No abstract

Analyte adsorption onto surfaces presents a challenge for many separations, often becoming a significant source of peak broadening and asymmetry. We have shown that surface adsorption has no effect on peak position or spatial broadening in micro free flow electrophoresis (μFFE) separations. Surface adsorption does affect the time it takes an analyte to travel through the μFFE separation channel and therefore contributes to temporal broadening. These results were confirmed using μFFE separations of fluorescein, rhodamine 110, and rhodamine 123 in a low ionic strength buffer to promote surface adsorption. Peak widths and asymmetries were measured in both the temporal and spatial dimensions. Under these conditions rhodamine 123 exhibited significant interactions with the separation channel surface, causing increased peak broadening and asymmetry in the temporal dimension. Broadening or asymmetry in the spatial dimension was not significantly different than that of fluorescein, which did not interact with the capillary surface. The effect of strong surface interactions was assessed using μFFE separations of Chromeo P503 labeled myoglobin and cytochrome c. Myoglobin and cytochrome c were well resolved and gave rise to symmetrical peaks in the spatial dimension even under conditions where permanent adsorption onto the separation channel surface occurred.

We have developed a high-throughput microdialysis-capillary electrophoresis (MD-CE) assay for monitoring branched chain amino acid (BCAA) uptake/release dynamics in 3T3-L1 cells. BCAAs (i.e., isoleucine, leucine, and valine) and their downstream metabolites (i.e., alanine, glutamine, and glutamate) are important indicators of adipocyte lipogenesis. To perform an analysis, amino acids were sampled using microdialysis, fluorescently labeled in an online reaction, separated using CE, and detected using laser-induced fluorescence (LIF) in a sheath flow cuvette. Separation conditions were optimized for the resolution of the BCAAs isoleucine, leucine, and valine, as well as 13 other amino acids, including ornithine, alanine, glutamine, and glutamate. CE separations were performed in <30 s, and the temporal resolution of the online MD-CE assay was <60 s. Limits of detection (LOD) were 400, 200, and 100 nM for isoleucine, leucine, and valine, respectively. MD-CE dramatically improved throughput in comparison to traditional offline CE methods, allowing 8 replicates of 15 samples (i.e., 120 analyses) to be assayed in <120 min. The MD-CE assay was used to assess the metabolism dynamics of 3T3-L1 cells over time, confirming the utility of the assay.