Removing t1 Noise from Homoonuclear 2D NMR Data - Video Tutorial

The often troublesome stripes of vertical noise in 2D NMR spectra are called t1 noise (i.e. noise originating in the t1 domain). When t1 noise occurs in homoonuclear 2D correlation experiments such as COSY, TOCSY, NOESY or ROESY, symmetrization can be used to remove a great deal of the noise and make the data more presentable. The technique was described in a previous post and is demonstrated in this video tutorial using a magnitude COSY spectrum as an example.

Cable Length and Probe Tuning

NMR probes can be tuned and matched on the bench or while in the magnet using a sweep generator and oscilloscope or a specialized tuning box such as the one available through Morris Instruments.  More typically, probe tuning and matching are monitored using the electronics in the NMR console and preamplifier.  In either case, it is important to realize that any filter or cable between the preamplifier and the probe is part of the rf circuit being tuned and should therefore be present while adjusting the tuning and matching capacitors of the probe.  This is illustrated in the figure below.  An NMR probe was tuned and matched using the wobble function of a Bruker AVANCE spectrometer. There was a bandpass filter and a short cable between the preamplifier and the probe.  The tuning curve is shown in the top of the figure.  The 90° pulse was measured at 11.25 μsec. The short cable was then replaced with a long cable and the probe tuning and matching capacitors were left unchanged.  The tuning curve is shown in the bottom of the figure below.  It is clear that the overall circuit is no longer tuned and matched.  The 90° pulse was measured at 13.75 μsec using the long cable.

Removing t1 Noise from Heteronuclear 2D NMR Data - Video Tutorial

The often troublesome stripes of vertical noise in 2D NMR spectra are called t1 noise (i.e. noise originating in the t1 domain). When t1 noise occurs in hereronuclear 2D correlation experiments such as HMBC, HSQC, HMQC or HOESY, there is a simple trick to remove a great deal of the noise and make the data more presentable. The technique was described in a previous post and is demonstrated in this video tutorial using a 19F - 1H HOESY spectrum as an example.

Exponential Line Broadening - Video Tutorial

Exponential line broadening is an important NMR data processing tool.  It involves multiplying the time domain signal by a decaying exponential function prior to Fourier transforming the data into the frequency domain.  It is used to improve the signal-to-noise ratio and is more fully described in a previous post.  The following short tutorial video demonstrates its use.

Phasing a 2D NMR Spectrum - Video Tutorial

The following video demonstrates how to phase a 2D NMR spectrum in TOPSPIN 3.

Thermal Noise in NMR Data

The University of Ottawa has recently been funded for a 600 MHz NMR spectrometer with a cryogenetically cooled probe.  Cryoprobes differ from conventional NMR probes in that the rf circuits and preamplifiers are cooled with cold helium gas while the sample is maintained at ambient temperature.  The benefit of cryogenically cooled electronics compared to room temperature electronics is that the thermal noise in the system is reduced at cryogenic temperatures while the NMR signal remains constant for the sample at ambient temperature.  The signal-to-noise ratio in an NMR spectrum acquired in a cryoprobe is therefore increased dramatically compared to a conventional probe, typically by a factor of 4.  This allows for data collection times on the order of 16 times shorter than those using conventional probes as well as lower detection limits.  This principle can be crudely demonstrated by replacing the NMR probe with a 50 Ω  load and collecting "NMR" data on the load at both high and low temperatures.  The "NMR spectra" in the figure below were collected (without using an rf pulse) on a 50 Ω load outside of the magnet at room temperature (left panel) and in a dewar of liquid nitrogen at 77 K (right panel).  The noise collected in the 77 K spectrum is 35% lower than that in the room temperature spectrum demonstrating the lower thermal noise at lower temperatures.

This effect is dramatically increased in a crypoprobe which cools the electronics of both the rf probe circuits and preamplifiers to temperatures much lower than 77 K.

Receiver Gain and Signal-to-Noise Ratio

The signal-to-noise ratio in an NMR spectrum can be affected drastically the choice of the receiver gain setting, so care should be taken to set the receiver gain correctly for optimum results.  At very low receiver gain settings, both the signal and the noise use only a fraction of the available digitization levels of the analog-to-digital concertor (ADC).  As a result, the intensity of each point in the FID is represented with only a few possible values and the FID is "choppy".  This is analogous to a black and white photograph being represented with a coarse gray scale of only a few shades of gray.  Just like such a poorly represented photograph, the NMR spectrum contains a great deal of digital noise and therefore a low signal-to-noise ratio.  As the receiver gain is increased, the FID is digitized with more available digitization levels.  Since the thermal noise in the FID at low receiver gain settings is smaller than or comaparable the size of the digitization step of the ADC, the noise (unlike the signal) will not be amplified by increasing the receiver gain until it exceeds the size of the digitization step of the ADC after which it will be amplified in the same way as the signal.  As a result, the signal increases more so than the noise as the receiver gain setting is increased therefore, the signal-to-noise ratio in the NMR spectrum increases steadily as the receiver gain setting is increased.  As the receiver gain is increased beyond the point where the thermal noise exceeds the size of the digitization step, both the sginal and the noise can be digitized properly and the signal-to-noise ratio increases much less as a function of receiver gain setting increase.  If the receiver gain is increased too much, the signal will exceed the limits of the ADC, the FID will be clipped at the beginning and the NMR spectrum will be severely distorted.  The first figure below shows a series of spectra plotted as a function of the receiver gain setting.  The spectra were scaled such that the signals were all of the same height.  It is clear that the signal-to-noise ratio increases initially and then levels off.  The data are plotted in the second figure.

