A method for the determination of the molar extinction coefficient of structure-linked chromophores

Synopsis This paper describes a general method for the determination of the molar extinction ceefficient of a chromophore covalently bound to structure-linked groups, without isolating the compound formed. The method is illustrated by the determination of the molar extinction coefficient of the reaction product of 2,4-dinitro-1-fluorobenzene (DNFB) with films of aminoethyl-cellulose (AE-cellulose). The method is based on the relation between the decrease in extinction of a DNFB staining solution and the increase in extinction of the AE-cellulose after staining as measured in a film-spectrophotometer. In addition, the value of the molar extinction coefficient was used in establishing reaction conditions for a quantitative staining procedure for determining amino groups with DNFB and picric acid. Conditions of optimum DNFB staining were determined and the measured extinction was converted into concentration of amino groups using the molar extinction coefficient. The amino group concentration of the same batch of AE-cellulose was also determined, after finding the optimum reaction conditions, by staining with picric acid. The results, when compared, showed a linear relationship with a slope of unity for batches of AE-cellulose of varying amino group concentrations. This is consistent with the same stoichiometry in both cases and indicates that in both procedures one chromophore molecule has reacted with one amino group and that this reaction has proceeded to completion. The general applicability of the method is discussed.