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Haverford College
Department of Biology

Users Guide for Haverford College ARX-300 NMR spectrometer

by R. Scarrow (updated March 2005)

This page was originally designed for the older UXNMR software. It contains some information on the spectrometers that goes into more detail than the Quicknotes (1D and 2D). I revised it to keep this additional information, but tried to get rid of all the outdated information.

Table of Contents:

  1. Safety
  2. Instrument Cautions
  3. A brief introduction to NMR on the ARX-300
  4. Sample Preparation
  5. Instrument Check Out
  6. Sample Changing
  7. The ARX-300 Computer System
  8. Navigating UXNMR
  9. Changing limits of spectral display
  10. Locking and shimming the magnet
  11. Setting up for acquisition
  12. Data acquisition
  13. Processing the spectrum
  14. Integration and Peak Listings
  15. Plotter Setup
  16. Plotting the Spectrum
  17. Simple Modifications to Plots
  18. Running proton-decoupled 13C spectra
  19. Running 13C DEPT experiments
  20. Before you leave
  21. After Power Outage
  22. About printing and plotting errors
  23. Additional Information
  24. Advanced Shimming Techniques
  25. The Variable Temperature Unit

A. Safety

Static magnetic fields by themselves pose no health danger to humans, unless they have implanted electronic devices such as pace-makers. However, if you bring a gas cylinder or heavy magnetic tool within several feet of the magnet, it will get rapidly pulled into the magnet, causing a nasty and expensive accident. (Note: if something gets stuck to the outside of the magnet, do not attempt to remove it yourself - notify the NMR Superuser immediately).

Before any person goes close to the magnet (beyond yellow tape on floor), they should answer NO to all of the following questions:

  • "Do you have a pace-maker or other electronic implanted medical device?"
  • "Are you carrying any magnetized MAC or VISA or copier cards that you ever want to use again?"
  • "Do you have a floppy disk, ZIP disk, or flash drive in your pocket containing files that you might want to use again in the future? "
  • "Are you carrying any tools on your belt or in your hands?"
  • "Are you wearing iron chains, earrings, noserings, etc. (and would you rather not have your nose stuck to the magnet)?"
  • "Is that a pair of metal handcuffs around your wrists?"

Make a habit of taking your keys and wallet out of your pocket before going across the yellow line, and make a habit of putting them back in when you return to the computer console.

B. Instrument Cautions

For the most part, the NMR spectrometer is fairly robust and difficult to break. The most common accident is tube breakage when performing a sample change. To avoid tube breakage:

  • Always check that dust cover is off top of magnet before changing the sample (i.e. before hitting the LIFT button on the SCM).
  • The NMR should always be left with a tube in the magnet. If the tube (in its spinner) does not appear after you hit the LIFT button, it is likely because of problems with the air supply (did you remember to open the valves on the wall?) rather than because of no sample in the magnet.
  • When removing the spinner/tube assembly from the top of the magnet, be sure to lift it up high enough so the bottom of the tube clears the top of the magnet.
  • When placing a new tube on top of the magnet, you should confirm that it is supported by the cushion of air before letting go completely.

C. A brief introduction to NMR on the ARX-300

  • Figure: Rotating Frame representation of the FT NMR experiment. The x and y axes of the figure are part of the "rotating frame"; that is, they rotate around the z axis at the RF frequency (i.e. 300 MHz for 1H). The precession shown in parts d) and e) of the figure are the result of the NMV's precessing slightly faster than the rotating frame (i.e. the NMR frequency of the two chemically distinct nuclei shown are slightly higher than the RF frequency).
    a) Net magnetization vector (NMV) at equilibrium t= 0 (moment that RF transmitter turned on)
    b) RF field along x causes NMV to rotate toward y. t = 5 µsec.
    c) RF transmitter turned of at t = 10 µsec. This is called 90° pulse. (excat duration depends on transmitter strength)
    d) t = 10 ms. Precession of NMV in xy plane causes observable RF signals (the FID).
    e) t= 50 ms. Chemically distinct nuclei precess around the magnetic field axis at slightly different rates.

The sample is placed in a magnetic field generated by an electromagnet to separate the energy of the spin states of 1H, 2H (D), 13C and other paramagnetic nuclei. While they sit within the static magnetic field, there will be a greater population of spins "with" the field than "against" it, due to the Boltzman distribution. The magnetic field is by convention defined to be the z-axis, and therefore the net magnetization vector from the spins is also along the z-axis. During an NMR experiment, this natural equilibrium is perturbed with a radio frequency (RF) pulse (see figure above).

If the RF pulse is a "90°" pulse (on our ARX-300 spectrometer this means the transmitter irradiates the sample for about 10 msec), the net magnetization vector will be rotated 90° from the z axis into the x-y plane. Magnetization in the x-y plane will precess at a frequency determined by the magnetic field (300.0 MHz for 1H, 46.1 MHz for 2H and 75.5 MHz for 13C in the 7.05 Tesla field of the ARX-300's magnet), and this oscillating magnetic field generates radio signals which are picked up by receiver coils which encircle the sample.

