Effect of the type of commercially available mixed-mode stationary phases on the retention of pharmaceutically important acidic and neutral compounds using different compositions of mobile phase

Grybinik, Sofiya; Bosáková, Zuzana

doi:10.1007/s00706-022-02960-6

Cited by 2 publications

(1 citation statement)

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“…Therefore, both "hydrophobic interaction" between the analyte and C18 ligand, and electrostatic interaction between the analyte and positively charged moiety of the stationary phase contribute to the overall retention [15]. Whether it will be electrostatic attraction or repulsion is determined mainly by the pK a of the functional groups of the analytes and by the pH of the aqueous part of the mobile phase [16]. Hence, it is clear that mixed-mode chromatography provides an increased number of tunable mobile phase parameters, which makes the method development more flexible, but also complex [17,18].…”

Section: Introductionmentioning

confidence: 99%

The benefits of mixed-mode chromatography columns for separation of peptides and protein digests

Kadlecová,

Boudová,

Kalíková

2023

Monatsh Chem

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In this work, the evaluation and comparison of mixed-mode chromatography and reversed-phase chromatography for separation of peptides and protein digests have been performed. The effects of pH of aqueous part of mobile phase as well as the effects of organic modifier on retention, resolution, and peak shape were investigated on several columns including three mixed-mode columns possessing reversed-phase/anion-exchange mechanism, two reversed-phase octadecyl columns, and one column with mixed-mode reversed-phase/anion-exchange character only in defined pH range. The set of peptides varying in their polarity, length, amino acid sequence, and charge state, namely dipeptides, N-blocked dipeptides, and oligopeptides, was selected to describe the chromatographic behavior under different conditions properly. These measurements showed the potential of mixed-mode chromatography columns for analysis of differently charged peptides in a single run. The applicability of the tested conditions has been verified by the analysis of cytochrome C digested fragments. Two types of samples were analyzed and compared, i.e., commercial cytochrome C digested standard and cytochrome C digested via trypsin spin columns. The obtained results point to the necessity of using mass spectrometry detection because of large number of unknown peaks in cytochrome C digested standard, probably originating from chymotryptic and miscleavage activities. Graphical abstract

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