance at expanded flow rates up to 1 [micro]L/min. The use of beveled emitters offers the potential for increased versatility in electrospray ionization interfaces. 10.1021/ac1017953

Mixing of two solutions into one is a spontaneous process with a net increase in entropy. However, the reverse of the mixing process is usually not possible unless certain conditions are met. A continuous solution stream containing a mixture of two compounds can be separated into two channels, each containing a pure compound, thus reversing the mixing process using a two-dimensional microfluidic electro-fluid-dynamic (EFD) device. When the electric field is strategically applied in the interconnecting channels of an EFD device, the pressure required to direct an analyte into a certain channel can be calculated by using the solutions of electric field and fluid dynamics in the mass balance equation. If the pressure and electric potential at various inlets and outlets satisfy these predetermined conditions, the reverse of a mixing process is observed. Conventional microfluidic devices have been used to introduce samples from interconnecting channels or efficiently mix different solutions into a single channel. The EFD devices expand the spatial separation of analytes from one dimension to two using both the differential migration behavior of analytes and the velocity field distribution in different channel geometries. The devices designed according to these basic physicochemical principles can be used for complete processing of minute samples and to obtain pure chemical species from complex mixtures. 10.1021/ac902938g

The specific binding characteristics of [[alpha].sub.v] [[beta].sub.3] integrins with an arginine-glycine-aspartic-acid (RGD) containing fluorescently labeled cyclic peptide is investigated with capillary electrophoresis-frontal analysis method. The new algorithm used to calculate the binding constants and binding stoichiometry was derived without the assumptions made in the commonly used Scatchard Plot method, thus enabling the determination of specific binding parameters in the presence of nonspecific binding. The [[alpha].sub.v][[beta]. sub.3] integrin, a membrane protein, was studied in solution, without the need of immobilization or any other kind of modification. An RGD containing fluorescently labeled cyclic pentapeptide is used as the ligand with both specific and nonspecific binding characteristics, and an argininealanine-aspartic-acid (RAD) containing pepfide is used as the control for nonspecific binding. While a typical specific binding isotherm has a shape of a rectangular hyperbola, a nonspecific binding isotherm is linear in the same ligand concentration region. A 1:2 specific binding stoichiometry was revealed with the second binding having a similar affinity compared to the first binding event.

Affinity capillary electrophoresis (ACE) has been used to estimate thermodynamic constants of binding interactions with linear or nonlinear regression methods. The accuracy of this approach relies heavily on the binding interaction mechanism, which is controlled by both the nature of the interaction and the experimental conditions. The development of a highly efficient computer-simulated ACE system makes it possible to demonstrate the detailed behavior of any interacting species of a given interaction under any conditions. The order of the mobilities of the complex and the two binding species in their free forms is a key factor to determine what molecules in what locations of the column are involved in the interaction, and the peak shape resulting from such interactions, of a given ACE experiment. In this paper and the supporting materials, 18 scenarios in 6 different combinations of migration orders of the free analyte, free additive, and complex formed are studied by a computer simulation program based on the mass transfer equation. From the study of these situations, we conclude high additive concentration (ensuring high capacity factor) and low analyte concentration (ensuring fast fill-in of the free additive in the analyte plug) are crucial for obtaining accurate results when using the regression methods. On the other hand, the approach to estimate binding constants with computer simulation can be much more accurate as long as accurate and efficient simulation models can be developed, especially when the ratio of the additive and analyte concentrations is not large enough.

With more accurate simulation models and more efficient algorithms becoming available, the binding constants of an affinity interaction can be obtained from much simpler experiments using capillary electrophoresis. With the enumeration algorithm, all possible combinations of the binding constant and the complex mobility in certain ranges that could result in the experimental migration time of an injected analyte are extracted from a 3-D surface, which depicts the migration times resulting from different values of the binding constant and the mobility of the complex formed between the interacting pair, to form a 2-D curve. When the experimental conditions are changed, the analyte migration time will also change. A new 2-D curve can be constructed from another 3-D surface on the basis of the pairs of binding constants and complex mobility values that could result in the new migration time. Because the true binding constant and complex mobility values have to be the same for both experimental conditions under the same temperature, there has to be a point where both 2-D curves will converge. The coordinates of the converging point give the values for a binding constant and a complex mobility that will fit all 2-D curves generated under certain experimental conditions, p-Nitrophenol is used as the analyte, [beta]-cyclodextrin is used as the additive, and a one-cell model is used to simulate affinity CE. The experimental conditions that can improve the accuracy of the binding constants are discussed.

