Analytical and Bioanalytical Chemistry

, Volume 408, Issue 28, pp 8079–8088 | Cite as

A new method for the determination of peak distribution across a two-dimensional separation space for the identification of optimal column combinations

  • Juri Leonhardt
  • Thorsten TeutenbergEmail author
  • Greta Buschmann
  • Oliver Gassner
  • Torsten C. Schmidt
Research Paper


For the identification of the optimal column combinations, a comparative orthogonality study of single columns and columns coupled in series for the first dimension of a microscale two-dimensional liquid chromatographic approach was performed. In total, eight columns or column combinations were chosen. For the assessment of the optimal column combination, the orthogonality value as well as the peak distributions across the first and second dimension was used. In total, three different methods of orthogonality calculation, namely the Convex Hull, Bin Counting, and Asterisk methods, were compared. Unfortunately, the first two methods do not provide any information of peak distribution. The third method provides this important information, but is not optimal when only a limited number of components are used for method development. Therefore, a new concept for peak distribution assessment across the separation space of two-dimensional chromatographic systems and clustering detection was developed. It could be shown that the Bin Counting method in combination with additionally calculated histograms for the respective dimensions is well suited for the evaluation of orthogonality and peak clustering. The newly developed method could be used generally in the assessment of 2D separations.

Graphical Abstract


Histogram Porous graphitic carbon Nano-LC Micro-LC Microscale liquid chromatography Serial column coupling Comprehensive two-dimensional liquid chromatography 



The authors would like to thank Harald Möller-Santner and Eike Logé from Sciex for the loan of the Eksigent NanoLC 425 system. Furthermore, we would like to thank Juergen Maier-Rosenkranz for organizing the packing of PGC columns. In addition the authors would like to thank Altmann Analytik GmbH & Co. KG and Merck KGaA for providing the HILIC column.


For the automated data evaluation, based on the in this work presented method, a Python 3.x based Script ( was developed. The script as well as an introduction is a part of the supplementary material and is available at

Compliance with ethical standards

The research in this manuscript did not involve human participants and/or animals. All authors of this manuscript were informed and agreed for submission.

Conflict of interest

The authors declare that they have no conflict of interest.

Supplementary material

216_2016_9911_MOESM1_ESM.pdf (1.2 mb)
ESM 1 (PDF 1206 kb)


