How Can I Get a Quantitative 13C NMR Spectrum?

With conventional acquisition parameters, ¹³C NMR spectra of liquids are rarely quantitative and usually not integrated. There are two main reasons for this. Proton decoupling is normally used to simplify the spectrum and as a way to build up sensitivity as a result of the nuclear Overhauser effect. Also, typical T₁'s for ¹³C are very long. One can still get quantitative data and a proton decoupled spectrum if a very long recycle delay (perhaps 5 minutes) is used and the ¹H decoupling is applied only during the acquisition time which is very short with respect to the recycle delay. Using such parameters however makes the collection of data prohibitively time consuming. To avoid this problem, you can add a paramagnetic relaxation agent to the sample to reduce the relaxation times.

In the spectra below the data were collected with a 10 second recycle delay. In the left hand spectrum, the duty cycle of the decoupler was 100 % and the spectrum exhibits an NOE. In the middle spectrum, the NOE is greatly reduced by decoupling only during the acquisition time (decoupling duty cycle = 10 %), however the long T₁'s prevent the spectrum from being quantitative. The spectrum on the right was acquired similar to that of the one in the middle except some Cr(acac)₃ was added to the sample to reduce the relaxation times for the carbons. This spectrum is quantitative.

Improving Your Baseline Correction

Spectral baselines are often corrected in Bruker's software by using the "abs" command after Fourier transformation. This correction is not always well suited to the data. Another method of getting flatter baselines is to define specific frequency limits "absf1" and "absf2" over which the baseline will be corrected and then use the "absf" command rather than the "abs" command. The smaller the difference between "absf1" and "absf2", the better the correction will be. Below is an example where "absf1" and "absf2" are defined by the region in pink.

31P - 109Ag HMQC

Silver has two spin I = 1/2 isotopes (¹⁰⁹Ag and ¹⁰⁷Ag) with natural abundances of 48.18% and 51.82%, respectively. Although ¹⁰⁹Ag has a lower natural abundance, it is the preferred isotope to observe as its gyromagnetic ratio is slightly higher than that of ¹⁰⁷Ag. Direct observation of ¹⁰⁹Ag is problematic in that the Larmor frequency is low, the NOE's to protons are negative and most importantly, the T₁'s are very long (minutes to hours). In some silver phosphine complexes with ¹⁰⁹Ag - ³¹P coupling, the problems of observing silver directly can be circumvented by using indirect detection via a ³¹P detected ³¹P - ¹⁰⁹Ag HMQC. The advantages of using this approach are that the directly observed ³¹P frequency is 8.7 times higher than that of ¹⁰⁹Ag and the relaxation delay in the experiment depends on the ³¹P T₁'s which tend to be orders of magnitude shorter than those of ¹⁰⁹Ag.

Below is an example of a ³¹P detected ³¹P - ¹⁰⁹Ag HMQC for a silver phosphine complex. These data were acquired without ¹⁰⁹Ag decoupling. In the ³¹P trace on the top of the spectrum one can see a single ³¹P resonance with coupling to both ¹⁰⁹Ag and ¹⁰⁷Ag. The ¹⁰⁹Ag is indirectly observed in the F1 domain.

Better 180 Degree Pulses

Many pulse sequences use 180 degree pulses. Standard hard pulses are not perfect at inverting all of the lines in a spectrum with a wide spectra width. In such cases a better choice may be a shaped adiabatic pulse. A comparison is shown below for the ¹³C spectrum of ethyl acetate. Each spectrum was acquired with the same number of scans. The trace on the left was acquired with a hard 180 degree pulse and the spectrum on the right was acquired with the CRP60comp.4 pulse from the Bruker pulse shape library. Note the better performance resulting in improved nulls.

Collecting 13C NMR Spectra of Highly Paramagnetic Compounds

I am sometimes asked to acquire ¹³C NMR spectra of highly paramagnetic compounds. Those of you attempting to acquire such spectra for the first time should be aware of the following:
1. You should use a large spectra width as the ¹³C chemical shifts can be very extreme. In order to get a wide uniform excitation, use very short high power pulses.
2. Both the T₁'s and the T₂'s are short, so you can use short acquisition times and short recycle delays.
3. The ¹H chemical shift range for paramagnetic compounds can span hundreds of ppm. The ¹H decoupling schemes used to collect ¹³C data do not permit such a broad decoupling bandwidth and you may have to collect several ¹³C spectra with different ¹H offsets. It is always best to collect a proton spectrum first to evaluate the ¹H decoupling needs in the ¹³C spectrum.
4. The lines are often very broad and therefore many scans are needed to build up the signal-to-noise ratio.
5. The ²H lock signal may be shifted by the paramagnetic compound, so don't be surprised if your automatic locking routines do not work.