   

DNP-NMR

Dynamic nuclear polarization (DNP) is a signal enhancement technique becoming more and more important in NMR studies of biological samples and materials.  Enhancement of NMR signals is accomplished by doping samples with stable free radicals.  The trapped free radicals in the cooled solid sample are irradiated continuously at the EPR microwave frequency.  Microwaves are generated by a gyrotron (which requires iits own superconducting magnet in addition to the superconducting NMR magnet).  The microwave radiation is introduced into the NMR probe by way of a wave guide.  While the unpaired electrons are irradiated, the population distribution of the Zeeman states of NMR active nuclei are modified providing more polarization and therefore a large NMR sensitivity enhancement.  Typically the polarized protons in the sample are used as a cross polarizatuion source for less abundant nuclides.  The data are typically acquired at low temperature with magic angle spinning.  The overall NMR enhancement is typically one or two orders of magnitude when one compares NMR spectra acquired with the microwave source on vs off.  Commercial DNP-NMR instruments are now available.

Thorsten Maly authors a very informative BLOG on all things DNP-NMR.  I encourage you to take a look at it.

A Useful Winter Emulsion

The winters in Ottawa are cold (and arguably miserable).  The cold can cause many detrimental effects on ones comfort.  One particularly uncomfortable condition usually disappears within a day or two on visiting the warm sunny Caribbean.  The proton and carbon NMR spectra below were acquired on a very useful emulsion used by many cold Canadians to keep this condition at bay.    What is the emulsion?

Happy Comfortable Holidays !!!!

NMR Tube Thickness and Signal-to-Noise-Ratio

The amount of NMR signal is expected to be proportional to the amount of sample inside the coil of the NMR probe.  As a result, the signal-to-noise ratio for samples run in NMR tubes with thick walls is expected to be lower than that for comparable samples run in NMR tubes with thinner walls due to a reduced filling factor of the NMR probe coil.  I was curious to see how much of a difference in signal-to-noise ratio there would be.  0.68 mL of  CDCl₃ (99.8 % D) was put in 5 mm NMR tubes with wall thicknesses of 0.38 mm and 0.80 mm.  The NMR tubes were New Era Entepprises NE-MP 5 (4.20 mm ID) and Norell S-300 (3.43 mm ID), respectively.  The samples are shown here:

The height of the sample column for the thick-walled tube is obviously higher due to the smaller inner diameter of the tube.  In this case, much of the sample will be "invisible" to the NMR measurement as it is outside of the active NMR probe coil volume and therefore "wasted".  Single scan proton NMR spectra were run for these samples on a 300 MHz instrument.  A third sample was prepared by removing some sample from the thick-walled NMR tube such that the column height was equal to the sample in the thin-walled tube.  The volume for this sample was 0.45 mL and it was run under identical conditions to the other two.  Care was taken to shim the magnet and tune and match the NMR probe reproducibly.  The data, processed with 0.5 Hz of line broadening, are plotted side by side in the figure below:

The 0.68 mL sample in the thin-walled tube (blue) gave a signal-to-noise-ratio of 566.  The 0.68 mL sample in the thick-walled tube (red) gave a signal-to-noise-ratio of 339 and the 0.45 mL sample in the thick-walled tube (green) gave a signal-to-noise-ratio of 369.  The difference in the signal-to-noise-ratios for the two samples in the thick-walled NMR tube may very well be the same within experimental error as the signal-to-noise-ratio is very sensitive to magnet shimming.  One would expect them to be similar based on the fact that both samples have volumes exceeding the active volume of the probe coil.  From the data, one sees a 35-40% loss in signal on going from a thin-walled to a thick-walled NMR tube.  It is instructive to look at the volume corrected signal-to-noise-ratio of the 0.45 mL sample in the thick-walled NMR tube compared to the 0.68 mL sample in the thin-walled NMR tube.  If the signal-to-noise ratio for the 0.45 mL sample is multiplied by (0.68 mL/0.45 mL), the corrected value is 557 which is very likely the same as the 566 value measured for the 0.68 mL sample in the thin-walled NMR tube within experimental error.  From these observations, one can conclude that the signal-to-noise-ratio loss is entirely due to the reduction in sample volume within the coil.         

