NMR Basics / FAQ.

All the really basic NMR questions you were too embarrassed to ask!

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Rolling baseline? Strange lineshapes? Go to Michigan State University's NMR Artifacts pages.
There is a comprehensive Basics of NMR document available at the Center for Imaging Science, Rochester Institute of Technology.
Michigan State University has a good introduction to NMR.
University of Saskatchewan has some good links.
Lecture notes on NMR theory (PDF format) by James Keeler.
The NMR Wiki has lots of information.
The ANZMAG youtube channel has many NMR lectures.

If you haven't used any NMR spectrometers at the NMR Centre before.
Ask a staff member to arrange a training session for you. Even if you have used Bruker spectrometers before, our setup here is likely to be slightly different, so in the long run you will save yourself time by having a short lesson. More information is available in the introduction to the NMR Centre. It is NMR Centre policy that you will not be given a licence to operate a spectrometer until we assess your competence.
How much solvent volume should I use?
To get good resolution you need about 0.7 ml of solvent in a 5 mm NMR tube. The minimum solvent volume on newer spectrometers is 0.55ml or 40mm length. If you have a limited amount of sample you can increase its effective concentration by reducing the solvent volume to 0.4 - 0.5 ml however you will need to spend more time shimming. If you have a sample that will give a proton spectrum in 15 minutes when disolved in 0.7 ml, there is little point reducing sample volume if it means you need to spend an extra 10 minutes shimming! On the other hand if you then want to run a carbon spectrum of that sample it would certainly be worth reducing the sample volume. For ¹³C HMQC or HMBC runs with very small amounts of material, a 4mm tube and rotor is available. 3mm spinners are available for the Bruker instruments.

What facilities are available for variable temperature operation?

Caution!

Users must not attempt variable temperature operation unless they have received training in this from an NMR staff member. Probe damage could result!
You should not raise the temperature of your sample higher than 10°C below the boiling point of your solvent. You should also not freeze samples disolved in H2O or D2O. Ask NMR staff about thick-walled NMR tubes if this is a problem.
The blue Bruker "POM" spinners can only be used for temperatures between 0°C and 80°C. (273°K to 353°K). Outside that range a ceramic spinner must be used.
Instruments must be returned to room temperature before the next user runs a sample.

The available / safe temperature range depends on which probe is used on the instrument in question.

Avance 700
This instrument can operate from -150°C to +150°C using the BBO probe.
There are instructions in the folder kept near this instrument.
Avance 400
The Avance 400 on the middle floor of building 137 has a limited variable temperature capability. However this instrument is heavily used so should not be used for long variable temperature runs. Also, given the high usage, it is even more important to ensure the instrument is returned to room temperature when you finish.
Under IconNMR, the achievable temperature range is approximately 5°C to 55°C
The Avance 400 in the NMR Suite can also be used for variable temperature.
Avance 600 and 800
These spectrometers use cryoprobes with a temperature range of 0°C to +80°C.
Users should not attempt to take the probe all the way to +80°C however, since a small transient rise in temperature above +80°C will cause the cryoplatform to warm the probe up.

