University of Ottawa NMR Facility Web Site

Please feel free to make suggestions for future posts by emailing Glenn Facey.



Tuesday, September 12, 2017

Optimizing the Signal-to-Noise-Ratio with Spin-Noise Probe Tuning

We have all been taught to tune our NMR probes to maximize the pulse power delivered to our sample (or minimize the reflected power back to the amplifier).  This prevents damage to the amplifiers and minimizes the duration of 90° pulses at fixed power levels.  This is typically done with the spectrometer hardware (eg, "atmm" or "wobb" on a Bruker spectrometer), with a sweep generator and oscilloscope or a dedicated tuning device.  Tuning a probe in this way optimizes the transmission of rf to the sample, however, the NMR probe must also detect signals from the sample to be amplified and sent to the receiver.  The "receive" function uses a different electronic path compared to the "transmit" function.  Since the electronic paths for the "transmit" and "receive" functions are completely different, they are expected to have different tuning characteristics.  A probe optimized to transmit rf to the sample is not necessarily optimized to receive the rf NMR signal from the sample.  As a result, one may not be getting the optimum signal-to-noise-ratio with a probe tuned and matched in the conventional manner.  The question then arises as to how can we tune an NMR probe optimized to detect and receive the NMR signals from the sample.  This can be done by measuring a spin noise spectrum of the sample - using no rf pulses whatsoever.  It has been shown1 that a probe is optimized to detect and receive the NMR signals when one observes an inverted spin noise NMR signal from the sample.  Since the spin noise signal is measured without any pulses from the "transmit" function of the spectrometer, it depends only on the electronic path of the "receive" function. To tune a probe for optimum "receive" function, one must adjust the tuning frequency and matching of the probe followed by the measurement of a spin noise spectrum until an inverted spin noise signal is observed.   The figure below illustrates an example of this using a 2 mM sucrose solution in 90% H2O/10% D2O.
The proton channel of a 600 MHz cryoprobe on a Bruker AVANCE III HD NMR spectrometer was tuned and matched at 10 different frequencies using the "atmm" function of the spectrometer.  The tuning offset frequencies were measured using the "wobb" display of the spectrometer.  For each tuning offset frequency, a spin noise spectrum of water was measured using 64 power spectra collected in a a pseudo 2D scheme and summed to produce the spin noise spectrum displayed.  The spin noise spectrum for the probe optimized for the "transmit" function is highlighted in pink and the spin noise spectrum for the probe optimized for the "receive" function is highlighted in yellow.  For every tuning offset frequency, the 90° pulse was measured with the "pulsecal" routine of the spectrometer which uses this method.  As expected, the minimum 90° pulse is obtained for the probe tuned to optimize the "transmit" function.  With all pulses optimized, a 1H spectrum of the sample for each tuning offset frequency was measured using excitation sculpting as a means of solvent suppression (pulprog= zgesgp).  The sucrose signal at ~ 3.9 ppm is displayed in the figure.  The maximum signal intensity (highlighted in yellow) is obtained at a tuning offset frequency of -695 kHz corresponding closely to where the spin noise spectrum is inverted (-895 kHz).  The noise levels in the spectra were found to vary somewhat at higher tuning offset frequencies.  As a result, the maximum signal-to-noise-ratio (highlighted in yellow) was observed at a tuning offset frequency of -488 kHz.  This represents a 21% improvement in the signal-to-noise-ratio compared to that observed for a probe tuned in the conventional manner (highlighted in pink).  The degree of solvent suppression using excitation sculpting was also found to deteriorate at higher tuning offset frequencies.  In conclusion, one can obtain spectra with higher signal-to-noise-ratios by using a tuning offset frequency other than zero.  One expects the specific optimum tuning offset frequency to be probe, instrument and sample dependent.  This phenomenon is described much more elegantly in the reference below.

