This paper reports the development and validation of an assay for the determination of acetonitrile in the recycled mobile phase using high performance liquid chromatography(HPLC). The method is based on that the retention in reversed-phase liquid chromatography increases with decreasing concentration of organic phase in the mobile phase. The natural logarithm of the capacity ratio for a given solute is linearly related to the volume fraction of the organic modifier in the mobile phase. For dimethylphthalate and diethylphthalate, the linearity range is 30%―60%, and for biphenyl and terphenyl, the range is 60%―95%. Precision values(RSD) were both <1% and the accuracy(RE) was in the range of ±1%. The assay was successfully applied to the determination of acetonitrile concentration of recycled mobile phase after the distillation of the column eluent in our laboratory.
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