Gradient Retention Factor Concept Applied to Method Development for Peptide Analysis by Means of RP-HPLC

Abstract

Using the van Deemter model, the efficiency of three stationary phase systems in the analysis of a mixture of synthetic peptides was evaluated: (i) monolithic, (ii) packed, and (iii) core−shell columns, and it was shown that the efficiency of the monolithic column is superior to the others, specifically using it, the lowest values of H min (0.03 and 0.1 mm) were obtained, and additionally its efficiency was not significantly affected by increasing the flow. Using the concept of the gradient retention factor (k*), a method for chromatographic separation of a peptide complex mixture was designed, implemented, and optimized and then transferred from a packed column to a monolithic one. The results showed that it was possible to separate all components of the mixture using both evaluated columns; moreover, the analysis time was reduced from 70 to 10 min, conserving the critical pair resolution (1.4), by the transfer method using the k* concept. The method developed was tested against a mixture of doping peptides, showing that this method is efficient for separating peptides of various natures. This investigation is very useful for the development of methods for the analysis of complex peptide mixtures since it provides a systematic approach that can be extrapolated to different types of columns and instrumentation.

An Automated Purification Workflow Coupled with Material-Sparing High-Throughput 1H NMR for Parallel Medicinal Chemistry

“…Typical analytical techniques such as UPLC can be coupled to ultraviolet (UV), charged aerosol detection (CAD), or evaporative light scattering (ELS) detectors indicating compound purity, quantifying the recovery of the compound, and measuring hydrophobicity, respectively. − When coupled to mass spectrometry (MS), these techniques provide molecular mass information but lack structure-based details essential to identify structures without ambiguity, especially when the potential for multiple products exists. In addition, isomers such as epimers, regioisomers, or atropisomers cannot be discriminated .…”