1.  M. Nausner, J. Schlagnitweit, V. Smrecki, X. Yang, A. Jerschow, N. Müller.  J. Mag. Res. 198, 73 (2009).

Friday, August 11, 2017

Boron Isotope Effects in Fluorine NMR Spectra

In previous posts on this BLOG, examples of  1H/2H and 12C/13C isotope effects were discussed.  The figure below shows an example of a 10B/11B isotope effect observed in the 19F NMR spectrum of tetrabutyl ammonium tetrafluorobarate.
The spectrum clearly shows two resonances separated by 0.05 ppm with an intensity ratio of approximately 20:80 corresponding to the natural abundances of  10B and 11B, respectively.  The low frequency resonance is due to 11BF4-.  Since  11B is a spin I = 3/2 nuclide we observe a 1:1:1:1 quartet with J = 1.25 Hz corresponding to the one bond 19F - 11B coupling.  The high frequency resonance is due to 10BF4-.  Since  10B is a spin I = 3 nuclide we observe a very poorly resolved 1:1:1:1:1:1:1 septet with J ~ 0.4 Hz corresponding to the one bond 19F - 10B coupling.  

Tuesday, July 4, 2017

Correcting NMR Spectra for Poor Shimming - Reference Deconvolution

The pleasingly symmetric and narrow Lorentzian resonances in a high resolution NMR spectrum are truly things of stunning beauty, appreciated by all NMR spectroscopists. Their majesty depends on the homogeneity of the NMR magnet around the sample. Inhomogeneous fields yield low resolution NMR spectra with broad asymmetric peaks pleasing no one. These repugnant, distasteful spectra are often obtained when automatic shimming routines are used on under-filled samples, samples with solids present (precipitates, floaters of suspended solids), samples with thermal gradients, poorly mixed samples etc…. Have you ever looked at such a spectrum and longed to recover the hidden beauty, resolution and information you know is present in the depths of its repulsive form? In many such cases reference deconvolution is a processing technique able to help. The distortions in a spectrum from an inhomogeneous magnetic field affect all peaks in the spectrum in the same way. The imperfect FID giving rise to the offensive spectrum, FIDexp(t), is essentially a perfect FID, FID(t) multiplied by an error function, E(t), resulting from the inhomogeneous field.

FIDexp(t) = FID(t) * E(t)

If we could find the error function and divide the experimental FID, by it, we could produce a perfect (or at least improved) FID, the Fourier transform of which would be a spectrum corrected from the effects of field inhomogeneity. In the reference deconvolution technique, one selects a high signal-to-noise ratio singlet peak as a reference in the experimental spectrum and sets all other points in the spectrum to zero. This spectrum is inverse Fourier transformed to produce an FID of the distorted singlet, FIDrexp(t). A synthetic FID, FIDrsyn(t) is constructed for what one would expect the time domain signal to be for a perfect reference peak (e.g. a sharp Lorentzian). The error function for the reference peak Er(t) is then determined by:

Er(t) = FIDrexp(t) / FIDrsyn(t)

Since all peaks in the experimental spectrum are affected equally by the inhomogeneity, E(t) = Er(t) and we can compute a corrected FID for the entire spectrum,

FID(t) = FIDexp(t) / Er(t)

The corrected FID is Fourier transformed yielding a much improved spectrum. This technique is available in newer NMR software processing packages and is particularly easy to implement in the MestReNova software package available to NMR users at the University of Ottawa. An example is shown in the figure below for a 300 MHz 1H NMR spectrum of a mixture of compounds.
The top two traces show portions of the spectrum obtained in a carefully shimmed magnet. The middle traces show the same portions of the spectrum obtained in a poorly shimmed magnet. The bottom traces were obtained by applying reference deconvolution to the spectrum obtained in the poorly shimmed magnet. Clearly, there is much improvement in the reference deconvoluted spectrum, allowing one to obtain much more information and recover some of the lost beauty. In fact, the corrected spectrum is very similar to the one obtained in the homogeneous field of a carefully shimmed magnet. The penalty paid is a lower signal-to-noise ratio, as the noise from the experimental reference signal is convolved into the error function which in turn gets convolved into the corrected spectrum. The loss in signal-to-noise ratio can be minimized by choosing a reference signal with a higher signal-to-noise ratio.

