Narrow, Open, Tubular Column for Ultrahigh-Efficiency Liquid-Chromatographic Separation under Elution Pressure of Less than 50 bar

We report that we can achieve extremely high separation efficiencies using a narrow, open, tubular (NOT) column for liquid-chromatographic separations, and we can carry out these separations under an elution pressure of no more than 50 bar. To improve the separation efficiency in packed-column liquid chromatography, one of the most effective approaches is to reduce the monodispersed-particle sizes. A direct consequence of reduced particle size is an increased elution pressure. High efficiencies have been obtained in ultrahigh-performance liquid chromatography (UPLC) using 1–2 μm or even submicron particles, and high elution pressures (greater than 1000 bar) are commonly used to carry out these separations. Open, tubular (OT) columns have been predicted to be the most efficient columns for high-efficiency liquid-chromatographic separations, as long as the column diameter is sufficiently small (1–2 μm). However, high efficiencies have not yet been publically reported, possibly because of the challenges (such as picoliter-volume detection, nanocapillary-column preparation, low sample loadability, etc.) of utilizing 1–2 μm diameter capillaries. In this paper, we show how we overcame these problems and achieved extremely high separation efficiencies using a 2 μm inner diameter capillary. We see 200+ apparent peaks with a peak capacity of 810 within 54 min when separating a sample from trypsin-digested cytochrome C, and we count 440 apparent peaks with a peak capacity of 1640 within 172 min when separating a sample from pepsin/trypsin-digested Escherichia coli cell lysate