13C - 14N J coupling

It has always puzzled me how students so willingly accept the fact that ¹³C is J coupled to a spin I = 1 nucleus like ²H (eg. the multiplets for the ¹³C spectra of deuterated solvents) however they do not question the fact that ¹³C almost never shows a J coupling to ¹⁴N (another spin I = 1 nucleus). The reason that ¹³C exhibits J coupling to ²H is that the relaxation among the three energy levels of ²H is rather slow and each ¹³C "sees" the ²H in each of its three Zeeman states, splitting the ¹³C resonance into 3 lines of equal intensity. The efficiency of the relaxation among the energy levels of quadrupolar nuclei (among other things) depends on the magnitude of the quadrupolar coupling constant - the larger the quadrupolar coupling constant, the faster the relaxation. Unlike ²H, which has a very small quadrupolar coupling constant, ¹⁴N (and most other quadrupolar nuclei) has a substantial quadrupolar coupling constant and therefore the relaxation among its energy levels is very fast. The ¹³C therefore "sees" the ¹⁴N in a single "average" state and as a result is a singlet. There are however compounds where the ¹⁴N is in a very symmetric environment. The high symmetry make the quadrupolar coupling constant much smaller and the relaxation therefore much slower. In these cases one can observe the ¹³C - ¹⁴N J coupling. Depending on the relaxation of the ¹⁴N, the ¹³C lines can vary from being a sharp 1:1:1 triplet, to a broad unresolved triplet, to a very sharp singlet. Below is an example of a case where the ¹⁴N is in a very symmetric environment and the ¹³C - ¹⁴N J couplings are resolved.

Enhancing 29Si NMR Spectra with DEPT

Measuring ²⁹Si NMR spectra of liquids can be very time consuming as ²⁹Si nuclei tend to have very long T₁ relaxation times and therefore very long delays must be left between scans to allow for relaxation. Also, since ²⁹Si has a negative gyromagnetic ratio, the NOE's with protons diminish the signals and inverse gated decoupling should be used. For molecules where there is a measurable ²⁹Si - ¹H J coupling, one can use a DEPT sequence to enhance the ²⁹Si signal. The advantages are two-fold: first, there is an enhancement on every scan from the DEPT sequence and second, the repetition rate of the experiment depends on the ¹H T₁ rather than the ²⁹Si T₁. The second advantage is very significant as the ¹H T₁'s can be shorter than the ²⁹Si T₁'s by two orders of magnitude. The example below demonstrates the advantages.

The traces on the left and right were both acquired in 1 minute with a 2 second recycle delay. One can see the tremendous signal-to-noise-ratio advantage of using a DEPT sequence. For comparison, the trace in the middle was acquired in 16.4 minutes with inverse gated decoupling and a 60 second recycle delay. (Note that the DEPT signal could have been enhanced even more if a 35 degree rather than a 24 degree flip angle was used for the final ¹H pulse in the DEPT sequence)

Shaped Pulses for Selective Excitation

Short rectangular pulses have broad excitation profiles (see BLOG entry for November 20, 2007) and are used for non-selective uniform excitation. These are often called "hard" pulses. Longer low power shaped pulses are used to excite specific spectral regions for selective excitation. An example of this is shown below.These are often called "soft" pulses.

Removing t1 Noise from Heteronuclear Correlation Data

Heteronuclear correlation data can often contain vertical stripes of noise, called t1 noise. This noise can be reduced by taking the projection a group of rows containing no signals (only noise) and subtracting the projection from every row of the data. This is illustrated below for an HMQC spectrum. The spectrum on the right is the data after Fourier transformation. The data contains a considerable amount of t1 noise. All of the rows highlighted in yellow, containing only noise, were used to prepare a projection. The projection was subtracted from every row of the data. The result is the spectrum on the right. The t1 noise is much reduced.

A New BLOG Dedicated to Processing and Predicting NMR Data

Carlos Cobas, president and co-founder MESTRELAB RESEARCH has started an excellent BLOG on processing and predicting NMR data. Given Carlos' expertise in this area, it promises to be a very valuable resource to the NMR community.

Visit Carlos's BlOG here.

http://nmr-analysis.blogspot.com/

Sensitivity Improvement from Cross Polarization

Yesterday's post showed the pulse scheme for cross polarization in solids. Below is a comparison of the single scan 39.7 MHz 29Si MAS spectra for solid tetrakis(trimethylsilyl)silane. The trace on the left was acquired with a simple one pulse sequence with high power CW proton decoupling during the acquisition (Bloch decay). The trace on the right was acquired with cross polarization and high power CW 1H decoupling. In both cases the sample was spinning at 2 kHz. The signal-to-noise improvement resulting from cross polarization is obvious.

Cross Polarization

The sensitivity of NMR spectra for solids containing protons can be increased dramatically by using cross polarization. Cross polarization is a technique where magnetization is transferred from an abundant proton source to a dilute (isotopically of chemically) nucleus, X, during a "contact" period. During the contact time, rf fields for both 1H and X are turned on. The ratio of power levels between 1H and X must be equal to the ratio of gyromagnetic ratios between X and 1H. The enhanced magnetization of the dilute isotope is then detected while the abundant protons are decoupled. The maximum gain in sensitivity is equal to the ratio of gyromagnetic ratios between 1H and X. The technique has the additional advantage in that the relaxation delay can be chosen based on the T1(rho) for 1H rather than the T1 of the dilute isotope, which is often larger by at least an order of magnitude.

