Liquid Chromatography Evaluation of a Novel Graphitic Carbon Stationary Phase Using Glucagon-Like Peptide‑1 Analogs

Innovations in liquid chromatography stationary phase media can address emerging peptide separation and purification challenges. Herein, we report the synthesis of novel carbon microbeads from natural micrographite flakes as the starting material (referred to as All Carbon microbeads) and their performance as reversed-phase liquid chromatography stationary phase media using the glucagon-like peptide-1 (GLP-1) analogs semaglutide and liraglutide as probe analytes. High-performance liquid chromatography (HPLC) performance metrics were characterized and validated, including column efficiency measured by theoretical plate count (N), as well as linear response, precision, limit of detection (LOD), limit of quantitation (LOQ), and loading capacity. Commercially available silica C18 media, the current industry standard, were used as controls for comparison. The results indicated that HPLC columns packed with All Carbon microbeads consistently separate the GLP-1 analogs with retention times similar to those of the reference silica C18 columns. Their performance metrics are comparable to those of silica C18 in terms of plate count (N), LOD, LOQ, and loading capacity. Additionally, they perform better in the precision of retention time and linear response compared with silica C18. The chromatographic performance of All Carbon microbeads was stable in the presence of ion-pairing agents, extreme pHs (pH 1 and 13), salt concentrations, under field conditions (liraglutide crude), and with 100% aqueous loading conditions. The results present opportunities for further development as a sustainable reversed-phase media for GLP-1 analog analytical characterization and manufacturing.