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Basic VnmrJ Manual for version 2.x - TAMU NMR Facility

1. complete printoff will close the output file and queue it to the printer device NMR USER S MANUAL November 9 2009 27 10 DATA STORAGE There are several data storage options available on the NMR workstations There is sufficient hard disk space available that every user account has its own disk space that may be used for routine data storage Quotas are enabled on the systems so that a few users cannot inadvertently fill all of the available disk space Users typically have a 100 Megabyte quota which can store quite a few spectra Even 2D data sets will typically occupy only 5 8 Meg of disk space This is enough space to work easily but small enough to be managable when it gets full If you have 100 Meg of space filled with data sets occupying less than 100K each this represents more than 1000 spectra Eventually you will have to archive or move data from this primary work area to someplace for long term storage The more often you do this the easier it will be It can be a very daunting problem to sort through several hundred data sets try to figure out what they are and where or if to store them Sorting your data into subdirectories as you collect them could pay real dividends later Regardless of which method or combination of methods you choose to use to archive your data verify that you can take your archived data and move it back to the workstation for further processing and plotting An archived data set that you canno
2. and weighting parameters for data processing Table 2 shows some of these parameters and the parameter names used for the various axes Everything for the third dimension and above is consistent with the numeric portion simply getting incremented for each additional dimension There is a Parameter Direct Detect Axis 1 Indirect Axis 2 Indirect Axis Frequency designation f2 fl f3 Time period t2 tl t3 Incremented delay time real time d2 d3 Sweep Width sw swl sw2 Number of Points np real ni complex ni2 complex Fourier Number fn fni fn2 Acquisition time at np 2 sw ni swl ni2 sw2 Line broadening Ib Ib1 162 Processing type proc procl proc2 Table 2 Multidimensional NMR nomenclature difference worth noting in the number of points collected In the directly detected dimension np is the number of real data points used in memory Since we are always collecting complex data points for quadrature detection half of these points are the real portion and half are the complex portion and we are collecting np 2 complex pairs In the indirectly detected dimensions ni and its successors indicates the number of discrete time points that are taken in the indirect dimension Typically for phase sensitive and quadrature detection complex points are required so two spectra will be taken at each value of ni with different phase cycles for each Consequently ni represents complex points already For a square data table in an auto correlated expe
3. enhancement is only enhancing resolution that was already present in the data set It cannot get resolution out of a poor sample or an improperly collected data set There is an interactive weighting command wti that can be very useful in visualizing and setting up weighting functions It displays the raw FID data the weighting function s and the transformed data and permits adjustment and selection of the weighting functions 6 4 FOURIER TRANSFORMATION There is really very little operator control over the fourier transform itself The only parameter affecting the fourier transform operation is the fourier number fn which defines how many data points will be transformed Increasing the fn past the number of points actually collected will result in more plotted points and perhaps a somewhat smoother spectrum but it will not increase the resolution of the data set which is defined by the acquisition time Transforming more points than are in the data table called zero filling is accomplished by adding zeros to the end of the data table which adds no information to the data T PLOTTING AND DISPLAY Having obtained a spectrum plotting and display are concerned with displaying the data in a usable manner and obtaining a hardcopy print of the data Generally there is a very good correlation between what you see on the screen and what will be plotted on the plotter The most obvious exception is simply in the area of resolution the screen displa
4. observe receiver and ADC overflow indicates clipping in the analog to digital converter Both of these conditions are errors that will introduce artifacts into your spectrum See gain section of the arrayed experiment discussion section 13 on page34 for more information on setting the receiver gain 3 INTERLOCK FAILURE and VT FAILURE These errors are interlocks designed and intended to stop data acquisition if a certain condition is lost The interlock failure indicates that the deuterium field frequency lock has been lost and VT failure indicates that the variable temperature unit is no longer regulating temperature These errors can be turned off by using inz n lock interlock and tinz n temperature interlock although normal practice is to leave them turned on Continued data acquisition after the error occurs will generally degrade the data already collected before the error occurred 4 AUTOGAIN FAILURE This indicates that the acquisition processor was unable to automatically adjust the gain The usual cause is running a proton sample that is too concentrated The system reduces the gain to zero the signal still overflows the analog to digital converter and generates an autogain failure error Two possible solutions are to reduce the excitation pulse width PW and to dilute the sample 5 AUTOLOCK FAILURE AUTOSHIM FAILURE Either the computerized locking or shimming failed These will generally be caused by a lock s
5. system measured intensities and plotting them manually at mid points of the acquisition periods Also valid are kinis and kinds to display the short form report 15 TWO DIMENSIONAL 2D NMR Two dimensional NMR is in many respects a logical extension of arrayed experiments A series of spectra are collected incrementing a time period in the pulse sequence for each spectrum The pulse sequence is written so that the peak intensities of the normal spectrum vary in either amplitude or phase as a function of the arrayed time period and some spectroscopic quantity of interest usually either a chemical shift or a coupling constant After the first normal fourier transform with a series of peaks of varying intensity the data table is conceptually rotated 90 and a second transform is performed to extract the frequency information from the peak modulation This generates a spectrum in two frequency dimensions one being the normal or directly detected dimension and the second being the indirectly detected dimension created by the time delays in the pulse sequence The directly detected axis of a 2D spectrum is ALWAYS the chemical shift of the directly detected nucleus normally proton in most current experiments The indirectly detected axis of a 2D spectrum is generally either a chemical shift axis or a coupling constant axis depending on the experiment being performed Again depending on the experiment a chemical shift axis could be the same as the o
6. unmount it Log in on the workstation and insert either a USB device in a USB port or a recordable CD in the CD R W drive You should get an icon on your desktop for that device In the case of a blank CD it is a generic Blank CD R Disc icon and in the case of a USB device it will have the manufacturers name for that device In either case you can double click on the icon to open the device if it did not open automatically The blank CD folder will be labelled as CD DVD Creator Folder and will have a grayed out Write to Disc button Once you copy data to that folder the button is no longer grayed out If the USB device does not automatically mount there may be a problem with the Linux daemon that does the aotomounting In this case remove the device from the USB port and run the command su fixusb in a terminal window This will stop and restart the appropriate daemon and you can then re insert the USB device It should mount at that point You then open your file manager the desktop folder named lt username gt home navigate that folder to the files that you want to backup and simply drag those files to the destination using the left mouse button You can create folder on the destination if you want to sort and organize your data while archiving it Once you have moved your data to the destination device you need to unmount and remove it In the case of the recordable CD disk click on the Write to Disc button to actually wri
7. Amrdata directory simply open a teminal window and do an IS of the pertinent directory to get it mounted If you want to archive entire directories you can do so from the xcdroast program directly If you want to pick and choose the data to archive it may be easier to create a new directory with only the files you want to archive This can include subdirectory structure to help organize the data There is a disk partition available as home1 scratch that may be used to collect the data that you want to archive Once you have decided on the data to be archived inset your CD R disk in the drive and start the CD burner software This is available on the System Tools menu of the Applications tab or it can be run from the command line with the XCdroast amp command The ampersand amp is important because it will run the commands in background leaving the Linux window available for running other commands The first time you run Xcdroast it will complain about the lack of a configuration file Enter the Setup section and go to the HD Settings tab At the bottom of the tab enter home1 images in the path window and click the Add button to add that path to the list of storage directories select Save Configuration to create a configuration file and Ok to exit the configuration panel This configuration file will be saved for subsequent invokations of the X CD Roast program The opening screen gives four menu buttons Setup Duplicate Create as
8. First users are required to be formally instructed on the instrument and obtain a username and password in order to operate the instrument Operators without adequate instruction are more likely to make mistakes that lead to instrument down time which creates a hardship for all the other instrument operators and they are also more likely to waste instrument time Sharing of passwords is not permitted and users should not give their password to others for any reason Improperly used passwords will be removed from the system Users are NMR USER S MANUAL November 9 2009 7 encouraged to use the passwd command as often as necessary to keep their passwords secure Also the security system is used to keep track of time used and generate monthly charges If someone else is using your password your research advisor is paying his bills The other form of security is more pragmatic Since all the magnets are quite vulnerable to damage and it is impossible to keep them totally isolated the doors to all the labs are kept locked at all times There are plastic chain barriers surrounding the magnets in order to provide a visual reminder to everyone who comes into the lab that they should not approach too closely and should not bring any iron near the magnets This is especially true of physical plant workers and the custodial staff who have no reason to know the nature of the magnets THUS IT IS IMPERATIVE THAT EACH AND EVERY USER BE CAREFUL TO REPLACE
9. MESSAGES 17 APPENDICES i 26 27 27 27 28 28 28 29 30 30 31 31 32 35 35 36 38 40 LIST OF FIGURES Annotated VnmrJ Screen Display Figure 1 Interactive Lock Display Figure 2 Shim Parameter Panel Figure 3 Integration Panel Figure 4 Parameter Array Window LIST OF TABLES Table 1 Representative Carbon 13 T1 relaxation times Table 2 Multidimensional NMR nomenclature NMR Solvent Information iii inside front cover 15 19 25 33 8 37 inside back cover FOREWORD This document is intended as both an instructional text for users new to the Texas A amp M University Department of Chemistry NMR facility and as a reference manual to be used routinely during normal operation of the instrument after formal instruction is complete Consequently it includes both material that is theoretical concerning why something is done as well as material that is pragmatic concerning how it is done It also contains material that duplicates information already available in the Varian manual While this volume is a good overview and start on understanding the Varian software the on line documentation is much more extensive and complete You should refer to that documentation for more complete descriptions of the various commands and parameters and the latest information The on line documentation is updated with every release of the software and will be the most accurate source for authoritative answe
10. University hands and are pushed forward by the hands as the lock level increases remembering the high water mark of the lock level as you shim On the right hand portion of the display are the various shim parameters Remember that only the Z gradients are adjusted with the sample spinning If you attempt to adjust any gradient with an X or Y term in its name the spinner must be turned off first The purpose of spinning the sample is to average out gradients in the XY plane and you cannot minimize those gradients if you are averaging them to zero As with the lock parameters the shim values are changed by clicking in the appropriate box with either the left decrease or right increase mouse button to change by the increment shown in the box The increment value can be changed to one of three values in succession by clicking on the middle mouse button The initial increment values are 1 10 and 100 These can be changed and per parameter basis by clicking with the middle button while holding down the shift key The current increment then has a dialog box where any value may be entered One very reasonable change to make is in the lock phase parameter Since the lock phase cannot be set more accurately than perhaps the nearest 10 it might by reasonable to set the increments to 5 20 and 45 SPECTRUM PROCESSING Spectrum processing is involved with converting a FID into a recognizable spectrum There are generally two steps involved
11. account will affect other users on the system Quotas in place so that individual users cannot fill up either inadvertently or intentionally an entire disk partition and cause problems for others It is possible however if a number of users are close to their quota limits that other users may not be able to access their full quota Your quota is a limit on maximum disk usage and not a guarantee of available disk space There are several different options for storing and archiving your NMR data from your account All of the systems have several USB ports available on the front panel These can be used to copy data directly to either memory sticks or USB disk drives The workstations also have CD DVD burners available for archiving larger amounts of data more permanently Finally data can be moved to your PC environment and archived there In the most general case you can use FTP to move you data sets to virtually any platform There is also a samba server set up so that you can access your home account from a Windows 2000 NT XP platform and simply drag n drop data sets to your PC file system Regardless of the mechanism you use for archiving your data you should verify that you can move it back to the spectrometer system and read it there for processing and plotting before deleting it An archive that you cannot access does you no good 1 8 SECURITY There are two security precautions that we take in order to help protect the NMR instruments
12. acqproc is not sufficient to regain communication it is necessary to restart the other end of the communication on the NMR console This is a much less common situation All that is necessary is to press the reset button on the master controller card in the electronics console Open the right hand console door push to open push to close and locate the master controller card on the far right side of the digital card rack The reset button is near the bottom of the card See the image on the right Press it once and wait for the console to restart itself This normally takes something on the order of 2 minutes or more so be patient Near the end of the process the phase lights on the remote status unit will cycle once each in turn NMR USER S MANUAL November 9 2009 43 Virtual Network Computing The nmrsun1 nmrsun2 and nmrsun3 workstations have been set up with automatic servers so that it is possible to login and do NMR processing without using Xwin32 or some other X windows emulator The X server runs on the Linux Workstation and the local PC uses only a small viewer For the Windows environment this viewer is only 176K and will easily fit on a floppy disk There are no shared libraries to install and it can be run directly from a floppy disk VNC viewers are available for Windows Macintosh systems and as source code for various unix flavors Several of these are available for ftp from the Sun workstations nmrsunl
13. adjusted they interact with Z1 and Z2 in the same manner making shimming quite involved In addition normal Z3 and Z4 error affect a smaller part of the sample and consequently have a less significant effect on the lock level even though they have a significant effect on the lineshape You will generally get a better results by either diluting your sample to a 5 cm depth and getting better resolution or by using gradient shimming to set the higher order gradients Figure 2 shows the screen used for manual shimming within the Shim parameter panel Although the lock scan is displayed it is not necessary or required for manual shimming Along the left edge of the shim panel are the lock parameters Varian continues to display the ZO value here although there is no basis for making a change in Z0 once the system is locked do not change it The lock power phase and gain may be adjusted to avoid saturation and to keep the lock level on scale The lock level meter shows the digital lock level beneath the meter as well as in the middle of the meter The lock level meter is like a clock display the hour hand indicates the 10 units and the minute hand the ones units The outer color band changes color with ever revolution of the minute hand to give an approximate range At the 4 00 position on the dial there is a small button that can be pressed to track the maximum lock level Two small dots are moved up against the two 20 User Copy Texas A amp M
