Capillary and Microfluidic Gradient Elution Isotachophoresis Coupled to Capillary Zone Electrophoresis for Femtomolar Amino Acid Detection Limits

In this work, gradient elution isotachophoresis was combined with capillary zone electrophoresis (GEITP-CZE) in a single microcolumn. The multistage approach addresses the issues of analyte resolution difficulties in GEITP, as well as poor concentration sensitivity in CZE. GEITP employs rapid electrophoretic focusing at a discontinuous ionic interface within a sample well generated through combined electroosmotic and hydrodynamic flows. The interface and enriched analytes are then pulled into a capillary or microchannel as the counter-flow is reduced for on-column detection. To transform GEITP-focused samples to CZE-based separation, the sample solution is replaced with CZE buffer solution while maintaining hydrodynamic flow to ensure migration toward the detector. The single solution switch and lack of polarity inversion allows for reproducible separations (typically < 6% relative standard deviation in peak heights and < 0.5% in migration times). Low-pressure hydrodynamic flow during CZE allowed for flexible resolution adjustment, with a linear increase versus the square root of migration time, without altering the separation column, field strength, or electrolyte system. As a first demonstration of the applicability of GEITP-CZE, a series of amino acids to be assayed for in future Mars exploration missions as indicators of biological life were studied. Separation of six amino acids, with limits of detection as low as 200 fM, were achieved using a capillary format with a total analysis time of 11 min. A glass-based microfluidic implementation is also demonstrated that can perform GEITP-CZE in 1 cm effective lengths.