What does the "ADC overflow" error message mean?
The signal recieved from the NMR sample is first amplified by the reciever and then digitised by the analog to digital converter (ADC). If the signal is too strong for them to handle, either the receiver or ADC will "overflow", causing a RECEIVER OVERFLOW or ADC OVERFLOW message to be displayed. The acquired FID is likely to be clipped, resulting in a distorted spectrum. The solution is to use autogain (type gain='n') or to type in a lower value for the receiver gain. If overflow still occurs when the gain is set to zero, reduce the observe pulsewidth (PW) to half its present value. If overflow still occurs dilute your sample, or if the solvent signal is causing the ADC overflow use a solvent suppression technique.
How do I shim / tune the spectrometer?
First of all, let's get our terminology straight. Shimming is adjusting the resolution of the signal by optimizing the homogeneity of the magnetic field. Tuning is adjusting variable capacitors in the probe to minimize its impedance at the frequency of interest. A poorly tuned probe reflects a lot of the power of the pulses, so that what should be a 90 degree pulse is in reality only (say) a 50 degree pulse. Probe tuning does not affect the resolution, however the signal to noise of a standard spectrum will be worse if the probe is poorly tuned. Also, experiments such as DEPT or COSY that rely on accurate 90 degree pulses may produce artefacts or not work at all. (Note however that it is possible to adjust the pulse width to give a 90 degree pulse on a poorly tuned probe). Poor shimming on the other hand, results in broad NMR resonances. People often talk about "tuning the resolution" which is where some confusion between shimming and probe tuning arises. Shimming is adjusting the homogeneity of the magnetic field, so that every part of the sample in the NMR tube experiences exactly the same field strength.
OK, so how do I tune the probe?
Tuning the probe is necessary because
- Frequency, solvent and sample height all affect probe tuning.
- You may not know what nucleus the previous user left the probe tuned to, or whether he/she completely messed up the tuning,
- A poorly tuned probe will reflect some of the pulse power, resulting in inaccurate 90° and 180° pulses. More sophisticated experiments such as COSY, HMQC, HMBC etc. require accurate pulses.
Tuning of the Bruker spectrometers is performed automatically under IconNMR, or by using the atma command.
Which spectrometer should I use for carbon?
When measuring carbon spectra, the main concern is usually signal to noise. You would expect higher field spectrometers to have a decisive advantage - for example a 500 Mhz spectrometer when compared to a 300 MHz spectrometer should have an advantage of (5/3) squared, or 2.8 times the signal to noise. However there are other considerations, including for example the type of probe. An indirect detection probe has the proton observe coil on the inside (that is, closer to the sample than the coil used for carbon). This improves the proton signal to noise, however if you use an indirect detection probe for directly observing carbon, the signal to noise will of course be worse than a probe which has the carbon coil on the inside. Regardless of the probe design, carbon and protons use different coils, and since the electronic circuit for the two nuclei is different it makes no sense to compare proton signal to noise on two instruments and extrapolate the results to carbon.
Also, signal to noise tests are usually performed by collecting a single scan on a concentrated sample, however this does not give the best indication of the results obtainable on "real" samples where the sample is scanned for several hours. When a sample is repeatedly pulsed, the relaxation times of the various carbons must be taken into consideration. Nuclei take longer to relax at higher fields, so the gain in signal to noise is less than expected. Also note that carbons that do not have directly bonded protons (i.e. carbonyls and quaternaries) have much longer relaxation times than protonated carbons.
Also remember that
- if there is not much sample available, you should reduce the amount of solvent. (See How much solvent volume should I use?)
- if you are interested in quarternary carbons, a longer D1 delay of 3 seconds or more is advisable.
- If the signal to noise of your carbon spectrum is too low, try running a short and/or long range proton-carbon 2D correlation experiment. It has been known for a long time that this can give dramatic improvements in S/N. See J. Am. Chem. Soc. 101, 4481 - 4484 (1979).
Why are some of the peaks in my APT missing?
The APT experiment relies as much on the size of the ¹J_CH coupling as the number of attached protons to generate the spectral pattern. This is because the delays in the experiment are matched to the inverse of the size of ¹J_CH. If ¹J_CH is much larger than the default ¹J_CH of the experiment (usually set to 140 Hz which is the average of ¹J_CH for sp³ and sp² carbons) then peaks will either disappear or appear with incorrect phase. Carbons that may show this behaviour are terminal ethynyl groups (¹J_CH = 250 Hz approx.), epoxide carbons (¹J_CH = 175 Hz), furan, pyrone and isoflavone carbons (¹J_CH = 200 Hz), 2-unsubstituted pyridine and pyrolle carbons (¹J_CH = 180 Hz) and 2-unsubstituted imidazole and pyrimidine carbons (¹J_CH > 200 Hz).
I can't lock on.
- You are using a deuterated solvent aren't you?
