NMR Spectrometer : Standard Operating Procedure

By: Pharma Tips | Views: 4590 | Date: 28-Nov-2013

General Procedures For All Spectra,Loading the Sample,Locking the Instrument,Running a 1H Spectrum,Acquiring the Spectrum,Transforming the Data,Manipulating the spectrum,Setting the Reference,Changing SW and O1 for Increased Resolution, Peak Picking,Plotting the Spectrum

Operating procedure for NMR spectrometer : Bruker Avance DPX-300 NMR spectrometer


Unless otherwise specified, statements such as “click on calib” mean to move the cursor on top of the calib button on the screen, and click with the left-hand mouse button. Consider the left-hand mouse button the default; if another button is required, it will be explicitly stated. 

Commands such as “Enter zg” mean to type zg followed by a return on the
keyboard. Commands must be followed by <return> before they are accepted.

Words written in bold Arial font, e.g., acqu, refer to buttons or pull down menus on the screen (items that can be selected with the mouse).  Multilevel menu choices written like this, Windows/lock, mean choose the first menu choice by left clicking with the mouse.  Choose the second menu choice from the menu that appears when the first choice is made.

Words written in bold Courier font, e.g., edsp, refer to commands that can be typed at the keyboard.  Commands must be followed by <return> before they are accepted.

General Procedures For All Spectra

 1.  Preliminary Notes

A.     This manual is a condensed and incomplete account of instrument usage.  It is in no way a substitute for reading the detailed instruction manuals available in the NMR room (M-02).  Please get to know them. This manual is written in a similar format to the Tufts AM-300 manual, to facilitate the transition from the AM to the DPX.

B.      All users should sign the logbook before they do anything else.

C.      Log into the computer.  The screen may be illuminated by hitting any key on the keyboard.  You should see a Welcome to IRIS window.  Enter your user name and password.  When the UNIX shell window appears, type xwinnmr.  The XWIN-NMR window will appear, as shown below.

D.  Only the active window will accept typing.  To make window active, position the mouse cursor inside it.  To bring a window to the front, left click in its title bar.

E.  Most commands are accessible from the menu bar.  Alternatively, any command may be typed into the command line below the spectrum window.  Most commands are the same as the AM-300.

F.  Parameters may be changed by typing the abbreviation for that parameter followed by the (return) key (hereafter <rtn>.  The computer will respond by popping a window with the current value of that parameter.  Typing the new value of the parameter followed <rtn>by will replace the old value with the new value.  Alternatively, groups of parameters can be edited with various ed commands.  For example, eda presents a window of all acquisition parameters, edp presents a window of all processing parameters.

G.  Sample control is provided through the BSMS (Bruker Smart Magnet System) control panel.  To access the BSMS Panel choose Windows/BSMS Panel from the menu. Lock, Shim, and Sample panels are similar in appearance to the hardware buttons on the AM-300, except control is through a slider bar and +/- button, rather than through the knob.  For all buttons the left mouse button decreases the value, the middle button increases.  Step size is changed by left (decrease) or right (increase)clicking on the :2/*2 button

2.  Loading the Sample

 A.  The probe should contain a sample at all times.  When you arrive at the instrument with your research sample, you should remove the standard sample from the magnet and replace it with your sample as follows:

From the XWIN-NMR menu choose Windows/BSMS panel.  Then click on the Shim button

-Click on the LOCK button to unlock the instrument.  The color of the button will change from yellow to gray

- Click on the SPIN button to stop the spinning of the sample The color of the button will change from yellow to gray

- Click on the  LIFT button.  The standard sample should rise out of the probe on a stream of air. Remove the standard sample from the top of the probe. Remove the standard from the spinner.

-Place your sample tube in the appropriately sized spinner and adjust the precise depth of the tube within the spinner by using the depth gauge.  Note that the depth gauge should be set at 10mm regardless of the tube size for a 10 mm probe. (and at 5 mm gauge for a 5 mm probe).  Note: 10 mm tubes cannot be run in a  5 mm probe. For best shimming set tube 1mm below the appropriate line to assure a uniform sample in the active region of the probe.

