The Use of Single Drop Microextraction and Field Amplified Sample Injection for CZE Determination of Homocysteine Thiolactone in Urine

Two cheap, simple and reproducible methods for the electrophoretic determination of homocysteine thiolactone (HTL) in human urine have been developed and validated. The first method utilizes off-line single drop microextraction (SDME), whereas the second one uses off-line SDME in combination with field amplified sample injection (FASI). The off-line SDME protocol consists of the following steps: urine dilution with 0.2 mol/L, pH 8.2 phosphate buffer (1:2, v/v), chloroform addition, drop formation and extraction of HTL. The pre-concentration of HTL inside a separation capillary was performed by FASI. For sample separation, the 0.1 mol/L pH 4.75 phosphate buffer served as the background electrolyte, and HTL was detected at 240 nm. A standard fused-silica capillary (effective length 55.5 cm, 75 μm id) and a separation voltage of 21 kV (~99 μA) were used. Electrophoretic separation was completed within 7 min, whereas the LOD and LOQ for HTL were 0.04 and 0.1 μmol/L urine, respectively. The calibration curve in urine was linear in the range of 0.1–0.5 μmol/L, with R2 = 0.9991. The relative standard deviation of the points of the calibration curve varied from 2.4% to 14.9%. The intra- and inter-day precision and recovery were 6.4–10.2% (average 6.0% and 6.7%) and 94.9–102.7% (average 99.7% and 99.5%), respectively. The analytical procedure was successfully applied to the analysis of spiked urine samples obtained from apparently healthy volunteers.

Fine-Tuning Electrokinetic Injections Considering Nonlinear Electrokinetic Effects in Insulator-Based Devices

The manner of sample injection is critical in microscale electrokinetic (EK) separations, as the resolution of a separation greatly depends on sample quality and how the sample is introduced into the system. There is a significant wealth of knowledge on the development of EK injection methodologies that range from simple and straightforward approaches to sophisticated schemes. The present study focused on the development of optimized EK sample injection schemes for direct current insulator-based EK (DC-iEK) systems. These are microchannels that contain arrays of insulating structures; the presence of these structures creates a nonuniform electric field distribution when a potential is applied, resulting in enhanced nonlinear EK effects. Recently, it was reported that the nonlinear EK effect of electrophoresis of the second kind plays a major role in particle migration in DC-iEK systems. This study presents a methodology for designing EK sample injection schemes that consider the nonlinear EK effects exerted on the particles being injected. Mathematical modeling with COMSOL Multiphysics was employed to identify proper voltages to be used during the EK injection process. Then, a T-microchannel with insulating posts was employed to experimentally perform EK injection and separate a sample containing two types of similar polystyrene particles. The quality of the EK injections was assessed by comparing the resolution (Rs) and number of plates (N) of the experimental particle separations. The findings of this study establish the importance of considering nonlinear EK effects when planning for successful EK injection schemes.

MÉTODOS DE INJEÇÃO DA AMOSTRA EM MICROSSISTEMAS ELETROFORÉTICOS

SAMPLE INJECTION METHODS IN ELECTROPHORETIC MICROSYSTEMS. Electrophoresis is by far the most popular separation method implemented in microscale, most probably due to its instrumental simplicity, low cost and portability. Due to the increasing use of miniaturized electrophoretic systems, the study of fundamental aspects can improve the development of methodologies for several applications. One of the major challenges related to electrophoresis chips refers to the sample injection mode, in this way, this study presents a review on sample injection methods for microchip electrophoresis covering electrokinetic and hydrodynamic approaches, describing theoretic and instrumental aspects. Since the sample volume affects the analytical performance, the precise and reproducible control of the sample amount to be introduced into the separation channel is highly desirable to ensure reliable chemical analysis. Electrokinetic modes based on floating, pinched and gated protocols are presented and discussed providing an overview about the electrokinetic control of the sample through three methodologies. In the same way, hydrodynamic techniques including the use of microfabricated valves and pumps, syringe pumps, electronic micropipettes, rubber suction bulb and acupuncture needle are approached, thus expanding the view of hydrodynamic injectors based on classical and alternative methods.