Compact Ultrahigh-Pressure Nanoflow Capillary Liquid Chromatograph.

ABSTRACT

A compact ultrahigh-pressure nanoflow liquid chromatograph (LC) was developed with the purpose in mind of creating a portable system that could be easily moved to various testing locations or placed in close proximity to other instruments for optimal coupling, such as with mass spectrometry (MS). The system utilized innovative nanoflow pumps integrated with a very low volume stop-flow injector and mixing tee. The system weighed only 5.9 kg (13 lbs) or 4.5 kg (10 lbs) without a controller and could hold up to 1100 bar (16000 psi) of pressure. The total volume pump capacity was 60 µL. In this study, the sample injection volume was determined by either a 60 nL internal sample groove machined in a high-pressure valve rotor or by a 1 µL external sample loop, although other sample grooves or loops could be selected. The gradient dwell volume was approximately 640 nL, which allowed significant reduction in sample analysis time. Gradient performance was evaluated by determining the gradient step accuracy. A low RSD (0.6%, n = 4) was obtained for day-to-day experiments. Linear gradient reproducibility was evaluated by separating a three-component polycyclic aromatic hydrocarbon mixture on a commercial 150 µm inner diameter capillary column packed with 1.7 µm particles. Good retention-time reproducibility (RSD < 0.17%) demonstrated that the pumping system could successfully generate ultrahigh pressures for use in capillary LC. The system was successfully coupled to an LTQ Orbitrap MS in a simple and efficient way; LC-MS of a trypsin-digested bovine serum albumin (BSA) sample provided narrow peaks, short dwell time, and good peptide coverage.