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1 - Department of Chemistry - University of Wisconsin–Madison

1. use Sunview to get normal acquisition setup acqi for the acquisition lock fid shim window and right button for acquisition status UWChem MR PFG Shimming With VNMR Page 71 Pulsed Field Gradient Shimming With VNMR cg fry created 99 01 27 updated 99 02 01 I General Discussion Pulsed field gradients allow analytically accurate automated adjustments of shims The technique uses gradient profiles more pictorially described as 1 dimensional images of the atomic density of either protons or deuterons see Section IM below for a description of how the profile is experimentally generated The picture below shows a sample tube with solvent in gray and a crude picture of a standard rf coil about the tube On the left side of the tube the density intensity of observed nuclei either H or 2H is shown as a function of position along the z direction with the assumption that the rf coil observes nuclei at equal intensities at all positions inside the coil boundaries and observes nothing outside the coil The profile to the right is a depiction closer to the real case where the rf coil loses intensity slowly outside its boundaries and has a slight loss of intensity in the center of the coil compared to the intensity closer to the horizontal wires ae oe ee 2 Z1 offset 22 Z2 A Z normal profile 7 Or 23 Z3 I nuclei I nuclei The normal profile is observed in the presence of a large
2. gt 0 Tip T e Usually a quick determination of the T is sufficient this allows proper setting the repetition delay d1 For most liquids use the rapid inversion recovery method in Section II below This method involves finding the time at which the magnetization goes through a null tp as shown in the figure on the right below More accurate determinations of T must utilize multi point fitting as described in section III Rapid Determination of T by Inversion Recovery Null Method good for any sample e g any liquid but is semi quantitative at best e Obtain a reasonable quality H spectrum Locate protons of interest for visual observation e 180 and 90 pulse lengths must be reasonably accurate If your sample is highly polar e g ionic water solution you must calibrate beforehand Otherwise if tuned correctly prior calibrations can be used e set pl 2 pw90 pw pw90 e d2 0 01 should give large negative peaks Lengthen d2 until proton peaks of interest are nulled slightly shorter d2 should give negative peaks slightly longer should give positive peaks For the nulled d2 T d2 In2 d2 1 44 e Find d2 for other protons of interest UWChem MR Varian NMR User s Guide Page 60 lll Quantitative measurement of T by Inversion Recovery Method A Comments e A reasonable estimate of T must be known to correctly setup use the Inversion Recovery Null Method Section II e 180 and 90 pulse
3. A Prepare a tar file see section I A above B Backup file to IOmega Zip drive Go to any One with a Zip is preferred of the PC s in 2224 or 2210 Insert your Zip disk in the drive you intend to use open WSFTP Winsock FTP 32 bit connect to the host computer of your VNMR account ex zhadum chem wisc edu as the remote system using your login name and password set the Zip drive as the local directory in WSFTP Select the tar file you wish to backup in the remote window hold the lt CNTL gt key down to select many files then transfer it to the local window using the left gt right arrow D Recovering backup ed up data see section I D above lll Backups to Pinnacle Optical Disk on Tango on 5 floor You must obtain specific permission from the Kiessling group to use this device A Format a New Optical Disk enter the following commands login as root see Director for root password exportfs a should re establish connection to opdisk if lost somehow cd usr local pmo pmo_format sun4 x usr local pmo pmo dat Enter Choice 0 sd0 esp0 slave 24 SCSI ID 3 slave 8 3 Enter Choice 0 PMO 650 format gt defect defect gt commit defect gt yes 0 defects usually defect gt quit format gt answer y lt small case format gt partition Partition gt Selecties tenann eoe ara dette les delire st aged 0 is whole disk partition gt label partition gt quit UWCheM HRI Data Backups Page 68
4. vnmr maclib manual menulib psglib seqlib shapelib shims move to home account for computer working at export home username moves to export home fry on vorlon workstation reads data fid file datafile name must have fid as suffix vnmr always appends as such with svf command all datafile fid files are actually directories containing LG shea eae a aide ea aed actual fid data NO Be uen ice ee eee cite 7 log file shows times for acquisitions PLOCD AMS occ scorers eer init processing parameters COKE eobre ine ene lata ces becstevecth ne a text file created by command text reads parameter file parfile name must have par as suffix vnmr always appends as such with svp command all parfile par files have only procpar and text files data acquisition area can go up to exp9 but can take significant disk space user macro library user manual help for pulse sequences directory user menu definitions directory user created or modified pulse sequences user compiled pulse sequences user created RF and gradient shapes e g for NOES Y1D user stored shim files system macro library system manual help for pulse sequences directory system menu definitions directory system pulse sequences system compiled pulse sequences system stored RF and gradient shapes e g for NOES Y1D system facility stored shim files IV Common Pulse Sequence Setup in VNMR this section is directed at students wantin
5. 1d Acquisition Parameters H G sweep width CO center or offset frequency tof solvent none change O1 thru solvent cdcl3 with solvent set correctly in VNMR tof 0 will center spectrum for jobfile normal organic compounds set spectrum window EP set window O set cursors movesw 1 set offset frequency EP set cursor O1 relaxation delay AM AC delay differs depending on ZG or AU to run experiment 1 common pulse width 90 length fixed by probe on AM AC s depends also on tpwr on Unity acquisition time AQ TD DW aq np sw dwell time DW 1 2 SW Bruker acquires complex pairs sequentially vnmr simultaneously of transients to acquire Bruker NS 1 which goes continuously vnmr nt 1e6 receiver gain RG larger gt gain larger gt on Unity if gain 0 still clips get ADC OVERFLOW message insert larger gain larger gain attenuator at preamp output observe transmitter power tpwr 52 higher AM AC s observe power is fixed Unity s have linear amplifiers on higher power both observe and decouple number of points acquired TD usually S np temperature see temp24 and similar macros written at UW decoupler transmitter power DP lt ret gt 20H lower dpwr 40 lower vnmr parameters are logical higher power lower power UWCheM HRI Intro to Unix and VNMR Page 18 1d Processing Commands and Parameters fouriertransform TPE number of points FT d zerio filling oc
6. format gt format takes 15 min format gt quit B Create File System on New Optical Disk enter the following commands mv usr etc mkfs usr etc mkfs orig cp p usr local pmo mkfs sun4 usr etc mkfs newfs c 336 d 0 dev rsd0c C Backup to Optical Disk insert the disk into the drive mount the optical disk with the command optmnt use the cp command to copy directories e g Cp rp home fry newdata opdisk newdata unmount drive when finished optdmnt IV Backups to Pinnacle CD Writer on Twiddle on g floor See Brad Spencer for specific questions about this device A Transfer Data to Twiddle Twiddle is the Dell with the 21 huge monitor in the far corner of the 9 floor computer room Twiddle OPEN or CONNECT and it will ask for your password you should see shadow in one window and the L drive empty in the other click on the directory s that you want transferred in the shadow window hold the SHIFT key down to select a range of directories or hold the CNTL key down to select random directories make sure BINARY transfer is selected press the lt or arrow button sending the data from shadow to the transfer runs at 0 5Mb s so 600Mb takes 20 min close WS_FTP32 when the transfers are finished Transfer Directories Files to Easy CD Window insert your CD using a CD caddy into the Pinnacle drive transparent side of caddy to right unhook the ethernet phone line connection at the
7. display stack horizontally return display of one spectrum to full width display stack using v0 and h0 as vertical and horizontal offsets UWChem 7 macros tuneh tunesi tunec loadshims axisp axish dsx invert tempoff tn H1 gain 0 su use UWMACROS TUNE H1 tn Si29 gain 0 su use UWMACROS TUNE S129 tn C13 gain 0 su load y su load n UWMACROS LOADSHIMS axis p axis h wft dscale 3 rp rp 180 inverts the 0 order phase of a spectrum vttype 0 temp N SU oo eee crucial command before probe change UWCheM HRI Quick Guide for New Users Page 13 temp24 vttype 2 temp 24 su vttype 0 su model temp macro settemp vttype 2 temp x su wait vttype 0 su intnorm normalizes peak to user specified value e g methyl peak to 3 disp2d sets up wp sc nicely for 2d plots pconpos plots contours of 2D spectra with positive peaks getting 10 contours negative peaks getting just 1 pconneg opposite of pconpos Ipforward sets up for forward linear prediction UWCheM HRI Intro to Unix and VNMR Page 14 lll VNMR Directory Structure There is a user vnmrsys area and a system vnmr area All files are looked for in user area first if not found there then the system area is checked cd cd vorlon fry rtf datafile rtp parfile vnmrsys exp1 maclib manual menulib psglib seqlib shapelib shims
8. 2225 Teaching Assistants Lizheng Zhang rm 8131 Ekasith Somsook rm 7363 Daniels 262 8828 262 3182 o 276 0100 h 262 7536 o 262 0563 262 8196 262 7948 262 0414 treichel chem wisc edu fry chem wisc edu cgfry facstaff wisc edu whittemo chem wisc edu marv chem wisc edu clausen chem wisc edu lizheng chem wisc edu esomsook chem wisc edu UWCheM HRI Varian NMR User s Guide Page 6 Quick Guide for New Users created 12 01 97 updated 9 27 99 I Login e Username practice Password do not use 9th floor password nothing from a dictionary at least one number case sensitive e datato zhadum practice other partitions europa ganymede starbase UNIX mkdir name VNMR FILE left click on name CHANGE will place data in practice name e shims macros etc to export home practice vnmrsys shims or vnmrsys maclib etc e exit VNMR before logging out ll Setup A 1 Time e MAIN MENU SETUP 1H CDCL3 this will put reasonable parameters in e phasing 100 Shows complete spectrum while phasing e MAIN MENU MORE CONFIG SELECT PLOTTER ngbar_plot or shadowp_plot SELECT PRINTER ngbar_print or shadowp_print B Parameters e Setup probe and pulsed field gradient parameters using macro with probe name e g type bbswg produces probe bbswg pfgon nny bbold probe bbold pfgon nnn hex gt probe hcx pfgon nny It is critical the probe parameter is set
9. check about the 360 value with an array increment size of 0 5 to 1 us plot arrayed spectra using the following commands wft dssh transforms all spectra and displays in a horizontal stack full resets plot area to full screen UWCheM HRI Varian NMR User s Guide Page 29 E 2 Order Shimming on a 500 MHz Spectrometer Make sure the lock is not saturating Check by watching that the lock level increases consistently with increasing LOCK POWER Once the lock level drops or stays steady with increasing power back off the power by roughly 20 I ve heard some Varian chemists look for a 50 decrease when dropping LOCK POWER by 6 dB when LOCK POWER is ok i e low enough to not be saturating but I ve not seen consistent results doing this I simply look for a bounce in the lock level and go 20 below the setting at which the bounce is last observed can be difficult for fast relaxing solvents like D O Adjust LOCK GAIN to give lock level in 25 65 range Optimize lock level using LOCK PHASE Optimize lock level with Z and Z2 Change Z in one direction enough to change lock level by 5 10 since optimized in previous step level will decrease Re optimize lock level again with Z If newly optimized lock level is lower than the previous one try changing Z in the other direction otherwise continue changing Z in same direction until re optimization does not improve the lock level Keep changing Z in the same direction and opti
10. check pw90 s of X and H observe not decouple e if numbers are close to facility values probe is likely OK e if pw90 is much less than facility value you are doing something wrong figure it out e if pw90 is much longer gt 1us than facility value find TA or facility staff perform calibrations for overnight or longer runs for HMQC HSQC experiments tune probe at 13C then H for 3C enriched sample 3CH3I Indirect 1 or NaOAc C calibrate pw90 for H as normal set tpwr to desired setting see vnmr probes probe probe for most current make sure you are on resonance set cursor on 2CH3 I singlet surrounded by 3CH I so looks somewhat like a triplet then movetof set di 1 at 1 for Indirect 1 samplpe use 360 pulse for final checks always once observe pw90 is obtained assuming in exp then jexp2 obtain 3C in exp2 move X cable to observe port and place 3C H3I quartet on resonance place cursor in middle or quartet and movetof jexp3 mf 1 3 wft put 73C on resonance by setting dof 75C tof from above setup decoupler calibration experiment with UW macro pwxdec90 check tof and dof check pwxlvl 58 and find pwx where antiphase doublet nulls this is decoupler hard 90 set pwxlvl 46 appropriately for decoupling probe dependent see Table on wall or vnmt probes probe probe and find pwx where antiphase doublet nulls set dpwr pwxlvl and dmf 1 pwx le 6 E Data Workup
11. died ns tte ui in sd A 9 VIII DIGI US ESS telat ieee dim chen acer A A obec nd chaste hed a Ne M tante 9 Introduction to Unix and VNMR ss ssssssnnnnnnssnseneneneeeeenennnnnnnnne 10 l Common Unix COMMAS 3 Sen detente nine 10 Il Common VNMR Commands Parameters and Flags ceeeeeeeeeeeeeeeeeeeees 11 IL VNMR Directory Structure 28 retarde than ed alter 14 IV Common Pulse Sequence Setup in VNMR eee eeeeeeeeeeeeeeeeeeenaeeeees 14 V VNMR to Bruker AM AC Parameter Conversion Table 16 MELE Troubles Shootings LE RL SR nn na tn ee ds 20 OpSratlon OF UNIES 00 nine ninia aenea rene rene rant tort cena eine 22 Proper Exiting and Logging In and Initial VNMR Setup 22 Il Commands for First Time and Novice Users 0 eeeceeeeeeee eee eeeeeeeeeeeeeeeenenaaeeees 22 lll Probe Changes ss coteeeactinetcacten a nn mena antennes 22 Table 1 Description of Probes on Unity 500 and Inova 500 23 Table 2 Calibrations of Probes on Unity 500 24 Iil Probe TUNNO AR D nn D lead 24 M LOCK And SIM Seeds es de nae cd ta tt tel ne et Re es AR At Sn en ea 25 Table 3 Field and Lock Power Settings for Unity 500 25 Table 4 Major Shim Interactions on Unity 500 26 Table 5 Shim Sensitivities on Unity 500 26 Experiments on Unity 500 csiiiicncnviiienciieeni naan 27 l Normal 1d A AC quisitionica acishonnccctteadartandsomaacioiantcenctaeneceanceaaies 27 D 1H pw90 Calibration 28 UWChem MR Tabl
12. or ppa for the major parameters only does plot out text which can be entered manually but easiest way is to use the CDE File Manager to go to vnmrsys exp1 assuming working in exp1 and double click on text file simple editing of the file can then be performed plots peak pick th controls threshold height used dpf will show peak picks on screen dll lists peak picks with intensities printon dll printoff will print ppf UWCheM HRI Experiments 1H Id Acquisition Page 30 pirn prints integral values under axis start with cz to clear integrals click MAIN MENU DISPLAY INTERACTIVE PARTIAL INTEGRAL type region if you don t like the regions start again with cz and use the RESETS button to enter regions with the mouse manually type intnorm oruse UWMACRO MORE NORMINT or DISPLAY NORMINT adjism is Varian s not so good command to adjust the peak amplitude enter vp 12 to get axis out of list area integrals will plot with pl when on screen pirn is needed to list values below axis UWCheM HRI Experiments X Id Acquisition Page 31 X Acquisition e g 31P or 13C 22 Jun 98 A Discussion The spectrometer acquisition time of an X nucleus experiment should be estimated from similar experiments on the same equipment if possible using eqn 1 or 2 below 3C and P experiment times on the 500 s and 360 can be estimated from similar experiments on the AC 300 Athena using S N cB
