---- I would definitely try hsqcetgpsisp.2 (all 180's replaced with shaped pulses). In my experience a nonzero p28 never helps anything, and in the case of samples in water, it is a giant leap backwards. ---- John - I am assuming these are H-C HSQC's? I think you will see a big improvement if you use either the si2 sequences which use gradients in the back inept to clean things up and/or use adiabatic 180's on C, especially at 750 but also at 500 too. I use them here on our 500 and I also do adiabatic 13C decoupling rather than garp. I am not in front of the spectrometer but I believe these are the sp.2 variants of hsqc. The adiabatic decoupling is great but you may have troubles with decoupling sidebands so be careful. ---- Several things come to mind as potential causes for this problem. 1) What software are you using to process your data? NMRPipe fails to recognize that Varian gNhsqc data is acquired with sensitivity enhancement, and require Rance-Kay F1 phasing instead of States or Complex. Could your offline processing software fail to recognize this in your Bruker data? 2) I've encountered oddly-shaped gNhsqc crosspeaks when the refocusing gradients aren't calibrated just right. On a Varian, this means gzlvl1 should be fixed, and gzlvl2 should be optimized to yield maximum intensity. Unfortunately, this can't be fixed in processing. 3) Lock phase could be off, which would also require re-acquisition. Hope this helps. I'll be interested to see how you resolve the problem. ---- I've encountered some phasing problems with HSQC experiments before, and it sounds like you have been on the right track when dealing with them. But for what it's worth, here is my take on it (in no particular order): invietgpsi ==> hsqcetgpsi is the new Bruker name for that pulse program. I'm a little surprised you are seeing the phasing issues on several different spectrometers and different fields. It sounds to me that it could be an imperfect pulse issue. Using adiabatic pulses for inversion and refocusing 13C pulses would be a very big improvement. In fact, at 500MHz and above, I have seen marked improvements when using adiabatic pulses versus the standard hard pulses. I would recommend you check out the following pulse program from the Bruker library: hsqcetgpsisp2.2 The pulse program gives suggested pulses for inversion and refocusing. At 750MHz, you should probably use the crp80 variations instead of the crp60. I'm sure someone at Bruker can provide more detailed advice on that. I'm not quite familiar with the old version of the pulse program, but the new one calculates the d4 delay based upon the value of the one- bond 1H-13C coupling constant that you provide. I think the default setting is 145Hz, which is a compromise. Depending on your particular molecule, you may need to play around with that a little bit. Regarding the prescan delay time, because you are using digital acquisition, it is not as critical to set "DE" as it was with analogue systems. To put it another way, I have generally found it best to leave DE=6us and let the digital filters deal with it. I have found that when DE was set too long, the phasing became a real issue, even for a simple 1D proton-NMR experiment. So check the DE, and if it has been changed, then set it to 6us. Of course, there is always the possibility that this could be a processing issue. But this may be best handled by looking at a dataset. Can you make a dataset available for download? ---- We have seen this effect in protein HSQCs where the intense (AND narrow) peaks can not be quite phased simultaneously with all other lower intensity (and broader) peaks. Although I have not tried to prove this experimentally, I am almost certain that this is caused by the "leakage effect" in discrete Fourier Transformation. See the very useful book by Hoch and Stern: "NMR Data Processing" - pages 25 -26. In your case it could be quite a nuisance. The remedy will be increasing the number of acquisition points. It may not completely eliminate the "phase error" but should reduce it. When there are only a few of such peaks (as is the case for proteins) one might be able to find a position for the carrier such that the inverse of the frequencies of the offending peaks is an exact multiple of the dwell time. Let me know what you find if you are going to experiment with the number of points or the carrier frequency. ---- I have all Varian, but I thought I would suggest a couple things: 1. Use the Bruker 2D test sample that they use for installation checks. Varian sends out an indanone sample and we routinely use it for troubleshooting and to verify that the 2D exps are working properly. If it works the way it should then the spectrometer is fine and so is the processing macros you are using. 