Simplified universal method for determining electrolyte temperatures in a capillary electrophoresis instrument with forced-air cooling.

Temperature increase due to resistive electrical heating is an inherent limitation of capillary electrophoresis (CE). Active cooling systems are used to decrease the temperature of the capillary, but their capacity is limited; and in addition, they leave "hot spots" at the detection interface and at the capillary ends. Until recently, the matter was complicated by the lack of a fast and generic method for temperature determination in efficiently and inefficiently cooled regions of the capillary. Our group recently introduced such a method, termed "Universal Method for determining Electrolyte Temperatures" (UMET). UMET is a probe-less approach that requires only measuring current versus voltage for different voltages and processing the data using an iterative algorithm. Here, we apply UMET to develop a Simplified Universal Method of Temperature Determination (SUMET) for a CE instrument with a forced-air cooling system using an Agilent 7100 CE instrument (Agilent Technologies, Saint Laurent, Quebec, Canada) as an example. We collected a wide set of empirical voltage-current data for a variety of buffers and capillary diameters. We further constructed empirical equations for temperature calculation in efficiently and inefficiently cooled parts of the capillary that require only the data from a single 1-min voltage-current measurement. The equations are specific for the Agilent 7100 CE instrument (Agilent Technologies) but can be applied to all kinds of capillaries and buffers. Similar SUMET approaches can be developed for other CE instruments with forced-air cooling using our approach.

Universal method for determining electrolyte temperatures in capillary electrophoresis.

Temperature increase in capillary electrophoresis (CE) due to Joule heating is an inherent limitation of this powerful separation technique. Active cooling systems can decrease the temperature of a large part of the capillary but they leave "hot spots" at the capillary ends which can completely ruin some CE analyses despite their short lengths. Here, we introduce a "universal method for determining electrolyte temperatures" (UMET) that can determine temperatures in both efficiently- and inefficiently-cooled parts of the capillary. UMET can be applied to all electrolytes, as it does not involve any probe; it requires only measuring current versus voltage for different voltages and processing the data using an iterative algorithm. To demonstrate the universality of UMET, we measured temperatures for electrolytes of different ionic strengths as well as for different capillary diameters. We further propose a "simplified universal method for predicting electrolyte temperatures" (SUMET) which only requires one measurement of current and voltage (that can be completed in 1 min) and uses two empirical equations to predict temperatures in the efficiently- and inefficiently-cooled parts of the capillary. The equations include several instrument-specific empirical parameters that are determined using a large set of current-voltage data obtained with UMET for a range of electrolytes and different capillaries. To demonstrate the utility of SUMET, we obtained the required data set for a Beckman MDQ CE instrument and produced all required empirical parameters that enable a user of this instrument to predict the temperature for every new experimental set in a matter of minutes. We confirmed the accuracy of SUMET by measuring the temperature-sensitive dissociation rate constant of a protein-DNA complex. We foresee that UMET will be used to produce instrument-specific empirical parameters for all CE instruments and then SUMET will be routinely used for temperature prediction in CE.

Milli-free flow electrophoresis: I. Fast prototyping of mFFE devices.

We coin a term of milli-free flow electrophoresis (mFFE) to describe mid-scale FFE with flow rates intermediate to macro-FFE and micro-FFE (µFFE). Introduced decades ago, mFFE did not find practical applications. We revive mFFE, as we view it as a viable purification complement to continuous synthesis in capillary reactors with product flow rates of ∼5 to 2000 µL/min, too small for macro-FFE but too large for µFFE. The development of the tandem of continuous synthesis/purification will require the production and evaluation of a large number of prototypes of mFFE devices. As the first step, we developed a fast (<24 h) and economical (∼$10) method for prototyping mFFE devices using a robotic milling machine. mFFE prototypes are constructed from two machined matching poly(methyl methacrylate) (PMMA) substrates, which are bonded in 10 min using dichloromethane to provide a strong and irreversible seal. Using the developed prototyping technology, we designed and evaluated 25 prototypes of mFFE devices. By optimizing the feed rates and rotational speeds of the drills, the depth of the electrode channels, the dimensions of the entrance and exit reservoirs, the sample flow rate, and the diameter and position of the sample input, we were able to achieve indefinitely long operation of the device with cycles of alternating 15-min electrophoresis and 0.5-min regeneration (bubble removal). The test analytes, rhodamine B and fluorescein, were baseline resolved by mFFE for flow rates ranging from 10 to 600 µL/min. These results prove that our prototyping approach is suitable for the challenging task of multi-parameter optimization of mFFE devices.

