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Since my university does not have a professional spectroscopist, I am a new learner of NMR, and I really have no one else to ask...

I have a very concentrated sample (small molecule, MW < 300, 100 mg / mL), and I am running a 2D NOESY experiment (pulseprogram = noesyph in Bruker 400 MHz). However, the NOE signals that I am looking for are very weak, hidden by serious t1 noise (since the signal concerned is a CH3 group, very strong proton signal). Besides tricks like symmetrization, what else can I do to reduce the t1 noise or strengthen my NOE signal?

Shall I: 1. Increase ns (no of scan, currently ns = 8)? 2. Increase d1 (relaxation delay, currently d1 = 3 s)? 3. Increase d8 (mixing time, currently d8 = 1.5 s)?

Thanks a lot for your help!

asked Oct 25 '14 at 10:03

Elvis's gravatar image


updated Oct 25 '14 at 10:04

5 Answers:
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Diluting your sample is a very good idea; 100 mg/ml with MW < 300 is a good concentration to run a carbon experiment, but not a noesy. 10 mg should be OK and if you look for a weak noe, never run less than 16 scans. I would try too shorter mixing times, maybe 500 ms or 700ms.


answered Oct 28 '14 at 04:51

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Thank you so much! I thought that NOESY are very insensitive and therefore used a pretty high concentration. Perhaps I should use a lower concentration. - Elvis (Oct 28 '14 at 10:08)

The problem isn't signal to noise ratio, but signal to artefact ratio. - Kirk Marat (Oct 28 '14 at 13:33)

I see, thanks a lot! - Elvis (Oct 31 '14 at 09:17)

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NMR software can erase t1 noise for you. Try ACD labs NMR proccessor. It's free for academics.


answered Oct 25 '14 at 16:04

Arkadiusz%20Leniak's gravatar image

Arkadiusz Leniak

I was using MestRenova, and it also contained "reduce t1 noise" function. However it also introduced artifects in the spectra, and I was a bit worried that it would affect my conclusion. Anyway, thanks a lot indeed! - Elvis (Oct 25 '14 at 22:01)

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You can try a different pulse program that has pulse field gradient. And if MW < 300, a 1.5mg/ml sample is > 5mM. You can dilute your sample at least 50 times and still get good signals. Maybe 1D NOE experiment also can be tried. Just for your reference.


answered Oct 25 '14 at 20:39

dejian's gravatar image


Thanks a lot for your suggestion! - Elvis (Oct 25 '14 at 22:00)

Unfortunately, it seems that the NMR machine I am using is not equipped with the hardware for gradient pulse field (is this possible?) - Elvis (Oct 28 '14 at 10:09)

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I'm not a big fan of 2D NOESY for small molecules in the extreme narrowing region. I find that the gradient selective pulse 1D NOESY experiments (available from the "Selective" panel in Topspin) are much better and can detect NOEs down to a small fraction of 1%. When you get to slightly larger molecules, at the cross-over between extreme narrowing and spin diffusion, then the corresponding 1D ROESY experiments (especially the modification by Julien Furrer J. Nat. Prod. 72, 1437, 2009). work very well.

If you really need the 2D experiment, then check for things that tend to cause T1 noise: vibration, room temperature stability, proper PID parameters in the VT unit, good sample, proper lock parameters, etc. I'm suspicious of processing algorithms, including symmetrisation, for reducing T1 noise.

Also, I think your mixing time might be a bit long. It's always a competition between NOE build-up and relaxation. I find that about 500 ms. tends to be a bit better for most things.

100 mg is probably a bit to high for concentration at this MW (10 to 20 mg would be fine), and it helps to degas your sample to ensure maximum NOE by reducing relaxation caused by dissolved oxygen.


answered Oct 28 '14 at 07:55

Kirk%20Marat's gravatar image

Kirk Marat

updated Oct 28 '14 at 08:42

What about 1D NOESY without gradient pulse? How bad is it compared with the gradient pulse one? I think the NMR machine that I am using cannot support gradient pulse... - Elvis (Oct 28 '14 at 10:11)

Haven't actually done one of those ion a long time. You can use a z-filter list to remove some of the artefacts that occur when you don't use gradients. - Kirk Marat (Oct 28 '14 at 11:21)

Thanks again for your detailed answers. - Elvis (Oct 31 '14 at 09:17)

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There are some tricks to optimize your spectrum once recorded but I think you should concentrate first on running an experiment with a better signal to noise ratio. A solution of 10 mg/ml should be sufficient wrt signal to noise. A mixing timne of 1.5 sec is much too long. For my experience you can use 250 – 500 msec as an initial delay, again depending on molecule and the interaction you want to observe. You can always optimize the delay but in this range you should be able to record a spectrum with a good signal to noise.

However, depending on your solvent, field strength and your molecule, you need to choose between a NOESY or ROESY, since the NOE effect will disappear and no cross-peaks will be observed in a NOESY spectrum when w*tc=1.

Back to your problem. You can do three simple things; 1) run the experiment with a mixing time of 500 msec. 1) try a ROESY using a 500 msec spinlock 2) change the tumbling rate by changing solvent, e.g. CDCl3 <-> DMSO.

Good luck

ps; like one of my friends always tells me when I am struggling to record good data: it is easier to optimize a signal then to find a signal ;-)


answered Nov 02 '14 at 07:16

RuudA's gravatar image


updated Nov 02 '14 at 07:22

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Asked: Oct 25 '14 at 10:03

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