14. command that is useful for plotting COSY type spectra and several other specialized plot macros in the system maclib The basic command for plotting a 2D contour plot is pcon Plot CONtours which has a number of options available to it The usual command for transforming a data set is wft2da Although it is normally useful to manually transform it adjusting the phase and weighting as you go by using the wti command 16 COMMON ERROR MESSAGES The following is a list of common error messages on the system what they mean and how to correct them It is certainly not complete but it should include most of the common routine errors The wording may not be exactly the same as the current software but the sense will be the same 1 Variable name Doesn t Exist The specified parameter name is not a valid system parameter Usually a typographical error NMR USER S MANUAL November 9 2009 39 2 RECEIVER OVERLOAD ADC OVERFLOW and LOW NOISE IN ACQUISITION These are all errors that have to do with the receiver gain being either too low or too high Normally the receiver gain is adjusted automatically with the gainz n parameter selection This is not possible with arrayed experiments where you must manually choose a gain setting The low noise message simply means that the gain may be too low It is not necessarily an error condition although low gain can lead to poor sensitivity Receiver overload indicates clipping in the
15. fourth order to correct samples which are shorter than the normal 5 cm sample column length Having chosen a shim method you can initiate the shim immediately by using the shim command or you can set the parameter wshimz e to cause shimming to be started when you begin the data acquisition If you change a parameter and restart the data acquisition the system will repeat the field shimming wshim is a parameter to tell the system when to shim e is at the start of an experiment is never and S is for each sample if you have an automatic sample changer which we do not The benefits of the automatic shimming are that the system will generally iterate to a reasonably good field but the disadvantage is that it tends to be quite slow An experienced operator can get homogeneity as good or better than the autoshim in less time but an inexperienced operator can degrade the field horrendously without realizing it 6 2 MANUAL SHIMMING As I noted above manual shimming can be risky for inexperienced operators but fortunately there is a fairly painless way for operators to gain experience The shim screen under the Start Tab provides a means of manually shimming the field using the computer controlled buttons These work the same way as the lock parameters described above except that they don t have the slider bar available When first learning to shim manually adjust the Z1 fine and Z2 fine until you believe it is the best that can be done Then
16. interface is not too high It is also necessary for the very highest quality spectra to insure that your sample is free of dissolved paramagnetic species These will hasten relaxation of your sample and thus give broader resonances Degassing samples to remove dissolved oxygen is often necessary and it may be necessary to treat your sample with a free radical scavenger of some sort in order to remove paramagnetic species This is especially true for experiments such as n O e Difference spectra that depend on having dipolar relaxation as the primarily relaxation mechanism 8 User Copy Texas A amp M University 3 RELAXATION TIMES There are two different relaxation times that are used to characterize NMR signals T is the longitudinal relaxation time measuring how fast z axis magnetization returns This is the time constant that governs the establishment of the Boltzman distribution between the various energy levels The second relaxation time is the T transverse relaxation time which governs how fast a coherent magnetization vector in the x y plane is dephased In some cases it is also necessary to consider T2 which is similar to T2 but takes into account magnetic field inhomogeneity T2 determines the natural line width of an NMR signal but T2 will govern the observed line width if itis shorter An inhomogeneous magnetic field will broaden a sharp line but not a broad one The relationship between these times is given by these inequali
17. not change the size of the data table Consequently the acquisition time sweep width and number of data points are excluded from arrays Also any number of parameters may be arrayed at once although carried to an extreme this could take a very large amount of time and disk space to collect To create a parameter array from the command line enter the parameter name and square brackets with a 1 for the starting subscript an equal sign and a list of values separated by commas Thus to set up an array of pulse widths to measure a 90 degree pulse width the command line would look like this pwz5 10 15 20 23 30 35 40 45 50 This creates an array of ten values for the pulse width that can be accessed as pw 1 through pw 10 To change the value of one entry simply assign it a new value pw 5 25 NMR USER S MANUAL November 9 2009 33 This corrects the mistake in the original entry sequence To add additional values simply name them as you did the first starting with the next subscript pw 11 55 60 65 70 The array now has 14 values Once you create an array you cannot reduce its size only increase it To clear the array simply give the parameter a normal value pw 15 While this is clearly a very workable system the Java software includes an array parameter window that can be accessed from several of the Aquire tab parameter panels This window Figure 4 allows you to setup up one or more parameter arrays control the numbe
18. offset value This parameter is used to establish the resonance condition initially The lock RF power lockpower controls how strongly the deuterium signal is excited If the power is too high the resonance will saturate not be able to relax adequately resulting in a weak oscillating lock If the power is too low the sensitivity of the lock is reduced resulting in a poor lock The lock phase lockphase is used to adjust the lock resonance signal for pure absorption and dispersion mode signals If the phase is improperly set the system cannot lock The phase should remain invariant and should not normally require adjustment Never change the phase without first noting its initial value so that you can restore it if necessary None of the automatic lock routines will adjust the lock phase it must be adjusted manually Finally the lockgain is used NMR USER S MANUAL November 9 2009 15 to adjust the amplitude of the signal after all the other parameters are set properly and is normally adjusted for approximately a 60 80 reading on the level meter Figure 1 shows the lock display screen used for manually locking the system This is obtained when you toggle the lock scan button on the lock or shim parameter panels under the Start tab Across the upper portion of the display is a reconstructed display of the analog signal out of the lock receiver This is flat at the far left where the receiver is blanked during the lock pulse and then dis
19. panels and toggle the Show Field check box NMR USER S MANUAL November 9 2009 5 1 5 DISPLAY SCREEN The VnmrJ display has a variety of distinct display areas that may be resized or removed as desired in order to have a display presentation most useful to you The inside of the front cover has an example of the default appearance of the NMR application and identifies the major display areas of the screen The menu bar across the top of the display is always displayed and provides to highest level access to a variety of functions The View pull down menu is especially useful in that it will permit you to turn on portions of the display that you had turned off The Tool bar has several useful Icon buttons for common functions duplicating in one click functions available from the text menus The User Toolbar below that has a pair of camera icons used to memorize and recall screen layout configurations When you have a display layout that you want to save left click on one of the camera icons for 5 seconds and it will memorize that layout Clicking on that camera later will restore that same layout The left side of the screen has a variety of vertical parameter panels that are accessed by the tabs at the top of the column This provides easy access to common parameters and some of the more advanced features such as the viewport capability Any of the tabs can be removed from the vertical panel by clicking on the x in the top right corn
20. peak Since these enhancements are small it is normal to calculate a difference spectrum subtracting a reference spectrum with off resonance irradiation from the irradiated spectrum This should show only the enhanced signals Spectra can be added and subtracted using experiment 5 as an add sub buffer The pertinent commands to look at include clradd spadd and addi Even easier however is a new technique using shaped pulses to achieve selective excitation of a given signal The Selective Excitation 1D submenu in the experiments menu has provision for running a reference spectrum and allowing you to choose one or more signals to irradiate It then calculates custom shaped pulses to excite those signals runs the Overhauser spectrum and does the subtraction as part of the pulse sequence so that you see only the enhanced signals in the final spectrum While most noe s are positive it is possible for some samples to observe negative Overhauser effects as well 14 ARRAYED EXPERIMENTS The Varian data systems permit you to enter an array of values for any of the acquisition parameters This permits the collection of a set of spectra that are related and can be processed and plotted together easily Common uses of arrayed experiments are measuring T times kinetic measurements 90 degree pulse width determinations and as a special case two dimensional NMR An array may have any number of values and can be created for any acquisition parameter that does
21. proton and 200 PPM for carbon In addition to the width it is necessary to have a value for tof the transmitter offset This parameter has the effect of offsetting your spectral window to higher or lower field The offset is in units of Hertz and is not normally set directly from the keyboard When you recall normal parameters and specify a solvent the system returns a value for tof that centers the spectral window on the normal chemical shift range The Default parameter panel under the Acquire tab has provision for specifying directly the limits of the observation window setting the sw and tof values for you A command that is useful for modifying the spectral window is movesw This will narrow the spectral window to the region between the cursors on a displayed spectrum This is useful when first looking for resonance The spectral window is opened fairly wide the resonance is found and the spectral window is then narrowed to the region of interest This command also adjusts the reference line information in addition to the spectral window and transmitter offset 4 3 ACQUISITION TIME NUMBER OF POINTS Next you need to select an acquisition time at which is how long the system will spend sampling the time domain response FID after each pulse The upper limit on digital resolution is always equal to 1 at Thus if you select a 2 second acquisition time you cannot observe better than 0 5 Hz per data point resolution regardless of the natu
22. subdirectory tree with separate files for the raw FID date the parameters and the descriptive text You could find that you transferred the subdirectory but not its contents Check the contents or sizes or disk usage and make sure that you have all of your data Be sure that you can move it back to the Linux workstation and access it so you know it is a good copy NMR USER S MANUAL November 9 2009 29 11 HOMONUCLEAR DECOUPLING Homonuclear decoupling is used to remove proton coupling from proton spectra in order to simplify or assign proton proton coupling patterns This can be done on any of the current spectrometer systems To perform homonuclear decoupling it is necessary to introduce an additional RF field at the frequency of the proton whose coupling you want to remove Consider for instance decoupling the methyl and methylene protons in ethylbenzene If you irradiate the methyl triplet with sufficient power the methylene quartet will collapse to a singlet The first step is to obtain a normal proton spectrum and to then generate a copy in another experiment If you run the proton in exp1 use mf 1 2 to move the FID and all the parameters from exp1 to exp2 and jexp2 to join experiment two To enable the decoupler set the decouple nucleus proton the homonuclear mode to true homo y and turn the decoupler on with dmz y Make sure the decoupler modulation is turned off with dmmz c that remains now is to set the
23. the recovery Very long and very short values provide little information about the recovery Once the data is collected and transformed select one of the longest d2 value spectra adjust the threshold to include all the peaks of interest and generate a line listing using nll or dll You can then request a analysis on any of the listed lines by referring to them by number For instance fp 2 3 6 t1 will perform a T1 analysis on the second third and sixth lines listed The fp command finds all the peak intensities for the requested lines and t1 does the actual exponential fit t1s will provide a short abbreviated listing of the analysis expl will display the exponential recovery data and pexpl will plot it on the plotter When looking at your analysis results compare the calculated values of S O and S INF the signal value at time zero and infinity If your data are good these should be equal in magnitude and opposite in sign If not it is a good indication that your data is suspect A 10 variation is not unusual and anything larger is a cause for concern If your calculated T1 is more than one third of the d1 relaxation delay it is significantly in error on the low side Insufficient relaxation delay will always make a T appear shorter than it really is Rerun the experiment using a significantly longer d1 value 14 2 KINETIC MEASUREMENTS Time evolution kinetics are rather simple to obtain using the pad acquisition parameter T
24. well as Exit choose the Create CD button You do not need to change anything in the Setup window The Create CD section initially will show information about images already created on the CD R that you inserted in the drive If there are previous sessions recorded they should be shown here After verifying that the CD R is correct you can move down to the Master Tracks button in order to create the list of data files to be archived The window on the right shows the entire disk directory tree You can navigate through that tree to locate your data either in nmrdata lt group gt home group unames or in home1 scratch if you created a special directory tree for your data Select the data directory that you 50 User Copy Texas A amp M University want to archive and press the Add button You will then get a dialog box requesting the path name that you want to use to store the data on the CD The default is to use the full path name which isn t normally going to be very useful You can specify either the root directory of the CD the last path component or a custom path Your choice will be the default for successive directory selections As an example suppose that I have service data in a vnmrl home data directory for userA that I want to archive The full path that I select in the right hand window may look something like home staff vnmr1 vnmrsys data userA and within that directory I would have the actual NMR data files pro
25. whether or not some peaks appear in your spectrum 41 STANDARD PARAMETERS Normally a standard set of parameters is recalled to the system and these parameters are modified as necessary in order to meet the needs of any given experiment The easiest way to do this is to select the desired nucleus from the Experiments pull down menu on the main menu bar The command line alternative is the setup or msetup command Two arguments must be supplied the nucleus to observe and the solvent being used for field frequency lock If these are not supplied the program will prompt for them The form the command takes is then setup C13 CDCL3 to set up parameters for a carbon experiment using deutero chloroform as the lock solvent Most common solvents are recognized by the system and other solvents can be added if necessary If you are having trouble finding your solvent you may want to examine the file vnmr solvents to see the current list of defined solvents 10 User Copy Texas A amp M University 4 2 SPECTRAL WINDOW OFFSET The first consideration is to choose a sweep width or spectral window sw which will define how wide a region you will observe If this is too small peaks of interest will be excluded and will fold or alias into your spectrum at unexpected positions If this is too large it will limit the ultimate digital resolution attainable It should typically be about 10 larger that the chemical shift range of your sample 12 PPM for
26. window to indicate that a plot file is open As you execute additional plot commands pscale pap the output is added to the output file When you are done plotting you issue a page command This closes the output file and sends it to the plotter device to be plotted when the device is free You can see that there will be a severe problem if you start plotting with one plotter device change the plotter device and continue issuing plot commands the output file will have mixed formats and will not print properly on either device If you find that you have started plotting with the wrong device defined the command page clear will clear the output file so that you can start over Also page filename will write the output to the named file instead of the selected device This will allow you to capture the plotter output file for import into other programs although there are now better was to do this 9 PRINT QUEUES Print queues and the printer parameter work in a manner very similar to the plot queue described above The primary difference is that there are a lot more ways to generate print text output Generally anything that writes text to the Text Output window on the bottom parameter panel of the Processing tab can be written to the printer instead The command printon redirects output from the text window to a print file Any commands that generate text output can then be executed parameter lists T1 analysis etc When the file is
