HMQC

Università di Firenze
		gHMQC/gHMBC
Department of Chemistry
(A PDF version of this page is available from the Handouts site)
Resources		Heteronuclear Multiple Quantum Coherence (HMQC) and Heteronuclear Multiple Bond Coherence (HMBC) are 2-dimensional inverse H,C correlation techniques that allow for the determination of carbon (or other heteroatom) to hydrogen connectivity. HMQC is selective for direct C-H coupling and HMBC will give longer range couplings (2-4 bond coupling). Our facility implements gradient-selected versions of both HMQC (gHMQC) and HMBC (gHMBC), which improves the acquired spectra by significantly reducing unwanted signal artifacts.
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Determine 1H-13C connectivity (HMQC, HMBC)

Running a Gradient Selected HMQC: Determine Direct Carbon to Hydrogen Connectivity. (Detailed PDF version)

Only directly bonded hydrogen and carbons will give cross peaks (quaternary carbons are not seen), which makes interpretation rather straight foreword. As seen in the simulated spectrum below, assignment is made by drawing two lines at a right angle from the 1H spectrum to the 13C spectrum through the cross-peak, which looks like a series of concentric ellipses. Thus, in the spectrum below, C1 is directly attached to H1.
- Acquire a standard 13C spectrum in exp1 and save it:
  - Note the left-most and right-most peaks.
- Acquire a standard 1H spectrum in exp2 and save it:
  - Note the left-most and right-most peaks.

NOTE: The above 1D spectra can be obtained earlier and retrieved for the HMQC experiment, but we recommend, at the very least, you should acquire the proton spectrum before every gHMQC run.

In exp3 load your 1H FID, transform, and phase the spectrum.
Make sure the temperature is regulated:
- type temp=24.6 (or 25.2) su. This sets the VT controller to 25 degrees Celsius. It is very important to have a stable temperature in order to minimize distortions due to convection. Allow 10 minutes for temperature equilibration if you set the temperature.
Turn off the spin:
- Lock and shim your sample. Set the lock power so that the lock level is about 50% or higher. Make sure that the signal is not saturated. A saturated signal is usually unstable or if you decrease the lock level and the lock level increases, you are saturating the lock. Decrease the lock power just below saturation. Increase lock gain if the lock level is below 40%.
Type gHMQC. This loads the gradient selected HMQC experiment with the standard parameters:
- Make sure that the pulsed field gradient is on by typing pfgon='nny'.
- adjust the 1H and 13C spectral windows
- Choose the number of transitions. For gHMQC, if you have sufficient sample, you should use a minimum of 2 scans. If your sample is dilute, increase nt to 8 or 16. You may want to consider using non-gradient methods, HSQC or HMQC, for dilute sample because they will give better S/N with the same number of scans (for these methods, you must set nt=8 or higher). Non-gradient methods, however, will have larger T1 noise.

Type nt=2 or higher.
Type time. This will display the total time required for your experiment. If you have more time, you can increase ni to 200 (i.e. type ni=200). Recalculate the time required.
Type go to start acquiring.

Data Manipulation:

Since this is a phase-sensitive technique, phasing may be necessary. If setup is OK, phasing should be limited to typing wft(1) to transform the first increment and phasing manually (click here for manual phasing procedure).
Type setLP1 gaussian wft2da to perform automated linear prediction, gaussian multiplication, and Fourier transform. Type d2d (macro that runs command dconi('dpcon',25,1.2) )to display an interactive contour map with 25 levels. This displays slower than the color map, but it is same as the printout.