This signal is amplified and digitized to give the Free Induction Decay (FID); it is a decay because after a few seconds the magnetization returns to the z axis and the RF signals disappear. Usually 8 or more FID's are summed to increase the signal-to-noise ratio. A Fourier Transform of the summed FIDs will result in an interpretable NMR spectrum. The spectrum may be plotted versus the frequencies of the RF signals contributing to the FIDs; it is, however, more common to plot vs. parts per million (ppm) offset from a reference frequency&endash;conventionally defined as the frequency for resonance for TMS, tetramethylsilane, or, in aqueous solution, for TSPS, sodium trimethylsilylpropane sulfonate

The magnetic field must be constant throughout the sample and in the time it takes to collect data for an NMR spectrum. In order to make the field constant in space, the spectrometer has a set of "shims", which are small electromagnets designed to compensate for inhomogeneities in the field of the large electromagnet. In order to keep the field constant in time, NMR spectrometers use a deuterium lock. The electronics associated with the deuterium lock make continuous adjustments of the current going through the electromagnet in order to maintain a constant NMR frequency of the 2H (deuterium) found in the solvents used for NMR.

Our ARX-300 spectrometer has electronics to irradiate the sample at two different frequencies. One of these is the proton frequency (300 MHz); the other frequency is variable, but is most often set to the 13C frequency (75.5 MHz).. We currently own two interchangeable RF probes which slip into the bottom of the magnet. A tag attached to the probe underneath the magnet identifies it. Usually the VSP-BB probe (also referred to as the "broadband" or "bb" probe) is in place; it has the tunable RF coils used for 13C inside the 300 MHz RF coils used for 1H. An INV or "inverse" probe is also available; because its 1H coil is on the inside, it gives better signal/noise ratios for proton spectra, which is useful for very dilute samples and for some 2-dimensional spectroscopic techniques.

If you need to use the "inverse" probe or need to observe a nucleus other than 13C or 1H, see the NMR superuser for instructions on changing the probe and tuning it. Any experiment can be done on either probe; however, the 90° pulse lengths are different on the two probes. That is the reason that parameter files (see below) have "bb" or "inv" appended to them. The instructions have been written assuming the VSP-BB probe is in place, so the "bb" suffix is used.

D. Sample Preparation

1. NMR Tubes

Recommended tubes are Wilmad 707-PP's. Inspect the tube you will be using for cracks (discard immediately if you find any) and dirt (special NMR tube cleaners can be used). The outside as well as the inside of the tube must be clean.

Clean your tube as soon as you are done with the NMR spectroscopy of your compound. Special tube cleaner which are used with an aspirator are available. Rinse your tube last with reagent-grade acetone or methanol, place the tube briefly (15 minutes) in an 80° drying oven, and use a stream of compessed N2 to blow out residual solvent vapors. Prolonged storage of NMR tubes in drying ovens can cause them to bend, resulting in failure to spin.

2. Deuterated solvent

Be sure to choose a solvent in which your compound is soluble. If in doubt, test with less expensive non-deuterated solvent. The sample must be completely dissolved. It is recommended that samples initially be prepared in a small, dried vial and filtered to remove undissolved sample and impurities.

Deuterated solvents are often supplied in 1.0 mL ampules. When using a bottle, cap it very tightly immediately after use, so it doesn't pick up H2O from the air.

The dissolved sample should occupy the bottom 3.5 cm (1.4") of the tube. This requires about 0.7 mL of solvent. There is little advantage to filling the tube to a greater depth, although if you are using a 1.0 mL ampule, use the entire amount. Expensive solvents sometimes come in 0.5 mL ampules. You will need to carefully center such a sample and spend more time shimming the sample. Vortex plugs are helpful with samples with volumes of 0.5 mL or less.

3. Recommended filtration procedure (if necessary)

a. Prepare a "mini-filter" by stuffing into a long glass Pasteur pipette either a wadded-up 1 square inch piece of KimWipe, or a pinch of glass wool. Use a wooden applicator stick or another long Pasteur pipette to stuff this material into the place where the pipette narrows down.

b. Support an NMR tube in an Erlenmeyer flask. Use a small clamp to prevent later movement, but don't tighten the clamp or you will break the thin glass of the NMR tubes.

c. Carefully rest the "mini-filter" pipette within the NMR tube. Transfer the material from the vial into your mini-filter. If it doesn't flow into the NMR tube by gravity, carefully push it through with a pipette bulb.

E. Instrument Check Out

When you first come in to the room, there are several things to check:

1. Sign in on the log book near the computer and SCM unit.

2. Magnet Inspection

Ice on the vent lines above the yellow sponges in normal. Condensation anywhere else on the magnet, or mist coming from the vent lines, indicates a serious magnet problem. Notify the NMR superuser immediately.

3. Air supply

    a. Review Safety Section A. Check the top of the magnet and pull off the orange cap with the brass screw if it is present on top of the bore hole.

    b. Turn ON the compressed air valves on the wall. A gurgling sound indicates a problem (turn off the air supply and notify the NMR superuser).

4. The NMR console

    a. Red power switch should be lighted (users should never turn this off unless smoke is coming out of the unit, or if there is an electrical outage -- see next page).

    b. The Eurotherm VT unit (right side of console): Heater switch should be off (not illuminated). Gas Flow should be set so the middle of the ball is at 1 (the lowest mark) on the right hand scale.

5. Shim Control Module

STD BY and FINE lights should be lit. If not, touch them once to turn them on.

6. If the computer screen is dark ...

    a. Move the mouse. This deactivates the screen saver.

    b. Turn on the monitor by pressing the big square button on the front of the monitor. It has a green light when it is on.

    c. Check to see if the + button below the screen brightens it.

7. Printers

These should be left on. The ready light should be on on the printer.

F. Sample Changing

Be sure to follow the previous section E, Instrument Check Out, before starting your first sample change of the day.

