Development of microfluidic chips for heterogeneous receptor-ligand interaction studies.

A simple microfluidic-based technique to quantitate the binding affinity between the glycopeptide antibiotics teicoplanin from Actinoplanes teicomyceticus and vancomycin from Streptomyces orientalis and 5-carboxyfluorescein-D-Ala-D-Ala-D-Ala (5-FAM-(DA)(3)) is described. In this work, (3-aminopropyl)triethoxysilane is used to modify the surfaces of a series of microchannels, and each channel is subsequently exposed to a solution of antibiotic for a few minutes. The antibiotic is retained after washing through electrostatic interactions, and the series of channels are subsequently exposed to an increasing concentration of 5-FAM-(DA)(3) followed by washing to exclude any nonspecific binding. The extent of fluorescence is quantified using a microscope fitted with a CCD camera. The binding constants for the interaction of teicoplanin and vancomycin with the fluorescent peptide were determined to be 6.03 +/- 0.97 x 10(4) and 4.93 +/- 1.13 x 10(4) M(-1), respectively, in good agreement with previous data. The ease of quantifying the extent of interaction in this microchip technique may prove powerful for exploration of a myriad of receptor-ligand pairs.

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.

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.

Separation of Nile Blue-labelled fatty acids by CE with absorbance detection using a red light-emitting diode.

The separation of fatty acids derivatised with Nile Blue (NB) by CE with detection using a red light-emitting diode (LED) was examined. NB was selected as the derivatisation agent due to its high molar absorption coefficient of 76,000 M(-1) cm(-1) at 633 nm, making it well suited for sensitive absorbance detection using a red 635 nm LED. NB-labelled fatty acids were separated by both MEKC using SDS micelles, i-PrOH and n-BuOH and by NACE in a number of solvents including MeOH, EtOH and ACN. The sensitivity of NACE was superior to MEKC, with detection limits of 5x10(-7)-7x10(-7) M obtained for each acid, approximately 20 times lower than the MEKC method. The NACE detection limits are approximately 100 times lower than previous reports on the separation of fatty acids by CE using indirect absorbance detection, ten times lower than using indirect fluorescence detection and are inferior only to those obtained using precapillary derivatisation and direct fluorescence detection. The efficiency of the NACE method was also superior to MEKC and allowed the separation of unsaturated fatty acids to be examined, although it was not possible to baseline-resolve linoleic (C18:2) and linolenic (C18:3) acids in a reasonable time. The method was used to analyse the fatty acid profile of two edible oils, namely sunflower and sesame oils, after alkali hydrolysis, where it was possible to identify both the saturated and unsaturated fatty acids in each sample.

Use of contactless conductivity detection for non-invasive characterisation of monolithic stationary-phase coatings for application in capillary ion chromatography.

A capacitively-coupled contactless conductivity detector (C4D) has been utilised as an on-capillary detector within a capillary ion chromatograph, incorporating a reversed-phase monolithic silica capillary column semi-permanently modified with a suitable ionic surfactant. The monolithic capillary column (150 x 0.1 mm i.d.) was modified using sodium dioctyl sulfosuccinate (DOSS), an anionic surfactant, for the separation of small inorganic and organic cations. With the use of the on-capillary conductivity detector, the longitudinal homogeneity and temporal stability of the coating were investigated. The approach allowed a detailed non-invasive observation of the nature of the ion-exchange coating over time, and an example of an application of the technique to produce a longitudinal stationary-phase charge gradient is shown. An investigation of the basis of the measured on-capillary conductivity was carried out with a counter ion study, clearly showing the on-capillary detection technique could also distinguish between chemical forms of the immobilised ion exchanger. The above method was used to produce a stable and homogeneously-modified monolithic ion-exchange capillary column, for application to the separation of inorganic alkaline earth cations and amino acids.

Evaluation of capillary ion exchange stationary phase coating distribution and stability using radial capillary column contactless conductivity detection.

