Nanocapillaries for open tubular chromatographic separations of proteins in femtoliter to picoliter samples

Abstract

We have recently examined the potential of bare nanocapillaries for free solution DNA separations and demonstrated efficiencies exceeding 10(6) theoretical plates/m. In the present work, we demonstrate the use of bare and hydroxypropylcellulose (HPC) coated open tubular nanocapillaries for protein separations. Using 1.5 microm inner diameter (i.d.) capillary columns, hydrodynamically injecting femto- to picoliter volumes of fluorescent or fluorescent dye labeled protein samples, utilizing a pneumatically pressurized chamber containing 1.0 mM sodium tetraborate solution eluent (typically 200 psi) as the pump, and performing on-column detection using a simple laser-induced fluorescence detector, we demonstrate efficiencies of close to a million theoretical plates/m while generating single digit microliter volumes of waste for a complete chromatographic run. We achieve baseline resolution for a protein mixture consisting of transferrin, alpha-lactalbumin, insulin, and alpha-2-macroglobulin.

Figure 1

Schematic diagram of the experimental setup

Figure 2

Chromatogram showing the elution of rAcGFP1. The sample (5.0 ng/μL) was injected for 10 s@100psi. The column was 47 cm long (44 cm effective) and 1.5 μm in diameter. The eluent was 1.0 mM Na₂B₄O₇ pressurized at 300 psi. The principal trace is the raw data from the LIF detector. Inset (a) shows a gaussian fit to the eluite peak. Inset (b) shows the same data after a 20-point moving average filter.

Figure 3

Protein separation and the effect of sodium tetraborate concentration. The elution order remains the same, from left to right (1) transferrin, (2) lactalbumin, (3) insulin, and (4) α-2-macroglobulin. Capillary i.d. 1.5 μm, 70 cm long (65 cm eff.) Eluent pressure 300 psi. The analyte concentrations are 0.6 μM, 6.0 μM, 39.5 μM, and 0.03 μM, respectively. Samples were injected for 8 s@100 psi.

Figure 4

Plot of log retention time vs. log eluent ion concentration. The slope changes are obvious in all cases above a borate anion concentration >1 mM, but is most notable for α-2-macroglobulin.

Figure 5

Effect of SDS as an additive on the separation of the four proteins Transferrin (T), α-Lactalbumin (L), Insulin (I), and α-2-Macroglobulin (M) in a 1.5 μm i.d. 70 cm long (65 cm eff.) capillary at 300 psi. Samples were injected for 8 s@100 psi. The inset shows log t_R-log [SDS] plots.

Figure 6

All separation capillaries had the same length (50 cm total and 45 cm eff.). Sample injection duration and pressure were adjusted to ensure injections of sample plugs of nearly equivalent length with 4 s@15 psi for 4.2 μm capillary, 5 s@40 psi for 2.4 μm capillary and 8 s@100 psi for 1.5 μm capillary. Elution pressure 150 psi.

Figure 7

The chromatograms were respectively obtained with a 155-cm long (150 cm eff.) x 4.2 μm i.d., a 70-cm long (65 cm eff.) x 2.4 μm i.d., and a 50-cm long (45 cm eff.) × 1.5 μm i.d. capillary. All sample injections were 8 s@100 psi and all separations were performed at 150 psi with 1.0 mM Na₂B₄O₇.

Figure 8

Resolution between transferrin and α-lactalbumin as a function of linear velocity (separation pressure) on a 70 cm long (65 cm effective) × 1.5 μm i.d. capillary. Sample injections 8 s@100 psi; 1.0 mM Na₂B₄O₇ eluent.

Figure 9

Resolution as a function of capillary length. All separations were carried out under a constant pressure gradient (4.3 psi/cm column length) using 1.5 μm i.d. capillaries of (A) 110, (B) 90, (C) 70, (D) 50 cm lengths, the effective lengths in each case was 5 cm smaller. The eluent was 1.0 mM Na₂B₄O₇.