Heat-associated field distortion in electro-migration techniques.

Electro-migration techniques, such as electrophoresis, are widely utilized in analytical sciences. If a single electrolyte is used, the field strength is typically assumed to be well-defined. Heat-associated field distortion (HAFD) has been suggested as a result of the nonuniform heat dissipation throughout the electrolyte; however, it has never been experimentally studied. Here, we experimentally demonstrated HAFD for the first time. We used capillary electrophoresis (CE) with a capillary having parts with different heat dissipation efficiencies. Our experiments showed a difference in field strength of approximately 1.5 times between the different parts of the capillary for a typical CE electrolyte. This result suggests that HAFD is a well pronounced phenomenon that can be a potential source of errors and instabilities in electro-migration experiments.

Non-orthogonal micro-free flow electrophoresis: from theory to design concept.

Micro-free flow electrophoresis (microFFE) is a technique that facilitates continuous separation of molecules in a shallow channel with a hydrodynamic flow and an electric field at an angle to the flow. We recently developed a general theory of microFFE that suggested that an electric field non-orthogonal to the flow could improve resolution. Here, we used computer modeling to study resolution as a function of the electric field strength and the angle between the electric field and the hydrodynamic flow. In addition we used our general theory of microFFE to investigate other important influences on resolution, which include the velocity of the hydrodynamic flow, the height of the separation channel, and the magnitude and direction of the electroosmotic flow. Finally, we propose four designs that could be used to generate non-orthogonal electric fields and discuss their relative merits.

Non-orthogonal-to-the-flow electric field improves resolution in the orthogonal direction: hidden reserves for combining synthesis and purification in continuous flow.

There is a pressing need for continuous purification of products of synthesis conducted in continuous-flow microreactors. An existing technique, micro free-flow electrophoresis (microFFE), could fulfill this niche if its resolving power for similar molecules was improved. MicroFFE continuously separates ions in the hydrodynamic flow by an electric field orthogonal to the flow. Here, we prove theoretically from first principles that the resolving power of microFFE can be greatly improved by the use of a nonorthogonal to the flow field. This result may be decisive in starting practical attempts to combine synthesis in continuous-flow microreactors with continuous-flow purification by microFFE.

Joule heating effects and the experimental determination of temperature during CE.

Joule heating is ubiquitous in electrokinetic separations. This review is in two major parts. The first part documents the effects of Joule heating on the physical properties of the electrolyte and efficiency of separations and the second part focuses on advances in the determination of electrolyte temperatures that have been described in the literature over the past 5 years. The focus is on methods that can be applied by practitioners without the need for elaborate experimental requirements. Although the emphasis is on CE, many of the conclusions also apply to microfluidic formats.

Determination of the surface heat-transfer coefficient in CE.

A knowledge of the heat-transfer coefficient, h(s), for the external surface of the capillary or the overall heat coefficient, h(OA), is of great value in predicting the mean increase in temperature of the electrolyte, DeltaT(Mean), during electrokinetic separations. For CE, traditional indirect methods of determining h(s) were time-consuming and tended to overestimate cooling efficiency; a novel method is introduced, which is based on curve-fitting of plots of conductance versus voltage to calculate several important parameters including DeltaT(Mean), h(s), the conductance free of Joule heating effects (G(0)) and the voltage that causes autothermal runaway, V(lim). The new method is superior to previously published methods in that it can be performed more quickly and that it corrects for systematic errors in the measurement of electric current for voltages <5 kV. These errors tended to exaggerate the cooling efficiency of commercial instruments so that the calculated increases in electrolyte temperature were smaller than their actual values. Axially averaged values for h(s) were determined for three different commercial CE instruments ranging from 164 W m(-2) K(-1) for a passively cooled instrument in a drafty environment to 460 W m(-2) K(-1) for a liquid-cooled instrument.

