Flow optimisations with increased channel thickness in asymmetrical flow field-flow fractionation

Highlights

• Usefulness of increased channel thicknesses in asymmetrical flow FFF was investigated.

• Ratio of crossflow to effective channel flow rates should be considered in selecting run conditions.

• A thick AF4 channel is useful to improve resolution without increasing crossflow rate.

Abstract

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.

Introduction

Flow field-flow fractionation (flow FFF), a variant of the FFF family, is an elution-based separation method capable of fractionating particles and macromolecules by size [[1], [2], [3], [4]]. Separation in flow FFF is carried out in a thin empty channel space without a stationary phase by applying two flow streams moving perpendicular to each other: a channel flow, which drives the sample components towards the end of a channel, and a crossflow, which moves across the channel wall to force the migrating sample components towards the accumulation wall. As the flow profile in a thin channel becomes parabolic, where the flow velocity becomes the lowest near the channel wall and maximum at the centre across the channel thickness, particles or macromolecules with a small diameter protrude further away from the wall as their diffusion is faster than that of components with large diameters. Therefore, the smaller components elute earlier than the larger ones. As flow FFF can utilise aqueous solutions including a biological buffer as the carrier liquid, it has been applied to various biological materials including proteins and protein aggregates [5,6], DNA [7], microRNA [8], plasma lipoproteins [9], exosomes [10,11], subcellular species [12], cells [13,14], virus-like particles [15], and water-soluble polymers [16,17].

Retention in flow FFF is affected by two important parameters: the rates of crossflow (external field strength) and channel flow [18], once other experimental factors such as the compatibility of sample components with the carrier solution and the type of channel membrane materials are assured. In a symmetrical flow FFF channel, which utilises two permeable frits at both channel walls (depletion and accumulation walls), channel flowrate (axial flowrate) remains the same throughout the channel and is equal to the outflow rate; therefore, the retention time is determined by the ratio of outflow rate to crossflow rate for a given channel thickness. Generally, the separation resolution can be improved by increasing crossflow rate, however it results in the increase in the analysis time. Consequently, a simultaneous increase in crossflow and outflow rates leads to a speedy analysis without loss of resolution. However, an increase in both flowrates often leads to a limitation in the channel pressure, which may lead to a leaking problem. In the case of an asymmetrical flow FFF (AF4) channel, only one permeable frit is utilised at the accumulation wall by replacing the permeable depletion wall with a solid impermeable block. Therefore, part of the flow entering the channel inlet is divided into crossflow and the remaining exits as outflow, and hence, the transport of the carrier liquid or channel flow is reduced along the channel axis. As a simple increase in the crossflow rate of AF4 accompanies the simultaneous increase in the axial flow along the channel axis, the outflow rate should be adjusted to be as low as possible (∼ a few tenths of 1 mL/min) with a sufficiently high crossflow rate to obtain a reasonable level of resolution for separation. Typically, for the separation of low-MW (MW: molecular weight) species such as proteins, a channel spacer with a reduced thickness (< 250 μm) offers high-resolution separation by using a high crossflow rate. Experimentally, it requires a careful selection of both crossflow and outflow rates. To increase the separation resolution of sample components with low MW, a very high rate of channel inlet flow should be introduced to maintain a high crossflow rate, which incurs the limitation of both channel pressure and pump system.

This study investigated ways to maintain or improve the resolution of separation in AF4 channels by increasing the channel thickness while examining the roles of crossflow rate and effective channel flowrate. AF4 channels of four different thicknesses (350, 490, 600, and 740 μm) were employed to examine the potential use of a thick channel for protein separation with an improved resolution instead of using a thin channel, which normally requires a high channel inlet flowrate. Focused were on the optimisation of flowrate conditions in AF4 by varying the channel thickness and the investigation of the peak recovery in channels of increased thicknesses at different field strengths. This study also demonstrated the usefulness of employing a thick channel in AF4 for the high-resolution separation of protein aggregates.