Short Spectrometer Manuals

Short Spectrometer Manuals

These notes include a general introduction to Bruker TopSpin software, and a step-by-step guide to the acquisition and processing of 1D spectra. It is by no mean a replacement for the short training course each user is obliged to take.

The Linux/UNIX operating system

The new spectrometers make use of the Linux operating system. It is a multi-user system, which means that each user has his own ‘account’ and is required to logon to the instrument using its own user name ( userid ). Each userid is password protected, so that only you should be able to logon to your account. The concept of user accounts is a very important one in Unix systems as this defines who created and "owns" the files, as well as where they are to be found and who has permission to alter and/or delete them. The NMR data sets you create (measure) are stored in directories and sub-directories, and each spectrometer user has his own directory in which his FID’s and spectra are to be found. For example, any data created by userid
‘tali ’ will be stored on the disk under the directory: /DISK/data/tali/nmr

(where /DISK specifies the particular disk partition on which the data is kept, e.g. /opt/topspin/data/tali/nmr).

Another important point is that Linux commands are case sensitive , and this therefore applies to commands you may use in TopSpin. Thus ft and FT will not be the same. Lowercase commands should be used at all times in TopSpin.
In the description on the following pages, text you should type is shown in bold italic, and responses from the system are shown in bold .
Getting started & Exiting: You should start at a monitor with the login screen. If this is not the case, exit (logout) as described later. Double click on your userid icon, and enter your password. Open a Unix shell and type topspin to start the program. To exit the program when you finished your session, type exit and confirm. This will return you to a Unix shell. To logout from Unix, use the right mouse button.

Interacting with the TopSpin program

Interaction with the TopSpin program is either by clicking on the icons with the mouse, by selecting a command from the pull-down menus or via keyboard commands (typing the same commands available in the pull-down menus). [A list of common Bruker commands and some basic nomenclature is available under the Instruction Manuals & FAQ section]. In addition, the mouse (left, right and middle) is used to interact with the spectrum directly. The basic TopSpin display (for 1d spectra) which you will usually get when you run the program is shown in the figure below. Other windows, which display other features (lock display, temperature control etc) may also appear at various stages. The main areas of the Main Display shown below include:

  • Pull-down menus, at the top, that contains the File, Acquire, Process, Analysis, Display, Window and the Help pull-down menus.
  • Identifier, which is the text that shows the data set currently in use, in the format of data set name (NAME e.g. btx1_g7_303_8), experiment number (EXPNO e.g. 1), processing number (PROCNO e.g. 2), disk partition (DISK e.g. /opt) and user name (USER e.g. tali).
  • Spectral window, the area used to display the FID and the spectra.
  • Icon panels (at the left), controls the display of the FID and the spectra at the spectral window (the size, range etc), as well as some additional subroutines.
  • Command line, just below the spectral window, where you could type the commands (starts with a >).
  • Information panel (below the command line), at which the current process or the development of the process is being described (e.g. fp: finished).

Data Acquisition

  • Defining data sets

    All data generated by the spectrometer are automatically saved to disk and stored in your directory. Therefore it is always necessary to define a name of your data before doing anything else; this should always be the first thing you do when you start a session on the spectrometer. There is a real chance of you overwriting a previous data set if you fail to do this. To define your new data set, type the command edc and complete the six variable entries (data set name, experiment number, processing number, disk partition, user name and the data type that is obviously NMR).

  • Reading and editing parameters

    Parameters are stored in four separate categories for acquisition, processing, plotting and output devices. All four may be loaded simultaneously with the rpar command. This will display a table of available parameter sets through which you may scroll and select your desired parameters. [Note that different probes may have e.g. different protons parameters so there is no universal parameter set for proton acquisition]. The second table asks which of the four parameter groups you wish to load; it’s better to Copy All .
    Very important information included in the parameter sets defines the routing of the rf through the spectrometer. Thus, it is not necessary to change the hardware set up, such as cables and pre-amplifier boxes, when you change experiment or nucleus. Thus, it is possible to change the rf routing via the command edasp. [Try to avoid using it as a new user].
    The parameters can be edited with the commands eda (edit acquisition), edp (edit processing), edg (edit graphics i.e. plotting), and edo (edit output device i.e. plotter). At this point it is necessary to point out two different parameter classes: “Set parameters”, are those you enter for use during acquisition, processing etc and can be viewed with eda, edp, edg etc. “Status parameters” that actually relate to the data already acquired or processed and can be seen only with dpa dpp , or dpg . For example, suppose you were to set initial parameters in eda to acquire 64 scans, but you halted acquisition after only 32 scans. The eda table will still indicate 64 scans, as would the parameter ns (number of scans) although this is clearly incorrect for the data you have acquired. However, the dpa table would show ns = 32, the actual number of scans for the data set.

