varian-dosy实验步骤.doc

NMR FacilityEquipment Protocols - NMRINOVA-399Collect a 1D 1H spectrum Log in Click the Vnmr icon Lock, shim and collect a 1D 1H spectrumtype gain? and write down the gain value Set temperature and spin go to start tab and spin/temp move slider to desired temperature (must be greater than room temperature) move spin slider to 0 and press spin off (make sure spin goes off) Get pulse sequence and optimize parameterstype Doneshot (or any of the other DOSY experiments)go to start tab and give file namecheck solvent change gain to the value found above by typing gain=# type gzlvl1=5000,15000,23000 type gatype nt=8set delay to 10s when data collection is finished type dssh to see all datatype ds(1)type f full*WARNING Poor signal to noise can result in incorrect diffusion values (high)*tupe aph type dsshmake sure that the last data set has about 5% -15% of the S/N as the first data setif there is not sufficient delay increas gt1 or del (type del? to find current value then increase a little- type ga to try again) Collect dosy datatype setup_dosyanswer: 30 500023000make sure spinner is off (press acqi press lock press spin off)type gawait for data collection to finishSAVE THE DATA by pressing the acquire tab, future actions and save fid nowData processing Press Main menu Dataand find and load the datatype fn=100000type wfttype ds(1)type f fulltype aph0 (if spectrum needs phase correction)integrate spectrumtype fbctype dosy or press calculate entire dosynote down the values for each resonance. The diffusion coefficient reported is the center of the peakand the error bar is half the width of the widest peak- up to 20% error is acceptableTrouble shooting and hintsCommon data collection problems:1) do not get decay down to 15%:adjust diffusion delay or gradient diffusion length (*careful adjusting the length- too long of a length can fry the probe) adjust these two in very increments (increase gt1 by .001 and del by .01) if you do not get all the way down to 15% you will not have a well defined curve and your signal will be broad2) data does not decay in a linear fashion:maybe too much gt- try lowering gt and raising delay (so you still get 15%) but be careful because raising the delay introduces additional error signals have weird phase problems there may be a temperature gradient across the sample- try turning heater off you may have a system in chemical exchange- this make DOSY very tricky and usual fails gt1 and del are wrong- try lowering del and increasing gt1 4) too wide a spread of diffusion rates from the same compound:increase d1 value (calculated rate depends on T1) but keep the D1 delay as short as possible- too long of a delay the greater the band of diffusion rates for different protons if you need a tighter band you need to try greater gradient strength spread or try using inept (very insnesitive)note: a compound moving in and out of a gel will have a wider band for different protons on the same molecule could be .4 m2/s difference in diffusion rates- not surprising- should be smaller difference if the same compound were free in solution5) final signal too broad (too big of an error):sample volume may be too much- the more sample volume the less EVEN the gradient along the Z axis will beCommon data processing problems: 1) Program bombs: check line broadening- it cannot be zero- should be 3-5 program will bomb if there is insufficient signal to noise peaks do not phase well and there is significant signal below the baseline-see Spectrum-does not phase (above) 2) Need to run fiddleplace cursor on reference peak ( a nice clean singlet)and type rlpress Ref and give it the correct reference value for that peakpress Th and move threashold close to baselinepress fullplace cursors on either side of the reference peaktype lb=-4 gf=.05make sure full spectrum is on the screentype fiddle(writefid,filename) and wait till it corrects every spectrumwritefid is types exactly and filename is a file you are going to write the data tointegrate each individual peak you want to include in the DOSY analysismake sure full spectrum is on the screenmake sure that threashold line is on screen and cursors suround reference peaktype fbctype dosy or press calculate entire dosyUncommon problems:1) full range of rates is not covered:you many need to recalibrate gcal for the probe2) one of the spectra does not phase well:remove that spectrum3) signal do not phase well:check to see that the solvent is correct- it must know the correct solvent since the 90s for each solvent are different in the probe file and the 90 being off will effect whethe