Analytical and Bioanalytical Chemistry

, Volume 408, Issue 14, pp 3751–3759 | Cite as

A novel four-dimensional analytical approach for analysis of complex samples

  • Susanne Stephan
  • Cornelia Jakob
  • Jörg Hippler
  • Oliver J. SchmitzEmail author
Research Paper


A two-dimensional LC (2D-LC) method, based on the work of Erni and Frei in 1978, was developed and coupled to an ion mobility-high-resolution mass spectrometer (IM-MS), which enabled the separation of complex samples in four dimensions (2D-LC, ion mobility spectrometry (IMS), and mass spectrometry (MS)). This approach works as a continuous multiheart-cutting LC system, using a long modulation time of 4 min, which allows the complete transfer of most of the first - dimension peaks to the second - dimension column without fractionation, in comparison to comprehensive two-dimensional liquid chromatography. Hence, each compound delivers only one peak in the second dimension, which simplifies the data handling even when ion mobility spectrometry as a third and mass spectrometry as a fourth dimension are introduced. The analysis of a plant extract from Ginkgo biloba shows the separation power of this four-dimensional separation method with a calculated total peak capacity of more than 8700. Furthermore, the advantage of ion mobility for characterizing unknown compounds by their collision cross section (CCS) and accurate mass in a non-target approach is shown for different matrices like plant extracts and coffee.

Graphical abstract

Principle of the four-dimensional separation


2D-LC CCS Ginkgo biloba IM-qTOF-MS Ion mobility LC+LC 



We are thankful to Agilent for the third Infinity pump system and Phenomenex for the HPLC columns.

Compliance with ethical standards

Conflicts of interest

The authors declare that they have no conflict of interest.

Supplementary material

216_2016_9460_MOESM1_ESM.pdf (531 kb)
ESM 1 (PDF 530 kb)


