The principle of inclined ultrafiltration, which can remove the filter cake continuously, was applied to the chiral separation of tryptophan enantiomers with bovine serum albumin (BSA) as the stereospecific macroligand by operating with the mode of single-pass using the hollow fiber membrane module vertically placed. The increase in the BSA concentration and the decrease in the racemic tryptophan concentration in the feed solution caused the increase in the separation factor of D-tryptophan, which preferentially permeated through the membrane, because of the strong stereospecific binding of the L-isomer to BSA at pH 7. The separation factor in affinity ultrafiltration was well evaluated based upon the Freundlich equation, in which the empirical constants were determined from the static binding experiments. The filtration rate was decreased with the BSA concentration, but was little influenced by the mass flow rate of the concentrate because BSA cake deposited on the membrane was efficiently removed by the gravitational effect even at the extremely low flow rate. Consequently, the decrease in the flow rate of the concentrate brought about the dramatic increase in the concentration ratio of BSA in the concentrate, serving the purpose of the performance increase of the next stage eluting the bound L-tryptophan from BSA. The concentration ratio of BSA was accurately estimated by considering the mass balance of BSA in the module at the steady state condition where the growth of the BSA cake on the membrane ceased. It is concluded that the method of single-pass ultrafiltration at the extremely low flow rate condition using the module placed vertically is distinctly efficient also in chiral separation by affinity ultrafiltration using the stereospecific macroligand. Additionally, the two-stage affinity ultrafiltration in which both the binding process of enantiomers to BSA and the elution process of the bound enantiomers were included was efficient for recovery of each tryptophan enantiomer. (C) 2008 Elsevier B.V. All rights reserved.