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Advanced 1D and 2D Experiments Guide

1. Name of frequency list 14 Make the followin Length of shaped pulse p12 Power Level of shaped pulse SP2 Name of shaped pulse SPNAM 2 15 Click on Click on 96 B7170 00 01 Figure 5 54 EM Select dataset for parameter update Target dataset NAME EXPNO 1 PROCNO 1 DIR C data3 0 O Do not ask again NOTE The Target data set window above is to verify the correct data set and can be switched off by enable the Do not ask again option 17 Click on 18 Use the Ctrl w keys to close the Shape Tool window 5 7 3 Setting up the acquisition parameters 1 Select the AcquPars tab by clicking on it 2 Make the following changes NS 64 DS 8 D9 0 080 5 7 4 Acquisition 1 Select Boo by clicking on it 5 7 5 Processing 1 Click on the Process tab in the TopSpin Menu bar Figure 5 55 Process Publish View Manage o Start Proc Spectrum v w Adjust Phase v Calib Axis v 1 Pick Peaks v IBI Integrate Advanced v 2 Click on the down arrow inside the J Proc Spectrum s button Acquire Analyse D 1 0 00 01 97 Figure 5 56 Window Multiplication wm Fourier Transform ft Start Automation AU Program xaup 3 Select Configure Standard Processing by clicking on it 4 Deselect the following options Auto Phasing apk
2. Control sample temperature edte 7 Select ej by clicking on it NOTE Wait till the sample lift air is turned on and remove any sample which may have been in the magnet 8 Place the sample on too the top of the magnet 9 Select Aj Samples by clicking on it Figure 7 5 Tum on sample lift air ej Control sample temperature edte 10 Select ij by clicking on it NOTE Wait till the sample is lowered down in to the probe and the lift air is turned off A licking sound may be heard 11 Select He Lock by clicking on it Figure 7 6 3 Solvents table EJ Solvent Description Acetic Acetone Acetone Hump C6D6 CD2CI2 CD3CN CDCI3 CDCI3_SENS CH3CN D20 D20 DEE Dioxane DME DMF DMSO EtOD acetic acid d4 acetone d6 acetone d6 CHCIS3 benzene d6 methylenechloride d2 acetonitrile d3 chloroform d chloroform d ETB HPLC Solvent deuteriumoxide diethylether d10 dioxane d8 dimethylether d6 dimethylformamide d7 dimethylsulfoxide d6 ethanol d6 Lock nucleus 2H 139 12 Select H20 D20 by clicking on it 13 Select by clicking on it NOTE This performs a atma automatic tuning and requires a probe equipped with a automatic tuning module Other options can be selected by clicking on the down arrow inside the Tune button 15 Select by clicking on it Figure 7 7 Turn sample rotation on ro on Change s
3. 45 40 3 5 3 0 2 5 ppm 10 Click on El to save the frequency list D 1 0 00 01 157 Figure 7 36 Write name of f1 list to acqu parameters 7 11 Click on 7 8 4 Setting up the acquisition parameters 1 Select the AcquPars tab by clicking on it 2 Change the following parameter PULPROG wetdw 4 Select 43C for F2 5 Click on Figure 7 37 ty Edit Spectrometer Paramete frequency logical channel amplifier preamplifier BF1 50026 NUC1 SFO1 500 262001 SGU1 X800W g oh 1H LNA OFS1 2001 0 He dH 00 XBB19F 2HS BF2 125 790477 NUC2 SFO2 125 795446 OFS2 4968 7 laac zj _ 1H 100 w_ seu BF3 500 26 MHz NUC3 SFO3 500 26 SGUS XS00W off OFS3 0 0 1H 100 W 2H 150 W cable wiring settings possible RF routing show receiver routing E show RF routing e cortab available m F show power at probe in Save Switch F 1 F2 Switch F 1 F 3 Add a logical channel Remove a logical channel f Info Param Close 6 Click on 7 Click on TE to display the pulse program parameters 158 B7170 00 01 Figure 7 38 NOTE The message in Figure 7 37 appears if there is no decoupling program entered in the CPDPRG2 parameter 8 Click on NS 16 DS 16 CPDPRG2 garp GPZ21 80 GPZ22 40 GPZ23 20 GPZ24 10 10 Click on gl to read in the Prosol parame