- place your sample in the top of the probe. Do not release the sample unless you are sure it is supported on the air current.

- Click on the LIFT button again.  Your sample should slowly lower into the tube as the air stream decreases. Click on the SPIN button.

B.  The rate of the sample spinning should be 20-25 rps.  To check this:

-Press the Sample  button on the BSMS panel.  Then click on the SPIN MEAS button on the Sample panel that pops up.  To change the spin rate, click on the SPIN RATE button, and usethe slider bar to change the value.

3.  Locking the Instrument

 A.  To display a swept lock signal, choose Windows/lock from the XWIN-NMR menu. You should now see a continuous wave deuterium NMR spectrum of your solvent.  This should be a trace across the screen with the signal in the middle of it.

B.  To lock the sample, type lock, then click on the solvent from the list that pops up.  Lock power, phase, and gain will be adjusted automatically.

C.  The lock can also be adjusted manually, using the buttons on the Lock panel from the BSMS panel.   Note: Appropriate power and gain levels are different from the AM-300.

 4.  Shimming the Magnet
 A.  To obtain a high resolution spectrum it is essential that all parts of the sample are exposed to the same magnetic field.  The homogeneity of the field is generally assessed by noting the intensity of the lock signal, the more intense the signal the better the field.  In practice, the shims most likely to require adjustment are the spinning ones, namely Z Z2 and Z3 shims, and the non-spinning shims X and Y.  To adjust the homogeneity by hand (often adequate for routine spectra and almost always faster than by automated computer shimming) you must first make sure that the lock trace is visible.  If it has gone off the screen during the locking procedure it can be brought back by lowering the LOCK GAIN level.  A sample procedure follows:

- Click on the LOCK GAIN button. Use the +/- button to adjust the lock gain until the trace is in the top two squares of the grid. Press the Z button. Use the +/- button to maximize the lock signal.  Change the step size if needed.  Repeat for the Z2 button. Continue to alternate between the these buttons maximizing the lock signal each time until no further improvement can be seen. You may have to adjust the Lock Gain if the trace goes off the screen.
-If the sample must be shimmed to highest standards, adjust the non-spinning shims by pressing the SPIN button and waiting until the sample stops spinning. Spin rate can be used to monitor this. Raise the lock level by adjusting the lock  gain until the level is in the upper half of the screen. Now press the X button and use the +/- button to adjust the lock level to maximum. Then press the Y button and repeat this procedure until no further improvement is possible. Start the sample spinning again by pressing the SPIN button and repeat the Z and Z2 shimming as above.

Running a 1H Spectrum

 5.  Setting the Parameters
 A.  From the XWIN-NMR menu choose File/New. A edc window will appear containing the file name parameters.  Enter a new NAME. Generally only the Name parameter needs to be changed. NOTE:  The first time you use XWIN-NMR you will need to change other parameters to EXPNO=1, PROCNO=1, DU=datanmr, User=<username>. Click on SAVE

B.  Data files containing typical parameters for a proton spectrum are stored in the hard disk for easy access.  To retrieve these:

Choose File/Copy/parameters from file from the menu, or type rpar. A list of standard parameter files will appear.  Use the scroll bar to access the entire list.  Click on proton. In the next window that pops up, click on Copy All.

Type eda to edit acquisition parametersAlways change Prosol to True by clicking on its value, which loads all default pulse lengths and also check that the solvent is correctly identified, otherwise calibration will be off. IMPORTANT:  Failure to set Prosol to True will result in all pulse lengths not set by you will be of zero length and rga will go off scale trying to find a signal.

Change any other acquisition parameters desired, then click on SAVE. Acquisition,  Ones you might want to change are
TD (time domain size)-Defines the number of data points of the fid.
NS (number of scans)
P1 (pulse length in microseconds)
SW, SWH (sweep width in ppm, Hz)
AQ (acquisition time in seconds)
RG (receiver gain)

B.  You may wish to tune the probe before beginning acquisition.  You can see this on another post Probe tuning.

C.  The last parameter to be set is the receiver gain (rg) which adjusts the gain of the signal coming out of the probe.  This will vary from sample to sample.  To set this gain type: rga (receiver gain adjust). The computer will automatically take a few sample pulses and alter the RG between each.  When the best value is found, the computer will type rga: finished.  You may see this value which the computer has selected by typing: rg. If you want this value then type: <rtn>.  If you wish to select another number then type in the new number  and then type <rtn>. Do not increase the value, but you may decrease it.