1. On the Shim Control Module (SCM) ...

    a. Turn off the lock (hit the lock button if the light is on).

    b. Hit the orange 2nd button, and then the lift off button. (The second meaning of this button is written below it - in this case it means lift).The previously-used NMR tube will now magically rise from the depths of the magnet and float on a cushion of air above the probe.

2. Changing the tube in the tube spinner

    a. Carefully take the tube and its spinner directly straight up and out of the magnet. Be very careful not to hit and break the tube. If a tube does break in or above tha magnet, notify the NMR superuser immediately.

    b. Replace the tube in the spinner. Use the plexiglass tube positioning device. Routinely put the bottom of your tube at 20.0 mm on the scale. Do not position the tube lower than this or your tube will hit the thermocouple installed under the RF probe. This could break your tube, or cause it not to spin. The black bands indicate positions of transceiver coils within the RF probe - your solvent should extend 1 cm above and below these. Center very small samples within the RF coils.

    c. Wipe your tube clean with a Kimwipe.

    d. Carefully place the tube and spinner on the air cushion in the magnet. Don't let go of the tube if you don't feel it supported by the air.

3. Back at the Shim Control Unit

    a. Press the lift off button, turning it off. The sample will slowly descend into the magnet. Note that no lock signal will be seen, nor will a spin rate be observed, until the tube is in the RF probe--about 30-45 seconds after lift off.

    b. Push spin rate. If it isn't around 20, adjust it with the dial.

    c. Push spin. The actual spin rate is shown when the Std By button is illuminated. If it is stuck at zero, then either (i) your tube is positioned too low, (ii) your tube or the spinner is greasy or otherwise dirty, (iii) the marks on the spinner have been washed off with acetone or (iv) the VT air gas flow is too high.

G. The ARX-300 Computer System

1. The Mouse

The mouse has three buttons on it. For dealing with pull-down menus, press and hold down the left button and things will work pretty much as on a MacIntosh. In these instructions, "Click" means rapidly press and release, whereas "Drag" or "Press and Drag" means press the mouse button down and hold it as you move the mouse. CLB means click left button, while CMB and CRB are similarly defined for middle and right buttons.

The three mouse buttons are used when you issue a command from one of the icons. Icons are generally at the bottom or sides of the screen, and usually contain three characters or short words corresponding to what will happen if you use the left, center, or right mouse button.

2. Common mistakes made by Mac users

    a. The cursor must stay on the active window for the keyboard to be active.

    b. Case matters. The name of the NMR program is xwinnmr, not UXNMR.

    c. When using a dialog box, hit return (ÿ) after changing any field. Otherwise, even though the field looks changed, it won't be.

3. Logging On and Off, and the UNIX shell

If the last person logged off, you will see a blue login box on top of a Bruker logo. If the person before you didn't log off, you can usually do so by typing exit to close the xwinnmr window, and then choosing the logoff option from the UNIX pulldown menu on the desktop..

a. Type in your login name (lower case) and password (if any--see below).

b. Open a unix shell from the UNIX pulldown menu. Then type xwinnmr to start the NMR program.

c. Typing exitÿ will return you to your UNIX 'shell'.

4. Passwords

To give yourself a password (or to change the password), type passwd from the UNIX shell. You will be promted for a password, which must contain at least 6 characters, one of which must be a "special" character such as #,!,? or $.

H. Navigating XWINNMR

1. Typing commands, and context specific help

At the bottom of the xwinnmr window there is a line (pink) where you may type commands, and below this a line that shows context specific help (usually telling you what the different mouse buttons do). In order to type in commands, the cursor must be positioned over the xwinnmr window.

2. Menus

The menu is at the top of the xwinnmr window, and are used with the left mouse button. Menus are pull-down, as on a Macintosh. Most menu items have keyboard equivalents, which, once learned are easier to invoke (just type them in). The keyboard equivalents are shown on the right sides of the menus to help you learn them.

3. Command buttons

Command buttons are generally located on the left side of the xwinnmr window. Use the left mouse button to activate the command. In a few cases, you need to push the left button down while holding it over the button, and then move the mouse up and down to adjust some parameter.

4. Subprograms

There are several subprograms which have different menus and icons. For subprograms, the right-most menu item is return, which gets you back to the main menu. (In contrast, for the main menu, the right-most menu item is exit which gets you out of uxnmr). With the exception of "show acquisition window" (which is accessed by typing acqu or by using the acquisition menu at the top), the subprograms are most easily invoked from clicking on the command buttons. When you are in a subprogram, the buttons on the left of the screen, and the display itself, will look different.

Subprograms often used include:

a. acqu - shows the contents of the data acquisition memory (the FID).

b. phase - allows phasing of the spectrum

c. utilities - Used to set the minimum and maximum peak sizes for peak picking.

d. integr - used for integration

Other subprograms include bsln, which can be used for fancy baseline correction, and winfunc, which nicely demonstrates effects of exponential multiplication and other "windowing functions" on the FID and the transformed spectrum.

I. Changing limits of spectral display

If your display says "No data available", you will have to collect data before trying out these commands.

1. Choosing the vertical scale of the display

    The buttons at the top left (*2, *8, /2, /8 and a full-scale button and scale-adjustment "slider" button) control the vertical scale.

2. Choosing a spectral region (horizontal scale)

    a. The easiest way to zoom in on part of the spectrum is as follows (this only works when the main menu is showing):

    A. Put the cursor on the spectrum and CLB. The cursor should now stick on the spectrum. (If not, you may have inadvertently double-clicked; the second click detaches the cursor from the spectrum. Try a gentle CLB again.)