13. resolution and sensitivity should increase by roughly a factor of 2 UWChem MR Experiments Other Hetero Correlation Exps Page 57 XVI Other Heteronuclear Correlation Experiments A Discussion e section not ready yet B Critical Parameters e section not ready yet C DEPT Acquisition e section not ready yet D Calibration e section not ready yet E Data Workup and Plotting e section not ready yet UWChem MR Spin Lattice Relaxation T Page 58 XVILFitting Partially Overlapping Peaks Deconvolution in VNMR e prepare baseline region by clearing all reset points type cz e click on PARTIAL INTEGRALS e click on DISPLAY NEXT RESET and pick integrals over all sections that are NOT good baseline regions i e this is not to get good integrals butto define regions of good baseline e resave the data to allow simple restarting at this point use svf command e use be 5 or similar I recommend you not use be which applies a spline fit this is a very dangerous command that can seriously modify the shapes and integrals of peaks e click on DISPLAY INTERACTIVE NEXT TH to change the threshold level you want to go just below the smallest obvious peak but not down to where a lot of noise from a large peak will be picked up e enter mark reset to ensure that all previous marks are deleted e move the cursor to each normal peak and press MARK this enter the center frequency and uses a d
14. sets the C F1 spectral axis set the next section as to how to set C HMQC Acquisition for long HMQC runs or lossy samples calibrate the 73C decoupler using a C enriched sample and PWXDEC90 sequence see section D below tune the probe at 73C then H for your sample acquire a standard H spectrum and optimize sw using movesw calibrate pw90 by arraying pw make certain d1 is not too small 1 5XT optimize the gain for pw pw90 and acquire a H high resolution spectrum at the optimized sw mp 3 assuming started in exp jexp2 and obtain just enough C X cable to observe port to see the solvent peaks nt 1 usually if in D20 you can rely on setref for approximate referencing or use decref if the H spectrum has a good reference optimize sw of 3C by estimating where protonated carbons will show for your compound and using movesw ga jexp3 and type gHMQC or gHSQC enter 2 when asked where 13C parameters are check that pwx pwxlvl dmf and dpwr are set correctly check j and d1 check ni and nt if running hmgc instead of gradient sequences optimize null set ni 1 and array null T or use null 0 7 T1 if you know T1 especially important for solvent if working in non deuterated solvent au UWChem MR Varian VNMR User s Guide Page 55 D Calibration general rule of thumb for these calibrations is short runs check proton pw90 for pwx pwxlvl use facility numbers if probe problems are suspected
15. t E 1 where c concentration B magnetic field strength t experimental time and amp probe filling factor To estimate the time under different conditions that would give identical S N then 2 Cold Bou new 3 fnew Lold 2 Boney A reasonable estimate although direct comparison to experiments on similar compounds is preferred on the UWChem 7 equipment can be made starting with the observation that a 0 1 M solution will typically give publication quality spectra in 20 min on the AC 300 Athena Standard decoupled NOE enhanced quantitative decoupled no NOE Bruker s INVGATE AU and NOE enhanced coupled spectra Bruker s GATEDEC AU can be obtained through use of the S2PUL sequence Nuclei with negative y values such as N and 29Si are best acquired using polarization transfer sequences DEPT in general is the preferred sequence over INEPT if more than one Jyp value is involved quaternary moieties of negative y nuclei are best obtained through long range coupling unless the 7 X values are known to be reasonably short id C Acquisition s2pul pl pw 180x 90x BC rf observe Yy dl d2 aq TH rf decoupler A B C UWChem MR Experiments 1H Id Acquisition Page 32 B Critical Parameters dl relaxation delay d1 gt T H to obtain optimum NOE dof usually 0 when solvent set correctly should be within 5ppm of H coupled to X nucleus of interest dpwr dec
16. 1 set cursor on solvent singlet nl dres should be lt 1 Hz in most cases highly dependent on tube 507 s stockroom will sometimes need Z3 Z4 adjustments 528 s typically should not IV Probe Tuning and 1H Calibration Use Hewlett Packard scope to tune probes disconnect H cable with small silver barrel filter attached from probe and hook HP scope into probe push H1 PROBE on the scope tune in TUNE capacitor bottom to center dip tune in MATCH capacitor upper to get dip down to bottom 2 3 squares readjust both to center and bottom dip appropriately disconnect scope cable and reconnect H tune 3C or other X nucleus if needed at this time check H pulsewidth using array command use pw 30 3 3 to set up first array check about 360 going negative to postive as pw increases pw90 pw 360 4 pw90 check is required for checkout also necessary before querying facility staff about probe V Acquisition Check that there is no external attenuation in line for C or other nuclei runs Set gain 40 and listen watch for an ADC OVERFLOW beep there is one beep for completion of the acquisition and a second beep if there is an ADC overflow turn gain down in 10 dB steps until ok this is recommended setup computer sees clipping better than you will in next example Can also perform a nt 1 acquisition then df if fid looks normal type gf wait gt 2s then can go into ACQI FID and obser
17. 13C or 9Si and J is the enhancing nucleus usually H but could also be 9F or 3 P The following generalizations can be followed e Polarization transfer is always recommended for nuclei having negative y values 295i 19N and 103Rh being three examples From eq 2 above the NOE enhancement for these nuclei could result in 0 signal PT is also always recommended for low y nuclei e g starting gt N and lower in frequency e DEPT is the best method for obtaining 3C spectra as well as spectra of other spin 1 2 X nuclei of typically protonated compounds DEPT is definitely preferred over INEPT if more than one Jy value is involved for the nuclei you want to observe e DEPT should be used to obtain coupled spectra turn the decoupler off during the acquisition dm yyn in general DEPT will give better S N than coupled NOE experiments e DEPT should be used even if no 1 bond coupling to protons are present for low y nuclei if long range couplings can be used e INEPT should be used only if one J value is involved and it s size is known e typically only a DEPT 135 is needed mult 1 5 but vnmr makes fully edited spectra easy to obtain accurate pulse widths are required for good methyl methine differentiation e For all these experiments delays will be dependent on Jy The better the coupling is known the better the experiment will work Make every attempt to measure the couplings from the isotope splittings in the
18. Calibration recalibrate 90 pulses for p1 p1lvl and pw tpwr see H section for instructions for longer experiments short quick experiments can be run with probe file calibrations and macro setup E Data Workup and Plotting same as dqcosy see DQCOSY section for phase sensitive workup UWCheM HRI Varian VNMR User s Guide Page 48 X Other COSY Variants 21 Jul 98 A Discussion e COSY can provide a good alternative to dqcosy for determining coupling constants E COSY has the advantage that fewer crosspeaks are present but all coupling information is still present E COSY has the disadvantage that the minimum phase cycle is very large 32 so the experiment can take much longer that necessary from S N considerations see van de Ven for an excellent discussion on E COSY and other variants gradient E COSY may provide a usable form of E COSY look for more details e see COSY for long range and relayed experiments B Critical Parameters e section not ready yet C Acquisition e section not ready yet D Calibration e section not ready yet E Data Workup and Plotting e section not ready yet UWChem MR Experiments NOESY 2d Acquisition XI NOESY 2d NOE and Exchange Spectroscopy 17 Jul 00 A Discussion Page 49 NOESY can be a powerful experiment for the correct types of compounds and with proper care during acquisition and analysis of the data Care is recommended during interp
19. a command similar to tar cvf destination dir tarfile tar directory names to be tarred wildcards ok I often move fid s to a separate directory to make a single tar file from all of them easier my r directory name tardir cd tardir tar cvf dest dir tarfile tar compress the file cd dest dir compress tarfile tar adds Z to end of filename Give file a DOS compatible filename mv tarfile tar Z tarfile taZ Move file to NT server Log into NT server ftp babylon username anonymous password lt email address e g fry chem wisc edu gt change to writable directory and image transfer type ftp gt cd waste ftp ftp gt binary put file in directory ftp gt put tarfile taZ C Backup file to Colorado Systems tape drive Go to the PC named Vulcan in rm 2224 exit Windows cd waste ftp type tape atthe DOS prompt and follow the prompts should be obvious from here D Recovering backup ed up data essential follow the previous steps in reverse use get tarfile taZ instead of the put statement in ftp remember to rename the file back to a compressed filename when you get it on UNIX my tarfile taZ tarfile tar Z uncompress tarfile tar use the following tar statement UWChem MR Varian VNMR User s Guide Page 67 cd lt directory you want fid s put into gt tar xvf tarfile dir tarfile tar Backups to IOmega Zip Drive on Windows NT Network currently installed on Morder Terminus Drazi
20. and Plotting work up essentially identically to dqcosy see end of DQCOSY section for phase sensitive work up UWCheM HRI Varian VNMR User s Guide Page 56 XV HMBC 2d Long Range Heterocorrelation Spec inverse detection 4 Aug 98 A Discussion HMBC and gHMBC are powerful assignment techniques B Critical Parameters pw 1H 90 pulse width at power tpwr e pwx X 90 pulse width at power pwxlvl e j J coupling for one bond couplings these will be suppressed 140 Hz taumb 0 5 0 6 for 13C experiments 1 2Jiong range s use 0 1 for 15N C HMBC Acquisition the macro should setup the following dm n HMBC cannot be acquired with C decoupling null 0 BIRD suppression cannot be performed so g HMBC should be superior increase at 0 3 to 0 4 s increase nt 8xnt from HMQC experiment ss 4 steady state pulses before every increment mbond y _ defines sequence as HMQC always perform linear prediction use button under UWMACROS MORE in F1 on HMQC and HMBC D Calibration excellent calibrations of both H pw90 tpwr should always be done before acquiring an HMBC and 3C pwx pwxlvi calibration should be recent are required Data Workup and Plotting HMBC is often worked in av phl mode even though both dimensions are acquired phase sensitive gaussian filters are typically applied in both dimensions linear prediction in F1 to 4xni is recommended
21. and enter sd obtain enough data to determine 1 if multiplet is being sufficiently saturated ii if any other close by multiplets are being effected by the saturation pulse if any are dpwr must be decreased until the close by multiplets are all completely uneffected this condition is essential interpretation of the data becomes much more difficult if the chosen multiplet cannot be saturated independent of the other protons if this happens consider performing a NOESY or ROESY experiment instead of the NOE diff ii currently I do not know how to plot or save difference spectra if dof is arrayed although this would seem to be the best way to acquire the data iii see cgfry about implementation of GOESY which should be a superior experiment to NOEdiff GOESY info should be available very soon D Calibration set dpwr low and raise by 3 dB to no more than dpwr 14 use the lowest power that saturates the selected multiplet E Data Workup and Plotting bring in reference spectrum with decoupler placed on baseline make sure expansion and vertical scale are what you want make sure exp5 is saved and not in current use then use clradd spadd to clear exp5 and transfer the current spectrum to it bring in next spectrum in another exp if desired but must have same expansion and vs more precisely sp wp vs must all be identical between spectra use addi to compare and subtract spectra see Vnmr manuals for more info UWChem MR Var
22. back of the PC this insures that no one will demand CPU cycles from the PC during the CD write and ruin your CD on the Microsoft Office Taskbar click on the ECD button again toward bottom in the Easy CD window click on CD R and the Properties change temp to c temp make sure 1X speed is selected Brad believe higher speeds are unsafe for writes UWCheM HRI Varian VNMR User s Guide Page 69 make sure Joliet filenames is selected not sure about TEST BEFORE WRITE button Prakash didn t use but Brad seemed to think it should be on e all should look ok at this point all items under CD R menu should be ready except WRITE e open MS Explorer window button on MS Office Taskbar that is a folder with a magnifying glass on it e click onto l drive select all your directories that were FTP ed click on top directory then hold SHIFT key down while clicking on last directory all directories should be selected e click and hold the mouse button down on any of the directories drag the mouse into the Easy CD window and let go all the directories should appear in the Easy CD window e select WRITE TO CD under the CD R menu e either stay next to the PC could take an hour or leave a sign on the PC for all to keep hands off e make sure to come back before the write finishes e recommend writing a second identical backup CD e MAKE CERTAIN THAT YOUR CD IS READABLE BEFORE DELETING ORIGINAL DATA e rehookup the ethernet cab
23. for correct parameters to be setup e MAINMENU SETUP Nuc Solv 1H should be ok check nt and gain 13C check decoupler settings e g probe bbswg make sure this is the probe installed dmm w dmf 10000 dpwr 40 dm yyy su turns decoupler on see also UWMACROS DECOUPLER ON C Variable Temperature e switch to N gas for 20 C gt temp gt 40 C e turn VT flow up to eject samples do not switch back to air unless close to ambient e Use UWMACROS SET TEMP to change temps or macro settemp or similar UWCheM HRI Quick Guide for New Users Page 7 UWMACROS SET TEMP should be used instead of manually setting temperature this macro avoids inadvertant temperature changes that can otherwise occur 20 changes take 15 mins or so before probe tuning and shims will be stable 50 changes take 30 mins 100 changes may take 1 h should be done in steps no bigger than 50 Itis the student s responsibility to finish early enough that their VT work does not affect the next user hcx probe 80 to 60 C bbswg probe 150 C to 80 C bbold probe 150 to 150 C h1f19 probe 150 to 150 C Locking and Shimming A Sample Prep Need gt 0 6ml 4 cm height solvent for Varian probes to attain good H shims without extraordinary shimming Set sample to 67 mm below bottom of spinner use ruler or center in rf coil region use Varian depth gauge if solvent lt 5 cm high On a 500 it is critic