EZNMR at the University of Ottawa

The NMR Facility at the University of Ottawa is equipped with eight NMR spectrometers and has on the order of 100 hands-on users at the graduate and post-doctoral level.  Like any university NMR facility, the users enter at varying knowledge and experience levels: from "What does NMR stand for?" to "How do I do a shearing transform for my 5QMAS data set?".  Also, the attitude of the user's supervisors varies considerably.  Some supervisors want their students to spend as little time in the NMR lab as possible by collecting all of their data in automation with a sample changer so they can maximize their time at the bench.   Others want their students to learn how to collect the best possible data and fully understand the NMR measurements they make.  There is no doubt that collecting NMR data under complete automation is incredibly time-efficient however, collecting data in this way teaches the student nothing about NMR measurements.  On the other hand, learning to use NMR spectrometers manually, at the most fundamental level, to collect the best possible data, requires a great deal of knowledge (both general and instrument-specific) and although it is the most educationally rewarding, it certainly provides less overall sample throughput.  In our facility, almost all students are first given 10 minutes of training, on how to collect NMR data under complete automation using our only fully automated instrument.  Running an NMR spectrometer in this way requires absolutely no knowledge of NMR spectroscopy.  Most users are also interested in using the other less automated instruments.  These students are provided with as much training as they desire.   The job of the NMR facility is to educate and satisfy the needs or each user.  Doing so, requires finding a "happy medium" between complete automation and complete manual spectrometer operation and using that medium as a minimum training standard.  For the last ten years or so, the "happy medium" used by the University of Ottawa to run four of it its Bruker NMR instruments is based on a customized button panel approach.  We have written button panels specific to each instrument and call the option EZNMR.  We have included EZNMR as an entry on the top TOPSPIN menu bar.  Clicking the EZNMR option opens up a button panel like the one shown below, used on our AVANCE 500 spectrometer. 

Each button either issues a command, runs a macro or runs an "au program".  Some of our students use exclusively this panel to collect their data.  The advantage to using such a system is that the student must at least learn all of the steps involved with collecting the data.  A typical EZNMR session involves simply following the button panel from top to bottom.  If the probe contains a sample, it is ejected with the EJECT button.  A new sample is lowered into the probe with the INSERT button.  The deuterium lock is established by pressing the LOCK button which prompts the user for the solvent and then establishes the lock.   The SHIM button first calls up a standard set of shims and then initiates a gradient shimming routine.  After the magnet is shimmed, the user presses any one of the green buttons depending on which NMR measurement they intend to carry out.  Pressing one of these buttons prompts the user to define a data set and then calls up a reasonable set of parameters into that data set.  If desired, the user can change the number of scans or some of the parameters by pressing the SCANS or PARAMETERS buttons.  The probe is then tuned using the TUNE button.  The START button optimizes the receiver gain and begins collecting the data.  Once started, the data can be processed at any time using the Proc 1D button or halted using the HALT button.  We use a similar button panel for the commonly used 2D NMR experiments which can be called up from the 1D panel.  It is shown here:

The advantages to using this system are:

-  It is highly customizable for the hardware of each instrument as it is based on macros and "au programs".

-  It can be added to as demands change.

-  All NMR spectrometers using EZNMR look pretty much the same so instrument specific training is less of an issue. 

-  Students can begin running NMR experiments very quickly.

-  Students are more likely to ask questions about each step and can learn at their own pace while maintaining high sample throughput.

-  Its use is entirely optional.

-  It is much more time-efficient than complete manual operation.

Students are of course encouraged to learn more about spectrometer operation than is available through the EZNMR buton panels.

19F NOESY

Two-dimensional ¹H NOESY data are routinely used to assign specific stereo-isomers based on the proton nuclear Overhauser effects (NOE's) which are strongly correlated to inter-proton distances through space.  For example, NOE's may be observed for cis- protons across a double bond but not observed for trans- protons.  The same technique can be used with ¹⁹F in fluorinated compounds to gauge the inter-fluorine distance and assign stereochemistry.  The figure below shows the ¹⁹F NOESY spectrum of a fluorine containing cobalt complex.

From the 1D-¹⁹F NMR spectrum, it is not clear which fluorine atoms are on the same or opposite sides of the four membered cobalt containing ring.  The 2D-¹⁹F NOESY spectrum, on the other hand, shows strong NOE cross peaks between fluorine C and both A and E indicating that C, A and E are on the same side of the ring.  There are also strong cross peaks between fluorine D, and both B and F indicating that D, B anf F are on the same side of the ring.

