I have a question about a phenomena I've been observing in an edited HSQC.
As can be seen in the attached figure [Doxycycline_Decoupling_issues], this particular methyl group of Doxycycline (blue) is splitting in the HSQC, but the 'splitting' is not equal in magnitude to the splitting observed in the proton spectra (aligned at the top). In the 1H (zg),(j=6Hz); in the HSQC, (j=41Hz).
However, when a different pulse (hsqcedetgpsp) is applied (shown in red) the problem reduces considerably, leading me to believe something is not 'set right' for the original pulse.
This is not the only time I've seen this problem, although in every case I have observed it, it has been a conformationally locked position on a ring (or fuzed ring). For reference, I've seen it on the 4-position of the Cladinose of Azithromycin (splitting 12Hz), a few methyls of Betulinic Acid (average j values 40Hz). The consistency of these j values leads me to believe it has a lot to do with the jC-H of each set of protons being detected (even though it is
This is most likely an issue with the 13C decoupling. It is weird that one sequence would show it an the other not. Assuming same O2 and same decoupling sequence. My HSQC of betulinic acid gave nice singlets for the methyls.
Clemens Anklin ,
Great to hear from you. I just checked, and the O1 is set the same in both sequences (3603.76Hz). Could it be something to do with the shape of the pulse, or the power setting? I looked through what I can understand in the pulse pattern, and it seems like hsqcedetgpsp doesn't use a match sweep pulse (I can't seem to find a note about it), whereas the hsqcedetgpsisp2.3 uses an adiabatic match sweep pulse, as it is explicitly stated in the pulse sequence file.
I also found a really nice "Which experiment should I choose" powerpoint (https://magnetlab.chemistry.utsa.edu/pdfs/6WhichExperimentShouldIChoose.pdf) which was talking about some problems with the adiabatic match sweep when j doesn't vary with the shift, which I can imagine might contribute to this.
Could you perhaps share the settings you would recommend to troubleshoot this?
Can you provide your experiment dependent acquisition parameters (type ased). My guess is similar to Clemens in that the CPD is not set up properly. One other thing you may want to try quickly is to run a simple HSQC without multiplicity editing or any shaped pulses to see what kind of peak splitting you get. Something like hsqcetgpsi.
Stephen Boulton , Thanks for reaching out!
I've checked and from what I can see, the decopuling is the same for both the hsqcedetgpsisp2.3 as well as the hsqcedetgpsp pulse sequence, it is 'garp'. I am going to re-acquire after shifting the O2 offset, just to see if I can get these peaks to decouple as expected.
Joseph, problems with the pulses in the sequence usually show up as phase twists and not as residual coupling. The issue with the matched sweep mostly affects carbons in cyclopropane or epoxy rings. In these there is a significant deviation from the typical correlation between shift and J. Moving O2 is the best approach to trouble shoot this. When yo move it closer to the methyl groups then the aromatic signals should begin to split.
Dear Joseph, have you checked matching and tuning on 13C (sometimes this "jumps" away...)
Kind regards
Alfred
Hello Everyone, it seems I've found a solution to this, but I still am not sure what actually caused it.
I moved my O2 from 100ppm to 75ppm, and it seems to have worked to solve the decoupling in this region. This leads me to believe the deoupling was 'incomplete' in these regions. Even with my O2 set here, I observe the correct peaks in the aromatic region, so this seems like a solid improvement. Oddly, between the two pulse sequences attempted earlier, the O2's were a match and the decoupling sequence was the same (as far as I could tell, the probe was tuned just before acquisition).
I am still very curious as to why this would happen for these particular resonances. For most applications, there have not been problems with the decoupling, and the 'areas' of where the decoupling was imperfect were somewhat inconsistent (but were very compound selective). For instance, with the Azithromycin decoupling, the C value was 34ppm and the problem was not observed for peaks that are upfield of this carbon value. With the Betulic acid, there are other methyls that were perfectly decoupled, but all of the ones shown here were not. If anyone has any ideas, theories, or information, I'd be very happy to hear it!
Thanks everyone for the suggestions, I will keep an eye out to see if I observe this happening again.
..hmm..strange for our setup we cover with about 3.6W (plw12) with a AQ of 140 ms at our 600 MHz machine a 13C decoupling width of at least +-90 ppm. I am wondering what your decoupling power is. We use garp4 with a pulse of 55 us. I do not think the phenomenon has anything to do with the molecule!
Hello Everyone,
Although I have solved the problem, I conducted an experiment that I found to yield some interesting results.
As can be seen above in the Doxycyline problem, the imperfect decoupling was assumed to be from the O2P not being set in the correct spot, and therefore did not cover enough of the collected range.
However, I also mixed Doxycyline (problem compound) with two other standards each at 15mg/mL, and ran the same pulse sequence that was causing the issue at hand (hsqcedetgpsisp2.3, with 02P set at 100ppm), I found that the signal itself [ green ] shifted (as one might expect, due to concentration/matrix effects), but that the 'left over' coupling seemed to be in line with that which was observed in the 'better behaved' pulse sequence [purpleish/brownish] (hsqcedetgpsp, O2P set at 90), more in line with the expected scalar coupling from the protons alone.
At this time, I've decided to push forward with my solution, but was curious to see what others thought might be going on. I can try to rationalize the difference through intramolecular interactions, but I feel as though this is a bit 'hand-wavy' and would like to know if anyone has any input as to what might be happening.
Cheers,
Joe
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