59Co : Temperature Dependent Chemical Shifts

⁵⁹Co is a very receptive, 100% naturally abundant, spin I = 7/2 quadrupolar nuclide with a chemical shift range spanning some 18,000 ppm.  The ⁵⁹Co NMR spectra of symmetric diamagnetic cobalt III complexes are characterized by relatively sharp resonances of a few Hz to tens of Hz.  The chemical shifts are extremely sensitive to temperature, pressure and solvent effects.  The temperature sensitivity of the chemical shift is largely due to the shortening or elongation of the chemical bonds between the cobalt and the surrounding ligands as a function of temperature.  The figure below shows ⁵⁹Co NMR spectra of K₃[Co(CN)₆] in D₂O on a 300 MHz NMR spectrometer collected as a function of temperature and time.  The spectrum in the bottom trace of the stacked plot was for a sample equilibrated at 21°C. The temperature was them set at 60°C and 80 single scan spectra were collected over a 9 minute period of time.  One can see that as the sample begins to warm up, the resonance moves to higher chemical shifts and broadens severely owing to a temperature gradient over the length of the sample.  As time passes and the temperature (read at the thermocouple in the probe) becomes stable, the chemical shift approaches a constant value while the line width narrows as the temperature gradient over the length of the sample becomes smaller.  The chemical shift change was measured to be 1.56 ppm/°C.  The data emphasize that temperature regulation is extremely important when collecting or reporting ⁵⁹Co NMR data.

1D 1H - 31P HOESY

2D Heteronuclear Overhauser Effect SpectroscopY (HOESY) is an effective way to determine whether or not a pair of heteronuclear spins are close to one another in space.  It is particularly effective for ¹H and ³¹P where both nuclides are 100% naturally abundant.  2D experiments, however, can be quite time consuming.  Alternatively, one can obtain 1D ¹H detected ¹H - ³¹P HOESY data to save data collection time.  When only one ³¹P resonance is present, the data can be obtained using nonselective ³¹P pulses.  An example of this, using the, using the pulse sequence from the reference¹ below, is shown in the figure.  The HOESY spectrum is on top while the simple ¹H spectrum is on the bottom.  One can see that heteronuclear ¹H - ³¹P NOE's are apparent on the bridging methylene protons and the ortho-aromatic protons.  Neither the meta- nor para-aromatic protons show significant heteronuclear NOE's.

1.  L.E. Combettes, P. Clausen-Thue, M.A. King, B. Odell, A.L. Thompson, V. Gouverneur and T.D.W. Claridge. Chem. Eur. J. 18, 13133 (2012).   

1D Selective 1H - 19F HOESY

2D Heteronuclear Overhauser Effect SpectroscopY (HOESY) is an effective way to determine whether or not a pair of heteronuclear spins are close to one another in space.  It is particularly effective for ¹H and ¹⁹F where both nuclides are 100% naturally abundant.  2D ¹⁹F detected ¹⁹F - ¹H HOESY data are typically obtained which provide all NOE correlations.  2D experiments, however,  can be quite time consuming, especially when only a few NOE correlations are sought after.  In such cases, 1D ¹H detected ¹H - ¹⁹F HOESY experiments¹ are very desirable and can save a great deal of time.  When only one ¹⁹F resonance is present, they can be obtained by using hard ¹⁹F pulses.  This was recently illustrated well by Dr. Michael Lumsden of Dalhousie University.  When more than one ¹⁹F resonance is present, one can use a selective ¹⁹F pulse and repeat the experiment selecting each type of fluorine.  An example of this is shown in the figure below.  Selective 1D ¹H detected ¹H - ¹⁹F HOESY spectra were collected for 2,3-difluoropyridine using a selective ¹⁹F pulse.  The simple ¹⁹F spectra are shown on the left with the selected ¹⁹F resonance color coded.  The upper two spectra on the right are the HOESY spectra while the spectrum on the bottom right is a simple ¹H spectrum.  One can see that when the fluorine in the 3-position is selected, there is a strong NOE to the nearest proton, C.  Alternatively, when the fluorine in the 2-position is selected, there are no strong NOE's as there are no adjacent protons.