Separation of inorganic anions on a high capacity porous polymeric monolithic column and application to direct determination of anions in seawater.

A commercially available 4.6 mm id x 50 mm polymethacrylate-based monolithic strong anion exchange column (ProSwift SAX-1S) designed for the separation of proteins has been successfully used to separate small inorganic anions in the presence of a seawater sample matrix. Using a hydroxide eluent with suppressed conductivity detection the ion exchange capacity of this column declined over time; however, using KCl as the eluent, the column performance was stable with a capacity of 530 microequiv. for nitrate. The optimum conditions for the separation of iodate, bromate, nitrite, bromide and nitrate were assessed by constructing van Deemter plots using 1.00 and 0.100 M KCl. Efficiencies of up to 26 700 plates/m were recorded using 1.00 M KCl, at a flow rate of 0.20 mL/min but iodate was not baseline resolved from the void peak. By reducing the concentration of the eluent to 0.100 M, efficiencies of up to 39 900 plates/m could be obtained at 0.35 mL/min. By employing a linear gradient ranging from 0.05 to 1.00 M KCl the ions dissolved in distilled water or a salt water matrix could be baseline separated in less than 3 min at a flow rate of 2.50 mL/min.

Reliable electrophoretic mobilities free from Joule heating effects using CE.

Ionic electrophoretic mobilities determined by means of CE experiments are sometimes different when compared to generally accepted values based on limiting ionic conductance measurements. While the effect of ionic strength on electrophoretic mobility has been long understood, the increase in the mobility that results from Joule heating (the resistive heating that occurs when a current passes through an electrolyte) has been largely overlooked. In this work, a simple method for obtaining reliable and reproducible values of electrophoretic mobility is described. The electrophoretic mobility is measured over a range of driving powers and the extrapolation to zero power dissipation is employed to eliminate the effect of Joule heating. These extrapolated values of electrophoretic mobility can then be used to calculate limiting ionic mobilities by making a correction for ionic strength; this somewhat complicated calculation is conveniently performed by using the freeware program PeakMaster 5. These straightforward procedures improve the agreement between experimentally determined and literature values of limiting ionic mobility by at least one order of magnitude. Using Tris-chromate BGE with a value of conductivity 0.34 S/m and ionic strength 59 mM at a modest dissipated power per unit length of 2.0 W/m, values of mobility for inorganic anions were increased by an average of 12.6% relative to their values free from the effects of Joule heating. These increases were accompanied by a reduction in mobilities due to the ionic strength effect, which was 11% for univalent and 28% for divalent inorganic ions compared to their limiting ionic mobilities. Additionally, it was possible to determine the limiting ionic mobility for a number of aromatic anions by using PeakMaster 5 to perform an ionic strength correction. A major significance of this work is in being able to use CE to obtain reliable and accurate values of electrophoretic mobilities with all its benefits, including understanding and interpretation of physicochemical phenomena and the ability to model and simulate such phenomena accurately.

Fluorinated ethylenepropylene copolymer as a potential capillary material in CE.

In this work, a new generation UV-transparent polymer, fluorinated ethylenepropylene copolymer (FEP) exhibiting a low degree of crystallinity, extruded in dimensions similar to the most commonly used CE capillaries of approximately 80 mum id and about 360 mum od was investigated for its use as a CE capillary. FEP is transparent down to the low-UV region, and as fluorinated polymers in general are good electrical insulators and exhibit reasonable heat conductivity, it has considerable potential as a material for electrodriven analysis in capillary or microchip formats. The FEP capillary has been characterised with regard to some important aspects for its use as a CE capillary, including its profile of EOF versus pH, as well as procedures for manipulating EOF by coating the inner capillary wall with various semipermanent and dynamic layers. The FEP capillaries were tested and compared with fused-silica capillary for the separation of inorganic and small organic ions using conditions involving direct and indirect detection in the low-UV region. Finally, advantages of the use of the FEP capillary for simultaneous detection of a mixture containing nine inorganic cations and anions using indirect photometric detection with a movable light-emitting diode (LED) detector and a novel electrolyte are demonstrated.

Identification of inorganic improvised explosive devices by analysis of postblast residues using portable capillary electrophoresis instrumentation and indirect photometric detection with a light-emitting diode.