24. from the right flutes clockwise looking down ma we looking down onto rotor at top rotor The bottom of the rotor goes into the stator first tighten lt _ the screws snug but not with excessive force e With the metal cap off the probe test the spinning by MU lt drive air i Turning the bearing air upper knob to 10 black scale bottom ii Slowly increase the drive air until spinning begins at half painted 5 on black scale sample should spin smoothly at 2kHz If seem to get stuck at 800 1200 or 1500 Hz DO NOT try to force spinning by turning air up Take sample out check cap tightness and fit tamp the rotor a bit try again to spin and repack if it still will not spin iii With metal cap on probe spin up to 2500 3000Hz Put probe into magnet and spin up to desired speed Do not spin faster than 6 kHz or raise bearing air above 20 on black scale with NMR Director s approval II Probe Tune Up This procedure MUST be performed for every sample change Even slightly untuned probes may prevent reasonable CP match e Check tn value by entering dg or tn Should get in C13 for 3C CP MAS e Set gain 0 and enter su for setup e Attach observe cable to middle tune BNC switch to tune at 50 or 60 Should be able to get 27 on meter by adjusting two large knobs on bottom of probe Switch back to observe e Tune decouple channel by entering tuneh macro tn H1 su Attach decouple cable to tune and adjust tw
25. in same direction positive change in Z4 results in positive change in Z2 Much of the table is not completed since new shims installed most interactions are now much weaker Adjusted Stong Weak Adjusted Stong Weak shim interaction interaction shim interaction interaction Le a Ea es 7 a a Ve Vu 2 Poze 3 ZY DE E a a Table 5 Shim Sensitivities on Unity 500 number following shim is normal adjustment when shim fairly close to correct Z 16 to 4 Z3 64to 16 Z2 16 to 4 Z4 64 Y 16 to 4 X 16 YZ 16 XZ 16 UWCheM HRI Varian VNMR User s Guide Page 27 Experiments on Unity 500 created 5 04 97 updated 12 21 97 l Normal 1d 1H Acquisition 21 Dec 97 A Discussion e 1d H acquisition on the Unity is simplified by the solvent solventname command When set appropriately tof 0 should center the transmitter correctly e With identical transmitter and decouple channels it is simple to decouple X nuclei on the Unity 500 while observing H e g for P set dn P31 and other parameters correctly see later section e calfa is an important baseline flattening timing correction macro on the Unity when setting up 2d experiments Note the use of this command in the acquisition section e Spin lattice relaxation T7 can be measured estimated with this sequence set p1 2 pw90 pw pw90 and d2 appropriately both p1 and d2 are normally are set 0 see later section for details on T estimates e Note t
26. roughly tune e switch off the power on the power strip not on the instruments and hookup to the Varian meter to precisely finish the tune III CP Match e The cross polarization experiment requires that The xpolar pulse program used by the spectrometer xpolar diagram IV Analyzing MAS Data to Obtain CSA Information There are three distinct steps involved in analyzing MAS data a prepare the 1d file to correct baseline roll find mark the isotropic peak and set threshold for spinning sideband setup and prepare three exp areas for display and plotting b simulate the spinning sideband pattern to obtain good CSA and ASYM parameters UWCheM HRI Varian NMR User s Guide Page 64 c perform a fit to obtain best values for CSA chemical shift anisotropy csa OF Gesa and ASYM asymmetry parameter n A Preparation of MAS spectra e readin the data use menu items MAIN MENU FILE SET DIRECTORY to change default dir e expand to exclude baseline i e to edges of SSB s e integrate spectrum to exclude all peaks from baseline correction routine type cz to zero all integral regions click on DISPLAY INTERACTIVE NEXT RESETS then click left button on each side of all peaks in spectrum the idea here is to remove everything from a baseline calculation all integral regions are ignored for baseline correction type be to correct baseline see VNMR online for more bc options e write down the isotropic peak compa
27. s Guide Page 74 First Time Use and Detailed Explanation of Parameters The pulse sequence used in the gmapsys system is gmapz and is shown schematically below This sequence performs a gradient echo Magnetization isochromats are dephased in frequency space according to the z location of the nucleus Assuming translational diffusion is small during the time gzt the dephasing of the isochromats will be refocused by the opposite signed gradient a time gzt later producing an echo The Fourier transform of the echo with absolute value processing will produce intensity versus frequency since the frequency is directly related to the z location the frequency axis is directly mapped to the z location This experiment is one example of how human and small animal images are obtained with MRI Gradient Echo Experiment gmapz pw tpwr H or at 2H rf dl d3 gzt amp G na A B c A Critical Parameters dl d3 ss gain nt experiment repitition rate should be set 2 3xT of the solvent H or 2H nuclei arrayed to two values the 2nd value should produce 0 8 to 0 4 the intensity of the 1st arrayed value use the SETUP GO DSSH buttons to see if d3 is arrayed correctly usually the 1st value 0 but see below for more explanation usually ss 0 but in cases where T is quite long it may be preferable to set ss 2 and pw 30 reduce the gain until the baseline portion is flat increase if
28. section not ready yet D Calibration e section not ready yet E Data Workup and Plotting e section not ready yet UWChem MR Experiments HMQC 2d Acquisition Page 54 XIV HMQC and HSQC 2d Heteronuclear Spectroscopy inverse experiment with 7H detection 20 Jul 01 A Discussion Two sequences for measuring H X chemical shift correlations gHSQC and gHMQC are very similar and both will work fine for most organic compounds HSQC will give sharper lines especially for CH since H H homonuclear coupling in the F1 axis is not suppressed in HMQC HSQC is more sensitive to pulse width calibrations has more m pulses but HMQC can suffer T sensitivity losses in viscous and high MW compounds Gradient sequences are typically superior in S N especially for long range HMBC B Critical Parameters pw H 90 pulse width at power tpwr pwx X 90 pulse width at power pwxlvl jixh J coupling for correlations of interest set to 140Hz for 1 bond HSQC and HMQC experiments see HMBC for long range experiments ni fixes the digital resolution in F1 dres1 swl 2ni because of the large J coupling in 1 bond experiments dres need only provide sufficient resolution for C chem shift and is therefore set larger than typical in a COSY experiment For COSY dres 4 Hz pt where in HSQC HMQC dres 2 30 Hz pt is usually sufficient di best set 2 2XT but not uncommon to run at 1 5xT swl dof
29. the signal to noise is poor The following should be setup correctly by a gmapsolvent macro pw E tpwr gzlvl sw gzt a 30 90 pulse use smaller angles for solvents having large T 90 if T is short it is absolutely critical tpwr not be gt 50 when using the lock channel to shim on 2H solvents normal values of pw tpwr can be used for H shimming 4000 works quite well for most situations 100 kHz for H shimming 15 kHz for 2H shimming actually set as at 2 d2 where d2 appears to be a gradient recovery time of 1ms not sure why it is needed UWCheM HRI PFG Shimming With VNMR Page 75 B Details of Correct Parameter Setup pw tpwr di d3 in cases where the solvent T is not known you must first obtain a calibrated 90 pulsewidth this is exactly the same as for other nuclei see for example Experiments on the UNITY 500 Section D H pw90 Calibration pg 26 of this manual When using the lock channel e callin a standard 2H parameter set using MAIN MENU SETUP e make sure you are on resonance to the H signal e set tpwr lt 50 if using the lock channel and array pw through a 360 rotation change tpwr such that pw90 400 500us on the lock channel after determining a correct 90 pulsewidth set p1 2 pw90 pw pw90 d2 0 1 take 1 scan and make sure the magnetization is inverted increase d2 until the magnetization nulls further increase in d2 make the magnetization go posi
30. us at power tpwr typically 46 to 52 used for MLEV spin lock calibration of this parameter is also recommended ni number experiments or number of points in f should be set ok by macro time allowing must have F1 digital resolution sw1 2ni lt 12 Hz pt but 6 Hz pt if time allows mix mixing time length of spin lock typically 30 to 100 ms nt multiple of 2 minimum multiple of 4 if time allows dl relaxation delay set 1 2 7 np number of points in fy usually want 1024 since costs nothing but disk space and gives better resolution in F2 phase 1 2 is recommended phase sensitive acquisition trim optimize by minimizing signal when d2 0 and phase 2 UWChem MR Varian VNMR User s Guide Page 47 C TOCSY Acquisition prior to final setup run ni 2 and carefully phase t spectrum then enter calfa and reacquire observe baseline flattening perform integrations on 1st increment if baseline correction is desired check that nt multiple of 4 2 can be used if time is critical check that F1 dig resol sw1 2ni lt 12 Hz pt desire 6 Hz pt if time allows check that p1 p1lvl are set appropriately should equal pw tpwr from 1d H experiment I will likely change this confusing definition during the DIPSI implementation and pw tpwr are set to give pw90 gt 20us set window 2pw clean tocsy only and optimize trim by mp current exp 1 phase 2 and d2 0 then choose trim giving minimized signal D
31. H spectrum or obtain good literature values Lacking both be prepared to run a series of experiments using different Jy values to find the optimum parameters e For small Jy couplings a compromise between signal loss from T inverse natural line width especially for low temp or high MW samples and PT must be made In some cases mainly when 77 x is not too large the non NOE decoupled Bruker s INVGATE may be the preferred experiment B Critical Parameters d1 relaxation delay typically 1 2s j 140Hz change if you want to observe X with Jgyy gt 180 Hz or lt 110 Hz pw tpwr observe X 90 pulse width pw at power level tpwr pp pplvl high power H 90 pulse width pp at power level pplvl mult determine type of DEPT 0 5 dept 45 1 0 dept 90 1 5 dept 135 see discussion below UWChem MR Varian NMR User s Guide 1d DEPT dept Page 36 pw pw 2pw pw 90x 90 180k 90 X rf observe If pp pp 2pp pp 90x 90x 180 90x 6 mult pp dwpr dmf 1 H rf dl 1 2J 123 M oiz ee decoupler C C DEPT Acquisition e for short runs use facility calibrations for pw tpwr pp pplvl dpwr lt 46 and dmf e FILE SETUP SEQUENCES DEPT will setup Dept correctly including for non 3C acquisitions mult is set by the number of coupled protons the interpulse delay is set according to j e for overnights or longe
32. Ip 0 if you do not rerun calfa and require again ifthe baseline still has some curvature either convex or concave then you might want to note the values of rof2 and alfa keeping the sum rof2 alfa constant change rof2 and alfa reacquire and note the effect of the change with rof2 and alfa one direction should move the baseline towards convex the other towards concave some combination should give optimal baseline flatness write down the values of sw rof2 and alfa these should always work for this sw e asa minimum estimate T using inversion recovery null method to provide setting for d1 D Calibration e always recalibrate 90 pulses for pw tpwr with NOESY spectra see H section for instructions e always work from a measured T estimate E NOESY Data Workup and Plotting e same as dqcosy see DQCOSY section for phase sensitive workup e baseline flatten fitting routines can be particularly useful for NOESY spectra workup it is recommended that the baseline is made as flat as possible prior to acquisition see section C but wftida be f2 wft2da be f1 can be particularly useful here fn1 fn is required for the integration regions in F2 to work for the F1 baseline correction UWCheM HRI Experiments ROESY 2d Acquisition Page 51 XII ROESY 2d NOE Spec Mixing via a Spinlock in the Rotating Frame 17 Jul 00 A Discussion e ROESY spectra will produce a maximum 0 5 enhancement for H
33. MMAP Each shim should show its characteristic polynomial dependence and less noise is of course better 8 Click on AUTOSHIM ON Z and wait for the procedure to finish Do this at least once more to ensure that the autoshim procedure has converged on good shims amp I have found that nearly every time the procedure fails to run 1 e get an error message and it doesn t even start it is because gzwin has been set slightly too large For example if gzwin 63 2 and the autoshim fails to run setting gzwin 61 will nearly always work Tf the autoshim fails to converge 1 e the convergence factor simply will not get lt 1 00 it is usually because the XY shims are very poor re read in the facility shim file or because your sample tube quality is poor check the tube quality especially for scratches check solvent clarity and filter if needed check solvent height PFG shimming can help a lot with solvent amounts less than recommended but cannot necessarily achieve a good shim for solvent heights lt 3 5 cm lt 0 45 ml In cases where autoshim fails to converge enter aa or click on ABORTACQ to stop the acquisition QUIT out of the gmapsys system and shim the XY shims make sure the spinner is turned off Then go back into gmapsys remake your shimmap and retry the autoshim Repeat this quit gmapsys and manually XY shim go back in and remake shimmap and autoshim until the shims are optimum UWCheM HRI Varian VNMR User
34. P SEQUENCES NOESY 2d NOESY and EXSY Spectroscopy ulnoesy dl 1H rf pw 90xx d2 pw 90 pw 90x mix atx x UWChem MR Varian NMR User s Guide Page 50 B Critical Parameters pw tpwr 90 pulse width at power tpwr recalibrate this parameter for noesy experiments ni number experiments or number of points in f should be set ok by macro time allowing want F1 digital resolution lt 6 Hz pt sw1 2ni nt multiple of 8 sspul y gives homospoil 90 homospoil preceding d1 d1 relaxation delay set 2 4 7 do not set too small or will get very bad f noise np number of points in t usually want 2 2048 since costs nothing but disk space and gives better resolution in F2 mix mixing time often is varied to provide build up curves Set close to T should provide maximized crosspeaks for high MW gt 2000 0 3s should work mixvar ulnoesy only variation in mix in percent 10 works reasonably well to remove cosy tocsy type crosspeaks will increase t noise so not recommended unless know is needed C NOESY Acquisition e set up similar to DQCOSY e make certain to optimize the gain e make certain to optimize baseline flatness for the particular value of sw you are working at acquire spectra with ni 2 and phase carefully run calfa which uses the Ip value from the phasing reacquire the ni 2 data and rephase you should get a good phase with
