**9. Specificity**

The specificity of analytical methods is typically assessed by examining system interference with the detection and quantification of analytes. Part of this evaluation is the determination of protein recovery from the column (Rossi, Pacholec et al. 1986; Eberlein 1995). The recovery determination requires the knowledge of the extinction coefficient for the protein, which can be calculated from its amino acid composition (Pace, Vajdos et al. 1995) or determined experimentally. It should be noted that the extinction coefficient of a protein may change as a function of pH (Eberlein 1995; Kendrick, Chang et al. 1997). Therefore, direct comparison of the recovery in the neutral pH, size exclusion method with the recovery in an acidic reversed-phase separation may not be valid due to differences in the operating pHs of the methods. The difference may not necessarily reflect the actual recovery, but rather shows pH dependent changes of spectroscopic properties of the protein. With such an approach, the specificity of the method can be assessed in every assay, and reflects dynamically the change in status of consumables (columns and mobile phases) and hardware.