Thank you to Graham Lee (of Dr. R.T. Baker's research group at the University of Ottawa) for kindly providing the sample and sharing his data. 

Isotope Effects and the 19F - 13C HMQC Spectrum of Trifluoroacetic Acid

The ¹⁹F - ¹³C HMQC spectrum of trifluoroacetic acid is shown in the figure below.

The data were collected with a delay appropriate for a ¹⁹F - ¹³C J  coupling constant between the ¹J_F-C coupling constant of 284 Hz and the ²J_F-C coupling constant of 44 Hz.  The top and side traces are the one-pulse ¹⁹F and ¹³C spectra, respectively.  Why are the HMQC responses not at the same ¹⁹F chemical shift and why aren't they correlated to the peak in the ¹⁹F spectrum?  In order to answer these questions one must take into consideration the ¹⁹F - ^{12, 13}C isotope effects.  The chemical shift of the fluorine depends on whether it is bound to a ¹²C or a ¹³C.  The effect is largest across one bond and gets smaller over multiple bonds.  The ¹⁹F NMR spectrum for trifluoroacetic acid is shown in the figure below with and without ¹³C broadband decoupling in the upper and lower traces, respectively.

Approximately 98% of the trifluoroacetic acid is the ¹²CF₃-¹²COOH isotopomer, giving rise to a large singlet plotted off-scale in the figure. Approximately 1% of the signal is from the ¹³CF₃-¹²COOH isotoponer giving rise to a doublet with ¹J_F-C = 284 Hz and approximately 1% of the signal is from the ¹²CF₃-¹³COOH isotoponer giving rise to a doublet with ²J_F-C = 44 Hz.  All of these signals are clearly present in the lower trace of the figure.  When ¹³C broadband decoupling is applied, the doublets collapse into singlets.  The singlets from each of the isotopomers are resolved in the top trace.  The one-bond ¹⁹F - ^{12, 13}C isotope effect is 0.13 ppm and the two-bond effect is 0.02 ppm.  The figure below shows the same HMQC data with the spectrum from the top trace used as a projection.

One can see that the HMQC responses are correlated to their respective isotopomers.  These effects are also present in ¹H - ¹³C HMQC spectra, but the ¹H - ^{12, 13}C isotope effect is much smaller than the ¹⁹F - ^{12, 13}C isotope effect.  

Measurement of Long Range C H Coupling Constants

The stereochemistry of compounds is assigned very often with proton - proton NOE's by applying the 2D NOESY technique or the 1D selective gradient NOESY technique.  These methods fail, however when the distance between protons is too large to measure an NOE.  When faced with this situation, it may be possible to measure long range proton - carbon coupling constants which are able to provide the necessary information.  Three-bond carbon - proton couplings follow a Karplus relationship where the magnitude of the coupling constant is related to the dihedral angle between the carbon and the proton.  In some cases, these dihedral angles may be used to assign the stereochemistry.  Coupling constants are largest for dihedral angles of 0° and 180° and smallest for dihedral angles of 90°. The simplest way to measure the long range coupling constants is to collect a ¹³C NMR spectrum without ¹H decoupling.  These spectra can be very complicated as can be seen from the figure below showing the C2 and C3 aromatic carbons of toluene.

Extracting specific long range carbon - proton coupling constants is quite tedious.  One way to simplify matters and obtain specific carbon - proton coupling constants is to apply the selective 2D heteronuclear J-resolved technique first introduced by Bax and Freeman in 1982 (JACS 104, 1099).  This method employs a ¹³C spin echo with a selective ¹H 180° pulse applied simultaneously with the ¹³C nonselective 180° pulse. A version of this sequence is shown in the figure below with a shaped adiabatic ¹³C 180° pulse.

In this sequence one obtains a 2D spectrum with ¹³C in the F2 domain and the long range couplings to the selectively inverted proton in the F1 domain.  An example is shown in the figure below for toluene where the methyl protons were selectively inverted with a 20 msec Gaussian pulse.

All of the carbons coupled to the methyl protons are split into quartets in the F1 domain and the long range coupling constants which were very difficult to obtain from the coupled ¹³C spectrum can simply be read directly from the 2D spectrum.    

Exact Simulaion of Quadrupolar Lineshapes in Solids

The NMR spectra for quadrupolar nuclei in solids contain a great deal of structural information.  The evaluation of quadrupolar coupling constants, asymmetry parameters, isotropic chemical shifts, chemical shift spans, chemical shift skews and the angles relating the electric field gradient tensor to the chemical shift tensor is typically done by simulating the NMR spectrum with suitable software and fitting the simulated spectrum to the experimental data.  Almost always, the spectra of quadrupolar nuclei in solids have been simulated using perturbation theory where the quadrupolar interaction is treated as a perturbation on the much larger Zeeman interaction.  With the recent developments in the collection of ultra-wide line NMR spectra, quadrupolar nuclei with larger and larger quadrupolar coupling constants are being studied by NMR and the perturbation approach may not be valid.  Errors between simulated and experimental spectra appear when the Larmor frequency is not significantly larger than the quadrupolar coupling constant.

