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

Application of sub‐/supercritical fluid chromatography for the fingerprinting of a complex therapeutic peptide

“…The amino-functionalized stationary phases (Figure 2A,B) showed an inferior selectivity resulting in an elution in a smaller Rt window, which disqualified these columns for further optimization steps. Much higher retention was observed on the zwitterionic than on the diol functionality, resulting in broader peaks, which is in line with the results reported in other studies earlier for other peptides [24]. Due to the higher retention, a better resolution of the peaks of the linear group was achieved, which on the other hand, caused a worse separation of the cyclic peptides.…”

“…The carbonate ions were proposed to cause a chaotropic effect enhancing the chromatographic separation of biomolecules [21]. Recently methanesulfonic acid (MSA) became a popular additive [22,24], even if its successful usage was already reported in 1999 [13]. Its high acidity and potential to mask amino functionalities of peptides were hypothesized to enable separation of the same by SFC through synergistic effects if combined with amino derivatized stationary phases [24].…”

A story of peptides, lipophilicity and chromatography – back and forth in time

“…100 Possible additives are TFA, methanesulfonic acid, ammonia acetate, ammonium hydroxide or iso-propylamine. 106,107 Due to its limited miscibility with CO 2 , water is not preferred as a modifier but was shown to improve the separation of uncapped charged 12mer peptides nevertheless. 108 TFA or the combination of TFA and ammonia as additives, each 0.1%, were shown to improve peak shapes of small macrocyclic peptides as well as of basic and acidic peptides up to 40 amino acids on various columns.…”

Sub/Supercritical Fluid Chromatography Versus Liquid Chromatography for Peptide Analysis

“…Additional advantages of UHPSFC derive from its easiness to be hyphenated to several detectors, such as ultraviolet (UV) and mass spectrometers (MS), demonstrating comparable performance to those of UHPLC-UV-MS [ 25 , 26 ]. More recently, a comparison of UHPSFC vs UHPLC in reversed-phase mode has been reported, together with an assessment of an unorthodox additive (methanesulfonic acid, MSA) under UHPSFC conditions, using a set of synthetic and commercially available peptides [25] . The authors concluded that UHPSFC offers an interesting complementarity in the separation profile to RPLC, while generating comparable chromatographic performance (e.g., separation efficiency and peak shape, etc.…”