Reversed phase liquid chromatography (RPLC) is a valuable analytical method for monitoring reaction progress and characterizing intermediates toward the synthesis of active pharmaceutical ingredients (APIs). Development of RPLC methods often demands extensive resources in addition to artistry. Herein, we present a case study for accelerated RPLC method development to monitor the synthesis of an isolated intermediate in the process to synthesize an API. Dramatic differences in hydrophobicity between the starting materials and product posed a significant challenge to efficient development. More importantly, the limiting starting material, SM-NH2, features a primary amine and does not contain a chromophore; while, the second component, SM-COOH, demonstrates greater UV activity, yet its ionizable carboxylic acid complicated retention. At the outset we selected a Waters ACQUITY HSS T3 column owing to its demonstrated retention of polar compounds; however, neither isocratic methods nor shallow gradients using conventional mobile phase additives afforded sufficient retention of SM-NH2. Therefore, we turned to heptafluorobutyric acid, an ion-pair reagent that could pair with the protonated form of our starting material, SM-NH3+, to form an electrically neutral species of increased hydrophobic character. With one challenge resolved, we observed a broad peak for SM-COOH with inadequate resolution from the product peak. We hypothesized that the equilibrium between SM-COOH and SM-COO– caused the peak broadening. Screening mobile phase pH demonstrated a suitable pH at which all peaks of interest were sharp and well-resolved. Finally, we were able to further improve peak shape by increasing column temperature.

Accelerated RPLC method development to separate highly polar molecules: A chromatography coming of age story

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