Recently, a new program called QUEST (QUadrupolar Exact SofTware) has been written by Frédéric Parras from the research group of David Bryce at the University of Ottawa.  As the name implies, this program is capable of simulating exactly the spectra of quadrupolar nuclei in solids without resorting to the assumptions of perturbation theory. QUEST is able to quickly calculate accurate lineshapes regardless of the ratio between the Larmor frequency and the quadrupolar coupling constant.  It even works in cases where the Larmor frequency is much less than the quadrupolar coupling constant (i.e. NQR). The figure below shows a series of spectra calculated for a spin I=3/2 nucleus as a function of the ratio of the Larmor frequency, ν_L , to the quadrupolar coupling constant, C_Q. The spectra near the top are NMR-like and those near the bottom are NQR-like.  QUEST is a fast, graphical, easy-to-use program able to handle multiple sites, export data in Bruker format, import experimental spectra for comparison to the simulations and simulate spectra as a function of the angle of the detection coil with respect to the magnetic field. The package also includes a very helpful well written pdf manual.  The program is reported and described fully here.  To take a look at the program in action, watch these tutorial videos prepared by the author.  The complete program is available for free download here.  I highly recommend it!

60 MHz NMR on the Bench Top

The development of bench top NMR spectrometers has certainly been exciting recently!  Nanalysis (a Canadian company) has recently introduced a 60 MHz bench top NMR spectrometer.  The NMReady^TM60P is capable of running both ¹H and ¹⁹F NMR spectra.  Like the PicoSpin spectrometer, this instrument should have a high impact on the NMR scene.

Weak Lock Signals and Distorted NMR Spectra

A good ²H lock signal with a high signal-to-noise ratio is a real advantage for maintaining a stable magnetic field for long data acquisitions and also for shimming the magnet using the lock signal.  Sometimes, however it is desirable to run NMR spectra for samples with only a only a very small quantity of deuterated solvent and therefore a very weak lock signal.  Such may be the case when one is monitoring a chemical reaction by removing aliquots and adding a drop or two of a deuterated solvent to help with magnet shimming using the ²H lock signal.  Although one may be able to shim a magnet using a very weak lock signal (with difficulty), running the spectrum locked may not be a good idea.  Running a spectrum while locked on a very weak lock signal can lead to distortions in the spectrum.  It is often better to use the weak lock signal to shim the magnet as best you can and then run the spectrum unlocked.  This is demonstrated in the figure below. 
The figure shows two single scan ¹H NMR spectra of a sample of acetone (one drop) in CCl₄ with a drop of CDCl₃.  The spectrum on the left was acquired using the ²H lock and the one on the right was acquired unlocked.  One can clearly see the distortion in the ¹H spectrum caused by locking on a very weak ²H signal.

The Extremely Complicated 1H NMR Spectrum of Ethane

It is often incorrectly assumed that simple compounds yield simple NMR spectra. The ¹H NMR spectrum of ethane is such an example. The complexity arises when one takes into account the inequivalence between methyl groups in the mono ¹³C isotopomer which accounts for 1% of the naturally occurring ethane. In this isotopomer, one methyl group experiences a one-bond ¹H - ¹³C coupling (¹J_H-C) while the other methyl group experiences a two-bond ¹H - ¹³C coupling (²J_H-C). Also, the effects of the three-bond ¹H - ¹H coupling (³J_H-H) are exhibited in the spectrum due to the inequivalence. These couplings have a dramatic effect on the spectrum. Furthermore, there is a very small isotope effect on the ¹H chemical shifts of each methyl group due to the presence of ¹³C vs ¹²C. This effect however, is very small (~0.002 ppm) and has very little effect on the spectrum. The left panel of the figure below shows a simulation of the ¹H NMR spectrum of the ¹²CH₃-¹²CH₃ which accounts for 98% of naturally occurring ethane. As expected, the spectrum is a singlet as both methyl groups are equivalent to one another. The middle panel of the figure shows a simulation of the ¹H NMR spectrum of the ¹³CH₃-¹²CH₃ isotopomer which accounts for 2% of naturally occurring ethane. In this case the spectrum is extremely complex due to the ¹J_H-C , ²J_H-C and ³J_H-H coupling. The panel on the right shows a simulation of a scaled up representation of what one would expect for naturally occurring ethane.
The parameters for the simulations are as follows: Δδ_H between -¹²CH₃ and -¹³CH₃= 0.002 ppm, ¹J_H-C = 125 Hz, ²J_H-C = -4.67 Hz, ³J_H-H = 8 Hz and LB = 0.5 Hz.

