Comparison of microcapillary column length and inner diameter investigated with gradient analysis of lipids by ultrahigh‐pressure liquid chromatography‐mass spectrometry

Biological samples in lipidomic studies can consist of extremely complex mixtures due to the diverse range of species and isomerism. Herein, highly efficient, in‐house packed microcapillary columns introduce the potential to better separate these complex mixtures. We compared the effects of changing column length (15, 30, and 60 cm) and inner diameter (75 and 100 μm) on lipid separation efficiency by reversed‐phase gradient analysis using ultrahigh‐pressure liquid chromatography coupled to mass spectrometry with operating pressures ranging from 450 to 2200 bar. Seven lipid standards composed of phosphatidylcholine and triacylglycerol species were analyzed at four different gradient rates to calculate conditional peak capacity. The longest column, 60 cm, at the shallowest gradient of 2% gave the highest peak capacity of 359 with a separation window of 2 h. The intermediate column length of 30 cm with 75 μm inner diameter provided a peak capacity of 287 with a separation window of 1 h. There was no significant difference in peak capacity between 75 and 100 μm inner diameter columns. This study showed that using highly efficient microcapillary columns increased peak capacity and resolution of lipids, and thus, this technique seems promising for enhancing lipid coverage and enabling better discovery of lipid biomarkers.