MCW Site Maphome
Biochemistrygraduate schoolresearchpeoplelinksInformation

Path

Home
 People
  Faculty
   Volkman
    Methods

See also:

Index
Talos
Xlist
Diffusion

Brian F. Volkman, Ph.D. - Lab Methods
Getting started on the Bruker DRX 600 NMR spectrometer
WARNING: Do NOT touch the magnet, RF console, cryoprobe, cryplatform or other instrument hardware EXCEPT as directed for inserting your sample or tuning the probe (see instructions below). All other adjustments are performed under computer control through the XwinNMR software. If you are unsure about the correct procedures for any aspect of data acquisition on the NMR, DO NOT PROCEED; ask for help from an experienced user. Call Brian Volkman (x8400) or the Volkman lab (x8081) and someone will be very happy to assist you.
  1. Log on to the SGI workstation with your username and password If this is your first time on the NMR, obtain this information from the system adminstrator and change your password immediately: In a unix shell window, type passwd and enter your old password and new password as prompted.

  2. Start the XwinNMR program In a unix shell window, type xwinnmr. Unless otherwise noted, all further command descriptions apply to the XwinNMR program and should be typed at the XwinNMR command line or accessed through its menu interface or related windows generated by XwinNMR. For more information on the unix command line environment, see the Appendix on XwinNMR/Unix filesystem organization.

  3. Check and set the sample temperature Type edte; a new window for controlling and monitoring the variable temperature unit should appear. Temperature is reported in Kelvin, and is systematically inaccurate by a few degrees. A calibration curve based on methanol temperature measurements is provided on the side of the console. Select the desired temperature on the vertical axis, and find its intersection with the calibration line to determine the appropriate set temperature. VALID CRYOPROBE SAMPLE TEMPERATURES ARE RESTRICTED TO THE RANGE FROM +10 TO +50 C. Enter the desired temperature in the edte window. If temperatures far from ambient (20-30 C) are needed, it may be necessary to adjust the maximum heater power between the range from 4 to 15%. DO NOT EXCEED A MAXIMUM HEATER POWER OF 15. Temperature stabilization may require 5-10 min, depending on the value.

  4. Putting your sample in the magnet First, locate the spinner and the sample depth gauge. If the spinner is not in plain sight, look at the indicator lights on the BSMS keypad (unit with round knob and shim controls) to see if a sample is currently in the magnet (green light - sample down; red light - no sample in magnet). Wipe the surface of your 5 mm NMR tube clean with a tissue and insert it into the spinner, using the gauge as a guide. When using a normal (non-microcell) sample tube, a minimum volume of 500 uL should be used to avoid shimming problems. Adjust the tube so that the bottom is even with the 18 or 19 mm line on the depth gauge (no lower than 20 mm). For shorter samples or Shigemi-style microcells, center the sample volume with respect to the field center. Next, check the top of the magnet to see if the bore tube cap (round black plastic) is inserted. If so, remove it. Depress the sample lift button on the BSMB keypad (upper left corner); within 5-10 sec, the sound of the lift air should be audible. Once you hear the lift air, place the sample in the top of the bore tube where it should 'float'. Finally, depress the sample lift button once more to lower the sample into the magnet. A 'clunk' sound when the sample reaches the probe is normal. Within a few seconds, the green 'sample down' light should be illuminated on the BSMS.

  5. Activate the deuterium lock First type lockdisp, to open a new window which will provide a continuous display of the deuterium lock signal intensity. Next, type lock to bring up a list of deuterated lock solvents; choose the one appropriate to your sample. Or type lock d2o, if your sample is of the biomolecular aqueous sort typically run on this instrument. In either case, a set of standard lock parameters are sent to the BSMS and the values for lock power and lock receiver gain are optimized automatically. When complete, a message will appear in the status line at the bottom of the main XwinNMR window. The lock display window should now show a horizontal line, moving back and forth. This is the indication of lock signal strength. If the signal is off the top of the window, or in the lower half of the window, depress the lock power button on the BSMS and use the dial to adjust the lock power (shown in the LED display on the BSMS) to somewhere between -15 to -10 dB. Then depress the lock gain button and adjust this value until the lock signal is somewhere in the upper third of the window.

  6. Tune the 13C channel Type dir to see a list of experiment directories that currently exist in your user directory (which in the unix environment corresponds to /u/data/username/nmr; see the Appendix on XwinNMR/Unix filesystem organization for more information). Select the experiment called tune13c. Type a to move to the acquisition screen. Type wobb to begin the 'wobble probehead' routine for tuning the 13C channel. After a few seconds the display should contain a V-shaped signal and a vertical line at the center. The probe coil circuit is optimally tuned when the bottom point of the V rest touches the bottom of the spectrum display window, and is centered on the vertical line. If this is the case, no adjustments are required. Often, however, minor adjustments are needed when a new sample is introduced or large changes in sample temperature are made. You will be instructed in the correct way to adjust the tuning capacitors at the probe, using a special screwdriver-like tool, but keep the following tips in mind: Do not force the tuning capacitors beyond their normal range (this is not hard to do with the tool), and take care not to bend them - these are the most common forms of probe breakage! Start by optimizing the match knob (labeled M), lowering the bottom of the V-shaped dip, then adjust the tune knob (labeled T). Iterate back and forth until the tuning is optimial. The preamplifier box next to the magnet has two banks of LED indicators to guide you in adjustments of matching and tuning (easier to see than the computer screen). The 1H/15N/13C cryoprobe has LOTS of tuning knobs; they are color coded by nucleus. Check the front of the probe body (where the RF connections are) if you forget - 13C is BLUE. When you are done making adjustments, type halt.