Friday, June 2, 2017

Testing an MAS Spin Detection Device

Recently, I had a problem with an MAS probe which would no longer allow measurement of an MAS spinning frequency.  I thought it might be instructive to describe the device and the steps I took to solve the problem.  An MAS spin detection device includes an IR LED source, an IR detector, a fiber-optic cable (split in two on one end), some electronics and an MAS speed controller.  Everything except the the MAS speed controller is shown in the figure below.
The spin detector is connected through a three-pin cable to the MAS speed controller from which it receives power and to which it sends information about the MAS rotor frequency.  The IR LED emitter inside the spin detector sends IR light through one leg of the split end of the fiber-optic cable.  The IR light passes through the fiber-optic cable where it is directed towards the bottom of the MAS rotor.  The position of the end of the fifer-optic cable with respect to the bottom of the rotor is very critical.  The IR light must strike the bottom to the rotor.  Half of the rotor bottom is marked with a black pen.  When the IR light strikes the dark side of the rotor, there is very little reflected IR light "seen" at the end of the fiber-optic cable near the rotor.  When the IR light strikes the white side of the rotor, most of the IR light is reflected back to the end of the fiber-optic cable and returned to the detector through one of the spit ends.  When the rotor is spinning an "off" - "on" binary pulse equal to the rotor spinning frequency is returned to the detector and sent to the MAS speed controller through the electronic cable.  If the device does not work, one possible problem could be that the fiber-optic cable is broken.  This can happen if the cable is fastened too tightly to a support rod in the probe.  It can be tested as shown in the figure below with a laser pointer or a flashlight.
Almost all of the light should pass through the fiber-optic cable.  Another possible problem could be a failing IR LED emitter.  Unfortunately, the IR light is not visible so you cannot just inspect it visually.  The IR light can however be detected by the front facing camera of an iPhone which does not have a built-in IR filter like the rear-facing camera.  Simply taking a picture with the front facing camera will indicate whether the emitter is working.  This is shown in the figure below, where the emitter is clearly visible when the spin detector receives power from the MAS speed controller but not visible when not connected to the MAS speed controller.
There could also be a problem with the detector.  This can be tested with a stroboscopic LED flashlight as shown in the figure below.
The strobe light is positioned at the end of the fiber-optic cable and one of the split ends is positioned at the detector.  When the spin detector is connected to the MAS speed controller, one should be able to observe the frequency of the strobe light on the rotor frequency display.  Other possible problems could be with the MAS speed controller or with the electronic cable.  I have two MAS probes for this instrument.  In one probe, the MAS spinning frequency could not be counted and in the other probe it could.  In the failing probe, the fiber-optic cable, IR LED emitter  and IR detector were all working properly.  The problem was with the connector on the cable between the spin detector and the MAS speed controller.  The connection was OK for one probe but not the other.  I will be happy if this post helps someone who may run into a similar problem.

Thursday, May 4, 2017

The Effect of the 2H Lock on Environmental Instability

The 2H lock of an NMR spectrometer continuously monitors the frequency of the 2H resonance of a deuterated solvent used to prepare the NMR sample.  If the frequency of the 2H resonance changes due to an environmental instability while the lock is engaged, a feedback mechanism is used to correct the magnetic field via a Bo shim coil, returning the 2H resonance to its original position.  It is very effective at reducing (if not eliminating) environmental instability from NMR data collected over periods of time spanning minutes or hours.  The effect of the lock is illustrated in the figure below.

Each panel in the figure represents a contour plot of a pseudo 2D 1H NMR data set for the residual protons of D2O on a Bruker Fourier 300 NMR spectrometer.  Each panel represents 2048 single-scan 1D 1H NMR spectra collected over 1.5 hours.  The data in the left-hand panel were collected during the day without the 2H lock.  The data in the center panel were collected in the middle of the night without the 2H lock.  The data in the right-hand panel were collected during the day using the 2H lock.  The 2H lock clearly compensates for environmental instability.  There was no student traffic in the lab during the collection of any of the data.  Outside of the lab are two construction sites which are busiest during the day and quieter at night.  This is reflected in a comparison between the left-hand and center panels of the figure.  The data collected in the middle of the night without the 2H lock show somewhat less instability compared to similar data collected during the day.      