  1. 1.
    Elsner V, Laun S, Melchior D, Köhler M, Schmitz OJ. Analysis of fatty alcohol derivatives with comprehensive two-dimensional liquid chromatography coupled with mass spectrometry. J Chromatogr A. 2012;1268:22–8. doi: 10.1016/j.chroma.2012.09.072.CrossRefGoogle Scholar
  2. 2.
    Marriott PJ, Chin ST, Maikhunthod B, Schmarr HG, Bieri S. Multidimensional gas chromatography. Trends Anal Chem. 2012;34:1–20. doi: 10.1016/j.trac.2011.10.013.CrossRefGoogle Scholar
  3. 3.
    Kanu AB, Dwivedi P, Tam M, Matz L, Hill HHJ. Ion mobility–mass spectrometry. J Mass Spectrom. 2008;43:1–22. doi: 10.1002/jms.CrossRefGoogle Scholar
  4. 4.
    Mukhopadhyay R. IMS/MS: its time has come. Anal Chem. 2008;80:7918–20. doi: 10.1021/ac8018608.CrossRefGoogle Scholar
  5. 5.
    Erni F, Frei RW. Two-dimensional column liquid chromatographic technique for resolution of complex mixtures. J Chromatogr. 1978;149:561–9.CrossRefGoogle Scholar
  6. 6.
    Bushey MM, Jorgenson JW. Automated instrumentation for comprehensive two-dimensional high-performance liquid chromatography of proteins. Anal Chem. 1990;62:161–7.CrossRefGoogle Scholar
  7. 7.
    Murphy RE, Schure MR, Foley JP. Effect of sampling rate on resolution in comprehensive two-dimensional liquid chromatography. Anal Chem. 1998;70:1585–94. doi: 10.1021/ac971184b.CrossRefGoogle Scholar
  8. 8.
    Marriott PJ, Wu Z, Schoenmakers P. Nomenclature and conventions in comprehensive multidimensional chromatography—an update. LCGC Eur. (2012);25Google Scholar
  9. 9.
    Hu L, Chen X, Kong L, Su X, Ye M, Zou H. Improved performance of comprehensive two-dimensional HPLC separation of traditional Chinese medicines by using a silica monolithic column and normalization of peak heights. J Chromatogr A. 2005;1092:191–8. doi: 10.1016/j.chroma.2005.06.066.CrossRefGoogle Scholar
  10. 10.
    Stoll DR, Carr PW. Fast, comprehensive two-dimensional HPLC separation of tryptic peptides based on high-temperature HPLC. J Am Chem Soc. 2005;127:5034–5. doi: 10.1021/ja050145b.CrossRefGoogle Scholar
  11. 11.
    De La Mata P, Harynuk JJ. Limits of detection and quantification in comprehensive multidimensional separations. 1. A theoretical look. Anal Chem. 2012;84:6646–53. doi: 10.1021/ac3010204.CrossRefGoogle Scholar
  12. 12.
    Thekkudan DF, Rutan SC, Carr PW. A study of the precision and accuracy of peak quantification in comprehensive two-dimensional liquid chromatography in time. J Chromatogr A. 2010;1217:4313–27. doi: 10.1016/j.chroma.2010.04.039.CrossRefGoogle Scholar
  13. 13.
    Lapthorn C, Pullen F, Chowdhry BZ. Ion mobility spectrometry-mass spectrometry (IMS-MS) of small molecules: separating and assigning structures to ions. Mass Spectrom Rev. 2013;32:43–71. doi: 10.1002/mas.CrossRefGoogle Scholar
  14. 14.
    Pringle SD, Giles K, Wildgoose JL, Williams JP, Slade SE, Thalassinos K, et al. An investigation of the mobility separation of some peptide and protein ions using a new hybrid quadrupole/travelling wave IMS/oa-ToF instrument. Int J Mass Spectrom. 2007;261:1–12. doi: 10.1016/j.ijms.2006.07.021.CrossRefGoogle Scholar
  15. 15.
    May JC, McLean JA. Ion mobility-mass spectrometry: time-dispersive instrumentation. Anal Chem. 2015;87:1422–36. doi: 10.1021/ac504720m.CrossRefGoogle Scholar
  16. 16.
    Campuzano I, Bush MF, Robinson CV, Beaumont C, Richardson K, Kim H, et al. Structural characterization of drug-like compounds by ion mobility mass spectrometry: comparison of theoretical and experimentally derived nitrogen collision cross sections. Anal Chem. 2012;84:1026–33. doi: 10.1021/ac202625t.CrossRefGoogle Scholar
  17. 17.
    Hofmann J, Struwe WB, Scar CA, Scrivens JH, Harvey DJ, Pagel K. Estimating collision cross sections of negatively charged N-glycans using traveling wave ion mobility-mass spectrometry. Anal Chem. 2014;86:10789–95. doi: 10.1021/ac5028353.CrossRefGoogle Scholar
  18. 18.
    Tao L, McLean JR, McLean JA, Russell DH. A collision cross-section database of singly-charged peptide ions. J Am Soc Mass Spectrom. 2007;18:1232–8. doi: 10.1016/j.jasms.2007.04.003.CrossRefGoogle Scholar
  19. 19.
    Counterman AE, Valentine SJ, Srebalus CA, Henderson SC, Hoaglund CS, Clemmer DE. High-order structure and dissociation of gaseous peptide aggregates that are hidden in mass spectra. J Am Soc Mass Spectrom. 1998;9:743–59. doi: 10.1016/S1044-0305(98)00052-X.CrossRefGoogle Scholar
  20. 20.
    Valentine SJ, Counterman AE. A database of 660 peptide ion cross sections: use of intrinsic size parameters for bona fide predictions of cross sections. J Am Soc Mass Spectrom. 1999;10:1188–211. doi: 10.1016/S1044-0305(99)00079-3.CrossRefGoogle Scholar
  21. 21.
