For the resolution of isomer-rich lipid extracts, it is common practice during RPLC method development to

evaluate column efficiency and mobile phase composition. However, further gains can be made by tuning other

parameters, such as the microstructure of the packing material. Today, lipid separations are mostly performed

with columns packed with particles having narrow pore sizes (80–120 Å). This is unsurprising, given the ex-

pected hydrodynamic radius of lipids up to of 23 Å. However, specific investigations of pore size influence on

lipid separations are scarce. Therefore, a systematic comparison of 150 mm columns packed with C18 particles

having pore sizes of 130 and 300 Å was performed with respect to selectivity, peak capacity and chromato-

graphic resolution. An RPLC/HRMS-based lipidomic analysis was developed on 150 mm columns. Similar

loadability, selectivity, sensitivity and average peak capacity were demonstrated by 300 Å column compared to

its narrow-pore counterpart. Overall, current experimental results indicate that wide-pore columns are equally

applicable for RPLC/HRMS-based discovery lipidomics and may be even beneficial for the analysis of lipids with

high structural complexity. In addition, these columns appear to be better suitable for high-throughput analysis

due to lower lipid retention via smaller surface area.