Leading Electrolyte Concentration Research Articles

Simultaneous optical and infrared thermal imaging of isotachophoresis

Joule heating in isotachophoresis (ITP) can limit minimum assay times and efforts to scale up processed sample volumes. Despite its significance, the dynamics of Joule heating on spatiotemporal temperature fields in ITP systems have not been investigated. We here present novel measurements of spatiotemporal temperature and electromigration fields in ITP. To achieve this, we obtain simultaneous and registered optical and infrared thermal images of the ITP process. We conduct a series of experiments at constant current operation and vary the leading electrolyte concentration to study and highlight the importance of buffer-dependent ionic conductivity on the resulted temperature rise. The measurements demonstrate a substantial increase of temperature in the adjusted trailing electrolyte region, and the propagation of a thermal wave in the ITP channel with a velocity equal to that of the electromigration front. We present scaling of the experimental data that indicates the dependence of front velocity and temperature rise on current density and ionic conductivity. The current study has direct application to the design and optimization of scaled-up ITP systems and the validation of numerical models of Joule heating.

Sample Zone Dynamics in Peak Mode Isotachophoresis

We present a theoretical and experimental study of analyte preconcentration via peak mode isotachophoresis (ITP). We perform perturbation analysis of the governing equations that includes electromigration, diffusion, buffer reactions, and nonlinear ionic strength effects. This analysis relaxes the inherent numerical stiffness and achieves a fast solution to the transient sample evolution problem. In this model, we have incorporated a semiempirical relation to capture dispersion phenomenon within ITP interfaces. We also present a simple, closed-form analytical model that identifies key parameters governing the preconcentration dynamics in peak mode ITP. We have validated our models through a detailed experimental study performed in constant current conditions. The relevant governing experiment parameters were varied independently; namely, the leading electrolyte concentration, trailing electrolyte concentration, and current. Through our experimental study, we have identified optimum conditions to achieve high preconcentration ratio and sample accumulation rates. Our approach to the theoretical problem and experimental study provides useful guidelines in optimizing parameters such as detector location, ITP duration, and electrolyte composition in ITP preconcentration and separation assays.

Gradient elution isotachophoresis with direct ultraviolet absorption detection for sensitive amino acid analysis

This work demonstrates coupling of the newly described electrophoretic enrichment technique of gradient elution isotachophoresis (GEITP) to a low-cost, conventional ultraviolet absorbance detector to realize sensitive measurements with a universal detector, eliminating the need for fluorescent analytes or derivatization. The effects of various parameters on enrichment were studied, including current density varied by leading electrolyte concentration, current density varied by applied electric field, and counter-flow acceleration across varying capillary inner diameters. Optimized parameters were applied to the enrichment and separation of the amino acids tryptophan (Trp) and tyrosine (Tyr). Limits of detection for Trp and Tyr were 51 and 215 nM, respectively, reflecting sensitivity enhancements of 860- and 1900-fold. Analysis times were less than 6 min, and peak height RSDs were less than 4%. A demonstration of enrichment and separation of these amino acids from artificial cerebrospinal fluid is additionally shown as a first step to realizing biochemical monitoring by GEITP.

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.

Zone shape and concentration in isotachophoresis–capillary electrophoresis

The relationship between injection volume of a dilute sample and its concentration at peak maximum in an isotachophoresis (ITP) zone is described. The concentration at peak maximum is predicted to increase linearly with injection volume for a given leading electrolyte concentration as long as the analyte mass injected is very small. In addition, the relationship between sensitivity of counterflow isotachophoresis–capillary zone electrophoresis and injection volume was investigated using angiotensins as test analytes. The results confirm that sensitivity is a nearly linear function of injection volume for dilute samples. ©1999 John Wiley & Sons, Inc. J Micro Sep 11: 341–345, 1999

Migration markers for capillary isotachophoresis of ribonucleotides

2-Chloropropionic, glyoxylic, and levulinic acids were evaluated as migration markers for isotachophoresis of ribonucleotides on Ucon-coated capillaries. The effects of leading electrolyte pH and concentration on the elution sequence of ribonucleotides were studied. 2-Chloropropionic and levulinic acids were effective at pH 4.50 for 10 mM leading electrolyte, while glyoxylic and levulinic acids worked best at pH 4.50 for 5 and 1 mM leading electrolytes. These migration markers divided the ribonucleotides into three groups: triphosphate, diphosphate, and monophosphate. At pH values lower than 4.50, mixed-analyte zones were formed. At pH values higher than 4.50, zones mixed with impurities were formed. The leading electrolyte concentration affected the effective mobility, elution sequence, concentration effects, and separation speed. Low leading electrolyte concentration (ca. 1 mM) provided high sensitivity and fast separation. The origin of peaklike bands in capillary isotachophoresis (CITP) is attributed to boundary overlap at the band edges. When a dilute sample is analyzed, the band width is mainly defined by the boundary widths. The boundary width is dependent on the electric field strength, temperature, and difference in effective mobilities of the two separands. High electric field strength, low temperature, and large difference in effective mobilities favor narrow boundaries, resulting in high sensitivity and fast separation. © 1998 John Wiley & Sons, Inc. J Micro Sep 10: 423–430, 1998