Determination of 9-fluorenylmethoxycarbonyl (Fmoc) resin loading in solid-phase synthesis by RP-HPLC internal standard method
Abstract
A simple RP-HPLC method based on an internal standard is proposed to determine the resin loading in solid-phase peptide synthesis. The method is applicable for the most common type of synthesis, where 9-fluorenylmethoxycarbonyl (Fmoc) is used as the protective group and piperidine as the deprotecting agent.
Both products of the deprotection reaction (dibenzofulvene and dibenzofulvene-piperidine adduct) are separated by RP-HPLC and quantified by a UV detector. Biphenyl serves as the internal standard for both analytes.
The resin loading is calculated using the equation resulting from the combination of internal standard method equations for each of the two analytes. The method provides true results, as evidenced by their comparison with the independent method.
The relative standard deviation of the method is 1.52%. A further advantage of the method is that it allows determination from the waste solution of the synthesis, and, therefore, can also be used for monitoring during multi-step synthesis.