Throwing Away Noise to Improve Your Data

It is no surprise that the Fourier transform of noise is noise, so getting rid of noise in you FID will get rid of it from your spectrum as well. If your FID decays long before the acquisition time is over then you have collected unnecessary noise and your spectrum will have a lower than necessary signal-to-noise ratio. You can throw away the noise and replace it with null points before carrying out the Fourier transform. This is shown in the figure below where the improvement in the signal-to-noise ratio is obvious.A better idea is to save spectrometer time, make the acquisition time shorter and avoid collecting the noise in the first place.

Nyquist Fold-backs in Magnetic Resonance Images

Nyquist fold-backs are not just limited to 1D and 2D NMR data. They can also be observed in magnetic resonance images. The figures below are magnetic resonance images of seedless grapes acquired on our AVANCE 500. The Nyquist fold-backs are circled in blue.

How Fast Should I Spin My Solid Sample?

The choice of MAS rate depends on the interaction to be averaged by the magic angle spinning. If you want to average out the chemical shielding anisotropy then you must spin the sample at a rate comparable to or greater than the span of the chemical shift tensor expressed in Hz. Although the spans of chemical shift tensors, measured in ppm, are independent of field strength they are linearly dependent on field strength, when expressed in Hz. As a result, an appropriate spinning speed in one magnet may not be an appropriate spinning speed for the same sample in another magnet. The spectra below illustrate this point. Both are 31P CPMAS spectra of dibasic ammonium phosphate with a spinning speed of 4 kHz. The lower trace was acquired at 11,75 Tesla while the upper trace was collected at 4.7 Tesla. One can see that there are many more spinning sidebands in the spectrum acquired at higher field despite the identical spinning speeds.One would have to spin the sample at 10 kHz in an 11.75 Tesla magnet to get a spectrum comparable to the one acquired with a spinning speed of 4 kHz in a 4.7 Tesla magnet.

HMQC Responses in HMBC Data

Despite the built in low pass J filters written into the HMBC pulse sequences, the much larger one-bond 1H - 13C coupling responses are not fully suppressed. These one-bond HMQC responses show up as a pair of responses in the acquisition domain separated by the one-bond 1H - 13C coupling. The responses are doubled since 13C decoupling is not typically used in HMBC sequences. An example of these artifacts is shown below.

31P Decoupling

All of you routinely use proton decoupling in your 31P NMR spectra, many of you without giving it a thought. Few people realize that it is a simple matter to decouple 31P in your 1H NMR spectra. This will allow you to determine which protons are coupled to phosphorus. An example of this is shown below. The lower trace is a standard 1H spectrum and the upper trace is a 1H spectrum with 31P decoupling.

Baseline Correction in Satellite Transition MAS Spectra of Quadrupolar Nuclei

When one acquires NMR data with extremely short dwell times (or very large spectral widths), problems are often encountered with severe baseline roll as the first few points in the FID are lost either to the receiver dead time or probe ringing. The satellite transition MAS spectra of quadrupolar nuclei are characterized by many sharp spinning sidebands and require the use of very large spectral widths to accommodate the entire sideband envelope. The FIDs for such samples contain rotational echoes at the period of the rotor. Due to the extremely short dwell times required to collect such data, baseline problems are very common. In such cases simply removing the initial few bad points or using backward linear prediction will not solve the problem satisfactorily due to the nature of the FID. There is however a very simple solution. One can simply remove all of the points in the FID before the first rotational echo and then carry out the Fourier transform. The figure below shows the FID's and spectra for the 27Al MAS NMR of kaolinite. The spectra show the satellite transitions. The resonance for the central transition is off scale in the figure. In the left hand side of the figure the raw FID was Fourier transformed and phase corrected. One can see extreme baseline roll. In the right hand side of the figure, all of the points prior to the center of the first rotational echo in the FID were discarded and the new FID was Fourier transformed and phase corrected. The baseline is much improved.

Magic Angle Spinning

The NMR spectra of solids generally have very broad lines due to a number of interactions which are averaged either to zero or an isotropic value in solution. It is the averaging of these interactions which leads to the narrow lines observed in liquids. The rapid random motion responsible for the averaging in solution is not present for solids, however, it can be mimicked by spinning the sample about an angle of 54.7 degrees with respect to the magnetic field at a rate comparable to or greater than the extent of the interaction being averaged. This technique is called magic angle spinning (MAS) and it allows the measurement of high resolution spectra of solids. When the spinning rate is less than the extent of the interaction, the spectrum is split into sidebands spaced at the spinning speed. In the figure below are 81 MHz 31P CPMAS spectra of ammonium dihydrogen phosphate with high power proton decoupling at various spinning rates. When the spinning speed is zero, one can see the powder spectrum characterizing the chemical shift interaction. As the spinning speed is increased, there is a centerband at the isotropic chemical shift with sidebands whose intensities can be used to calculate the chemical shift tensor. When the spinning speed exceeds the span of the chemical shift interaction the sidebands are very small and a liquid-like spectrum is obtained.

Excitation Profiles

The range of frequency over which NMR resonances are excited depends on the duration of the monochromatic radio frequency pulse applied to the sample. Very long pulses will excite a very narrow range of frequencies whereas very short pulses will excite very wide frequency ranges. For example, presaturation pulses used for solvent suppression, are typically several seconds long while non-selective pulses are typically micro seconds in duration. It is essential to use very short pulses to provide even excitation over large spectral widths. The shape of the excitation profile is related to the Fourier transform of the pulse.

