In our study, we observed that higher column temperatures lead to shorter ON retention times and smaller peak widths (Table ). The latter can possibly be explained by decreased diastereomer separation at higher temperatures, and/or an increase in mass transfer kinetics. The separation of the impurities from the FLP also improved at higher temperatures. At 5 °C, the N-1 and DA impurities coeluted with the FLP, while a resolution of approx. 1 was achieved for the N+1 and PO impurity with respect to the FLP. In contrast at 80 °C, resolutions of 1.5 or higher from the FLP were obtained for the N-1, N+1, DA, and PO impurities. Interestingly, the retention order of the N+1 and PO impurities swapped when the temperature was above 45 °C, where the resolution of the PO impurity and FLP increased less than the N+1-FLP resolution when the temperature was raised. The steeper increase in resolution of N+1 from FLP as compared to PO might be related to the conformational differences of PO at low temperatures that are lost or reduced at high temperatures and/or perhaps different temperature dependencies of the pKa values of PO and PS. A column temperature of 80 °C was chosen as optimal due to the overall highest separation resolution of each impurity from the FLP.