Max gets NOESY
(A PDF version of this page is available from the Handouts site)
The Nuclear Overhauser Enhancement Spectroscopy (NOESY) experiment is the 2-D equivalent of the transient-NOE experiment (NOESY1D). It is important to remember that the NOE's generated in this experiment are NOT equilibrium values and thus will be less than those measured using a traditional 1D steady-state difference NOE experiment. Furthermore, the % enhancement obtained from this experiment and from the NOESY1D are NOT directly comparable to those obtained from steady-state measurements. NOE's are plotted as the cross-peaks between two coupled nuclei. NOE's arise between two spins that possess dipolar coupling (i.e. through-space coupling) and not from scalar coupling (spin-spin coupling which produces the multiplets in proton spectra). Since NOE depends on the inverse of the distance to the sixth power, short-range dipolar couplings are heavily weighted. This dependence on distance allows for an estimation of the internuclear distance. Care must be taken, however, as many processes lead to reduced NOEs including spin-lattice relaxation, temperature, increased solvent viscosity, increased molecular weight, and dissolved paramagnetic impurities including oxygen. Furthermore, it is difficult to completely remove unwanted COSY-like cross-peaks (due to zero-quantum coherences), which at best makes interpretation difficult. In fortunate cases, these COSY-like cross-peaks, which have the same phase as the diagonal while NOESY cross-peaks have the opposite phase, will not overlap with NOESY cross-peaks making interpretation less cumbersome. At other times, however, the COSY-like cross-peaks can coincide with the NOESY peaks, which will cancel the NOE signal.
Note: Bold text represent boxes you should click. italic text represent text you should type and hit RETURN.
Determine Through-Space Interactions for Stereochemistry (NOESY, NOESY1D):
Running a NOESY1D. (PDF Version)
NOESY1D is a selective 1-D experiment that uses shaped pulses to selectively excite specific resonances in order to observe isolated dipolar couplings. This is a transient NOE technique and will have NOE's that are less than those obtained from steady-state experiments. The NOE signals will have opposite phase to the inverted peak (i.e. the inverted peak, when phased properly, will be pointing down in the spectrum while the NOE signal will be up). Signals with the same phase as the inverted peak are typically a result of exchange coupling (see EXSY), but can arise from relayed NOE's (NOE's alternate sign and lose intensity as they are relayed down a chain; i.e., positive, negative,positive). With the shaped pulses and gradient selection, there are essentially no interference peaks, which makes identification of small NOE's feasible. Interpretation of NOESY1D spectra is straightforward and quite a bit easier to understand than NOESY. When you are looking at NOE's from a relatively small number of signals (less than 10) or for very small long range NOE's, NOESY1D is preferred.
It must be noted, however, that the % enhancement obtained from this technique is not directly comparable to those obtained from traditional steady-state NOE experiments (i.e. the classic NOE difference experiment). Furthermore, the % enhancement is dependent on the parameter settings (careful calibration and multiple experiments are necessary to obtain accurate values), which means that you should either note and report those settings if you intend to site % enhancement using this technique or simply report relative values (i.e. small, medium, or large enhancement).
- Setup a 1H NMR experiment in exp1 and type gain='n'.
- Make sure your sample is free from particulates.
- It is best to deoxygenate your NMR sample to ensure the maximum NOE. A simple somewhat crude method of degassing the sample is to slowly bubble nitrogen through the solution using a thin hard plastic tube (PTFE 1/32 inch diameter). Make sure you have a slight excess of solvent to conpensate for evaporation. Bubbling for 5 minutes should suffice. A more thorough method is freeze-pump-thaw.
- Acquire a normal 1H spectrum. Phase it well and save the FID fur future use.
- Set the temperature to the desired value and let it equilibrate for at least 20 minutes.
- Determine your 90 degree pulse if desired or type pw90? to get an approximate value and record this value.
- Type tpwr? and record this value.
- Type mf(1,2) to move the FID to exp2.
- Type jexp2 to join experiment 2.
- Type wft to Fourier Transform the spectrum.
- Type NOESY1D('ds') Note: Use capital letters for NOESY1D.
- Type pfgon='nny'. This ensures that the pulsed field gradient is on.
- Turn off the spinning and increase lockgain and lockpower so that the lock level is about 70. Be sure not to use too much lockpower. You have too much lock power if the lock level is not stable (decrease lockpower until lock level becomes stable).
- Move the 2 cursors to bracket the first peak of interest.
- Click Select.
- Move the cursors to the second peak (if needed). Click Select. Repeat for all peaks of interest.
- Click Proceed.
- At this point, you may be required to answer the following questions:
- Enter reference 90 degrees pulse width (usec):
- Type the value you determined previously (from pw90? in exp1)
- Enter reference power level:
- Type the value of tpwr from exp1 (from tpwr? in exp1)
- Type nt=32 or higher. Be sure to use multiples of 8.
- Type gain='y'.
- Type go. This will start your acquisition.
- Type wft to Fourier transform your data. This will transform all of your NOESY1D('ds') spectra.
- Type vp=80 to move the spectrum up.
- Phase the largest peak (this is the irradiated peak) so that it is negative (i.e. it points down). The small NOE peaks will be opposite phase to the irradiated peak.
- To view all spectra, type dssa.
- To view individual spectra, type ds(#), where # is the number of the desired spectrum. For example, I decided to do 5 different NOE experiments irradiating at 9, 6, 5, 3, and 1 ppm. I selected the peaks in the order given, which means that typing, ds(1), will give me the spectrum with irradiation at 1 ppm. Typing ds(4) would give me the spectrum with 3 ppm irradiation.
- Integration and peak picking are done exactly like 1H spectra.
Printing your NOESY1D Spectra:
- To print all spectra in the array:
- Type dssa and look to see if the spectra overlap. If so, type ds(1) and reduce the scale of the spectrum. Type dssa. If necessary, readjust by typing ds(1), adjusting scale, and typing dssa.
- When satisfied with spectra, type pl('all') pscale page.
- To print individual spectra:
- Type ds(#), where # is the spectrum number you wish to print.
- Phase, peak pick, and integrate as desired.
- Type pl pir pll pltext(150,150) page, for example.
Running an EXSY Experiment:
The EXSY (EXchange SpectroscopY) experiment is used for the investigation of dynamic processes within a given molecule. One is able to discern which resonances are exchanging by quick inspection of the 1-D or 2-dimensional spectrum that is obtained. Extraction of rate constants is possible, but can be a bit tedious. Qualitative interpretation, however, is rather straightforward as long as one knows the COSY spectrum. The pulse sequence is identical to NOESY for 2-dimensional and NOESY1D for 1-dimensional.
For the 2-D EXSY, the cross-peaks due to EXSY will have the same phase as the diagonal and are generally quite large as compared with the NOESY peaks. For the 1-D EXSY, the EXSY peaks will have the same phase as the irradiated peak (i.e. the irradiated peak and the EXSY peak will both be down).
*Portions of this page were adapted from procedures by Long Lee and Kermit Johnson.
Last Updated: June 16, 2011 - WebMaster
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