Interacting with the 2-D Color Map/Contour Map
To do the following...	You should...
Increase/Decrease the scale	Click on either vs+20% or vs-20% or type vs2d=vs2d1.5* and click Redraw. The typed command increases the display by a factor of 1.5. You can use a larger number if you like (e.g. vs2d=vs2d2*, increases by a factor of 2.
Change the number of color levels	Use the middle mouse button to click on the color scale to the right of the color plot. Click on the smaller number to increase the number of colors displayed.
To expand on a region	Ensure that you are in the interactive mode; if not, click Main Menu=>Display=>Color Map. Click with the left mouse button on the left-most point of your desired region. Click with the right mouse button on the right-most point. Click on Expand.
To expand an exact region	Type sp=#p wp=#p (for the F2 dimension, usually vertical) and sp1=#p wp1=#p (for the F1 dimension, usually horizontal), where # are the numbers in ppm for the region of interest. sp designates the start of plot and wp is the width of the plot. You will need to click on Redraw to update the screen. For example, I want to expand the region between 1 and 4 ppm in F1 and between 2 and 4 ppm in F2, I would type sp=2p wp=2p sp1=1p wp1=3p, then I click Redraw to see the result.
To reference the 2-D spectrum	Expand the region of interest. Click Hproj(max) for the horizontal projection and Vproj(max) for the vertical projection. Place the cross-hair cursor on the diagonal position you wish to reference (the projections will help you to orient the cross-hair). Type rl(#p) rl1(#pdfrq/sfrq)*, where # is the value in ppm you want to be the reference. rl sets the F2 dimension reference and rl1 sets the F1 dimension reference.
Redisplay the spectrum	Click on Redraw.
Display a projection of the 1D spectrum on the side of the 2-D plot	Click Proj, then click Hproj(max) for the horizontal projection or Vproj(max) for the vertical projection. Use the middle mouse to adjust the scale.
Display a trace of the 2-D plot	Click Trace and use the left mouse button to drag the cursor.
View the contour plot	Click Main Menu=>Display=>Contour
Increase number of levels on contour plot: Interactive plot	Type, for example, dconi('dpcon',15,1.2). The dpcon flag is for displaying the contours. The first number (15, in this case) is the number of contour lines (default is 4). The second number (1.2, in this case) is the relative spacing intensity (default is 2). You can input different numbers if you wish, but the second number must be greater than 1.
Increase number of levels on contour plot: Non-interactive plot	Type, for example, dpcon(15,1.2). The dpcon flag is for displaying the contours. The first number (15, in this case) is the number of contour lines (default is 4). The second number (1.2, in this case) is the relative spacing intensity (default is 2). You can input different numbers if you wish, but the second number must be greater than 1.

The processed spectrum should look similar to the one pictured below:

pic of gHMQC spectrum

If, on the other hand, your spectrum looks like this one, then 2-D phasing is necessary:

pic of poorly phased gHMQC

The spectrum above has a phasing issue in the F1 or indirect dimension. Since the description of the phasing approach is rather lengthy, I leave it to the user to go through the detailed method for phasing 2-D spectra, which is available in PDF format from here. It is not that difficult, but it does require a little caution so as to avoid creating a roll in the baseline.

Autoprinting your gHMQC with Projections generated from HMQC (Easy to do, but NOT RECOMMENDED):

Display the region of interest by expanding and click Autoplot: This plots your gHMQC with the projections generated from the 1-D data subsets. The indirectly detected dimension will have low resolution and thus, the projection for that dimension (usually F1) will have broad peaks. For better resolution projections, use the following procedure.

Printing your gHMQC with 1-D Spectrum as Projections: Open your gHMQC Spectrum. Click Main Menu=>Display=>Size=>Center. Join another experiment and retrieve the 1-D spectra:

Type jexp1 or any other experiment number.
Load the 1-D proton spectrum; Fourier transform (wft), and phase (aph).
Type jexp3 or any other experiment number that is not your proton or gHMQC spectrum.
Load the 1-D carbon spectrum; Fourier transform and phase.

Type jexp2 or join the experiment number where your gHMQC resides:

Click Display=>Contour and expand, scale, etc. the region of interest (refer to table for interacting with the color or contour map).
Type plghmqc and follow the directions on the screen: This is our in-house macro that prints your desired 2-D spectrum with high-resolution spectra as the projections. You will be required to respond to the following:
- Enter experiment number containing the 1H spectrum:
- Enter experiment number containing the 13C spectrum <0=none>:
- Enter experiment number containing contour spectrum:
If you would like to generate a postscript file of this plot, type plghmqc_ps. See Inserting NMR Spectra... for more information.

Running a Gradient Selected HMBC: Determine Long-range Carbon to Hydrogen Connectivity.

The gHMBC is used to help establish the carbon skeleton through the multiple bond carbon to hydrogen connectivity. This experiment is relatively insensitive as compared to gHMQC because multiple bond correlations are less efficient than one bond correlations. Typical one-bond coupling constants are around 150 Hz whereas multiple-bond coupling constants fall in the range of 5-15 Hz, which is similar to the range for H,H-homocoupling.

The experimental protocol is similar to gHMQC, just type gHMBC and use the default values:
- The default values are nt=8 and ni=400. This will require about 1 hour 10 minutes.
Perform data manipulation and plotting as with gHMQC except use setLP1 sinebell wft2d in place of wtf2da.

*Some sections of this page were adapted from procedures by Long Lee and Kermit Johnson.