Elution Behavior of Protein and Pullulan in Asymmetrical Flow Field-flow Fractionation(AsFIFFF)

摘要

An AsFIFFF channel was designed and built,and then tested for analysis of pullulans and proteins.Pullulans and proteins having various nominal molecular weights were injected at various conditions of the cross-flow rate(F_c)and the channel-out flow rate(F_(out)).The retention(measured by the retention ratio R)and the zone broadening(measured by the plate height H)were measured,and then compared with theory.When the incoming flow rate,F_(in)(and thus F_(out))was varied with F_c fixed at 2.5 mL/min,the plate height measured for the pullulan with nominal molecular weight(M)of about 100,000 showed the trend expected by the"longitudinal diffusion"theory(H decreases with increasing flow rate).In contrast,when F_(out)was varied with the flow rate ratio,F_c/F_(out),fixed constant at 5,the plate height measured for the same sample showed the trend expected from the"non-equilibrium"theory(H increases with increasing flow rate).Calibration plots(log D vs.log M)obtained with pullulans and proteins were not coincide,probably due to the difference in molecular conformation,suggesting the analysis of pullulans and proteins using AsFIFFF requires independent calibration.It was found that the linearity of the protein-calibration plot was improved by using a buffer solution as the carrier.