Needle in a Hay Stack: Novel Approaches for Peptide Analyses Using Traditional and Innovative Reversed-Phase Chromatography

Available on demand

The ability to effectively separate peptides at substantially different concentrations is important in applications ranging from the characterization and quality control of biotherapeutic proteins to the discovery of biomarkers or surrogate peptides. Consequently, chromatographers continue to search for easy to implement approaches that address concerns of peptide retentivity and peak shape for various LC and LC-MS applications.

Historically, peptide separations have been performed using shallow gradients of increasing acetonitrile concentration with eluents containing strong ion pairing reagents such as 0.1% trifluoroacetic acid (TFA). However, while the use of 0.1% TFA addresses retentivity and peak shape concerns, its deleterious effect on MS-signal suppression frequently limits its use in this data rich application.

As protein digests can contain hundreds of species of various sizes, charge profiles, and concentrations, this webinar begins by comparing and contrasting different C18 phases in TFA and formic acid (FA) containing eluents. Data will be shown revealing the effective use of an innovative, Charged Surface Hybrid (CSH™) reversed-phase column offering whose performance is less dependent of the ion-pairing reagent used, making it possible to detect that “needle in a haystack” in the LC-MS analysis of a primary biotherapeutic compound and its undesired contaminants.

In this webinar, Stephan M. Koza PhD, Principal Applications Chemist, and Dr Brooke M. Koshel, Senior Scientist, at Waters Corporation, will discuss:

• How to take advantage of selectivity and retentivity differences offered by different reversed-phase column chemistries.
• Novel C18 columns that offer excellent peak shape with TFA and FA mobile phases, making it highly compatible with both optical as well as LC-MS applications.
• The importance of column loadability for accurate detection and quantitation of low abundant peptide species.
• The benefits of incorporating mass detection into a routine peptide monitoring workflow to enhance decision-making information of gathered LC-MS data.