  • Setting the temperature

    within the probe is done using the edte command. It is highly recommended to set the temperature before you insert your sample.

  • Important acquisition parameters

    In order to interact with data acquisition you should go from the main display (used for interacting with processed data) to a similar display for acquisition by selecting from the pull-down menu Acquire -> observe FID window (in most of the spectrometer, the alias a given from the command line, exist). The return icon will always take you back to the main display. The pulse program you will be using contains a list of commands regarding rf pulses, delays etc. The name of the pulse program, as well as of the different acquisition parameters can be edited using the eda command. A reduced list of parameters to be used by the selected pulse program is obtained via the ased command.

    • PULPROG- the pulse program you wish to run (e.g. zg, zgpr, noesygrtp19 etc).
    • D1- the relaxation delay between scans (sec).
    • P1- the 90 deg pulse length, hard pulse (µsec).
    • PL1- the power level, the power attenuation of the transmitter pulse (in dB).
    • SW- the spectral width (ppm). Typically, in the range of 10-12 ppm for 1.
    • SFO1- the transmitter frequency in MHz. Could be set by setting the frequency at the center of the spectral window (O1, in Hz). For aqueous samples, it is usually set on the water resonance.
    • TD- the time domain data size. Typically 16-32k points.
    • NS- the number of scans to be acquired.
    • DS- the number of dummy scans before data is collected, to obtain steady-state (2-16).
    • RG- the receiver gain, could be set automatically by the rga command.
       

  •  Locking and Shimming

    To see the lock display go in the pull-down menu: Windows -> lock display. If you have changed the probe, the solvent or the type of sample tube, you have to recall an appropriate set of shim parameters by typing rsh. A list of saved shim files will appear from which you should select the appropriate one. A digital lock system is used on most of the instruments. To lock on the deuterium signal of your solvent, type lock and select your solvent from the list. The lock power, gain and phase should be set correctly automatically. The shim file you have recalled using rsh will not update them, but you can still view them using the vish command. Shimming is done via the shim keyboard located near the workstation keyboard. Further information regarding  shimming procedures can be found under the Instruction Manuals & FAQ section.

  • Tuning the probe

    Prior to acquiring the data, the probe must be tuned and matched to optimize its performance at the frequency of the studied nucleus (defined via eda or edasp ) and to maximize the power that is transmitted to the coil. To start the procedure type wobb (in most instruments you can use the alias ) and after ~30 seconds enter the acquisition display ( acqu ). Approach the probe, and using the tuning and matching rods or sliders tune and match the probe. The results are shown both on the LED display of the preamp as well as in the acquisition submenu of TopSpin. A well tune probe will show a signal exactly at the center, with its minima (dip) as deep as possible; a minimum number of illuminated LEDs will further support the quality of the wobbling. When tuning is complete, click on the stop icon, type ii (initial interface) and wait for the routine to finish. When tuning and matching a probe with multiple resonant circuits, it is best to tune and match the lowest frequency circuit first. Thus when wobbling a probe for both 1H and 13C, first do the 13C and then adjust the 1H. When tuning and matching a nucleus that its routing does not go through the preamp, it is required to physically change the cable connections so it would pass through the preamp. Please ask the NMR staff is you are not sure how to do it. After tuning & matching the probe, check once more if further improvement in the shimming is possible.

  • Calibrating the pulses

    . In case you want to calibrate your pulses, or use previously-calibrated values, please check the     Pulse Calibration Procedures , as well as the Calibrated Pulse-Length Values .

  • Acquiring the data

    To start data acquisition, enter zg. To see the acquired FID enter the acquisition display (acqu). As long as the acquisition is in progress, information regarding the number of scans, the actual number of scans that have already been acquired and the time remaining till the end of the experiment, will appear on the screen. To terminate acquisition immediately use the stop command, but be aware that it will not save your data (which will be lost). To stop acquisition and view the scans acquired so far, use halt , which will write your data at the end of the current scan and allow processing. If you want to view the data acquired till now, without terminating the acquisition process, use the transfer command, tr . This forces the data from the computer memory (where it is held during the acquisition) onto the disk. Go back to the Main Display by pressing the return icon.

  • Important note

    When leaving a long experiment to run, NEVER leave it on the acquisition window- ALWAYS return to the main menu.
    Procedures for calibrating pulse lengths can be found under Instruction Manuals& FAQ.

Data Processing

Once you have your data on disk, you may process it and interact with the result using the Main Display. The parameters that define the processing may be edited with the edp command that will open them in a table.

  • Zero Filling (ZF). In case no zero filling is required, the size of the spectrum (SI) should be set to half the size of the time domain data, the FID (TD). Setting SI=TD means that zero filling will occur.