    Bush MF, Hall Z, Giles K, Hoyes J, Robinson CV, Ruotolo BT. Collision cross sections of proteins and their complexes: a calibration framework and database for gas-phase structural biology. Anal Chem. 2010;82:9557–65. doi: 10.1021/ac1022953.CrossRefGoogle Scholar
  22. 22.
    Paglia G, Williams JP, Menikarachchi LC, Thompson JW, Tyldesley-Worster R, Halldórsson S, et al. Ion mobility-derived collision cross-sections to support metabolomics applications. Anal Chem. 2014;86:3985–93. doi: 10.1021/ac500405x.CrossRefGoogle Scholar
  23. 23.
    Paglia G, Angel P, Williams JP, Richardson K, Olivos HJ, Thompson JW, et al. Ion mobility-derived collision cross section as an additional measure for lipid fingerprinting and identification. Anal Chem. 2015;87:1137–44. doi: 10.1021/ac503715v.CrossRefGoogle Scholar
  24. 24.
    May JC, Goodwin CR, Lareau NM, Leaptrot KL, Morris CB, Kurulugama RT, et al. Conformational ordering of biomolecules in the gas phase: nitrogen collision cross sections measured on a prototype high resolution drift tube ion mobility-mass spectrometer. Anal Chem. 2014;86:2107–16. doi: 10.1021/ac4038448.CrossRefGoogle Scholar
  25. 25.
    Liu X, Valentine SJ, Plasencia MD, Trimpin S, Naylor S, Clemmer DE. Mapping the human plasma proteome by SCX-LC-IMS-MS. J Am Soc Mass Spectrom. 2007;18:1249–64. doi: 10.1016/j.jasms.2007.04.012.CrossRefGoogle Scholar
  26. 26.
    Malkar A, Devenport NA, Martin HJ, Patel P, Turner MA, Watson P, et al. Metabolic profiling of human saliva before and after induced physiological stress by ultra-high performance liquid chromatography-ion mobility-mass spectrometry. Metabolomics. 2013;9:1192–201. doi: 10.1007/s11306-013-0541-x.CrossRefGoogle Scholar
  27. 27.
    Kurulugama RT, Darland E, Kuhlmann F, Stafford G, Fjeldsted J. Evaluation of drift gas selection in complex sample analyses using a high performance drift tube ion mobility-QTOF mass spectrometer. Analyst (Cambridge, U K). 2015;140:6834–44. doi: 10.1039/c5an00991j.CrossRefGoogle Scholar
  28. 28.
    Dück R, Sonderfeld H, Schmitz OJ. A simple method for the determination of peak distribution in comprehensive two-dimensional liquid chromatography. J Chromatogr A. 2012;1246:69–75. doi: 10.1016/j.chroma.2012.02.038.CrossRefGoogle Scholar
  29. 29.
    Li D, Schmitz OJ. Use of shift gradient in the second dimension to improve the separation space in comprehensive two-dimensional liquid chromatography. Anal Bioanal Chem. 2013;405:6511–7. doi: 10.1007/s00216-013-7089-5.CrossRefGoogle Scholar
  30. 30.
    Stoll DR, Talus ES, Harmes DC, Zhang K Evaluation of detection sensitivity in comprehensive two-dimensional liquid chromatography separations of an active pharmaceutical ingredient and its degradants. Anal Bioanal Chem; 2014. 265–277. doi: 10.1007/s00216-014-8036-9.Google Scholar
  31. 31.
    Filgueira M, Huang Y, Witt K, Castells C, Carr PW. Improving peak capacity in fast on-line comprehensive two-dimensional liquid chromatography with post first dimension flow-splitting. Anal Chem. 2011;83:9531–9. doi: 10.1021/ac202317m.CrossRefGoogle Scholar
  32. 32.
    Ding S, Dudley E, Plummer S, Tang J, Newton RP, Brenton G. Quantitative determination of major active components in Ginkgo biloba dietary supplements by liquid chromatography/mass spectrometry. Rapid Commun Mass Spectrom. 2006;20:2753–60. doi: 10.1002/rcm.CrossRefGoogle Scholar
  33. 33.
    Giddings JC. Two-dimensional separations: concept and promise. Anal Chem. 1984;56:1258A–70A. doi: 10.1021/ac00276a003.CrossRefGoogle Scholar
  34. 34.
    Gilar M, Olivova P, Daly AE, Gebler JC. Orthogonality of separation in two-dimensional liquid chromatography. Anal Chem. 2005;77:6426–34. doi: 10.1021/ac050923i.CrossRefGoogle Scholar
  35. 35.
    Liu Z, Patterson Jr D. Geometric approach to factor analysis for the estimation of orthogonality and practical peak capacity in comprehensive two-dimensional separations. Anal Chem. 1995;67:3840–5. doi: 10.1021/ac00117a004.CrossRefGoogle Scholar
  36. 36.
    Dolan JW, Snyder LR, Djordjevic NM, Hill DW, Waeghe TJ. Reversed-phase liquid chromatographic separation of complex samples by optimizing temperature and gradient time I. Peak capacity limitations. J Chromatogr A. 1999;857:1–20. doi: 10.1016/S0021-9673(99)00765-7.CrossRefGoogle Scholar
  37. 37.
    Taylor L, Tichy S. Ion mobility technology for LC/MS reveal greater detail. Separation - Innovations in Mobility Techniques.pdf. 2013 Accessed 22 Jan 2016.

Copyright information

© Springer-Verlag Berlin Heidelberg 2016

Authors and Affiliations

  • Susanne Stephan
    • 1
  • Cornelia Jakob
    • 1
  • Jörg Hippler
    • 1
  • Oliver J. Schmitz
    • 1
    Email author
  1. 1.Applied Analytical ChemistryUniversity of Duisburg-EssenEssenGermany

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