The differential equation describing electrophoretic migration can be evaluated with various finite difference schemes (FDSs). However, the accuracy and efficiency can be dramatically different depending on the FDS chosen and the way the algorithm is implemented in a computer simulation program. The monotonic transport scheme is used as the algorithm for the hyperbolic part of the differential equation, and the first-order fully explicit scheme is used for the parabolic part of the equation. The combination of these algorithms minimizes the errors and maintains high efficiency. A circular arrangement of the cells in the computer's memory is used in the implementation of the algorithms, and the use of concentration thresholds to enable and disable cells along the capillary makes the new algorithm highly efficient. Either thermodynamic or kinetic constants can be used in this program to simulate binding interactions between two species for equilibrium and nonequilibrium affinity CE. Simulation results with various parameters are presented. The simulated peak with proper parameters for an equilibrium affinity CE experiment has shape and position similar to that of the experimental peak. The simulated electropherograms for a nonequilibrium affinity CE experiment also show characteristics of the experimental electropherograms.

Individual analytes can be isolated from multicomponent mixtures and collected in the outlet vial by carrying out electrophoretic purification through a capillary column. Desired analytes are allowed to migrate continuously through the column under the electric field while undesired analytes are confined to the inlet vial by application of a hydrodynamic counter pressure. Using pressure ramping and buffer replenishment techniques, 18% of the total amount present in a bulk sample can be purified when the resolution to the adjacent peak is ~3. With a higher resolution, the yield could be further improved. Additionally, by periodically introducing fresh buffer into the sample, changes in pH and conductivity can be mediated, allowing higher purity ([greater than or equal to] 99.5%) to be preserved in the collected fractions. With an additional reversed cycle of flow counterbalanced capillary electrophoresis, any individual component in a sample mixture can be purified providing it can be separated in an electrophoresis system.

In capillary electrophoresis, the relative orders of mobilities of analyte, additive, and the complex formed determine the analyte peak shape in a way similar to the way the binding isotherms determine the peak shapes in chromatography. The three mobilities allow six possible orders; each produces a characteristic peak shape in CE. Equations describing the analyte migration in a CE system with the presence of mobility-changing additives can be implemented into computer programs to predict the migration times of the analyte peak maximums, and the predicted migration times agree well with the experimental results.

A capillary electrophoresis method for measuring the effective mobilities of proton and hydroxide ions was developed. Photodiode array detection coupled with pH-indicating dyes enabled the detection of proton and hydroxide ions using a conventional CE instrument. The effects of ionic strength and pH of the background electrolyte on the measurement of effective mobility of the proton were first investigated. Mobility measurements were found to be independent of applied voltage. Hydroxide ion mobility was also measured and found to be in agreement with literature values. The effects of pH indicator and proton source on the ion mobility were determined and are described herein.

Most affinity studies in capillary electrophoresis assume that the analyte concentration is much smaller than the additive concentration so that the migration of the analyte has no effect on the concentration of the additive in the capillary. However, in most medium- to high-affinity interactions, the additive concentration has to be kept rather low to observe the changes in analyte mobility before saturation is reached. In this paper, a mathematical model is developed to describe the migration behavior of the analyte in a system where the complex formed becomes concentrated to levels much greater than the original concentration of the additive due to the differences in the mobilities of the analyte, additive, and complex. The analyte is flurbiprofen, the additive is transthyretin, and the stoichiometry of the reaction between the two is 1:2. This study also provides a new algorithm to determine medium- to high-affinity binding interaction constants by CE.

Velocity-difference induced focusing (V-DIF) of nucleotides was achieved by using a dynamic pH junction in capillary electrophoresis (CE) with UV detection. The influence of specific analyte properties, such as nucleotide base structure, sugar structure, and degree of phosphorylation, is examined. The pK(sub a) values and borate complexation with vicinal diols are important factors that caused the focusing. Therefore, the pH and borate content in the sample and background electrolyte can be adjusted to optimize the focusing effect. This method allows the injection of large volumes of sample (approximately 300 nL), resulting in at least 50-fold improvement in concentration sensitivity. The detection limit of 4.0 x 10(super -8) M for nucleotides can be achieved in favorable conditions. V-DIF can be also applied to nucleotide pool analysis from cell extracts to improve the concentration sensitivity of CE and to reduce the time-consuming steps of desalting and off-line preconcentration that are often required for assays of nucleotides from biological samples.

A systematic study of selective analyte focusing in a multisection electrolyte system by capillary electrophoresis (CE) is presented. It was found that a dynamic pH junction between sample and background electrolyte zones can be used to focus zwittefionic catecholamines and weakly acidic compounds without the use of special ampholytes. Differences in pH and concentration of complexing agents, such as borate, in the sample and background electrolyte zones were determined to cause focusing through changes in the local velocity of the analyte in two different segments of the capillary. Velocity-difference induced focusing (V-DIF) of analytes using a dynamic pH junction allowed the injection of large sample volumes and significantly improved the concentration sensitivity of CE. Under optimized conditions, the limit of detection for epinephrine was determined to be about 4 x 10(super -8)M (the original sample) with conventional UV absorbance detection. Moreover, separation efficiencies greater than a million theoretical plates can be achieved by focusing such large sample volumes into narrow zones. Multisection electrolyte systems, which lead to the formation of a dynamic pH junction, can be tuned toward improving the concentration sensitivity of specific analytes if their chemical properties are known.