Increasing the Signal-to-Noise Ratio in Solids MAS Spectra

Often MAS or CPMAS NMR spectra of solids will have several spinning sidebands. In such spectra, where there are no other complications, one can increase the signal-to-noise ratio for the isotropic signals by simply adding the sideband intensity to the isotropic spectrum. Below is a 50.68 MHz, room temperature, 15N CPMAS spectrum of a clay sample which absorbed some 15N labelled pyridine and then was heated to 400 degrees. The spinning speed was set to 2.5 kHz. The full spectrum is shown in the bottom panel. The isotropic region is shown on the top left panel. The spectrum in the top right panel was obtained by shifting the spectrum by multiples of the spinning speed and adding the shifted spectra to the original spectrum. Note that the isotropic region of the spectrum has a much improved signal-to-noise ratio.

DEPT and DEPTQ

Many of you run both standard 13C and 13C DEPT-135 spectra of your compounds. This may take a considerable period of time (and cost a considerable amount of money) if your sample is dilute. You can run a single DEPTQ-135 spectrum of your compound and get the same amount of information. This may save a great deal of spectrometer time. A DEPTQ-135 spectrum is the same as a DEPT-135 spectrum except the quaternary carbons are present and 180 degrees out of phase with respect to the CH and CH3 carbons. Note also that the commonly used deuterated solvents will show up in a DEPTQ spectrum and can be used for chemical shift referencing. Below is the standard 13C spectrum, the DEPT-135 spectrum and the DEPTQ-135 spectrum of ethylbenzene.

Zero Filling

Zero filling is a data processing technique where zero points are appended to the free induction decay before Fourier transformation. The effect of zero filling is to increase the digital resolution in the spectrum. Since the zeros contain no new information, there is no new information added to the spectrum. You can think of it as artificially increasing the acquisition time after the data collection without adding any new information or true resolution to the spectrum.

To zero fill on a Bruker spectrometer, the "si" parameter is increased to a value greater than the number of points collected in the free induction decay. On a Varian spectrometer, the corresponding parameter is "fn".

HSQC and Edited HSQC Spectra

Many of you use a simple magnitude HMQC sequence to establish heteronuclear one-bond 1H -13C correlations. One can also use the phase sensitive HSQC sequences to obtain the same information. Although the data must be phased by the user, the phase sensitive sequences will provide data with higher resolution as absorption line shapes are much narrower than the magnitude signals obtained in non-phase-sensitive sequences. There is also an edited HSQC sequence available which provides multiplicity information similar to that of a 13C DEPT-135 sequence where CH and CH3 signals are phased up and CH2 signals are phased down. In the figure below is the HSQC and edited HSQC spectra of 3-heptanone. The red CH2 signals in the edited HSQC are negative with respect to the black CH3 signals. Using this sequence may save you time (and money) as there will be no need to run a 13C DEPT spectrum.

What Mixing Time Should I Use for My 2D-NOESY Measurements?

NOEs are often very small and the appropriate choice of mixing time is a critical factor in achieving good NOESY spectra. There are two opposing factors which must be considered when choosing the most appropriate mixing time. Firstly, you would like the NOE to build up for as long as possible during the mixing time and secondly, you want to loose as little signal as possible to relaxation during the mixing time. The best compromise is to choose a mixing time equal to the average T1 relaxation times for the signals in which you are interested. (See the BLOG entry for October 31, 2007, to learn how to estimate T1's.)

If the standard pulse programs are used, the mixing time is the "d8" parameter on a Bruker spectrometer and the "mix" parameter on a Varian spectrometer.

Testing a Magnet for Field Drift

One can test a magnet for drift by measuring a spectrum unlocked with multiple scans using a very long recycle delay. The entire spectrum will shift between scans according to the magnet drift, therefore you should have as many multiples of the spectrum as the number of scans. In the figure below, the drift rate for a 500 MHz wide bore magnet was measured. A 16 scan 1H spectrum was acquired for CHCl3 in acetone-d6 without locking. The recycle delay was 1 hour. The resonance shifts to lower frequency as a function of time. One can also see that the shim currents (either the cryoshims or room temperature shims) have drifted over the course of the measurement accounting for the broadening and multiplicity at later times.

Learn How to Play Music with Your NMR Spectrometer

I always thought this site by Walter Bauer was a lot of fun. The difference frequency between an NMR resonance and the carrier frequency is used creatively to make music. Listen to your spectrometer!

http://www.chemie.uni-erlangen.de/oc/research/NMR/music.html

NMR spectrometers are really just very expensive musical instruments!

Increasing the Signal-to-Noise Ratio in Solid State Wideline 2H Spectra

At low magnetic field strengths where chemical shielding anisotropy is not a problem, the wide line 2H spectra of solids produce symmetrical spectra. One can increase the signal-to-noise ratio in these spectra by a factor of the square root of two by reversing the spectrum and adding it to itself as illustrated below for the 2H spectrum of perdeuterated poly-methyl methacrylate.

Why Does My Quaternary Alkyne Carbon Show Up in My 13C DEPT Spectrum?