4. 11 Select by clicking on it Figure 3 8 3 Solvents table t3 Acetic acetic acid d4 Acetone acetone d6 Acetone Hump acetone d6 CHCI3 C6D6 CD2CI2 CD3CN CDCI3 CDCI3_SENS CH3CN D20 D20 DEE Dioxane DME DMF EtOD H20 D20_ Lock nucieus 2H B benzene d6 methylenechloride d2 acetonitrile d3 chloroform d chloroform d ETB HPLC Solvent Acetonitril D20 deuteriumoxide diethylether d10 dioxane d8 dimethylether d6 dimethylformamide d7 ethanol d6 90 H20 and 10 D20 E 35 12 Select DMSO by clicking on it 13 Select by clicking on it NOTE This performs a atma automatic tuning and requires a probe equipped with a automatic tuning module Other options can be selected by clicking on the down arrow inside the Tune button 15 Select by clicking on it Figure 3 9 Turn sample rotation off ro off Change sample rotation rate ro MAS Pneumatic Unit masdisp 16 Select ro on by clicking on it NOTE Rotation may be turned off for probes such as BBI TXI TBI and for small sample probes 17 Select amp Shims by clicking on it NOTE This executes the command topshim To select other options click on the down arrow inside the Shim button 18 Select fl Prosol by clicking on it NOTE This will load the pulse width and power levels in to the parameter set 3 2 3 Acquisition 1 Select Gains
5. LAYOUT 2D_ hom _xwp w Lexecute cancel NOTE To avoid the message shown in Figure 3 14 the option Auto Phasing apk2d may be disabled for magnitude like 2D experiment D 1 0 00 01 45 Figure 4 15 EEE proton_exp 2 1 C data3 0 Spectrum ProcPars AcquPars Title PulseProg Peaks Integrals Sample Structure Plot Fid Acqu 2D gradient COSY experiment 30 mg Menthy Anthranilate in DMSO d6 F1 ppm 2 F2 ppm 4 3 5 Plotting 1 Use the 4 buttons to adjust for a suitable contour level 2 Click on the Publish tab in the TopSpin Menu bar Figure 4 16 4 Start Acquire Process Analyse Publish View Manage o ign Copy Printy jPlotLayoute PDF v E Mail 6 Click on 7 Select the Plot tab by clicking on it 46 B7170 00 01 Figure 4 17 proton_exp 2 1 C data3 0 Spectrum ProcPars AcquPars Title PulseProg Peaks Integrals Sample Structure Plot Fid Acqu i lt u uo Ej 2D hom xwp 2D gradient COSY experiment 30 mg Menthyl Anthranilate in DMSO d NOTE If desired any changes can be administered by clicking on the icon to open the Plot Editor m 8 Click on the Bis to plot the spectrum B7170 00 01 47 4 4 Observations 48 B7170_00_01 4 5 2 D Multiple Quantum Filtered COSY experiment The COSY Multiple Quantum Filtered COSY MQF experi
6. 42 B7170 00 01 NOTE This will load the pulse width and power levels in to the parameter set 9 Select Figure 4 8 ee setlimits Close this dialog box after setting frequencies o 1 Open 1D dataset from Browser 2 Zoom into region of interest 3 Click OK to set frequencies and return to original dataset J cance 10 To open the 1D Proton spectrum right click on the dataset name in the browser win dow e g proton exp 1 and select Display or click and hold the left mouse button for dragging the 1D Proton dataset in to the spectrum window 11 Expand the spectrum to display all peaks leaving ca 0 2 ppm of baseline on either side of the spectrum NOTE The solvent peak may be excluded if it falls outside of the region of interest Dig ital filtering however is only applied in F2 and the solvent peak is folding in F1 Figure 4 9 proton exp 1 1 C idata3 0 TER Spectrum ProcPars AcquPars Title PulseProg Peaks Integrals Sample Structure Plot Fid Acqu f Proton experiment 30 mg Menthy Anthranilate in DMSO d6 4 2 ppm D 1 0 00 01 43 12 Click on to assign the new limit Figure 4 10 1H spectral limits copied for F1 and F2 dimensions c i he Pewee 13 Click on TC imm NOTE The display changes back to the 2D data set 4 3 3 Acquisition by clicking on it 1 Select 2 S
