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

An AsFlFFF channel was designed and built, and then tested for analysis of pullulansand proteins. Pullulansand 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 pullulanwith 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 pullulansand proteins were not coincide, probably due to the difference in molecular conformation, suggesting the analysis of pullulansand proteins using AsFlFFF requires independent calibration. It was found that the linearity of the protein-calibration plot was improved by using a buffer solution as the carrier.