Skip to main content

Selection of the optimum electrospray voltage for gradient elution LC-MS measurements


Changes in liquid composition during gradient elution liquid chromatography (LC) coupled to mass spectrometry (MS) analyses affect the electrospray operation. To establish methodologies for judicious selection of the electrospray voltage, we monitored in real time the effect of the LC gradient on the spray current. The optimum range of the electrospray voltage decreased as the concentration of organic solvent in the eluent increased during reversed-phase LC analyses. These results and related observations provided the means to rationally select the voltage to ensure effective electrospray operation throughout gradient-elution LC separations. For analyses in which the electrospray was operated at constant voltage, a small run-to-run variation in the spray current was observed, indicating a changing electric field resulting from fouling or degradation of the emitter. Algorithms using feedback from spray current measurements that can maintain the electrospray voltage within the optimum operating range throughout gradient elution LC-MS were evaluated. The electrospray operation with voltage regulation and at a constant, judiciously selected voltage during gradient elution LC-MS measurements produced data with similar reproducibility.


  1. Aleksandrov, M. L.; Gall, L. N.; Krasnov, V. N.; Nikolaev, V. I.; Pavlenko, V. A.; Shkurov, V. A.; Baram, G. I.; Gracher, M. A.; Knorre, V. D.; Kusner, Y. S. Direct Coupling of a Microcolumn Liquid Chromatograph and a Mass Spectrometer. Bioorg. Khim. 1984, 10, 710–712.

    CAS  Google Scholar 

  2. Whitehouse, C. M.; Dreyer, R. N.; Yamashita, M.; Fenn, J. B. Electrospray Interface for Liquid Chromatographs and Mass Spectrometers. Anal. Chem. 1985, 57, 675–679.

    CAS  Article  Google Scholar 

  3. Aebersold, R.; Mann, M. Mass Spectrometry-Based Proteomics. Nature 2003, 422, 198–207.

    CAS  Article  Google Scholar 

  4. Domon, B.; Aebersold, R. Mass Spectrometry and Protein Analysis. Science 2006, 312, 212–217.

    CAS  Article  Google Scholar 

  5. Marginean, I.; Kelly, R. T.; Page, J. S.; Tang, K. Q.; Smith, R. D. Electrospray Characteristic Curves: In Pursuit of Improved Performance in the Nanoflow Regime. Anal. Chem. 2007, 79, 8030–8036.

    CAS  Article  Google Scholar 

  6. Jackson, G. S.; Enke, C. G. Electrical Equivalence of Electrospray Ionization with Conducting and Nonconducting Needles. Anal. Chem. 1999, 71, 3777–3784.

    CAS  Article  Google Scholar 

  7. Ramanathan, R.; Zhong, R.; Blumenkrantz, N.; Chowdhury, S. K.; Alton, K. B. Response Normalized Liquid Chromatography Nanospray Ionization Mass Spectrometry. J. Am. Soc. Mass Spectrom. 2007, 18, 1891–1899.

    CAS  Article  Google Scholar 

  8. Valaskovic, G. A.; Murphy, J. P.; Lee, M. S. Automated Orthogonal Control System for Electrospray Ionization. J. Am. Soc. Mass Spectrom. 2004, 15, 1201–1215.

    CAS  Article  Google Scholar 

  9. Staats, S. L. T.; Fogiel, A. J.; Suna, A. Active Spray Control with Electric Field Optimization for Online NanoLC with Polymeric Spray Tips. In Proceedings of the 56th ASMS Conference on Mass Spectrometry, Denver, CO, June 1–5, 2008.

  10. Gapeev, A.; Berton, A.; Fabris, D. Current-Controlled Nanospray for the Analysis of Less Than Ideal Samples. In Proceedings of the 56th ASMS Conference on Mass Spectrometry, Denver, CO, June 1–5, 2008.

  11. Marginean, I.; Kelly, R. T.; Prior, D. C.; LaMarche, B. L.; Tang, K.; Smith, R. D. Analytical Characterization of the Electrospray Ion Source in the Nanoflow Regime. Anal. Chem. 2008, 80, 6573–6579.

    CAS  Article  Google Scholar 

  12. Kinter, M. M.; Sherman, N. E. Protein Sequencing and Identification Using Tandem Mass Spectrometry. New York: Wiley-Interscience.

  13. Shen, Y. F.; Tolic, N.; Zhao, R.; Pasa-Tolic, L.; Li, L. J.; Berger, S. J.; Harkewicz, R.; Anderson, G. A.; Belov, M. E.; Smith, R. D. High-Throughput Proteomics Using High Efficiency Multiple-Capillary Liquid Chromatography with On-line High-Performance ESI FTICR Mass Spectrometry. Anal. Chem. 2001, 73, 3011–3021.

    CAS  Article  Google Scholar 

  14. Shen, Y. F.; Zhao, R.; Belov, M. E.; Conrads, T. P.; Anderson, G. A.; Tang, K. Q.; Pasa-Tolic, L.; Veenstra, T. D.; Lipton, M. S.; Udseth, H. R.; Smith, R. D. Packed Capillary Reversed-Phase Liquid Chromatography with High-Performance Electrospray Ionization Fourier Transform Ion Cyclotron Resonance Mass Spectrometry for Proteomics. Anal. Chem. 2001, 73, 1766–1775.