7. PENTI li F E TI Uryleribapez A Dm suena Lae TopSpin 3 x Advanced NMR Methods User Manual Version 001 NMR Spectroscopy Innovation with Integrity Copyright by Bruker Corporation All rights reserved No part of this publication may be reproduced stored in a retrieval system or transmitted in any form or by any means without the prior consent of the publisher Product names used are trademarks or registered trademarks of their respective holders This manual was written by Peter Ziegler December 2 2010 Bruker Biospin Corporation Billerica Massachusetts USA P N B7170 For further technical assistance on the TopSpin 3 x unit please do not hesitate to contact your nearest BRUKER dealer or contact us directly at BRUKER Biospin Corporation 15 Fortune Drive Billerica MA 01821 USA Phone 978 667 9580 ext 5444 FAX 978 667 2955 E mail applab bruker biospin com Internet www bruker com Table of Contents 1 La LE 6 10 1063 1 Ce e 7 1 1 Ea E ee ee ee E 7 1 2 Bisera EEEE Dm 7 2 Pulse CANDEAUOM ne mnnn 9 2 1 MOU 10 1 eaen EE EE E E E 9 2 2 SD 9 2 3 1H 900 transmitter pulse M E 9 2 3 1 Preparation experiment ss ssisssieeserereseeeeesreeeeseenees 10 20 2 FMC IE PSE CUP aa ame TE 10 2 3 3 Determine the 1H 900 transmitter pulse 13 2 4 FOS NON S ee ee tes occ Sec ect annie a eee 17
8. Ex 6 Select by clicking on it Figure 5 44 EM Parameter Sets rpar File Options Help Find file names enter any string Class Any Reset Filters SELCOGP SELMLGP SELMLZF 1H SELRO1H SELROGP sELU 13C SELU 1H sELU COSY SELU HMBC SELU HSQC SELU ROESY SELZG1H NOTE Enter SEL in to the Find file names window and hit Enter to display all selec tive parameter sets shown in figure 5 45 7 Select SELMLGP 8 Click on 9 Select the acqu proc and outd parameter options only 10 Click on the down arrow next to the Keep the following parameter window 11 Select P1 O1 PLW1 from the pull down menu D 1 0 00 01 93 Figure 5 45 source Parameter Set C BrukenTopSpin3 D b 44XexpstannmnpansSELNOGP Destination Data Set sel_noesy 1 1 C data3 0 1 Select the desired file types of the source parameter set 2 Press OK to copy them to the destination data set acqu prac Keep the following parameters P 1 01 PLW 1 v 12 Click on 13 Select the Title tab by clicking on it 14 Make the following changes 1 D Selective TOCSY experiment 30 mg Menthy Anthranilate in DMSO d6 15 Click on to store the title 16 Select the Spectrum tab by clicking on it 17 Click on the Aquire tab in the TopSpin menu bar Figure 5 46 E Start Acquire Process Analyse Publish View Manage o V Samples 3 Lock V Tune v dk Spin amp
9. Figure 2 25 No acquisition running C data pz nmr pulse_calibration 3 pdata 1 Spectrum ProcPars AcquPars Title PulseProg Peaks integrats Sample Structure Fia Acqu Carbon 90 degree pulse calibration 10 4M 15 Urea and O 1M 13C CH3OH in DMSO d6 10 20 3 4 3 3 3 2 3 1 3 0 2 9 ppm 4 Increase p3 in increments of 1 or 2us execute zg followed by the command efp until the signals go through a null or a phase change This will be the 13C 90 decoupler pulse 22 B7170_00_01 Figure 2 26 Spectrum ProcPars AcquPars Title PulseProi P3 3us less then 90 degree pulse calibration 4 1 C pz Ini x Spectrum ProcPars AcquPars Title PulseProi P3 fOus 90 degree 10l x Spectrum ProcPars AcquPars Title PulseProt 4 P3 15us greater then 90 degree B7170 00 01 23 2 6 Observations 24 B7170 00 01 2 7 15N 90 decoupler pulse Figure 2 27 fl channel I 90 f channel S p3 The pulse program used in this procedure is the decp90 sequence shown in Figure 2 22 The sequence consists of two channels f1 1 and f2 S where in this case f1 is set for 1H and f2 to 15N Channel f1 shows a recycle delay d1 followed by a 90 pulse and a delay d2 1 2J44 for the creation of antiphase manetization A 15N pulse on channel f2 is been executed after the delay d2 and then the 1H signal is detected When the 15N pulse is exactly 909 th