  1. 1.
    Haun J, Leonhardt J, Portner C, Hetzel T, Tuerk J, Teutenberg T, et al. Online and splitless NanoLC × CapillaryLC with quadrupole/time-of-flight mass spectrometric detection for comprehensive screening analysis of complex samples. Anal Chem. 2013;85(21):10083–90.CrossRefGoogle Scholar
  2. 2.
    Leonhardt J, Teutenberg T, Tuerk J, Schlüsener MP, Ternes TA, Schmidt TC. A comparison of one-dimensional and microscale two-dimensional liquid chromatographic approaches coupled to high resolution mass spectrometry for the analysis of complex samples. Anal Methods. 2015;7(18):7697–706.CrossRefGoogle Scholar
  3. 3.
    Krauss M, Singer H, Hollender J. LC-high resolution MS in environmental analysis: from target screening to the identification of unknowns. Anal Bioanal Chem. 2010;397(3):943–51.CrossRefGoogle Scholar
  4. 4.
    Zedda M, Zwiener C. Is nontarget screening of emerging contaminants by LC-HRMS successful? A plea for compound libraries and computer tools. Anal Bioanal Chem. 2012;403(9):2493–502.CrossRefGoogle Scholar
  5. 5.
    Neue UD. Peak capacity in unidimensional chromatography. J Chromatogr A. 2008;1184(1–2):107–30.CrossRefGoogle Scholar
  6. 6.
    Nägele E, Vollmer M, Hörth P. Improved 2D nano-LC/MS for proteomics applications. A comparative analysis using yeast proteome. J Biomol Tech. 2004;15(2):134–43.Google Scholar
  7. 7.
    Ralston-Hooper KJ, Turner ME, Soderblom EJ, Villeneuve D, Ankley GT, Moseley MA, et al. Application of a label-free, gel-free quantitative proteomics method for ecotoxicological studies of small fish species. Environ Sci Technol. 2013;47(2):1091–100.CrossRefGoogle Scholar
  8. 8.
    Zhao L, Liu L, Leng W, Wei C, Jin Q. A proteogenomic analysis of Shigella flexneri using 2D LC-MALDI TOF/TOF. BMC Genomics. 2011;12:528.CrossRefGoogle Scholar
  9. 9.
    Willemse CM, Stander MA, Vestner J, Tredoux AG, de Villiers A. Comprehensive two-dimensional hydrophilic interaction chromatography (HILIC) × reversed-phase liquid chromatography coupled to high- resolution mass spectrometry (RP-LC-UV-MS) analysis of anthocyanins and derived pigments in red wine. Anal Chem. 2015;87(24):12006–15.CrossRefGoogle Scholar
  10. 10.
    Ouyang X, Leonards PEG, Tousova Z, Slobodnik J, de Boer J, Lamoree MH. Rapid screening of acetylcholinesterase inhibitors by effect-directed analysis using LC × LC fractionation, a high throughput in vitro assay, and parallel identification by time of flight mass spectrometry. Anal Chem. 2016;88(4):2353–60.CrossRefGoogle Scholar
  11. 11.
    Mondello L, Lewis AC, Bartle KD. Multidimensional chromatography. Chichester: Wiley; 2001.CrossRefGoogle Scholar
  12. 12.
    Groskreutz SR, Swenson MM, Secor LB, Stoll DR. Selective comprehensive multi-dimensional separation for resolution enhancement in high performance liquid chromatography. Part I: principles and instrumentation. J Chromatogr A. 2012;1228:31–40.CrossRefGoogle Scholar
  13. 13.
    Wang S, Qiao L, Shi X, Hu C, Kong H, Xu G. On-line stop-flow two-dimensional liquid chromatography-mass spectrometry method for the separation and identification of triterpenoid saponins from ginseng extract. Anal Bioanal Chem. 2015;407(1):331–41.CrossRefGoogle Scholar
  14. 14.
    Li D, Jakob C, Schmitz O. Practical considerations in comprehensive two-dimensional liquid chromatography systems (LCxLC) with reversed-phases in both dimensions. Anal Bioanal Chem. 2015;407(1):153–67.CrossRefGoogle Scholar
  15. 15.
    Li D, Schmitz OJ. Comprehensive two-dimensional liquid chromatography tandem diode array detector (DAD) and accurate mass QTOF-MS for the analysis of flavonoids and iridoid glycosides in Hedyotis diffusa. Anal Bioanal Chem. 2015;407(1):231–40.CrossRefGoogle Scholar
  16. 16.
    Murphy RE, Schure MR, Foley JP. Effect of sampling rate on resolution in comprehensive two-dimensional liquid chromatography. Anal Chem. 1998;70(8):1585–94.CrossRefGoogle Scholar
  17. 17.
    Stoll DR, Cohen JD, Carr PW. Fast, comprehensive online two-dimensional high performance liquid chromatography through the use of high temperature ultra-fast gradient elution reversed-phase liquid chromatography. J Chromatogr A. 2006;1122(1):123–37.CrossRefGoogle Scholar
  18. 18.
    Dugo P, Cacciola F, Donato P, Mondello L. Comprehensive two-dimensional liquid chromatography applications. In: Mondello L, editor. Comprehensive chromatography in combination with mass spectrometry. Hoboken, NJ: Wiley; 2011. p. 391–427.CrossRefGoogle Scholar
  19. 19.
    Dugo P, Cacciola F, Donato P, Mondello L. Comprehensive two-dimensional liquid chromatography combined with mass spectrometry. In: Mondello L, editor. Comprehensive chromatography in combination with mass spectrometry. Hoboken: Wiley; 2011. p. 331–90.CrossRefGoogle Scholar
  20. 20.
    Stoll DR, Wang X, Carr PW. Comparison of the practical resolving power of one- and two-dimensional high-performance liquid chromatography analysis of metabolomic samples. Anal Chem. 2007;80(1):268–78.CrossRefGoogle Scholar
  21. 21.
    Francois I, Sandra K, Sandra P. History, evolution, and optimization aspects of comprehensive two-dimensional liquid chromatography. In: Mondello L, editor. Comprehensive chromatography in combination with mass spectrometry. Hoboken: Wiley; 2011. p. 281–330.CrossRefGoogle Scholar
  22. 22.
    West C, Elfakir C, Lafosse M. Porous graphitic carbon: a versatile stationary phase for liquid chromatography. J Chromatogr A. 2010;1217(19):3201–16.CrossRefGoogle Scholar
  23. 23.
    Leonhardt J, Hetzel T, Teutenberg T, Schmidt TC. Large volume injection of aqueous samples in nano liquid chromatography using serially coupled columns. Chromatographia. 2015;78(1–2):31–8.CrossRefGoogle Scholar
  24. 24.
    Li D, Dück R, Schmitz OJ. The advantage of mixed-mode separation in the first dimension of comprehensive two-dimensional liquid-chromatography. J Chromatogr A. 2014;1358:128–35.CrossRefGoogle Scholar
  25. 25.
    Semard G, Peulon-Agasse V, Bruchet A, Bouillon J-P, Cardinaël P. Convex hull: a new method to determine the separation space used and to optimize operating conditions for comprehensive two-dimensional gas chromatography. J Chromatogr A. 2010;1217(33):5449–54.CrossRefGoogle Scholar
  26. 26.
    Dück R, Sonderfeld H, Schmitz OJ. A simple method for the determination of peak distribution in comprehensive two-dimensional liquid chromatography. J Chromatogr A. 2012:69–75Google Scholar
  27. 27.
    Rutan SC, Davis JM, Carr PW. Fractional coverage metrics based on ecological home range for calculation of the effective peak capacity in comprehensive two-dimensional separations. J Chromatogr A. 2012;1255:267–76.CrossRefGoogle Scholar
  28. 28.
    Camenzuli M, Schoenmakers PJ. A new measure of orthogonality for multi-dimensional chromatography. Anal Chim Acta. 2014;838:93–101.CrossRefGoogle Scholar
  29. 29.
    Gilar M, Olivova P, Daly AE, Gebler JC. Orthogonality of separation in two-dimensional liquid chromatography. Anal Chem. 2005;77(19):6426–34.CrossRefGoogle Scholar
  30. 30.
    Gilar M, Fridrich J, Schure MR, Jaworski A. Comparison of orthogonality estimation methods for the two-dimensional separations of peptides. Anal Chem. 2012;84(20):8722–32.CrossRefGoogle Scholar

Copyright information

© Springer-Verlag Berlin Heidelberg 2016

Authors and Affiliations

  • Juri Leonhardt
    • 1
    • 2
  • Thorsten Teutenberg
    • 1
    Email author
  • Greta Buschmann
    • 2
  • Oliver Gassner
    • 1
  • Torsten C. Schmidt
    • 2
  1. 1.Institut für Energie- und Umwelttechnik e. V.IUTA (Institute of Energy and Environmental Technology)DuisburgGermany
  2. 2.Instrumental Analytical ChemistryUniversity of Duisburg-EssenEssenGermany

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