Retention in flow field-flow fractionation (flow FFF) is generally governed by the combination of crossflow and migration flowrates. Especially for an asymmetrical flow FFF (AF4) channel in which the channel-inlet flow is divided into crossflow and outflow, the separation of low-molecular-weight proteins or macromolecules requires a relatively high crossflow rate along with a very low outflow rate for a reasonable level of resolution, which often leads to a limitation in channel pressure. In this study, the performances of AF4 with increased channel thicknesses have been investigated by adjusting the effective channel flowrates in the asymmetrical channels according to the variation of channel thickness. Four AF4 channels of different channel thicknesses (350, 490, 600, and 740 μm) were employed to examine the potential usefulness of employing a thick channel in the high-resolution separation of low-molecular-weight proteins (< 100 kDa) and to determine the relationship between higher channel thickness and the recovery of elution. Experiments showed that the ratio of crossflow rate to the effective channel flowrate should be considered in the selection of a run condition at an increased channel thickness. The study also demonstrated that a thick AF4 channel can be useful for the high-resolution separation of low-molecular-weight species such as protein aggregates without using extremely high crossflow rates.
Keywords: Asymmetrical flow field-flow fractionation; Channel thickness effect; Protein separation; Resolution.
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