  7. Tune the 1H channel Type dir; select the 1puls experiment, type a to go to the acquisition screen. Type wobb and look for the V-shaped dip. This one often has a little bit of a squiggle near the bottom of the V which can confound the tuning a bit, so it may help to look a the computer screen more frequently when making adjustments to the tuning knob. Adjust the YELLOW 1H match and tune knobs, referring to the instructions given above for 13C as necessary; the overall process is the same. When you are done making adjustments, type halt

  8. Shim using automated gradient shimming First save the current shims: Type wsh; a window will appear; type a name for the shim file in the space provided, and click OK. Type gradshim. The first time gradshim is run, the program will probe the hardware configuration to determine which shims are actually installed. Unfortunately this has the effect of resetting all the shim values. Fortunately, you just saved your shims to a file. Now you can read them back in: type rsh; select your shim file. As part of your training session, we will configure a complete set of default parameters for the gradshim program so that on subsequent occaisions you can simply start gradshim and click the start button after selecting either 1D (Z-shims only) or RT (all shims) gradient shimming. Make sure the username field contains your username and not 'gradshim'. A 1D shimming run with 2 or 3 iterations should only take a minute or so to complete. 3D shimming takes longer (~5 min per iteration), but may enable better H2O solvent suppression. For more information on defining an iteration control file, shim groups, size values, etc., consult an experienced user in the Volkman lab (or the Bruker documentation, should it ever materialize).

  9. Calibrate a 1H 90 degree pulse Type dir; select the 1puls experiment. Type p1; in the parameter window that appears, set the value of p1 to 3 microseconds (typing 3 or 3u will work equally well; hit return). In a similar fashion, set pl1 (1H power level) to the appropriate value, typically -2 dB for the 1H/15N/13C cryoprobe (type pl1; in the window that appears, type -2 and return). Type zg; various lights will blink on the BSMS keypad, including the red ADC light. When the red ADC light goes off, type ft. You should see a spectrum with one single broad line (if this is an aqueous sample - 90% H2O/10% D2O). Find the button in the left side of the window labeled PHASE and click it. Find the button labeled BIGGEST and click it. The spectrum should be phased fairly well. If additional adjustment is required, click and hold the left mouse button while over the PH0 button on the screen and scroll up or down to make interactive adjustments to the phase. When phasing is complete, click the RETURN button, and select 'save and return'. A typical 90 degree pulse is ~10 us in duration. Calibration is obtained by performing a 360 degree pulse to give a null spectrum and dividing the resulting pulse width by 4. Set p1 to ~ 40 us; zg; fp (does FT and applies phase params). If a negative peak is observed, increase p1 and repeat until the null is found. If a postive peak is observed, decrease p1 instead. Note the value of p1 which gives the null and calculate the 90 degree pulse width.

  10. Acquire a 1D 1H spectrum Use dir to go to the 1puls experiment. Type edc; in the window that appears, type a new experiment name and click OK. Check to see that p1 and pl1 are set to the values which you calibrated above. Type ns to see how many scans will be acquired; set it to an appropriate number (16 or 128, e.g.). Type rga to run an automated routine for optimizing the receiver gain value. When it is finished, type rg and note the value (if running an H2O sample, it is probably still 1). Start the acquisition with zg. You can process the data while acquisition is ongoing by typing tr to store the current data to a file. Then ft and phase as usual. The complete acquisition is automatically stored to the same file upon completion.

  11. Acquire a 1D 1H spectrum with H2O solvent suppression Use dir to go to the experiment called 1dp19. Set p1 and pl1 as before according to your calibrated values. Set ns to the desired value, and run rga to set the receiver gain. Check rg afterwards; it should be in the range from 64-1024. Start the acquisition with zg. Transform (ft) and phase as above.

  12. Acquire a 1D 13C spectrum Use dir to go to the experiment called 1d_c13. You have not done your own calibration of 13C pulse widths, but a typical pulse width (p1) is 16us at a pl1 of -3 dB. Set ns to the desired value, and run rga to set the receiver gain. Check rg afterwards; it should be 512 or higher, depending on the strength of your 13C signals. Start the acquisition with zg. Process as above.

  13. Plot a 1D spectrum using XwinPlot Use dir navigate to the spectrum to be plotted. Process and phase as desired. Type xwinplot to bring up plotting window. Click on icon for 1D spectrum plot (looks like a 1D NMR spectrum, left side). Click and drag a box on the plotting window; a spectrum should appear. Click the selection icon (green boxes, upper left), click on the spectrum to select it, and click on the edit icon (top row). Edit various parameters to define horizontal and vertical scaling, axes, etc. Other display adjustments can be made in the 1D/2D edit dialog. When a satisfactory spectrum is displayed, select print from the File menu.


Appendix A. XwinNMR and Unix file and directory organization:
TopGraduate ProgramPeopleResearchLinksMapSearchHome
Last modified on: Monday, 13-Oct-2003 16:33:52 CDT