10. NOTE The reference spectrum is necessary to adjust the spectral limits of the sweep width in the F2 dimension and to use it for the projection The HSQCEDETGPSP 3 parameter set has a default sweep width in the F1 dimension of 165ppm If a carbon DEPT135 or DEPT45 spectrum of the same sample is available the F1 sweep width can D 1 0 00 01 111 be limited using the setlimit AU program 6 1 5 Setting up the HSQC experiment 1 Click on the Start tab in the TopSpin Menu bar Figure 6 5 Publish o 1 Create Dataset S Find Dataset Open Dataset r Paste Dataset E Read Pars 2 Selec reale Dalase by clicking on i 3 Enter the following information in to the New window Acquire Process Analyse View Manage Figure 6 6 Prepare for a new experiment by creating a new data set and initializing its NMR parameters according to the selected experiment type For multi receiver experiments several datasets are created Please define the number of receivers in the box below NAME shape hsqc exp EXPNO 1 PROCNO 1 DIR C data3 0 v Solvent DMSO v Experiment Dirs C Bruker TopSpin3 0 b 44 exp stan nmripar v Experiment HSQCEDETGPSP 3 ADIA v TITLE D edited HSQC experiment with adibatic pulses 30 mg Menthyl Anthranilate in DMSO de C Show new dataset in new window 1 Receivers 1 2 16 NOTE The directory DIR is specific to how the
11. Calculate Bandwidth for Excitation analyze bandw2 Calculate Bandwidth for Inversion analyze bancwzi special Bandwidth Calculations Calculate gammaB max analyze calc mo Calculate gammabimax for Adiabatic shapes analyze calcbladia Calculate Blach Siegert Shift analyze Osiegerts Calculate average Power Level analyze calcpas Integrate Shape analyze integra Integrate Adiabatic Shape analyze integradia Integrate and compare to Reference analyze integandcomp Simulation analyze simulate D 1 0 00 01 95 11 Type the value from step 4 e g 29 6 in to the Calculator window Delta Omega Hz and hit the Enter key Figure 5 51 r Calculatar i ee ZE DeltaGmega Hz 287973 DeltaT usec NOTE The value for Delta T usec is calculated after executing step 11 12 Click on the down arrow inside the Options button Figure 5 52 13 Select Define Parameter Table by clicking on it Figure 5 53 3 Define Parameter Table Parameter Table IP 4 Length of hard pulse PL 1 Power level of hard pulse IP 12 Length of shaped pulse v Power level of shaped pulse SPNAM2 Name of shaped pulse Length of ref shaped pulse Power level of ref shaped pulse SPNAM3 Name af ref shaped pulse
12. Figure 5 2 5 3 1 D Selective COSY 5 3 1 Introduction The hard pulses used in all the experiments from the previous chapters are used to uni formly excite the entire spectral width This chapter introduces soft pulses which selec tively excite only one multiplet of a 1H spectrum Important characteristics of a soft pulse include the shape the amplitude and the length The selectivity of a pulse is measured by its ability to excite a certain resonance or group of resonances without affecting near neighbors Since the length of the selective pulse affects its selectivity the length is selected based on the selectivity desired and then the pulse amplitude i e power level is adjusted to give a 90 or 270 flip angle NOTE The transmitter offset frequency of the selective pulse must be set to the fre quency of the desired resonance This transmitter frequency does not have to be the same as o1p the offset frequency of the hard pulses but for reasons of simplicity they are often chosen to be identical Most selective excitation experiments rely on phase cycling and thus subtraction of spectra to eliminate large unwanted signals It is important to minimize possible sources of subtraction artifacts and for this reason it is generally suggested to run selective experiments using pulse field gradients and non spinning Section 5 3 describes the acquisition and processing of a one dimensional 1H selective gradient