- Can you see a lock signal? If not, click the lock off button in the BSMS display, increase the lock power and lock gain, and look for the lock signal by adjusting the field. If you find the lock signal, adjust the field so that the signals in each sweep direction appear symetrically around the centre of the display. Then reduce the lock power (to avoid saturating the lock) and click the "ON" button.
- If it loses lock as soon as you try to lock on, turn the lock off and adjust the lock phase as shown in the manual.
- Can your tube spin? It might not be able to spin because it broke when being inserted. Take the tube out and check that it is in one piece. While you have it out, use a depth gauge to check that the sample is centred in the probe.
It won't shim.
- To start with, gradient shimming is performed automatically by the "topshim" routine. If this fails it can be because the shims were very poorly adjusted to start with, or because there is something wrong with the sample or NMR tube.
  If you are attempting to shim manually, make sure the lock power is not turned up so high that the lock is saturated. If you reduce the lock power and the lock level does not drop, or it increases, you are saturating the lock. Keep reducing the lock power until the lock level drops.
- Check the linewidth of the narrowest line in your spectrum. If there are some broad lines and some narrow lines, the broad lines are probably broad because they are undergoing chemical exchange, not because the resolution is poor. Broad lines may also be caused by quadrupolar broadening if your compound has a transition metal.
- If you have not already done so, load a recent shim set using the rsh command. You don't know what sort of state the previous user left the shims in! Make sure to pick a shim set that applies to the probe currently in use.
- If it still won't shim, take the tube out and inspect your sample. Is the tube scratched? Is there anything floating in the sample? Is the sample centred in the coil? If you are using a small amount of solvent to improve the concentration, you may need to add some more solvent to make it easier to shim.
- Do you have paramagnetic ions in your sample?
- Have other people been getting poor resolution? If so, report it to a member of the NMR staff. If not, change NMR tubes, filter your sample, and try again. If changing tubes solves the problem, throw the old tube away.
- Have you placed your NMR tube in an oven to dry? If so, throw the tube away as it has distorted. (Remember, glass is a liquid. It flows at high temperature). The correct way to dry a tube is via a stream of dry nitrogen through a glass wool filter.
My tube broke when I inserted it into the magnet.
If this happens, it is absolutely essential to leave a note to warn others not to use the spectrometer. If someone lowers their tube on top of your broken tube, not only will it break their sample, but it will force your samle further into the probe, and the probe will then have to be sent away for repair. A single broken tube is unlikely to damage the probe, but two broken tubes will!
During the day, phone an NMR staff member. Tell them the solvent and any hazards posed by your compound. After normal working hours email an NMR staff member after leaving the note.
I can't phase correct my spectrum.
The apk (automatic phase correction) command usually does a good job of correcting the phase, and should be the first thing you try. Sometimes (for example in noisy spectra) the apk command is unable to correct the phase. In these situations you will have to correct the phase manually. First a couple of obvious things: if you ran a DEPT or APT experiment or something similar, there will be some positive and some negative peaks, so don't try and phase them all positive! Similarly in a 1:1 binomial solvent suppression sequence, half the spectrum will be positive and half negative.
Having established that you are not running an exotic pulse sequence that produces strange phases, the next thing to consider is foldback. Are you sure that you used a large enough spectral width when acquiring the spectrum? If one or more resonances occurred outside the observe region, the method used to digitise the signal results in these resonances appearing within the observed spectral width, but with a phase error. If in doubt, double or triple the spectral width, run the spectrum again, and see if the resonance that could not be phase corrected remains at the same chemical shift as before.
I need to run my spectrum at a higher field to get better resolution.
No you don't! The resolution of a high field spectrometer may even be worse than a low field spectrometer. What a high field instrument has more of is dispersion. This means that resonances with different chemical shifts are further apart. Multiplets due to coupling will not show any improvement unless the higher field instrument separates overlapping multiplets with different chemical shifts, or the multiplet showed strong coupling effects at lower field. Some nuclei such as ³¹P may have worse resolution because of a property called chemical shift anisotropy which increases with field strength.
There are no parameters for the solvent I want to use.
If you're running a proton spectrum, set up for ¹H / CDCl₃, double the spectral width, run a quick spectrum, then expand the result so that only about 1ppm of baseline appears at each end of the spectrum. Then click on the icon to reset O1 and SW to the displayed region and acquire the final spectrum. If you're running a carbon spectrum, set up for ¹³C / CDCl₃, increase the spectral width by 20 percent, and run as normal. If your solvent has carbon nuclei which show up quickly, reference the solvent and check that the observed spectral range is correct.
