IU NMR user guide

HMQC – Heteronuclear Multiple-Quantum Coherence Experiment

(Frank Gao, 8/1/01)

The HMQC experiment establishes correlations between protons and other nuclei such as ¹³C or ¹⁵N while detecting high sensitivity protons. The correlations between protons and heteronuclei proceed via double and zero quantum coherences.

Note: 1) On I500, you have to recable the spectrometer when you change the detected nucleus between proton and ¹³C (or other X nuclei). The other spectrometers do not require hardware changes. 2) The gradient version of HMQC/HMBC experiments is preferred if the spectrometer is equipped with a gradient accessory (e.g., I500). 3) The HMQC/HMBC experiments are best-done using the inverse probe.

In order to minimize residual non-¹³C-bound proton signals and “t1-noise”, a number of operating conditions that can influence the stability of the system should be controlled: 1) run experiments non-spinning; 2) optimize the lock signal; 3) use proper RF filters; 4) use VT (variable temperature) regulation; 5) use a moderate air flow through the probe; and 6) run experiments at night or weekend if possible.

1.1 Setting air pressure, temperature, RF filter, etc.

1. Set the air pressure to 20 PSI
2. Use a ¹³C band-pass filter (e.g., BE109-22-8BB for I400 and VXR400) in the X channel (if you are not sure, ask the NMR staff for assistance)
3. Set up a standard ¹H experiment in expn (e.g., exp1)
4. Type temp=25 spin=0 su                – set temperature and turn off the spinning
5. Wait until the temperature displayed in the Acquisition Status Window is regulated at 25 ºC

1.2 Locking and shimming

1. Make sure the spinning is turned off.
2. Lock and shim as usual (i.e., use lock power given on solvent sheet)
3. Optimize the lock power:
• For non-experienced user, increase the lock power by 3;
• For experienced user, find the highest lock power at which the lock is stable and then lower the lock power by 3;
4. Set the lock gain such that the lock level is about 50.

2.1 For ¹³C

1. If on I500, make sure the spectrometer is cabled for ¹³C detection
2. Join another experiment (e.g., exp2 by typing jexp2)
3. Set up a standard 1D ¹³C experiment (note: use the same solvent name as for ¹H)
4. Enter temp=25
5. Type nt=1 ga to acquire a quick 1D ¹³C spectrum. You should see the solvent peaks (unless the solvent contains no carbon, such as D₂O).
6. Type dscale to display the chemical shift scale. Reference the spectrum to the chemical shifts of solvent peaks (e.g., CDCl₃ peaks should be at ~ 77 ppm).
7. Narrow the ¹³C spectral window to leave about 10% on either side of the peaks of interest by using two cursors and typing movesw (although these peaks are usually not seen in the quick spectrum, they can be expected from a previously collected ¹³C spectrum for this sample or estimated from similar samples).
8. Write down the tof and sw. They will be the dof and sw1, respectively, in the HMQC experiment.

2.2 For ¹H

1. If on I500, make sure the spectrometer is cabled for ¹H detection
2. Type jexp1                – go back to exp1
3. Type nt=1 ga to collect a quick 1D ¹H spectrum
4. Narrow the ¹H spectral window to leave about 10% on either side of the peaks of interest by using two cursors and typing movesw
5. Move the parameters to the experiment in which HMQC will be performed (e.g., enter mp(1,3) to move the parameters from exp1 to exp3)

1. Type jexp4                – join an experiment for the calibration
2. Type mp(1,4)             – copy the ¹H parameter set from exp1
3. Enter temp=25
4. Increase the relaxation delay d1 e.g., d1=10
5. Enter pw=5 (or any other value less than 90º pulse width)
6. Enter gain=’y’           – disable the auto-gain setting
7. Type ga                     – acquire a spectrum
8. Phase the spectrum
9. Enter an array of pw values around the estimated 360º pulse width (i.e., 4*pw90, where pw90 is a pre-calibrated 90º pulse width for the standard sample). For example, enter pw=38.4, 39.2, 40, 40.8, 41.6, 42.4 assuming pw90=10.
10. Type da                     – list the pw array you entered
11. Type ga                     – acquire an array of spectra
12. Type wft dssh            – display all spectra horizontally
13. Find the value of pw for which the spectrum has minimum intensity, and divide this value by 4 to get the accurate ¹H 90° pulse width for your sample.

