Question

I am running BioPack on a Varian INOVA 800MHz on a newly aquired 13C15N1H sample and I have come across an issue that I was wondering if anyone could shed some light on.

When it got to the CBCAcoNH, the experiment it setup to calibrate the phi7cal is set to take ~3hours! with nt=520. I have reasonable signal (my sample is 0.8mM), so why is it doing this? Does it determine a S/N value at somepoint that maybe went awry?

I set up BioPack in the following manner:

create new experiment, load ghn_co and set parameters:

cexp(100) jexp100 ghn_co
TROSY='n' gzlvl1=10000 gzlvl2=9600 mag_flg='n' 
np=1024, tpwr=62, pwClvl=63, pwNlvl=62

set pw and tof as manually determined

Main Menu ->Setup->Proteins->Manag Probe Files=> Update Probe Files with these Parameters

Back->Calibrate->Full

Thanks for any insights!

Answer 1

Hi,

If you got to the stage of gcbca_co_nh you've already done all the major probe calibrations. The s/n is measured is at the beginning and it aborts if the s/n on the first experiment is too low. There are a couple of other cases where s/n is measured and nt re-calculated (for the gNhsqc d1 array to set nt for the gNhsqc_1D data set (BP7a macro) and BP10 for the ghn_co data set.

You should recall ~/vnmrsys/AutoTripRes/ghn_co_ca_1D.fid. This has the nt value that is used also for the gcbca_co_nh phi7cal calibration. It should be a small number, not something that would take hours.

You can also recall the ghn_co_1D data set. This has the nt set by the macro BP10 to achieve a s/n > 20. If something prevented normal behavior this experiment might not work and the s/n would be too low for normal nt. You can also check the behavior of the ghn_ca_1D, ghn_ca_co_1D and ghn_co_ca_1D fids - all of these should be using the nt determined by the BP10 macro and used for the ghn_co_1D data set. Note that the gzlvl2 and compC calibrations had already been done and should have taken a normal time.

Check the printouts (if you did them) and look at the nt value in the parameter printout. Otherwise, you could recall the data in the order it was acquired (use a shell and cd to vnmrsys/AutoTripRes. Then do "ls -altr". This will show the fids in order, with the last acquired fid at the bottom. Recall the data and check the s/n and the nt number.

-George Gray, Varian Inc.

Answer 2

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There may be other reasons, but if "hard" 13C 90 pulse is longer than probe specification - you can check this value yourself, Biopack calibration may set decoupler power to a value above the limit set in the pulse sequences. Then routine will fail with an error something like "dpwr too high, don't fry the probe".

Other reasons might be trivial - your sample being too dilute, cables not connected properly or "other hardware issue" - which can of course include lot's of things. If you can confirm this failure with a reliably good 15N, 13C-labeled sample and you are confident in cabling of the instrument - you should probably ask your facility engineer to check the spectrometer and the probe.

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answered Jan 17 at 14:59

Evgeny Fadeev
54●1

updated Jan 17 at 15:41

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