If you are running a phosphorus spectrum, set up for ³¹P / CDCl₃, increase the spectral width by 20 percent, and run as normal. Then supply an NMR tube containing the solvent to the NMR staff so that they can set up H₃PO₄ referencing parameters for you.
How can I suppress a strong solvent resonance in a proton spectrum?
If the solvent signal is less than two or three times the size of the largest signal from your compound, it may not be worth bothering. One method is to presaturate the solvent signal. Although it is simple, this method has the disadvantage that NH or OH protons that are exchanging with water also have their signals reduced or eliminated. Another method is watergate solvent suppression. If chemical exchange is very rapid, watergate may not be suitable, in which case a binomial pulse sequence is the best choice.
What is nuclear spin?
All nuclei carry a charge. In some nuclei this charge "spins", causing the nucleus to behave like a tiny bar magnet. This is why it aligns with or against the magnetic field of an NMR spectrometer. However unlike a bar magnet, the low energy state is aligned with the field and the high energy state is aligned against the field. Up to now we have been talking about nuclei with a uniform spherical charge distribution. These nuclei are said to have a spin of ½. Protons, ¹³C and ³¹P are all spin half nuclei. Note that the most common isotope of carbon, ¹²C, has no spin and can therefore not be observed using NMR. Nuclei with a non-spherical charge distribution have a spin number I of 1, 3/2 or higher (in steps of ½ ), and are referred to as quadrupolar nuclei. Spin ½ nuclei have two orientations (with or against the field). Spin 1 nuclei have three orientations, spin 3/2 nuclei have 4 orientations, etc. Deuterium is an example of a spin 1 nucleus. Although deuterium is chemically the same as hydrogen, for the purposes of NMR it is completely different. For example a carbon spectrum of CDCl₃ is a 1:1:1 triplet regardless of whether you turn on the proton decoupler. This is because the deuterium attached to the carbon can have three orientations, and occurs at a different frequency to protons.
What is a double quantum coherence?
When you put your sample in the magnet, all the spin half nuclei align either with or against the magnetic field. The population difference between these two orientations (known as the Boltzman distribution) is field dependent, and is determined by their energy difference. An NMR signal is observed when nuclei flip from one orientation to the other. This is a single quantum coherence. When two nuclei are coupled, they can flip together as though they were a single unit. If they flip in opposite directions, the flips "cancel each other out" (sort of) resulting in a zero quantum coherence. If they both flip the same way, you get a double quantum coherence. The frequency of a zero quantum coherence is between zero and a few kilohertz, so it is not directly observed. Similarly the frequency of a double quantum coherence is roughly twice the normal observe frequency, so that is not observed directly either. You can also have triple quantum coherences from groups of three coupled nuclei. The effect of double and triple quantum coherences can only be observed by inserting pulses or delays into a pulse sequence to convert them to single quantum coherences before acquisition of the NMR signal. Do not confuse double quantum coherences with coupling in a normal spectrum. A doublet for example, arises when there are two coupled spins, but only one of these spins flips.
What are pulsed field gradients?
Imagine if you could really mess up the Z1 resolution for a few milliseconds then restore it to its proper value during the course of the pulse sequence. This is an oversimplification, since pulsed field gradients do not use the normal shim circuits. A special PFG probe, and a PFG amplifier are necessary. By applying a gradient to the magnetic field, the top of the sample experiences a slightly different magnetic field to the bottom of the sample. Since magnetisation precesses at different rates in different fields, it is possible after a 90 degree pulse and a PFG of a few milliseconds to have the magnetisation vectors along the length of the tube pointing in all directions instead of nicely aligned along one axis of the rotating frame. Obviously if the magnetisation vectors are pointing in all directions, there is no net signal. The vectors are said to be dephased. If you now apply a PFG of opposite sign for the same time, you will rephase the magnetisation, and get your signal back. You could achieve the same thing by giving the dephased vectors a 180 degree pulse, then applying a PFG of the same sign. The other thing to be aware of is that double quantum coherences dephase at twice the rate of normal single quantum coherences, so by adjusting the strength or duration of pulsed field gradients, you can select single, double or triple quantum coherences. The "old fashioned" way of selecting certain types of coherences is to use elaborate phase cycles which cause the unwanted signals to cancel out on successive scans. The PFG method acquires only the desired signal on each scan, resulting in fewer artifacts and allowing fewer scans. The old method can be thought of as "cancellation of unwanted signals over time" whereas the PFG method can be thought of as "cancellation of unwanted signals over space" where "time" refers to successive scans, and "space" refers to the physical length of the sample in an NMR tube.
What is the Nuclear Overhauser Effect?
Glad you asked. Have a look at our NOE guide.
How do I run a quantitative spectrum?
