Effect of Surface Adsorption on Temporal and Spatial Broadening in Micro Free Flow Electrophoresis

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 (mu FFE) separations. Surface adsorption does affect the time it takes an analyte to travel through the mu FFE separation channel and therefore contributes to temporal broadening. These results were confirmed using mu FFE 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 mu FFE 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.