    B. Move the cursor to the left edge of the desired region and CMB

    C. Now move the cursor the the right edge and CMB again. The display should zoom in on the region you have selected.

    D. CLB to detach the cursor from the spectrum.

J. Locking and shimming the magnet

Note: refer to the NMR Quicknotes 1D for recommended settings and sequence of shimming commands.

(a)
(b)
(c)

1. Get the lock display on the screen

Within xwinnmr, type lockdisp. The lock signal should look like (a) when moving from left to right, and like (b) when moving from right to left. (Sometimes, the lock signal doesn't appear at all. In this case, try quitting the lockdisp window and restarting. That usually fixes the problem.) When overlapped, the left-to-right and right-to left signals should be centered on the screen (c).

If the lock display fills the screen, drag its left margin to the right (using the left mouse button). CLB on the xwinnmr window to return to the main program. If you wish, close the lock display window by CLB on its icon.

2. Finding lock using the SCM (shim control module).

    a. Press Field and adjust with the black dial to center the lock signal. The field value (shown under "actual") should be within a few hundred of the values showin in the 1D NMR QUICKNOTES, even if you are using an oddball solvent. Press Std By button after you have made the adjustment (in order to deactivate the black dial). (If you press Field a second time, before pressing any other button, you will revert to the "previous" value shown in the display. Do this if you think you've gone too far or the wrong way.)

    b. Press lock power and adjust with the dial to the "lock off" value shown in the Quicknotes.

    c. If necessary (it usually isn't) adjust phase so the height of the lock signal is the same going left to right as it is going right to left. In each direction the lock signal should go up to a maximum before it goes down and starts "ringing".

    d. Press auto lock. The lock gain is adjusted automatically as part of the autolock sequence, which takes about 30 seconds. When the lights have stopped flashing on the autolock and lock gain buttons on the SCM unit, click the lock button to maintain lock but turn off autolock (on the 200 MHz NMR, this step is not needed because the lock button automatically comes on when the autolock procedure is finished).

3. Shimming the Z and Z2 shims.

    a. Push lock gain and with the dial lower the straight locked signal to about 1 1/2 inches below the top of the screen.

    b. Push Z to adjust the current to the Z shimming magnet. Use the dial to maximize the lock signal; if it pegs at the top of screen, reduce the lock gain.

    c. Adjust the Z2 shim. Then go back and touch up Z if necessary.

    d. Push lock gain and with the dial raise the signal to just below where it is pegged near the top of the screen. Press stdby button.

K. Setting up for acquisition

1. Specify experiment name and number

WARNING: it is imperative that you change at least the number of the experiment to one which is previously unused before you execute the zg command, or you will lose your previous data.

a. Type edcÿ (for edit current data set).

b. Change the name or experiment number to a combination not used before. Remember to hit the return key (ÿ) after making any change to the fields in the dialog box.

  • Note: the user should always be the name you logged in as ("superlab" for instance)
  • If you obtain multiple types of spectra on the same sample, it is suggested that you give them all the same name, but different experiment numbers. Each experiment number has its own set of NMR data (free-induction decay data).
  • The processing number is usually 1. It can be changed later in order to generate different spectra from the same data set (for instance, you could change the line broadening parameter to see what effect this has on the NMR spectrum).

c. CLB on save. The screen should say "NO DATA AVAILABLE" - if not, you have accidently chosen a name/expno combination that you already used.

2. Read in acquisition and processing parameters

    a. Type rparÿ

    b. Select the parameter set proton8 and then CLB the COPY ALL button.

    c. Some of the acquisition parameters depend on the probe and solvent that are being used. To set these to the correct values, type edaÿ (edit acquistion parameters) and scroll down until you find the SOLVENT and PROSOL fields. Set the correct solvent, and then click on the box next to the words PROSOL (the words "true" should appear, to indicate that you have changed the appropriate parameters to their correct values). Exit the eda dialog box.

3. Alternate Procedure, starting from previous data set

If you have previously taken a proton spectrum, it is usually easier to just do the following:

a. Click on Data/Browse in the top menu and drag right to user names, and pick a spectrum with parameters the same as what you want. Click is succession on your username, the data set name, the experiment number, and the process number (this is usually 1).

b. Type edcÿ. Change the name or experiment number to a combination not used before. [Note: the user should always be your loginID]. Select save. The screen should then say "NO DATA AVAILABLE." The parameters, however, will be the same as for the previous data set.

4. Editing parameters

The param menu contains many commands used to edit the acquisition, processing, plotting and output parameters. These are in the Data/Acqu/Proc and Plot/Print submenus. You may want to try these commands to see how many parameters you can identify.

L. Data acquisition

1. Receiver gain adjustment

Type rgaÿ [receiver gain adjustment]. Look at the bottom of the screen and wait for the computer to say finished.

2. Optimizing sweep width (optional; proton NMR only).

For highest resolution, you should set SW and SFO1 parameters so that the spectral region you are observing extends only about 2 ppm beyond your upfield-most and downfield-most peaks. This is easy to do for a proton spectrum.

a. Type nsÿ1ÿswÿ25ÿ [number of scans = 1 and sweep width = 25 ppm]. This sweep width is wider than desired for final data collection but gives a range which will certainly include your proton spectrum.

b. Type zgÿ [zero (the data memory) & go (collect data into memory)].

c. When acquisition is complete (about 15 seconds; look for a message at the bottom of the uxnmr window), type efÿ [exponential multiplication followed by a Fourier transform]. Don't worry about phasing the spectrum this time.

d. Expand the spectrum vertically so the large peaks are offscale and the noise in the baseline is visible (see page entitled "Changing limits of spectral display").

e. Expand the spectrum horizontally so that all peaks (including impurities) show. Allow about 2 ppm of baseline on either side of the spectrum.

f. Now CLB on the SW-SFO1 button. This changes the transmitter frequency (SFO1) and the sweep width (SW) so that just the region on the screen will be recorded next time.