27. D but once you have 20 30 MB of data to archive the overhead becomes more acceptable If you have a large amount of data on remote data storage somewhere this can be burned in a single session without any additional overhead There are three basic steps involved in creating a data CD with your NMR data The first is to decide what data is to be archived and perhaps to collect it in a single location if it is spread out across several directories The second step is to create an image of the 1509660 file system that will be burned on the CD when it is recorded The third step is to actually write the image to the CD Do not attempt to either write the CD on the fly one of the option buttons in X CD roast or to run other programs or applications while actually burning the CD The computer needs all of its clock cycles to keep the CD burner buffer full This will save you both CD R media and aggravation There is nominal provision for buffer underrun protection and newer systems are faster and more robust but treat this as emergency prevention rather than something to rely on To begin making a data CD login on to one of the nmrsun system consoles you cannot run this remotely It is necessary to access any remote data directories you want to use in order to have the operating system mount them before you try to access them with the CD burner software Your home directory is automatically mounted when you log in If you have remote data in the the
28. NMR TRAINING MANUAL LINUX BASED SPECTROMETERS And VnmrJ 2 x TEXAS A amp M UNIVERSITY DEPARTMENT OF CHEMISTRY November 9 2009 1 13 1 2 1 3 1 4 1 5 1 6 1 7 1 8 4 1 4 2 4 8 44 4 5 4 6 5 5 1 5 2 5 3 6 6 1 6 2 6 3 6 4 7 7 1 7 2 7 7 4 8 TABLE OF CONTENTS OVERVIEW WHAT IS FTNMR MULTITASKING DUAL PROCESSORS KEYBOARD MOUSE DISPLAY SCREEN PRINTER PLOTTER DATA STORAGE SECURITY SAMPLE PREPARATION RELAXATION TIMES ACQUISITION PARAMETERS STANDARD PARAMETERS SPECTRAL WINDOW OFFSET ACQUISITION TIME NUMBER OF POINTS PULSE WIDTH RELAXATION DELAY DECOUPLER CONTROLS ACQUISITION FLAGS FIELD FREQUENCY LOCK AUTOMATIC LOCKING MANUAL LOCKING STEP BY STEP LOCKING FIELD SHIMMING AUTOMATIC SHIMMING MANUAL SHIMMING APODIZATION AND WEIGHTING FOURIER TRANSFORMATION PLOTTING AND DISPLAY PHASING REFERENCING INTEGRATION DISPLAY PLOT QUEUES o O Iv 10 10 11 12 13 14 14 16 17 18 20 21 21 22 23 24 25 26 9 PRINT QUEUES 10 DATA STORAGE 10 1 On Board Storage Devices 10 2 CD Burner 10 3 Samba File Server 10 4 Network Directories 10 5 FTP File Transfers 11 HOMONUCLEAR DECOUPLING 12 EDITED CARBON SPECTRA 12 1 ATTACHED PROTON TEST 12 2 DEPT SPECTRA 13 NOESY ROESY and NOE DIFFERENCE SPECTRA 14 ARRAYED EXPERIMENTS 14 1 T MEASUREMENTS 14 2 KINETIC MEASUREMENTS 15 TWO DIMENSIONAL 2D NMR 16 COMMON ERROR
29. THE CHAIN BEFORE LEAVING THE LAB EVEN FOR A BRIEF PERIOD It only takes a second for someone to come in and cause magnet damage that could require up to 3 months and 15 000 or more to repair 2 SAMPLE PREPARATION samples for routine proton and carbon spectra are prepared in 5 mm diameter tubes It is important that samples be prepared properly in order to obtain high quality spectra All samples should be dissolved completely in a deuterated solvent The deuterated solvent is necessary for two reasons first it provides the deuterium reference signal for the field frequency lock system and second it eliminates the large solvent resonance in proton spectra There are times when it is possible and or necessary to use solvent mixtures and non deuterated solvents but this is not generally recommended For optimum resolution samples should be completely dissolved and free of any suspended particulate matter It takes only very small variations in the bulk susceptibility of the sample to distort the magnetic field and degrade spectral resolution For this same reason it is important that the sample column in your NMR tube be at least 5 centimeters tall Shorter sample columns bring the solvent air interface closer to the center of the receiver coil and the resulting change of the magnetic susceptibility results in degraded resolution For the very highest quality samples this sample column should be exactly 5 cm so that the solvent air
30. The three stand alone workstations all include a CD DVD burner available for burning recordable CD and DVD media This provides a reasonably large 600 Mbyte 4 Gbyte data storage capability with a very 28 User Copy Texas A amp M University reliable medium Writing data CDs can be done two different ways the entire 650M capacity can be written at once in a single session or smaller amounts of data can be written over a period time in several separate sessions on a multisession disk Writing multisession disks requires a certain amount of overhead reducing the total capacity of a disk but given the cost of the media 0 50 each and the convenience of writing smaller amounts of data as it is available this seems to be a small price to pay There are detailed notes available describing how to prepare and write a data CD with your NMR data There is an easy drag n drop interface available for storing data on a recordable CD but unfortunately it does not support multi session recordings This means that it will use an entire 650Meg CD R even if you are only storing 50 or 100Meg of data The Xcdroast utility is also available on the workstations While it a little geeky and troublesome to learn at first it does give you the opportunity to create multi session disks 10 3 Samba File Server Samba is a Linux utility that makes a Unix file system or directory available to PCs operating with the Microsoft Windows NT 2000 XP operating systems When
31. as yh2 pw90 pw360 or dmf The only requirement is that you use the same units for both power levels Note that increasing the value of tpwr dpwr will increase the power and increase the field strength as measured by the yh2 value This will correspond to a larger dmf value and a shorter pulse length 46 User Copy Texas A amp M University Using Linear Prediction Linear prediction is a software processing tool that examines a periodic function such as an NMR free induction decay extracts a set of coefficients and extrapolates either forward or backward to predict what the data would have done had it been collected This is a potentially valuable technique for extracting useful information from marginal data but it is also easily subject to abuse as well It is not possible to collect a very small data set and then extrapolate that data set in order to get high resolution just as if you had collected the data If the data is not sampled sufficiently long to determine the difference in two frequencies they will extrapolate as a single frequency with no improvement in resolution There are two cases where linear prediction is very useful The first is to backward calculate the first several data points of a data set that are severely distorted by either receiver recovery problems or shaped excitation pulses In this case somewhere between 2 and 6 points are calculated to replace the faulty data If the first several data points are bad t
32. ation does not The delay period is chosen to be 1 J so that the outer lines of triplets will precess exactly 1 revolution in the rotating frame and refocus with the center line At the same time however the lines of a doublet are precessing only half as fast since they are only J 2 Hertz away from the chemical shift frequency and the two lines of the doublet refocus along the negative X axis instead of the positive X axis Quartets behave in a manner almost identical to that of doublets The net result is that singlets and triplets are apparently unaffected by the pulse sequence and doublets and quartets have their phase inverted This then provides a very simple means of distinguishing between methyl methine carbons and methylene quaternary carbons The primary advantage of this sequence is that you see all the carbons of your spectrum in a time comparable to the time needed for a normal carbon spectrum In fact however quaternary carbons are often missing or severely attenuated in intensity This sequence also depends on a good estimate of the Jy c value since this determines how completely the signals refocus 135 140 Hertz is a good compromise between aromatic 160 Hz and aliphatic 120 Hz couplings If you have a better estimate of the coupling constants in your compound you should use that value The easiest way to run an APT spectrum is from the Proton Carbon Multiplicity submenu in the experiments menu If you prefer to run your s
33. bserved nucleus in the case of homonuclear experiments auto correlated or different in the case of heteronuclear experiments Likewise if the second axis is a coupling constant it could be either homonuclear or heteronuclear The cross peak intensity correlating frequencies on the two axes are also a function of the experiment being performed There are a variety of proton proton auto correlated experiments for instance and the cross peak intensity may arise from short range scalar coupling long range scalar coupling dipolar cross relaxation etc In each case the cross peak intensity will give different information about the chemical system NMR USER S MANUAL November 9 2009 37 Perhaps one of the more confusing aspects of 2D NMR is simply the nomenclature used for the various parameters involved There is no standard nomenclature and the names that Varian uses are a combination of old historic values and newer consistent values Initially nobody considered anything past two dimensions so nomenclature wasn t as critical Now 3D is becoming more common and high dimensions are conceptually very simple For the general nD NMR spectrum there will be one directly detected frequency axis which will always be chemical shifts of the observe nucleus and n indirectly detected axes with attributes determined by the pulse sequence of these axes have the same requirements in terms of sweep width associated parameters for acquisition
34. ction Decay FID the first order exponential decay of the induced magnetization precessing freely The Fourier Transform is a mathematical procedure that interconverts a time domain function and a frequency domain function The fourier transform of the FID time domain signal is the normal intensity versus frequency NMR spectrum that you are used to seeing a frequency domain function Application of more complex pulse sequences permit the operator to establish a variety of non equilibrium conditions that permit the measurement of a variety of NMR and chemical parameters 1 2 MULTITASKING The spectrometer system is multitasking on two separate levels First of all it is multitasking in the sense that it has two separate processors so that data acquisition can occur in background in the acquisition processor uninterrupted by other data processing tasks occurring in foreground in the host processor The host processors Linux operating system is also inherently multitasking allowing several different things to happen simultaneously For instance the operating system maintains queues for the printer and plotter allowing them to run in background as the printer resource is available Likewise it is possible to run the NMR software while also running a file manager program or editor This is now very standard operation in windowed systems The Linux systems are also multitasking in that more than one user may log on at once with each user performing
35. data set to correct the first 2 data points in order to improve the baseline The second example shows the parameters necessary to forward predict a 2D data set in order to more fully used the increments collected Example 1 1D backward Linear Prediction Data Points L 2 037 4 sb 06 28 9 WOO IL 12 I 4 125 np 2 lpopt b lpfilt lpnupts 2 First point to use in calculation strtlp 3 First point to calculate strtext 2 Number of points to calculate lpext 2 Last Point to use in calculation lpnupts 2 Use lpopt b lpalg lpfft lpfilt 16 strtlp 3 lpnupts 256 strtext 2 lpext 2 Example 2 2D Forward Linear Prediction Data Points 254 255 256 ni 257 258 259 260 strtlp 256 strtext 257 Last point to use in calculation 256 lpnupts l Last Point predicted 256 lpext lpoft f lpfilt lpnupts 2 Use lpopt f lpalg 2 lpfft lpfilt 8 strtlp 256 lpnupts 128 strtext 257 lpext 128 or 256 48 User Copy Texas A amp M University Drag N Drop Data Storage TheRedHat workstations can easily store NMR data on either CD R W disks or USB memory devices using a simple drag n drop user interface It is important that if you are going to use either a recordable CD or a USB device that you login on the workstation before inserting your device If you are not logged on to the system console first the device is owned by root and you cannot then
36. db scale is logarithmic a difference of three units in the dpwr parameter results in a factor of 2 difference in RF power Normal values of dpwr will range from 8 or 10 up to about 20 A value of 10 is generally a good starting point Increasing it to 13 will double the power to the probe and increasing it to 16 will double the power again Start with a value around 10 or 12 and move up or down depending on the results you see Power levels over 25 should be avoided 30 User Copy Texas A amp M University 12 EDITED CARBON SPECTRA There are currently two popular pulse sequences used to obtain edited carbon spectra By edited spectra I mean spectra that can distinguish between methyl methylene and methine carbons The first of these is the attached proton test APT sequence which modulates the phase of a carbon line using scalar J coupling information The second method uses a special case of distortionless enhancement by polarization transfer DEPT sequence which transfers polarization from the protons to the carbons through the scalar J coupling Both sequences have their advantages and it is advisable to be familiar with both of them 12 1 ATTACHED PROTON TEST The APT spectrum is reasonable simple and straight forward to understand and interpret The pulse sequence is a simple spin echo sequence with the proton decoupler turned on for only one of the delay periods The result is that the chemical shifts refocus but the J coupling inform
37. different tasks It is possible for the whole system to slow down if too many things are occurring at once since there is a finite amount of computing power available on any given system For this reason remote Note that this manual is printed with wide outside margins so that you may make personal notes to yourself in the manual itself 2 User Copy Texas A amp M University access for data processing is not permitted on the spectrometer systems and several stand alone workstations are available for processing data away from the spectrometer Another aspect to the multitasking capability of the instrument is the use of multiple experiments As defined by the system an experiment is a file somewhere on the file system that contains all the information necessary to carry out a complete NMR experiment including space to store both the collected data FID and the transformed spectrum It is quite possible and common to have a proton spectrum in experiment 1 a carbon spectrum in experiment 2 an edited carbon spectrum experiment in experiment 3 and a two dimensional experiment in experiment 4 It is then possible to jump back and forth between the various experiments measuring data from each of them in turn as necessary Experiment 4 can be collecting data in the acquisition processor while experiment 1 is plotting the proton spectrum through the plot queue and you are adjusting the carbon data for plotting in the host processor The
38. e spectrum and measuring the integration increase the d1 period and repeat the spectrum Compare integration ratios and make sure that the relative values didn t change This assures you that the nuclei were completely relaxed between pulses 4 5 DECOUPLER CONTROLS Normally the decoupler parameters will not require readjustment from the default values returned by the standard parameters The decoupler offset dof works in a manner analogous to the transmitter offset tof and is used to identify the center of irradiation in the proton spectrum The decoupler mode dm is used to define when during the pulse sequence the decoupler is turned on and off There are three periods identified in the standard pulse sequence the relaxation delay d1 an interpulse delay d2 that is not normally used and the acquisition time at dm is simply a list of y s and y is used to indicate that the decoupler is on during the specified period and n to indicate that it is off Thus dmz nny would gate the decoupler off during the relaxation delay d1 the first n off during the interpulse delay d2 the second and on during the acquisition time at the final y The decoupler modulation mode dmm is used with the decoupler modulation frequency dmf to determine how the decoupler is modulated to provide broadband decoupling in carbon spectra These parameters are not used for homonuclear proton decoupling dmm is used much the same way as dm above
39. e right phase depending on the mode The left button will be a coarse adjustment and the right button a fine adjustment of the parameter The middle button always adjusts the vertical scale as usual When you select a peak region for phasing both the horizontal and the vertical position of the cursor are important 20 region of the spectrum symmetrically placed around the cursor frequency is highlighted as the area to be interactively phased horizontal line is drawn at the vertical position and this defines the zero point for phase corrections Clicking dragging with the left or right mouse buttons above this line will increase the selected phase parameter and moving below the line will decrease the parameter Therefore if you do the initial selection too close to the top or bottom of the screen you may not be able to fully adjust the phase parameter To phase the spectrum press the Phase button to enter the interactive phase routine and with the left mouse button choose a large peak near the right side of the spectrum and phase it Phase this peak as accurately as possible You will notice that only the right phase parameter is changing and that the phase of the entire spectrum is changing by the same amount This is the zero order phase parameter Once the selected peak is phased properly select a peak area to the left of this to phase You will notice that as you phase this peak both the lp and rp phase parameters will change The syst