A commercial portable capillary electrophoresis (CE) instrument has been used to separate inorganic anions and cations found in postblast residues from improvised explosive devices (IEDs) of the type used frequently in terrorism attacks. The purpose of this analysis was to identify the type of explosive used. The CE instrument was modified for use with an in-house miniaturized light-emitting diode (LED) detector to enable sensitive indirect photometric detection to be employed for the detection of 15 anions (acetate, benzoate, carbonate, chlorate, chloride, chlorite, cyanate, fluoride, nitrate, nitrite, perchlorate, phosphate, sulfate, thiocyanate, thiosulfate) and 12 cations (ammonium, monomethylammonium, ethylammonium, potassium, sodium, barium, strontium, magnesium, manganese, calcium, zinc, lead) as the target analytes. These ions are known to be present in postblast residues from inorganic IEDs constructed from ammonium nitrate/fuel oil mixtures, black powder, and chlorate/perchlorate/sugar mixtures. For the analysis of cations, a blue LED (470 nm) was used in conjunction with the highly absorbing cationic dye, chrysoidine (absorption maximum at 453 nm). A nonaqueous background electrolyte comprising 10 mM chrysoidine in methanol was found to give greatly improved baseline stability in comparison to aqueous electrolytes due to the increased solubility of chrysoidine and its decreased adsorption onto the capillary wall. Glacial acetic acid (0.7% v/v) was added to ensure chrysoidine was protonated and to enhance separation selectivity by means of complexation with transition metal ions. The 12 target cations were separated in less than 9.5 min with detection limits of 0.11-2.30 mg/L (calculated at a signal-to-noise ratio of 3). The anions separation system utilized a UV LED (370 nm) in conjunction with an aqueous chromate electrolyte (absorption maximum at 371 nm) consisting of 10 mM chromium(VI) oxide and 10 mM sodium chromate, buffered with 40 mM tris(hydroxymethyl)aminomethane at pH 8.05. All 15 target anions were baseline separated in less than 9 min with limits of detection ranging from 0.24 to 1.15 mg/L (calculated at a signal-to-noise ratio of 3). Use of the portable instrumentation in the field was demonstrated by analyzing postblast residues in a mobile laboratory immediately after detonation of the explosive devices. Profiling the ionic composition of the inorganic IEDs allowed identification of the chemicals used in their construction.

Temperature profiles and heat dissipation in capillary electrophoresis.

While temperature control is usually employed in capillary electrophoresis (CE) to aid heat dissipation and provide acceptable precision, internal electrolyte temperatures are almost never measured. In principle, this limits the accuracy, repeatability, and method robustness. This work presents a fundamental study that combines the development of new equations characterizing temperature profiles in CE with a new method of temperature determination. New equations were derived from first principles relating the mean, axial, and inner wall electrolyte temperatures (T(Mean), T(Axis), T(Wall)). T(Mean) was shown to occur at a distance 1/ radical3 times the internal radius of the capillary from the center of the capillary and to be a weighted average of (2/3)T(Axis) and (1/3)T(Wall). Conductance (G) and electroosmotic mobility (mu(EOF)) can be used to determine T(Mean) and T(Wall), respectively. Extrapolation of curves of mu(EOF) versus power per unit length (P/L) at different temperatures was used to calibrate the variation of mu(EOF) with temperature (T), free from Joule heating effects. mu(EOF) increased at 2.22%/ degrees C. The experimentally determined temperatures using mu(EOF) agreed to within 0.2 degrees C with those determined using G. The accuracy of G measurements was confirmed independently by measuring the electrical conductivity (kappa) of the bulk electrolyte over a range of temperatures and by calculating the variation of G with T from the Debye-Hückel-Onsager equation. T(Mean) was found to be up to 20 degrees C higher than the external temperature under typical conditions using active air-cooling and a 74.0-microm-internal diameter (di) fused-silica capillary. A combination of experimentally determined and calculated temperatures enables a complete temperature profile for a fused-silica capillary to be drawn and the thickness of the stationary air layer to be determined. As an example, at P/L = 1.00 Wm(-1), the determined radial temperature difference across the electrolyte was 0.14 degrees C; the temperature difference across the fused-silica wall was 0.17 degrees C, across the poly(imide) coating was 0.13 degrees C, and across the stationary air layer was 2.33 degrees C.

