2D 1H-1H NOESY

1D and 2D NOESY

One-dimensional NOESY relies on selective inversion or saturation of one peak and observing NOE peak at another position. The reliance on selective pulse reduces the sensitivity of the experiment and limits NOE to one 1H at a time. Among the versions of 1D NOE experiments, the NOE difference experiment (noedif or cyclenoe) relies on perfect cancellation of spectra done with and without 1H saturation, a very demanding quality for any spectrometer, particularly older ones. Temperature stability, vibration, hardware instability from scan to scan all contribute artifacts to the cancellation spectrum. Additionally, through-bond coupling artifacts are not suppressed, and are often shown as dispersive signals in the final spectrum, complicating data analysis. NOEDIF or CYCLENOE is NOT the recommended 1D NOESY experiment unless quantitative NOE (% enhancement) is desired. The recommended, gradient enhanced 1D NOESY experiment (NOT based on difference and cancellation) is similar to the 2D experiment shown below, with gradient pulses and zero-quantum suppression.

Whenever possible, the gradient-based 2D NOESY (~ 30mins) is strongly recommended. 2D NOESY has the advantage of using hard pulses to excite all signals at once, more efficient and sensitive. It is also more reliable in distinguishing real NOE peaks from artifacts because of the automatic crosspeak symmetry present.

2D Gradient Enhanced NOESY

The following figure is the gradient based 2D NOESY pulse sequence with the main parameters displayed (NMR500). This sequence uses Z-gradient to crush zero-quantum (via J-coupling) artifact contribution, often shown as dispersive peaks. This is the best choice of mordern 2D 1H-1H experiments.

Procedure (on NMR600)

Default means the settiong is already in place by default, unless changed by a user.

• Lock and shim your sample as usual. Keep lock at ~80% after shimming by increasing gain or power.
• Find proper sw and tof to use. Note that the whole signal region must be enclosed in the spectral window.
• Under exp1 (type jexp1 to go there), collect a standard 1H spectrum with nt=1, process and reference the spectrum. Alternatively, you can also load a previous 1H experiment of this sample and process it.
• Put the box cursor (two cursor lines) to enclose the signal region and to include ~10% flat baseline area at each end of the peak region. Type movesw. This command sets a new spectral width sw and spectrum center tof.
• Run another 1H spectrum with nt=1 with the new setting. Reference the spectrum carefully or enter setref.
• Load standard 2D NOESY parameter file
• Type jexp2 to goto exp2. From exp2, type mf(1,2) to move fid from exp1 to exp2. Type wft f full to display spectrum. Check and reference spectrum.
• Type setexp('noesy2d') su to load standard NOESY parameters. Solvent, and 1H spectral width and center are copied over from 1D spectrum.
• Enter time to display experiment duration
• Use default parameters values (~20-30 mins experiment) in most cases. The default number of scans (nt) is 4 and NOE mixing time (mix) is 0.5 sec. For dilute sample or to get stronger NOE signals, increase nt or increase mix.
• nt=8 (or 16, 24, ... 4*n)
• mix=1.0 (in most cases, confine mix to 0.5 to 1.0 sec. Longer mixing time may further reduce undesired through-bond signals but may bias the NOE accuracy.)
• Type time to check experiment time.
• Type go to collect data.
• Watch for ADC or receiver overflow:
• In arrayed experiments such as 2D's, a fixed gain (not autogain) must be set. Watch the receiver overflow light on the VT monitor next to the workstation and the ADC overflow error message immediately after the experiment starts. The default (gain=26) works most of the time unless there is an extremely strong 1H signal in your sample. With a gain above 20, it makes only small differences.
• If the receiver overflows (red light blinks on VT display) or ADC overflow message is seen above the command area anytime during the experiment except during the dummy scan period, it is likely the data are ruined. Stop the experiment (aa), check the current gain (gain?) and reduce gain by 2-4 at a time (gain=22, etc), enter go until the overflow disappears.

Default parameters

The default setting (modified from Varian's default values) in our parameter set uses the following for a quick 2D NOESY:

• mix=0.5 (NOESY mixing time is 500msec, sufficient for most molecules of < 1000 Da)
• ni=128 (128 complex points along indirect dimension)
• d1=1 (recycle delay between scans is 1 sec)
• ss=16 (16 dummy scans before actual data collection)
• nt=4
• phase=1,2 (phase-sensitive, hypercomplex mode in indirect detection)
• zqflg='y' (turns on zero-quantum signal suppression to crush through-bond (J) contributed artifacts, often shown as dispersive signals)
• gain=26 (reduce it by 2-4 at a time if receiver or ADC overflows)

Data Processing

You can use vnmrJ to process the data, but vnmrJ's graphics display for 2D spectrum leaves much to be desired. A separate program is strongly recommended for detailed processing and analysis. Mnova and nmrPipe/nmrDraw are installed on NMR600 and the data processing station.

Example spectrum collected on NMR500

• Data collected September 2010 (~30 mins)
• Processed and displayed with nmrPipe/nmrDraw
• NOE crosspeaks are shown as negative (red) peaks. Diagonal peaks are black.
• Some artifacts are typical. Some are due to the compromise made for a quick experiment Most can be easily identified.

Expansion of the 1.4ppm to 4.7ppm region

H.Zhou updated April 2012