4.6. 2D 1H-13C HSQC-TOCSY-Band Selective Experiments

2D ¹H-¹³C HSQC-TOCSY is a hybrid NMR experiment which combines the HSQC and TOCSY pulse sequences in order to relay the magnetization along with a proton network, using the greater dispersion of the ¹³C chemical shifts. This experiment is very useful to elucidate assignments in overcrowded ¹H-NMR spectra since its row, for a particular ¹³C chemical shift, contains a ¹H TOCSY sub-spectrum of the molecular fragment. The 2D ¹H-¹³C HSQC-TOCSY experiment has been successfully employed to correlate carbon signals with the adjacent allylic protons of unsaturated fatty acids [89,90] and to analyze the positional distribution of unsaturated chains in TAG model compounds [91]. Of particular interest is the application of gHSQC-TOCSY experiment for mixture analysis in DAG oils [59]. Carbon - proton pairs connected over two bonds were observed for the allylic and bis-allylic carbons of the unsaturated chains which resulted in the unambiguous assignment of several carbons of the acyl chains of oleic, linoleic and linolenic acids (Figure 20). For example, with the use of the pairs H7, C17 of linolenic acid (LN) direct assignment of the allylic carbons C17, C16, C14, C11 and C8 could be achieved with a mixing time of 80 ms (for the notation system for ¹H resonances see the original article).

600 MHz gHSQC-TOCSY spectrum of DAG oil in CDCl₃ solution, showing consecutive connectivities between carbons and protons along a common coupling pathway (for the notation system for ¹H resonances see the original article). Adopted, with permission, from [59]. Copyright 2011, Springer AOCS.

A significant increase in the digital resolution in the ¹³C dimension can be achieved with the use of band selective ¹H-¹³C HSQC-TOCSY experiment. The method has been successfully applied by Willker et al. [86] to investigate multiple ¹³C labeled biofluids and to identify and quantify blood plasma lipids [89] (see Section 5.6.4).