35. UNITY 5 If you are using a different deuterated solvent than the last time you PFG shimmed run the macro to setup parameters for the solvent you are now using E g for CDCl enter gmapcdcl3 or for acetone enter gmapacetone To see a list of available macros for various solvents and temperatures type the following in the UNIX terminal window Is vnmr maclib gmap Parameters for deuterated solvents will be very temperature dependent See Section III below for a detailed discussion of how to setup parameters for individual cases 6 Click on SETUP on the gmapsys menu and then on FIND GZWIN gzwin is a very important parameter for proper PFG shimming see item 8 below gzwin is the percentage of the spectral width that contains usable data so it would 70 in the schematic shown above 7 If you have a recent Shim Map in place for the probe you are using skip to step 8 Shim Maps done on protonated solvents will not work for deuterated solvents and vice versa A Shim Map made with DMSO will work for CDCl3 however Since DMSO provides much better S N with 2H than CDCly it is a much better choice for making a Shim Map for all deuterated solvents Otherwise click on SHIMMAP MAKE SHIMMAP Depending on the solvent the shim map will take 2 to 10 mins to create As noted above it is often best to use a DMSO d sample to make the shim map You should check the quality of the map after it finishes by clicking on RETURN DISPLAY DISPLAY SHI
36. University of Wisconsin Madison Chemistry Department VARIAN NMR USER S GUIDE by Charles G Fry updated December 19 2001 Note This guide provides an introduction to use of Varian equipment at the UWChem 7 This guide is not intended in any way to be a replacement to the excellent Varian documentation All students should refer regularly to the Varian VNMR Liquids Users Guide for learning and the Varian VNMR Command Reference Guide for specific guidance All the Varian documentation is available in both hardcopy and on line Copyright 1996 2001 Charles G Fry All Rights Reserved Table of Contents Page i Table of Contents UW Chemistry Magnetic Resonance Facility UWChemMRP ssssss0 4 Facility Layout 2 floor Matth WS 4 SGIls NATOTH Avance host computer and GQUAN for off line data workup 5 Il Facility Personnels a de en ge eta tae a 5 Quick Guide f r New USET S nn ionerne riaient irakar riaa aai 6 le J EogiNierea a coh esd a nn a aah ce dits et 6 HE RS TUN me a e al ee a a a e chal 6 A 1 Time 6 B Parameters 6 C Variable Temperature 6 llil Locking and SUMING Reese fr enr ee ta Tailed Ses 7 A Sample Prep 7 B Locking 7 C Shimming 7 IV Probe Tuning and FC AlIDrAtION Sedan ah Bi ment array 8 Mis ACQUIS ON siiani te Een Re EN AR de E te 8 VI Saving and Deleting Data and Backups 9 VII Logging OUT
37. VNMR Setup if an experiment is running and your time is clearly in effect use svf savename while experiment is running will save last bs dataset use FileManager to check that file wrote correctly unix command df better exit vnmrx right click and hold on background and release on EXIT to exit CDE do not save the workspace while VNMRX is open Commands for First Time and Novice Users Some of the following commands procedures may have to be performed when first starting many of them will not be needed again or only occasionally phasing 100 or 60 on a Sparc1 if the data is gt 64k cexp 2 cexp 3 create additional experiment areas see also MAIN WORKSPACE CREATE click MAINMENU MORE CONFIG PRINTER and keep clicking PRINTER until set to Shadowp_LJ laserjet portrait printing repeat above except for PLOTTER and click unter set to Shadowp_LJR landscape plotting gf following correct setup to give good fid spectrum shimming inside ACQI FID window Probe Changes ONLY FOR TA S AND FEW STUDENTS OK ED FOR PROBE CHANGES see Table 1 on the next page for a description of probes make sure acquisition is complete and data saved by previous user stop temperature control by using macro tempoff in vnmr maclib physically switch the temp controller off eject sample type eject at command prompt or click eject inside shimming acqi window type insert to turn the air back off disconnect rf cabling VT line and prob
38. Z1 shim offset produced by the pulsed field gradients but otherwise identical in nature to the RT shim s Z1 shim setting When higher order Z shims are misset from the perfectly shimmed case denoted as the normal profile above identifyable changes occur in the profiles Exagerated changes are shown for Z2 and Z3 missets The gradient shimming software analytically expresses these changes when a shim map is made each RT z axis shim is changed independently providing data upon which an analytical expression can be calculated for each Z shim From this shim map analytically accurate changes to the Z shims can be performed to minimize shim missets getting as close to a perfect shim as possible in the presence of noise and inaccurate non radial X Y shims amp Keep in mind that linewidths are sample dependent PFG or gradient shimming cannot overcome aggregating or suspended samples solvent bubbles too little solvent height use susceptibility inserts scratched sample tubes etc And many samples simply will not shim to better than 1 or even 2 or higher Hz linewidths due to their intrinsic relaxation properties in the solvent and temperature being used i e their natural linewidths are gt 1 or 2 Hz UWChem MR Varian VNMR User s Guide Page 72 amp Students needing to perform PFG shimming must make a commitment to learning the proper procedures and pitfalls involved with this method facility staff will not entertain ext
39. a see Varian Subject manual Adv Id section 1 Expand about the peak s of interest Adjust the threshold with the Th 2 Enter dpf or dll then fp to locate the peak s and then find them through the relaxation set fp writes a text file fp out in the current experiment e g vnmrsys exp1 ft out 3 If the dataset is a T set enter t1 for T enter t2 or for a solid state T p enter analyze expfit p3 t2 list 4 tl and t2 will display the information on the bottom of the screen To print repeat using e g printon t1 printoff or print the file vnmrsys exp analyze list text file same information from the File Manager or in vnmr with ptext analyze list 5 Show a plot of the data using expl and plot with pexpl page Note that you can look at specific peaks using expl 3 and pexpl 3 page as an example for the 3rd peak To make a smaller plot use the sequence expl center pexpl page To delete a point from the analysis use dels lt data gt fp t1 or similar command UWCheM HRI Varian VNMR User s Guide Page 66 Data Backups from UNIX Workstations created 4 10 96 updated 4 10 96 Backups to Colorado Systems Tape Drive on Windows NT Network A Perpare tar file on UNIX workstations cd to the directory that the fid directories reside in you must not use full paths for the tar statement when bringing back data tar tries to follow the path explicitly tar the fid s into a single or more tar sets using
40. al that the sample be clear no particulate floating if possible and the tube be of high quality Wilmad 506 minimum 528 better with no nicks or scratches Keeping within these standards will allow excellent quality shims to be attained in lt 5 min shimming Reasonable shims can be attained in other conditions but with longer shimming sessions and no guarantee that good quality lineshapes can ever be achieved Locking use UWMACROS LOADSHIMS or manually use rts or rts hex shim loadshims loadshims load y su load n su rts bbswg shim loadshims rts your shims loadshims FILES SAVE SHIMS or svs will save in export home practice vnmrsys shims in ACQI window set FIELD until fid on resonance no oscillations and positive LOCK POWER see START suggestion but be aggressively higher if needed LOCK GAIN NOTE IF ACQI WINDOW DOES NOT APPEAR USE ACQI TO CALL IT UP should lock up now turn SPIN ON if routine 1D not if 2D or selective 1D experiments C Shimming shim LOCKPHASE Z1 Z2 to achieve maximum lock signal lower LOCK POWER approaching FINAL suggestion but keep lock level gt 15 UWCheM HRI Varian NMR User s Guide Page 8 spinning LOCK PHASE gt Z1 Z2 2 order Z3 usually not necessary non spinning X gt Y gt Xz 2 order YZ XY gt X2 Y2 then back to above 2D do all without spinning check shims using standard H setup with nt
41. amount of material he 5mm 1H 13C X gt 80 C inverse triple with PFG lt 60 C good for 1H and H X heterocorrelation excellent 1H S N 800 bbold 5mm broadband standard probe for direct 13C SIP 295i observation 1H signal to noise S N and line shape are poor with this probe bbswg 5mm broadband switchable standard probe for direct 13C SIP 29Si i e 1H observe with pfg observation 1H SIN is adequate with this probe so probe switching for H observation is not needed for best H S N use inverse or h1f19 probe bbswg 1H S N 350 h1f19 5mm H 9F for best sensitivity H work when concentration is limited 1H S N is good 540 on EB nal3mm 3mm nalorac INOVA for H 1D and 2D heterocorrelation only CIN only when sample amount is limited or need best water suppression 1H SIN is very good 5mm probes are better for concentration limited samples invx Smm inverse broadband for 2D hetercorrelation work HMQC HMBC HSQC 1H signal to noise is very good with this probe X S N is poor with this probe do not do 13C observe with this probe triple 5mm triple for 2D H 3C_ N work HMQC HMBC HSQC 1H SIN is very good 13C and N S N is poor VT range is limited 50 to 80 C UWChem MR Operation of Unity 500 Page 24 Table 2 Calibrations of Probes on Unity 500 Use the following guidelines for probe calibrations short runs use facility numbers see vnmr shims probes file for up to date number
42. code so common code must be placed in the user s vnmrsys psglib and compiled The compiled code is automatically placed into the user s vnmrsys seqlib which must then be copied into vnmr seqlib and have the owner set to vamrl nmr e g chown vnmrl nmr vnmr seqlib relayh root permission is required for the copy and chown unless the user is vnmri When writing or editing a pulse sequence it is easiest to begin with a premade parameter set New parameters must be created with the create parameter lt type lt tree gt gt command See section 5 2 in the Pulse Programming manual It is important to correct define the parameter when creating it e g when making a new variable dpw2d for a decoupler power setting that should not go above 35 to prevent probe damage use the command create dpw2d integer and follow that with setlimit dpw2d 35 0 1 Note that pulse length parameters should be defined with create pwsl pulse to insure the units for the pulse are in us See the create command information in the vnmr documentation for other parameter types Some newer sequences being written at Varian usually now use a phase table named the same as the sequence and located in vnmr tablib although the newest sequences avoid the use of tablib by specifying the table within the pulse sequence itself rather than externally Typically the macro finishes by setting some parameters directly and then displaying the help file
43. curs here e g np 1024 fn 2048 will zero fill once line broadening parameter EB interactively set weighting none wtia in vnmr middle button still control intensity in all windows left parameters button sets parameter apply exponential line broad in vnmr wft applies whatever weighting function is setup rl Op ponies Meee ean ee ommend nl linear prediction bs 5 in vnmr bs is not recommended default spline fit Al lt ret gt 0 nm in vnmr vs 100 will fill screen absolute intensities Al lt ret gt 1 jai 1d Plotting Commands plot spectrum PX pl plot parameters plot axis in vnmr axis p sets axis to ppm plot coordinates sc is mm from right side vp is mm vertically up from bottom vs plot integrals plot peak picks in DPO setup dil or ppf printon dll printoff prints table to separate page do before any plot size CX CY we is mm width of chart vs is vertical scale plotting commands ppf plots on spectrum new page UWCheM HRI Varian NMR User s Guide Page 19 2d Acquisition Parameters sweep width for F1 Swi AC AM SW1 is 1 2 of observed sweep width vnmr sw1 observed sweep width increments experiments IN in vnmr phase determines total experiment 1x or 2xni type of 2d acquisition MC2 AC AM only absolute value and TPPI are available in software vnmr absolute value phase 0 States Habercorn phase 1 2 TPPI phase 3 2d setup ST2D in vnmr type in sequence macro then dps to
44. cursor on multiplet then movetof set d1 20s or longer depending on sample T use 360 pulse for final checks always e once the observe pw90 is obtained assuming in expl then jexp2 move H cable to observe port and find resonance for benzene doublet take one scan move cursor to middle of doublet and use movetof write this value of tof down as H dof e assuming C still in expl and H in exp2 do jexp1 mp 3 jexp3 put H on resonance by setting dof H dof from above setup decoupler calibration experiment with UW macro pwxdec90 set pwxlvl 60 and find pwx where antiphase doublet nulls this is decoupler hard 90 set pplvl pwxlvl and pp pwx for hetcor and dept experiments set pwxlvl appropriately for decoupling probe dependent see Table 2 on wall and find pwx where antiphase doublet nulls set dpwr pwxlvl and dmf 1 pwx 1e 6 E 1d X H Data Workup and Plotting e typically will want Ib 2 or 3 for C experiments e see 1d H section E for plotting description UWCheM HRI Varian VNMR User s Guide Page 35 lll DEPT Distortionless Enhancement by Polarization Transfer 22 Jun 98 A Discussion of PT versus NOE experiments and DEPT versus INEPT Summarizing Derome see Chap 6 for very good discussion p 129ff Polarization transfer experiments can offer sensitivity enhancements of Polarization Transfer Yi 1 Yx NOE Ma 2 27 x where X is the nucleus being observed e g
45. detect 29Si and J si H 2 Hz then d2 1 4x2 Hz 125 msec o and d3 st gi l 12 27msec 360 x 2Hz Vo 360 x 2Hz Vo 180 2 af 1l 2x19 47 or mult sin 0 216 same as in DEPT setup 180 These values are calculated internal in the vnmr psglib ineptrd c pulse sequence code E Data Workup and Plotting e workup is similar identical to C 1d and DEPT UWCheM HRI Experiments NOEDIFF Id Acquisition Page 40 V 1d NOE Difference Spectroscopy 24 Dec 97 A Discussion See the Bruker User s Guide introduction for discussion on this non trivial but powerful experiment Sanders amp Hunter also contain excellent discussions on NOE experiments highly recommended Look for instructions on use of GOESY which should be significant improvement over NOEdiff B Critical Parameters dl relaxation delay needs to be 2 3 T see later section for measuring T7 dpwr must not be set above 14 typically 1 4 works well for saturating multiplets dm yyn typical but dm nyn and d2 can be used to vary saturation within fixed di d2 delays dof set by command sd preceded by placing cursor on desired multiplet C NOE Diff Acquisition read in a normal H setup MAINMENU SETUP NUCLEUS SOLVENT SETUP SEQUENCE NOEDIFF will setup the following parameters dn H1 homo y dmm c dpwr 1 dm yyn place the cursor in the middle of the multiplet to be saturated
46. e cooling tygon disconnect temp heating cable using blue nonmagnetic screwdriver unscrew two probe thumbscrews and guide probe out insert correct probe use care with last 1 you may have reseat aluminum bore tube by pushing gently downward pressure at top of magnet necessary if sample won t spin reconnect cables keep Nalorac cable and filters separate and use only for that probe power up and restart temp control with UWMACROS SET TEMP or macro similar to temp24 read in new shims and load e g rts triple loadshims better use UWMACROS LOADSHIMS change probe and pfg settings appropriate for probe probe hcx pfgon nny probe bbold pfgon nnn probe 1h19f pfgon nnn probe 3mm pfgon nny UNITY pfgon yyy INOVA UWChem AF Varian VNMR User s Guide Table 1 Description of Probes on Unity 500 and Inova 500 Use the following general rules for probe selection e concentration limited samples use the largest diameter probe appropriate to the experiment hex or H 9F best for 1d or homonuclear H experiments e quantity limited samples use the smallest diameter probe appropriate to the experiment Nalorac 3mm probe best for all H experiments in this case strongly consider using susceptibility matched inserts for 3mm 7H or 5mm X experiments 3x saving in amount of material needed to obtain a particular S N in a fixed amount of time or 10x decrease in time for fixed