The BLOG entry for September 20, 2007 illustrated how some signals from protonated carbons can be either missing or of much reduced intensity in 13C DEPT spectra. This is due to unusual one-bond 13C-1H coupling constants. There are also cases where quaternary carbon signals will show up in 13C DEPT spectra due to unusually large two-bond 13C-1H coupling constants. Such is the case for the alkyne in the figure below. A standard 13C DEPT-135 sequence optomized for a 13C-1H coupling of 145 Hz (the average one-bond 13C-1H coupling constant) was acquired and compared to the standard 13C spectrum with 1H decoupling. One can see that the protonated alkyne carbon has less intensity than expected as its one-bond 13C-1H coupling constant is ~ 250 Hz. Also the quaternary alkyne carbon is present in the spectrum as its 2-bond 13C-1H coupling constant is ~ 50 Hz. Although neither of these couplings is close to 145 Hz, they are both of a magnitude where one would expect to see a signal in the DEPT 135 spectrum.

Getting Incorrect Lineshape Specifications

One can easily fool themselves by getting line shape specifications (see BLOG entry for Tuesday October 23, 2007) that are better than the truth. Erroneous specifications will be obtained on the standard line shape sample (1% CHCl3 in acetone-d6) if sufficient time is not allowed for complete relaxation to occur and the height of the 13C satellites is used as a guide to measure the line width. This error results because the T1 of the 13CHCl3 isotopomer is shorter than that of the 12CHCl3 isotopomer. Therefore, if the signals are partially saturated, the 13C satellites will be over-represented with respect to the singlet. If the height of the satellites is used as a guide to measure the line width on the singlet, the line shape specifications will be narrower than they would be if complete relaxation was allowed to occur. This is illustrated in the figure below. The trace on the left is a fully relaxed single scan spectrum using a 90 degree pulse with an 8 second acquisition time. The trace on the right was acquired under identical conditions however the spectrum was partially saturated by collecting 15 dummy scans with an inter-pulse spacing of 8 seconds immediately prior the receiver being turned on. The spectra were scaled such that the singlets had the same height. Note the difference in signal-to-noise ratio and the relative heights of the satellites compared to the singlet.

One can still obtain the correct line shape specification in a partially saturated spectrum by measuring the linewidths at 0.55% and 0.11% of the height of the singlet if the satellites are not used as a guide.

19F - 1H HOESY Experiment

Many of you use proton NOESY spectra to establish whether or not two types of protons are close to one another in space. The same type of experiment is also possible between nuclei of different isotopes. This technique is called HOESY (Heteronuclear Overhauser Effect SpectroscopY) and can be used to assess the spatial proximity between 2 heteronuclei. Below is an example of a 19F detected 19F - 1H HOESY spectrum of a sugar. (sample courtesy of Jennifer Chaytor)

How Many Scans Should I Collect?

Many students ask this question. You should collect enough scans until the signal-to-noise ratio is adequate for your needs. For NMR measurements (where saturation is not a problem) the signal -to-noise ratio increases as the square root of the number of scans. In order to double the signal-to-noise ratio in a particular spectrum one must collect 4 times as many scans as were collected in the original spectrum. Take a look at the figure below where spectra are plotted on an absolute intensity scale against the number of scans collected.
If the signal-to-noise ratio is only 2 after 1 hour, it will be < 7 after 12 hours and only 16 after 64 hours. If you do not see a signal in 1 hour it is probably not worth running the sample overnight!

FID Truncation and Spectral Distortion

Have you ever seen a spectrum like the one in the lower trace of the figure below?

This sin(x)/x distortion at the base of an NMR line is the result of truncation of the FID. The acquisition time was not long enough to capture the entire time domain signal. In addition to the distortion at the base of the lines, there is also a loss in spectral resolution as "sharp" features in a spectrum are defined by later times in the free induction decays. If you see this, you should re-run your spectrum with a longer acquisition time. If you do not have the option of re-running the spectrum, increase the line broadening (LB) and re-transform the data. This will get rid of the distortion but will not help with the resolution.

NMR to Distinguish Solid Polymorphs

Some solids have a number of different polymorphs (or phases). Being able to distinguish different polymorphs is very important for the pharmaceutical industry. Solid State NMR is able to distinguish between them. In the figure below is the 13C CPMAS spectrum of the solid amino acid, methionine acquired on a Bruker AVANCE 500 NMR spectrometer. The spectrum clearly shows two signals for each type of carbon.

When the temperature is varied the intensity ratio of the peaks changes as one polymorph becomes more favored than the other.

T1 Measurements and Estimation

T1 relaxation time measurements are usually done with a simple 180 -tau -90, inversion recovery pulse sequence (see figure). Tau is varied from a small value to a large value and a nonlinear regression is carried out to fit the best T1 value.These measurements can be very time consuming. One can get a reasonable estimate of the T1 much more quickly. Follow these simple steps:

1. Call up the pulse sequence "t1ir1d" (Bruker) or "s2pul" (Varian).

2. Set p1 and p2 (Bruker) or PW and P1 (Varian) to the 90 degree and 180 degree pulses , respectively.

Set the recycle delay, d1 (Bruker and Varian) to something you believe is much longer than the T1.

3. Set tau to a very small value (3 microseconds for example). Tau is d7 on a Bruker spectrometer or d2 on a Varian spectrometer.

