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Tuesday, March 11, 2014

Chemical Exchange Agents to Simplify NMR Spectra

One can simplify 1H NMR spectra by eliminating exchangeable proton signals.  This is most commonly done by adding a drop or two of D2O to the NMR sample.  An example of this can be seen in a previous post.  The deuterium from the D2O replaces the exchangeable protons (-OH, -NH, -NH2, -COOH) of the sample and their 1H signals disappear.  The disadvantage of this technique is the introduction of a strong HDO signal which may overlap with other signals in the spectrum and thereby hinder the interpretation.

An alternative of the "D2O shake" is to add a drop or two of concentrated trifluoroacetic acid (TFA) to the sample.  TFA has a single exchangeable proton at ~ 14 - 16 ppm.  The -COOH proton of the TFA exchanges with the exchangeable protons in the sample.  The exchange rate is usually fast enough on the NMR time scale such that the resultant spectrum has a single broad resonance representing all of the exchangeable protons at a chemical shift between the chemical shift of the pure TFA and that of the exchangeable protons in the sample (usually >10 ppm depending on the sample and the amount of TFA added).  The broad peak at a shift > 10 ppm is not likely to overlap with other resonances in the spectrum and therefore will not hinder the interpretation of the data.  An example of the use of TFA is shown in the figure below.


The bottom spectrum is that of sucrose dissolved in DMSO-d6.  One can observe all of the -OH protons in addition to all of the other sugar protons.  The middle spectrum is that of pure TFA in DMSO-d6.  The -COOH resonance appears at ~ 15.6 ppm.  The top spectrum is that of sucrose in DMSO-d6 with a drop of TFA added.  One can see that all of the -OH protons of the sugar (highlighted in yellow) have combined with the -COOH resonance of the TFA yielding a single broad resonance at ~ 13 ppm as a result of the exchange.  In addition to moving the -OH resonances out of the way, one can see simplifications to the other sugar protons as the result of loosing the J coupling between the -OH protons and the remaining sugar protons.

A comparison of the use of TFA compared to D2O as an exchange agent is shown in the figure below.


Both methods produce similar results except that the spectrum with added D2O has a large HDO peak (off-scale in the figure) which overlaps with other signals.

Thursday, March 6, 2014

Variable Temperature to Improve NMR Resolution

Many millions of dollars have been spent on high field NMR magnets to improve both sensitivity and chemical shift dispersion.  Many younger NMR users have had the good fortune to use only high field spectrometers where chemical shift resolution is often not an issue.  These users are not familiar with some of the "tricks" used to improve resolution which were needed on lower field instruments where chemical shift resolution was frequently a problem.  With the current helium shortage and the increasing popularity of low field permanent magnet spectrometers, these "tricks" will again become more and more common.  Among them are; the use of paramagnetic chemical shift reagents, the use of aromatic solvents or solvent mixtures and the use of variable temperature.  In this post, I would like to demonstrate the incredible power of simply changing the temperature at which the NMR data are collected.

The 1H chemical shift is a sensitive parameter related to the conformation of a molecule.  In solution, small molecules may adopt a number of conformations whose populations depend on the potential energy profile.  Furthermore, the molecules are often in fast exchange between the available conformations and the observed chemical shift is the weighted average chemical shift of all of the conformations present.  As the temperature is changed, the populations of conformations are altered and the observed average chemical shift value may change.  The changes in chemical shifts at different temperatures are often enough to resolve resonance which may have overlapped with one another at room temperature.

The chemical shift of exchangeable protons ( -OH, -NH or NH2) depends dramatically on the degree of both inter-molecular and intra-molecular hydrogen bonding.  When molecules with exchangeable protons are dissolved in aprotic solvents, one is often able to observe the exchangeable protons as well as their associated J couplings.  Such is the case with sucrose dissolved in DMSO-d6 where all of the -OH protons can easily be observed.  When the temperature is changed, the populations of available conformations change and the degree of intra-molecular hydrogen bonding is affected with dramatic changes in the chemical shifts of the -OH resonances.  The figure below shows the anomeric and -OH region of the 500 MHz 1H NMR spectrum of sucrose in DMSO-d6 collected as a function of temperature.


All of the protons can be assigned with standard 2D NMR methods.  As the temperature is increased, the anomeric proton (1) moves to higher frequencies while the -OH protons (2-9) all move to lower frequencies to different extents.  Note that the resonances in the highlighted region of the spectrum at 21°C are overlapped with one another but at higher temperatures are fully resolved.  The resolution has increased by simply increasing the temperature.