40. e the gradient shimming system You will be looking for a shim map file named probe lk date fid which will be specific for the probe installed on the system use probe to determine which probe is installed and choose 18 User Copy Texas A amp M University the one with the latest date The gmapsys command can also be run from the Tools gt Calibration Experiments gt Setup Gradient Shimming menu button Gradient shimming does a one dimensional imaging experiment on the Z axis and does not depend on being able to optimize the lock level and the shimming is done with the lock off and the sample non spinning since is it pulsing on the deuterium signal This means that in the case of very poor homogeneity it is possible to do gradient shimming before attempting to lock the deuterium signal Automatic shimming can be also be done by optimizing the lock level This is the older more traditional way of autoshimming While being significantly slower than gradient shimming it has the advantage of being able to get sligthtly better homogeneity and to optimize the transverse gradients that gradient shimming cannot adjust The special display group dgs contains a parameter method which is a name parameter containing the name of a shim method file Currently the only useful options here are method Z1Z2 which does a simple first and second order correction for normal samples and methodcz hires which does more complex correction through the
41. e to the maximum permitted by the available memory The system will never change the spectral width in order to maintain a permissible combination of SW at and np You can also specify a value for np and let the system calculate the required value for at This is sometimes useful when doing arrayed experiments where it is necessary to limit the total data memory used 4 4 PULSE WIDTH RELAXATION DELAY The next pair of acquisition parameters is the excitation pulse width pw and the relaxation delay d1 pw defines the angle through which the macroscopic magnetization vector is rotated in order to generate an observable magnetization component in the XY plane and d1 defines the period during which it will be allowed to return to equilibrium If the pw value is too small you get very little induced signal If it is too large it takes a long time for the system to come back to equilibrium If d1 is too small the system cannot come back to equilibrium and if it is too large the data acquisition is inefficient The relaxation times of the nuclei of interest determine what is too long or too short The system parameter pw90 defines the pulse width necessary to tip the magnetization 90 degrees The angle generated by an arbitrary pulse pW is then simply a linear function as given by equation 1 a pw pw90 90 degrees 1 After the magnetization is excited it returns to equilibrium by a first order process whose time constant is called T1 the longitud
42. ean and how to calculate the power necessary for various applications Maximum power is 63 db and minimum power is 16 There is no absolute limit on the power used for different experiments but an estimate of the normal range of values for various applications is Power level tpwr dpwr application 50 gt 63 Hard pulses 35 49 Heteronuclear Decoupling 20 gt 35 Shaped pulses 5 gt 20 Homonuclear decoupling water presaturation 16 gt 10 Spin tickling n O e difference spectra Since the amplifiers are linear if we know the power at any one point we can calculate the power elsewhere It is advisable however to calibrate the power at a value near the value you are going to use to avoid extrapolating too far Also calibrated power is normally only needed for the first three applications hard pulses heteronuclear decoupling and shaped pulses Finally the linear amplifiers become non linear at very high power levels above 58 or 60 Consequently power calibrations obtained at values greater than 55 should never be extrapolated for use at other power levels The tpwr and dpwr parameters are in db which is a logarithmic scale A change of 3db will double or halve the power into the probe Since pulse excitation depends on the voltage at the probe rather than the power and power is proportional to voltage squared a change of 6db is required to double or halve the yh2 value measured at the probe The yh2 value is measure of t
43. em is calculating the contribution of the left phase parameter at the frequency of the original zero order peak and subtracting that contribution to keep the phase of that peak constant You can then select other peak areas and repeat this procedure to refine the frequency dependent phase correction If you need to readjust the zero order correction you must restart the process by pressing the Phase mode button again It should be noted that the zero order phase correction that affects the entire spectrum equally is limited to a 360 degree range This value may be displayed with values of 180 or 360 but 360 360 are all the same phase correction The first order correction on the other hand can take on any value You can think of it as being the phase difference between the left edge and the right edge of the spectrum A value of 360 would mean no change on the left edge and an inversion 180 shift in the middle A 720 value would mean no shift at the left edge or in the middle 360 shift and inversions at points 25 180 shift and NMR USER S MANUAL November 9 2009 23 75 540 shift through the spectrum You could also picture this as twisting one end of a flexible picket fence getting all of the pickets lined up in the same direction These phase parameters can only correct for first order phase distortions which are normal due to time delays through audio filters and the receiver section They cannot cope wit
44. er when the tab is selected Deleted tabs can be restored in the view menu Deleting all of the tabs will remove the vertical panel area but it will be automatically restored when a tab is re enabled Tabs can also be hidden on the left side of the panel area to automatically pop up when the mouse hovers over it The main Graphics Canvas is where all of the spectral display is done The command line appears directly above the graphics canvas along with a command history The parameter pages appear beneath the graphics canvas There are 3 tabs to select different groups of parameter panels The Start group has all of the lock and shimming functions to get started with a new sample The Acquire group has all of the parameter panels associated with the data acquisition these are the parameters that must be correct before the data acquisition is started The Process group has all of the parameters and actions associated with processing and plotting the data Just to the right of the tabs selecting the parameters are action buttons that are pertinent to the parameter tab currently selected At the very bottom of the display is the Hardware status bar that has information on the state of the acquisition console To the right of the Graphics Canvas are the Graphic Control Buttons that are used for easy manipulation of spectral data The tool bar can be picked up and moved with the small 3 dot handle at the top of the bar This will permit
45. est of circumstances NMR integration accuracy will probably not be better than 3 5 J m eum T _ Cy 9 8 7 6 5 3 2 ppm io cr is delta os ETE l nx Start Acquire Process Full Clear Screen Default Integration Set Integral Area Display List of Integrals Weighting Integral Display Mode Display Normalized Integrals Display eic OX Normalize Area To More 1D i Single Peak Sum region start ppm end integral Integration Scale display to fit 1 8 38842 8 16707 0 0171479 Lists Integral Area 10 00 2 8 16676 6 89452 2 77917 P l 6 38495 6 09276 0 0039506 Plot Set Integral Regions Set Integral Value 4 5 53037 5 23452 0 0008601 Text Output Auto Find Integrals E i 5 4 21263 3 86763 0 0101143 ow Integrals 6 3 73298 3 52293 0 00549827 Interactive Resets Show Integral Values 7 3 52262 3 039 0 875607 Clear Integrals Snow Normalized 8 3 039 2 18778 1 69827 Add Rece at cunt 9 2 18748 0 629763 4 65468 Remove Reset at Cursor Show Quantitation Tools Panel Figure 3 Integration Panel 7 4 DISPLAY The display canvas is where all of your spectral data is displayed Upon the right hand side of the Menu Bar are two icons a stylized FID and a stylized spectrum used to display either the time domain or the 26 User Copy Texas A amp M University frequency domain data When either i
46. factor There is a very simple relationship between SE and 16 SE LB z 1 The sensitivity enhancement is a time constant in seconds e USE and ideally should be equal to or one third of the acquisition time assuming at 3 T5 A smaller value will result in additional line broadening but a larger NMR USER S MANUAL November 9 2009 21 value will not result in significantly better resolution since the resolution will be limited by the acquisition time see section 4 3 on page 10 The other less commonly used weighting process is resolution enhancement This is the opposite of sensitivity enhancement and uses a positive exponential e RE to enhance the end of the FID with the resolution information at the expense of the beginning with the sensitivity This can only be done on spectra that have very good signal to noise to begin with since much of the signal is thrown away and the noise is multiplied Since the weighting function is expanding it is usually necessary to use an explicit apodization function to keep the tail of the FID from getting too large simple Gaussian factor is normally used for this The resolution enhancement time constant is achieved by using a negative value for the line broadening An appropriate value of gf is then used to force the tail to zero The command resolv is a good place to start It assumes that at 3 T5 and selects starting values for Ib and gf It should be noted as well that this resolution
47. for writing multisession DVDs so you would have to write a full 4 7 GB at once to use the entire DVD NMR USER S MANUAL November 9 2009 49 Recording CD R Disks with X CD Roast The nmrsun workstations have CD R W drives available for storing NMR data to CD In addition to the operating system Drag n Drop interface which will not burn multisession CDs there is also the X CD Roast software that can burn multisession disks While this requires more effort than the Drag n Drop interface it allows storing data at different times on the same CD X CD Roast will not burn multisession DVDs and the DVD driver is missing on the current systems so DVD burning is best left to the Drag n Drop interface Data in the Amrdata mount points removable USB media such as memory sticks as well as home home directory space are accessible for burning your CD A recordable CD can hold 650 700 MB of data depending on the media that you use Since home directory space is limited it is unlikely that you will be burning a full 650 MB disk at one time Multisession recording permits you to record a session and access that data and then go back at some time in the future and add additional sessions to the disk This process can be repeated until the disk is full Multisession recording has some overhead associated with it 22 MB for the first session and 13 MB for each additional session Consequently it does not make sense to add just a few 100K of data to a C
48. freely to establish the order of evaluation For instance you can set the matched filter line broadening with something like Ib 1 at 8 A number of the acquisition and processing parameters have a boolean on off mode as well as their normal value The line broadening parameter Ib for instance takes a numeric value denoting the amount of line broadening to be applied to the spectrum By setting Ib n note the string function the line broadening function is turned off or disabled and the spectrum will not have any line broadening applied The numeric value is still retained however and setting Ib y will restore the numeric value and apply the line broadening with the next Fourier Transform If a parameter is disabled this way and you interrogate it s value on the command line the system will report both that it is not used and show the stored value of the parameter Ib Not Used 1 2 The same parameter when displayed in a parameter panel will have a check box to enable the parameter and a field entry box with the value The entry box is grayed out and inactive if the parameter is inactive Parameter changes and commands that are typed at the keyboard and echoed on the screen ALWAYS require a carriage return character Enter Key before they are be executed The mouse is used to select items from the various menus and to adjust the spectrum display in the graphics window in the middle of the screen three mouse buttons are u
49. frequency on resonance and select a power level for the irradiation The frequency selection is done by displaying a cursor and positioning it on the line of the spectrum you wish to irradiate In the above example display the spectrum position the cursor on the center line of the methyl triplet and type Sd to set the decoupler frequency The system will report back the value for the decoupler offset dof and set the parameter for you The parameter dpwr is then used to select the strength of the decoupling irradiation If the power level is too low the peaks will remain coupled or be only partially decoupled Too much power will introduce various birdies glitches and other spectral artifacts The optimum power is a function of the degree of coupling magnitude of J the chemical shift separation and the relaxation times involved Larger coupling constants and shorter relaxation times will require more power to collapse the coupling pattern Close chemical shifts limit the amount of power due to the various sidebands present near the irradiation frequency The optimum power needs to be determined empirically for each sample by trial and error All of these parameters can be set easily in the Channels panel of the Aquire tab The dpwr parameter represents the amount of power supplied to the decoupler amplifier expressed in db This means that the higher the dpwr parameter value is the higher the power that you are supplying to your sample Since the
50. h doesn t depend on accurate calibration This Varian s older swept square wave modulation which is not as efficient but it may still be a useful alternative in case of trouble 4 6 ACQUISITION FLAGS There is a boolean flag that affect how normal data is collected The interlock flag in determines what happens in the case of a lost lock signal If inz y then the data acquisition will stop if the field frequency lock is lost This can be especially useful on long term overnight acquisitions If something happens to lose the lock the acquisition will stop with whatever data there is instead of adding only noise to the existing signal Finally one more parameter that affects data acquisition is a combination flag and value parameter If the block size parameter bs has a numeric value not set to n not used then the system will write data from the acquisition processor to the host processor at the end of every data block comprised of bs pulses This can be especially useful for overnight data acquisitions in that the data is saved to disk periodically If power is lost or the system crashes then at least part of the data will still be available The value for bs should be chosen so that a block completes every 15 30 minutes 5 FIELD FREQUENCY LOCK Field frequency lock describes an instrumental condition that locks the magnetic field and the various RF frequencies together to eliminate drift in the spectrum Since the resonant frequency of a g
51. h higher order distortions which usually only arise from folded peaks in the spectrum If you have a very large value for the left phase with extra twists in peaks you will also see a lumpy rolling baseline Set 1 0 and start over 7 2 REFERENCING Referencing a spectrum is necessary if you want to measure absolute chemical shifts The spectrometer system will have default referencing based on the solvent that you chose and for many applications this is sufficient Chemical shifts are affected by the bulk magnetic susceptibility of the sample however so explicit referencing to an internal reference is required for the most precise chemical shift determinations The next best referencing is an external reference The last choice is the default referencing Internal referencing is done by adding TMS or some other chemical shift reference to your sample Ideally the concentration of the reference will be comparable to that of the sample so that it is easy to see without overwhelming the sample signals There are two parameters that control the referencing rfl and rfp The reference position parameter rfp defines the chemical shift in Hertz of the reference line For primary shift references like TMS this will be zero For secondary references such as the solvent line this will be the literature value of the reference The reference line parameter rfl defines where in the spectrum the reference line is observed This is measured as
52. he fourier transformed spectrum will have a large baseline offset or roll making it difficult to phase and integrate spectra The second case is to forward calculate data at the end of the fid in 2D data sets where the acquisition time is arbitrarily short The parameters in either case are almost identical Linear prediction parameters are added to a data set with the parlp command Parlp is equivalent to parlp 1 and adds parameters for the first dimension Parlp 2 adds parameters for the second dimension etc The dglp command will display all of the defined linear prediction parameters in a tabular form In order to perform a linear prediction calculation it is necessary to calculate a set of coefficients describing the frequencies present Ipfilt defines the number of frequency components to analyze for and Ipnupts defines the number of data points to use to calculate these coefficients Ipnupts must be at least twice Ipfilt and should probably be 4 8 times Ipfilt depending on the noise level in the data Ipopt is the option parameter that defines whether you are doing forward or backward calculations It can take only f or b as values The value of Ipopt defines the sense of the strtlp and strtext parameters strtlp is the data point number to use to start calculating the necessary coefficients This will be the first data point used in the case of a backward calculation and the last data point used in the case of a forward calculation The
