X–PULSE
2D experiments Inverse Heteronuclear Correlation

Application Note 14

Introduction
One of the big advances in NMR since its inception
has been the introduction of 2-dimensional (2D) NMRexperiments. Their introduction greatly increased the powerof NMR for structural elucidation and broadened therange and complexity of problems that could be tackled.
These experiments are a series of 1-dimensional (1D)
experiments that differ only through a time increment whichis introduced through the pulse sequences, resulting in a2-dimensional array with two separate time evolutions - thedirect measurement (t2) and the indirect (t1). A 2D Discrete
Fourier Transform of the data generates a 2D spectrum withfrequency axes F1 and F2. At high-field, the use of 2D NMR
experiments has become routine and within the limits ofpracticality this should be the same for benchtop NMR.
Experimental Set-Up
An important family of 2D experiments are theheteronuclear correlations experiments. These experiments
provide correlations between hydrogen and carbon
signals and are crucial for determining the structure of an
unknown molecule. Here, we show two such experiments,
ME-HSQC and HMBC. These experiments are often
described as inverse experiments because the signals fromthe hydrogen nuclei are measured directly to maximise thesensitivity of the experiments. This allows heteronuclear
correlations to be performed much faster than with atypical HETCOR experiment, reducing the time required
from multiple hours to tens of minutes. We illustratethese experiments using a 1M sample of the molecule
gemfibrozil (figure 1), a medication used to treat abnormal
blood lipid levels.
ME-HSQC (Multiplicity Edited Heteronuclear Single QuantumCoherence) Experiment•	
HSQC: This experiment provides a method to directly linkhydrogen and carbon spectra. The signal from a hydrogennucleus can be correlated to the signal from a carbon that
it is directly bound to. In the HSQC spectrum this isexhibited as cross peaks (peaks that appear in boththe carbon and hydrogen spectra). Signals in the1D carbon spectrum with no cross peak areidentified quickly as quaternary carbons.

Figure 1: Chemical
structure of gemfibrozil

X–PULSE
2D experiments Inverse Heteronuclear Correlation

Application Note 14•	Multiplicity Edited: A popular extension to the basic HSQC experiment is the multiplicity edited experiment, sometimes
known as DEPT-edited. As with the HSQC experiment, the signals from carbon and hydrogen nuclei that are directly bound areconnected through a cross peak in the ME-HSQC spectrum. In addition, the multiplicity of the hydrocarbon group, i.e., CH3,CH2, CH, can be directly determined by the phase of the peak. The CH3 and CH peaks are positive (red in figure 2) and the CH2peaks are negative (blue in figure 2). The ability to quickly identify the multiplicity is important for structural elucidation. Thecarbon spectrum in figure 2 shows only amplitude and not phase which is why the CH2 peaks still appear positive. The full MEHSQC spectrum for gemfibrozil is shown in figure 2.

Figure 2: ME-HSQC for 1M gemfibrozil. 4 scans with total experimental time 35 mins. The numbers on the cross peaks correlate to thenumbered carbon positions in figure 1.

HMBC (Heteronuclear Multiple Bond Correlation) Experiments•	HMBC: The HSQC experiment provides valuable information about the hydrocarbon functional groups, which is usually
enough for verifying a known structure. However, it is not enough for the structural elucidation of an unknown. In general,structural elucidation requires more information about the connectivity along the carbon backbone of the molecule. The HMBC
experiment provides this information.	In this experiment, the signals from hydrogen nuclei are correlated with signals from carbon nuclei 2 and 3 bonds away.
If we consider part of a hydrocarbon chain;
C1 and Ha, C2 and Hb and C3 and Hc.

the ME-HSQC would show cross peaks between

The cross peak between C2 and Hb would be negative (blue). In the HMBC spectrum, the HSQC cross peaks would be suppressed
and instead cross peaks of C1 with Hb and Hc, C2 with Ha and Hc and C3 with Ha and Hb would be observed. The combination of thetwo spectra allow the NMR user to build up a picture of the carbon backbone structure and the associated hydrocarbon groups.
The full HMBC spectrum of gemfibrozil is shown in figure 3.

Figure 3: HMBC for 1M gemfibrozil. 16 scans with total experimental
time 1 hour 40 mins
Figure 3: HMBC for 1M gemfibrozil. 16 scans with total experimental time 1 hour 40 mins

Conclusion
Heteronuclear correlation experiments allow users to determine the structure of completely unknown molecules bycorrelating hydrogen atoms with the carbons they are bound to (ME-HSQC) and with carbons 2 and 3 bonds away
(HMBC). Inverse experiments such as HSQC and HMBC use the greater sensitivity of proton measurements to help
elucidate the structure of an unknown compound in under 3 hours.

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