47. e fairly certain you know J reasonably accurately pw tpwr observe X 90 pulse width pw at power level tpwr pp pplvl high power H 90 pulse width pp at power level pplvl mult multipler for d3 delay where mult 1 all positive 2 CH2 nulled 3 CH2 inverted from CH3 and CH lt C INEPT Acquisition the INEPTRD macro run by the menu SETUP SEQUENCES INEPT is not the standard Varian sequence but in my opinion is superior it incorporates all aspects of Bruker s INEPTRD and INEPTP sequences for decoupled and coupled acquisition respectively for short runs use facility calibrations for pw tpwr pp pplvl dpwr lt 46 and dmf UWChem MR Varian VNMR User s Guide Page 39 e FILE SETUP SEQUENCES INEPT will setup Inept correctly including for non 3C acquisitions mult is set by the number of coupled protons and j d3 mult 4j the d2 interpulse delay is set according to j e for overnights or longer runs recalibrate at least observe and best decoupler pulse widths e set mult as needed see below D Calibration see 3C section for nominal 3C X and H decoupler calibrations e The delay d2 and mult d3 are calculated by the DEPT macro as follows d2 aT echo period involving two d2 delays creates H antiphase state It 1f af 1 1 af 1 d3 __ sin rad si S deg 4J Ont vn 360 J Vn where n I nuclei coupled with Jy example suppose have trimethylsilyl and want to
48. e of Contents Page ii E 2 Order Shimming on a 500 MHz Spectrometer 29 F 1d Data Workup and Plotting 29 ll X Acquisition e g PORT PO ot csc cel ces ate oak nine titre 31 Table 6 Common Decoupler Parameter Settings 32 D 13C X pw90 and 1H Decoupler Calibrations 33 Ill DEPT Distortionless Enhancement by Polarization Transfer 35 IV INEPT Insensitive Nuclei Enhanced by Polarization Transfer 38 V 1d NOE Difference Spectroscopy 40 VI Homonuclear 1d Decoupling us scse cescvetesne coneccsteesteceg doten coun tenedennaedd denneeesveereecuve 41 VII COSY 2d Homonuclear Correlation ccccccesccccceeeeeeeseceeeeeeeeesnseeeeeeeeeetens 42 E 2d Data Workup and Plotting 43 VIII DQ COSY Double Quantum Filtered COSY Phase Sensitive 44 IX TOCSY or HOHAHA Total Correlation Spectroscopy 46 X gt Other COSY Variants eeann a e a E 48 XI NOESY 2d NOE and Exchange Spectroscopy ccceeceeeeeeeeeeeeeeeeeeeeeee 49 XII ROESY 2d NOE Spec Mixing via a Spinlock in the Rotating Frame 51 XIII HETCOR 2d Heterocorrelation Spec normal X nucleus detection 53 XIV HMQC and HSQC 2d Heteronuclear Spectroscopy inverse experiment with 1H detection 54 XV HMBC 2d Long Range Heterocorrelation Spec inverse detection 56 XVI Other Hete
49. efault linewidth e move right and left cursor to the approximate half height points of broad peaks and click MARK this enters the average of right and left cursors for the center position and the difference between right and left cursors as the width e by opening a shell tool and entering cd vnmrsys exp1 more markld out you can see the file that the MARK command built and will use e clickon MAIN ANALYZE DECONVOLUTE USEMARK FIT to perform the fit e if you geta too many points error message enter FN FN 2 WFT BC 5 and click through to the FIT command again keep reducing FN until it will fit e use the PLOT button to get a quick plot e use the commands CLRADD SPADD ADDI to compare two spectra UWChem MR Spin Lattice Relaxation T Page 59 XIX 7H Spin Lattice Relaxation T1 updated 00 07 19 I Discussion e Spin lattice relaxation T is nominally an exponential process and is important as the determining factor in all experiments repetition delay d1 Suggestions for how abusable borrowed from Varian d1 is for various experiments is provided below T becomes critically important for any quantitative exchange or cross relaxation type of experiment such as NOE The relaxation from zero magnetization and from an inversion pulse are both shown in the figures below Recovery from M 0 0 Recovery from M 0 M T T ln2 T T In2 M t M 1 exp t T M M t M 1 2exp t T
50. ended questioning or training sessions to assist students beyond a brief introduction Moreover it is my opinion cg fry that PFG shimming is not as efficient for shimming as standard methods for most samples I and the facility staff therefore will not answer any questions about PFG shimming unless students can provide a good reason as to why they are not manually shimming their sample in Some common reasons for needing PFG shimming 1 When working with a wide range of temperatures PFG shimming in my experience is very helpful in adjusting to the changes in Z shims when temps change gt 40 degrees When working with D20 or DMSO or similar deuterated solvent that simply will not lock shim well students might should be FID shimming in these cases Anyone FID shimming should look into PFG shimming as large time savings in getting to good line shapes are likely When filter of the sample is not possible so cloudy solutions or some suspension etc are present that substantially pushes the shims away from the facility settings When forced usually by cost to reduced solvent quantities such that air solvent interfaces get close enough to the rf coil such that shims are pushed well away from the facility settings I recommend in these cases that students try susceptibility inserts in some cases these cannot be used and PFG shimming will assist in obtaining optimimum lineshape for very small solvent quantities good line shape
51. equence expl center pexpl page To delete a point from the analysis use dels lt data gt fp t1 or similar command UWCheM HRI Varian VNMR User s Guide Page 61 D T Abusability vs Experiment Repetition d1 from Varian course notes Likelihood of artifacts increases as go down column normal 1d T at 30 pulse COSY magnitude mode lt 1XT INADEQUATE gt 1 1 5x Ty O HMQC amp HMBC gt 1 5 xT HOM2DJ HET2DJ HETCOR COSYPS TOCSY NOESY amp ROESY qualitative 2 3 x T NOESY amp ROESY quantitative gt 3 5XT DQ COSY amp MQ COSY gt 3 x T homospoil Quantitative T 25xT UWCheM HRI CP MAS NMR Page 62 XIX Cross Polarization Magic Angle Spinning NMR 5 Mar 96 I Computer Set Up e Login enter acqi in the dialog box use right mouse on background to bring up acquisition status box e Typically all zero shims see with dgs will work fine for solids e Readin the file cpmas setup fid in usr fry directory for first run e set gain 0 temp n vttype 0 su better use tempoff macro II Sample Set Up e Pack sample into rotor with special care for keeping top radial symmetry during packing The caps must fit all the way on even a bit too much UUA dgive air sample keeping the cap slightly off will prevent smooth spinning ba e Note the flutes on both caps face the same direction with drive air coming
52. es exponential broadening of 5 hertz with wft or ga UWChem MR Varian NMR User s Guide wft aph np 4k fn 16k Isfid 2 movesw CZ be be 5 pl text text goes here atext more text pap ppa axis p pscale 3 axish pscale 6 page clradd spadd addi sl rl md 3 4 dssh full dss Page 12 weighted depending on parameters set FT good automatic phase to at least start phasing often set by sw this directly sets to 4096 points acquired changes aq number FT to with previous command does two zero fills left shift two points before FT increase if getting baseline rolling macro sets sweep width sw to right and left cursor positions zero all integral resets use menu button reset and mouse for resets baseline correction with default spline fit depends on integral resets baseline correction using 5th order polynomial fit plot spectra um as displayed text header plotted on top of page adds lines to text header plot all parameters plot only a few parameters set axis to ppm h for hertz k for kilohertz plot scale in current units macro to set Hz scale and plot axis 6 cm below spectrum go to next page for plotting clear exp5 and place current spectrum in exp5 setup for addi add sub dual display like routine save display parameters can use s1 through s9 recall display 1 and display as previously saved with s1 move saved display regions s1 to s9 from exp3 to exp4
53. ff always finish with this command su dm nny su inverse gated Bruker s INVGATE AU mode gives quantitative assuming d1 gt 5T decoupled spectra dm yyn su gated spectra Bruker s GATEDEC AU gives coupled spectra but with NOE buildup DEPT is necessary for 295i 15N and other y nuclei dmm ccp normal setup for DEPT and INEPT most 2d 3C experiments does hard dseq waltz16 _dres 90 dmf 1 pw90 sets decoupler pulsewidth for composite pulses where pw90 is the H decoupler 90 pulse width at the dpwr setting used for decoupling dpwr typically 42 decoupler power in dB typically want 90 pulsewidth 100 150us dmm p sd normal setup for 3C acquisition 8 1 97 dmm w works fine UWCheM HRI Varian NMR User s Guide Page 33 1d C Variations in VNMR standard setup coupled noe acquis quantitative dec acq Bruker s GATEDEC AU Bruker s INVGATE AU dm yyy dm yyn dm nny BC dl d2 aq dl d2 aq dl d2 aq 1H A B C A B C A B C e use dm yyy su to turn on decoupler or UWMACRO DECOUPLER ON see Table 6 and figure below for more decoupler information e use go to start acquisition ga will automatically apply a wft weighted Fourier transform following acquisition use wft to manually transform dscale will display the axis dsx will apply wft dscale 3 e gain 30 to 40 should usually work for 73C acqu
54. for the sequence that resides in vnmr manual via the command man relayh as and example for cosy Keep in mind that all vnmr usage looks for macros parameters pulse sequences etc in the userlib vnmrsys first and uses that copy if it exists prior to looking in the system area vnmr Thus every user can have their own version not recommended of the same sequence User s are encouraged to copy a sequence into a new name in their own directory Finally I am beginning to implement an intermediate area for facility developed software e g maclibpath vnmr maclib path VNMR will look first at vnmrsys then at vnmr maclib path and last vnmr for a macro I will update documentation here when this implmentation is done UWCheM HRI Varian VNMR User s Guide Page 16 V VNMR to Bruker AM AC Parameter Conversion Table comments apply to VNMR unless specifically mentioned otherwise Parameter Comments Experiments standard 1d H ZG si is s id gO Spl go zeros memory and starts acquisition seqfil s2pul homonuclear decoupling set the cursor on the peak and use sd to get the decoupler frequency 1d NOE difference s2pul homo y array can be used to run multiple decoupler frequencies in one exp dm yyn standard 1d decoupled X 3C NOE enhanced coupled 13C DEPT is preferred unless you need quat s quantitative decoupled 13C X nucleus T s can be quite long so this experiment can be ardu
55. g to know details of the vnmr psg setups 1 Always start by reading in standard parameters using MAINMENU SETUP UWChem MR Quick Guide for New Users Page 15 10 Then continue by running a macro e g cosy runs the cosy macro in vnmr maclib or use SETUP SEQUENCE Cosy reads in the same macro The better macros read in only the parameters specified in psgset macro statements within the primary macro routine only ProteinPack macro do not and use rtp instead Typically the parameter file found in vnmr parlib has the same name as the macro but not always e g macro hmqc has statement psgset hmqc13c dg dg1 pwx pwxlvl dpwr which reads the specified parameters in from vnmr parlib hmqc13c Reading in dg and dg1 setup the appearance of the parameter display in the dg and dg1 screens The macros for 2d sequences call another macro set2d that sets up np fn ni fn1 based on the desired digital resolution e g set2d dqcosy 3 6 sets up 3 Hz pt in F2 and 6 Hz pt in F1 Usually but not always set2d sets the actual pulse sequence used e g inside set2d isa statment seqfil 1 where 1 is the first parameter passed into the macro i e in this case the sequence name seqfil is the compiled pulse sequence psg residing in vnmr seqlib The pulse sequence source C code resides in vnmr psglib The source code is compiled with the statement seqgen e g for cosy seqgen relayh c Only users and vnmr1 can compile source
56. hat Varian sequences commonly hide some delays In this sequence a delay rofl prior to and delays rof2 and alfa following pw are not shown See Varian s documentation on pulse programming for more details 1d H Acquisition s2pul pl pw 180 90 1H rf y dl d2 aq B Critical Parameters dl relaxation delay assuming pw 30 d1 gt T7 to obtain quantitative integrations aq acquisition time determines ignoring sample effects resolution 1 aq pw amp twpr critical for pw90 calibration and many other experiments C Id 1H Acquisition e to setup parameters method b is recommended a either read in a data file rt or FILE eft click LOAD or parameter file rtp for normal H acquisition or amp b click on MAIN MENU SETUP and select nucleus and solvent UWChem MR Experiments 1H Id Acquisition Page 28 c alternatively run s2pul macro but normally will need to reset spin n gain 0 check temp and vttype and check tpwr 58 typically and pw 5 8us typically use UWMACRO TUNE PROBE and tune probe use go to start acquisition ga will automatically apply a wft weighted Fourier transform following acquisition use wft to manually transform dscale will display the axis dsx willapply wft dscale 3 optimize sw by setting right click or drag right mouse button and left drag left mouse button cursors and use movesw set reference by placing cursor close to
57. iac on e run VT outside 80 to 60 C on hcx probe 120 to 80 C on bbswg probe UWCheM HRI Varian VNMR User s Guide Page 10 Introduction to Unix and VNMR created 3 5 95 updated 12 19 01 Il Common Unix Commands cd change directory to home dir you first go to on login cd vnmrsys change to your vnmr directory pwd show current location path df k display file system will show free space amp paths see section III on directory structures mkdir make directory rmdir remove directory man cmnd show manual pages for cmnd Is list files Is la list files with options lga alias dir Is la in your cshrc file cp r name copy recursively from name down path 1 e including all subdirectories mv fname tname move fname to tname 1 e a rename unless a change in path is specified rm r name remove from name down path including all subdirectories use care this is a dangerous command Use the text editor in the CDE windowed environment if possible much easier than vi but always finish with a carriage return at the end of the file when creating macros In addition you can use fextedit filename at a unix prompt to bring up the more usable textedit editor vi filename edit filename using vi editor cgfhas couple pages of Vi Quick Reference i insert must use ESC or double up down arrow to exit insert mode a append insert after cursor pos
58. ian VNMR User s Guide Page 41 VI Homonuclear 1d Decoupling 1 Jul 98 A Discussion e HOMODEC has for the most part been replaced by COSY and cosy variants but still has utility for multiplets that are well resolved HOMODEC can provide actual J couplings whereas fast COSY usually does not B Critical Parameters d1 dpwr dm yyy dof C Acquisition relaxation delay needs to be 2 3 T see later section for measuring T7 must not be set above 24 typically 1 14 works well for saturating multiplets correct for homodecoupling set by command sd preceded by placing cursor on desired multiplet can array this variable to obtain all homodec s in same experiment e setup using SETUP SEQUENCES HOMODEC or just type in homodec to run the macro dn H1 homo y dmm c dpwr 1 dm yyy e array the dof values to acquire all in one experiment e once decoupler is on simply use go or ga D Calibration e adjust dpwr lt 14 to reduce multiplet without spilling into close by multiplets E Data Workup and Plotting e workup as a standard H e dssa should be useful for plotting see vnmr documentation UWChem MR Experiments COSY 2d Acquisition Page 42 Vil COSY 2d Homonuclear Correlation 21 Jul 01 A Discussion Absolute value mode COSY often provides sufficient homonuclear correlation data in a very efficient robust experiment Ji 2 4 Hz can typically be observed but