13. e An evolution delay optimized to 1 2 J XH to achieve antiphase proton magnetiza tion IxSz e Simultaneous 180 1H and 90 X pulses The proton pulse will allow to refocus 1H chemical shift evolution while the carbon pulse creates multiple quantum coher ence e During a second delay also optimized to 1 2 J XH heteronuclear coupling is not evolving e Simultaneous Y 1H and 90 X pulses The carbon pulse refocus 13C chemical shift evolution while the Y proton pulse creates a different functional dependence as a function of carbon multiplicity e CH 2lzSysin Y CH2 Alzl zSysin Y cos Y CH3 8lzl zl zSysin Y cos2 Y A final evolution delay also optimized to 1 2 J XH to achieve in phase 13C magnetization e 13C acquisition is performed under broadband proton decoupling Figure 5 63 The 90X pulse can be replaced with a adiabatic shaped pulse to achieve better phasing over the whole spectrum range This is specially useful on higher field instruments where the phasing of a normal DEPT spectrum can be a problem Figure 5 64 below shows the selective pulse DEPT 135 sequence using a proton 1359 pulse D 1 0 00 01 103 Figure 5 64 5 9 2 Experiment setup 1 Click on the Start tab in the TopSpin Menu bar Figure 5 65 Publish View Manage oe Acquire Process Analyse Create Dataset S Find Dataset J Open Dataset Paste Dataset Read Pars _ by clicking on it 3 En
14. hsqcetgpsp shaped pulses for inversion on f2 D 1 0 00 01 109 hsqcetgpsp 2 shaped pulses for inversion and refocusing on f2 hsqcetgpsp 3 shaped pulses for inversion and refocusing on f2 for 13C labeled mole cules hsqcdiedetgpsisp 1 shaped pulses for all 180 pulses on f2 multiplicity editing during selection hsqcdiedetgpsisp 2 shaped pulses for all 180 pulses on f2 inversion of directly cou pled protons hsqcdiedetgpsisp 3 shaped pulses for all 180 pulses on f2 multiplicity editing during selection 6 1 2 2 D edited HSQC experiment using adiabatic pulses For improvement of the phasing the pulse sequence using matched sweep adiabatic pulses Figure 4 2 is used in this chapter If desired the sequence hsqcedetgpsisp2 4 can be used to suppress the COSY peaks Figure 6 2 6 1 3 Sample A sample of 30mg Menthyl Anthranilate in DMSO d6 is used for all experiments in this chapter 110 B7170 00 01 Figure 6 3 a uu i 15 5 A WS A RR 17 8 x TA A 3 10 o M ue ES V om 6 1 4 Reference spectrum 1 Run a 1D Proton spectrum following the instructions in 1 D Proton experiment Chapter 3 Figure 6 4 Acquisition finished C data3 0 proton_exp 1 pdata 1 Spectrum ProcPars AcquPars Titie PuiseProg Peaks integrals Sample Structure Plot Fia Acqu 1 Proton experiment 30 mg Menthy Anthranilate in DMSO d6 rel 15 10 5 0 ppm
15. 6 3 5 Processing 1 Click on the Process tab in the TopSpin Menu bar Figure 6 32 El Start Acquire Process Analyse Publish View Manage oe Proc Spectrum v w Adjust Phase v A Calib Axis v 3 Pick Peaks ff Integrate Advanced 2 Select EERE cicking on Figure 6 33 3 apk2d spectrum has no imaginary part MC2 F1 QF PH maod F1 2mc Could not phase real spectrum NOTE This executes a standard processing program proc2 The message shown in Figure 4 33 pops up in case of a magnitude 2D experiment and the apk2d option is enabled To configure the processing program follow the steps below To avoid the mes sage shown in Figure 4 33 the option Auto Phasing apk2d may be disabled for mag nitude like 2D experiment Figure 6 34 shape hmbc exp 1 1 C data3 0 spectrum ProcPars AcquPars Title PulseProg Peaks Integrals Sample Structure Plot Fid Acqu ET 2D selective HMBC experiment E 50 mM Gramicidin S in DMSO d6 amp B 8 B o S 8 6 4 2 F2 ppm 126 B7170 00 01 6 3 6 Optimizing the parameters on the carbonyl region 1 Type wrpa on the command line Figure 6 35 EN wrpa Copy data set If NAME ends with top the destination will be a 1 file dataset no expno procho required Please specify destination NAME shape hmbc exp EXPNO PROCNO 1 DIR Cata 2 Change the EXPNO to 2