The use of radial (across) capillary column capacitively coupled contactless conductivity detection is demonstrated as a simple and rapid technique for visualisation of stationary phase longitudinal coverage and coating stability in capillary ion exchange chromatography.

Sensitive determination of carbohydrates labelled with p-nitroaniline by capillary electrophoresis with photometric detection using a 406 nm light-emitting diode.

p-Nitroaniline was explored as a derivatising reagent for UV absorbance detection of carbohydrates after separation by CE. This derivatising agent has three advantages: first, it has excellent water solubility; second, it has high molar absorptivity; and third, it is possible to obtain sensitive detection using a UV or blue light-emitting diode (LED) as the light source. The labelling reaction took less than 30 min to complete with high reaction yield. The separation process was modelled and optimised using an artificial neural network. Nine carbohydrates were separated by a CE system within 16 min using a 0.17 M boric acid buffer at pH 9.7. On-column LED detection at 406 nm allowed the detection of carbohydrates with good detection limits (<1.1 microM or 8.8 fmol) and reproducible quantification in the concentration range of 2.6-200 microM. This method was applied successfully to the determination of component carbohydrates in some food samples.

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.

Preparation and characterisation of dual-layer latex-coated columns for open-tubular capillary electrochromatographic preconcentration of cations combined in-line with their separation by capillary electrophoresis.

A dual-layer ion-exchange latex-coated column was prepared and characterised for on-capillary preconcentration of cations using an open-tubular ion-exchange CEC format. After preconcentration, the analyte cations were eluted with a transient isotachophoretic gradient and separated by CE. The latex double layer was established by first coating the negatively charged wall of the capillary with a layer of cationic quaternary ammonium anion-exchange Dionex AS5A latex particles (60 nm diameter), and then coating a layer of anionic sulphonated cation-exchange Dionex CS3 latex particles (300 nm diameter) onto the underlying AS5A layer. The adhesion of layers is based on electrostatic attractions. Several dual-layer capillaries were characterised for their EOF and ion-exchange capacity and this showed that coatings could be prepared reproducibly by a simple flushing procedure. The dual-layer columns exhibited a moderate, pH-independent EOF (ca. 26 x 10(-9 )m2V(-1)s(-1)) and an ion-exchange capacity of 57 microequiv./g (or 2.69 nequiv./column). Using an 8 cm length of coated capillary combined with a 72 cm length of untreated capillary, a method for on-line preconcentration and separation of monovalent organic bases, alkali metal ions and alkaline earth metal ions by CE was developed. Recoveries for the preconcentration step were 48% for 4-methylbenzylammonium, 43% for benzylammonium, 30-32% for alkali metal ions and 71-75% for alkaline earth cations. In all cases, recoveries were reproducible with RSDs being less than 6.2%. The influences of the ion-exchange selectivity coefficient of the analyte and the sample-loading rate on analyte recovery were also examined. The proposed method was utilised for the determination of alkaline earth cations and low microM detection limits were obtained.

Preparation and characterisation of anion-exchange latex-coated silica monoliths for capillary electrochromatography.

Silica monoliths coated with functionalised latex particles have been prepared for use in monolithic ion-exchange capillary electrochromatography (IE-CEC) for the separation of inorganic anions. The ion-exchange monoliths were prepared using 70 nm quaternary ammonium, anion-exchange latex particles, which were bound electrostatically to a monolithic silica skeleton synthesised in a fused silica capillary. The resulting stationary phases were characterised in terms of their chromatographic performance and capacity. The capacity of a 50 microm diameter 25 cm latex-coated silica monolith was found to be 0.342 nanoequivalents and 80,000 theoretical plates per column were typically achieved for weakly retained anions, with lower efficiency being observed for analytes exhibiting strong ion-exchange interaction with the stationary phase. The electroosmotic flow (EOF) was reversed after the latex-coating was applied (-25.96 m2 V(-1) s(-1), relative standard deviation (RSD) 2.8%) and resulted in anions being separated in the co-EOF mode. Ion-exchange interactions between the analytes and the stationary phase were manipulated by varying the ion-exchange selectivity coefficient and the concentration of a competing ion (phosphate or perchlorate) present in the electrolyte. Large concentrations of competing ion (greater than 1M phosphate or 200 mM perchlorate) were required to completely suppress ion-exchange interactions, which highlighted the significant retention effects that could be achieved using monolithic columns compared to open tubular columns, without the problems associated with particle-packed columns. The latex-coated silica monoliths were easily produced in bulk quantities and performed reproducibly in acidic electrolytes. The high permeability and beneficial phase ratio makes these columns ideal for micro-LC and preconcentration applications.

