University of Ottawa NMR Facility Web Site

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



Tuesday, March 11, 2014

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.
 

Thursday, March 6, 2014

Variable Temperature to Improve NMR Resolution

Many millions of dollars have been spent on high field NMR magnets to improve both sensitivity and chemical shift dispersion.  Many younger NMR users have had the good fortune to use only high field spectrometers where chemical shift resolution is often not an issue.  These users are not familiar with some of the "tricks" used to improve resolution which were needed on lower field instruments where chemical shift resolution was frequently a problem.  With the current helium shortage and the increasing popularity of low field permanent magnet spectrometers, these "tricks" will again become more and more common.  Among them are; the use of paramagnetic chemical shift reagents, the use of aromatic solvents or solvent mixtures and the use of variable temperature.  In this post, I would like to demonstrate the incredible power of simply changing the temperature at which the NMR data are collected.

The 1H chemical shift is a sensitive parameter related to the conformation of a molecule.  In solution, small molecules may adopt a number of conformations whose populations depend on the potential energy profile.  Furthermore, the molecules are often in fast exchange between the available conformations and the observed chemical shift is the weighted average chemical shift of all of the conformations present.  As the temperature is changed, the populations of conformations are altered and the observed average chemical shift value may change.  The changes in chemical shifts at different temperatures are often enough to resolve resonance which may have overlapped with one another at room temperature.

The chemical shift of exchangeable protons ( -OH, -NH or NH2) depends dramatically on the degree of both inter-molecular and intra-molecular hydrogen bonding.  When molecules with exchangeable protons are dissolved in aprotic solvents, one is often able to observe the exchangeable protons as well as their associated J couplings.  Such is the case with sucrose dissolved in DMSO-d6 where all of the -OH protons can easily be observed.  When the temperature is changed, the populations of available conformations change and the degree of intra-molecular hydrogen bonding is affected with dramatic changes in the chemical shifts of the -OH resonances.  The figure below shows the anomeric and -OH region of the 500 MHz 1H NMR spectrum of sucrose in DMSO-d6 collected as a function of temperature.


All of the protons can be assigned with standard 2D NMR methods.  As the temperature is increased, the anomeric proton (1) moves to higher frequencies while the -OH protons (2-9) all move to lower frequencies to different extents.  Note that the resonances in the highlighted region of the spectrum at 21°C are overlapped with one another but at higher temperatures are fully resolved.  The resolution has increased by simply increasing the temperature.

Friday, February 28, 2014

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.

Monday, February 24, 2014

Deternining 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.

Friday, February 21, 2014

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.

Thursday, February 20, 2014

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.


Tuesday, February 18, 2014

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!
 

Monday, December 16, 2013

Echoes, T2 Measurements and Diffusion

In a perfectly homogeneous magnetic field, the T2 relaxation time constant can be measured directly from the free induction decay in the time domain or the full width at half height of the resonance in the frequency domain.  The magnetic field however, is never perfectly homogeneous.  Each microscopic volume element of the sample resides in a slightly different magnetic field and therefore the offset frequencies of the resonance in each volume element are slightly different from one another.  The net effect on the spectrum of the entire sample is that the NMR resonances are broader than what one would expect from the T2 relaxation process alone.  The distribution of offset frequencies due to magnetic field inhomogeneity is referred to as inhomogeneous broadening.  In an inhomogeneous magnetic field, the FID decays faster, with time constant T2* where 1/T2* has a contribution from the natural relaxation rate, 1/T2, of the resonance and that due to the field inhomogeneity.  In other words, when a sample is in an inhomogeneous magnetic field, the resonances are homogeneously broadened by the natural T2 relaxation process and inhomogeneously broadened by the non-uniform magnetic field.  The measurement of T2 relies on separating the homogeneous broadening from the inhomogeneous broadening.

One of the first pulse sequences typically introduced in NMR textbooks is the spin echo or Hahn echo.  This sequence consists of a 90° pulse followed by a delay, τ, during which offsets frequencies evolve.  A 180° pulse is then applied after which another period of time, τ is allowed where offsets continue to evolve, producing an echo at 2τ. The spin echo sequence has the ability to refocus the distribution of offset frequencies due to magnetic field inhomogeneity (inhomogeneous broadening) however it cannot refocus the natural distribution of frequencies due to the T2 relaxation process (homogeneous broadening).  It would seem as if the spin echo sequence has the ability to separate out the homogeneous broadening from the inhomogeneous broadening and therefore should be able to be used to measure the T2 relaxation time constant in a scheme like the one shown in the figure below.



where the intensity of the signals as a function of 2τ is fitted to an exponential decay to give T2.  Can this sequence really be used to measure T2?  Let's look a bit deeper.