4. Setting up a HMQC experiment

1. Type jexp3                – join the experiment for HMQC
2. Type iuhmqc             – read in the default parameter set for HMQC
3. Enter temp=25
4. Enter spin=0
5. Make sure that the spinning is turned off
6. Enter dof=…             tof from ¹³C spectrum that you wrote down in 2.1.8
7. Enter sw1=…            sw from ¹³C spectrum that you wrote down in 2.1.8
8. Make sure that the following parameters are set to their up-to-date values listed at the console:
• pw                     – ¹H 90º pulse width
• tpwr                  – ¹H pulse power
• pwx                   – ¹³C 90º pulse width
• pwxlvl               – ¹³C pulse power
9. The following parameters may be changed upon your sample:
• j1xh                  – average ¹J_CH coupling constant (default 145 Hz)
• d1                     – relaxation delay (default 1.5 s)

5. Optimizing the “null” delay (skip this if you deal with a macromolecule)

1. Enter phase=1 nt=1 ni=1 ss=4 dm=’nnn’
2. Enter an array of null values with one very short value (e.g., 0.001). For example, null=0.001, 0.1, 0.2, 0.3, 0.4, 0.5, 0.7, 1, 1.5, 2
3. Type da                     – list the null array you entered
4. Type ai                      – select the absolute-intensity display mode
5. Type ga
6. Type wft dssh
7. Select the value of null for which either most of the peaks, or the biggest peaks, or the peaks you are most interested in, are approximately zero.

6. Presaturation set up (if desired)

Presaturation can be used to suppress the unwanted big peak (e.g., solvent peak, water peak) in a HMQC experiment. Set it up in the following manner:

1. Enter null=0 ss=0
2. Type ga                     – acquire a spectrum
3. Type mf(4)                – copy the data in the current exp (exp3) to exp4
4. Type jexp4                – join exp4
5. Type wft
6. Set the cursor to the peak that is to be removed by presatuation
7. Type nl movetof
8. Write down the value of tof. It will be the satfrq in the HMQC experiment
9. Type jexp3                – go back to exp3 (for HMQC)
10. Enter satfrq=…         the tof value you determined in the step 8
11. Enter satmode=’y’
12. Enter satdly=1.5       a significant time compared with T₁ of the peak to be presaturated
13. Enter d1=0.05
14. Enter satpwr=10,7,4,1
15. Type ga                     – acquire a series of spectra with different saturated power levels
16. Type wft dssh
17. Find the minimum satpwr for which the peak will be removed, and set satpwr to this value

7. Finalizing the set up and starting the HMQC experiment

1. Reset the following parameters:
• phase=1,2
• dm=’nny’
• ss=32             – number of steady-state scans
• nt=16             – number of scans per FID, must be a multiple of 8
• ni=160           – number of data points on the ¹³C dimension
2. Enter null=…           the optimized value determined in 5.7
3. Type time                – estimate the experiment time
4. You may change nt, and ni to suite your time, nt will cost you signal/noise, ni resolution in the ¹³C dimension
5. Type au                   – start the HMQC experiment

