Automated frit inlet/frit outlet flow field-flow fractionation for protein characterization with emphasis on...

A flow field-flow fractionation (FFF) unit fitted with a 254-nm spacer, frit inlet (FI), and frit outlet (FO) was automated for protein analysis by addition of a system controller, autosampler, and computer software to control pumps, sample loading, and data capture. Standard molecular size marker proteins and polymeric wheat storage protein extracts were used to assess the performance of the automated unit. Optimum resolution for these proteins was obtained with a sample inlet flow of 0.2 mL/min, a frit inlet flow (recirculating) of 1.4 mL/min, and a cross-flow (recirculating) of 5 mL/min using 0.05 M acetic acid containing 0.002% FL-70 as a carrier. Use of the FIFO FFF eliminates the requirement for stop-flow relaxation and pressure balancing, results in better reproducibility, and generates a 7-10-fold increase in sensitivity at the detector by concentrating fractions eluting from the channel. These improvements resulted in superior resolution of polymeric wheat protein fractions compared to those obtained previously using a standard channel with manual load and stop-flow relaxation, allowing accurate integration of peak or size range areas. Automation of this system allows unattended sample fractionation and hence markedly increases potential for sample throughput.