An equation was derived to describe the variation of the gas number density within the region between the orifice and the skimmer of an electrospray ionization mass spectrometer. The equation was used to develop a semi-quantitative model to predict the value of orifice voltages that lead to ion fragmentation within this region. This model made it possible to predict the types of solvent adducts observed for analytes at various orifice voltages. In addition, it is shown that a small number of high-energy collisions is equally effective for collision-induced dissociation as compared to a large number of low-energy collisions. Finally, this model is tested with different background electrolyte solutions and a different electrospray mass spectrometer. It is demonstrated that controlled fragmentation studies can be performed on single-quadrupole mass spectrometers, and the proposed model gives a reasonable description of the fragmentation process in both spectrometers.

When the properties of an analyte are known, the separation system can be designed to make the analyte of interest migrate at either a much faster or a much slower velocity compared to other molecules in the sample matrix. A simple and sensitive method to analyze the [Gamma]-carboxyglutamic acid (Gla) content of protein, urine, and plasma was developed using capillary electrophoresis with laser-induced fluorescence detection (CE-LIF). The separation method is designed according to the specific properties of three amino acids of interest. The number of Gla residues from three vitamin K-dependent proteins were estimated by quantifying the amount of fluorescein thiocarbamyl derivative of Gla after alkaline hydrolysis and fluorescein isothiocyanate labeling. Human prothrombin, blood coagulation factor X, and bovine osteocalcin were calculated to have 10.0 [+ or -]: 0.7, 11.0 [+ or -] 0.6, and 2.1 [+ or -] 0.1 Gla residues per mole of protein, respectively, which agreed well with amino acid sequencing data. The analysis of free Gla content in urine and plasma was also demonstrated by this method. It was demonstrated that submicrograms of protein can be characterized by CE-LIF.

Research was conducted to examine the equilibrium constants and to describe chemical processes dictated by equilibria using rectangular hyperbolae. To compare the distributions of binding constants calculated by several estimation methods under various experimental conditions, simulated experiments were used in a Monte Carlo analysis. Results indicate the reliability of the binding constant estimated under a specific set of conditions.

This paper presents a quantitative investigation into the effect of analyte-additive interactions on analyte migration behavior in capillary electrophoresis (CE) when both 1:1 and 1:2 stoichiometries are present. Equations based on the individual capacity factors for each interaction are derived to account for the effect of both first- and second-order equilibria. The analyte migration behavior is described using these equations with a full account of how the microscopic equilibrium constants and microscopic mobilities are combined to give the macroscopic values. The binding isotherms of interactions with both 1:1 and 1:2 stoichiometries are compared with those of a 1:1 stoichiometry. 4,4[prime]-Biphenol and 4-phenylphenol were chosen as analytes that undergo complexation with one and two hydroxypropyl-[Beta]-cyclodextrin (HP-[Beta]-CD) molecules; phenol was used as an analyte that interacts with only one HP-[Beta]-CD molecule. The process of calculating higher order equilibrium constants and complex mobilities from the binding isotherms is demonstrated. The effects of experimental conditions, such as the additive concentration range and the number of data points, on the error in the calculated constants and the ability of the equations to accurately describe the experimental data are discussed. A comparison of the linear transformations of the binding isotherm with respect to their ability to detect higher order equilibria is made, and the advantage of using the capacity factor in CE is illustrated.

Techniques in applied mathematics are used to characterize the unique attributes of multivariate binding isotherms in capillary electrophoresis (CE). Dengsu concentration can be easily identified when two additives are utilized in the CE. The binding stoichiometries of the additives as well as of the analyte can be confirmed using the shape of the binding isotherms found in the profile plots.

A water-soluble tetraethylsulfonate derivative of 2-methylresorcinarene (TESMR), an aromatic-based, bowl-shaped macrocycle, was used as an additive in capillary electrophoresis. Several phenol derivatives are used as analytes to demonstrate the effect of this highly charged additive. TESMR is observed to interact differently with a mixture of positional isomers and other types of phenol derivatives. A comparative study of separations with two charged additives, TESMR and sulfobutyl ether-[Beta]-cyclo-dextrin (SBE-[Beta]-CD), provided insight into the selectivity exhibited by these additives. The influence of buffer pH, ionic strength, and organic modifier content on separation and peak shape is investigated. Peak asymmetry caused by the use of highly charged additives at lower pH is minimized by the addition of small amounts of a polar aprotic organic solvent to the run buffer. The effects of mixing a charged additive with a neutral additive are also discussed.

The impact of the maximum-response range on the accuracy and precision of estimated binding constants for chemical equilibria was investigated. Findings indicated that the nonlinear regression method produced the most accurate and precise estimates for the binding constant as the maximum-response range was decreased. It was also shown that the difference in the slope of the binding isotherm at the lowest and highest additive concentrations is a good indicator of the precision of a binding constant estimated using a certain set of conditions.