Advances in frit-inlet and frit-outlet flow field-flow fractionation

Abstract

Flow field-flow fractionation (flow FFF), a method that provides direct measurement of particle size and diffusion coefficient, has been improved by the introduction of frit-inlet hydrodynamic relaxation and frit-outlet sample concentration operations. Using a combination frit-inlet/frit-outlet flow FFF channel, the overall system is dramatically simplified as the sample relaxation procedure is automated, the detector baseline interruption caused by flow disturbances in the conventional stopflow relaxation is completely eliminated, and the system detection sensitivity is considerably increased by enhancing the concentration of material in the channel. In this article, the general theory of flow FFF and the principles of frit-inlet relaxation and frit-outlet sample concentration are introduced. The system relaxation efficiency, sample recovery, detection sensitivity, and separation efficiency are described and discussed. Theoretical and experimental results are presented for protein components showing that the frit-inlet hydrodynamic relaxation can produce separation efficiency similar to that of the conventional stopflow operation. It is also shown that frit-outlet operation can improve the system detection sensitivity by more than a factor of 10 without losing resolution and sample recovery. Flow rate control and adjustment of the frit-inlet and frit-outlet flow FFF system are further simplified by using recycled carrier delivery loops. After demonstrating relaxation efficiency, sample concentration enhancement, and recovery, the system is applied successfully to the separation and characterization of proteins and lipoproteins. This new flow FFF system exhibits great ability for the separation of a wide variety of biological macromolecules with high resolution and sensitivity and for the accurate determination of hydrodynamic size and diffusion coefficient. © 1998 John Wiley & Sons, Inc. J Micro Sep10: 7–18, 1998