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

Area of Science:

• Analytical Chemistry
• Separation Science
• Biophysical Chemistry

Background:

• Retention in flow field-flow fractionation (flow FFF) depends on crossflow and migration flow rates.
• Asymmetrical flow FFF (AF4) requires high crossflow and low outflow for separating low-molecular-weight species, often limited by channel pressure.

Purpose of the Study:

• Investigate AF4 performance with increased channel thicknesses.
• Evaluate the utility of thicker AF4 channels for high-resolution separation of low-molecular-weight proteins (< 100 kDa).
• Determine the relationship between channel thickness and elution recovery.

Main Methods:

• Utilized four AF4 channels with varying thicknesses (350, 490, 600, and 740 μm).
• Adjusted effective channel flowrates in asymmetrical channels based on thickness variations.
• Examined the impact of channel thickness on separation resolution and elution recovery.

Main Results:

• The ratio of crossflow rate to effective channel flowrate is crucial for optimizing run conditions in thicker channels.
• Increased channel thickness in AF4 enables high-resolution separation of low-molecular-weight species, including protein aggregates.
• Thicker AF4 channels mitigate the need for excessively high crossflow rates.

Conclusions:

• Thicker AF4 channels offer a viable strategy for high-resolution separation of low-molecular-weight proteins and aggregates.
• Optimizing the crossflow to effective channel flowrate ratio is key for thicker AF4 systems.
• This approach overcomes pressure limitations associated with traditional AF4 methods for small biomolecules.