8. Processing a HMQC spectrum

1. Type gaussian         – set up the weighting function for the ¹H dimension
2. Type wft(1)              – Fourier transform the 1st FID for ¹H dimensional phasing
3. Phase this 1D spectrum in the usual way. Be aware that there may be some negative peaks in the spectrum. In some case, the 2nd FID is better suitable for phasing – just type wft(2) instead of wft(1).
4. If the data acquisition is completed, type iuhmqcproc – automatically process the 2D HMQC data and display the 2D spectrum.
5. If the data acquisition is in progress (but at least 4 FIDs have been acquired),
• Enter gf1=’n’                     – disable the Gaussian function for the ¹³C dimension
• Type sinesq(2, xx, ’f1’)     – set up a cosine-squared weighting function for the ¹³C dimension. xx is the number of FIDs that have been acquired, which can be found in the Acquisition Window, e.g., sinesq(2, 64, ’f1’)
• Type wft2da                       – perform a 2D Fourier Transform
6. Usually the phase correction on the ¹³C dimension (f1) is not required. In case that you need to phase the ¹³C dimension, follow the steps below:
• Make sure that the 2D spectrum is displayed in “F1 mode” – display the f1 axis (¹³C dimension in HMQC/HMBC) horizontally. If not, enter trace=’f1’ and click the Redraw button.
• Click the phase2D button, the trace to which the horizontal cursor is positioned will be displayed on the top of the 2D spectrum.
• Click and drag the horizontal cursor up and down to select a trace which have a peak toward the right-hand edge of the spectrum, then click the spec1 button
• Select one other trace which have a peak toward the left-hand edge of the spectrum, then click the spec2 button
• Click the Phase button, the spectrum 1 (1st trace) will be displayed on the screen. Click on the peak displayed near the right edge of the spectrum and phase it. Do NOT click in the left part of the spectrum at this time.
• Click the spectrum 2 button to display the 2nd trace. Click the mouse near the right edge of the spectrum and do NOT rephase, then click the mouse on the peak at the left edge of the spectrum and phase it.
• Click the spectrum 1 button to recheck the 1st trace. Repeat the process again if necessary.
• Click the DONE and then Return button to quit from the 2D phasing menu.

The same approach can also be used for f2 phasing with the 2D display in “F2 mode” (trace=’f2’)

9.1 Adjusting the vertical scale and threshold

• Enter the value of the parameters vs2d (vertical scale) and th (threshold) directly (e.g., vs2d=500 th=2), or
• Adjust them by properly using the mouse.
• A macro nm2d can be used to set up vs2d and th automatically.

9.2 Setting the references for a 2D spectrum

1. Make sure you are in the interactive display mode – a pair of cursors (cross-line) or two pairs of cursors appear in the 2D display. To switch to the interactive display mode, click Redraw (if this button is in the menu) or click Main/Display/Color Map (or Contour).
2. If two pairs of cursors are displayed (i.e., in the box mode), click the Cursor button in the interactive 2D display menu to change the display to the cursor mode (i.e., one pair of cursors are displayed).
3. Move the F2 cursor to the position (usually a peak) to be referenced for the F2 dimension by clicking and dragging the mouse (left button), then type rl(x.xp) where x.x is the chemical shift in ppm (e.g., rl(7.27p)).
4. Move the F1 cursor to the position to be referenced for the F1 dimension, then type rl1(x.xd) where x.x is the chemical shift in ppm (e.g., rl1(120.5d)).

9.3 Expanding a 2D display

• In the interactive display mode, use the left mouse button to determine the lower left corner of the region to be expanded and the right mouse button to determine the upper right corner, then click expand
• To display full spectrum, click Full (in cursor mode)

9.4 Plotting a 2D spectrum

1. Type full to adjust the position and size of a 2D spectrum to fill the entire screen (and page).
2. Type dpcon(’pos’, 10, 1.5) to see how your 2D spectrum looks if plotted with 10 contours spaced 1.5 level apart. Try other numbers if you wish.
3. The macro command iuplhxcor can be used for plotting a 2D HMQC spectrum (see the manual for this macro by typing man(’iuplhxcor’)), for instance
• iuplhxcor(’pos’, 10, 1.5, 0, 0) to plot the 2D spectrum solely with 10 contours spaced 1.5 level apart;
• iuplhxcor(’pos’, 10, 1.5, 1, 2) to plot the 2D spectrum with 1D ¹H (in exp1) and ¹³C (in exp2) spectra along the respective axes, those 1D spectra should be put in the respective experiments previously;
• iuplhxcor(’pos’, 10, 1.5, 1, 0) to plot the 2D spectrum with 1D ¹H spectrum (in exp1) only;
• iuplhxcor(’pos’, 10, 1.5, 0, 2) to plot the 2D spectrum with 1D ¹³C spectrum (in exp2) only.
4. Type pltext to plot the experimental text file.
5. Type iupage (or click page) to send the plot to the printer.