Tuesday, April 25, 2017

HMBC vs. H2BC

NMR spectroscopy is an indispensable tool for assigning the structure of organic compounds.  One very useful method in the NMR toolbox is the Heteronuclear Multiple Bond Correlation (HMBC) experiment.  HMBC data are 1H detected and provide a 2D correlation map between 1H and 13C similar to HMQC or HSQC except that the correlations are between protons and carbons separated by two, three and sometimes even four bonds.  This long range information is very helpful in elucidating chemical structures, especially those with non-protonated carbons.  The problem, however with HMBC data is that the correlations depend only on the magnitude of the long-range 1H-13C coupling constants.  Two- or three- bond coupling constants are very similar in magnitude to one another and therefore it is not possible to distinguish between two- and three- bond correlations.  Also, since many long range 1H-13C coupling constants (including two-bond coupling constants) are near zero, some correlations may be absent.  These problems may make structure elucidation frustrating or impossible.  The Heteronuclear 2 Bond Correlation (H2BC) experiment1 provides an HMBC-like correlation map with (almost) exclusively two-bond 1H-13C correlations.  Unlike the correlations in the HMBC measurement, which rely exclusively on long range 1H-13C coupling constants, the 1H-13C correlations in the H2BC experiment rely on three-bond 3JH-H coupling between the protons on adjacent carbons.  It is a combined HMQC-COSY experiment.  The size of the H2BC correlations depends on the magnitude of the 3JH-H coupling constant.  Three-bond 1H-13C correlations are possible only if four-bond 4JH-H coupling is significant.  One disadvantage to the H2BC experiment is that all correlations between protons and non-protonated carbons are necessarily absent because of the absence of H-H coupling.  In general, two-bond 1H-13C correlations that are weak or absent in HMBC spectra are strong in H2BC spectra and three-bond 1H-13C correlations which are strong in HMBC spectra are absent or very weak in H2BC spectra.  The techniques are very complimentary.  The figure below illustrates  the complimentary nature of the two methods for styrene.

The HMBC spectrum in the left panel was scaled up until some of the HMQC artifacts (color coded in blue) were visible.  The data show only one 2-bond 1H-13C correlation (color coded in pink). The three-bond 1H-13C correlations are color coded in yellow.  In comparison, the H2BC spectrum in the right panel shows exclusively two-bond 1H-13C correlations with the exception of those involving the C1 non-protonated carbon.

1. Nyberg, Duus, Sorensen.  J. Am. Chem. Soc. 127, 6154 (2005).

Tuesday, January 24, 2017

Improved 1H Resolution with 14N Decoupling

The J coupling between 13C and quadrupolar nuclides can be resolved, for example, in the cases of the 13C NMR spectra of deuterated compounds, some cobalt complexes and some tetraalkyl ammonium salts.  The ability to resolve the coupling depends on the relaxation rates among the Zeeman levels of the quadrupolar nuclide with respect to the reciprocal coupling constant.  When the relaxation is slow, the J coupling can be resolved and when it is very fast, the 13C is a sharp singlet and said to be "self decoupled".  When the relaxation rates among the Zeeman levels of the quadrupolar nuclide are on the same order of the coupling constant, the NMR resonance of the 13C will be broadened.  This is a very common observation for the 13C resonances of nitrogen bearing carbons.  It is also possible to see broadened 1H or 19F resonances due to coupling to 14N.  Such is the case for the resonances of the proton on C6 and the fluorine on C2 in 2,3-difluoropyridine as can be seen from the figure below which clearly shows these resonances broadened compared to the resonances of 1H or 19F further removed from the nitrogen.
The broadening of the resonance of the 1H on C6 can be reduced by applying 14N decoupling during the acquisition time, thus providing much improved resolution.  This is  demonstrated in the figure below.