59. independent of MW The experiment is still transient as in NOESY however so enhancements are always smaller than 0 5 As always with NOE ROE experiments absence of a crosspeak does not confirm lack of proximity see Sanders amp Hunter for a good introduction on this subject e An ROE is produced by inverted magnetization created via chemical shift during f d2 and locked to the y axis by the pulsatile spin lock during the mix time pw should be set at 30 and ratio set to 6 for tpwr 52 on bbswg probe 2d ROESY Spectroscopy ulroesy pl pl pl 90x 90 i ratio 90 dl d2 Y a 1H rf D o SL mix A B C B Critical Parameters pl pllvl 90 pulse width at power pllvl recalibrate this parameter for roesy experiments pw tpwr 30 pulse width at power tpwr recalibrate this parameter for roesy experiments ratio number of pw with no rf between actual pw pulses usually set 6 should be 25 The spinlock field strength used in this type of experiment Kessler spinlock is B i PW360 ratio 1 Type slroesy to get the spinlock power for a particular ratio and pw ni number experiments or number of points in f should be set ok by macro time allowing want F1 digital resolution sw1 2ni 6 Hz pt nt multiple of 2 multiple of 8 preferred sspul y gives homopoil90 homospoil preceding d1 d1 relaxation delay set 2 3 7 do not set too short or will get very bad f n
60. isition if always getting ADC OVERLOAD beep then receiver gain is too high itis important the receiver gain be optimized for X nucleus experiments see H section above for how to set the gain accurately e optimize sw by setting right right mouse button and left left mouse button cursors and use movesw e set reference by placing cursor close to peak then type nl rl command e g rl 77p D BC X pw90 and 1H Decoupler Calibrations e general rule of thumb for these calibrations is short runs use facility numbers if probe problems are suspected check pw90 s of X and H observe not decouple e if numbers are close to facility values probe is likely OK e if pw90 is much less than facility value you are doing something wrong figure it out e if pw90 is much longer gt 1us than facility value find TA or facility staff always perform calibrations for overnight or longer runs for PT type experiments for standard decoupled experiments calibrations are rarely needed e 13C and other X nuclei pw90 calibrations require concentrated or labeled samples for C use 50 benzene in acetone d do not degas these samples addition of GdCl or Cr acac can improve T s dramatically UWCheM HRI Experiments 1H Id Acquisition Page 34 set tpwr to desired setting see Table 2 on lab wall for most current for calibrations for probes in UWChem 7 facility make sure you are on resonance set
61. ition use at end of line x delete single character 10x will delete 10 characters dd delete single line A move to beginning of line then use i to insert at beginning of line move to end of line then use a to insert at end of line wq write and end vi session q quit discard changes text lt ret gt go to next occurrance of text set number gives line numbers help with debugging UWChem MR Intro to Unix and VNMR Page 11 Common VNMR Commands Parameters and Flags particularly useful commands dg dg1 dgs dps da array full wysiwyg n svf filename rtf filename svp filename rtp filename svs filename rts filename wexp svf filename explib cexp delexp temp24 vttype 0 or 2 temp n vttype 0 temp n su vttype 2 temp 24 su tn Si29 d e g dl 1 pw 8 p e g p2 4000 tpwr tpwrf nl rl 9 86p rl1 9 86p Ib 5 display group normal parameters display second processing plot parameters display shim group display pulse sequence display array powerful array command for stacked acquisition kinetic exps pw checks uses full screen needed after dssh and other commands is NOT the same as the DISPLAY INTERACTIVE FULL button sets display so scaling does NOT match printer save file 1d or 2d with associated data phasing etc FILE SAVEFID reads file men
62. le at the back of the PC e delete your files from the 1 drive select all directories in Explorer window and hit DEL key UWCheM HRI Varian VNMR User s Guide Page 70 Administration of Varian Spectrometers created 5 6 96 updated 5 6 96 Rebooting Sun s on Spectrometers requires root privileges A Shutting down UNIX exit current login by assuming in SunView VNMR click on EXIT in acqi and acquisition status windows type exit in Vnmr command window hold right mouse button while mouse on background and let go on EXIT and confirm at Unix gt prompt type logout login as root gt username root password see charlie shutdown system ed vnmr acqbin killacqproc should get a message back that Acqproc Terminated cd ete mvacgqpresent acqpresent old or could use shorter filename like joe shutdown h now be patient takes a couple minutes for filesystem to stop power down system turn off hard disks then computer then diff box B Powering Up UNIX power up turn on computer then hard disks wait for gt prompt to come up then turn on diff box type b and lt enter gt and wait for system to come all the way up to login prompt login as root see above cd ete mvacgpresent old or joe acqpresent cd vnmr acqbin startacqproc wait for Acquisition Ready hit enter once unix prompt comes back login as user could use username fry password
63. lengths must be accurate e For precise measurements use d1 10 T d1 5 T will provide reasonable values if experimental times get long The number of d2 values nd2 can also be decreased to lessen experimental times nt nd2 2 d1 B Acquisition Set up use the macro dotl some randomization of the selected d2 values might be preferable ensure d1 gt 5 Ty De Table I A reasonable grouping of VD delays for Inversion Recovery T sequence where 77 is the T estimate from inv rec estimate or a repetition rate experiment 0 017 C T Analysis see Varian Subject manual Adv Id section 1 Expand about the peak s of interest Adjust the threshold with the Th 2 Enter dpf or dll then fp to locate the peak s and then find them through the relaxation set fp writes a text file fp out in the current experiment e g vnmrsys exp1 ft out 3 Ifthe dataset is a T set enter t1 for T enter t2 or for a solid state T p enter analyze expfit p3 t2 list 4 tl and t2 will display the information on the bottom of the screen To print repeat using e g printon tl printoff or print the file vnmrsys exp analyze list text file same information from the File Manager or in vnmr with ptext analyze list 5 Show a plot of the data using expl and plot with pexpl page Note that you can look at specific peaks using expl 3 and pexpl 3 page as an example for the 3rd peak To make a smaller plot use the s
64. less than a minute unless nlat is large e once done click on SIMULATE DISPLAY can be replaced with command wft nodc e to compare to the experimental spectrum type addi e change CSA and ASYM until the simulation is fairly reasonable C Least squares fitting of spinning sideband patterns e join a new experiment e g jexp3 e clickon MAIN MENU ANALYZE SOLIDS SETUP FOR SOLIDS MAS e enter the correct values for csa asym isotr srate e enter same values for np and fn as used for the simulations e the first time I like to use nlat 18 once the fit looks OK then I increase nlat e clickon FIT MARK ISOTROPIC then click left button line onto isotropic peak and click MARK UWCheM HRI CP MAS NMR Page 65 e clickon FIT FIND SSB if threshold was set right and good s n spectrum only the correct peaks should be labelled if they aren t use the EDIT SSB to delete extra labelled peaks e clickon FIT CALC the program will ask for a file name use something descriptive and unique e then open a window click and hold right button on background and slide to WINDOW and type simulate e when finished join yet another experiment and use MAIN MENU FILE click on fit filename LOAD e use wft nodc to transform and display and addi to compare the fit to experimental e the Jog file in the fit filename directory has n and 611 6 633 info at the end of the file use the unix command more log V Analyzing Relaxation Dat
65. mizing lock level with Z until an overall maximum has been found Set Z and Z to this maximum setting Keep lock signal value between 25 65 using the LOCK GAIN check that LOCK PHASE is set correctly on regular intervals especially after any large changes in 22 Other 2 order shim combinations that would require a similar shimming iterative scheme such as described above i e find a simple maximum in lock level change the high order shim in a particular direction lock level decreases then re optimize with lower order shims to see if lock level improves from what started with Z3 Z Z will require 2 order corrections at Z to obtain overall maximum in lock signal Z4 Z 2 order correction involving 2 order optimization of Z2 thus each change in Z4 can require a significant effort to see if there s any improvement at all XZ X YZ Y XZ2 XZ 2 order X YZ2 YZ 2 order Y F Id Data Workup and Plotting de will correct any linear baseline shifts be 5 will correct fifth order baseline assuming region command run automatically on a be can find peak and baseline regions be is not recommended for 1d but is ok for 2d workup default spline fit see manual for details display the axis using dscale 3 and axis p or use the macro dsx plot spectra using the following commands pl plots spectrum with se being cm in from right side we being width in cm pap plots all parameters along left hand side
66. nnot get good pw90 calibration check that probe is properly tuned check that tpwr is set correctly or pwxlvl for decoupler calibration check that external attenuator is not placed in H observe position UWChem MR Varian NMR User s Guide Page 21 e waits a long time before acquisition starts have one of the following flags set spin 40 if spin is set to a number the spectrometer will regulate the spinning taking time before acquisition to make sure the spinning is regulated gain n for this setting spectrometer will perform an autogain recommend setting the gain to a specific value manually and not using autogain wshim a autoshimming will occur should not be used except possibly between kinetic runs simply too inefficient and wastes spectrometer time use the flagsoff macro to set all these flags to appropriate values e No acqi window Type acqi in vnmr command line Can t Connect to spectrometer Pressing connect button on acqi doesn t work check that magnet leg is set to observe not tune Try in UNIX terminal window su acqproc twice once to kill once to restart this should re enable connect to acqi e FIFO Underflow Error Check sweep width an excessive sweep width gt 80 000 Hz can show this error try reducing sw and re acquiring UWCheM HRI Operation of Unity 500 Page 22 Operation of Unity 500 created 11 7 95 updated 10 16 08 Proper Exiting and Logging In and Initial
67. o small knobs to get 27 on meter Replace cable to decouple and switch to observe Enter tunec macro tn C13 gain 0 su check temperature control with vttype 2 for control 0 if control unit off and temp enter su UWChem MR CP MAS NMR Page 63 X tune silver De 1H tune X match amp match red magic angle adjust don t touch tuning knobs on bottom of Doty CP MAS probe Use of Wavetek Sweep Unit Use this setup if you cannot find the resonance with the Varian tuning meter e switch oscillascope Wavetek and synthesizer on using the power strip switch at back of cabinet e use appropriate tunex command e g tunec and note the sfrq reading exactly e set the synthesizer to the sfrq setting and the Wavetek also to sfrq e turn the level up on the synthesizer to 0 5 dBm e hook the BNC from the mixer to the input of the probe e turn the Wavetek s sweep width control clkwise to increase the sweep and center the synthesizer marker on the oscillascope e adjust the tune cap to center the resonance on the marker the marker size can be reduced with the marker width control small knob on the Wavetek e turn the match cap to make the resonance as deep as possible it will hit the bottom line only when the sweep width on the Wavetek is very narrow spreading out the resonance very wide at this setting it will be difficult to keep the resonance on screen e 2 order tune the tune and match caps to only a
68. oise np number of points in t usually want 2 2048 since costs nothing but disk space and gives better resolution in F2 mix mixing time often is varied to provide build up curves Set close to T should provide maximized crosspeaks for high MW gt 2000 NOESY is preferred C ROESY Acquisition e see NOESY section adjust baseline similarly and make certain to use actual ROESY sequence do this by setting ni 2 when performing the calfa correction UWChem MR Varian NMR User s Guide Page 52 D Calibration calibrate pw tpwr p1 pilvl for all ROESY experiments e watch out for coupling partners that are centered in the spectrum these will give TOCSY crosspeaks in a ROESY in this case change tof to get the coupling pair off center E Data Workup and Plotting e same as dqcosy see DQCOSY section for phase sensitive work ups UWCheM HRI Varian VNMR User s Guide Page 53 XII HETCOR 2d Heterocorrelation Spec normal X nucleus detection 27 Dec 97 A Discussion e typically want to run HMQC see next section or HSQC as dramatic improvement in S N is obtained in the inverse experiments e HETCOR is still run when high digital resolution is needed in the X dimension a common example would be 3C H correlation spectroscopy where resolved closely grouped aromatic 3C are needed to improve assignments of the aromatic protons B Critical Parameters e section not ready yet C HETCOR Acquisition e
69. on TRACE and select strong intensity trace trace f1 changes columns rows trace f2 goes back wtia interactive phasing on t trace left button sets Ib gf wft2da performs second or both transform s pick off two traces that have crosspeaks downfield trace save number as rl upfield trace save number as r2 ds r1 do 0 order phase only ds r2 do 1st order phase only click left mouse button on downfield position sets toggle pt iterate between ds r1 and ds r2 to get good phase dconi Should now have good phasing trace f2 dconi allows phasing along F2 similar to above if needed if integrals have been setup as above and only if fnl fn then be f1 can sometimes work wonders here to plot plot2dhr is a new macro that works quite well if you want 1d projections load the high resolution 1d into a separate experiment before issuing the macro command Otherwise I like parameters wc 130 we2 we sc 0 this leaves room for a vertical projection or to print parameters on the page use disp2d to set these maximum printable parameters on 8 5x11 paper are wc 230 wc2 150 rl p gives F2 referencing rll p gives F1 reference use plot2dhr preferred or pconpos or pconneg to plot phase sensitive spectra UWCheM HRI Experiments TOCSY 2d Acquisition Page 46 IX TOCSY or HOHAHA Total Correlation Spectroscopy 17 Jul 00 A Discussion e TOCSY provides COSY like spectra but with all H connec