4. Collect a spectrum and phase it such that all peaks are negative (one scan is often enough for protons). Store the phase correction.

5. Repeat step 3. increasing d7 (Bruker) or d2 (Varian) until the peak of interest is nulled. If the peak is negative, tau is too short. If it is positive, tau is too long.

6. The T1 of the peak of interest is the tau value for the null divided by the natural log of 2.

Spectra Acquired with a Sweeping Field

Have you ever seen spectra like those in the top and middle traces below and wondered what was the problem?

These spectra were acquired while the magnetic field was sweeping. This will happen on a Bruker spectrometer if you do not bother locking the field (or fixing the field when running unlocked). On rare occasions in automation, if there is difficulty in locking your sample, I have seem the spectrometer run a spectrum while sweeping the field. This however is very infrequent.

Quadrature Images

Quadrature images are caused by an imbalance in the magnitude of signals in the x and y channels of the receiver. Such an imbalance will create a quadrature spike in the Fourier transformed spectrum (see entry for October 11, 2007) as well as quadrature images. An example of quadrature images is shown below for the 1 scan proton spectrum of a 10% ethylbenzene in CDCl3. Quadrature images are small reflections of large signals in the spectrum about the center. They are much less of a problem with newer instruments than they were with older ones. They become smaller as more scans are collected due to receiver phase cycling. You should be aware of quadrature images if you are searching for very small signals in the presence of very large ones.

Why Are Some of My HMQC Correlations Doubled?

HMQC and HSQC signals are 1H detected with 13C decoupling. The sequences essentially throw away all of the signals for protons attached to 12C and retain all of those for protons attached to 13C. The coupling between 1H and 13C is used to establish a correlation but while the 1H signal is being detected, the 13C is decoupled. Occasionally you may find that some of your HMQC/HSQC correlations appear to be doubled or have a much lower than expected signal-to-noise ratio. Often this is due to poor 13C decoupling and is a problem for correlations at the extremes of the 13C chemical shift scale. At moderate magnetic field strengths, 13C "garp" decoupling is efficient over about 150 ppm or so. If the correlations are near the limits of the center of the 13C axis plus or minus (150 ppm/2), then you will find a loss in signal-to-noise ratio and perhaps some signal doubling due to partial 13C decoupling. Below is an example of the partial HMQC spectrum of 3-heptanone. In the left panel the 13C offset was set near the 13C resonances. In the right-hand panel the 13C offset was set at 110 ppm and the terminal methyl signal is poorly decoupled.

If you encounter this problem, center the 13C offset closer to the correlation which is poorly decoupled.

New Solids NMR Spectrometer for the University of Ottawa

Congratulations are in order for Dr. David Bryce whose successful CFI proposal will fund a new solids NMR spectrometer for the University of Ottawa. Way to go Dave!!

To Spin Or Not To Spin?

For liquids NMR one can average out the transverse inhomogeneity of the magnetic field by spinning the sample tube about the direction of the magnetic field. This will lead to sharper lines and a higher signal-to-noise ratio however artifacts called spinning sidebands will appear at multiples of the spinning frequency on either side of an NMR line. The intensity of these artifacts depends on the degree transverse inhomogeneity of the magnet, the quality of the NMR tube and the NMR probe. The decision to spin depends on how well the transverse shims of the magnet (i.e. x, y, xy, (x2-y2).... etc..) are set, and your tolerance for spinning sidebands. In a very well shimmed magnet it may not be worth spinning at all. In a poorly shimmed magnet spinning will provide a substantial line narrowing as well as a huge boost in the signal-to-noise ratio. For 2D experiments, one should never spin as sidebands will introduce undesirable artifacts in the spectra. Below are two comparisons of spinning vs non spinning in a well shimmed and a not-so-well shimmed magnet. What do you think? Should you spin?

Measurement of 13C-19F Coupling in a 1H-13C HMBC

It is often difficult to find the 13C signals in fluorinated organics by direct 13C observation. It may sometimes be easier to find these signals by way of a 1H-13C HMBC spectrum if there are nearby protons in the molecule. As shown below, it is even possible to measure the 13C-19F coupling constants.

Measuring 1H Line Shape - The Line Shape Sample

It is important for NMR spectroscopists to know how well their magnets are shimmed and how well their probes are performing as far as line shape and width are concerned. A quantitative specification is needed so one magnet-spectrometer-probe configuration can be compared meaningfully to another. We use a standard sample of 1% chloroform in acetone-d6. The resonance of CHCl3 is examined. The width of the line (in Hz) is measured at 50% of the line height, 0.55% of the line height and 0.11% of the line height. 0.55% of the line height was chosen as it is the height of the 13C satellites. The specification is usually stated as x/y/z where x = the width at 50%, y = the width at 0.55% and z = the width at 0.11%. Often just y/z is given. These numbers are often quoted with and without spinning the sample. In the example below from our Bruker AVANCE 400 spectrometer on a non-spinning sample, the line shape was 0.27 Hz / 5.28 Hz / 8.72 Hz.