16. Find Dataset Open Dataset Paste Dataset Read Pars Acquire Process Analyse View Manage by clicking on it 3 Enter the following information in to the New window B7170 00 01 41 Figure 4 5 Prepare for a new experiment by creating a new data set and initializing its NMR parameters according to the selected experiment type For multi receiver experiments several datasets are created Please define the number of receivers in the box below NAME cosy exp EXPNO PROCNO DIR v Solvent DMSO v Experiment Dirs C BrukenTopSpin3 0 b 44 exp staninmnpar v Experiment COSYGPSW v TITLE D gradient COSY experiment 30 mg Menthyl Anthranilate in DMSO de CI Show new dataset in new window 1 Receivers 1 2 16 NOTE The directory DIR is specific to how the data are stored and therefore may show different entries as the one in Figure 3 5 above Click on the down arrow button to browse for a specific directory 4 Click on 5 Click on the Aquire tab in the TopSpin menu bar Figure 4 6 E Acquire Process Analyse Publish View Manage oe Options v 6 Select db spins by clicking on it Figure 4 7 Turn sample rotation on ro on Change sample rotation rate ro MAS Pneumatic Unit masdisp 7 Select ro off by clicking on it NOTE 2 D experiments should be run non spinning 8 Select AV Prosol by clicking on it
17. by clicking on it NOTE To adjust rg manually click on the down arrow inside the Gain icon 2 Select Boo by clicking on it NOTE Other options are available by clicking on the down arrow inside the Go button 36 B7170_00_01 3 2 4 Processing 1 Click on the Process tab in the TopSpin Menu bar Figure 3 10 E 9 9 Pick Peaks Integrate ais v Start Acquire Analyse Publish Proc Spectrum v Adjust Phase v Calib Axis v NOTE This executes a processing program including commands such as an exponen tial window function em Fourier transformation ft an automatic phase correction apk and a baseline correction abs Other options are available by clicking on the down arrow inside the Proc Spectrum button Process View Manage Figure 3 11 Acquisition finished C data3 0 proton_exp 1 pdata 1 rel 1 Proton experiment 30 mg Menthy Anthranilate in DVISO d6 15 10 5 0 ppm D 1 0 00 01 37 38 B7170 00 01 4 2 D gradient experiments 4 1 Introduction The vital importance of NMR in chemistry and biochemistry relies on the direct relation ship between any given NMR experiment and the molecular information that can be extracted from it Thus every experiment is based on some NMR parameter usually coupling constants or NOE which is related to a specific molecular
18. p90 proton EXPNO PROCNO DIR 6 Change NAME p90 proton 7 Click on 8 Expand the region between 3 5ppm and 2 8ppm NOTE Normally a single peak set to on resonance is used to determine the 90 trans mitter pulse For practical reason the Methanol signal region from 3 5ppm to 2 8ppm is used to measure the 1H 90 transmitter pulse since the same signals will also be used in determining the 13C 90 decoupler pulse D 1 0 00 01 11 Figure 2 8 No acquisition running C data pz nmr pulse_calibration 1 pdata 1 Spectrum ProcPars AcquPars Title PulseProg Peaks integrals Sample Structure Fia Acqu Proton 90 degree pulse calibration 0 1M 15 Urea and 0 1M 13C CH3OH in DMSO d6 9 Click on de to set the sweep width and the O1 frequency of the displayed region Figure 2 9 amp x New setting of SW SFO1 from current region G SW 0 7000 ppm SWH 210 080 Hz O1 945 40 Hz SFO1 300 1309454 MHZ 10 Click on 11 Select the AcquPars tab by clicking on it 12 Make the following changes PULPROG zg TD 4K NS 1 DS 0 D1 10 13 Select the ProcPar tab by clicking on it 14 Make the following changes SI 2K PH_mod pk 12 B7170_00_01 15 Select the Spectrum tab by clicking on it 16 Select by clicking on it 17 Select Boo by clicking on it 18 Process and Phase correct the spectrum 19 Display the full soectrum
