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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.

6 comments:

Fred said...

Very cool stuff. Recently Kupce and Freeman showed that such VT data could be processed with a Radon transform to generate 2D NMR spectra with chemical shifts and chemical shift temperature dependence on the other axis. In that way the whole set of data could be used to get the best possible resolution of the peaks. (JACS 135 2871)

Glenn Facey said...

Fred,

Yes. I saw Freeman give a great talk at the last ENC about that. Thanks for the reference though. I have not read it yet.

Glenn

Anonymous said...

Dear Dr Facey,

May I ask the influence of T1 and T2 on signal width and intensity with reagards to temperature for small molecules and large molecules?

Glenn Facey said...

Dear Anonymous,

Thank you for the questions.

The line width at half height for an NMR resonance for a dissolved substance (in the absence of exchange or quadrupolar effects) is 1/(pi*T2). A plot of log T2 vs correlation time first decreases and then comes to a plateau at longer correlation times. The correlation time is defined as the time a molecule takes to rotate by one radian. At higher temperatures the correlation time is shorter and at lower temperatures the correlation time is longer. Therefore at as the temperature is decreased one expects the correlation time to increase and the T2 to decrease. Since the line width at half height is 1/(pi*T2), the line width increases as temperature is decreased.

For a given temperature, larger molecules have a longer correlation time than smaller molecules and therefore larger molecules have wider lines than smaller molecules at the same temperature.

Macromolecules such as large proteins generally have correlation times in the plateau region of the log T2 vs correlation time plot and have line widths less sensitive to temperature.

It is always true that T1 is greater than or equal to T2. For many small molecules T1 is approximately equal to T2 and line width estimates can be made based on T1 (or visa versa) in such cases. For macromolecules or solids T2 is much shorter than T1 and estimates of line width based on T1 cannot be made.

The area of an NMR resonance is constant regardless of line width. If you define intensity as the "height" of an NMR line then the intensity decreases for shorter T2's. I do not recommend that you define "intensity" in this way.

I hope this helps.

Glenn

Dammy said...

Thanks that was really helpful. I have a question though. Could it be d same reason why for my DNA proton NMR at room temp The DOH water region signal had an overlap with the 3' regions of d sugars in the DNA, but when I increase the temp to 35degree celcius the water signal seperated from the the 3' signals and I was able to assign ghat region. Can you give a reason why that happened? If not the same reason you already discussed. Thanks

Glenn Facey said...

Dammy,

The chemical shifts for exchangeable proton signals are very dependent on the degree of hydrogen bonding, concentration, pH and exchange with other exchangeable protons. Two of these effects are altered by changing the temperature. Any or all of them may affect your spectrum.

See also:

http://u-of-o-nmr-facility.blogspot.ca/2014/03/chemical-exchange-agents-to-simplify.html

http://u-of-o-nmr-facility.blogspot.ca/2012/03/protic-samples-in-aprotic-solvents.html

Glenn