Researchers from Ecole Normale Supérieure in Paris, France develop novel two-field NMR spectrometer.
Advances in technology have meant that the strength of magnetic fields used in nuclear magnetic resonance (NMR) spectroscopy has continually increased. This has resulted in major improvements in the resolution and sensitivity available to researchers. Indeed, some research institutions have begun to use ultra-high field NMR involving one-GHz magnets.
However, as magnetic field strength increases, so too does the likelihood of severe line broadening in NMR. This effect is caused by chemical exchange or relaxation by chemical shift anisotropy (CSA).
The first of these, chemical exchange, is common in biological macromolecules. This occurs when a biomolecule can exist in more than one conformation or interacts with another molecule, resulting in it shifting between conformations over time.
When the rate at which the molecule switches conformations is slower than the rate of the NMR experiment, separate peaks will form representing the molecules that were in each state during the experiment. But when the rate of exchange increases, this can result in individual molecules switching conformation during the course of the experiment, so the peaks begin to reflect the amount of time that the molecules spent in each conformation. This results in peak broadening. Depending on the rate of exchange and the nature of the conformations, it may produce just one peak that is an average of the conformations, or it may produce a peak so broad it is undetectable.
Chemical shift anisotropy is caused by the fact that the chemical shift of a nucleus is dependent on its orientation in relation to the magnetic field. The fluctuations of this effect due to molecular motions cause relaxation, resulting in line broadening. In macromolecules, line broadening from CSA increases as the square of the magnetic field strength. For some nuclei, this can actually mean high-field spectrometers are a disadvantage. For example, 13C nuclei in carbonyl groups suffers from fast relaxation at high fields due to CSA.
Both the issues of chemical exchange and CSA are exacerbated at high magnetic fields. This has meant that researchers have typically had to choose between the enhanced intrinsic signal intensity and resolution offered by high field strengths for some nuclei and the better line widths and relaxation rates available at lower fields for other nuclei.
In an attempt to overcome this conundrum, a team of researchers modified a 600MHz (14.1T) NMR spectrometer to allow multiple fields to be explored in one experiment.
They devised a two-field NMR spectrometer containing two magnetic centers. To do this, they used a shimming system to generate a plateau of magnetic field at 0.33 T in the stray field of the 14.1 T superconducting magnet, and equipped the system with a low-field triple-resonance probe. A pneumatic system shuttles the sample between the two field strengths in a time of ~100 ms.
The researchers applied a technique, which they refer to as two-field heteronuclear zero-quantum correlation (2F-HZQC), to correlate zero-quantum 13C–1H coherences at low field with single-quantum 1H coherences at high field. This approach is intended to minimize the effect of magnetic field inhomogeneities on line broadening, as linewidths of single-quantum coherences are directly proportional to overall field homogeneity.
The team, led by Fabien Ferrage from Ecole Normale Supérieure in Paris, France, found that the technique allowed them to maintain the high levels of sensitivity and resolution associated with high magnetic fields, while significantly reducing exchange-related line broadening seen at low fields.
For example, they studied a compound called triazene, which contains two exchanging methyl groups and one non-exchanging methoxy group. They showed that at 14.1 T, as they increased the temperature, and therefore the rate of exchange, the resonances of the two exchanging methyl groups became so broadened as to become undetectable. By contrast, the peak of the resonance of the non-exchanging methoxy group was over 1000 times more intense at a temperature of 31°C.
They repeated the experiment using the two-field correlation. This time around, the signal from the exchanging methyl group was easily seen and was barely affected as they raised the temperature from 21°C to 26°C through to 31°C.
Ferrage and colleagues say that the approach could help overcome the limitations of CSA by harnessing the benefits of high-field NMR as well as the advantageous properties of low fields. For example, the optimal magnetic field to minimize relaxation of 13C nuclei in carbonyl groups in proteins is likely to be between 2 T and 5 T. As a comparision, for amide 15N-1H pairs, the optimal field is closer to one-GHz. Two-field NMR spectroscopy, the authors explain, means that spins such as 15N, 13C or 31P can be manipulated in the field most appropriate for them, while others like 1H, or other 13C can be detected at higher fields, to make use of the greatest resolution and sensitivity.
What’s more, they say that almost any high-field NMR system could be turned into a two-field spectrometer, provided that the low-field center is below 0.5 T.
The team, who report their findings in Angewandte Chemie International Edition, conclude that the study “marks the beginning of a new generation of multiple-field NMR experiments, and opens the way to the characterization of a wide variety of systems.”
- Cousin SF, Kaderavek P, Haddou B, Charlier C, et al. Recovering Invisible Signals by Two-Field NMR Spectroscopy. Angewandte Chemie International Edition 2016; 55: 9886-9889.
- Higman VA, 2012. Chemical Exchange. Available at: http://www.protein-nmr.org.uk/general/chemical-shifts/chemical-exchange/
- Reich H, 2016. Relaxation in NMR Spectroscopy. Available at: https://www.chem.wisc.edu/areas/reich/nmr/08-tech-01-relax.htm