Eliminating t1 Noise in 2D-Homonuclear Data

2D experiments often have t1 noise (not to be confused with T1 relaxation). This noise is evident as "stripes" perpendicular to the directly detected domain (see the left panel of the figure below). Several factors such as instrumental instability and temperature instability can lead to t1 noise. In homonuclear experiments one can remove the t1 noise by symmetrization of the data about the diagonal. In this process all regions symmetric about the diagonal are compared. The region having the largest signal is thrown away and the region with the smallest signal is put on both sides of the diagonal. This procedure will retain all symmetric signals (i.e. cross peaks) and eliminate t1 noise. Below is an example of the 1H COSY spectrum of ethylbenzene.

One should use this method with care as artificial cross peaks will appear for uncoupled signals with excessive t1 noise. Before symmetrization, one should look for the smallest real off-diagonal signal. Make a mental note of the signal. Symmetrize the spectrum and then scale it such that the smallest real off-diagonal signal noted above is the smallest signal in the symmetrized spectrum.

On a Bruker spectrometer one can symmetrize the data with the commands "sym" and "syma" for magnitude and phase sensitive data, respectively. On a Varian spectrometer the command is "foldt".

Excellent Resource for Solids NMR

If you are a beginner at solids NMR, you should take a look at this EXCELLENT link from Durham University.

http://www.dur.ac.uk/solid.service/information/

It is full of valuable and practical knowledge.

I Only Have 0.5 mg of Sample. What Can I Do?

Here are your options on collecting NMR data when you have only a limited amount of sample.

1. Use a standard setup and get more sample or collect more scans. Yeah yeah..... I know ..... if you could do this you wouldn't have a problem.

2. Reduce the volume of the solution keeping the same mass and lift the NMR tube such that all of the sample is in the receiver coil. This is not a very desirable option as the magnetic susceptibilities of the solution inside the coil and the air outside the coil are very different and will result in a severe distortion of the magnetic field. It is very difficult to shim the magnet to compensate for this effect and the resolution and signal-to-noise ratio in your spectrum will suffer greatly.

3. Reduce the volume of the solution keeping the same mass and use a "Shigemi" tube. These tubes have plastic parts which go above and below the sample which is centered in the receiver coil. The plastic parts are chosen to have the same magnetic susceptibility as the solvent used to prepare your solution.

4. Reduce the volume of the solution keeping the same mass and use an NMR tube of a reduced diameter with a probe designed for such a tube. This is an option we have at Ottawa U. on the Varian INOVA 500 for which we have a 3 mm probe.

5. Use a Cryogenically cooled probe. These probes have cryogenically cooled coils and electronics which dramatically reduce the amount of noise thereby increasing the signal-to-noise in the spectrum. They are very expensive to both purchase and maintain and not currently an option at Ottawa U. Maybe we can ask Santa.....

Proton Probe Tuning for 13C Detected Experiments

I am sometimes asked: Do I have to tune the proton channel of the probe for my 13C detected experiments? The answer is yes. If you are just running a simple 13C with proton decoupling then the efficiency of the decoupler will depend on the tuning of the proton channel. If the proton channel of the probe is very badly tuned, you may see broadening and even splittings in your carbon signals. The signal-to-noise ratio will also suffer. For experiments like DEPT or INEPT, the proton tuning is critical as these sequences require proton pulses of specific flip angles. If the proton channel of the probe is not tuned and matched then the flip angles for the proton pulses in the sequence will be less than they should be and you will not get the results you expect. In the figure below is an example. The bottom trace is a 13C DEPT-135 spectrum of menthol with the proton channel of the probe properly tuned. The spectrum in the upper trace is also a DEPT-135 spectrum of menthol run under the same conditions except that the proton channel of the probe was detuned. It looks more like a DEPT-90 as the proton pulses are closer to 90 degrees than 135 degrees.

Remember to tune the proton channel!

The Proton Decoupled 31P NMR Spectrum of Triphenyl Phosphate

I was asked about the origin of the small peaks in the 31P NMR spectrum of triphenyl phosphate posted to this blog on October 4, 2007. The small peaks are 13C satellites. The phosphorus is coupled to the 13C in the ipso (J=7.47 Hz), ortho (J=4.98 Hz), meta (J=0.93 Hz) and para (J=1.32 Hz) positions on the aromatic rings. The coupling constants were measured in a high resolution 13C NMR spectrum. The figure below shows the proton decoupled 31P NMR spectrum with the couplings color coded. The satellites due to the meta and para couplings overlap. The displacement of the doublets is due to the isotope effect (see blog entry for September 13, 2007).

U of O NMR Facility Blog Receives a Positive Review

The University of Ottawa NMR Facility Blog was started less than two months ago. Even though it was intended for the NMR users of the University of Ottawa, it is being read by others interested in NMR as well. It has received a positive review from Ryan Sasaski of Advanced Chemistry Development. Check it out here.

http://acdlabs.typepad.com/my_weblog/

Ryan's NMR Blog is a very valuable resource for those interested in ACD's NMR prediction software as well as other issues related to NMR.

Thank you Ryan!

The Consequences of Setting the Receiver Gain Too High

The receiver gain of an NMR spectrometer is much like the volume control on a radio. When it is set too high there will be distortion in the NMR signal. The FID will be clipped near the beginning of the signal. The Fourier transform of this distorted signal is a distorted NMR spectrum. The figure below shows what this distortion looks like.

