Protein Analysis

The NanoLC™ product line from Eksigent uses direct (splitless) pumping in combination with microfluidic flow control to deliver precise gradients even at nano flow rates (20 nL/min-1000 nL/min). For protein digests, the retention time repeatability is shown to be better than 0.5% for all resolved peaks with either UV or MS detection.

Limited amounts of sample available for proteomics research have created the need for nanoscale (column ID 50-150 μm) chromatography featuring flow rates between 20 nL/min and 1000 nL/min. This is far below the 0.1-5 mL/min flow rate range of a typical HPLC instrument used for conventional chromatography (column ID 1.0-4.6 mm).

Various approaches exist to deliver flow rates in this range. One approach uses conventional HPLC in combination with a splitter. For example, delivery of 200 nL flow rates is accomplished by taking a conventional pump, operating at 200 μL/min, and splitting it through a tee where 0.1% of its total flow would be routed to the NanoLC column while the remaining 99.9% goes to waste. This approach typically suffers from the difficulty of maintaining a constant split ratio. As the back pressure on the NanoLC column changes, the split ratio of the setup changes as well. In such a case the conventional pump, well upstream of the column, will still accurately be delivering its 200 μL/min flow, but the flow through the NanoLC column itself will not be accurate. This causes the retention time of the separated analytes to drift.

A second approach uses displacement pumps, specifically designed for low flow rates. In this case, mobile phase from a servo driven pump is displaced and routed to the NanoLC column at pressure. Careful initial calibration of the displacement will give reasonable results with equipment of this sort. However, without feedback, the user cannot be sure that the delivered flow rates remain accurate as column pressures and mobile phases change.

The Eksigent NanoLC product line uses a third technique, microfluidic flow control, which ensures high quality, repeatable chromatography even when conditions downstream of the pumps are changed. With microfluidic flow control, nano flow rates are delivered using direct pumping. A dynamically adjusted pressure source delivers fluid through a flow module which monitors the actual flow rate. Any discrepancy between the actual and programmed flow rates are corrected for by a control loop.

The benefits of using microfluidic flow control can be seen in the quality of the chromatography. By using real-time closed loop control, retention time variation can be kept to a minimum. Splitless flow also saves up to 1000× the amount of solvent that would be required with a split system.

The Eksigent NanoLC system effectively uses microfluidic flow control to produce highly repeatable chromatograms in both NanoLC/UV and NanoLC/MS applications. For the NanoLC/UV runs the average RSD for the 17 peaks studied is 0.27%, while the value for the 14 peaks in NanoLC/MS runs is 0.35%. While both of these values are substantially lower than the specification of 0.5% on the instrument they are somewhat different. The most likely reason for this is the fact that in the NanoLC/UV case the column is placed in a temperature controlled oven, while for NanoLC/MS the column is at ambient.

It is shown here that relative standard deviations can be kept below 0.5% throughout the runs even while doing shallow gradients that demand flow rate accuracy of better than 500 pL/min.

Read the Application Note in its entirety.