Determination of equilibrium constant of alkylbenzenes binding to bovine serum albumin by solid phase microextraction

Solid phase microextraction (SPME) coupled with GC has been applied to stud y the binding properties between bovine serum albumin (BSA) and volatile or ganic compounds such as benzene, toluene, ethylbenzene, propylbenzene and b utylbenzene. Their protein-ligand equilibrium constants have been determine d. The measurement of free and bound ligand concentrations in the aqueous s olution was based on the equilibrium among the analyte in the fiber coating (C-f), headspace (C-h) and aqueous solution (C-s). The work demonstrated t hat SPME is a simple and effective method in the study of protein binding t o measure the freely dissolved analyte concentration as well as the equilib rium constant. The theoretical aspect of the SPME applied to the equilibriu m constant measurement in two-phase (liquid sample-fiber coating) and three -phase (liquid sample-headspace-fiber coating) systems has been thoroughly discussed. The results demonstrated that the interpretation of the calibrat ion data is crucial to the determination of freely dissolved analyte concen tration and the equilibrium constant especially when the sample volume is s mall. The error in the experimental system is discussed. It is demonstrated in this study that for the three-phase system the amount of the analyte pa rtitioned in the headspace could be ignored only in certain circumstances, where the Henry's law constant and the ratio between headspace volume and s ample volume are sufficiently small.