Variation of zeta-potential with temperature in fused-silica capillaries used for capillary electrophoresis.

The temperature variation of electroosmotic mobility corrected for the effects of Joule heating (muEOF0) was employed to investigate the variation of the zeta-potential (zeta) with temperature in fused-silica capillaries. Experimentally determined values for zeta increased at 0.39% per degrees C, a rate that is about four to five times smaller than reported previously. Experimentally determined values of zeta were directly proportional to the absolute temperature although values were also influenced slightly by changes to the dielectric constant. It was found that the effective charge density at the inner surface of the capillary was independent of temperature.

Internal electrolyte temperatures for polymer and fused-silica capillaries used in capillary electrophoresis.

Polymers are important as materials for manufacturing microfluidic devices for electrodriven separations, in which Joule heating is an unavoidable phenomenon. Heating effects were investigated in polymer capillaries using a CE setup. This study is the first step toward the longer-term objective of the study of heating effects occurring in polymeric microfluidic devices. The thermal conductivity of polymers is much smaller than that of fused silica (FS), resulting in less efficient dissipation of heat in polymeric capillaries. This study used conductance measurements as a temperature probe to determine the mean electrolyte temperatures in CE capillaries of different materials. Values for mean electrolyte temperatures in capillaries made of New Generation FluoroPolymer (NGFP), poly-(methylmethacrylate) (PMMA), and poly(ether ether ketone) (PEEK) capillaries were compared with those obtained for FS capillaries. Extrapolation of plots of conductance versus power per unit length (P/L) to zero power was used to obtain conductance values free of Joule heating effects. The ratio of the measured conductance values at different power levels to the conductance at zero power was used to determine the mean temperature of the electrolyte. For each type of capillary material, it was found that the average increase in the mean temperature of the electrolyte (DeltaT(Mean)) was directly proportional to P/L and inversely proportional to the thermal conductivity (lambda) of the capillary material. At 7.5 W/m, values for DeltaT(Mean) for NGFP, PMMA, and PEEK were determined to be 36.6, 33.8, and 30.7 degrees C, respectively. Under identical conditions, DeltaT(Mean) for FS capillaries was 20.4 degrees C.

Conductivity detection for conventional and miniaturised capillary electrophoresis systems.

Since the introduction of capillary electrophoresis (CE), conductivity detection has been an attractive means of detection. No additional chemical properties are required for detection, and no loss in sensitivity is expected when miniaturising the detector to scale with narrow-bore capillaries or even to the microchip format. Integration of conductivity and CE, however, involves a challenging combination of engineering issues. In conductivity detection the resistance of the solution is most frequently measured in an alternating current (AC) circuit. The influence of capacitors both in series and in parallel with the solution resistance should be minimised during conductivity measurements. For contact conductivity measurements, the positioning and alignment of the detection electrodes is crucial. A contact conductivity detector for CE has been commercially available, but was withdrawn from the market. Microfabrication technology enables integration and precise alignment of electrodes, resulting in the popularity of conductivity detection in microfluidic devices. In contactless conductivity detection, the alignment of the electrodes with respect to the capillary is less crucial. Contactless conductivity detection (CCD) was introduced in capillary CE, and similar electronics have been applied for CCD using planar electrodes in microfluidic devices. A contactless conductivity detector for capillaries has been commercialised recently. In this review, different approaches towards conductivity detection in capillaries and chip-based CE are discussed. In contrast to previous reviews, the focus of the present review is on the technological developments and challenges in conductivity detection in CE.

Determination of inorganic ions using microfluidic devices.

The separation and detection of inorganic ions on microfluidic devices has received little attention since the 'lab-on-a-chip' concept has revolutionised the field of electrokinetically driven analysis. This review presents a summary and discussion of the published literature on inorganic analysis using microfluidic devices and includes sections on electromigration separation methods, namely isotachophoresis (ITP), capillary electrophoresis (CE), and hyphenated ITP-CE, together with a brief account of flow injection analysis. The review concludes with the authors' perspective on future directions for inorganic analysis on microfluidic devices.