A quantitative spectrum is simply a spectrum where you can trust the integral ratios. In other words, if the integral of resonance A is twice the height of the integral of resonance B, you can say with certainty that resonance A is due to twice the number of nuclei as resonance B. Why do we use integrals? Because it is the area of the resonances that is proportional to the number nuclei. The height of a broad line may be less than that of a sharp line, but its area may be greater. How do we get accurate integrals? By ensuring that all resonances are equally excited, well digitised, and properly relaxed.
- Equally excited : if the pulse power is not high enough, some resonances far from the observe frequency may experience a reduced flip angle, resulting in a smaller observed signal.
- Well digitised : if the number of data points in the spectrum is too low, there will not be enough points to accurately define each resonance, resulting in inaccurate integrals (and peak heights).
- Properly relaxed : resonances that are not fully relaxed give a weaker signal than fully relaxed resonances. The nuclei in your compound will not all relax at the same rate, so if you pulse too rapidly the quickly relaxing resonances will appear stronger than the slowly relaxing ones. To be sure of obtaining accurate integrals, you need to measure the relaxation times of your compound, and set a delay equal to 5 times the longest relaxation time. Fortunately it is easy to run an inversion - recovery experiment to measure relaxation times.
It is harder to obtain quantitative carbon spectra, because carbon relaxes more slowly than protons, is less intense, and steps have to be taken to eliminate the Nuclear Overhauser Effect which builds up when protons are decoupled.
Further notes are available on the Internal NMR web site..
What is digital resolution?
Digital resolution is simply the separation in hertz between each data point in your spectrum. It has nothing to do with shimming. Say, for example, you set the number of points td to 32,768 and acquire a normal 1 dimensional FID. The number of points in the spectrum you see will be 16384, since half the data points are imaginary. Now if the spectral width (sw) is 6000, the digital resolution will be 6000/16384, or 0.366 hz per point. (Before you grab your calculator to measure your own digital resolution, note that the number of points in the spectrum is not always simply td/2. See the section below on the SI parameter). If the natural linewidth of a resonance is comparable to the digital resolution, the resonance may only be defined by one or two data points. If you expand a line like this, it will look more like a spike than a proper Lorentzian line. Consequently the height of the line may appear less then it really is, the integral will be inaccurate, and even the chemical shift value will be less accurate than it should be. Also, if the separation between two resonances is comparable to the digital resolution, they may appear as a single resonance in the spectrum, because no data point falls in the space between the tops of the two peaks.
What is the SI parameter?
Mathematicians can do a Fourier transform of any number of points. NMR spectrometers speed things up by using the Cooley-Tukey fast fourier transform algorithm. As implemented on NMR spectrometers, this requires the number of points to be a power of two. So what happens if the number of points td is not a power of two? The number of points used for the Fourier transform is controlled by the SI parameter which is always a power of 2. If SI is less than td, some points on the end of the FID are not used in the Fourier transform. If SI is greater than td, the end of the FID is padded with zeros to increase the number of points. This is referred to as zero filling. Zero filling does not write extra zeros on to the end of the FID file on the disk where the FID is stored, it merely adds the zeros in memory just before doing the transform.
What is the relaxation time?
It would be an oversimplification to say that the relaxation time is the time taken for a nucleus to relax to equilibrium. After a pulse, a nucleus relaxes toward its equilibrium value at an exponential rate. The value quoted as the relaxation time is actually the time constant of this exponential curve. It takes five time constants for the magnetisation to relax to 95% of its equilibrium value. There are two basic types of relaxation, T₁ and T₂. In the T₁ process, the magnetization remaining along the z-axis relaxes back to its equilibrium value. This is also known as spin-lattice relaxation because relaxation occurs by the loss of energy from the excited nuclear spins to the surrounding molecular lattice. In the T₂ process, the magnetization in the x-y plane fans out out until the net magnetization is zero. This is also known as spin-spin relaxation because it is due to the excited spins exchanging energy with each other.
What NMR Simulation Programs are Available?
- Bruker Topspin and Varian VnmrJ programs both have spectral simulation routines. To simulate a normal (non-exchanging) spin system, the first step is to decide what sort of spin system you have - AB, A2X, ABCXY etc. The letters are not important, so it doesn't matter whether you tell it that you have an ABC or an AMX system. The software only needs to know the values of the chemical shifts and coupling constants.
- To simulate a dynamic (exchanging) spin system, the usual procedure is to run a series of normal spectra at different temperatures, and then simulate the lineshapes. This is done using the DNMR5 program, an implementation of which is available in Bruker's Topspin program, version 2 or later.
  Further notes are available in the Internal NMR web site.

Australian National University NMR Centre home page.