1.  L.E. Combettes, P. Clausen-Thue, M.A. King, B. Odell, A.L. Thompson, V. Gouverneur and T.D.W. Claridge. Chem. Eur. J. 18, 13133 (2012).   

1H Decoupled 1H NMR Spectra

¹³C NMR spectra acquired with ¹H decoupling are particularly simple to interpret as every symmetrically unique carbon atom gives rise to a peak in the NMR spectrum.  One is usually able to simply count the number of carbons in a molecule by counting the peaks in the ¹³C NMR spectrum.  ¹H NMR spectra, on the other hand, are complicated by homonuclear ¹H - ¹H coupling such that many ¹H resonances are complex multiplets spread over a frequency range of some tens of Hz.  Furthermore, multiplets often overlap complicating the interpretation of the data.  Historically, this problem has been tackled by using higher and higher magnetic field strengths which disperse the chemical shifts over a wider frequency range without affecting the value of the coupling constants.  The effect is higher chemical shift resolution at higher fields.  In the limit of infinite field, the width of the ¹H multiplets would be insignificant with respect to the chemical shift differences and one would obtain ¹H NMR spectra containing essentially singlets.  Of course, we do not have access to infinite fields however, it would be very desirable to collect ¹H decoupled ¹H NMR spectra consisting of a singlet for each ¹H resonance, much like the ¹³C signals in proton decoupled ¹³C NMR spectra.  It is not possible to collect proton decoupled ¹H NMR spectra in the same way as it is to obtain proton decoupled ¹³C NMR spectra since one would have to both observe and decouple all of the protons at the same time.  There are however very clever techniques to obtain such pure shift ¹H spectra.^1,2  They are based on selective refocusing pulses applied simultaneously with weak field gradients and hard 180° pulses allowing all chemical shifts to be measured at the same time but from different slices of the column of sample in the NMR tube.  For each resonance, the coupling from all of the coupling partners is refocused simultaneously.  The data are collected in a conventional 2D matrix with an incremented evolution time.  An FID is constructed by concatenating a chunk from each of the individual 2D time domain signals.  The Fourier transform of the reconstructed FID is a ¹H decoupled ¹H NMR spectrum.  An example of this is shown in the figure below for a sample of menthol using a Bruker AVANCE II 300 MHz NMR spectrometer.³  The lower spectrum is the conventional ¹H NMR spectrum.  One can see that it consists of broad complex multiplets some of which overlap with one another.  The upper spectrum is the pure shift spectrum.  It is greatly simplified compared to the conventional spectrum in that all of the multiplets are collapsed into singlets and each of the 14 types of protons of menthol can be identified.

Obtaining such spectra comes at the cost of much reduced sensitivity and much greater data collection times.  There is however, interest in improving this with modifications in the sequence and the way in which data are collected.⁴

1.  Zangger and Sterk. J. Mag. Reson. 124, 486 (1997).
2. Aguilar, Faulkner, Nilsson and Morris. Angew. Chem. Int. Ed. 49, 3901 (2010).
3. Bruker User Library  http://www.bruker.com/ppg .
4. Castanar, Nolis, Virgili and Parella. Chem. Eur. J. 19, 17283 (2013).