53. he field strength of the irradiating field This value can be used to determine both the pw90 value necessary for pulse experiments as well at the dmf value necessary for decoupling experiments Shaped pulses with usually specify explicitly the yh2 value needed for proper operation It is easiest to think about yh2 in units of hertz as the frequency that the macroscopic magnetization vector is driven around the co linear axis in NMR USER S MANUAL November 9 2009 45 the perpendicular plane A yh2 value of 1000 applied along the x axis in the rotating frame implies that it take Imsec for the magnetization to complete one revolution around the x axis in the y z plane This would correspond to a 360 degree pulse It follows immediately then that pw90 the time for a 90 degree pulse is one quarter of that 1 msec or 250usec Varian always defines dmf the decouple modulation frequency as 1 pw90 since it is always used to time 90 degree pulses From this then we can write the basic relationship between these various quantities pw360 1 yh2 pw90 1 4 yh2 dmf 1 pw90 4 yh2 Also when calibrating power at a new field strength it doesn t make any difference if we think and calculate in terms of pulse widths field strengths or dmf values since they are all proportional and we only need the ratio of the two Adb 20 log P1 P2 Where Adb is the db change in either tpwr or dpwr and P1 and P2 are the two reference power levels either
54. his Pre Acquisition Delay is the amount of time that the system delays before starting a data acquisition Kinetics experiments work especially well when the pad time is long compared to the actual acquisition time NT AT D1 For instance collecting a proton spectrum for 60 seconds every 15 minutes keeps the data acquisition time short compared to the time between points The results can be less meaningful however if you 36 User Copy Texas A amp M University are collecting a 15 minute carbon spectrum every 20 minutes Note that the values in the pad array will be pre acquisition delays the time between the end of one acquisition and the start of the next They are NOT absolute times indicating when the acquisition will start For this type of experiment the interleave flag must be turned off il n After the data is collected and transformed analysis is almost identical to that for T measurements described above The appropriate commands for analysis are kini and kind for KINetics Increasing and KINetics Decreasing You just select the proper command depending on whether the peak intensity in question is increasing or decreasing with time In either case the system fits only to a first order exponential time constant Also the system plots each peak intensity as if it occurred at the beginning of the acquisition period If your acquisition period is long compared to the delay period you will probably get better results by taking the
55. ignal that is too weak for the system to find or keep There is generally no problem with using normal deuterated solvents although CDCl3 will sometimes cause problems You can have trouble however if you are attempting to use a mixed solvent system such as adding just a little deutero benzene to THF This error generally indicates insufficient deuterated solvent 6 SPINNER FAILURE The computer attempted to set a regulated spin rate and failed This generally indicates a dirty spinner turbine and or spinner bearing 40 User Copy Texas A amp M University Cleaning the spinner turbine with a Kimwipe and water will sometimes correct this error but it generally requires the disassembly and cleaning of the spinner bearing Note the error in the log book but do NOT attempt to disassemble the bearing 17 APPENDICES The follow pages have several of the NMR Notes that have been written over the past several years to aid and assist users These are included as a convenience only There is no new or additional information in these versions than in those previously distributed These are believed to be accurate and up to date These notes are currently included e Rebooting Acqproc Inova and Mercury consoles e Rebooting Acqproc VNMRS consoles e Using Virtual Network Computing VNC e Calculating Transmitter Power Levels e Using Linear Prediction e Drag N Drop Data Storage e Burning CD R Discs with X CD Roast NMR USER S MANUAL No
56. in this the first being to multiply the FID by a mathematical function in order to emphasize some aspect of it and the second being the actual fourier transformation from the time domain to the frequency domain There are only three commands associated with the processing Wt performs whatever weighting is specified by the various weighting parameters ft performs the fourier transform of the raw unweighted data and wft performs both the wt and the ft Using Wt you can observe the weighted FID before doing any additional processing but you must use Wft for the transformation since ft always goes back to the original data not the weighted data 6 3 APODIZATION AND WEIGHTING Apodization refers to the multiplication of the FID by cosine type function that forces the data table to Zero at the end in order to eliminate truncation type artifacts in the spectrum This is done automatically as part of the fourier transform and need not concern us further here The weighting function usually takes the form a decreasing exponential function which emphasizes the signal at the beginning of the FID at the expense of the resolution data at the end of the FID Thus it trades resolution in order to get sensitivity and is controlled by the line broadening parameter Ib which is the amount of additional line broadening that will be added to all the lines in your spectrum The data is actually multiplied by a function e USE where SE is a sensitivity enhancement
57. inal relaxation time This spin system relaxes during both the acquisition time at and the relaxation delay d1 It is critical for proper spectroscopy to choose a proper or optimum value for the combination of the pulse width pw and the relaxation delay d1 The problem comes in defining what we mean by optimum conditions For most proton spectra optimum conditions will imply quantitative results with accurate integrations For virtually all carbon spectra and most other nuclei optimum conditions will generally refer to qualitative results with the best possible signal to noise ratio in a given experimental time These two cases are mutually exclusive and we must choose pulse conditions accordingly Consider first the carbon experiment and the need for qualitative results For a given value of T at and d1 the optimum pulse angle alpha is given by the Ernst equation 2 Note that this pulse angle is optimized for sensitivity per unit time not for quantitative results Consequently it is not intended for use with proton spectra T arccos e 71 Q Since this expression is not one that is readily estimated by inspection there is a program on the system which will take as input your estimate of T look up the current values for at d1 and pw90 and 12 User Copy Texas A amp M University calculate the optimum value for alpha and set pw accordingly Simply type ernst T to calculate and pw Since the d1 delay period does nothing except pe
58. ing accurate integrations There are two different mechanisms available for doing a baseline correction A simple Drift Correction dC command does a simple level and tilt adjustment based on the baseline levels on the edges of the current spectral display This should be sufficient for most spectra Also available is a baseline correct command that calculates a high order spline function to NMR USER S MANUAL November 9 2009 25 force all of the blanked integral regions to zero This can result in significant distortions of the remaining baseline areas and should be used with extreme caution The easiest way to approach base line correction is from the Display panel under the Processing tab On the right hand side are three buttons associated with baseline correction The first is the simple drift correct command which is normally sufficient If after looking at the integral of the drift corrected spectrum you decide you need something more try applying the baseline correction Use the Autofind integrals to find all of the peak intensity in the spectrum This will help avoid erroneous baseline corrections Then use the baseline correct button to calculate the baseline correction If the baseline correction is not right and helpful for your spectrum simply repeat the fourier transform Wft to get the uncorrected spectrum back The simpler drift correction can be removed with the cde cancel drift correction command on the command line Under the b
59. ing or starting the acqproc process Normally once the acqproc process has been restarted there is normal communication with the console and the acquisition processor status is again idle If restarting acqproc is not sufficient to regain communication it is necessary to restart the other end of the communication on the NMR console This is a much less common situation that is necessary is to press the reset button on the acquisition console cpu card in the electronics console On the Inova console open the left hand console door pull to open push to close and locate the cpu card on the far left side of the digital card rack slightly recessed from the other cards The reset button is near the top of the card labeled RST just below another button labeled ABT and half hidden by an ethernet cable See the image on the right On the Mercury console open the smoked plastic door The reset button is the red button on the right side labeled System Reset Press the reset button once and wait for the console to restart itself This normally takes something on the order of 2 minutes or more so be patient Near the end of the process the phase lights on the remote status unit will cycle once each in turn 42 User Copy Texas A amp M University REBOOTING ACQPROC VNMRS CONSOLES VnmrJ uses an ethernet connection between the host computer and the NMR electronics console to allow control of the spectrometer The co
60. iven nucleus is a function of the magnetic field strength it is imperative that the field strength not change during the course of the experiment Since typical line widths are 1 Hertz or less and typical resonance frequencies are on the order of tens and hundreds of megahertz we require stability on the order of 1 part in 109 The current technology to achieve this type of stability is to use the magnetic resonance condition itself to control the magnetic field AII current generation spectrometers use the deuterium signal of the deuterated solvent for this purpose 14 User Co Texas A amp M Universit 5 1 AUTOMATIC LOCKING of the spectrometers have provision for automatic locking of the magnetic field There are now two different tools available for performing the automatic locking function There is new tool findz0 that is a very robust and fast mechanism to lock a sample This method does a deuterium pulse on the sample measures the frequency off resonance and changes the 70 value according putting the field very close to on resonance It then turns on the lock switch and the system will normally capture lock quite readily This is now the preferred method for locking most samples This is always available on the Standard panel under the Start tab The traditional autolock that has been available for at least a decade is a computerized emulation of what you would normally do manually The advantage to use the automatic locking i
61. l thing is to adjust it for zero frequency As you move the field farther from resonance the frequency of this signal will increase and the amplitude will decrease due to low pass filter circuits in the receiver Moving toward resonance will reduce the frequency and increase the amplitude of this signal After the lock is established the lock power must be adjusted to avoid saturation and the lock gain is normally adjusted for a lock level of 60 80 Saturation is the condition where the RF power powerlevel is too high and nuclei are excited to their upper energy level faster than T processes can relax them back to their lower energy level When the RF power is properly adjusted a small increase in the power will result in an increase in the lock level and a small decrease in power will result in a decrease in the lock level As you increase the power to the point of saturation a small change in power will not affect the lock level and as the saturation becomes very pronounced increases in the power will decrease the lock level and the lock level will become unstable and will bounce up and down The spin system will respond to changes in the lock RF power at a rate determined by the solvent T not instantaneously so make changes slowly and wait for a new equilibrium 5 3 STEP BY STEP LOCKING This is a simple guide to locking the spectrometer and should not be used as a guaranteed method but rather as an indication of the normal seque
62. ld be automatically mounted on media mount point of your sessions should be shown there When done do a Cd to get back to your home directory unmount your CD R remove your image file s from home1 images and any temporary data in home1 scratch directory exit xcdroast logout and you are done Congratulations
63. lues real numbers and text strings Real numbers are used to store any numeric value integer or real Integers are stored as real numbers and converted back before use Text strings are enclosed within single quote characters This is a sample Text String The backslash character can be used to introduce a special character in a text string The most common combinations are V to introduce a single quote JohnVs Reference sample and to introduce a new line 2 Command and parameter names are all case sensitive both in Linux and in the VnmrJ program 3 Spaces are used to separate individual items on the Vnmr command line Spaces cannot be incorporated into any command or parameter except when part of a literal text string argument 4 Some commands require arguments to specify how they will operate Arguments will always be included in parenthesis following the command If more than one argument is supplied the arguments are separated by commas i e setins 3 5 5 Parameter values are changed by simply giving the parameter name an equal sign and a new value i e at 2 4 6 When more than one item appears on the command line the individual items are executed in the order typed 7 Parameter values and arguments can be specified as mathematical expressions which may include parameter values symbolically Again no spaces may be 4 User Copy Texas A amp M University included but parenthesis can be used
64. mat screen which is closer to the display size on the system consoles The useful commands therefore are vncviewer nmrsun2 1 for the normal 1024 x 768 desktop on nmrsun2 vncviewer nmrsun2 2 for a larger 1280 x 960 desktop on nmrsun2 In all of these commands nmrsun3 or nmrsunl can be substituted for nmrsun2 There is no difference between the three workstations In the Windows viewer you may right click on the title bar to get an options menu The most useful option is the full screen mode which eliminates the scroll bars and other Window dressings and leaves you with a screen that looks the same as you see on the workstation when you log in CTRL ESC followed by ESC will get you back to a task bar so that you may remove the full screen mode or chose other options If your Xserver screen is larger than your PC screen size e g 1280x960 on the Xserver and 800x600 on your Windows desktop your desktop will act as a view port onto the Xserver display scrolling automatically as you move the mouse cursor off the edge of the screen This can be rather disconcerting and hard to get used to but does let you have a virtual NMR display larger than your physical screen display 44 User Co Texas A amp M Universit Transmitter Power Calculations The Varian consoles all use linear amplifiers with precision attenuators to provide a range of output power on both the transmitter and decoupler channels It is important to understand what these powers m
65. nce of events 1 TURN OFF THE LOCK CONTROL WITH THE LOCK OFF BUTTON This is essential to proper locking 2 Turn the lock gain gain to its maximum value and set the lock power powerlevel to 35 40 NMR USER S MANUAL November 9 2009 17 3 Adjust field position 20 for the zero frequency resonance condition Within about a half cycle on the screen is close enough 4 Turn on the lock control with the Lock On button You should see the normal lock display 5 Reduce the lock power below the point of saturation 15 20 for acetone 20 25 for benzene or water 25 27 for chloroform about half of these values on the Mercury 300 and adjust the lock gain to 60 80 level 6 If necessary adjust optimize the lock phase for maximum signal intensity 6 FIELD SHIMMING Field shimming is the process of adjusting the homogeneity of the magnetic field so that all nuclei in the sample experience the same static magnetic field strength Just as the lock is necessary to keep the main field position fixed very accurately so also field shimming is necessary to make the field uniform to approximately 1 part in 10 over the active sample volume in order to observe individual resonance lines Just as it is difficult to regulate the magnetic field strength it is also difficult to measure its uniformity Again we will use the magnetic resonance condition itself to measure the field homogeneity Once a stable lock signal is established the in