Monday, January 23, 2017

PSYCHE to Evaluate 1H-19F Coupling Constants

Even small molecules can yield very complex 1H NMR spectra as the result of spin - spin coupling.  This is particularly true for small molecules that contain fluorine.  It can sometimes be challenging to determine which splittings are due to 1H-1H coupling and which are due to 1H-19F coupling.  One can collect a 1H spectrum with 19F decoupling to give a spectrum with only 1H-1H coupling present.  Even with this data, it may be difficult to evaluate the 1H-19F coupling constants by comparing the 1H[19F] spectrum to the 1H spectrum due to the complexity of the multiplets.  The 1H-19F coupling constants can however be read directly from a 1H PSYCHE spectrum.  The PSYCHE spectrum provides a 1H decoupled 1H spectrum, leaving only the 1H-19F coupling behind.  The bottom trace of the figure below shows the 300 MHz 1H NMR spectrum of 2,3-difluoro pyridine.  The spectrum is quite complex, making it difficult to assign 1H-1H and 1H-19F couplings.  The middle trace shows the 1H[19F] spectrum which allows the evaluation of all of the 1H-1H coupling constants (3JH5-H4 = 4.8 Hz, 4JH5-H3 = 1.6 Hz and 3JH4-H3 = 8.0 Hz.  The top trace shows the 1H PSYCHE spectrum which allows one to evaluate all of the 1H-19F coupling constants.  For this compound, 4JH3-F1 = 3JH3-F2 = 9.8 Hz, 4JH4-F2 = 3.2 Hz and 4JH5-F1 = 1.8 Hz.

Friday, January 20, 2017

Pure Shift 1H NMR - PSYCHE

Much effort has been directed to obtain broadband 1H decoupled 1H NMR spectra.  The subject has been reviewed recently.1  One technique used to obtain such spectra is the pseudo-2D Zangger - Sterk method2,3 based on a selective refocusing pulse applied simultaneously with a weak field gradient centered in the t1 evolution period 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.  The data are collected in a conventional 2D matrix however, a single 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 pure shift, 1H decoupled 1H NMR spectrum.  The PSYCHE (Pure Shift Yielded by CHirp Excitation) modification4 of the Zangger - Sterk method uses a pair of small flip angle, frequency swept chirp pulses rather than a selective 180° pulse applied simultaneously with the weak spatially selective field gradient.  This modification offers improved sensitivity.  The details of implementing this technique are kindly provided on-line by the Manchester NMR Methodology Group.  As an example, the figure below shows the 600 MHz PSYCHE spectrum of sucrose in in DMSO-d6, collected in less than 3 minutes.  One can observe the collapse of all multiplets into singlets.
The PSYCHE technique can dramatically simplify complex 1H NMR spectra as shown in the figures below. The second figure is an expansion of the low frequency region of the first.


1.  Castañar and Parella. Mag. Res. Chem. 53, 399 (2015).
2.  Zangger and Sterk. J. Mag. Reson. 124, 486 (1997).
3.  Aguilar, Faulkner, Nilsson and Morris. Angew. Chem. Int. Ed. 49, 3901 (2010)
4.  Foroozandeh, Adams, Meharry, Jeannerat, Nilsson, Morris. Angew. Chem. Int. Ed. 53, 6990 (2014).

Thursday, January 12, 2017

Exchange Effects in HSQC Spectra

The effects of chemical or dynamic exchange on NMR spectra are very well known.  Exchange is often studied by observing line shape changes as a function of temperature, by 2d EXSY, inversion transfer or saturation transfer methods.  Effects due to exchange can also be observed in 1H - 13C HSQC spectra.  The HSQC method works by transferring 1H magnetization to 13C magnetization via an INEPT transfer through the one-bond J coupling across the 1H - 13C chemical bond.  The 13C magnetization evolves during the incremented delay, t1, of the 2D pulse sequence according to its chemical shift.  The 13C magnetization is then transferred back to 1H magnetization where is observed during t2.  HSQC spectra thus exhibit cross peaks between 1H resonances and the resonances of their attached carbons.  If there is exchange between nonequivalent carbon sites during t1, some 1H resonances may appear to be correlated to two carbon resonances.  An example of this is shown in the figure below.
The 13C spectrum of cannabidiol has equally intense broad, resolved aromatic resonances for non-protonated carbons 2 and 6 (not shown) as well as for the protonated carbons 3 and 5.  The 1H spectrum has broad resolved resonances for both aromatic protons.  This indicates that either the aromatic ring undergoes 180° flips about the 1 - 4 axis or it has two equally probable rotomers defined by a rotation about the 1 - 4 axis.  In either case, the dynamic exchange is slow enough on the NMR time scale to produce resolved resonances yet fast enough to cause significant line broadening.  For each of the two aromatic protons, the HSQC spectrum shows correlations to both C3 and C5; a strong correlation to the carbon to which it is chemically bonded and a weaker correlation to the carbon site in exchange with its attached carbon.