Routine 2D NMR on DPX300 instruments

Acquiring 2D experiments using ICONNMR or the automated sample changer is almost as straightforward as collecting 1D data sets.
Processing is performed automatically and no knowledge of 2D processing is required. Manual processing can be applied, however, and will improve the data quality in many cases.

Plotting is more involved for 2D experiments and is best achieved using the XWIN-NMR software package. The 2D WINNMR package is also available but has fewer features. The XWINPLOT package is a WYSIWYG option.

Each experiment is automatically preceded by a standard ¹H NMR spectrum, so there is no need acquire this separately. The spectral width is automatically optimised in the proton dimension(s) to include only regions where there are signals of "significant" intensity.

For experiments involving ¹³C, The carbon dimensions can only be changed manually and only if permission has been granted. For routine experiments changing the carbon spectral width is generally not necessary.

Which Instrument to use?

Always remember that these experiments are of an "off the shelf" nature that can be improved upon significantly by optimising conditions and possibly changing instrument.

FLIP300 is configured to acquire all of the basic 2D experiments with higher sensitivity than GYRO300 and so FLIP300 is generally the preferred instrument. This is because the "inverse" probe in FLIP300 is optimized for proton sensitivity and all the 2D experiments detect protons. GYRO300 has the advantage of automation with the sample changer and does not need to be tuned.

GYRO300, with its "normal" probe arrangement, has higher sensitivity than FLIP300 for carbon and phosphorus observe experiments.

Often, a good strategy is to acquire 2D experiments on FLIP300 and carbon and DEPT experiments on GYRO300, particularly when a limited amount of sample is available. Obviously, a shift to higher frequency, 500 or 600 MHz, will allow both increased resolution and higher sensitivity.

Available Experiments

Single Experiments:

• 1HCOSY - Quick COSY with gradients.
• 1HNOESY - Quick NOESY with gradients. 600 ms default mixing time.
• 1HTOCSY - Quick TOCSY. 200 ms default mixing time.
• 1H13CHMQC - One bond HMQC correlation with gradients
• 1H13CHMBC - long range H-C correlation with gradients

All experiments default to have the normal 1D proton spectrum plotted along F2 (horizontal). Homonuclear experiments are set to have the normal 1H spectrum plotted along F1 (vertical axis) as well. Multiple experiments that include acquisition of a 13C spectrum automatically plot this 13C 1D spectrum along the F1 dimension of the HMQC and HMBC experiments. Otherwise a projection internally generated from within the 2D data can be plotted along the F1 dimension in the heteronuclear experiments.

Experiment times

Do not take up large amounts of time during the week nights with multiple 2Ds on the sample changer instrument! ONLY put your sample on after 3 pm in the afternoon. You can book FLIP300 for longer periods, with better 2D results.

Acquiring the data

GYRO300: This is really no different to acquiring 1D data sets. Just pick the 2D experiment name instead of the 1D experiment name. You will see multiple experiment numbers inserted into the queue. The experiments that are to be used as projections also appear in the list. They are shaded blue and have a red arrow on the left.

FLIP300: The only modification required here is that it is possible to tune the probe to your sample before starting. Otherwise, the procedure is just like using ICONNMR. Just pick the 2D experiment name instead of the 1D experiment name.

Parameters may be modified in the same way as for 1D experiments on both instruments. Use the Parameters --> User Specific Parameters/Commands buttons of the Routine or Automation flow chart window to pick experiment and parameter

Tuning Pulse - OPTIONAL!

Consult Facility staff to learn how to do this or you may do a lot of expensive damage! Place sample in probe and proceed as for routine operation. After selecting the ICONNMR experiment(s), open the XWINNMR window (by clicking on the icon of the same name if necessary):

The XWINNMR Icon

Type tune1H. This macro will bring up the tuning window and automatically run the wobb command. On occasion it may be necessary to manually enter the acqu window if the tuning dip does not appear. If this occurs, left click the return button in the bottom left corner of the XWINNMR window:

Click return again if a small new panel appears then type acqu at the XWINNMR command line prompt. Adjust the proton tuning controls under probe. See the Bruker manual for further details on the wobb command. The tuning dip should be as deep as possible and centred when correct:

When finished click the stop button in the XWINNMR control window.

After completing your experiments, retune the probe to a sample with chloroform solvent!

Effect of gradients

Almost all of the 2D experiments employ gradients during the execution of the experiment. This causes the lock signal to "bounce":

If the lock signal does not bounce like this it may mean the gradients are not functioning. This is very rare except for when they have been physically disconnected.

Transferring the data and starting XWINNMR on the PC

See the notes in the XWINNMR under Windows NT document.

Processing and Plotting routine 2D spectra

N.B. These notes are in no way a substitute for reading the Bruker manuals. These are available online in all Bruker software under the Help menu found in the top right hand side of the software window.

i) Read in your data and run the macro

Use the diro command to locate your own data directory.