6.  Acquiring the Spectrum

 A.  To start the acquisition type zg to zero the memory and go (start the acquisition).  You can watch the progress of the acquisition by choosing Acquire/observe fid window from the menu, or by typing acqu.

B.  The acquisition will automatically end when the number of scans taken equals ns.  However if you wish to stop it prematurely or abort the run then you will have to click on the STOP button to the left of the FID in the acquisition window.

7.  Transforming the Data

 A. A complete description of processing parameters and their meaning can be found in the  XWIN-NMR Software 

BThe time domain data can be processed by typing bc, then em, then ft (or type ef to do all three at once).

C.  It is not necessary to wait for the acquisition to end before transforming the data.  Type tr to transfer the data to disk at any time during the acquisition.  Once written to disk, the data can be transformed.  Time domain (fid) and frequency domain (spectrum) data are always stored in separate files. All data acquired so for can be processed while the acquisition continues in the background.

8.  Manipulating the spectrum

 A.  The spectrum can be expanded vertically and horizontally using the mouse, clicking on the buttons to the left of the spectrum.  *2, /2, *8, /8 multiply or divide the vertical scale by 2 or 8.  To expand the horizontal scale, left click anywhere in the spectral window. Then middle click on the desired expansion limits.  Left click again to unlock the mouse from the expansion.  To expand the vertical scale by an arbitrary amount, left click and hold the double arrow button to the right of /8.  Move the mouse vertically until the peaks are desired height.  Release the mouse button.  

 9.  Phasing the Spectrum
 A.  The spectrum will usually not have all the signals upright in an absorption phase. To correct this type apk (automatic phase correction).  For spectra with well separated narrow lines, apk will usually work well.

B.  If the automatic phase correction does not work well, click on the phase button to the left of the spectrum, then click on biggest.  A marker will appear under the biggest peak in the spectrum. Left click and hold the PH0 button.  Move the mouse vertically until the largest peak is phased correctly.  Release the mouse button and click and hold the PH1 button. .  Move the mouse vertically until the other peaks are phased correctly.  Release the mouse button.

 10.  Setting the Reference

 A.  The spectrometer cannot provide an exact chemical shift without being calibrated to a standard (usually TMS).  Click on the calibrate button to the left of the spectrum.  A small arrow will appear on the spectrum.  move the arrow to the reference peak with the mouse, and click the middle mouse button.  A window will appear with the current frequency value.  Enter the reference frequency. A chart of chemical shift values for solvents is on the console.

11.  Changing SW and O1 for Increased Resolution

The default parameters display the spectral range 16 to –4 ppm.  This range can be changed for greater resolution.  Click the left mouse button somewhere in the spectral window to tie the cursor to the spectrum. Position the cursor at the left limit of the desired spectral width. Click the middle mouse button to set a marker at this frequency. Move the cursor to the desired right hand limit and click the middle mouse button to expand the spectrum. The expanded region now appears in the window. Click the left mouse button to release the cursor from the spectrum. Click on sw-sfo1 while the expanded region is displayed. This adjusts sw so that it has the same value as the expanded region and also adjusts o1 (and thus sfo1) so that the carrier frequency lies in the center of the expanded region. (You can verify these changes by checking the eda table.) Notice that by reducing the spectral width, the acquisition time aq is increased while the parameter fidres is reduced. Finally, now that the acquisition parameters are optimized, it is a good idea to repeat the automatic receiver gain adjustment (rga).  Notice that since the spectral width has been changed, it may be necessary to readjust the phase correction.
 12.  Integrating the Spectrum
 A.  It is often useful to integrate 1H spectra. The simplest way to define the integral range is by entering abs. The command abs performs an automatic baseline correction and also automatically defines the integral ranges.  The integrals will not appear on the screen, but will appear on the plot.

