Sample stacking in CZE using dynamic thermal junctions I. Analytes with low dpKa/dT crossing a single thermally induced pH junction in a BGE with high dpH/dT.

The possibility to compress analyte bands at the beginning of CE runs has many advantages. Analytes at low concentration can be analyzed with high signal-to-noise ratios by using the so-called sample stacking methods. Moreover, sample injections with very narrow initial band widths (small initial standard deviations) are sometimes useful, especially if high resolutions among the bands are required in the shortest run time. In the present work, a method of sample stacking is proposed and demonstrated. It is based on BGEs with high thermal sensitive pHs (high dpH/dT) and analytes with low dpK(a)/dT. High thermal sensitivity means that the working pK(a) of the BGE has a high dpK(a)/dT in modulus. For instance, Tris and Ethanolamine have dpH/dT=-0.028/ degrees C and -0.029/ degrees C, respectively, whereas carboxylic acids have low dpK(a)/dT values, i.e. in the -0.002/ degrees C to+0.002/ degrees C range. The action of cooling and heating sections along the capillary during the runs affects also the local viscosity, conductivity, and electric field strength. The effect of these variables on electrophoretic velocity and band compression is theoretically calculated using a simple model. Finally, this stacking method was demonstrated for amino acids derivatized with naphthalene-2,3-dicarboxaldehyde and fluorescamine using a temperature difference of 70 degrees C between two neighbor sections and Tris as separation buffer. In this case, the BGE has a high pH thermal coefficient whereas the carboxylic groups of the analytes have low pK(a) thermal coefficients. The application of these dynamic thermal gradients increased peak height by a factor of two (and decreased the standard deviations of peaks by a factor of two) of aspartic acid and glutamic acid derivatized with naphthalene-2,3-dicarboxaldehyde and serine derivatized with fluorescamine. The effect of thermal compression of bands was not observed when runs were accomplished using phosphate buffer at pH 7 (negative control). Phosphate has a low dpH/dT in this pH range, similar to the dK(a)/dT of analytes. It is shown that mid R:dK(a)/dT-dpH/dTmid R>>0 is one determinant factor to have significant stacking produced by dynamic thermal junctions.

Sample stacking in CZE using dynamic thermal junctions II: analytes with high dpKa/dT crossing a single thermal junction in a BGE with low dpH/dT.

In a previous work [M. Mandaji, et al., this issue] a sample stacking method was theoretically modeled and experimentally demonstrated for analytes with low dpK(a)/dT (analytes carrying carboxylic groups) and BGEs with high dpH/dT (high pH-temperature-coefficients). In that work, buffer pH was modulated with temperature, inducing electrophoretic mobility changes in the analytes. In the present work, the opposite conditions are studied and tested, i.e. analytes with high dpK(a)/dT and BGEs that exhibit low dpH/dT. It is well known that organic bases such as amines, imidazoles, and benzimidazoles exhibit high dpK(a)/dT. Temperature variations induce instantaneous changes on the basicity of these and other basic groups. Therefore, the electrophoretic velocity of some analytes changes abruptly when temperature variations are applied along the capillary. This is true only if BGE pH remains constant or if it changes in the opposite direction of pK(a) of the analyte. The presence of hot and cold sections along the capillary also affects local viscosity, conductivity, and electric field strength. The effect of these variables on electrophoretic velocity and band stacking efficacy was also taken into account in the theoretical model presented. Finally, this stacking method is demonstrated for lysine partially derivatized with naphthalene-2,3-dicarboxaldehyde. In this case, the amino group of the lateral chain was left underivatized and only the alpha amino group was derivatized. Therefore, the basicity of the lateral amino group, and consequently the electrophoretic mobility, was modulated with temperature while the pH of the buffer used remained unchanged.

Performance of a sound card as data acquisition system and a lock-in emulated by software in capillary electrophoresis.

The performance of fluorescence detectors in capillary electrophoresis is maximized when the excitation light intensity is modulated in time with optimal frequencies. This is especially true when photomultiplier tubes are used to detect the fluorescent light. The photomultiplier tube amplified raw output signal can in principle be captured directly by a personal computer sound card (PCSC) and processed by a lock-in emulated by software. This possibility is demonstrated in the present work and the performance of this new setup is compared with a traditional data acquisition system. The results obtained with this "PCSC and lock-in emulated by software" were of the same quality or even better compared to that obtained by conventional time integrators (Boxcars) and data acquisition boards. With PCSC the limits of detection (LOD) found for both naphthalene-2,3-dicarboxaldehyde-derivatized tyrosine and alanine were 3.3 and 3.5fmol (injection of 5nL of samples at 0.66 and 0.70micromol/L), respectively. This is at least three times better compared to conventional systems when light emitting diodes (LEDs) are used as the excitation source in fluorescence detectors. The PCSC linear response range was also larger compared to conventional data acquisition boards. This scheme showed to be a practical and convenient alternative of data acquisition and signal processing for detection systems used in capillary electrophoresis.