66. nmrsun2 or nmrsun3 You can go to the directory usr local pub VNC Viewers and find an appropriate file vncviewer exe Windows vnc 3 3 3beta2 68k mac sit Macintosh vnc 3 3 3beta2 ppc mac sit Power PC vnc 3 3 312 unixsrc tgz Unix source tarred and gzipped The Macintosh files are compressed and will have to be unpacked to be used The Windows file can be copied and executed directly Unix source code is must be gunzipped detarred and compiled When you run the vncviewer program you can enter the server name on the command line If you don t a dialog window will be opened and ask which server you wish to use This is in the form hostname display where hostname is the host that you wish to connect to and display is the display number Display 0 is the system console display and display numbers 1 through 99 are available for users Rather than explicitly starting a vncserver in order to get a display number the NMR workstations have been set up to automatically start a server when you request a connection The display number is then used only to choose a screen resolution The display value defaults to 0 but since display 0 is reserved byRedHat for the system console it is unavailable for remote use and you must explicitly specify the display that you want to use Display 1 is used to select 1024 x 768 pixel display size This is the smallest practical display size to run VnmrJ Display number 2 will choose a larger 1280 x 960 for
67. nnection requires two way communication between the computer and the console and each end of the communication has a process running to conduct this communication In the normal course of operation it is not usually necessary to do anything with this process it goes on in background very reliably When there is a problem with the communication it is necessary to manually restart the communication process This is evidenced most commonly by an Inactive status message in the acquisition processor status window at the bottom of the screen but it may also be required any time there is a problem with console access running acquisition that cannot be stopped unable to start an acquisition or eject a sample etc The acqproc process is the process on the host computer that controls this communication Usually all that is necessary is to stop the process and then restart it This is done readily using the command su acqproc this is a toggle type command that looks for a running acqproc process If it finds one it stops it and if it doesn t find one it starts it This is run in a Linux shell window right click on the desktop area and select Open Terminal to get a shell window When you run SU acqproc it tells you whether it is stopping or starting the acqproc process Normally once the acqproc process has been restarted there is normal communication with the console and the acquisition processor status is again idle If restarting
68. normal convention used by some of the older the automated software is to use experiment 1 for proton spectra experiment 2 for normal carbon spectra experiment 3 for non standard carbon APT DEPT etc and experiment 4 for two dimensional spectra COSY HET2DJ etc This is only a convention and much of the newer software does not follow any particular convention Any experiment can be used for the various spectra and the size of the experiment file is adjusted dynamically to accommodate any given experiment The only experiment that is special is experiment 5 which is used as an ADD SUB buffer for adding and subtracting spectra as well as for some other special data processing procedures If you are not using these procedures it will make no difference if you use experiment 5 for data acquisition Be warned though that if you do use any of these routines the data in experiment 5 could be lost The command explib will display a summary of the experiments currently defined on the system their current size and an indication of which ones are either running or waiting to run You may define an arbitrarily large at least 999 experiments but bear in mind that these all occupy disk space and will count against your quota 1 3 DUAL PROCESSORS These systems are equipped with two separate computer systems The host processor is the Linux workstation and this is the computer that controls the keyboard CRT display printer and plotter and data st
69. note the values of the 71 and Z2 gradients the lock level and the lock gain Set methodz Z1Z2 and run shim on the command line to let to the automatic mode shim the Z1 and Z2 gradients for you When the automatic shim is done make user the lock power and lock gain levels are the same and compare the lock level with your recorded value If there is substantial improvement as a result of the computer shimming go back to the shimming panel note the new values of Z1 and Z2 restore your old values for 71 and Z2 and see what it takes to manually shim to the correct values NMR USER S MANUAL November 9 2009 19 Start Acquire Process Setup Hardwar Show Time Standard Lock Scan 1 23 2 3 1923 10 185 10 10 10 daran Yi Ya 23Y ng 125 _ 10 5 10 10 4854 j e F sto Lk Power Yz Yz2 22XY 24 21 Ea cud 10 5 10 5 10 RO xY 10 2 E 10 Lk Gain 1 Suy SUB wx RS ser ang sag Lk Phase p Lock 98 0 Spin On Spin Regulated Recr gain spn oma 20Hz 60 Figure 2 Shim Parameter Panel Remember that Z1 and Z2 interact quite strongly and that you cannot simply optimize one and then the other You must move Z2 an arbitrary amount optimize Z1 and repeat looking for an absolute maximum on the lock level Z3 and Z4 should not normally require adjustment at least at 300 MHz unless the sample column is quite short When Z3 and Z4 are
70. omatic gain selection by setting gainz n When a data acquisition starts the acquisition status will show Autogain as it adjusts the gain to an optimum value In an arrayed experiment the desire is usually to compare intensities between the various spectra in the array and this precludes having the system change the gain for each FID in the experiment As a result gainz n is forbidden by the software in the case of arrayed experiments The easiest way to set the gain is to select ntz1 and do a go to let the autogain software select a gain value Then set gainz y to disable the autogain set nt the desired number of transients set up your array and go After the autogain is disabled with gainz y gain will report a numeric value between 0 lowest gain and 60 highest gain If peaks are going to be getting larger or sharper during the course of your experiment it may be advisable to reduce the gain by 2 or 3 units gain z gain 3 If the receiver gain is too low you will have poor sensitivity and if it is too high you will clip the peaks off of the FID and introduce harmonic lines in your spectrum This can also generate ADC OVERFLOW errors and or RECEIVER OVERLOAD errors at each pulse These are error conditions that must be corrected The gain parameters are most easily accessed now on the acquisition panel of the Aquire tab After the data are transformed individual spectra can be viewed with the DS command and a subscript ds 1 fo
71. orage devices This is the only computer that you will interact with directly The acquisition processor is a dedicated slave processor that is controlled by the host processor over an Ethernet connection and it controls all the instrumental control functions associated with running the acquisition console and collecting data This includes controlling the RF pulses signal averaging to memory setting decoupler modes adjusting the audio filter bandwidth and receiver gain controlling the locking and shimming operations and handling any other hardware associated with data acquisition NMR USER S MANUAL November 9 2009 3 1 4 KEYBOARD MOUSE There is nothing especially noteworthy about the system keyboard except to note that none of the function keys or special editing keys are used to any significant extent in the NMR software The mouse is a three button mouse that is used extensively for the operation of the NMR program The keyboard can be used to enter commands and parameter changes on the command line but many of these operations are now performed more easily with the mouse There are some operations however that can still be done only on the command line The command line appears directly above the graphic canvas see inside front cover and virtually any number of commands can be entered on a single line There are several points that should be noted about commands and parameters 1 There are just 2 different types of parameter va
72. pace required will be the product of the FID size and the number of elements in the array The second consideration to make is the state of the il flag IL Y instructs the system to InterLeave the data acquisition using the block size 6 parameter This will collect bs transients on the first array value then bs transients on the second value etc until there are bs transients done for each array element The system then cycles back and does the second bs transients on the first array value repeating until all nt transients have been done for all elements of the array The benefit of this is two fold it helps eliminate systematic errors in long acquisitions field homogeneity drift overnight for instance and it permits you to see some of all of your data before the experiment is finished permitting you to stop acquisition early if necessary Half way through an experiment for instance you could see all of your array elements with half of the transients done on each FID giving 40 less signal to noise than the final spectra Without interleave you would have all of the transients collected on half of your FIDs Some experiments such as kinetic measurements will produce meaningless data with the interleave turned on however Acquisition is then started normally with either the or au command The third decision to be made is to choose a value for the receiver gain In the case of normal single fid experiments the system uses aut
73. pectrum manually prepare a normal carbon parameter set preferably by running the normal carbon experiment Use the command apt to modify these parameters for the APT experiment adjust J and any other parameters as needed and do a normal acquisition In particular it is important to be sure that p1 is set to be a 180 pulse otherwise refocusing will be either poor or non existent Due to the spin echo nature of the pulse sequence phasing is liable to be significantly different from the normal spectrum and may require extra attention and patience Note also that it is possible with the rp parameter to completely invert a spectrum so that the methylene quaternaries are upright instead of inverted This is not a problem only the relative phases are significant NMR USER S MANUAL November 9 2009 31 12 2 DEPT SPECTRA The DEPT sequence is much more involved than the APT sequence This sequence varies a proton pulse angle to modulate the carbon intensities based on the J coupling The basic data from this experiment is very similar to that obtained in the APT sequence except that it is now possible to run several spectra with different proton pulse angles and to analyze the resulting spectra in more detail The normal DEPT sequence runs four separate spectra A provided analysis routine calculates linear combinations of these spectra to yield fully edited subspectra showing separate traces for the methyl methylene and methine carbons Since thi
74. plays the output of the receiver This signal is a sinusoid signal whose frequency indicates how far off resonance the signal is and whose amplitude indicates the strength of the signal As you move the magnetic field farther from resonance the frequency of this signal will increase and the amplitude will decrease due to low pass filter circuits in the receiver At the resonance position the frequency will be zero and the display becomes a flat line as shown On the VNMRS consoles there are two traces displayed one for the real and one for the imaginary receiver signal Only the real signal is of interest The imaginary portion is 90 out of phase and should not show any intensity when the lock phase is properly adjusted mx Start Acquire Process Seuup Hardwar Standard Lock off Insert Eject Lock Scan Spin On Spin Regulated Hock Spin Off 20 Hz Shim Status Regulated o Spin Tem 47 N MM P 4791 cia 4791 ower Leve gt Lock Level E 1 30 1 Gain 1 25 62 1 bz ugs Phase PU 9 o 96 10 96 Find zo Gradient Shim x Load Shims Into Hardwaress Figure 1 Interactive Lock Display In the lower portion of the display are the computer controlled switches for turning the lock and spinner on and off ejecting the sample and toggling the lock scan on and off Below that are the
75. r of elements as well as their values and to remove the array if necessary The area on the lower right side allows for changing individual array element values In the top portion listing the arrayed parameters is a column indicating order this is the order of precedence which parameter will be incremented first It is possible to give two arrayed parameters the same order IF the arrays are the same size and thereby increment the two parameters simultaneously generating the diagonal of the array matrix This might be useful for instance if you wanted to run several different decoupling m Array Parameter Param Name Description Size Order On Off dpwr Power level for first decoupler wit 5 2 On Array Size 25 Total Time 0 24 0 UnArray New Array Active Param dof Current Value lo Position Value 1 0 Array Size 5 2 2 3 4 First Value 0 4 6 Increment 2 0 5 8 Last Value 8 Inc Style Linear Randomize Eit Undo Close Abandon Figure 4 Parameter Array Window 34 User Copy Texas A amp M University experiments using a different decoupler power for each frequency Before starting an acquisition with an arrayed acquisition parameter there are three considerations that need to be made The first is the size of the data table Each FID in the arrayed experiment will require a certain amount of disk space and the total disk s
76. r the first spectrum ds 2 for the second etc dssh will display all of the spectra stacked horizontally NMR USER S MANUAL November 9 2009 35 end to end and dssa will do an automatic stack plot adjusting VO and ho the vertical and horizontal offsets between spectra pl will plot the current spectrum given by the indices in the upper right hand corner of the screen and pl all will plot all the spectra using current values of ho and vo Experience is the best teacher in determining how you want to display your arrayed data The ArrayedSpectra tab in the left hand vertical parameter panels has some very useful buttons and options for viewing arrayed data sets 14 1 Ti MEASUREMENTS T1 relaxation time measurements are normally done by the inversion recovery method 180 t 90 sequence The p1 pulse is set to twice the value of pw90 in order to get a 180 degree inversion pulse The d2 delay is arrayed to allow a variable amount of recovery before the DW observe pulse which should be set equal to pw90 The d1 delay must be at least 3 times and preferably 5 times the T value being measured in order to allow the system to come back to equilibrium between pulses There should be at least four t values in your d2 array and preferably 6 8 for a good T determination The d2 values are the times that are plotted on the time axis of the exponential recovery and these should cover a range from roughly 0 1 T to to properly characterize
77. ral regions There are two benefits to be gained by breaking your integral up into regions First a baseline correction can be performed by assuming that all the blanked pieces of integral represent flat baseline and fitting a straight line through those spectral regions The second benefit is that the remaining sections can be normalized so that for instance a methyl peak has an integral of 3 For those who have done integration on the older VnmrX 6 x software all of the old command line methods still work and can still be used These same commands are now also imbedded in the integration parameter panel shown in Figure 3 This panel consolidates all of the various commands and generally makes integration a much easier task To begin select the partial integral display mode in the first column and click Clear Integrals to remove any existing reset points The Auto Find Integrals will do an automatic search for peaks and integral regions You can use this as a starting point modifying the resets if necessary or go directly to the Interactive Resets mode In the interactive mode the left mouse button will add a reset at the cursor location and the right button removes one at or nearest to the cursor When you have selected all of the reset points you want click the Scale Display to fit button This will exit the interactive routine and autoscale the integral display amplitude A good flat baseline is critical to gett
78. re of your sample However you cannot improve the resolution of a poor sample simply by increasing the acquisition time You will simply increase the number of data points defining the peak Ideally the acquisition time should be about 3 T2 where T2 is the observed transverse relaxation time and is related to the natural line width of your sample by the relationship LW 1 n T2 If your acquisition time is longer than that the FID has decayed to zero and you are simply adding noise into the spectrum If it is shorter you are throwing away resolution information The spectral width and the acquisition time combine to define how many data points will be required to store your data set The Nyquist theorem from information theory says that a sine wave must be sampled at least twice per cycle in order to adequately characterize it Thus to sample a frequency Sw the maximum sweep width we must sample 2 SW data points per second If we sample for at seconds the total number of data points required is NP 2 SW AT There is a maximum number of data points possible however based on the amount of memory available in the system This is typically 256K complex points although it may be possible to modify a configuration to get more if necessary The system will attempt to comply with any NMR USER S MANUAL November 9 2009 11 acquisition time you request by increasing the number of points up to the maximum It will then limit the acquisition tim