  • Window Functions. In order to increase sensitivity or resolution of the spectrum, we often apply a suitable window function to the FID before the Fourier Transformation. Sensitivity enhancement is done by multiplying the FID with an exponential weighting function, which forces the end of the FID towards zero. This exponential multiplication, done via the command em (prior to the ft ), improves the S/N ratio, but leads to some line broadening as well. The sensitivity enhancement is controlled by the parameter lb which defines the resulted line broadening, in Hz. [It is possible to use the ef, which does the two operations ( em + ft ) consecutively]. For resolution enhancement, one can use the sine-square window, qsin , along with the parameter ssb that defines its shape. One can adjust the parameters for window functions interactively, using the menu Process -> Manual window adjust .

  • Fourier Transformation (FT). To produce a spectrum (frequency domain data) from the time domain data (FID), the raw data must be Fourier transform by the command ft. The spectrum is saved with the processing number defined with edc, and the raw FID also remained (unchanged) on the disk, and could be viewed using the FID icon.

  • Phase Correction. After the FT the spectrum does not usually appear normally, it is not a pure absorption spectrum. Zero (non-frequency dependent) and first order (frequency dependent) phase-distortion appears. These may be corrected automatically or manually. The automatic correction is done via apk. The manual correction is done by clicking on the phase button. Usually, one selects the largest peak to define zero-order phase correction by clicking biggest . Now position the cursor over the PH0 icon, press the left mouse and drag to correct the phase of the selected peak (dotted vertical line). When done, repeat the procedure for the PH1 icon and correct all other peaks in the spectrum. In order to terminate the process, click on the return icon, save and return to store the new phase. Spectra can be processed identically afterwards using the command pk, or fp that combines ft + pk . [In cases you want to define another peak- not the largest, manually, define it with the cursor icon, or do it manually without definition].

  • Spectrum Calibration. Defining the origin of the spectrum can be done automatically using the menu Analysis -> sref. Manual calibration is done using the calibration icon, and selecting the top of the reference peak with the middle mouse. Type then the desired reference valuein ppm, in the popping window.

  • Base-Line Correction. Distortion in the base line is observed when the noise in the signal-free regions of the spectrum are not scattered around the zero line, but around a polynomial or some other curve. Automatic base line correction is done with the abs that subtracts a polynomial from the spectrum. The manual correction is done using the menus Process -> Special Processing -> Baseline correction . Five polynomial functions (A to E) should be adjusted, by clicking and dragging on each. Start with A (linear offset) and go through till E. When suitable fit has been found, click on diff to see the result. To save values use return , then save and return . [You can do FID base line correction prior to the window function and FT, by using the command bc].

  • Integration. To enter the manual integration routine click on the integrate icon. Place the cursor over the spectrum and click with the left mouse button: the cursor will slide along the spectrum (left mouse terminates this). Go along the spectrum and use the middle mouse to mark the ‘start’ and ‘finish’ of each integral region, after which each new trace is displayed. The icon calibrate can be used to give a numerical value to a specific (current) integral trace, while all others will be scaled accordingly, so the figures will not have an arbitrary value. To define the ‘current/active’ integral trace, click on the left mouse, moves the cursor to under the required trace and click the left button again. An arrow will indicate that this is the ‘current/active’ trace, and all buttons under current will apply to this trace. Now calibrate this trace. Integrals can be saved via return, and save as ‘intrng’ & return . The list of integrals can be printed with li (or may be included in the plot of the spectrum).

  • Peak Picking. In order to avoid picking noises, it is recommended to set the minimum threshold for the picking procedure. First ensure that the Y-axis units are in centimeters (use the Y icon). Select the utilities button and click on MI. Move the white horizontal line and define the minimum level (MI in cm) using the left mouse. The maximum cut off may also be defined using the MAXI button and repeating the procedure. To view all peaks you have picked on screen, type pps (and plot them using the print button).

Other Useful Tasks

  • Title. A title may be set using the command setti (use the alias t).
  • Dual display of two spectra: use the command edc2 to define the second data set. Press the icon dual to display the current and the ‘second’ data set, one on the top of the other.

A brief summary of the steps

  1. Login to the system (your account)
  2. Run topspin
  3. Define your data set using edc (read appropriate parameters if required using rpar)
  4. Set the temperature within the probe (edte)
  5. Insert your sample
  6. Recall a shim file (rsh), lock and shim (BSMS keyboard)
  7. Tune and match the probe (wobb or atmm)
  8. Calibrate the pulses if required
  9. Set acquisition parameters (eda)
  10. Acquire the data (zg)
  11. Process the FID (window, ft, bc, peak picking, integr etc).