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.

Graphic abstract

中文翻译：

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RP-HPLC 内标法测定固相合成中 9-芴基甲氧基羰基 (Fmoc) 树脂的负载量

提出了一种基于内标的简单 RP-HPLC 方法来确定固相肽合成中的树脂负载。该方法适用于最常见的合成类型，其中 9-芴甲氧羰基 (Fmoc) 用作保护基团，哌啶用作脱保护剂。脱保护反应的两种产物（二苯并富烯和二苯并富烯-哌啶加合物）通过 RP-HPLC 分离并通过 UV 检测器进行定量。联苯作为两种分析物的内标。使用由两种分析物各自的内标方法方程组合得到的方程计算树脂负载量。该方法提供了真实的结果，与独立方法的比较证明了这一点。该方法的相对标准偏差为1.52%。

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