Use the dir command to locate your data set name.

To load each experiment number, type re xx where xx = the number of the experiment you wish to view.

When the 2D data are loaded type pc2d. MAKE SURE YOU DO THIS IN EVERY 2D EXPERIMENT NUMBER. This runs a macro that sets some defaults for the PC.

ii) Transform if required

Basic processing is performed by the spectrometer workstation prior to transfer. Expert users may wish to investigate the edp command and reprocess. If the data are a few days old after they are transferred (slacker!) then the processed data may have been autodeleted. Reprocess using the xfbc macro.

iii) Reference your spectra

Start with the 1D spectra. Reference them correctly using the Calib button and middle mouse button. After referencing, type sr to display the spectral referencing parameter for that nucleus. Write this number (in Hz) down.

To reference the F2 (horizontal) dimension type in 2 sr then enter the appropriate number obtained earlier from the 1D spectra for that nucleus. In the basic experiments, F2 is always proton. To reference the F1 (vertical) dimension type in 1 sr then enter the appropriate number obtained for that nucleus. The F1 dimension is proton for COSY, NOESY and TOCSY experiments and carbon for HMQC and HMBC experiments.

iv) Define your plot region

The hardest part of the whole operation! To do this you need to use several mouse buttons:

Positive/negative levels:

Use to display positive (yellow/orange/red) or negative (blue/green) or both types of levels. Click with left mouse button to toggle through options. Usually want positive levels only for HMQC, HMBC and TOCSY spectra. Both levels for COSY and NOESY.

Set threshold:

Use to define the minimum peak height that will be plotted. Click and drag up and down with left mouse button. Sometimes it is necessary to reduce the number of colours to allow the threshold intensity to be increased (see below). The trick is to get all of your peaks above the threshold without getting any noise. Sometimes this is not possible and you will need to plot some t1 noise in order to see all your peaks.

Number of colours:

Use to change number of levels for the plot. Click and drag up and down with left mouse button.

Expand region:

Use to select region by drawing a box around it. First, click once with LEFT mouse button in the main data window where the spectrum is displayed. A cross hair appears. Move cross hair to bottom left corner and click MIDDLE button once. This locks one corner. Move active cross hair to the top right corner and click MIDDLE button once again. Next, click once more with LEFT mouse button in the main data window where the spectrum is displayed. This cancels cross hair selector. A box should be drawn around your desired region. Finally, click once with LEFT mouse button on the expand region button.

DefPlot:

Use to make currently displayed window the plotted window. Click once with left button. Answer y to change levels question. Enter the number of levels you want plotted up to the maximum indicated. (If you have problems plotting, keep this number small or better yet, see the manual method of defining contours in the troubleshooting section.) Answer y to contours question. The contours visible on the screen will now appear on the plot.

All:

Resets display to the whole spectrum. Click once with left button.

intensities/contours:

Switch display between intensities and contours. Use left mouse. Must be using intensities to use the set threshold and number of colours buttons.

v) Change the projections if required

Usually, the defaults will work OK!

For HMBC or HMQC experiments where you have no normal 1D, change the option to an internal projection. To do this type pf1ext then select positive from the list.

For HMBC or HMQC experiments where you do have a normal 1D to use as a projection, first ensure you have transferred the required 1D spectrum to the local D: drive. Next, define the 1D experiment parameters i.e., username (your 3 letter code, e.g., jab), name (e.g., Jun12jab) experiment number (e.g., 10) projection. To do this type edg then click the ed button next to EDPROJ1. Fill in these parameters, called PF1USER, PF1NAME and PF1EXP respectively and use the mouse to set PF1EXT to external(1r):

vi) Set the title

Use the setti command to edit the title with the notepad editor.

vii) Plot

Save a tree! Use the view command to preview your spectrum. If it looks OK type plot. If you want to make further modifications you can use the edg command in consultation with the Bruker manual. Experiment with plotting several expansions and different contour levels.

XWINPLOT

This is alternative WYSIWYG method of plotting data that many people like. See the Bruker manual.

Troubleshooting

Popup windows appear off the bottom of the screen: Move main XWINNMR window up and reduce size if necessary.

Plotting:

Error messages of the type "plotpf: Error During plot processing…….levels not correct continuation with next plot is tried" resulting in not all contours or parameters being plotted can be avoided using the manual method for defining contours. Use DefPlot button to define the plot window. Use the lev0 parameter to define your minimum threshold as a percentage of the highest peak, the nlev parameter to define the number of levels (default = 20), then execute the levcalc command. Experiment with different values of lev0 and nlev then check with view to make sure it looks good

Error messages of the type "....specified plotter not available....." are fixed using the edo command. Select an available plotter from the pull down menu of the CURPLOT parameter.

Error messages of the type "plotpf: Windows API: Open Printer: Open the specified printer. The printer name is invalid" The printer is set incorrectly and probably means the pc2d macro was not executed. Can be fixed using edo command.

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