The Major Constituents of Natural Gas

The major constituent of natural gas is methane however, other gaseous hydrocarbons are also present. One way to identify other components is to dissolve some natural gas in a solvent and examine the ¹H NMR spectrum. The spectrum in the figure below was acquired on a sample prepared by bubbling natural gas through benzene-d₆ for several minutes. The spectrum clearly shows the presence of methane, ethane, propane and water. The spectrum also indicated other impurities at much lower levels (not shown in the figure).

Protic Samples in Aprotic Solvents

The appearance of the ¹H NMR signals of protic samples in aprotic solvents depends critically on the concentration of the sample. The -OH, -NH₂ or -COOH signals can have chemical shift values and line widths over a wide range due to varying extents of hydrogen bonding and chemical exchange. The concentration can also determine whether or not one is able to observe J coupling between and -OH proton and other protons in the sample. An example of this is illustrated below. The figure shows the spectrum of methanol in deuterated acetone. The spectrum on the top is that of concentrated methanol and it consists of two singlets. In this case the methanol molecules are hydrogen bonded to one another and the -OH protons are undergoing fast exhange with one another. The spectrum on the bottom is that of very dilute methanol. It is a second order spectrum with two signals approximating a doublet and a quartet due the J coupling between the methyl protons and -OH proton, respectively. Note that the chemical shift of the -OH proton is much lower for the dilute methanol compared to the concentrated methanol. In this case the methanol molecules are not hydrogen bonded to one another and there is no (or very slow) exchange among the -OH protons between molecules allowing for the observation of the J coupling.

Double Presaturation

Presaturation is a common method of reducing the water signal in the ¹H NMR spectra of aqueous samples. Sometimes, a sample may contain more than one undesirable resonance which a user may want to presaturate. In such a case, one must presaturate at multiple frequencies simultaneously. On a two-channel Bruker spectrometer, two signals can be presaturated. This is accomplished by using both Signal Generation Units (SGUs). One of the undesirable signals is put on-resonance and is presaturated with the signal from SGU1 after which the hard pulse is given (also through SGU1). The second undesirable resonance is presaturated using SGU2. The configuration is as follows:If one has a three-channel system, one can presaturate three resonances using three SGUs. The figure below shows an example of double presaturation on a two-channel system. The sample consisted of phenylalanine dissolved in D₂O contaminated with methanol. The one-pulse spectrum in the bottom left panel shows the intense HDO and methanol signals. The double presaturation spectrum in the top left panel is on the same vertical scale as the one on the bottom left. One can see that both solvent signals have been almost completely eliminated. The spectra on the right-hand side are the same data as on the left except the vertical scale has been increased by a factor of 100. You can try this on your Bruker spectrometer using the pulse program "lc1prf2".

Sorting Out NOE's for Exchanging Rotamers

2D NOESY spectra contain cross peaks from both NOE interactions and peaks due to rotamers in slow exchange with one another on the NMR tine scale. For small molecules, the cross peaks resulting from slowly exchanging rotamers are of the same sign as the diagonal peaks. The NOE cross peaks, on the other hand, are of opposite sign compared to the diagonal peaks. When both types of correlations are present there may be more NOE correlations than expected. What follows is an example of this. The figure below shows a color coded chemical structure of a ruthenium complex with a color coded partial ¹H NMR spectrum.It is obvious from the NMR spectrum that all of the signals from the color coded protons are doubled in the spectrum. One possible explanation for this is that there is a slow rotation about the ruthenium carbon bond indicated with the red curly arrow allowing for two possible nonequivalent rotamers. This is confirmed with the 2D ¹H NOESY spectrum shown in the figure below with a 0.9 second mixing time. The spectrum clearly shows exchange peaks between corresponding pairs of ¹H signals from each rotamer.The interesting thing to note from the NOESY spectrum is that each aromatic proton (pink) from a single rotamer shows NOE correlations to the methyl groups (blue and yellow) of both rotamers - not just those from a single rotamer. With this data, it is not possible to assign the subspectrum of a single rotamer. Presumably, the assignment could be made by collecting a 2D NOESY spectrum at low temperature where the rotation was completely frozen out or by collecting a 2D NOESY spectrum with a very short mixing time where the rotation would be limited. The problem with the former approach is that the solvent may freeze at a temperature too high to stop the bond rotation. The problem with the latter approach is that the NOE's would be much reduced due to the short mixing time and collecting a 2D data set with sufficient signal to noise ratio would take a great deal of time. Another approach is to collect selective 1D gradient NOESY spectra with selective excitation of the aromatic proton from each rotamer individually. These data are shown in the figure below for two different mixing times.Each spectrum is displayed in two parts. The left-hand panel is the aromatic region with the selectively excited resonance colored red and the right-hand panel is the aliphatic region showing the NOE correlations to the methyl groups. From the spectra collected with a 2 second mixing time, one can see that the selective excitation is no longer selective due to bond rotation during the long mixing time. One can see inverted peaks for the aromatic protons of both rotamers despite the fact that the ¹H signal of only one rotamer was selectively excited. Furthermore, NOEs to the methyl signals from both rotamers are present. The spectra collected with only a 0.2 second mixing time, on the other hand, show very selective excitation. The time scale of the bond rotation is obviously longer than the 0.2 second mixing time. The spectra show only the NOEs between the selectively excited aromatic proton and the methyl groups from a single rotamer. The NOEs build up fast enough to be observed during the 0.2 second mixing time before rotation occurs. These data allow for the assignment of signals from each of the rotamers.