79. relaxing carbon nuclei The rate of molecular tumbling in the magnetic field will affect how efficient any relaxation mechanism is Relaxation tends to be more efficient at higher molecular weights where the tumbling rate is slower and the correlation times are longer Small molecules molecular weights 250 tend to have long relaxation times However carbons that are locked into very rigid environments with no molecular flexing can have very long relaxation times Table 1 gives a few representative relaxation times and can be used as a guide This list is not at all exhaustive but does indicate the range over which carbon relaxation times can vary If you do not make an explicit estimate of the relaxation times in your system you are simply using a default value that someone else chose for you It is much more advisable to make your own estimates and at least know the assumptions being made Also it is fairly easy to measure relaxation times so that you can in fact run optimized experiments on a given class of compounds and know that the conditions are the best possible 4 ACQUISITION PARAMETERS The acquisition parameters are those parameters which define how data is collected These parameters must be set properly in order to generate a good data set and cannot be changed or corrected after the data set has been collected These parameters define the spectral region observed the ultimate digital resolution attainable and can even affect
80. requires that the CD with the prior sessions be loaded before continuing so that the older directory information can be appended to the new session After the image file is created you are ready to actually write it to the CD R If this is an additional session on a multisession CD go back to the CD Image Info button select update to refresh the information go back to Write Tracks section again Go to the Layout Tracks tab The window on the right will show the available track image files and you can select your file and add it to the list of tracks to write Note that the list of available tracks shows a complete pathname to the image file Once you are satisfied with your track selection you can move to the Write Tracks tab either directly or with the Accept Track Layout button Check the Create Multisession CD button if this is a multisession track check the write speed remove the selection to pad tracks make sure that the Write mode is Track at Once TAO and NOT Disk at Once DAO and press the Write Tracks button to begin recording If you are recording a multisession disk there will be a dialog box to confirm that If there is an error box saying that the media and the session are inconsistent do not proceed until you refresh the CD information again Do not do anything to interrupt the writing once you start it will ruin the CD After the write is completed the CD will be ejected You can re inset the CD R in the drive and it shou
81. riment such as a COSY np will be twice ni In all cases the fourier number represents the number of complex points that will be transformed The normal situation where fn np means that there is one level of zero filling twice as many points are being transformed as collected so another np zeros are added to the data set to fill it out Management of the disk space used by a 2D experiment is done through the np ni fn and fn1 parameters A single complex data point occupies 8 bytes since all of Varian s NMR data is now stored in as 38 User Copy Texas A amp M University floating point values The disk space required for the raw FID data is then calculated as np 2 ni 8 bytes A fairly common set of parameters might have npz1024 and niz512 giving 256K complex points and using 2 Meg of disk space Transformed data is always 8 bytes per complex point floating point representation and the size of the transformed data table is fn fn1 In the above situation both fourier numbers would likely be set to 1024 zero filling in both dimensions and the resulting table is the 8 Megabytes for a total of 10Meg of disk space for this experiment One additional consideration for the amount of disk space used is the phase mode pmode parameter Normally the phase mode is set to partial so complex data can be phased only in the f1 dimension If you set the mode to full you save an extra data table for the f2 dimension and you can then pha
82. rmit the system to return to equilibrium it should ideally be Zero or very short However if it is too short the excitation pulse pW will also be short resulting in very little signal on each pulse since the induced signal is proportional to I recommend the following procedure for selecting values of pw and d1 Having already chosen at for resolution set d1 to zero and use ernst T1 to calculate an optimum pw If pw lt 1 5 uSec or a lt 10 degrees increase d1 and recalculate the optimum pw Repeat until a reasonable set of values are generated Generally this means that d1 will be as short as possible There is not usually a good reason for using longer d1 values except when measuring T values Now consider the case where quantitative results are desired i e proton spectra It is now necessary to allow for complete relaxation of the nuclei between pulses This typically means using a very short pulse 1 2 usec and a long relaxation delay d1 10 20 sec In the case of proton spectra there is an additional complication in that the strongly coupled spectra normally associated with proton spectroscopy have strong second order effects due to cross relaxation and consequently do not relax by simple first order processes This makes an analytical solution to optimum pulse conditions even more difficult The pragmatic approach to this problem is to simply adjust the pulse conditions until quantitative results are obtained After running th
83. rs Comments suggestions and criticisms from users of this document are welcome and solicited since this is written for the benefit of the users Any suggestions to make this document more useful are most welcome iv 1 OVERVIEW The VNMRS Inova and Mercury spectrometer consoles are integrated systems with all the resources necessary to perform virtually all common NMR experiments both 1 and 2 dimensional The routine 300 Mhz systems are dedicated to proton carbon spectroscopy with a nominal proton frequency of 300 Mhz They can be switched automatically by computer between proton and carbon operation without any operator intervention The host computer system is a Dell PC workstation running RedHat LINUX as its operating system These computers rarely perhaps once every 3 months require rebooting The NMR application program is one of the applications available to the operating system and runs under the X windows system Throughout this manual system commands will be shown in bold arial font face help Optional or user supplied arguments will be shown in bold italic arial font face svf filename 1 1 WHAT IS FTNMR Fourier Transform Nuclear Magnetic Resonance is a technique used to rapidly perform a variety of NMR type experiments using Radio Frequency pulses An RF pulse is used to excite all the nuclei of a given isotope in the sample and their return to equilibrium is monitored as a function of time This generates the Free Indu
84. rtant when performing these experiments to be sure that the sample is clean and degassed to remove any dissolved oxygen Any paramagnetic relaxation mechanism will be much more efficient than the dipolar relaxation and will obscure the Overhauser interaction The two dimensional noesy and roesy experiments can be run readily using the Homonuclear 2D submenu in the experiments menu The biggest problem in setting up one of these experiments is in choosing a value for the mixing time This is the amount of time that the spins are allowed to cross relax to generate the information of interest If the mixing time is too short there is little cross relaxation and little information in the form of cross peaks If it is too long the correlations begin to decay and dephase and the information is lost again It may be necessary to run several experiments to find the optimum value for a given sample Generally 32 User Copy Texas A amp M University low molecular weight samples will have longer relaxation times slower relaxation and require a longer mixing time to generate cross peak intensity Typical values for mixing times will be between 100 and 500 or more milliseconds One dimensional versions of the n O e experiment are also available Traditional n O e Difference spectroscopy consists of performing a low power decoupling experiment on a peak of interest and looking for enhanced intensity for signals that are cross relaxing with the irradiated
85. s is a population transfer experiment quaternary carbons are not observed at all and like the APT sequence it is susceptible to problems if the J value is selected improperly The final drawback to this sequence is that it requires four separate data acquisitions thus taking more time than the APT Its primary advantage however is the simplicity of the interpretation Again the easiest way to run this experiment is from the Proton Carbon Multiplicity submenu in the experiments menu If you desire to run this experiment manually establish a normal carbon parameter set and use the command dept to modify the parameter set for the DEPT experiment Modify any parameters you want and start the acquisition After the acquisition is complete phase the first spectrum using the commands ds 1 aph Display a threshold adjust it to include all the peaks in your spectrum type nll to force the system to find the lines and then perform the automatic analysis using the adept command This will require a minute or two to run be patient The spectra can then be plotted automatically using the command pldept 13 NOESY ROESY and NOE DIFFERENCE SPECTRA The noesy roesy and n O e Difference techniques all generate information about molecular conformation using dipole dipole through space interactions These techniques depend on dipolar relaxation as being the primary relaxation mechanism available to the spins of interest This means that it is impo
86. s selected the appropriate data is displayed along with a menu bar of Graphic Control buttons These are initially displayed on the right hand edge of the graphic canvas but you can grab the three dot handle and drag it to what ever location is convenient On the plot parameter page under the Processing Tab is a button to toggle the wysiwyg What You See Is What You Get parameter on and off If this is turned off wysiwyg n then the entire display area is used to display your spectrum This is normally the most useful mode If you turn this on Wwysiwygz y then the system uses only a portion of the display area and maintains the proper aspect ratio ratio of horizontal and vertical dimensions for your selected output device Since the laser printer is more square and the graphics display area more rectangular this means that only a portion of the available screen is used 8 PLOT QUEUES The plotter parameter defines both the physical device that will be used for plot output and the logical format that is generated for that device PCL HPGL PostScript Since the output devices printers and plotters both may be used by more than one user at a time a plot job is collected as a file and queued to the output device when it is complete When you first issue a plot command such as pl an output file is opened output is written to that file in a format specified but the selected output device and a flag is set up on the top of the command line
87. s that the system can relieve the operator of the chore of manually locking the instrument and can optimize both the lock power and the lock phase for the inexperienced operator The disadvantages are that the autolock can fail with samples that are either weak poorly made or have short sample columns and autolocking can take considerably longer to accomplish than manual locking by an experienced operator There are two different ways that autolocking can be accomplished The simplest is to use the lock command which simply causes the system to execute its automatic routine immediately The other possibility is to set the parameter alockz a which will cause the system to automatically lock before beginning the data acquisition The caution here is that the system does not reset this parameter after successfully locking the sample and if you change a parameter and re acquire data the system will repeat it s autolock procedure unless you explicitly reset it with alockz n Although still available there is no particular advantage to using this autolock function instead of the newer 20 operation 9 2 MANUAL LOCKING Manual locking of the field gives you the most control over what is happening and may be required in especially difficult situations weak lock mixed solvents There are four parameters which control the field lock and these are controlled by computer controlled sliders during manual locking The first is the 20 the magnetic field
88. se your spectrum in both dimensions The price for this is a doubling of the disk space to 16 Meg in the above example It is also possible to set the phase mode to none and reduce the complex data to real phased data and the disk space requirements are then halved to 4 Meg The only problem here is that you will not be able to phase in either dimension This can be useful to reduce disk usage after you have determined the proper phase parameters Since you have a temporary 20Meg overdraft on the disk quota it is possible to set the phase mode to full transform and phase the 2D spectrum set phase mode to none and retransform This will reduced your disk usage and prevent inadvertent changes to the phasing Since the raw FID data is never modified you can always go back retransform and rephase the data if necessary Be careful with some of the parameters generated by Varian s automatic 2D routines They sometimes use unreasonably large values of fn and fn1 and chew up large amounts of disk space to no purpose There is no good reason to have ni 512 and 11 16384 It just uses a lot of disk space and slows down display and plotting routines The normal display command is dconi Display CONtours Interactive and this provides the same sort of functionality as the normal ds command does for 1D spectral There is no good easy way to plot all 2D data although the autoplot button on the menu bar does a good job for many spectra There is a plcosy
89. sed for spectrum adjustment and their function will change depending on what you are doing There is a parameter display in yellow on the bottom of the graphics screen showing which parameters are being controlled by the three mouse buttons The middle button is almost always used for a vertical scale adjustment while the left and right buttons are typically used for starting and stopping values In addition to the normal functions the middle mouse button can have a separate function when used at the left edge of the graphics display There is a fourth parameter displayed on the bottom left side of the graphics window for this additional function If you look at the spectrum display in on page you will see that values for the cr VS and delta parameters are displayed on the bottom right of the display The left button will adjust the left cursor position the right button will adjust the difference delta value between the cursors and therefore indirectly the right cursor position and the middle button will adjust the vertical scale On the far left side of the screen the vp parameter is displayed Using the middle button near the left edge of the graphics window within 5 of the displayed spectral width perhaps will cause the vertical position rather than the vertical scale to change This parameter display can be turned off usually to facility clean screen print output if desired To turn it on or off go to the Frame tab in the vertical
90. sliders that control the various parameters used in locking the system In all cases the parameter name and value will be displayed on the left side and the slider will show proportionately where that value falls between the minimum 16 User Copy Texas A amp M University and maximum allowed values for the parameter and the parameter value box will let you increase left mouse button or decrease right mouse button the value by the increment shown in the box The increment can be changed by clicking the middle button You can pick up the slide bar with the left mouse button and move it back and forth changing one of the parameters interactively This is sometimes useful with the 20 parameter but not usually with the others The proper method for locking the system is to insert the sample into the probe using the Eject and Insert buttons adjust the spin rate if necessary and to turn the Lock Off opening the feedback loop If this is not done large compensating corrections can be introduced into the system resulting in an unstable lock Once the feedback is turned off the Z0 lock power and lock gain are adjusted for a good resonance condition zero frequency sine wave and the lock is turned back On closed lock feedback loop The good resonance condition is recognized more by experience than by description but generally it will look very similar to a normal lock signal although the phase may vary and even be inverted The critica
91. strtext parameter defines the starting data point number for the extrapolated data In the case of a forward calculation this will be the first data point added and in the case of a backward calculation it will be the last data point calculated The Ipext parameter defines the number of data points to be calculated and included in the data set This number should be no more that Ipnupts the number of points used to calculate the coefficients The lpalg parameter defines the algorithm used to calculate the coefficients and should always be Ipfft The only other valid value is which can handle noisier data and broader lines but cannot model narrow lines Finally the proc parameter defines the processing used by the wft command A value of ft does the normal fourier transform and a value of 1 will do the linear prediction instead NMR USER S MANUAL November 9 2009 47 The following diagrams are a visual representation of the inter relationship of these various parameters of the parameters are measured in complex data points When performing 2D hypercomplex data acquisitions the ni parameter counts the number of complex points collected in the t1 f1 dimension In both 1D and 2D spectroscopy the np parameter indicates the number of computer data points collected real and imaginary not complex points Consequently there are np 2 complex points collected The first example demonstrates doing a backward prediction on a 1D
