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High-Resolution Charge-Based Electrokinetic Separation of Almost Identical Microparticles.

Well-established techniques, e.g., chromatography and capillary electrophoresis, are available for separating nanosized particles, such as proteins. However, similar techniques for separating micron-sized particles are still needed. Insulator-based electrokinetic (iEK) systems can achieve efficient microparticle separations by combining linear and nonlinear EK phenomena. Of particular interest are charge-based separations, which could be employed for separating similar microorganisms, such as bacterial cells of the same size, same genus, or same strain. Several groups have reported charge-based separations of microparticles where a zeta potential difference of at least 40 mV between the microparticles was required. The present work pushes the limit of the discriminatory capabilities of iEK systems by reporting the charged-based separation of two microparticles of the same size (5.1 μm), same shape, same substrate material, and with a small difference in particle zeta potentials of only 3.6 mV, which is less than 10% of the difference in previous studies. By building an accurate COMSOL Multiphysics model, which correctly accounts for dielectrophoresis and electrophoresis of the second kind, it was possible to identify the conditions to achieve this challenging separation. Furthermore, the COMSOL model allowed predicting particle retention times (tR,p) which were compared with experimental values (tR,e). The separations results had excellent reproducibility in terms of tR,e with variations of only 9% and 11% between repetitions. These findings demonstrate that, by following a robust protocol that involves modeling and experimental work, it is possible to discriminate between highly similar particles, with much smaller differences in electrical charge than previously reported.