Separation of biomolecules using electrophoresis and nanostructures.

A large number of nanostructures have the potential to be used together with electrophoresis as separation media or separation additive in capillary electrophoresis, micellar electrokinetic chromatography, capillary electrochromatography, and other analytical techniques. Among those structures are nanotubes, nanocavities, nanowires, nanoposts, nanocones, nanospheres, molecular imprints, nanoparachutes (conical monodendrons), and general nanoparticles with random structures. This review is focused only on publications describing experimental works using molecular imprints, nanoposts, and nanospheres that are fabricated and applied for the purpose of separation media in electrophoresis-driven separations. The review follows an approximate chronological order in each section. As shown, the most popular are those resulting from molecular imprinting technologies. These biomimetic receptors are used in a great variety of fields, which includes electrophoresis, micellar electrokinetic chromatography, capillary electrochromatography, and other fields not reviewed in this work. A few examples of these other fields are, e.g., liquid chromatography, membranes, extractor or preconcentration techniques, immunosorbent assays, and sensing devices. The second topic scanned in the present work is the nanostructures that are used as obstacles to replace gels or polymers solutions in electrophoresis. Finally, the nascent field of nanospheres of gold and other materials as separation media is also reviewed.

Method development for the determination of iron in milligram amounts of rice plants (Oryza sativa L.) from cultivation experiments using graphite furnace atomic absorption spectrometry.

The amount of sample that is available for analysis in laboratory plant cultivation experiments is usually very limited. Highly sensitive analytical techniques are therefore required, even for elements that are present in the plants at mg g(-1) concentrations, and graphite furnace atomic absorption spectrometry (GFAAS) was chosen in this work because of its micro-sampling capability, and its relatively simple operation. Four micro-methods were investigated for the determination of iron in roots and leaves of rice plants: i) a micro-digestion with nitric and hydrochloric acids, ii) a slurry procedure using tetramethylammonium hydroxide (TMAH) tissue solubilizer, iii) a slurry prepared in 1.4 mol L(-1) nitric acid, and treated in an ultrasonic bath, and iv) the direct analysis of solid samples. The micro-digestion was suffering from high blank values and contamination problems, so that it could not be recommended for routine purposes. The TMAH method exhibited poor precision and occasional low recoveries, particularly for real samples. Direct solid sampling analysis gave results similar to those obtained with the slurry technique with ultrasonic agitation for the determination of iron in certified reference materials with iron content up to about 100 microg g(-1), but was too sensitive for the investigated rice plants, which had an iron content up to several mg g(-1). The slurry technique with ultrasonic treatment of the samples, suspended in dilute nitric acid, was finally adopted as the method of choice. The method was then applied for the determination of iron in the leaves and in different compartments of the roots of two rice cultivars, one sensitive to iron toxicity, an important nutritional disorder, and the other one resistant to iron toxicity. The results suggest that the higher resistance to iron toxicity of the second cultivar is due to a smaller uptake of iron from the soil, resulting in lower iron levels in all compartments of the plant.

The propagator (retarded Green function) formalism as a new calculation method to predict the time evolution of bands in capillary electrophoresis and microchannels.

Capillary electrophoresis (CE) and microchannel (MC) techniques are important tools in chemical and biological sciences, mainly in the study of genomes, transcriptomes, proteomes, metabolomes, and organic as well as inorganic ions. The speed of DNA sequencing increased significantly during the last decade with the use of capillary electrophoresis (CE). The time evolution of the bands' spatial profile inside capillaries and channels is of paramount importance, since the main goal of these techniques is to maximize resolution (the ratio between the spacing of the peaks and their mean standard deviations). In the present work, the propagator (retarded Green function) formalism is applied to solve a few problems which are typical for CE and MC. We also apply this mathematical method to the problem of velocity gradients along the capillary, i.e., the time evolution of the bands is analyzed when they enter regions where they migrate with different velocities.

Performance of an ultraviolet light-emitting diode-induced fluorescence detector in capillary electrophoresis.

The performance of a fluorescence detector in capillary electrophoresis (CE) using a light-emitting diode (LED) as excitation source is reported. An ultraviolet LED pulsed at a repetition rate of 500 Hz, combined with a time-discrimination and averaging acquisition system, was used. Limits of detection of 3 and 18 fmoles (at a signal-to-noise ratio equal to 3) were achieved for fluorescamine-derivatized bradykinin and lysine, respectively. This system exhibited a linear response for a concentration range between 54 and 417 microM for derivatized lysine, and between 1.81 and 23.58 microM for derivatized bradykinin. This detection system showed to be very convenient for routine analytical applications.