Rapid breakthrough measurement of void volume for field-flow fractionation channels

A peak breakthrough technique is described and evaluated for measuring the void volume of field-flow fractionation (FFF) channels, particularly those used for flow FFF. This technique uses a high-molecular-mass macromolecular or particulate probe that can be displaced rapidly by flow through the FFF channel with minimal transverse diffusion. The particles that emerge first are those carried through the entire length near the channel centerline at the apex of the parabolic flow profile. These particles generate a sharp breakthrough profile. The measured breakthrough time is two thirds of the void time, thus making it possible to calculate both the void time and the associated void volume. This method, although applicable to all FFF channels (and capable of extension to open tubes), is particularly useful for flow FFF because conventional low-molecular-mass void probes can diffuse into the permeable walls and thus distort void measurements. The theoretical basis of the breakthrough technique and an explanation for the sharpness of the breakthrough front are given. A method for compensating for deviations from perfect sharpness is developed in which the breakthrough time is identified with the time needed to reach 85-88% of the breakthrough peak maximum. Preliminary experimental results are shown using various protein probes in four different FFF channel systems.