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DOSY

Diffusion Ordered SpectroscopY
2D presentation of relative diffusion rates
Bruker Avance AV 500 / Topspin 2.1

  1. Preliminaries
    1. Sign the logbook (name and phone number)
    2. Login to the computer
        user name
        password
    3. Open Topspin (double-click the icon)
        topspin icon
  2. Acquire a 1H spectrum
    1. lock, tune and match, shim, rga, zg, proc
    2. Is the shimming okay?
    3. Does the sample appear as expected?
    4. Optimize NMR acquisition paramters: sw, o1p, td, ns,
      • d1 and p1 - optional but recommended

  3. Read in the DOSY paramter file
    new window
  4. Update AcquPars
    Enter the optimized values into the new data set:
    • rg
    • sw
    • o1p
    • td
    • ns (8 * n)
    • d1
    • p1

  5. Optimize the gradients
    1. Acquire two spectra with different gradient strengths (2% and 95%)
      • type: dosy
      • ignore any error message
      • enter first gradient amplitude: 2
      • enter final gradient amplitude: 95
      • enter number of points: 2
      • rampe type: l(inear)
      • do you want to start acquisition? yes
    2. While the acquisition runs, click on the ProcPars tab and change SI(F2) to 2
    3. Process the setup spectra
      • rser 1
      • wrpa 2
      • rser 2
      • proc
      • multiefp
      • current expno = 1
      • number of expnos = 2
    4. Measure the signal attenuation
      • Enter multiple display mode .md

      • Select expno 2 (2% gradient, usually drawn in red) and
        decrease the signal intensity to match that of
        expno 1 (95% gradient, usually drawn in blue).

      • Check the factor of the peaks of interest.
        It should be around 0.05 ±0.04

      • If the residual signal (95% gradient) is too small or totally gone (decay was too fast), then reduce d20 (ms timescales).

      • If the residual signal is still too big (decay was incomplete), then increase d20.

      • (An advantage of the led pulse sequence is storage of magnetization on +z, to reduce T1 effects from long d20's).

  6. Acquisition
    1. Go to a new experiment number, for example, type: iexpno
    2. Setup paramters for DOSY:
      • set TD(F1) to 32
      • set SI(F2) to 32
    3. Type (without the part in parenthesis): dosy 2 95 32 l(inear) n(o)

  7. Processing
    1. When the acquisition is finished, type the following to process the dosy spectrum:
      • setdiffparm
      • set line broadening for good sensitivity enhancement, 1 - 5 Hz
        • eddosy menu
          type: lb 5
      • eddosy

        • Parameters automatically set during acquisition:
        • ExpVar = Gradient
        • Xlist=difflist
        • Nstart=0
        • Ndata=32

        • Parameters auotmatically set during setdiffparm:
        • Gamma[Hz/G]=4257.64
        • Grad[G/cm]=
        • Gdist[ms]=
        • Glen[ms]=

        • Other eddosy parameters:
        • PC=4
        • SpiSup=1
        • LWF=1
        • DISPmin=1e-010
        • Dispmax=1e-008
    2. phase correction
    3. automatic baseline correction: abs2
    4. calculate 2D dosy spectrum: dosy2d

  8. More options / tips and tricks
    1. Use the same instructions as above to calculate absolute values for D.
      These "diffusion experiments" require careful calibration,
      • unlike DOSY, which is a relative measurement
      Good temperature control is required.
      Good spectrometer gradient strength calibration is required.
      Known compounds should be run to determine accuracy.
      Use the relaxation guide which opens by typing: t1/t2
      (NOTE: There's a good step-by-step guide in section 2 of the Advanced Training Courses folder by the hood.)

    2. If you don't see any peaks at all in the DOSY spectrum:
      • Is the correct values for big delta (d20) in the eddosy table?
      • Is the correct values for little delta (p30) in the eddosy table?
      • Do the display limits (DISPmin and DISPmax) correspond to the expected diffusion rates?
      • Check the peak picking paramter PC.

    3. If the peak separation is poor:
      • Perhaps the diffusion rates are too similar.
      • Run the experiment again with a lager ns (8 * n).
      • Try to play around with the eddosy processing parameters:
        lb - avoid overlap but maximize the signal-to-noise ratio
      • SpiSup - smaller values may increse resolution.
      • LWF - values <1 will give better resolution, but truncation artifactsmight appear.

    4. Use the ceramic spinner and high gas flows to avoid temperature gradients at high PFG (pulsed field gradient) powers.
      Try using a pulse program with bipolar gradients to reduce heating effects.

    5. More elaborate pulse programs:
      include water suppression, such as watergate (Bruker: stebpgp1s19)
      Pseudo-3d with TOCSY mixing (Bruker ledbpgpml2s3d)
      NOTE: for processing, to calculate the 3D dosy spectrum: dosy3d

    6. Some examples of interest:
      JACS(2005)127:5714
      JACS(1993)115:4291
      Concepts in Magnetic Resonance(1997)9:299
      Concepts in Magnetic Resonance(1998)10:197
      JACS(2011)133:7640 for "LOCODOSY"

  9. Plotting
    For a simple printout of the screen, type: prnt
    To export pdf files of your plot, or have control of colors, fonts, sizes
    enter the topspin plot editor by typing: plot
    In brief, a description of the plotting elements:
    “Data” contains the list of expno's listed in the plot editor's portfolio
    The green squares icon allows data window selection
    “Edit” to change colors, fonts, displayed regions, etc.
    “1D/2D Edit” to interactively change intensity and plot region
    File, Print to get a hardcopy (control p)
    File, Export to save a picture (formats include: pdf, jpg, emf, and bmp)
    DO NOT SAVE (this changes the file called by “layout” in topspin)

  10. Save your data onto another computer
    DATA MAY BE DELETED at any time WITHOUT WARNING
    1. Open the my computer file icon on the desktop (double-click nmr500)
    2. Click the “start” icon to get to the nmrlab4 network shortcut
    3. Drag and drop files to nmrlab4.
      Preserve the hierarchy:
      directory/data/user/nmr/name/ expno

  11. Remove your sample
    1. on the BSMS keyboard:
    2. TURN OFF: spin and lock (no light = off)
    3. press “lift on/off” (green = on)
    4. gently remove your sample from the magnet – lift it up to clear the magnet
    5. press “lift on/off” (no light = off)

  12. Log off

  13. Open your files on another computer
    To open Topspin files on the other computer:
    Topspin icon
    1. Open the program Topspin
    2. Navigate to your data using the Browser
    3. Drag you data file into the display window

  14. Report problems and bugs to Yael or Shifi - 3748