3. Collecting your keeper FID data

a. Type nsÿ8ÿ or nsÿ16ÿ to set the number os scans (which should always be a multiple of 8 to allow for phase cycling which reduces artifacts in the spectrum). For very dilute sample, you may later want to increase the number of scans in order to increase the signal/noise ratio, but to double the signal/noise ratio, you must quadruple the number of scans (S/N µ ). It is often quicker to make up a new, more concentrated sample.

b. Type zgÿ [zero (the data memory) & go (collect data into memory)]. If you wish to view the FID accumulating, click on acqu from the top menu. Wait for the nmr to complete the scans (if you are in the acqu subprogram, there are no messages to tell you acquisition is done; instead the number of scans which have been collected is shown on the screen).

M. Processing the spectrum

1. Exponential Multiplication and Fourier Transform

Exponential multiplication is controlled by the LB parameter (type lb to see what the current value is, in Hz). If you wish to see the effects of exponential multiplication, you may type emÿ while the acquisition window is open. Then type ftÿ to perform the Fourier transform (this will automatically take you out of the acquistion display routine).

You can do both of these steps together by simply typing efÿ [exponential multiplication followed by a Fourier transform]. If you have been in the aquisition display subroutine, you are automatically returned to the main program.

See the page "Additional Information" for more on exponential multiplication.

2. Phasing

See Quicknotes 1D for information on phasing.

3. Calibration

a. Expand the spectrum around TMS or other reference peak.

b. CLB on the calibration button.

c. Move cursor to the top of the calibration peak and CMB. You will be prompted for the correct ppm for the peak.

N. Integration and Peak Listings

1. Automatic baseline correction (recommended before integration)

Type absÿ [automatic baseline]. See page 156 of the manual for more details. This routine also automatically creates integral regions, so step 2 below is optional. Usually, a user can do a better job than the computer at determining integration regions, so step 2 is recommended.

2. Integration sub-program (optional--see above).

3. Peak and Integral Listings on Printer (optional)

These steps are optional because plots can contain peak and integral listings. However, peak listings on the printer are in both ppm and Hz, which is useful for determining J splitting s for multiplets.

a. Select the correct printer (see Selecting the output device on the next page).

b. To list peaks on the printer, type ppÿ

c. If there are too many or too few peaks in the listing, go to the utilities subroutine and set the MI (minimum intensity).

d. To list integrals, type liÿ

O. Printer and Plotter Setup

1. Selecting the output device

The command edoÿ can be used to select the output device. Usually we use the same networked laser printer as the output device for both the printer and plotter.

P. Plotting the Spectrum (recommended procedure)

The procedures for making plots are described in the quicknotes for 1D and 2D spectra. The following notes give additional information.

1. Set a title (optional)

    a. Type settiÿ

    b. Edit the title, and exit.

2. View and Plot

The view command (type it) allows you to preview what the output will look like. Use this before typing plot.

Q. Simple Modifications to Plots.

1. Other plot parameter sets

When you read in standard parameters sets using the rpar command, a set of recommended plotting parameters is read in. You can also use rpar to read in some other standard plot parameter sets. A number of other standard plot parameter sets have been set up by the NMR superuser; all start with "plot", followed by various combinations of letters indicating what features come with this parameter set.

    H axis tic increment is 0.1 ppm

    C axis tic increment is 2.0 ppm

    Par Put parameters on right side of plot

    Int Plot integrals at bottom of plot

    PP Peak pick in ppm at the top of the plot

    PH Peak pick in Hz (useful for determining J of multiplets)

The only difference between plotH... and plotC... files is the tic increment (xticdis -- see below); thus plotH... would be appropriate for a small region of a 13C spectrum.

2. Modifying plot parameters using edg (edit graphics)

If you want to change how your plot looks, you need to use edgÿ. The dialog box which appears contains a number of fields (such as EDSPECT) which contain "ed". CLB on these fields brings up another dialog box with more parameters.

Rather than using edg, you may just type in the name(s) of the parameter(s) you wish to modify, and you will get a box allowing you to change the current value of that parameter. Some of the parameters you may wish to change are:

dhei height (in cm) of data window. There is a maximum value for this, and uxnmr will not let you set it higher.

szero position (in cm from bottom of data window) of y=0. Normally this is 0.5 cm. If there are negative peaks (as in a DEPT 135), set szero (and cy) to dhei/2.

cy Height (in cm) of largest peak. Normally cy=dhei-szero. If you wish to vertically expand your plot, multiply cy by the expansion factor. The largest peaks will be chopped off at the top of the data window.

pscal This determines whether the "largest peak" for cy is the largest in the spectrum (pscal=global), or just within the plot region (pscal=preg). For expansion plots, use preg. (This is actually an edp rather than an edg parameter).

ihei Height (in cm) of largest integral trace.

xticdis Distance between tic marks. Should be in the range of 1% of the total range.

plunit Hz or ppm, for peak picking.

3. Plots with expansion insets.

(I'm not sure if this still works with the xwinnmr software, but I've left it in so you can try. Let me know how it goes if you try this.)