2H NMR on a Bruker AVANCE Spectrometer

The acquisition of high resolution ²H NMR data on a Bruker AVANCE spectrometer is done differently than that for ¹³C, ³¹P or any other heteronucleus.  Most heteronuclear data are collected using a broadband amplifier, a broadband preamplifier and the high sensitivity coil of a broadband probe.  This configuration cannot be used to collect ²H NMR data as the broadband preamplifier on AVANCE spectrometers has a built in ²H stop filter.  There are at least two options for collecting ²H NMR data on a Bruker AVANCE spectrometer using a broadband NMR probe: one, requiring no re-cabling with low sensitivity and another, requiring some re-cabling with high sensitivity.  The low sensitivity option uses the 20W ²H amplifier (normally used for ²H gradient shimming), the lock preamplifier and the ²H lock coil of the probe.  Although convenient, since no re-cabling or reconfiguration is necessary, the sensitivity is low because the lock coil often has a very low filling factor and the 20W ²H amplifier has limited power.  This method can be used to observe ²H labelled compounds at high concentration where sensitivity is not an issue. The high sensitivity option uses the higher power  (300 W in my case) broadband amplifier, the lock preamplifier and the broadband coil of the probe tuned to ²H.  This method requires a bit of re-cabling and re-configuration but has a large sensitivity advantage.  It is suitable for cases where the deuterium is in low concentrations where sensitivity is an issue, for example to observe ²H at natural abundance or very low concentrations of ²H labelled compounds.  The figure below shows an example of both cases on a 500 MHz AVANCE spectrometer using a triple resonance (BB, ¹H, ³¹P) probe.  The sample is neat tap water where the ²H is at natural abundance (0.015 %).  The spectra were collected with 90° pulses, 2 sec recycle delays, 1.8 sec acquisition times and 128 scans.  The pulse programs used were zg2h and zg for the low and high sensitivity cases, respectively.

For this NMR probe, there is a 26 times gain in signal-to-noise ratio between the two methods.  This will depend strongly on the type of NMR probe used as the filling factor of the lock coil compared to the broadband coil must be taken into account.  

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.

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.

Dirty NMR Probes

In a previous post I emphasized the importance of cleaning the outside of your NMR tube before putting samples in the NMR magnet.  The "stuff" from your fingers (on the outside of your NMR tube) accumulates on the inside of the NMR probe coil inserts and can cause spinning problems, shimming problems and problems with inserting or ejecting samples.  Furthermore, the accumulation of "stuff" causes a significant background signal.  The figure below shows the top of an NMR probe before and after cleaning.

The probe was in use for several months before cleaning.  Notice the sticky black "stuff" present in the photo on the left.  Please take care in cleaning your NMR tubes before putting them in the spectrometer.

Determining 90° and 180° Soft Pulses

Many modern NMR pulse sequences (e.g. the 1D gradient selective NOESY experiment) depend on shaped pulses for selective excitation or inversion of specific resonances.  Since the width of the excitation profile of a shaped pulse is determined by its duration, the pulse duration is chosen by the user for the selectivity needed.  The longer the pulse, the higher the degree of selectivity.   Some spectrometer software will calculate the pulse duration based on a selected region in a spectrum containing the desired resonance for excitation.  The calculation depends on an initial pulse calibration.  The standard calibration method for hard pulses involves incrementing the pulse duration at a fixed power level.  The 90° pulse is at the first maximum and the 180° pulse is at the first null.  Since the duration of a the selective pulse is fixed by the desired selectivity, the 90° and 180° pulses must be found by varying the pulse power rather than the pulse duration.  The figure below shows the calibration for three different 50 msec shaped pulses on a Bruker AVANCE spectrometer.

An on-resonance water signal was observed as a function of pulse power using a selective one-pulse sequence.  The scale is in units of decibels of attenuation.  Maximum power is at -6 dB so the scale goes from low power on the left to higher power on the right.  The 90° and 180° pulses are indicated with arrows.  The intensity profiles are not sinusoidal due to the logarithmic dB scale.  The 90° and 180° pulses are separated by 6 dB of attenuation as expected.