    CAS  Article  Google Scholar 

  15. Kelly, R. T.; Page, J. S.; Luo, Q. Z.; Moore, R. J.; Orton, D. J.; Tang, K. Q.; Smith, R. D. Chemically Etched Open Tubular and Monolithic Emitters for Nanoelectrospray Ionization Mass Spectrometry. Anal. Chem. 2006, 78, 7796–7801.

    CAS  Article  Google Scholar 

  16. Jaitly, N.; Mayampurath, A.; Littlefield, K.; Adkins, J. N.; Anderson, G. A.; Smith, R. D. Decon2LS: An Open-Source Software Package for Automated Processing and Visualization of High Resolution Mass Spectrometry Data. BMC Bioinformatics 2009, in press.

  17. Jaitly, N.; Monroe, M. E.; Petyuk, V. A.; Clauss, T. R. W.; Adkins, J. N.; Smith, R. D. Robust Algorithm for Alignment of Liquid Chromatography-Mass Spectrometry Analyses in an Accurate Mass and Time Tag Data Analysis Pipeline. Anal. Chem. 2006, 78, 7397–7409.

    CAS  Article  Google Scholar 

  18. Livesay, E. A.; Tang, K. Q.; Taylor, B. K.; Buschbach, M. A.; Hopkins, D. F.; LaMarche, B. L.; Zhao, R.; Shen, Y. F.; Orton, D. J.; Moore, R. J.; Kelly, R. T.; Udseth, H. R.; Smith, R. D. Fully Automated Four-Column Capillary LC-MS System for Maximizing Throughput in Proteomic Analyses. Anal. Chem. 2008, 80, 294–302.

    CAS  Article  Google Scholar 

  19. Nilsson, S.; Svedberg, M.; Pettersson, J.; Bjorefors, F.; Markides, K.; Nyholm, L. Evaluations of the Stability of Sheathless Electrospray Ionization Mass Spectrometry Emitters Using Electrochemical Techniques. Anal. Chem. 2001, 73, 4607–4616.

    CAS  Article  Google Scholar 

  20. Chen, M. L.; Cook, K. D. Oxidation Artifacts in the Electrospray Mass Spectrometry of A β Peptide. Anal. Chem. 2007, 79, 2031–2036.

    CAS  Article  Google Scholar 

  21. Geromanos, S.; Freckleton, G.; Tempst, P. Tuning of an Electrospray Ionization Source for Maximum Peptide-Ion Transmission into a Mass Spectrometer. Anal. Chem. 2000, 72, 777–790.

    CAS  Article  Google Scholar 

  22. Marginean, I.; Kelly, R. T.; Page, J. S.; Tang, K. Q.; Smith, R. D. Toward a Stable Electrospray. Proceedings of the 55th ASMS Conference on Mass Spectrometry, Indianapolis, IN, June 3–7, 2007.

  23. Evans, C. A.; Hendricks, C. D. Electrohydrodynamic Ion-Source for Mass-Spectrometry of Liquids. Rev. Sci. Instrum. 1972, 43, 1527–1530.

    CAS  Article  Google Scholar 

  24. Chen, D. R.; Pui, D. Y. H.; Kaufman, S. L. Electrospraying of Conducting Liquids for Monodisperse Aerosol Generation in the 4 nm to 1.8 µm Diameter Range. J. Aerosol Sci. 1995, 26, 963–977.

    CAS  Article  Google Scholar 

  25. Old, W. M.; Meyer-Arendt, K.; Aveline-Wolf, L.; Pierce, K. G.; Mendoza, A.; Sevinsky, J. R.; Resing, K. A.; Ahn, N. G. Comparison of Label-Free Methods for Quantifying Human Proteins by Shotgun Proteomics. Mol. Cell. Proteomics 2005, 4, 1487–1502.

    CAS  Article  Google Scholar 

  26. Ono, M.; Shitashige, M.; Honda, K.; Isobe, T.; Kuwabara, H.; Matsuzuki, H.; Hirohashi, S.; Yamada, T. Label-Free Quantitative Proteomics Using Large Peptide Data Sets Generated by Nanoflow Liquid Chromatography and Mass Spectrometry. Mol. Cell. Proteomics 2006, 5, 1338–1347.

    CAS  Article  Google Scholar 

  27. Kelly, R. T.; Page, J. S.; Marginean, I.; Tang, K. Q.; Smith, R. D. Nanoelectrospray Emitter Arrays Providing Interemitter Electric Field Uniformity. Anal. Chem. 2008, 80, 5660–5665.

    CAS  Article  Google Scholar 

Download references

Author information

Authors and Affiliations


Corresponding author

Correspondence to Richard D. Smith.

Additional information

Published online December 13, 2008

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Marginean, I., Kelly, R.T., Moore, R.J. et al. Selection of the optimum electrospray voltage for gradient elution LC-MS measurements. J Am Soc Mass Spectrom 20, 682–688 (2009).

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI:


  • Anal Ysis
  • Electrospray Voltage
  • Valco Instrument
  • Spray Current
  • Electrospray Ionization Fourier Transform