A sample of tetrakis-trimethylsilyl silane ( Si(Si(CH3)3)4 ) was dissolved in CDCl3.  The magnet was shimmed such that the full line width at half height of the 1H resonance was 2 Hz.  A standard one-pulse proton spectrum and a Hahn echo spectrum (with τ set to 1 second) were collected.  The same measurements were made after adjusting the magnetic field shims such that the full width at half height was 5 Hz and 13 Hz.  The results are shown in the figure below.

 

The top panel shows the NMR spectra resulting from the one-pulse measurement.  The spectra all have the same integrated area as expected.  The middle panel shows the FID's from the one-pulse measurements.  The initial intensity of each FID is the same since the initial intensity of the FID is proportional to the integrated area of the resonance in the frequency domain.  The bottom panel shows the Hahn echoes collected with a value of τ =1 second (a value substantially shorter than T2).  The receiver was turned on immediately after the 180° pulse to collect the entire echo.  Unlike the one-pulse FID's which remained constant as a function of magnetic field inhomogeneity, the height of the Hahn echoes decreased as the magnetic field inhomogeneity increased, all other parameters being constant.  This should convince you that the simple Hahn echo is not always suitable for T2 measurements as the intensity of the echo depends on the degree of inhomogeneous broadening.  This is so because of molecular diffusion.  During the one second τ delays, molecules move from one volume element to another in the sample and therefore change their offset frequencies over the course of the measurement.  The net result for the entire sample leads a loss in echo intensity due to destructive interference in the time domain signal from the sum of all volume elements.  The loss in echo intensity is worse the more inhomogeneous the field.  The simple Hahn echo would be expected to work as a means to measure T2 only in cases where the diffusion is insignificant with respect to τ (solids or dissolved macromolecules). 
 
How then are T2's measured for small molecules in solution where diffusion is fast?  One uses a train of Hahn echoes where the τ delays for each echo are chosen sufficiently short such that diffusion is not a problem (typically on the order of msec or tens of msec).  The T2 is calculated from a series of spectra collected as a function of the number of echoes in the train based on the overall time between the initial 90° pulse and the collection of the signal.  Such a scheme is called a Carr Purcell Meiboom Gill (CPMG) sequence and is shown in the figure below.
 
 
 

Wednesday, October 23, 2013

FSLG CP HETCOR

Solid-state 1H MAS NMR spectra with resolution comparable to that obtained for liquids, are difficult (if not impossible) to obtain. The main problem is that magic angle spinning is unable to average the homonuclear 1H dipolar coupling interaction to zero.  The combined use of MAS and multiple pulse decoupling schemes (CRAMPS) can be used to improve the resolution.  In this case, the 1H FID is sampled during windows of the multiple pulse decoupling scheme where pulses are not being delivered, however the attainable resolution is still much less that that observed for liquids where the rapid molecular tumbling reduces the homonuclear dipolar interaction to zero.  Furthermore, CRAMPS experiments can be difficult to setup and run.  An alternative method of obtaining "high resolution" solid-state 1H NMR spectra (with resolution comparable to that of a CRAMPS spectrum) is a frequency switched Lee-Goldburg cross polarization heteronuclear correlation experiment (FSLG CP HETCOR) where the 1H spectrum is obtained in the indirect dimension of a 2D experiment.

In this pulse scheme, used in conjunction with MAS, 1H magnetization is aligned at the magic angle and subjected to FSLG decoupling where it is forced to precess about a field oriented at the magic angle by using 2π pulses with carefully chosen offset frequencies.  The ideal effect is to average the homonuclear dipolar coupling to zero.  The FSLG decoupling train serves as the evolution time (t1) in a 2D data collection scheme.  During the variable evolution period the 1H chemical shifts evolve while the heteronuclear dipolar coupling is averaged by MAS and the homonuclear dipolar coupling is averaged by both the MAS and the FSLG pulse train.  The 1H magnetization is then returned to the transverse axis and cross polarization (CP) is used to transfer the frequency encoded proton magnetization to 13C.  The 13C FID is observed while 1heteronuclear decoupling is applied.  If CP contact times are chosen sufficiently short, one obtains a 2D 13C-1H dipolar correlation map with correlations present between carbon resonances and the protons to which they are most strongly dipolar coupled.  If longer contact times are used, more correlations will appear resulting from longer range dipolar couplings and 1H spin diffusion.  In either case, the 1H projection of the data represents a high resolution 1H spectrum of the sample with resolution comparable to or better than a CRAMPS spectrum.  The figure below shows FSLG 13C-1H CP HETCOR spectra for Dianin's compound acquired on a 200 MHz spectrometer using a spinning speed of 5 kHz.