Thank you to Justin Lummiss of Dr. Fogg's research group for aharing this interesting system.

13C NMR of a Delicious Christmas Treat

As my Santa Claus-like belly may indicate, I love holiday treats. The solid-state ¹³C NMR spectra below were collected from a special sample of a holiday treat prepared by my wife, Patty from her Great Grandma Jennings lab book. The sample was prepared from only four ingredients as follows:

To 227 g of softened butter, 65 g of fructose was added while stirring with a spatula. Slowly, 199 g of flour and 1.26 g of sodium chloride were stirred into the mixture until it became difficult to mix with a spatula. The mixture was kneaded gently until cracks in the surface began to appear after which it was rolled to a thickness of 38 mm and cut into round samples of approximately 51 mm in size. The samples were heated in an oven at 436 K - 450 K for approximately 600 seconds until gold in color.

The bottom trace is a ¹³C CPMAS spectrum and the top trace is a ¹³C MAS spectrum. Both spectra were acquired with high power ¹H decoupling. This pair of spectra serves to illustrate the different types of information available from each of these techniques. The sample is a mixture of rigid and mobile components. The ¹³C CPMAS technique detects mainly the more rigid components as it relies on the dipolar coupling between protons and ¹³C for the cross polarization. The dipolar coupling is averaged to nearly zero for the mobile constituents and therefore they do not appear in the spectrum. The ¹³C CPMAS spectrum therefore, shows primarily all of the rigid constituents (mainly flour and sugar). The ¹³C MAS spectrum with high power ¹H decoupling shows both rigid and mobile constituents. The resonances from the mobile constituents (mainly butter) have sharp lines while the broader lines from the rigid constituents show up at very low intensity as the sensitivity is not enhanced by cross polarization.

Now, you too have enjoyed Patty's delicious shortbread.

Enlightening NMR Videos on YouTube

The Journal of Magnetic Resonance (JMR) has been publishing important contributions from NMR spectroscopists since 1969. Some of these contributions have become "classic papers" and have been sited thousands of times. A play list of videos has been assembled on YouTube highlighting some of the most significant contributions in NMR spectroscopy over the last few decades. The research is described anecdotally (and in some cases very humbly) by the original authors in the videos. I think you will enjoy putting faces to the names of the authors of these highly cited papers.

The Effect of Viscosity on 1H NOESY Spectra

For small molecules, ¹H 2D NOESY spectra exhibit positive NOE's between protons close to one another in space and the off-diagonal correlations are opposite in phase to those of the diagonals. As molecules become larger and larger the motional correlation times become longer and longer. As the correlation times become longer and longer, the NOE's become less positive, cross zero and then become negative. For large molecules, like proteins, the NOE's are negative and the off-diagonal correlations between close protons are of the same phase as the diagonal peaks. When the correlation times are extremely long, for example in rigid macromolecules or solids, the dipolar coupling among all of the protons is inefficiently averaged by molecular motions and spin diffusion becomes efficient. Spin diffusion allows all of the protons in a dipolar coupled network to exhibit correlations with one another. The sign of the correlations is similar to that observed for chemical exchange or negative NOE's. This phenomenon is demonstrated in the figure below. In the figure, positive contours are represented in black and negative contours are represented in red. The NOESY spectrum on the left is that of a solution of menthol in CDCl₃. The off-diagonal correlations between proximate protons is opposite in phase compared to the diagonal responses, typical of small molecules with short correlation times. The NOESY spectrum on the right is that of the same solution of menthol dissolved in a very viscous fluorinated oil. The extreme viscosity of the sample is sufficient to make the correlation time for the molecules very long such that the menthol behaves like a very large macromolecule where spin diffusion is efficient. As a result, off-diagonal responses are of the same phase as those of the diagonal and are observed between all protons in the molecule.This technique has been cleverly applied* to mixtures of molecules immersed in viscous oils where intra-molecular correlations are observed whereas inter-molecular correlations are not observed. The data allow for the observation of the constituent components of complex mixtures.