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.

On-line preconcentration of organic anions in capillary electrophoresis by solid-phase extraction using latex-coated monolithic stationary phases.

Quaternary ammonium functionalised polymeric latex particles were coated onto the wall of a fused-silica capillary or onto a methacrylate monolithic bed synthesised inside the capillary in order to create ion-exchange stationary phases of varying ion-exchange capacity. These capillaries were coupled in-line to a separation capillary and used for the solid-phase extraction (SPE), preconcentration and subsequent separation of organic anions by capillary electrophoresis. A transient isotachophoretic gradient was used for the elution of bound analytes from the SPE phase using two modes of separation. The first comprised a low capacity SPE column combined with a fluoride/octanesulfonate discontinuous electrolyte system in which peak compression occurred at the isotachophoretic gradient front. The compressed anions were separated electrophoretically after elution from the SPE preconcentration phase and resolution was achieved by altering the pH of the electrolyte in which the separation was performed. In the second approach, a latex-coated monolithic SPE preconcentration stationary phase was used in combination with a fluoride/perchlorate electrolyte system, which allowed capillary electrochromatographic separation to occur behind the isotachophoretic gradient front. This method permitted the removal of weakly bound anions from the SPE phase, thereby establishing the possibility of sample clean-up. The effect of the nature of the strong electrolyte forming the isotachophoretic gradient on the separation and also on the preconcentration step was investigated. Capillary electrochromatography of inorganic and organic species performed on the latex-coated monolithic methacrylate column highlighted the presence of mixed-mode interactions resulting from the incomplete coverage of latex particles onto the monolithic surface. Analyte preconcentration prior to separation resulted in compression of the analyte zone by a factor of 300. Improvement in the limit of detection of up to 10400 times could be achieved when performing the preconcentration step and the presented methods had limits of detection (S/N=3) ranging between 1.5 and 12 nM for the organic anions studied.

Non-aqueous capillary electrophoresis with red light emitting diode absorbance detection for the analysis of basic dyes.

Non-aqueous capillary electrophoresis was evaluated for the separation of five hydrophobic basic blue dyes for application in forensic dye analysis. The use of a red light emitting diode as a high intensity, low-noise light source provided sensitive detection of the blue dyes while also allowing the evaluation of solvents that absorb strongly in the UV region. Excellent peak shapes and separation selectivity were obtained in methanol, ethanol, acetonitrile and dimethylsulfoxide, however water, tetrahydrofuran, dimethylformamide and acetone were unsuitable as solvents due to poor peak shapes and a lack of sensitivity, most likely due to adsorption onto the capillary wall. Due to the known compatibility of methanol with capillary electrophoresis-mass spectrometry, this solvent was examined further with the relative acidity/basicity of the electrolyte being optimised with an artificial neural network. The optimised method was examined for the separation of ink samples from 6 fibre tip and 2 ball point blue or black pens and showed that a unique migration time for the main dye component in seven of the eight pens could be obtained.

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.

Contactless conductivity detection of synthetic polymers in non-aqueous size-exclusion electrokinetic chromatography.

