Flow optimisations with increased channel thickness in asymmetrical flow field-flow fractionation
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.
Section snippets
Theory
The retention ratio, R, in FFF is defined as the ratio of channel void time, t°, to retention time, tr, and is simply expressed aswhere kT is the thermal energy, η is the viscosity of the carrier liquid, w is the channel thickness, U is the transverse velocity of sample components across the channel driven by an external field, and d is the particle diameter [1,2]. In the case of flow FFF, U becomes the transverse velocity of crossflow represented as where is the
Experimental
Eight protein standards were purchased from Sigma–Aldrich (St. Louis, MO, USA): ferredoxin (11 kDa), myoglobin (17 kDa), carbonic anhydrase (CA, 29 kDa), α-1 acid glycoprotein (AGP, 41 kDa) from human, ovalbumin (OVA, 43 kDa), bovine serum albumin (BSA, 66 kDa), transferrin (78 kDa), and alcohol dehydrogenase (AD, 150 kDa). An AF4 channel (model LC) from Wyatt Technology Europe GmbH (Dernbach, Germany) was utilised with the regenerated cellulose membrane (MWCO 10 kDa) from Merck Millipore
Results and discussion
Four different channel spacers with thicknesses of 350, 490, 600, and 740 μm were utilised in this study, and the actual thickness of each channel system was calculated as 320, 467, 571, and 702 μm, respectively, from the theory (Eq. 6) using the experimental retention time of BSA obtained at . Based on the calculated values of channel thickness, separation of the four protein standards (ferredoxin, CA, BSA and AD) was accomplished by decreasing the ratio according
Conclusion
In this study, the separation in an AF4 with thick channels was optimised by varying the effective channel flowrates in an asymmetrical channel. In the case of an AF4 channel system whose effective channel migration flowrate simultaneously increases with the crossflow, the ratio of crossflow rate to the effective channel flowrate should be considered for the selection of suitable conditions in order to achieve the desired resolution. To improve the resolution of separation, a decrease in the
Acknowledgement
This study was supported by the grant NRF-2018R1A2A1A05019794 from the National Research Foundation (NRF) of Korea.
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