92. t access doesn t do you any good This is especially true if you are using a PC or MAC to burn your data to CD R media There are many options and modes available for burning CDs and not all of them are able to be read back on the spectrometer system Truncated names extra periods and capitalized names are some of the problems that have been encountered The procedures established for burning CDs on the Linux system drives are known to work well and create disks that can be read back on the workstation as well as on PCs and MACs 10 1 On Board Storage Devices The host processors all have a couple of different options for storing your data on removable media The nmrsunl workstation has a 3 1 4 floppy disk drive which is probably not very effective or efficient for storing data but it is an option if that format is useful to you All of the other hosts have a multi format card reader instead of the floppy disk drive This card reader will read virtually all of the various flash memory cards SM xD SD etc that are often used in cameras and other portable equipment This can provide a lot of memory in a very small form factor The other storage option available on all of the hosts are front panel USB ports that will support more traditional memory sticks and USB disk drives All of these media can be used on either the spectrometers or the workstations to store and access NMR data separate from the NMR file server environment 10 2 CD Burner
93. t it is not desirable to add 24 User Copy Texas A amp M University it as an internal reference To do this referencing it is necessary to run a reference spectrum of the reference material at the exact same magnetic field strength as the analytical sample The normal procedure is to insert the analytical sample lock and shim the field and set up the desired observation parameters for the sample The sample is then unlocked and the 20 value carefully adjusted to be exactly on resonance The sample is replaced with the reference sample a few pulses collected with the lock still off and the reference set as above The analytical sample is then replaced and the lock turned back on The spectrum of the analytical sample is then collected with the referencing already properly set The key to this working is that the 20 value is not changed while the reference sample is being measured Default referencing is set every time a setup command is run This referencing is based on the deuterated solvent and is accurate only if the proper solvent is specified 7 3 INTEGRATION Integrating a proton spectrum is perhaps one of the harder tasks for new users The integration routine provides a means of optionally blanking alternate sections of the integral When integral blanking is turned on pmode partial the odd sections of integral first third fifth are not displayed and the remaining regions second fourth sixth etc are the active integ
94. te the data to the CD and eject it In the case of a USB device simply close the folder right click on the desktop icon and select unmount volume and the device will be unmounted and the icon removed from the desktop You may then remove it from the USB port You can also left click the File menu on the USB folder to get a menu item to unmount the volume Either one will work When you write to a CD the disc is closed and you cannot write any additional data at a later time Note that it is not necessary to have VnmrJ running while storing or archiving this data everything is done by the Linux operating system To access your data on either a CD or a USB device login to workstation and insert the device just like above A CD with data will act the same way as a USB device does except that you cannot write additional data to the disc You can open VnmrJ and use the file browser or the file open menu to navigate to your device to open files The devices are all mounted in the media directory so navigate up the directory structure until you get to the root directory then move down into media to find your device Since you can have both a USB device and a CD R disc mounted at the same time you can archive data from a memory stick your nmrdata directory if you have one as well as your home directory onto the CD R disc It is also possible to use the same procedure to write DVD s using DVD R media There is currently no reliable method
95. tegrated intensity of the lock resonance will be constant As we improve the magnetic field homogeneity so that all the molecules in the sample experience the same magnetic field strength all the nuclei of a given type will resonate at the same frequency and the signal will become narrower and more intense Thus the lock level will be a measure of magnetic field homogeneity It is essential for this process that the lock signal not be saturated With the current software there are two fundamentally different mechanisms for doing automatic shimming in addition to provision form manually shimming the field In addition the older lock level automatic shimming can be done either explicitly or automatically with a data acquisition 6 1 AUTOMATIC SHIMMING The preferred method of automatic shimming on systems with a Pulsed Field Gradient amplifier and probe check for gradtypez nnl or gradtypez nnp to see if these are present is to let the computer do a gradient shimming on the deuterium signal This is a relatively new technique and when it works properly it can generate very good homogeneity in very little time Once your account is set up for gradient shimming by loading a shimmap file all that is necessary to shim is to run the gmapshim command on the command line The gmapshim can be run from the Gradient Shim buttons on the Standard and Lock panels of the Start tab The command gmapsys will let you choose an appropriate shim map the first time you us
96. the distance in Hertz from the upfield end of the spectral window Internal referencing is done by placing the cursor line on the reference line signal using the nl command nearest line to be sure that the cursor is exactly on the line and using the rl command to define this as the reference line The rfp parameter is then set to the appropriate value for this reference material There is a REF button on the menu bar for the interactive display that will do this for you as well External referencing is very similar to the internal referencing except that the reference material is external to the analytical sample This means that the reference material does not experience the same bulk susceptibility as the sample and the referencing will be wrong by a small amount The external reference may be a small capillary of material inserted into the analytical sample or a separate sample altogether In the case of an inserted capillary the referencing procedure is identical to that described above for internal referencing Using this technique has two problems First the inserted capillary will introduce spinning noise and artifacts that degrade the quality of the spectrum Second it is virtually impossible to control the concentration of the reference material present External referencing with a second sample is most often done for nuclei other than proton and carbon where the reference compound is more exotic and perhaps chemically reactive so tha
97. ties Ti2T2T It is necessary to make some estimate of the T relaxation time since underestimating this relaxation time can cause severe attenuation or even suppression of signals Generally estimating proton relaxation times is not a problem since they tend to be fairly short and proton acquisition times tend to be long for good resolution The primary effect of long proton relaxation times will be seen when attempting to obtain quantitative integrals Carbon on the other hand poses a definite problem since the wide sweep widths used prohibit long acquisition times and carbon relaxation times can run to several hundred seconds in unfavorable cases What is necessary then is to understand the conditions that promote efficient relaxation so as to recognize problem systems compound T1 Seconds compound T1 Seconds Benzene 30 Pyrene Benzonitrile CH on axis 5 C CN 130 CH off axis 6 5 C CN 110 Cx off axis 130 CH ortho 10 Cx on axis 230 CH para 6 Adamantane Cholesterol CH2 11 ring CH2 0 25 CH 20 ring CH 0 5 Methyl cyclohexane ring CH3 1 CH3 12 side CH3 1 6 CH2 14 CH 22 Table 1 Representative Carbon 13 T relaxation times NMR USER S MANUAL November 9 2009 9 The single most effective mechanism for carbon relaxation is a dipolar interaction with a proton Since the relaxation due to dipolar interaction is inversely proportional to the sixth power of the distance between the nuclei only directly bonded protons are very effective in
98. tions tab The options that should be selected by default are Rock Ridge anonymous allow 31 character names Joliet extentions and allow ISO names starting with a period Verify that these are selected and select Save Defaults if necessary Under 7509660 Header tab enter a volume ID for your CD This should not contain any spaces and the number of characters is limited to 32 You may also enter Publisher and Preparer ID information if you want and may also choose to save these default values Next move to the Create Session Image tab There will be a delay as the system calculates the size of the session you are attempting to create The Session Information will show both a size data CDs as well as the equivalent time audio CDs for the new session as well as the space used and space available on the CD R The Create Session window shows the available free space on the disk for creating the ISO9660 image file This space must be larger than the size of the session In the window on the right select Create multisession CD unless you are creating one large single session disk Eject after Write Do not pad tracks used for audio Cds and enable Buffer underrun protection Press the Master to Image file button to start creating the ISO9660 disk image file If you are creating a multisession image you will get a dialog box asking whether this is the first session of a multisession CD or an additional session If it is an additional session it
99. to specify the type of modulation used during each of the periods in the pulse sequence dmm can take on a variety of different values to specify the types of modulation Most of them are dependent on the hardware NMR USER S MANUAL November 9 2009 13 being used but at least one may be software driven depending on the decoupler present A value of C is used for continuous wave or single frequency irradiation i e no modulation This is the only permissible value for homonuclear decoupling and is sometimes used for single frequency decoupling experiments in carbon The so called waltz modulation mode is selected with dmmz w The waltz mode provides decoupler modulation over a very wide range with minimum power but it requires an accurately calibrated value of the modulation frequency The waltz sequence is actually a series of 90 degree pulses on the proton channel and the dmf is used to time these pulses The requirement is that dmf 4 y 21 Ho where 2 2 is a measure of the decoupler power in Hertz The decoupler is normally calibrated and a valid combination of values for dpwr the decoupler power setting and dmf is set in the probe file so that these values are setup properly with each set of new parameters It should not normally be necessary to change either the decoupler power setting or dmf from the default values If you are having trouble getting good decoupling and suspect a calibration problem try changing to dmmz f whic
100. ton fid carbon fid dqcosy fid etc After I select the userA directory from the tree and choose Add if I choose the root directory for the path name then the full path name is removed and when I look later at the CD the CD root directory will have proton fid carbon fid etc If I choose the last path component then the CD root directory will have a directory with the data files contained within it This is probably the most useful option for most applications If I choose Someg roup UserA as a custom path then I specify that the data files in the USerA directory be placed in a directory Someg rou p USerA on the CD You can choose as many different data directories from various locations as you want in order to create your session Furthermore you can choose a directory from the session list and either remove it or change the pathname that will be used for it on the CD The only caveat here is that as you create the session no calculation is done as to how much space is being used You may want to use the du summary command ahead of time on your data directories to decide how much of your data will fit on a CD The information box underneath the session window showing used space available space is referring to space already recorded on the CD R that is currently loaded in the drive not the session you are creating Once you have chosen the data for your session you can move from the Master Source tab to the ISO9660 op
101. vember 9 2009 41 REBOOTING ACQPROC INOVA and MERCURY CONSOLES VnmrJ uses an ethernet connection between the host computer and the NMR electronics console to allow control of the spectrometer The connection requires two way communication between the computer and the console and each end of the communication has a process running to conduct this communication In the normal course of operation it is not usually necessary to do anything with this process it goes on in background very reliably When there is a problem with the communication it is necessary to manually restart the communication process This is evidenced most commonly by an Inactive status message in the acquisition processor status window at the bottom of the screen but it may also be required any time there is a problem with console access running acquisition that cannot be stopped unable to start an acquisition or eject a sample etc The acqproc process is the process on the host computer that controls this communication Usually all that is necessary is to stop the process and then restart it This is done readily using the command Su acqproc this is a toggle type command that looks for a running acqproc process If it finds one it stops it and if it doesn t find one it starts it This is run in a Linux shell window right click on the desktop area and select Open Terminal to get a shell window When you run su acqproc it tells you whether it is stopp
102. y only has enough resolution for 1000 1200 horizontal channels which is not adequate for a data set of 8 32K or more data points The laser jet printer can support closer to 6000 points permitting much better fidelity in the reproduction of your experimental data 22 User Copy Texas A amp M University 7 1 PHASING Spectral phasing is accomplished most often with the autophase command aph This works well for most spectra but it sometimes has trouble with spectra containing broad resonances and resonances close to the edge of the spectral window It is possible to manually adjust the two phase parameters and the mechanism for doing so is fairly straight forward There are two phase parameters used to phase a spectrum rp and lp rp the right phase parameter is a zero order correction that affects the entire spectrum equally Ip the left phase correction is a first order correction that applies a linear phase shift across the spectrum It has no effect on the right edge and maximum effect on the left To manually phase a spectrum select Phase mode button from the graphic control buttons menu The bottom labels show that the left button controls the right phase the right button controls the left phase that is not a typo they are swapped and that the middle button controls the vertical scale This is not actually the case although these are the parameters being controlled At any one time you will be adjusting either the left or th
103. you open the Network Neighborhood on a Windows microcomputer you should see an NMR group You will need a separate Samba NMR password to login on the server and then you should be able to open your spectrometer directory space from Windows You can then use the Windows File Manager to drag and drop data files from the Linux environment to the Windows platform and use your favorite archive utilities there There are some administrative issues with the current samba server utility and this may not be operating as well as it should 10 4 Network Directories If your group has a networked Linux workstation it is possible to use NFS to export a directory or file system from that workstation to the NMR spectrometers There is provision for automatically mounting such a directory as nmrdata lt group gt so that your workstation directory is directly available for storing your NMR data as you collect it You are limited only by the space on your workstation file system and you can archive your data from that system at your convenience as you collect it 10 5 FTP File Transfers Finally the File Transfer Protocol FTP utility is available on the Linux workstations so that you can use ftp either from the workstation or from a remote system to move your data sets to virtually any platform for storage and archiving Be careful with FTP however since it does not inherently or automatically descend into subdirectories Varian NMR data sets are stored as a
104. you to locate it to wherever is convenient on the screen Placing it in the empty space of the User Tool bar just above the command line is convenient in that you need make only small vertical moves between the spectrum and the tool bar rather than longer horizontal moves to the edge of the window 6 User Copy Texas A amp M University 1 6 PRINTER PLOTTER Printing and plotting on the spectrometer systems is all done to a local laser jet printer There is no provision for plotting to other remote printer devices on other workstations since all processing and work on the spectrometers should be local The three standalone workstations can have provision for printing to remote networked printers elsewhere in the building If your group has a computer room with several computer systems and a networked printer it may be possible to set it up for remote plotting and printing if it is not already The VnmrJ software has provision for plotting to both Black and White and Color Laserjets There is also provision now through the screen print and plot utilities to generate graphic files of plots in a variety of different graphic formats GIF JPG TIFF This permits easy incorporation of spectra in your thesis or publications 1 7 DATA STORAGE Each user account has it s own data storage quota typically 100 Mbytes and storage space It also has its own set of VnmrJ configuration files and experiments so that no mistake or problem with one user s

Basic VnmrJ Manual for version 2.x - TAMU NMR Facility

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