The parameter sets "plotexpH", "plotexpHPP" and "plotexpHPH" (accessable by rpar) are for expansion insets for the upper half of your spectrum plot, something like that shown at the right. (As before, PP and PH indicate peak picking in ppm or Hz respectively). Each expansion inset is 5 cm wide.

a. Start by making a regular plot, but don't include peak picking, and you may wish to omit parameters as well (thus, rpar "plotH" or "plotHInt"). You may want do decrease cy so that the vertical scale is reduced.

b. After typing plotÿ to make the regular plot (which you use to measure where you want your expansion plots), type plotsÿ (suspended plot) to generate another copy of the plot in the computer's memory.

c. rpar plotexpH plotÿ

d. Select on the screen the first region you wish to expand.

e. Define the plot limit by CLB on the icon.

f. Type sxlleftÿ and set the left offset for the expansion inset. This is the number of cm from the left edge of the main spectrum to the left edge of the expansion inset. You may wish to use sxlleft=1 for the first expansion, sxlleft=7 for the second, and so on.

g. Type plotsÿ. This adds to the plot image in the computer memory.

h. Repeat steps d - g for additional expansions.

i. Type flplotÿ to "flush the plot" from the computer memory to the plotter.

4. Plotting on 11 by 17 paper ("US B" size)

This only works on the hp7550a plotter, connected to the nmr2 computer.

a. Set up the plot using a parameter set "plotB..."

b. Load the paper in manually: Turn off the autofeed mode (use the button at the top left of the plotter LCD to undisplay the *). Press the sheet feed (bottom left) button , and press it again after placing the 11 x 17 paper in the plotter.

c. Type plotÿ.

d. As a courtesy, return the plotter to autofeed mode and load in a 8.5 x 11 sheet when you are done plotting.

R. Running proton-decoupled 13C spectra

Instructions are given for this in the quicknotes. Here are some additional notes

    a. if you want to do a receiver gain adjustment (rgaÿ) it won't hurt. It usually sets gain to the maximum value of 45000.

    b. The experiment is set up for 512 scans, which will take about 1/4 hour. If you want, you can change ns to a larger number (i.e. type ns 5120) and then monitor the progress of the spectrum until an adequate signal to noise ratio is obtained. If you are curious about the total time for an experiment, type exptÿ.

    • Wait for a few scans to be acquired; you may want to use the acqu subprogram to see the FID. Then type trÿ [transfer data to file], followed by efÿ. (The latter command operates only on data already saved to the file).
    • Phase the spectrum. (Review section M on processing 1H spectra). It is normal for 13C spectra to be noisier than 1H spectra, but the peaks should be sharp. Find TMS or a solvent peak (which will be a 1:1:1 triplet, 1:2:3:2:1 quintet, or 1:3:6:7:6:3:1 septet depending on how many D's are attached - splitting will be ca. 35 Hz) and calibrate on it.
    • As time goes by, the number of scans will increase and the signal to noise ratio (S/N) will increase. You can see how your spectrum is doing by typing trÿ and then, after a few seconds, efpÿ (ef with previous phasing).
    • Once the S/N is satisfactory, type stopÿ. After acquisition has finished, type efpÿ, so you are using all the data that was collected. Then plot the spectrum or get a peak listing using the techniques desribed for 1H spectra. Remember, integrals aren't reliable for 13C spectra.

    c. If you finish taking the spectrum and you decide you need more scans to get better signal-to-noise, you may append additional scans to the data already collected by just typing go.

S. Running 13C DEPT experiments

See quicknotes.

T. Before you leave

1. Routine shut-down

    a. Replace your sample with the sample which was present when you arrived or with next users sample.

    b. If no user is waiting, lock the magnetic field (no need to shim).

    c. If no user is waiting, shut of the air supply with the valves on the wall and place the orange dust cap on top of the magnet bore hole.

    d. exitÿ the xwinnmr program, and use the UNIX desktop pulldown menu to logout of UNIX.

    e. Remove all your printouts and plots from the printer and plotter.

2. Shutting down the computer (before power outage, for instance)

    a. Login to the computer and exitÿ xwinnmr in order to return to the login shell.

    b. Type powerdownÿ. A password may be needed. Answer yÿ to the prompt about an express powerdown. When the screen says the system is down (about 30 seconds), you may turn the computer and monitor off.

    c. If a power outage is expected, turn off both the computer and also turn off the red switch on the front of the NMR console.

3. Problems?

Make a note of all problems and give the note to the NMR superuser. Include as many details as possible (error messages, what you tried to solve problem, etc.). In case the computer you are working at freezes up (mouse is unresponsive, and control-/ doesn't work), shut the computer down remotely, using the following procedure (this is safer than just turning the computer off).

4. Shutting down the computer remotely

    a. At the auxilliary workstation (nmr2), login as guest (no password needed).

    b. Type telnet nmr1ÿ. You should get a login prompt from the nmr1 computer (if not, it is really perplexed, and turning it off is the only thing left to try).

    c. Login as guest and then follow step 2b above to do a powerdown.

A similar procedure will shut down nmr2 from nmr1. You may also login remotely via any Mac, using telnet. The internet address is nmr1.chem.haverford.edu. Caution: these computers are not configured to accept mail.

U. After Power Outage

1. If you happen to be in the lab when there is an electrical outage...

We have UPS's to maintain power for about 45 minutes. If the outage goes on for more than a few minutes, follow the instructions above to power down the computers and NMR. If the power outage has already gone on too long and the UPS's have run out of juice (the computers and NMR have shut down), turn off the main red switch on the front of the NMR console and also turn off (or pull the plugs) on the computers in the room. This reduces the chance of damage from a power spike when the power returns.

Wait five minutes after power returns (power is often unstable in the first few minutes after power returns) before turning console and computers back on.

