Retention and Efficiency in Frit-Inlet Asymmetrical Flow Field-Flow Fractionation

Abstract

Sample relaxation in the frit-inlet asymmetrical flow field-flow fractionation (FIA-FlFFF) channel is accomplished hydrodynamically. The focusing/relaxation procedure, which is essential to the efficient operation of conventional asymmetrical flow FFF, is therefore avoided. It follows that for FIA-FlFFF there is no requirement for valve switching to achieve convergent (focusing) channel flows, and sample relaxation and separation proceed without interruption. This represents a major advance in the facility of operation of asymmetrical flow FFF. In this work, we present the derivation of equations to describe the variation in mean channel flow velocity along the full length of the frit-inlet asymmetrical channel. Equations for channel void time (the time for a nonretained material to pass through the channel) are also presented. The equations are sufficiently general so as to be applicable to both rectangular and trapezoidal channel designs. On the basis of these equations, the retention time for a sample component can be calculated by applying basic flow FFF theory. Simple equations are also presented to determine conditions consistent with complete relaxation of samples within the frit-inlet region. Such conditions ensure maximization of separation efficiency during subsequent elution. The retention and resolving power of FIA-FlFFF are demonstrated using sets of standard latex spheres and proteins. Experimental data show that retention in FIA-FlFFF follows the general principles of field-flow fractionation, provided the variation in flow velocity is correctly accounted for. Finally, effects of sample load and injection volume are also discussed.