70. osy macro sw1 2ni gives usable resolution must have lt 12 Hz but want 6 Hz if time allows for best results nt 4is minimum phase cycle sw set using movesw macro preceeded by boxing selected area of spectrum swl must equal sw for foldt macro commonly used recommended for fast COSY UWChem MR Varian VNMR User s Guide Page 43 tau dl set 50 150 ms typical for long range COSY best set 2 2x7 but often run with d1 T C COSY 2d Acquisition e for COSY setup as in H 1d and optimize sw with the movesw macro then retake the data to ensure Sw is set correctly move the parameters assuming you ran the H in exp and have nothing important in exp2 the command mp 2 jexp2 entered from exp will work from exp2 then use SETUP SEQUENCES COSY or enter the macro cosy or gcosy check that ni swl 2ni lt 12Hz pt 6Hz pt if time allows d1 and np are correct check time then enter au to run cosy D Calibrations e can use facility pulse width calibrations COSY is very forgiving with pulse widths e even so performing a pw90 calibration is always recommended for all 2d experiments E 2d Data Workup and Plotting e COSY data can be processed with the do2d macro or use the wft2d command see also man cosy or man gcosy do2d applies symmetrization with the foldt macro e general display and plot commands dconi displays 2d s with color map faster than contours plot projections before iss
71. oupler power larger number is higher power typically never gt 46 dmf decoupling strength pw90 H at dpwr dmm decoupler modulation mode either dmm ccp dseq waltz16 dres 90 or dmm cew both are equivalent is best for typical compounds dm decoupler on off flag see Table 6 C Id X 1H Acquisition e start by setting the probe parameter appropriately e to setup parameters method b is recommended a either read in a data file rt or parameter file rtp for standard X H acquisition or amp b clickon MAIN MENU SETUP and select nucleus and solvent c alternatively run s2pul macro but normally will need to reset spin n gain 0 check temp and vttype and check tpwr 58 typically and pw 5 8us typically e use UWMACROS TUNE PROBE TUNEC13 or tunec macro and tune C channel reattach C X cable to X Obs BNC make sure 4 wave cable is correct for C make sure low pass brown filter is in line make sure correct probe cap is inserted none in bbold probe make sure no external attenuator is in line e use UWMACROS TUNE PROBE TUNE H1 or use tuneh macro will have to reset gain after tunec if UWmacro is not use and tune decoupler channel reattach H decoupler cable to decoupler BNC Table 6 Common Decoupler Parameter Settings Parameter Settings dm yyy dpwr 46 su typical for 13C acquisition full on decoupler keep dpwr lt 48 fdm nnn su sd turns decoupler o
72. ous DEPT 3C editing homonuclear correlation 2d sw ni 2 gives usable digital resolution usually need lt 6 Hz pt long range cosy double quantum cosy complete phase cycling is crucial for the dq filter nt muliple of 8 phase sensitive noesy flat baselines are important for observing small noe s use calfa N Q phase sensitive roesy flat baselines are important for observing small noe s use calfa total correlation WOHAHA 222 toesy O O useful for mixtures or separated spin systems heteronuclear correlation use only if need very high 13C resolution inverse hetero correlation TI multiple bond hetero correl hmqc bond 0 important experiment for observing through linkage bonding i Read and Save Commands read data file rt filename in VNMR use also MAIN MENU FILE click on filename and LOAD save data file svf filename in VNMR no menu selections for this read parameter file rtp filename save parameter file svp filename rts filename load shim file noneneeded loadshims loadshims is UW written macro having load y su load n su UWChem MR Varian NMR User s Guide Page 17 tune H observe tune 72C channel zero and go automation run AU autom nam au all VNMR programs run from compiled routines halt acquisition with data H_ ssa in VNMR svf can be issued during acquisition to save data resume acquisition abort acquisition CRE or SK faa data is ot automation setup 1 S
73. peak then type nl and rl command e g rl 7 24p if always getting ADC OVERLOAD beep then receiver gain is too high gain can be set three ways number 1 recommended amp 1 simply lower gain until ADC OVERLOAD warning goes away if warning stays with gain 0 insert attenuators into bottom BNC preamp output not into the probe connections make sure gain is within 10B of overload warning 2 reduce nt 1 and enter gf make sure to wait a few seconds menu will flash before clickon acqi and then FID make sure SPECTRUM is not selected and click on DOWN until you see the red horizontal lines that indicate the ADC clipping limits adjust gain until the fid fills 1 2 of the region to the clipping voltage switch nt back to original value 3 set gain n which will implement autogain adjustment not recommended especially for 2d where huge artifacts can result D 1H pw90 Calibration normally can perform calibration on sample best to not perform it on a clean solvent since T for solvents can be very long set tpwr to desired setting typically 58 for 5mm probes 52 for 3mm probes set d1 5 10s depending on sample T d1 5 is usually ok recommended perform a coarse check of pulse widths using array lt ret gt variable to array pw number of increments 20 starting value 3 size of increment 3 set nt 1 and use go should see a sinusoidal response if not usually d1 needs to be longer to obtain an accurate pw90
74. r runs recalibrate at least observe and best decoupler pulse widths e set mult as needed dept 135 has mult 1 5 for full editing use array mult 0 5 1 0 1 0 1 5 e use au to acquire for full editing ga is ok for dept 135 D Calibration e see 3C section for nominal 3C X and H decoupler calibrations e Often the best way to calibrate the decoupler is to run a DEPT 90 on a compound having a known methylene this carbon should be nulled in a DEPT 90 Change pp can use an array to obtain minimum signal at the methylene and use on unknown or less concentrated sample e The delay d2 and the final decoupler pulsewidth mult pp are calculated by the DEPT macro as follows 1 D2 zJ use Jc 150 if olefinic present 130 otherwise 6 pulse mult pp sn 2 4 Thus mult sin T T je radians pp af 1 d 00 D in J 1 I af l d d in ra ian 90 D z in e vn UWCheMm HRI Varian VNMR User s Guide Page 37 For trimethyl silyl J5 _77 2 Hz d2 250 msec 19 47 mult 0 216 o These values are calculated internal in the vnmr psglib dept c pulse sequence code E Data Workup and Plotting for full editing try autodept or padept macro for dept 135 use wft and phase sl s and rl r are enormously useful for comparing data in different workspaces use these in combination with md 1 2 for example to move the save regions in e
75. re two spectra at non multiple srates the peak that does not move is the isotropic peak in ppm use macro axisp e write down the spinning speed using BOX in hertz use macro axish and check parameter DELTA e write down the value of fn e set the threshold click on MAIN MENU DISPLAY INTERACTIVE NEXT TH to just under the smallest spinning sideband peak to be included in the fit e save the new spectrum using svf command e to setup for comparing simulations and fits to the data use the commands clradd and then spadd for more info look under addi in VNMR online to move the experimental data to exp5 B Simulation of spinning sideband patterns You normally will always run simulations of the MAS data to gain reasonable estimates of CSA and ASYM Only if you are very confident of these two parameters should you go directly into a fit If the data is messy you might use simulation alone to give estimates of CSA and ASYM e join another experiment assuming section A done in exp1 e g jexp2 and load the file in that you just saved at the end of section A e clickon MAIN MENU ANALYZE SOLIDS SETUP FOR SOLIDS then MAS e enter the values written down for isotr srate and fn and make best guesses for csa and asym e set np equal to 2048 up to 8192 lt fn e set nlat 18 to 72 bigger is more accurate but takes longer for estimates to put into the fit algorithm I recommend nlat 18 e clickon SIMULATE CALC FG and wait should take
76. retations See Sanders amp Hunter Chap 6 for an excellent introduction to NOE s see the Bruker User s Guide for more details about NOE experiment particulars Often combinations of MW solvent and temperature conspire to make NOESY crosspeaks non realizable during nominal mix time in this transient experiment ROESY will often be better alternatives for small lt 2000 MW Performing at the least an inversion recovery null estimate of H T values is highly recommended prior to acquiring NOESY spectra This method of obtaining H T s is quick and simple to setup Perform full T analysis for best results see dot1 macro Degassing leads to longer T values for smaller MW compounds and is recommended for 1D variants The longer T values which can lead to greatly enhanced NOE build ups sometimes cannot be maintained for 2D experiments due to limitations in time for the experiment NOESY crosspeaks for small MW will be positive opposite to the inverted diagonals as will exchange crosspeaks use temp variation to differentiate exchange from NOE crosspeaks For larger MW crosspeaks will be negative same sign as diagonals Multiple mix times lt 77 will be required for quantitative work in using NOESY build up curves for obtaining distance information ulnoesy is recommended but currently unavailable over the vnmr standard sequence noesy For now use tnnoesy if needing presaturation otherwise noesy or equivalently SETU
77. ronuclear Correlation Experiment ccceeeeeeeeeeeeeeeeeeeeeeeeee 57 XVII Fitting Partially Overlapping Peaks Deconvolution in VNMR 58 XIX TH Spin Lattice Relaxation T1 59 Il Rapid Determination of T by Inversion Recovery Null Method 59 Ill Quantitative measurement of T by Inversion Recovery Method ee 60 A Comments 60 B Acquisition Set up 60 C T Analysis see Varian Subject manual Adv 1d section 60 XIX Cross Polarization Magic Angle Spinning NMR 62 A Preparation of MAS spectra 64 B Simulation of spinning sideband patterns 64 C Least squares fitting of spinning sideband patterns 64 Data Backups from UNIX Workstations ses 66 Backups to Colorado Systems Tape Drive on Windows NT Network n s 66 A Perpare tar file on UNIX workstations 66 B Move file to NT server 66 C Backup file to Colorado Systems tape drive 66 D Recovering backup ed up data 66 Il Backups to Omega Zip Drive on Windows NT Network currently installed On MOord r Terminus Drazi esee ninenin i i 67 A Prepare a tar file 67 B Backup file to IOmega Zip drive 67 D Recovering backup ed up data 67 Ill Backups to Pinnacle Optical Disk on Tango on 5 floor cescsceeeeseeseeeeseeee 67 A Format a New Optical Disk 67 B Create File System on New Optical Disk 68 C Backup to Optical Disk 68 IV Backups to Pinnacle CD Writer on Twiddle on 9 floor c c ce
78. s 1H pw90 checks are always recommended time permitting for all experiments if probe problems are suspected check pw90 s of X and H observe not decouple always perform calibrations at minimum H pw90 check for overnight or longer runs for PT type experiments for standard decoupling calibrations are rarely needed even for long runs although having pw lt 90 is best See file on line narn vnmr5 3b shims probes eseese if not logged onto narn lexport home vnmr shims probes secere if logged onto narn or preferable to the following is UWMACROS LOADSHIMS or FILES DATA SHOWSHIMS shim files that are available can be listed by entering the following commands in a UNIX window facility shim files type dir vnmr shims your shim files type dir vnmrsys shims user shim files type dir home user vnmrsys shims or simply go to path in the FileManager load shims with UWMACROS LOADSHIMS Probe Tuning recommended method UWMACROS TUNE PROBE enter macro similar to tuneh in vnmr maclib gain 0 is necessary for tuning UWmacros restore original gain setting make sure decoupler is off dm n su if necessary e move cable e g H from obs or dec BNC to tune BNC e switch knob from obs to tune e adjust tune and match to achieve 0 on meter in most cases getting needle lt 10 is sufficient On many of the probes there will be three capacitors It is essential that the
79. s will not be feasible When performing water suppression I have not had great success with having PFG shimming always improve my water suppression but Varian and Bruker for their systems insist PFG shimming can do better than the best spectroscopists even for these most demanding samples So it is worth trying and experimenting with Normal PFG Shimming Procedure Save your shims if they are important use svs lt enter gt or click FILE SAVESHIMS Type gmapsys to start the gradient mapping system in the software If you lose the gmapsys buttons at anytime as happens for example after a wft or ds simply re enter the gmapsys command to get them back You can run gmapsys from any experiment but you must end the session by clicking on QUIT to properly exit back to your original data set parameters Check that the parameters are setup correctly for your solvent e g if you are working in a deuterated solvent tn Ik INOVA or tn H2 UNITY for a protonated solvent 90 10 H O D 0 tn H1 See Section III below for 1ST TIME USE and a detailed explanation of parameter setup UWCheM HRI PFG Shimming With VNMR Page 73 4 Ifyou are using a deuterated solvent on the UNITY only disconnect the lock cable at the bottom of the 2nd lock filter attached to the back of the magnet preamp box and connect it to the X Observe BNC No cable switching is needed on the INOVA or for protonated solvents on the
80. sescseestssteseeeeeen 68 UWCheM HRI Table of Contents Page iii A Transfer Data to Twiddle 68 B Transfer Directories Files to Easy CD Window 68 Administration of Varian Spectrometers ss 70 Rebooting Sun s on Spectrometers requires root privileges eeeeees 70 A Shutting down UNIX 70 B Powering Up UNIX 70 Pulsed Field Gradient Shimming With VNMR ss 71 l G n ral DISCUSSION Rss dass nn a a eerie Meee td 71 Il Normal PFG Shimming Procedure cccccceesececeeeeeeeeeessaeaeeeeeeeeeeeaaeeeeeees 72 Ill First Time Use and Detailed Explanation of Parameters 74 A Critical Parameters 74 B Details of Correct Parameter Setup 75 UWCheM HRI Quick Guide for New Users Page 4 UW Chemistry Magnetic Resonance Facility UWChem 27 I Facility Layout 2 floor Matthews _ neil whittemore Matthews 2nd floor 2237 marv kontney D x ao Sun Wkstn NMR PCs As of July 2001 ATHENA HOMER PHOENIX VIR AC 300 AC 300 AC 250 UNITY 300 Phoenix routine H 19F 31P 13C routine H 3C routine BB VT solid state NMR auto sample changer quad nucleus probe 1H 3C dedicated routine BB 79Si B 7H 99H etc variable temperature conformational motions solid state packing catalysts amorphous and glassy compounds UWChem MR Quick G
81. t F1 digital resolution lt 6 Hz pt sw1 2ni number of points in ty usually want 2 2048 since costs nothing but disk space and gives better resolution in F2 usual to not zerofill in F2 fn np and give one zerofill in F1 fn1 2ni fn1zfn is ok for DQCOSY but to baseline correct in both dimensions set fnl fn multiple of 8 y gives homospoil 90 homospoil preceding d1 C dqcosy Acquisition DQCOSY is setup essentially the same as COSY use dqcosy macro except want fn np2 2048 and typically ni 350 ni is set up by macro check that F1 dig resol lt 6 Hz pt sw1 2ni D Calibration always recalibrate pw90 90 pulse for dqcosy experiment is sensitive to quality of this easily calibrated parameter see H section for instructions macro uses pw90 to set pw with high res 1d or perhaps better ni 2 run carefully baseline correct then run calfa UWChem MR Varian VNMR User s Guide Page 45 E Phase Sensitive 2d Data Workup and Plotting often should not need to phase DQCOSY data at all phase sensitive data should be processed something like the following set pmode full allows phasing along F2 in 2d spectrum wft 1 transform just first spectrum wtia interactive phasing middle button scales left sets Ib wftida perform first transform on t dimension if integrals have been setup best on high res 1d done prior to setting up dqcosy then be f2 can sometimes work wonders here click
