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Measurement of Long-Range H,C Couplings

One of the experiments proposed to measure long range H,C couplings invlolves the acquisition of two spectra: a Constant Time HMBC and a reference HSQC acquired under the same conditions. See:

Journal of Magnetic Resonance 163 (2003) 257

Each cross-peak in the reference HSQC should show a large one-bond anti-phase coupling and several small couplings. The cross peaks thus appear as pairs of multiplets. Each half of the pair is split by the same small couplings, while the distance between the two multiplets corresponds to the one-bond coupling. The sign of the two halves is reversed (anti-phase relation).

We can take one of this halves and add, computationally, a small trial coupling. The value of this trial J is modulated until the result (a simulated multiplet) becomes just like a related signal in the HMBC. The signal to fit is a cross-peak between the same hydrogen and another carbon. We can easily find a value of J that minimizes the difference between the HMBC multiplet and the simulated one. At this point we can assume that the HMBC cross-peak correlates two atoms whose mutual coupling is equal to the trial coupling.

The intensity of the HMBC multiplets, if compared with the HSQC ones, is scaled by the factor: α sin( π J Δ ); where Δ is the constant evolution time for the multiquantum coherence (a pulse sequence related parameter) and α accounts for experimental errors (α = 1 in theory).

How to Use iNMR to Extract the Couplings:

Step 1

Process the two experiments. The number of points in the proton dimension must be as high as possible for optimal resolution.

Step 2

Select the command: Simulate > 2D H-C Coupling. The module for the calculation appears. It contains an upper panel to host the reference HSQC and a lower panel for the CT-HMBC. Now we want to connect the two panels to the respective spectra.

Step 3

Click on the reference HSQC window to make it active. The calculator remains the topmost window. Click the button called “Set reference HSQC”. The path to the file will appear into the upper panel.

Step 4

Click on the CT-HMBC window to make it active. Click the button called “Set CT-HMBC”. The valur of Δ will be extracted for you and shown at the top of the calculator. Should it fail to appear, enter it manually. Steps 3 and 4 can be performed in any order.

Step 5

Select the multiplet to fit into the HMBC. Expand the surrounding region. A good amount of empty space, but no other signal, should be visible at the sides of your multiplet. Locate, inside the HSQC, the cross peak belomnging to the same hydrogen. Expand one half of it, in the same way. Create an horizontal mark in each window (with alt-click) to select the rows that will be used for calculation.

Step 6

From now on we will work with the calculator module only. Click on the two buttons called “Import Row Fragment” to import the selected multplets.

Step 7

Move the upper vertical slider to add a trial coupling and start the simulation. The lower panel will show both the experimental and the simulated multiplets. Move the horizontal slider until their centers coincide. Move the other vertical slider to compensate for any intrinsic difference of intensity between the two spectra. This is the factor α cited in our introduction. Read the residual error at the bottom of the module. Keep moving the sliders until you have minimized this error. Annotate the final value of J on your notebook.

Step 8

To move on to another multiplet, return to step 5 and repeat. When repeating the procedure with other multiplets, the amplification slider should not be touched again, unless for minimal cosmetic touches.

If you find out that you have chosen the wrong half of your HSQC multiplet, you can invert the sign with the button “Invert”. Thanks to this trick, you are free to select the cleanest half of your multiplet, regrdless of the sign.

Related Topics

Simulate and Fit a Multiplet Under All Conditions