* Andre J. Simpson, Gwen Woods, and Omid Mehrzad, Anal. Chem, 80, 186-194 (2008).

Resolution During a Liquid Nitrogen Refill

Just as it is not advisable to collect high resolution NMR data during (and shortly after) a liquid helium refill, it is similarly not advisable to collect data during a liquid nitrogen fill. The figure below illustrates this point. A 300 MHz NMR spectrometer was set up to collect an NMR spectrum every minute before, during and after a liquid nitrogen refill. The resolution deteriorates as soon as the one-way valves are removed from the nitrogen cryostat. One can see that the resolution during the fill is much worse than before the fill. Even 60 minutes after the fill, the magnet has not fully recovered the resolution attained before the fill. The last spectrum of the series was collected after re-shimming the magnet and is comparable to the spectrum collected before the fill. The resolution profile during a fill may be different for every magnet. For this particular magnet, it is advisable not to collect high resolution data for at least 90 minutes after a nitrogen refill.

"The Resonance" - A New Web Site from Bruker

Bruker has introduced a new web site called "The Resonance". This well organized site has NMR/MRI news, posts related to specific areas of research, hardware and software information and more. The site is frequently updated and permits posts from users. It will be a great resource for users of Bruker NMR instruments as well as those interested in how magnetic resonance is applied in research.

Probe Tuning and 90 Degree Pulses

In order to get meaningful results from multiple-pulse NMR pulse sequences, it is essential that the 90° and 180° pulses are calibrated at the power levels used in the sequences (see this post for example). The calibrations are usually done on a standard sample in a well tuned and matched probe. The calibrations are typically stored in a file which is called up when setting up particular NMR experiments. It is important to know that these calibrations are correct for the particular sample of interest only when the probe is well tuned and matched. For samples of high ionic strength, it may not be possible to properly tune and match the probe and the 90° and 180° pulses for these samples will be longer than those previously calibrated, resulting in questionable data. In these cases, the pulses must be calibrated on the problematic sample. The figure below addresses the question of how important proper tuning and matching are with respect to the 90° pulse duration. The ¹H 90° pulses for a sample HDO in a 500 MHz broadband probe were measured by the fast nutation method for various states of probe tuning and matching. In the left-hand side of the figure, pulses were calibrated for a perfectly matched probe as a function of tuning frequency. One can see that the 90° pulse is at a minimum when the probe is perfectly tuned and increases as the probe is detuned in either direction. In the right-hand side of the figure, pulses were calibrated for a perfectly tuned probe as a function of probe mismatch. One can see that the 90° pulse is at a minimum in a perfectly matched probe and increases as a function of the degree of mismatch (in units of screen divisions on the spectrometer display). It is interesting to note that the 90° pulse duration is more forgiving to mismatch than to errors in probe tuning.

Resolution During a Liquid Helium Refill

Students sometimes ask whether or not they can collect NMR data during a liquid helium refill. The answer depends on the type of NMR experiment. For most solids experiments, where high resolution ( <~ 100 Hz) is not required, one can often collect data without a problem. In experiments for liquids, where high resolution is almost always required, the quality of data are affected drastically by the disturbance from the liquid helium refill and it is not advisable to collect data during this time. Furthermore, after a liquid helium refill, the magnet must recover in order to obtain the same resolution prior to the fill. The figure below illustrates this point. A 300 MHz NMR spectrometer was set up to collect an NMR spectrum every minute before, during and after a liquid helium refill. One can see that the resolution during the fill is much worse than before the fill. Even 15 minutes after the fill, the magnet has not fully recovered the resolution attained before the fill. The last spectrum of the series was collected after re-shimming the magnet and is comparable to the spectrum collected before the fill.
The resolution profile during a fill will probably be slightly different for every magnet. For this particular magnet, it is advisable not to collect high resolution data for at least 30 minutes after a helium fill.

Nitrogen Pressure and Spectral Resolution

Super-conducting NMR magnets are cooled with liquid helium. The boil off rate of the liquid helium is minimized by surrounding it with a high vacuum and a liquid nitrogen cryostat. The liquid nitrogen cryostat may either be vented directly to the atmosphere or it may be maintained at a pressure slightly above atmospheric pressure with the use of pressure relief valves. NMR users should be aware that the homogeneity of an NMR magnet is affected drastically by the pressure in the liquid nitrogen cryostat. The left-hand panel of the figure below shows a well resolved 400 MHz ¹H NMR spectrum of the methyl triplet of ethylbenzene in a well shimmed magnet where the nitrogen cryostat was maintained slightly above atmospheric pressure with pressure relief valves for several days. The spectrum in the right-hand panel was run under identical conditions after the cap to the nitrogen fill port was removed, relieving the pressure in the nitrogen cryostat.