Many spectrometers will calculate the receiver gain automatically however you should be aware that this automatic calculation is not always perfect and that the receiver gain may have to be set manually. On a Varian spectrometer the receiver gain is the "gain" parameter. On a Bruker spectrometer the parameter is "rg". In both cases higher numbers mean a higher receiver gain.

Nyquist Fold-Backs in HMBC Spectra

The digital filters of modern NMR spectrometers have not only improved the signal-to-noise specification quoted by instrument companies but they have also eliminated Nyquist fold-backs for signals outside of the spectral window. This is not the case however for the indirect dimension of 2D experiments. In the left panel of the figure below is a properly recorded HMBC spectrum of 3-heptanone. In the right panel is a spectrum acquired with the 13C spectral window set too small. The carbonyl correlations are folded into the low frequency end of the 13C axis.
If you inadvertently collect a spectrum with a Nyquist fold-back you can still calculate the correct chemical shift, as the signal will be the same number of ppm away from the wrong end of the axis as it is outside of the correct end of the axis.

Quadrature Spikes and How to Get Rid of Them

Have you ever seen a small sharp peak in the exact center of your NMR spectrum? This is called a quadrature spike and is due to a small offset in the two channels used for quadrature detection. It can sometimes be confused with a real NMR peak. You can get rid of this peak very simply by reprocessing your data. In XWINNMR or TOPSPIN do the following:

Type "bc_mod qfil"

Type "bcfw 0.02"

Type "efp" to reprocess your data

This will null a region 0.02 ppm in width at the center of the spectrum. In the unlikely event that you have a real signal at the exact center of the spectrum, you should not use this technique as it will null out the real signal as well. Below is an example of removing such a spike in a 13C NMR spectrum.

Why is the Tuning Display Unstable When I Tune the Probe?

On the Varian INOVA 500 you may notice that the tuning display is unstable while you are tuning the probe. On the Bruker spectrometers, this may show up as ripples or waves on the "wobble" tuning curve. This phenomenon is due to Q modulation in the probe as a result of spinning the NMR tube. The tuning of the probe is dependant on the orientation of the NMR tube within the probe. This problem is much worse for cheap NMR tubes or NMR tubes which have been damaged or lack perfect radial symmetry. The effect can also be seen for solids samples spinning in MAS probes.

To avoid the problem, simply stop spinning the sample.

13C NMR of Fluorinated Organics

Many NMR users (of the organic variety) come to believe that all 13C resonances are singlets as the most common way to collect the data is with proton decoupling, which indeed reduces most 13C resonances to singlets. It is a surprise to some to find multiplicity in the 13C resonances when a nucleus such as 31P or 19F is present in the molecule. Below is the aromatic region of the proton decoupled 13C NMR spectrum of alpha, alpha, alpha-trifluoro-p-tolualdehyde (the aldehyde peak is not shown). The assignments are given by the colors in the figure. Note the multiplicities in the 13C resonances due to the 1, 2 and 3 bond J coupling to 19F.

Some people find it surprising that the trifluorinated methyl group comes in the aromatic region of the spectrum. Fluorinated carbons are often difficult to find in 13C spectra with low signal-to-noise ratios as the signal is spread over multiple lines and can be buried in the noise.

The Width of Your Water Line - Radiation Damping

The width of the water resonance in a proton NMR spectrum depends critically on the amount of water present. When the concentration of H2O is very low the NMR resonance is very narrow. When the concentration is very high the width is many times greater. The reason for this is that the strong magnetization of the water signal induces currents in the NMR coil which generate magnetic fields which broaden the line. This phenomenon is called radiation damping. The width of the water line is a function of the strength of the water signal which depends on the amount of water, probe tuning, field strength, coil size etc... Radiation damping can also affect the symmetry and phase of the peak. Below are the 500 MHz proton NMR spectra of two samples of H2O / D2O with different concentrations of H2O. In both cases the magnet is well shimmed.

The Effect of Proton Decoupling on the Signal-to-Noise Ratio for 31P NMR

I have seen some students collect 31P NMR data without proton decoupling. Their reason was that there were no directly bound protons to the phosphorus. What they have not taken into account are the remote protons which have small couplings to the phosphorus. There are usually many of these remote protons and the cumulative effect of the couplings is to broaden out the 31P resonance. This broadening causes a severe loss in the signal-to-noise ratio. The upper trace of the figure below is a single scan 31P spectrum of triphenylphosphate where the closest protons are 4 bonds away from the phosphorus atom. The lower trace was run under identical conditions with the proton decoupler turned on. In this case, the gain in signal-to-noise using proton decoupling is 17.

Proton NMR Assignment Tools - The D2O Shake

You can identify exchangeable protons in your proton NMR spectrum with a very simple technique called a "D2O shake". For some reason this simple technique, used frequently years ago, seems to be used so much less today. All you do is run a 1H NMR spectrum of your sample then put a single drop of D2O in the tube, shake it, and run another spectrum. The exchangeable protons will exchange with the deuterium in the D2O and disappear from the spectrum. The D2O does not have to be miscible with the solvent.

Below is a partial proton spectrum of menthol in CDCl3. The bottom trace is the spectrum before the addition of D2O and the top trace after 1 drop of D2O was shaken with the sample. The -OH peak has clearly disappeared.