, Volume 404, Issue 5, pp 1427-1437
Date: 19 Jul 2012

Resolving the problem of chromatographic overlap by 3D cross correlation (3DCC) processing of LC, MS and NMR data for characterization of complex glycan mixtures

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Chromatographic overlap is a common problem in the analysis of complex mixtures. As a result, it is not possible to identify the components because each resulting NMR or MS spectrum contains multiple components. We introduce three-dimensional cross correlation (3DCC) that dissects NMR spectra of a mixture into spectra of the individual components without actually separating them. Correlation of peaks from MS and NMR profiles along a common LC time domain yields 3DCC NMR spectra of pure components correlated with a mass and a retention time. The method requires an LC run followed by fractionation and recording of MS and NMR spectra. The method is applicable to mixtures of any classes of molecules. Here, we demonstrate its application to a mixture of complex glycans obtained from a glycoprotein. Fourteen glycans eluting within only 3 min showed heavy overlap in the chromatographic run. 3DCC allowed their direct characterization without separation. Some of these structures from the glycoprotein bovine fibrinogen had not previously been described. The 3DCC procedure has been implemented in standard software. Actually, 3DCC can be used for any combination of separation techniques, like LC or GC, combined with two characterization methods like UV, IR, Raman, NMR or MS.


LC runs of complex mixtures often result in overlap of several analytes and prohibit thereby characterization by NMR. The new 3DCC method allows extraction of pure NMR spectra directly from mixtures by correlating NMR and MS data from an LC run.

Henning N. Behnken and Meike Fellenberg contributed equally to this work.