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2D Experiments – Inverse Heteronuclear Correlation

Introduction

One of the big advances in NMR since its inception has been the introduction of 2-dimensional (2D) NMR experiments. Their introduction greatly increased the power of NMR for structural elucidation and broadened the range 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 which is introduced through the pulse sequences, resulting in a 2-dimensional array with two separate time evolutions – the direct measurement (t2) and the indirect (t1). A 2D Discrete Fourier Transform of the data generates a 2D spectrum with frequency axes F1 and F2. At high-field, the use of 2D NMR experiments has become routine and within the limits of practicality this should be the same for benchtop NMR.

Experimental Set-Up

An important family of 2D experiments are the heteronuclear  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 from the hydrogen nuclei are measured directly to maximise the sensitivity of the experiments. This allows heteronuclear correlations to be performed much faster than with a typical HETCOR experiment, reducing the time required from multiple hours to tens of minutes.

We illustrate these experiments using a 1M sample of the molecule gemfibrozil (figure 1, left), a medication used to treat abnormal blood lipid levels.

ME-HSQC (Multiplicity Edited Heteronuclear Single Quantum Coherence) Experiment

HSQC: This experiment provides a method to directly link hydrogen and carbon spectra. The signal from a hydrogen nucleus can be correlated to the signal from a carbon that it is directly bound to. In the HSQC spectrum this is exhibited as cross peaks (peaks that appear in both the carbon and hydrogen spectra). Signals in the 1D carbon spectrum with no cross peak are identified quickly as quaternary carbons.

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 are connected 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 CH2 peaks are negative (blue in figure 2). The ability to quickly identify the multiplicity is important for structural elucidation. The carbon spectrum in figure 2 shows only amplitude and not phase which is why the CH2 peaks still appear positive.

The full ME-HSQC spectrum for gemfibrozil is shown in figure 2 (above).

HMBC (Heteronuclear Multiple BondCorrelation) 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 a hydrocarbon chain; the ME-HSQC would show cross peaks between C1 and Ha, C2 and Hb and C3 and Hc. 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 the two 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 (above).

Conclusion

Heteronuclear correlation experiments allow users to determine the structure of completely unknown molecules by correlating 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.

Date: 12th June 2019

Category: Technical Article

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