2. Reboot the computers

3. To reset the NMR console (after the ASPECT workstations have rebooted):

Open the left door of the console (lift handle up and to the left). Check that there are no red lights on in the "power control" panel of the power supply near the bottom. If there are, use a pen to push the reset button for the power supply.

Hit the red reset buttons on CPU/4 and AQI boards in the top rack inside the left door of the console. The yellow lights should flash on the CPU/4 and the RES light should be on on the AQI board. (If the lights aren't on, there is a power supply problem for the rack that houses these boards; in this case, open the back panel and hit the reset button to clear the red lights on the power supply; if this doesn't work, turn the power to the rack off, wait 5 seconds, and turn it back on.)

Inside the right door, hold down for five seconds the big red SCM reset button (about half way down, on the left side). There is also an RS232 reset button accessible with a pen tip.

4. To reset the pre-amplifier (on floor near magnet):

Use a pen tip to hit the recessed red reset button on the back of the preamp unit.

5. To verify things are working OK:

Type iiÿ from within the UXNMR software, and then try rgaÿ. If "Error during open of serial port /dev/tty15" appears, try resetting SCM again. If "Error in Preamp Unit -Error Code 13: Power Fail" appears, repeat typing iiÿ and rgaÿ, and then try resetting the pre-amp again. If things still aren’t working, repeat steps 3 and 4 again, and then contact the NMR superuser.

V. About printing and plotting errors

The computer has a printer queue that gets backed up if something is wrong with the printer or the plotting instructions. If no plot appears after attempting a plot from UXNMR, try the following steps in order until the problem is fixed.

1. Printing Errors

Make sure edo settings are correct (section O1).

2. Plotting Errors

    a. Check that the plotter is on. If the LCD on the plotter says "Load paper to plot", then push the lower left sheet feed button (and reload paper tray if necessary). Check that autofeed is on (* in upper left of LCD).

    b. The problem may be with the plot instructions you or someone else generated. Type uxlpstatÿ. A blue screen appears with info. At the bottom is a listing with one or more lines beginning hp7550a-### or hplj3p-###. These are job numbers. Write down the number of the job which the listing says is currently being printed. Hit return (ÿ). Then type uxcancelÿ, and then type in the job number: hp7550a-###ÿ or hplj3p-###ÿ.

    d. If there are multiple plots in the queue, and the previous step did not result in the plotter coming to life, then repeat the previous step.

    e. Only after uxlpstatÿ indicates no jobs in the printer queue should you try plotting again. If there is still no plot, and you can't find the NMR superuser, then use uxcancelÿ to delete your plot from the queue (the problem may be some incorrect parameter in your data files which won't affect other users, unless you leave your plot in the queue).

    f. If you still can't get the plotter to work, the serial port probably needs to be re-initiallized, which requires you to reboot the computer. If the NMR superuser is available, ask him or her to reboot the computer.

W. Additional Information

1. About UNIX, XWindows, and xwinnmr

UNIX is an operating system dating from the 1960's used on many mid- to large-sized computer systems. Computer jocks like it becuase of its power (you can do a lot of things with single commands which would be much more difficult in MS-DOS, MacIntosh operating systems, or even VMS, the operating system used on VAX mainframe computers), but is difficult to learn because of a large number of commands whose names are non-intuitive.

In the last five years, XWindows has become the standard terminal display interface to UNIX. It controls how windows appear on the screen and how the computer interprets keyboard input and mouse movements. It is just enough similar to the MacIntosh operating system to get the Mac user totally confused.

xwinnmr is the program which controls the spectrometer and allows you to manipulate data. It is supplied by Bruker Instruments, the NMR manufacturer.

2. About exponential multiplication and line broadening

Exponential multiplication is a noise reduction technique which also results in a broadening of all the peaks in your spectrum. The degree of line broadening is usually set to 0.30 Hz for a proton spectrum (to verify or change this, type lbÿ). If you have very closely overlapping peaks, you may wish to change this parameter to 0; if you have a very noisy spectrum, you may wish to increase it to 1 or 2 Hz. If you make changes to lb you must redo efÿ for the changes to take effect.

Note that efpÿ is just like efÿ, except that the efp command also phases the spectrum the same way as it was phased previously. This is useful if, after phasing the spectrum and zooming in on a particular peak or cluster of peaks, you wish to investigate the effect of changing the lb parameter.

The winfunc subprogram can also be used to investigate the effects of various line broadening values on the appearance of the spectrum. It graphically shows how the FID is multiplied by a "windowing function" (e-(lb)t; t is time after the RF pulse), and allows you to use icons to increment the value of lb and see the effects on the windowing function and spectrum. It also allows you to try out alternative windowing functions (which are mostly used for 2-D NMR).

X. Advanced Shimming Techniques<

In most cases, adjustment of Z and Z2 shims is all that is necessary (see section J).

The X, X2, Y, Y2 and XY shims should always be adjusted with spinning off. Spinning side-bands (peaks ±20, ±40, ±60 Hz, etc. on either side of any big peak (particularly noticeable for singlets) are a sure sign that these "non-spinning" shims need to be touched up.

[See manual for more on shimming]

Y. The Variable Temperature Unit

Note: the VT unit on the 300 MHz spectrometer is currently not operational. It may be fixed at some point in the future, and these instructions (contributed by L. Durney) should be checked at that point to make sure they are still current.

The variable temperature unit can be used to both heat and cool your sample. Be sure the temperature(s) at which you wish to work are safely above the freezing point and/or below the boiling point of your solvent.