82. tal time of experiment interleaved acquisition depends on routine in vnmr acquire bs scans per increment loop until nt completed 2d Processing Commands and Parameters FT size in F1 AC AM square requires SI1 SI 2 SI2 2 reference in FI FT and weight full set in vnmr for absolute value sets use do2d or wft2d FT and weight t dimension counter intuitive commands but mean Ist transform interactive weighting can be done on t fid e g wft 1 wtia and on t fid e g wftida TRACE wtia display color map 2d Plotting Commands plot contours in vnmr see also pconpos and pconneg UW written macros plot size in vnmr sc still controls distance in mm from right hand side Plot 2d using high res 1d o pot have 1d high res already worked up in separate exp follow prompts peak picking and volumes UWCheM HRI Intro to Unix and VNMR Page 20 VI Trouble Shooting wrong parameters make sure probe parameter matches probe in magnet sample won t spin if probe has been changed find TA to try reseating spin collar tube at top of magnet push down the aluminum tube guiding sample in check that tube is not inserted too far into spin collar check that VT air is not turned up too high sample won t eject try turning the VT air up to 80 turn it back down after inserting check that VT air is hooked up rather than N gas which has lower pressure check that air pressure gauge in southeast corner is turned up
83. ted appropriate with the mix time Thus mixtures and small molecular fragments can often be more easily identified with TOCSY e TOCSY spin lock mixing provides Hartman Hahn matching for coherence transfer Strong coupling is required and off resonance effects can be troublesome Use long range COSY or DQCOSY to observe correlations involving small J couplings e ROE s can be a problem with TOCSY spectra especially as mix gets large Clean TOCSY a variant not implemented in Varian s standard sequence can offset ROE s by providing a similar NOE build up but this variation will only work for large MW where NOE s are negative e Watch the manual page man tocsy for changes in the TOCSY sequence s Implementation of DISPSI 2 spin lock is underway as well as gradient homospoils e Multiple mix times are often useful These cannot be run arrayed with phase 1 2 phase 3 TPPD must be selected if arraying mix is desired Queuing multiple experiments with different mix times and phase 1 2 is another method for this type of data collection and is recommended 2d TOCSY Spectroscopy MLEV 16 Spin Lock pl pllvl trim 90 pw tpwr dl d2 ee at 1H rf Jaa MLEV 16 spin lock A C B Critical Parameters pl pllvl 90 pulse width at high power pllvl typically 52 to 60 recalibrate this parameter for all tocsy experiments uses pw tpwr from Id to set p1 p1lvl pw tpwr 90 pulse width 20 to 40
84. this is dependent on sw and ni 1 digital resolutioninF2 2X digital resolutionin F1 np 2ni assuming zero filling only in F1 not common in F2 For very large ni very small couplings can be observed often well below the linewidth of a high resolution 1D experiment A common rule of thumb is that Jyp 2 dres1 3 will be observed in a COSY spectrum this estimate is definitely a guideline only dres digital resolution in F1 defined above The most efficient method of observing small Jj is with long range COSY which involves this sequence and tau gt 0 typically tau 50 200 ms cosy loses sensitivity from the strong resolution enhancing sinebell and sinebell squared apodization functions used to overcome the absolute value processing Even so minimum phase cycling nt 4 usually gives sufficient sensitivity GCOSY is even faster nt 1 nt 2 is better DQF COSY next section removes all singlets and gives much cleaner diagonals Use this sequence after a COSY if crosspeaks close to the diagonal or on a strong singlet region are needed gcosy is preferred if a PFG e g hcx or bbswg probe is installed nt 1 can be used with this sequence reducing total acquisition time by 4 2d Absolute Value COSY relayh pw pl 90x 8 dl d2 tau tau at IH rf y A B C B Critical Parameters p1 90 for maximum sensitivity 45 typical to reduce intermultiplet crosspeaks ni usually set satisfactorily by c
85. tive T 1 4xd2 n set dl 2 3 x T start with d3 0 0 6xT then click in gmapsys SETUP GO DSSH if the ratio of intensities is 1 0 8 to 0 4 the d3 array is ok Increase d3 if the 2nd profile is gt 0 8 x the 1st decrease d3 if the 2nd profile is lt 0 4 x the 1st or if the 2nd profile is severely distorted compared to the first sometimes for solvents with different 2H nuclei e g pentane d chemical shift and or J coupling of the H cause intensities for d3 gt 0 to be bigger than d3 0 if these cases you just have to experiment with different d3 values to select the best most sensitive array UWCheM HRI
86. to mark sample won t shim read in proper shim file use UWMACROS LOADSHIMS check that you have enough solvent 20 6 ml and are 68mm down or centered in rf region check that lock power is not too high and that lock phase is correctly adjusted let magnet warm for quite a while up to 1h after a cold experiment shims are more sensitive to this than on AM 500 if previous student didn t stop early enough you will need to adjust especially the lock phase fairly often during the warmup and wack the previous person as hard as possible with a wet noodle and also reshim somewhat over 30 min to 1 hour command doesn t work type return and try it again some mistypes carry over to next line S N seems poor most likely an attenuator has been left in line at the output of the preamp leading back to the ADC if your sample is not very concentrated remove this attenuator and adjust the gain setting check pw90 at least on the observe side if unusually long check with TA or facility staff spectrum on screen is only an inch long or so type full to reset plot window needed after dssh command says exp locked enter the command unlock where is the exp number that s locked won t let jexp probably have not created the experiment see WORKSPACE explib will list all experiment areas cexp will create experiment area delexp will delete experiment area saves disk space ca
87. two similar capacitors stay at nearly the same capacitance i e same number of turns from end so make sure to move them together For example on the bbold probe the H channel has a gold and two silver rods all small diameter connected to capacitors The silver are both match capacitors and must therefore be turned together if you move one clockwise by 4 turn the other should also be turned clkwise 1 4 turn e switch knob back to obs e move cable back to obs or dec BNC e tune other channels as needed inverse 1H X probe for H channel tune the gold match and silver tune1 knobs first then make sure black knob tune2 is within 4 turn of silver knob UWChem MR Operation of Unity 500 Page 25 IV Lock and shim Use care when clicking on CONNECT on the acquisition window fast clicking can crash the computer requiring up to 30 min to correctly reboot so use patience when going to acqi click into the LOCK panel in the acqi window and turn off the lock change ZO until there is no oscillation in the lock signal do not hesitate to turn up lock power and lock gain achieve lock but lower LOCK POWER as soon as possible to avoid lock saturation set the LOCK POWER to recommended settings only go up to potentially safe setting if shims are poor set back once shims have improved and use LOCK GAIN thereafter to adjust amplitude adjust LOCK PHASE analogous to a shim to get positive going signal turn on LOCK adj
88. u alternative MAIN MENU FILE save only parameters 1d or 2d significant savings in disk space reads parameters save shim settings to user directory vnmrsys shims FILE SAVESHIM reads shims follow with loadshims macro UWMACROS LOADSHIMS saves data to filename at completion of experiment V required for quotes within the main quotes requires au used to start acquisition lists all experiment areas menu MAIN WORKSPACE LIBRARY creates experiment area number see also WORKSPACE deletes experiment area number save disk space see also WORKSPACE UW macro to set temp to 24 C cp into your vnmrsys maclib rename and edit for other temperatures UWMACROS SET TEMP variable temp controller not present 0 or present 2 no temp specificied for control command preceeding probe change see macro tempoff command to re establish temp control at 24 C after probe change see macro temp24 set transmitter to 2 Si frequency uses look up table see macro tuneh set delay time in seconds use dps to make sure about timing common pulse width in usec use dps pulse width in usec use dps observe transmitter power set in dB from 0 to 63 larger is more power observe transmitter fine power control from 0 to 4095 3db spread first set cursor close to peak then finds nearest line first use nl to set cursor exactly on then sets reference expand about correct reference on diagonal peak use to ref f7 axis giv
89. uide for New Users Page 5 NATOTH Avance 360 non routine BB VT long term VT kinetics concentration limited samples 5 and 10 mm BB probes 5 mm inverse probe NARN UNITY 500 non routine H BB VT high sensitivity sample limited H lt 5 mg C lt 15 mg short run sophisticated experiments e g HMQC DQCOSY gCOSY gNOESY limited access VORLON INOVA 500 inverse exps 2D studies long term sophisticated gradient enhanced experiments combi chem MAS probe limited access XIRTH INOVA 600 long term 2D studies long term most sophisticated gradient enhanced experiments e g NOESY ROESY HMQC DQCOSY limited access ESR ESP 300 electron spin resonance paramagnetism free radical chemistry PC s DEATHSTAR TERMINUS MORDOR VULCAN surround the main printer GKAR KOSH has special software BABYLON 128 104 70 61 is the Win NT server Sun s for data workup UltraSparc 1 M170 s CENTAURI and ZHADUM are located in rm 2010 VORLON is in rm 2201a NARN and SHADOW Sparc 5 s are hosts for the respective instruments SGIs NATOTH Avance host computer and GQUAN for off line data workup ll Facility Personnel Director Chemistry Instrument Center Prof Paul M Treichel Rm 6359A Director Magnetic Resonance Facility Dr Charles G Fry Rm 2128 Charlie Associate Director MRF Dr Neil Whittemore Rm 2132 NMR Engineer Marv Kontney Rm 2210 ESR Engineer Roger Clausen Rm
90. uing the following command using menus use the PLOT button pcon plots contours pconpos plots phase sensitive contours with positive peaks having 10 contours and neg peaks having 1 contour pconneg Opposite of pconpos plot2dhr plots with high resolution traces must have 1d in another experiment UWCheM HRI Experiments DQ COSY 2d Acquisition Page 44 Vill DQ COSY Double Quantum Filtered COSY Phase Sensitive A Discussion 17 Jul 00 DQCOSY is currently accepted by most in the NMR community as the most powerful form of COSY it is recommended for any experiment beyond the FAST COSY or GCOSY removes singlets important for strong solvent peaks diagonal in pure adsorption mode with crosspeaks cleans diagonal for close in crosspeaks direct coupling for crosspeak at V V is J 2 is anti phase whereas indirect coupling at V V is J 3 or J 4 for example is in phase allowing for J assignments in complex molecules alternative experiment is E COSY see section X 2d Double Quantum Filtered COSY dqfcosy 1H rf pw pw pw 90 90xx 90 Gage dl d2 at A B C B Critical Parameters pw tpwr di ni np fn fnl nt sspul 90 pulse width at power tpwr recalibrate this parameter for all dqcosy experiments relaxation delay set 2 3 T artifacts will result if d1 is too small number experiments or number of points in t should be set ok by macro time allowing wan
91. ust LOCK PHASE as a shim to maximize lock signal make sure to return to LOCK PHASE fairly often when shimming especially after large changes in Z2 click into SHIM window and shim normally start by 1 order shimming Z and Z2 when finished take nt 1 acquisition to check line shapes use nl dres or if S N is excellent use nl res to get indication of line shape target 50 full linewidth lt 1 Hz for most samples spinning or non spinning now 2 order shim Z2 choose a direction to move Z2 this will decrease lock signal 1 order shim had lock signal maximized at current Z2 see if Z1 improves if so continue if not go other direction in Z2 shim X Y XZ XY XY X2 Y2 all 1 order then repeat 2 order Z Z2 shim check line shape if not at target try spinning sample if improves considerably turn spin off and work on X Y shims if did not improve much with spinning then need to target higher order Z s Table 4 shows shims dependencies for the Unity 500 2nd order shimming is required on all 500 MHz instruments i e you simply cannot expect to get a good shim without it Table 3 Field and Lock Power Settings for Unity 500 ZO field as FINAL STARTING solvent_ un of 97 12 01 lock a RE lock RE CE 1 936 Te ae oe dimethysulfoxide d6 2 4965 ZO will change by 100 units each week UWCheM HRI Varian NMR User s Guide Page 26 Table 4 Major Shim Interactions on Unity 500 means shim move
92. ve fid directly while changing gain go or ga will automatically wft or au will perform additional commands useful for 2D or auto saving data 1H acquisition usually needs only a check of nt movesw similar to O in EP on AM AC s movetof similar to changing O1 on AM AC s 13C turn on decoupler dm yyy su UWCheM HRI Quick Guide for New Users Page 9 remember to turn off when done set nt 1e6 if don t know needed number of scans can wft aftereach bs scans Ib 2 4 needed for 13C use sa_ to stop acquisition once S N is good enough e cursor close to peak nl rl 77p will correctly reference CDCl3 peak rl1 77p for fI in 2d s e dsx wft dscale 3 e ppa pscale pl page typical plot VI Saving and Deleting Data and Backups e FILES SAVEFID or svf data name saves only raw fid but with all parameter s intact e MAIN MENU FILE SAVEFID type in name without quotes e MAIN MENU FILE left click on data name DELETE will delete data e use FTP program on PC s and connect to ZIP s to backup VII Logging Out e exit VNMR first e right click on background exit Vill Don ts e use Unity without VT regulation run at 26 C if just want ambient e use too little solvent Varian probes require more solvent than Bruker probes too little solvent will just give you a terrible shimming session e run VT outside 20 to 40 C without switching over to N gas e run VT below 10 C without Var
93. xp1 to exp2 use clradd spadd to move a spectrum into exp5 then addi to compare that spectrum with currently displayed spectrum use dss with proper vo and ho to give a stack pl all to plot the stack UWCheM HRI Experiments INEPT Id Acquisition Page 38 IV INEPT Insensitive Nuclei Enhanced by Polarization Transfer 22 Jun 98 A Discussion of INEPT See the discussion section in the previous section on DEPT INEPT should be used if one J value is involved and it s size is Otherwise use of DEPT is recommended The previous statement is not intended to dissuade students from experimenting with the different version of polarization transfer After all the fearless facility manager has been known to give not such good advice on occasion Please do let me know your observation preferences if you try both INEPT and DEPT on same similar compounds Refocused Decoupled INEPT ineptrd c written by cgfry from Bruker s INEPTRD AU 90 180 90 y 180 90 y lt 1 4J gt k 1 4J ke m 8J k m8 gt 1H rf pp 2pp pp 2pp 1808 90 180 X rf 2pw pw 2pw dl lt d2 gt lt d2 es lt d3 gt B Critical Parameters dl relaxation delay typically 1 2s j 140Hz change if you want to observe X with Jy y gt J180 Hz or lt 110 Hz INEPT is sensitive to accurate J coupling being entered use DEPT unless you ar

1 - Department of Chemistry - University of Wisconsin–Madison

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1 - Department of Chemistry - University of Wisconsin–Madison