A capacitively coupled contactless conductivity detection (CCD) system has been applied for the detection of neutral synthetic polymers in capillary size-exclusion electrokinetic chromatography (SEEC). Polystyrene standards, that were used as a model compounds, were separated on a capillary column packed with porous 10 microm silica particles with an electrokinetically driven mobile phase, and detected by CCD and UV detection simultaneously. Mass-calibration curves for polystyrene were constructed. Satisfactory results were obtained for the linearity, the run-to-run repeatability (<0.2% for the relative retention and <4% for the peak area) and the robustness of the detector. One of the major issues in this preliminary study was to investigate the origin of the peaks observed for the polystyrene standards. The effect of the molar mass of the polystyrenes on the sensitivity was small. Therefore, the signals obtained could not be explained as the result of an increased viscosity and a decreased solution conductivity of the solute zone. An alternative hypothesis is suggested, and recommendations for further research are given.

Latex-coated polymeric monolithic ion-exchange stationary phases. 1. Anion-exchange capillary electrochromatography and in-line sample preconcentration in capillary electrophoresis.

A sulfonated methacrylate monolithic polymer has been synthesized inside fused-silica capillaries of diameters 50-533-microm i.d. and coated with 65-nm-diameter fully functionalized quaternary ammonium latex particles (AS18, Dionex Corp.) to form an anion-exchange stationary phase. This stationary phase was used for ion-exchange capillary electrochromatography of inorganic anions in a 75-microm-i.d. capillary with Tris/perchlorate electrolyte and direct UV detection at 195 nm. Seven inorganic anions (bromide, nitrate, iodide, iodate, bromate, thiocyanate, chromate) could be separated over a period of 90 s, and the elution order indicated that both ion exchange and electrophoresis contributed to the separation mechanism. Separation efficiencies of up to 1.66 x 10(5) plates m(-1) were achieved, and the monoliths were stable under pressures of up to 62 MPa. Another latex-coated monolith in a 250-microm-i.d. capillary was used for in-line preconcentration by coupling it to a separation capillary in which the EOF had been reversed using a coating of either a cationic polymer or cationic latex particles. Several capillary volumes of sample were loaded onto the preconcentration monolith, and the analytes (inorganic anions) were then eluted from the monolith with a transient isotachophoretic gradient before being separated by electrophoresis in the separation capillary. Linear calibration curves were obtained for aqueous mixtures of bromide, nitrite, nitrate, and iodide. Recoveries of all analytes except iodide were reduced significantly when the sample matrix contained high levels of chloride. The preconcentration method was applied to the determination of iodide in open ocean water and provided a limit of detection of 75 pM (9.5 ng/L) calculated at a signal-to-noise ratio of 3. The relative standard deviation for migration time and peak area for iodide were 1.1 and 2.7%, respectively (n = 6). Iodide was eluted as an efficient peak, yielding a separation efficiency of 5.13 x 10(7) plates m(-1). This focusing was reproducible for repeated analyses of seawater.

Isoelectric buffers for capillary electrophoresis. 2. Bismorpholine derivative of a carboxylic acid as a low molecular weight isoelectric buffer.

A new compound class of synthetic isoelectric buffers is introduced, designed as a small molecule with one fully or prevailingly dissociated acidic group (such as sulfonic or carboxylic) and two partly pronated (buffering) basic amino groups attached onto a hydrophilic UV-transparent backbone. As an example, a new isoelectric compound 2,2-bis(4-morpholinylmethyl)propanoic acid (BMMPA) was synthesized by attaching two morpholine groups onto a molecule of pivalic acid. It was characterized as having an isoelectric point pI = 6.5 and exhibiting satisfactory buffering capacity at the pI. Solutions of BMMPA are transparent down to the low-UV spectral region, thus making it a potentially suitable buffer for a number of separation methods. Its use in capillary electrophoresis was demonstrated in a separation system for indirect photometric detection of anions based on an electrolyte with the anionic dye Orange G as the indirect detection probe and using BMMPA as a buffer. The use of an isoelectric buffering compound brings the advantages of a buffered electrolyte without the concomitant introduction of co-ions that would be detrimental to the indirect detection process. Submicromole per liter limits of detection for a number of inorganic and small organic ions were achieved. Optimal structural properties of the isoelectric buffer with respect to its buffering properties are discussed.

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.