Measurement of 13C 90° Pulses in Solids via Cross Polarization

The direct measurement of ¹³C 90° pulses in solids under MAS conditions by the conventional method suffers from the very low inherent sensitivity of ¹³C and is very time consuming due to the typically long ¹³C T₁'s.  These problems can be at least partially overcome by using ¹H - ¹³C cross polarization which has a potential four-fold sensitivity gain and also a time advantage as the repetition rate depends on the ¹H T₁ rather than the ¹³C T₁, the former typically being less than the latter by a factor of ten.  The 90° pulses are measured by carrying out the usual cross polarization contact which leaves the ¹³C magnetization along the -y axis.  The contact is followed by a ¹³C -x phased pulse (φ_-x) which rotates the magnetization towards the z axis in the -yz plane.  The acquisition follows with high power ¹H decoupling.  The sequence is illustrated in the figure below.

When φ_-x = 0°, one observes the usual positively phased CP spectrum.  As φ_-x is increased the signal decreases until φ_-x = 90° at which point the ¹³C magnetization is on the z axis and a null signal is observed.  As φ_-x is increased further, +y magnetization is created and a negative signal is observed until φ_-x = 180° at which point the magnetization is on the y axis and a maximum negative signal is observed, etc..... The 90° pulse can be read directly from the first null or 1/3 of the second null at 270°.  The vector diagrams and a typical measurement (where φ_-x was increased from 0.5 µsec to 20 µsec in 0.5 µsec steps) are illustrated in the figure below.

Getting (x,y) ASCII Data from TOPSPIN

TOPSPIN is able to export graphical data in a number of formats for import into word processing, presentation or graphics programs.  It is also able to export a spectrum into the ASCII JCAMP format for those programs able to import such data. Often, however, one would like to have simple ASCII data as (x,y) coordinates representing an NMR spectrum for use in other computer programs.  There are two ways to do this.  The simplest way is to process the raw data as usual, producing the real NMR spectrum in the "1r" file then run the au program "convbin2asc" by simply entering "convbin2asc" on the TOPSPIN command line.  An ASCII text file like the one shown below will be created in the same directory as the "1r" file with the name "ascii-spec.txt".

This file contains a one-line header followed by four columns of comma delimited text.  The first through fourth columns contain the point number, the intensity (y values), the frequency in Hz (x values) and the chemical shift in ppm (x values), respectively. These data are very easily imported into other programs.

As described in a previous post, another way to obtain a simple ASCII file is to process the raw data as usual and display the region of the spectrum for which you would like ASCII data.  Right-click within the spectrum window and select "Save Display Region To..." from the pop-up window.  Another window will open from which you should select "text file for use with other programs" and then click "OK".  A third window will open where you can input the name of the file you wish to create, the directory in which you would like to have it stored and whether or not you would like the imaginary data stored as well (usually only the real data are desired).  You must then click "OK".

A shorter way to accomplish this is to simply enter the command "totxt" in the TOPSPIN command line.  This will take you directly to the bottom window of the figure above.  After clicking "OK" a text file like the one shown below is created with the name you have chosen in the directory you have chosen.

The file contains a header with information including the number of data points, the chemical shift of the left-most point and the chemical shift of the right-most point.  The main body of the file consists of a single column of intensities (y values) which are easily imported into other programs.  The x values must be generated separately using the information in the header.

Educational NMR App for the iPad

Tim Burrow from the NMR Facility of the University of Toronto has released a free iPad app called "Learn NMR FID" highlighting the key concepts for processing NMR data.  The app presents the user with the FID and NMR spectrum of a two peak spectrum.  The controls of the app allow the user to interactively change the frequencies of and coupling between the resonances, the apodization function, phase, zero filling, noise, number of scans etc... while observing changes in the real and imaginary FIDs and Fourier transformed spectrum.

This is a great tool for new NMR users to investigate how changing parameters affects the time and frequency domain NMR data.  Tim has also released an iPad/iPhone app for power conversions for NMR pulses called "Attenuator".

This too is very useful.  Both apps are available at the Apple App Store.  Great job Tim!