A simple and sensitive spectrophotometric method for the quantitative determination of solid supported amino groups

Abstract

A simple and sensitive method for the quantitative determination of free amino groups on solid support is described. This approach is a modification of Ngo's [(1986)] J. Biochem. Biophys. Methods 12, 349–354] method reported earlier. The method is based on the reaction of the solid support with an excess of 5′- O-(4, 4′-dimethoxytrityl)-thymidine-3′- O-(2, 4-dinitrophenyl) succinate (DTDS) in the presence of a catalytic amount of 4-dimethylaminopyridine. After removing the excess reagent, solid support is treated with perchloric acid to release 4, 4′-dimethoxytrityl cation into the solution. The released 4, 4′-dimethoxytrityl cation, which has a strong absorption at 498 nm ( ε 498 = 70 000), is then determined spectrophotometrically. A comparative study of DTDS, N-succinimidyl-3-(2-pyridyldithio)propionate and 4, 4′-dimethoxytrityl chloride is also included. The method was found to be very useful to determine those amino groups which are available for functionalization of solid supports, especially, monitoring the functionalization of solid supports for affinity chromatography and synthesis of biopolymers.