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Oct 1, 2005 (Vol. 25, No. 17)

Nanoscale Gradient Chromatography for LC-MS

Using Microfluidic Flow Control to Improve LC-MS for Drug Discovery Proteomics

  • In the search for viable drug targets and compounds, drug discovery researchers are adopting high-resolution, high-content protein identification and functional analysis methods. Liquid chromatography combined with mass spectrometry (LC-MS) offers the resolving power and structural information about proteins needed to address these applications and others in phosphopeptide analysis, biomarker discovery, and metabolomics.

    For example, mass spectral comparison of protein mixtures from diseased versus healthy tissue samples can provide deep insight into the molecular nature of diseases and uncover biomarkers and biomarker patterns of therapeutic and diagnostic value. Sensitive, high-resolution LC-MS can lead directly to the identification of drug target proteins, and enable researchers to detect and quantify therapeutic and toxic effects of potential new chemical entities.

    Efforts to optimize LC-MS technology to meet the escalating requirements of proteomics researchers for analytical speed, sensitivity, and reproducibility have led to the development of nanoscale proteomics and nanoflow chromatography, a method that takes advantage of small sample quantities and low chromatographic solvent flow rates.

    Low-volume, low-flow-rate chromatography improves peptide identification and simplifies mass spectrometry analysis by delivering separated components at high peak concentrations to a nano electrospray ionization source. This enhances the sensitivity of mass spectrometry, enabling the analysis of smaller samples of purified protein preparations. When implemented with components specifically matched to the requirements of nanoflow chromatography, nanoscale HPLC separation systems also reduce solvent waste by orders of magnitude.

    The main challenge of nanoflow chromatography is maintaining flow stability at low flow rates. Optimal flow rates for nanoscale LC columns are between 100 and 500 nanoliters per minute. Conventional HPLC pumps are optimized for flow rates of three to four orders of magnitude higher than this, and instrument manufacturers initially entered the market for nanoflow chromatography with systems based on the use of these pumps combined with flow splitters, which bleed off all but a small amount of the flow.

    For routine separations at standard HPLC flow rates, conventional, piston-driven HPLC pumps provide accurate flow rates, but can suffer from pulsing due to piston restroke and typically require additional hardware to dampen this effect. The flow rate stability of systems based on flow-splitting delivery formats is further degraded by backpressure changes, which can cause the actual flow rate to the column to deviate substantially from the set point.

    The cumulative effect of these limitations is reduced separation reproducibility and an inability to perform precise gradients at the low flow rates needed to optimize the sensitivity advantages of nanoscale chromatography systems.

  • Achieving Stable, Low Flow Rates

    Microfluidic Flow Control (MFC), developed by Eksigent Technologies (Livermore, CA), employs a continuously variable pressure source to produce accurate and stable flows for nanoscale chromatography. In an MFC system, an in-line flow meter feeds real-time flow rate information back to an electronically controlled pressure source to maintain programmed rates (Figure 1). Low internal system volumes enable nanoscale chromatography systems driven by MFC pressure sources to respond to programmed flow rate set point changes in seconds.

    When used in tandem, two MFC pressure sources can be programmed to form precise, accurate, and reproducible elution gradients for LC-MS. The tandem system delivers binary gradients at flow rates ranging from 501,000 nL per minute without the use of flow splitting. Flow rate precision of the system is <0.5% RSD at 500 nL per minute. Stable flow rates of each mobile phase are maintained at their set points regardless of downstream changes in viscosity or other factors affecting flow resistance.

    Precise column flow rates produced by the tandem MFC system are directly compatible with 50 to 100 micron internal diameter separations columns that feed directly into nano electrospray ionization systems for mass spectrometry.

  • Peak Parking

    Peak parking is a method used to extend the amount of time available for MS analysis of the components in a peak of interest. A typical nanoscale HPLC flow rate of 500 nL per minute will deliver sharp peaks from compounds that will then be analyzed for a few seconds by the mass spectrometry instrument. The brief time during which the compound is being analyzed may not be long enough to acquire sufficient spectral data for conclusive analysis.

    In peak parking, the flow rate is reduced to an ultra-low level at the moment the peak of interest begins to be analyzed and held at the lower flow rate for the duration of compound elution (Figure 2). At the end of the peak parking period, the flow rate is restored to the original programmed flow rate. The extended peak analysis time ensures optimal data collection from co-eluting peaks and low-abundance peptide fragments.

    MFC pressure sources programmed for nanoflow gradient elution can rapidly reduce flow rate by an order of magnitude during the course of gradient separation. Peak parking can be initiated by a mouse click from the instrument operator, or the pressure sources can be programmed to initiate peak parking and restore the original flow rate automatically based on a data feedback loop from the MS system. When the original flow rate is restored, the MFC pressure sources for each mobile phase resume the programmed gradient profile (Figure 3) and elution proceeds without loss of any separation fraction.

    Nanoscale HPLC with MFC for precise low flow rates, accurate gradient mixing, and peak parking has been commercialized by Eksigent Technologies in the NanoLC-1D Plus instrument. The system incorporates a direct-pumping binary gradient system optimized for flow rates of 201,000 nL per minute without flow splitting.

    The instrument addresses the potential problem of slow sample loading at nanoscale HPLC flow rates with an integrated high flow rate pump and autosampler control system, which loads samples more rapidly. NanoLC-1D Plus has been successfully integrated with a variety of analytical systems, including a nanoflow-optimized ESI source and a hybrid triple quad/linear ion trap mass analyzer to meet the high analytical performance requirements of LC-MS for drug discovery



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