Recovery, overloading, and protein interactions in asymmetrical flow field-flow fractionation

Area of Science:

• Analytical Chemistry
• Separation Science
• Biochemistry

Background:

• Quantitative results in asymmetrical flow field-flow fractionation (AF4) depend on mass recovery and overloading.
• Understanding these factors is crucial for accurate protein analysis.

Purpose of the Study:

• To systematically investigate mass recovery and overloading effects in AF4 for globular proteins.
• To evaluate the influence of membrane chemistry and protein properties on AF4 performance.

Main Methods:

• Systematic AF4 study using five globular proteins (36.7–669 kDa) and two membrane types (regenerated cellulose and polyethersulfone).
• Analysis of adsorption and mass overloading effects under standard phosphate-buffered saline (PBS) conditions.
• Derivation of theoretical equations to explain protein concentration-dependent migration velocity due to non-ideal viscosity.

Main Results:

• Actual molecular weight cutoff (MWCO) exceeded nominal values and varied by membrane chemistry.
• Membrane adsorption depended on chemistry (lower for RC than PES) but not protein type.
• Mass overloading effects were more pronounced for gamma-globulin, linked to local viscosity changes.

Conclusions:

• Membrane chemistry significantly influences adsorption in AF4.
• Protein properties, particularly those affecting local viscosity, are key to understanding overloading phenomena.
• Developed theoretical models provide insights into non-ideal viscosity effects on protein migration velocity in AF4.