1. Inserting the self-tune sample.

    a. Use one of the ceramic spinners. They are usually found in the drawer where the clean NMR tubes are kept. They are opaque, and heavier than the plastic spinners.

    b. Insert the ethylene glycol sample (found in the tube holder) sample as usual.

    c. Place the orange cap securely in place on top of the magnet.

2. Using liquid nitrogen to work below room temperature.

    a. Fill the large dewar kept near the magnet with liquid nitrogen. The dewar has two handles and a blue cap. Using the shorter hose, fill it at least half-full. Replace the cap, and return it to its position near the magnet.

    b. Insert the heating unit into the dewar. First place the metal ring with the black O-ring at the mouth of the dewar. Then slowly insert the heating unit. This is a long thin heater that fits into the dewar and causes the nitrogen to boil off. The gas then flows through the corrugated black tube into the magnet. Be careful, if the dewar is very full, liquid nitrogen will overflow. Finally, use the round metal clamp to attach the heater to the dewar. Wait until the gas coming from the black tube is very cold to be sure the boil-off has purged the black tube of air.

    c. Remove the green tube from the gas inlet. First, take off any metal objects and mechanical watches. Then disconnect the green tube and its accompanying clamp from the gas inlet underneath the magnet.

    d. Clamp the black liquid nitrogen tube in place. Move the dewar as needed to make the tube straight as it connects with the inlet.

    e. At the computer, change the temperature set point. Type teÿ and then enter the desired temperature. Make sure this is safely above the freezing point of your solvent. Then type tesetÿ. Or type edteÿ and change the first parameter, called Set Temperature. This will take effect as soon as you hit return.

    f. By using the computer to set the temperature, you've disabled the keys on the temperature unit itself. To switch back to manual control, type edteÿ. Disable the Digital Input Lock and turn off the Key Lock.

    g. At the temperature control unit, turn on the heater in the liquid nitrogen. The on/off button is under LN2; use the up and down arrows to control the flow-rate of the nitrogen vapor. The gauge above these buttons shows the flow rate, 10-15 is about right.

    h. When the temperature on the display reads close to your setpoint (within .3 of a degree) turn on the heater.

    i. Allow the temperature to equilibrate for 10 minutes. Then continue with step 4.

3. Working above room temperature

    a. Adjust the gas flow to its maximum. Make sure the sample is spinning. At the temperature control unit, turn the gas flow knob counterclockwise to raise the black ball until you've lifted the sample out of position, and the spinning stops. Then turn the knob down (clockwise) a quarter of a turn. Usually the center of the ball will be just above four on the left-hand scale. Check that the sample is spinning.

    b. Enter the desired set point. Type teÿ and then enter the desired temperature. Make sure this is safely below the boiling point of your solvent. Then type tesetÿ. Or type edteÿ and change the Set Temperature parameter. This will take effect as soon as you hit return. Then check the overshoot limit (HB), change this to your desired temperature. The undershoot limit (LB) ought to be about 70 K. Also change the ramp rate (RR) to 35 K/min for temperatures above 330 K or 25 K/min for temperatures below 330 K.

    c. By using the computer to set the temperature, you've disabled the keys on the temperature unit itself. To switch back to manual control, type edteÿ. Disable the Digital Input Lock and turn off the Key Lock.

    d. At the temperature control unit, turn on the heater.

    e. Allow the temperature to equilibrate for about 10 minutes, then continue with step 4.

4. Self-tuning the temperature control unit

    a. Perform a self-tune. At the computer, type edteÿ to edit the temperature parameters. Then click on Self Tune , and turn it on. Quit the menu. The small green "SP" light on the temperature display will blink for one minutes. Then the "At" light in the upper right corner will blink until the self-tune is done.

    b. To determine the exact temperature of your sample, spin and hit the sweep off button. There is no deuterium in the ethylene glycol sample and so you cannot lock and shim. Change NS to 8, and type zgÿ. (If you will be doing this repeatedly, it is worth using the SFO-SW1 button to select the area around the two peaks). efÿ, phase, then type ppÿ. The distance between these two peaks varies with temperature (because of hydrogen bonding) as follows:

Ethylene Glycol: Temp (K) = 466.5 - 101.4*Æppm

c. For temperatures below room temperature, use a methanol thermometer:1

Methanol: T(K) = 429.2 - 62.26*Dppm - 13.85*(Dppm)2

5. Continuing with data acquisition

    a. Replace your sample. Remove the orange cap before ejecting the self-tune sample. Use the ceramic spinner. Be sure the temperature you wish to work at is safely above the freezing point and below the boiling point of your solvent. Replace the orange cap.

    b. Allow the temperature to equilibrate to the set point. For long-term stability you should wait 90 minutes. To run one brief scan the temperature will be reasonably stable after 20 minutes.

    c. Lock and shim the magnet at this temperature, and continue with your acquisition.

6. When finished.

    a. Reset the set point to room temperature. Type teÿ, and 295.5ÿ. Then type tesetÿ.

    b. Turn off the liquid nitrogen heater (if using) and the heater at the variable temperature unit.

    c. Immediatly remove the black liquid nitrogen tube from the gas inlet and reconnect the green air hose (if necessary). Disassemble the heating unit and dewar and replace the blue cap, the liquid nitrogen will last longer.

    d. Return the gas flow rate to its usual position (if necessary). Wait as long as possible before doing this, the probe will cool much more quickly that way. Turn the knob clockwise until the the center of the ball is at the 1 on the right-hand scale.

    e. Remove your sample (remove the orange cap first) and replace the lock sample.