Background
The purpose of this study was to evaluate calcium salts as privileged additives for the purification of highly polar ammonium and oligopeptides on normal-phase silica. Challenges in the purification of highly polar compounds are of particular importance as these motifs are commonly present in drug active pharmaceutical ingredients (APIs). Typically, when compounds become too polar for normal-phase silica gel, less general and much more expensive reverse-phase separation media is required. The ability to use normal-phase techniques to purify these active APIs would offer significant cost savings and allow for more rapid purification at pre-clinical scale.
Figure 1: HPLC UV chromatogram of compounds A, B, and C both individually and in a three-component mixture.
Approach
The separation of test compound mixtures and crude reaction mixtures in the presence of various calcium additives and solvent systems was evaluated at an analytical scale using high performance liquid chromatography (HPLC) (Figure 1), and at a preparative scale using thin-layer chromatography (TLC) and medium-pressure liquid chromatography (MPLC) (Figure 2).
Figure 2: A) TLC of crude HLö7 (left), purification using Biotage MPLC (middle), and TLC of collected fractions (right). B) Biotage UV chromatogram of collected column fractions.
Accomplishments
Calcium salts were found to be privileged additives which provided improved separations not seen with other cations. A variety of commonly available or easily prepared calcium salts were evaluated with varying levels of success. Calcium chloride and calcium trifluoroacetate were among the best in our analytical assay. We have identified new calcium additives and chromatography conditions which have improved upon previously discovered conditions. A workflow for rapid condition screening and preparative sample purifications has been established. This workflow was utilized for the purification of multiple highly polar ammonium and polyamine compounds which could not previously be purified by simple chromatographic methods. We also have preliminary results that indicate that ion assisted chromatography is appliable to the purification of peptides on normal phase silica which would be a major breakthrough in the purification of peptides. Future studies of ion assisted peptide purification are warranted.
Presentations
Calcium-Enhanced Chromatography for the Purification of Polar Ammonium and Peptide Compounds Travis Menard, William Cantrell, Andrey Malakhov, Paul Miguel, Shawn Blumberg To be presented at AAPS 2025 PharmSci 360 conference, San Antonio TX, November 11th
Patents
SwRI Invention Disclosure docket number 4313