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.

Temperature Calibration - An Alternative Method

It is well known that the actual temperature of a sample in an NMR probe is not necessarily the same as that read from the variable temperature unit on the spectrometer. This is because the thermocouple used by the variable temperature unit is below the sample tube and not in the center of the rf coil where the NMR measurements are made. One normally must make a calibration plot for the actual temperature vs. the set temperature. For temperatures above room temperature this can be done by employing the known temperature dependent chemical shift difference between the two proton resonances of ethylene glycol (see this link). At temperatures below room temperature, the same measurement can be made for the known temperature dependent chemical shift difference between the two proton resonances of methanol. The actual temperature is determined from the chemical shift difference and plotted against the temperature read from the variable temperature unit. One potential problem with this method is that the resistance of the magnet shim coils change slightly with temperature affecting the shim currents and the NMR line shapes of the resonances. This makes it difficult to measure a precise chemical shift difference. In order to obtain reliable results, the magnet must be reshimmed at each temperature.

I have used is a very simple alternative method for calibrating the temperature of the sample compared to that of the variable temperature unit. This is illustrated in the figure below.A "sample" is prepared by pushing a NONMAGNETIC thermocouple through an NMR tube cap. The depth of the thermocouple is adjusted such that when the cap is put on the NMR tube, the tip of the thermocouple sits in the center of the rf coil. The NMR tube should contain a suitable liquid filled to the correct depth. The tube is placed in the spinner and set to the proper depth with a depth gauge. While holding onto the thermocouple, the sample is lowered into the magnet until it sits correctly in the NMR probe. The thermocouple is connected to a digital thermometer (some of these devices can use a second thermocouple in an ice water bath as a reference). The desired temperature is set on the variable temperature unit. When the temperature on both the variable temperature unit and digital thermometer have stabilized (~ 10 minutes), the values from each are recorded. This is repeated for temperatures over the desired temperature range and a calibration plot is constructed. Shimming is not an issue. Note that no NMR measurements are made and that the sample tube is not spinning.

Field Homogeneity and VT Gas

In order to obtain optimum resolution, NMR spectroscopists always correct the inhomogeneity of the magnetic field around the sample by adjusting the current in the the room temperature shim coils. The magnetic field homogeneity around the sample depends not only on the quality of the superconducting magnet but also on the magnetic susceptibility of the materials in the vicinity of the coil and the sample. The careful selection of materials in probe manufacturing and their use around the coil are essential for being able to produce a homogeneous field in the vicinity of the sample using the shim coils. This is one of the reasons why high resolution NMR probes are very expensive. One "material" near the coil which is often overlooked by the NMR user is the VT (variable temperature) gas being passed over the sample. The two most common VT gasses are air and nitrogen. One might think that these are very similar to one another as dry air is approximately 80% nitrogen. The magnetic susceptibility between the two however, is quite large and they will distort the magnetic field around the sample to differing extents. This is demonstrated in the figure below. A sample of CHCl₃ in acetone-d₆ was placed in a 500 MHz magnet equipped with a probe using air as the VT gas. The magnet was shimmed and the spectrum acquired is shown in the top trace. The air source was then replaced by a source of nitrogen gas at the same flow rate. The spectrum was measured again without re-shimming the magnet and is displayed in the lower trace. The difference in line shape and width is due to the difference in magnetic susceptibilities between the two gases. It should be noted that a spectrum of similar quality to the one obtained using air can be obtained after re-shimming the magnet to correct for the susceptibility difference.

Variable Temperature NMR - Thermal Equilibrium

When doing variable temperature NMR, students often ask me how long they should wait for thermal equilibrium in their sample before collecting NMR data. The answer depends of course on the amount of gas flow around the sample and the temperature difference between the current and desired sample temperature. The position of the thermocouple in an NMR probe is typically right below the sample. It takes time between when the thermocouple reports the desired temperature and when the sample is at the desired temperature. During this time there is a large thermal gradient across the sample as well as convection currents which will affect the line width of NMR resonances. These effects are demonstrated in the figure below. For this measurement, the temperature of the probe was set to 50°C with an air flow of 800 L/hour. Once the thermocouple read 50°C, a sample of D₂O was placed in the probe and 30 minutes was allowed to pass, after which the sample was presumed to be at thermal equilibrium. The lock was established and the magnet was then shimmed. The sample was removed and allowed to sit at room temperature for 30 minutes. It was then reintroduced to the probe at 50°C. ¹H NMR spectra of the residual HDO were then collected at 30 second intervals for a period of 10 minutes. As soon as the room temperature sample is reintroduced to the warm probe, it begins to warm up. During the this time, the thermal gradients and convection currents are large and the line width is adversely affected. As the sample temperature approaches 50°C the thermal gradients are smaller and the line becomes narrower. After approximately 6 minutes the width of the line changes very little. The sample appears to be at thermal equilibrium after 10 minutes.

Temperature Calibration in an NMR Probe

Often it is desirable to collect NMR data at temperatures other than ambient temperature. Most NMR spectrometers are equipped with a variable temperature accessory. The user sets the desired temperature and the variable temperature unit regulates the temperature by continually adjusting the current in a resistive heater within the probe. The probe heater is inside a dewar into which a gas (air or nitrogen) is directed. The gas must be cooler than the desired temperature. It is heated by the heater and directed over the sample tube. The temperature is typically measured at the bottom of the NMR tube with a thermocouple. The set temperature may be different than the true temperature of the sample in the coil due to thermal losses, poor thermocouple calibration, or undesirable gas flow characteristics. Although the precision of the set temperature is typically 0.1 degrees Celsius, the accuracy (i.e. difference between the set temperature and the true temperature) can be several degrees Celsius. The problem is then to know the true temperature of the sample in the coil. For temperatures above room temperature, this can be done by collecting proton NMR spectra of ethylene glycol as a function of the set temperature. The chemical shift difference between the -OH and methylene protons is linearly dependant on temperature (Stefan Berger and Siegmar Braun, 200 and more NMR Experiments (2004), p. 146) and can be used to construct a calibration plot for the set temperature vs. the true temperature. The figure below shows such data collected as a function of temperature. Methanol shows similar behavior and is commonly used to calibrate temperatures below room temperature.