Sub Topic | Secondary Topic: Analytical Methodology | HPLC (Biopharmaceutical Molecule)
Authors: Ken Tseng, Nacalai USA Inc. (Main Author, Presenting Author); Toshi Ono, Nacalai USA Inc.; Tsunehisa Hirose, Nacalai USA Inc.
Presenting Author: Ken Tseng
Purpose: As pentafluorophenyl (PFP) and pentabromobenzyl (PBr) phases become popular in providing alternate selectivity to C18, it is imperative that we understand their separation mechanisms for better method development. In this poster, we compared 9 compounds on three PFP and one PBr core-shell HPLC columns. Both PFP and PBr phases are mixed-mode columns with p-p, dipole-dipole, dispersion (dipole-induced dipole), and ion-exchange interactions.
Methods: 9 small molecule compounds were divided into basic, neutral, and acidic groups. The retention time vs. % acetonitrile were plotted to show selectivity differences among compound classes and columns. A: 0.1% formic acid in water B: 0.1% formic acid in acetonitrile 0.4mL/min, 40 deg-C, UV 254nm
Results: All PFP columns exhibited the characteristic U-shape with positively-charged compounds. This is caused by dual separation modes of reversed-phase and ion-exchange in PFP. However, the retention time is markedly different for each brand. The peak shape in PFP ion-exchange mode is very broad and tailing, making the analysis in this region impractical. PBr lacks such ion-exchange mechanism, rather the reversed-phase interaction dominates, making method development straight forward. PBr retains non-charged compounds stronger than PFP in reversed-phase. It is, therefore, possible to retain very polar compounds in 100% aqueous mobile phase with PBr. All three PFP columns exhibit similar selectivity for non-charged compounds.
Conclusion: - For positively-charged compounds, PFP columns exhibited both reversed-phase and ion-exchange separation, although each brand retains them very differently. The peak shape in PFP ion-exchange mode is very broad and tailing, making the analysis in this region impractical. PBr showed mainly reversed-phase separation with little or no ion-exchange. PBr retains less of positively-charged compounds compare to PFP. - For non-charged compounds, all three PFP columns produced almost identical retention. PBr showed consistently higher retention compare to PFP, sometimes significantly. - Because PBr retains non-charged compounds much stronger than PFP and C18, it can be used for polar molecule analysis in reversed-phase mode, in high aqueous condition even in 100% water. This means PBr can compliment or replace HILIC for very polar compound analysis. - PBr can be useful in preparative-scale purification of polar molecules by taking advantage of high solubility, consequently high sample-loading capacity, in aqueous solvent. - PBr is great for mixture profiling analysis, e.g., natural product and cannabinoid analyses.
See attached abstract pdf for images.