NOE effect is a powerful tool to probe spatial relationship between atoms in a molecule. 2D NOESY is a relative easy experiment to set up. However, samples with small concentration have challenging signal sensitivity for 2D NOESY. In such cases, 1D NOE Difference Spectroscopy is an excellent alternative.
- For best NOE, sample need to be degassed to remove dissolved oxygen gas which is paramagnetic and will compete with NOE. Nonpolar solvents are particularly capable of dissolving a large amount of oxygen gas, which could make NOE vanish. Several cycles of freeze-pump-thaw of an uncapped NMR tube would remove most of the dissolved oxygen gas.
- Run a standard proton spectrum
- Take note of the chemical shift values of (A)the peak that you want to irradiate and (B) an irrelevant peak (eg. Solvent or TMS)
- With this proton file on screen, create a new file by edc. Then type rpar and choose NOEDIFF. Then input the o2p value of the targeted proton peak. Collect a spectrum (A). Phase correct it.
- Use spectrum A to create another 1D NOE new file and input the o2p value of the irrelevant peak. Do not do rga – you need to use the same rg as in A. Collect a spectrum (B).
- Integrate spectrum B and save the integrals.
- Load spectrum A. In Multiple Display mode, load spectrum B and subtract the two spectra. Click “Save”, and you will be asked a “PROCNO”. Type 2. This will save the difference to processing number 2 within the same experiment (assume it is exp # 1).
- Read in the difference file by typing re 1 2. The difference spectrum should have the irradiated peak negative and the NOE-enhanced peaks positive.
- Integrate the difference spectra. Right click on the NOE-enhanced peak integration curve and select “Use last scale for calibration”. The ratio between this area and the area obtained in Step 6 is the NOE enhancement ratio.