The spectrum on the right was acquired with a 50 µsec contact time and shows the aromatic carbon resonances correlated to aromatic proton resonances and the aliphatic carbon resonances correlated with the aliphatic proton resonances.  The spectrum on the left was acquired with a 300 µsec contact time and shows all of the 13C resonances correlated to all of the 1H resonances.  In both cases the 1H projection is a high resolution 1H NMR spectrum.

Friday, July 19, 2013

Understanding NMR Spectroscopy

Undergraduate students are typically introduced to the subject of NMR spectroscopy through the organic chemistry curriculum where, after a brief introduction to the technique, they learn how to interpret chemical shifts, coupling constants and NOE's in terms of chemical
information. Unfortunately, this is often the extent of a students training in NMR despite the fact that many who pursue graduate studies use NMR spectroscopy every day.  These students learn to operate NMR spectrometers and will agree that NMR spectroscopy is by far the most valuable technique for characterizing their chemical compounds yet most lack a fundamental understanding of the technique.  It cannot be disputed that an understanding of the fundamentals of NMR enables the chemist to become a confident, knowledgeable NMR user able to gain the maximum amount of information from NMR results.

In my opinion, by far, the best NMR book devoted to the fundamentals of NMR spectroscopy published in the last 10 years is James Keeler's book, Understanding NMR Spectroscopy (my copy is well worn).  Although it is limited to spin-1/2 nuclides and does not cover solid state NMR, it covers the fundamentals of NMR in a very clear understandable way.  Keeler has a talent for teaching and makes the material accessible to all with a basic science background.  After studying this book the reader will gain a much better understanding of one- and two-dimensional pulse sequences, product operators, relaxation, nuclear Overhauser effects and coherence selection through both phase cycling and pulsed field gradients.


In addition to the book, a detailed set of notes is available on Dr. Keeler's web site and recently, an entire course given by Keeler, consisting of 14 lectures, has appeared on YouTube.  Links to the lectures are as follows:

1.                 Energy levels
2.                 The Vector Model
3.                 Fourier Transformation
4, 5, 6          Product Operators
7, 8              Two-Dimensional NMR
9, 10, 11      Relaxation
12, 13, 14    Coherence Selection

I highly recommend the book, and the lectures.  Never has understanding NMR spectroscopy been more accessible.

Thursday, June 13, 2013

Manual Phase Correction of 1D Spectra - Video Tutorial

In some cases where there are baseline issues, automatic phase correction may not do a satisfactory job.  It then becomes necessary to correct the phase manually.  The following video demonstrates how to manually phase correct a 1D NMR spectrum in TOPSPIN.

Tuesday, June 4, 2013

Bruker Fourier 300 NMR Spectrometer in the Undergraduate Lab

The Department of Chemistry undergraduate laboratory at the University of Ottawa is equipped with a Bruker Fourier 300 NMR spectrometer which is used by each of the undergraduate chemistry students requiring NMR data.


To minimize the amount of training for new students and maximize the throughput during the busy laboratory periods, we have implemented a simplified data collection scheme called EZNMR.  The following tutorial video was prepared for the undergraduate students who have yet to run their first 1H NMR spectrum.

Friday, April 5, 2013

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, TOCSYNOESY 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.


Thursday, April 4, 2013

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.

Tuesday, March 26, 2013

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.

Monday, March 18, 2013

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.



Wednesday, March 13, 2013

Phasing a 2D NMR Spectrum - Video Tutorial

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

Thursday, March 7, 2013

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.

Thursday, February 14, 2013

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.
   

Wednesday, January 23, 2013

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.

Friday, December 21, 2012

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 !!!!

Friday, December 7, 2012

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.         

Monday, November 19, 2012

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.

Thursday, November 15, 2012

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. 

Monday, October 22, 2012

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 1JF-C coupling constant of 284 Hz and the 2JF-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, 13C 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 1JF-C = 284 Hz and approximately 1% of the signal is from the 12CF3-13COOH isotoponer giving rise to a doublet with 2JF-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, 13C 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, 13C isotope effect is much smaller than the 19F - 12, 13C isotope effect.  

Friday, August 17, 2012

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.    

Monday, June 25, 2012

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, νL , to the quadrupolar coupling constant, CQ. 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!

Wednesday, May 2, 2012

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 NMReadyTM60P 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.

Friday, April 20, 2012

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.

Friday, March 30, 2012

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 (1JH-C) while the other methyl group experiences a two-bond 1H - 13C coupling (2JH-C). Also, the effects of the three-bond 1H - 1H coupling (3JH-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 1JH-C , 2JH-C and 3JH-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: ΔδH between -12CH3 and -13CH3= 0.002 ppm, 1JH-C = 125 Hz, 2JH-C = -4.67 Hz, 3JH-H = 8 Hz and LB = 0.5 Hz.