Non-porous thin dense layer coating: Key to achieving ultrahigh peak capacities using narrow open tubular columns

Abstract

Chromatographic theory has predicted that narrow open tubular (OT) columns are essential toward achieving ultrahigh-efficiency liquid chromatographic separations. It is also well-recognized that low loadability is a major issue for capitalizing the benefits of open tubular liquid chromatography (OTLC). To mitigate this issue, researchers have tried to increase surface area of or create a thick porous coating on the column wall. In this work, we hypothesize that a thin dense non-porous coating is well-suited for us to achieve ultrahigh efficiency or ultrahigh peak capacity if the inner diameter of the OT column is small (e.g., 1−2 µm). We first discuss the rationale of this hypothesis. To provide evidence to further support the hypothesis, we prepare, optimize and characterize the coating for high-resolution separations. We utilize columns prepared under the optimized conditions to separate a peptide sample form E. coli lysate digested first by pepsin and then by trypsin, and we show ultrahigh-peak-capacity results (770 within 47 min using a 45-cm-long column and 2000 within 155 min using a 155-cm-long column). The coating is stable, and the coating method is reproducible. Ultrahigh-resolution separations and ultra-sensitive analyses are critically important and currently challenging for analyses of complex proteomics samples; employing narrow OT columns can alleviate this problem effectively.