B.  To display the integral on the screen:

Choose Analysis/Manual integration from the menu, or simply click on the integrate button of the button panel at the left side of the XWIN-NMR window.  Additional buttons, like those that control vertical and horizontal scale for the spectrum, will appear in the lower left.  The upper part of the button panel is identical to the standard layout, and allows you to shift and scale the integral data on screen. In addition, there are three special sections headed by current:, all:, and mouse:. The command buttons in these sections work on the current integral marked by the user, on all integrals on the screen, and on the mouse sensitivity, respectively.

Defining integration regions
Move the cursor into the data area of the XWIN-NMR window. Click the left mouse button. The cursor is now bound to the spectrum, and moves along the spectrum trace when you move the mouse. It can be released from there by clicking the left button again. Clicking the middle button will mark the current position (the mark can be removed using the right button). Clicking the middle button a second time at a different cursor position will define the area between the mark and the current cursor position as the integration region, and the corresponding integral trace is displayed along with the value of the area under the integral. By default, the first integral region defined will be assigned the value 1.0. This procedure can be continued for all desired regions, and need not proceed left to right. Click the left button to release the cursor from the spectrum when you are finished.

In order to mark one of the defined integrals as current integral, move the cursor into the data area of the XWIN-NMR window. Click the left mouse button. The cursor is now bound to the spectrum, and moves along the spectrum trace when you move the mouse. Select the integral you want to make the current integral by moving the cursor under it, and release the cursor by clicking the left button again. The integral will be marked with an asterisk. All button panel commands in the section current: can now be applied to this (and only this) integral.

To calibrate the integrals, mark an integral as the current one, then click on calibrate.  Enter the integral value in the dialog box.

When all integral regions have been defined, click on return. Click on save as ‘intrng’ and return to save the regions to a disk file.

13.  Peak Picking

The most straightforward way to produce a peak list is through the command line.  First display the Y axis in cm, if it is not already displayed.  The Y button to the left of the spectrum window will toggle the y-axis, the YU button will change the units from cm to absolute.   Choose the region for peak picking by setting the parameters f1p and f2p to the ppm values for the left and right limit, respectively. (If the x-axis is in Hz, use f1, f2 instead of f1p, f2p.) Set the parameter mi to the desired minimum intensity in cm.  Type edo and check that CURPRIN is set to hplj5l. The command pp will produce a peak listing on the printer.  Use pps to print to the screen.

14. Plotting the Spectrum

A straightforward way to plot 1D spectra is by using most of the plotting parameters found in the plot parameter file standard1D. Read in the file by choosing File/Copy/parameters from file from the menu, selecting standard1D from the menu of parameter file names, and then selecting plot from the menu of parameter file types that appears.  Then click on Copy. This sets the plotting parameters to values appropriate for most 1D spectra.  1D and 2D Plotting Parameters’. For basic 1D spectra no changes need to be made within the parameter menu edg itself; however, the spectral region and the integral range must be defined, and the spectrum title must be written. To select the spectral region (full or expanded) to be plotted, first make sure the spectrum appears as desired on the screen, and then click DP1and simply hit return in response to the following three (3) questions:

F1 = <return>
F2 = <return>
Change y-scaling on display according to PSCAL?<return>

You may change the values of F1 and F2 if you wish.

Other plot parameters can be changed by typing edg (edit graphics) and changing the values, mostly yes/no, which determine whether integrals, parameters, etc appear on the plot. The edg window is analogous to the DPO command on the AM-300. Some plot features have a button labeled ed.  Clicking on this button will open another window, with parameters that control the position and style of these features.  If simply editing the yes/no parameters in the edg dialog box will not give the plot you want, it is usually easier to use XWIN-PLOT, rather than to change other plot parameters.  XWIN-PLOT is a graphical plot layout program.

Next create a title for the spectrum. Enter setti to use the editor to open the title file. Write a title and save the file.  The title must end with a <return>.

To preview the plot, as it will appear on paper, type view.

To plot the spectrum, type plot (provided the correct plotter is selected in
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