Expanding the range of sub/supercritical fluid chromatography: Advantageous use of methanesulfonic acid in water-rich modifiers for peptide analysis

The aim of this work was to expand the applicability range of UHPSFC to series of synthetic and commercialized peptides. Initially, a screening of different column chemistries available for UHPSFC analysis was performed, in combination with additives of either basic or acidic nature. The combination of an acidic additive (13 mM TFA) with a basic stationary phase (Torus DEA and 2-PIC) was found to be the best for a series of six synthetic peptides possessing either acidic, neutral or basic isoelectric points. Secondly, methanesulfonic acid (MSA) was evaluated as a potential replacement for TFA. Due to its stronger acidity, MSA gave better performance than TFA at the same concentration level. Furthermore, the use of reduced percentages of MSA, such as 8 mM, yielded similar results to those observed with 15 mM of MSA. The optimized UHPSFC method was, then, used to compare the performance of UHPSFC against RP-UHPLC for peptides with different pI and with increasing peptide chain length. UHPSFC was found to give a slightly better separation of the peptides according to their pI values, in few cases orthogonal to that observed in UHPLC. On the other hand, UHPSFC produced a much better separation of peptides with an increased amino acidic chain compared to UHPLC. Subsequently, UHPSFC-MS was systematically compared to UHPLC-MS using a set of linear and cyclic peptides commercially available. The optimized UHPSFC method was able to generate at least similar, and in some cases even better performance to UHPLC with the advantage of providing complementary information to that given by UHPLC analysis. Finally, the analytical UHPSFC method was transferred to a semipreparative scale using a proprietary cyclic peptide, demonstrating excellent purity and high yield in less than 15 min.