The 1D-nOe experiment measures the distance-dependent nuclear Overhauser effect from one ¹H proton to other protons within 5Å. The idea is to irradiate one peak and change the size of the peak(s) coupled through space relative to a reference spectrum. One resonance in the spectrum is perturbed by saturation. The net intensities of other resonance may change due to spins close in space to those directly affected by the perturbation. A measure of the change in amplitude can be used to determine the geometry of the molecule.

In a ¹H - ¹H nOe the percent change is small (2-10%), using the nOe difference experiment. ¹H - ¹H nOe is used to determine the proximity of pairs of protons. A 2-D NOESY experiment gives a measure of the through-space coupling for all the protons at once.

Acquiring 1D nOe data

1. Acquire a proton spectrum. Set the appropriate window. Type dgain and record this value.
2. Adjust the following parameters:

3. Normally a dpwr value of 10 is a reasonable value. The post-run spectrum should show the irradiated peaks to be about 10-20% of the non-irradiated peaks. You can set dpwr=10 as a test, run a few scans and then measure the delta of the irradiated peak and the non-irradiated peaks. You can then adjust the dpwr value so that the irradiated peak is no larger than 10% of its original height. NOTE: the greater the dpwr value, the wider the irradiated band becomes and could produce undesirable results by partially irradiating peaks next to the main irradiated peak.

4. Acquire a standard 1D-proton spectrum and then place the cursor on the peak to be irradiated, type nl and then sd (this sets the decoupler offset to the cursor position). This is the "on-resonance decoupler frequency".
5. Place the cursor in a blank region, near the end (downfield), of the spectrum (at least 1/2 ppm away from any peaks) and type sda. This adds the cursor value to the decoupler offset array. This is the "off-resonance decoupler frequency" and is termed the sda-blank in this document.
6. If additional peaks are to be irradiated, you place the cursor on the next peak, type nl and then sda again. Do this for all of your peaks of interest. If you have more than 3 or 4 peaks that you want to irradiate then you may want to set a second sda-blank in your experiment to allow you to check the system stability. You could set this at any blank space on your spectrum. Typing da will display a list of the peaks and off-resonance frequencies you have selected.
7. nt should be set from 10 to 100 times the number of transients normally collected for a ¹H spectrum of this sample. nt should be a multiple of 32. Quantitative nOe measurements require excellent signal/noise while a qualitative nOe is a little more lenient.
8. You can type time to see how long your experiment will take. This will help you in scheduling your time for the spectrometer.
9. Type ai ga to set the absolute intensity mode and start your acquisition. If you receive an ADC overflow error message, you will want to reduce your gain setting and start the experiment again.
10. At the end of your run be sure to save your data with the file - save FID selection. All spectra can be saved in 1 file under 1 name

Processing nOe data

1. Log on to a Sun data-station.
2. Recall your nOe data like any other fid into any experimental area except exp5.
3. Set your lb (line broading) parameter between 1.0 and 5.0, type ai and then wft. This increases the signal-to-noise ratio of the spectrum (at the expense of resolution) which is important for accuracy.
4. Type ds(#) f full and aph to start processing your first spectrum (NOTE: # is the peak you irradiated).
5. Type bc. This performs a baseline correction to your spectrum.
6. You reference your solvent peak next.
7. Type ds(2) f full to process the second spectrum (DO NOT phase. The phasing values from step #4 are used) NOTE: 2 is the sda-blank region you irradiated. You may have made the sda-blank your third, fourth, or fifth irradiated region in which case you would use ds(3), ds(4), or ds(5).
8. Type bc. This is the same as step #5.
9. NOTE: (see #1 in the last section below.) Type clradd ds(#) spadd ds(2) addi. This will clear experimental 5 area, add ds(#) to that area, and then place ds(2) there and subtract ds(2) from ds(#) and show the results. Always use the sda-blank in the second half of the above command statement.
10. Select sub from the interactive menu.
11. Click on the select button twice (the visible yellow spectrum is active now). Increase the scale of the yellow spectrum with the middle mouse button. DO NOT touch the green or blue spectrum.
12. Select the Save button to save your worked-up data at this point.
13. Join exp5 (jexp5) to view and analyze this different spectrum. NOTE: If you receive an error message to the effect that experimental 5 area can't load the processed data then load the FID and WFT the data. Next join another experimental area and start at step #2 again.

You will type the following commands to print your nOe spectrum.

1. text('name') This will place a name on your printout.
2. ds(#) vsadj vs=vs/2 vp=12 pl pltext pscale NOTE: # is the irradiated peak of interest, also, record the vsadj value.
3. ds(2) vp=60 pl NOTE: ds(2) is the irradiated blank area (sda-blank) in this write-up. Do not change vertical scale here
4. NOTE: If you want to plot out more than two spectrums on one page, please refer to "How do I print Several Different Spectra onto one page?" in the online IUNMR Users Guide.
5. jexp5 ds vp=120 Adjust your vertical scale to the value obtained above (step #2).
6. pl page This sends your plot to the printer.

A simplified way to quantitate your nOe data is as follows:

1. Follow the instructions above (the Processing nOe data section) up to but not including step number 9.
2. Print out a spectrum so you can write and place remarks on it.
3. Zoom in on your area of interest on the spectra.
4. Type vsadj to set the vertical adjustment of your displayed spectra. You should write this value down. This is the only time you will issue this command. All other views of this or other spectra needs to have this vertical scale value in order for you to obtain meaningful data.
5. Place the cursor on a peak and type nl. You will be given a value for the height of the peak. Write this value on your printout by the peak. Also annotate whether this value is from an irradiated peak or non-irradiated peak. You repeat this step for all peaks of interest.
6. Type ds(x) where x is the next irradiated peak or sda-blank (irradiated blank region). You repeat step number 5 for this spectrum and write down all values on your printout (You may want to print out a fresh spectrum to write your next set of values on). REMEMBER: DO NOT typevsadj at anytime other than the first time you typed it in step number 4. This would cause you to change the vertical scale in the middle of your data gathering and would makes the data worthless. Repeat step 6 until all irradiated peaks and the irradiated blank area (sda-blank) have been measured and recorded. You should be able to produce a nice table with these values. (See Table 1.)
7. With this set of data you can now do the following calculations:

• To estimate the amount of saturation by the irradiation on your peaks of interest: Starting with your irradiated peak, divide the height value of the irradiated peak by the height value of the same peak in the sda-blank spectrum. You will obtain a numerical value of Y. Subtract Y from 1 to obtain a value Z where Z is the saturation efficiency value. i.e. (1-Y = Z).
• For all other peaks, you subtract the value of a non-irradiated peak measured in the sda-blank spectrum from the value of this same peak measured in the irradiated peak column. You then divide that answer with the non-irradiated peak value measured in the sda-blank again. With this semi-final value you divide with the Z value from the above step. Multiply by 100 and you now have the percentage enhancement value for the peak.
• A descent nOe will have a value of around 5%, very strong will be 10%, and a weak one is less than 2%. A positive value or a negative value can be obtained.

The following is an example of a calculation for the nOe on Peak #2 when Peak #1 is irradiated.

nOe =