2. Do you check your proton pw90 on the sample after tuning? There are samples that the tuning of the probe will still not give you the same value of pw90. ---- It would be very nice to see a software program be able to phase sections of a 2D data set independent of other sections. That might solve your problem with already obtained data. It seems like this would be easy to do with minor adjustments, and something I've wished for on occasion in the past. I'll mention this to the Mestre-C people, who are busy about to make available a big new release that is Mac, PC and Linux compatible; we may purchase a site license if they've improved the stack plotting. ---- I am not familier with Bruker sequences. However, "unphasable [i.e., small phase error that could not be corrected]" contours in a "pure-absorptive gradient coherence selected experiment" could mean the p-type [for example, 4:1 ratio of gradients] and the n-type [corresponding 4:-1 ratio gradients] selections are not exactly the same. This might mean that the positive and an equivalent negative gradients do not produce the same G/cm. You may want to superimpose a positive gradient profile on top of a negative gradient profile and see how well they match. In an ideal situation, they should superimpose. ---- I am aware of the phasing issue. One can usually phase the majority of peaks but some are out of phase. There is no processing procedure, that I am aware of, that would solve this. With a large range of intensities the problem always becomes more apparent, because you can no longer cut off high enough not to see the problem. I think some of the sequences with adiabatic pulses do give cleaner spectra but I do not have real comparison data it is just my impression. ---- The main problem with the phase is the non ideal and offsett dependent 180° pulse on 13C. You must take a pulse program with a "sp", that is applying an adiabatic (frequency swept) chirp pulse, otherwise you will not succeed. See experimental description in Exp. 12.10 of "200 Basic NMR experiments" ---- > ----- Original Message ----- > From: John Ralph > To: ammrl_at_chemnmr.colorado.edu > Sent: Friday, November 24, 2006 12:44 AM > Subject: AMMRL: Contour peak phasing issues in hsqc spectra > > We have been having a problem for a while and, especially now when > we are trying to apply chemometrics methods to 2D NMR data, it is > becoming a real issue. > > It has to do with contour peak phases in 2D HSQC spectra. > > It seems we can never phase every peak in the spectrum -- if you > phase some, others are necessarily out of phase in a distinctly non- > linear way, particularly the intense contours. > > This is NOT an issue with a single spectrometer -- it is something > to do either with the types of sample and/or the parameters, and/ > or ?? The same "problems" are on 500-750 cryo machines in two > countries (and also on non-cryo at 360 MHz). We're using the ea si > hsqc expt (Bruker standard -- sorry, forgot the new name for a bit, > but used to be invietgpsi). > > The samples have a range of component intensities, are fast > relaxing (proton fid is dead in about 100 ms). We have tried the > obvious things (is relaxation delay too short -- no, 1s for > something that relaxes in 100 ms, AQ is 200 ms), turing p28 off or > not, and various other things. But we have not yet tried the > Adiabatic variants that may help if it is a J-mismatch problem > (with 180s). > > Anyone know how we might be able to solve this? (after acquisition > (by processing) would be great (e.g. is it that the early part of > the FID is messed up, but with digital we don't know how to > manipulate that the way we could in the old days), but before would > also help tremendously for the future). Any suggestions? Does > anyone need to see a dataset? > > Cheers and thanks in anticipation. > > John ------------------------------------------------------------------------ ----------------------------------------------------- John Ralph US Dairy Forage Research Center, USDA-ARS and Dept. of Biological Systems Engineering 1925 Linden Drive West Univ. of Wisconsin, Madison Madison, WI 53706-1108 (608) 890-0071 FAX (608) 890-0076 E-mail: jralph_at_wisc.edu http://www.dfrc.ars.usda.gov **Let me know if you want my vCard!** Full-text Publications available directly from: http://www.dfrc.ars.usda.gov/DFRCWebPDFs/pdfIndex.html ------------------------------------------------------------------------ -----------------------------------------------------Received on Mon Nov 27 2006 - 10:13:21 MST
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