19. 4 4 S ole M m 48 D 1 0 00 01 3 Table of Contents 4 5 2 D Multiple Quantum Filtered COSY experiment 49 4 5 1 Preparation experiment cccccccccceesccceseecceeeecceueeeceececeuseeseueeessageessueeesseesensageenes 50 4 5 2 Setting up the MFQ COSY experiment 50 4 5 3 PCO ION RT 53 4 5 4 POCO SING iee TT E E 53 4 5 5 gu a PER ET EEEE TEE EEEE P 54 4 6 ODBSeOrn allofiS aana E E E E E 56 4 7 2 D HMBC experiment nanas ea 57 4 7 1 Preparation experiment ccccecccccsescccseccceeeecceeeeceuececsueeeseaeeessagseseueeesseseessgeenes 59 4 7 2 Setting up the HMBC experiment ss 60 4 7 3 PCOS MONA NOI 64 4 7 4 PO OS I PR 64 4 7 5 giu 66 4 8 Observati riS as aa eme ns dan eee demie ee ee 68 5 1 D experiments using shaped pulses 69 5 1 iglifere B eiio saoe EE a EEE E 69 5 2 vc s m X ne 69 5 3 1 D Selective COSY cccccccccccccccccceseeeeeeeceaeeeeceeeeeeeeeeeeeeseaeeeeeeseeeeaeeeeeeesaaeeeeeeesaas 70 5 3 1 Jgif ere Breliie PENNE REEL 70 5 3 2 Reference spectrum cccceecccsesccceessecceeeeecsueeeceaeecsauseecsuseessaeeessageessaeeesseseesseneeess 71 5 3 3 Selective excitation region set Up 71 eh OMS OnO O ee m T Tm 71 5 3 4 Setting up the Selective COSY sir 73 5 9 5 PCAN e MR T 75 5 3 6 POCOS SING
20. For multi receiver experiments several datasets are created Please define the number of receivers in the box below EXPNO PROCNO DIR CAdata3 0 v solvent DMSO v Experiment Dirs CABrukenTopSpin3 0 b 44 exp stannmnpar Experiment PROTONT1 TITLE Proton T1 experiment 30 mg Menthyl Anthranilate in DMSO d6 Show new dataset in new window 4 Receivers 1 2 16 NOTE The directory DIR is specific to how the data are stored and therefore may show different entries as the one in Figure 4 6 above Click on the down arrow button to browse for a specific directory 4 Click on 5 Click on the Aquire tab in the TopSpin menu bar Figure 8 6 3 Start Acquire Process Analyse Publish View Manage o Samples Lock V Tune db Spin S Shime Prosol CSetLimits i Gain gt Gow 6 Select db spins by clicking on it Options D 1 0 00 01 169 Figure 8 7 Turn sample rotation on ro on Change sample rotation rate ro MAS Pneumatic Unit masdisp 7 Select ro off by clicking on it NOTE T1 experiments should be run non spinning 8 Select A Prosol by clicking on it ES Fi NOTE This will load the pulse width and power levels in to the parameter set 9 Select Figure 8 8 HA setlimits Close this dialog box after setting frequencies o 1 Open 1D dataset from Browser 2 Zoom into region of interest 3
21. Go v Options 14 Select db spin by clicking onit Figure 5 13 Turn sample rotation on ro on Change sample rotation rate ro MAS Pneumatic Unit masdisp 15 Select ro off by clicking on it NOTE 1 D selective experiments should be run non spinning 16 Select A Prosol by clicking on it NOTE This will load the pulse width and power levels in to the parameter set 5 3 5 Acquisition 1 Select Boo by clicking on it 5 3 6 Processing 1 Click on the Process tab in the TopSpin Menu bar Figure 5 14 E Start Process Publish View o JL Proc Spectrum v Adjust Phase v Calib Axis v A Pick Peaks j Integrate v Advanced v 2 Click on the down arrow inside the Proc Spectrum button Acquire Analyse Manage D 1 0 00 01 75 Figure 5 15 Window Multiplication wm Fourier Transform ft Start Automation AU Program xaup 3 Select Configure Standard Processing by clicking on it 4 Deselect the following options Auto Phasing apk Set Spectrum Reference sref Auto Baseline correction abs Warn if Processed data exist Figure 5 16 e procid Press Execute to process the current dataset Press Save to just change the processing options Changed options will be effective when pressing the one click Proc Spectrum button Exponential Multiply em LB HJ 0 1 Fourier Tra
22. Set Spectrum Reference sref Auto Baseline correction abs Warn if Processed data exist Figure 5 57 e procid Press Execute to process the current dataset Press Save to just change the processing options Changed options will be effective when pressing the one click Proc Spectrum button Exponential Multiply em LB Hz 01 Fourier Transform ft Auto Phasing apk Set Spectrum Reference sref CO Auto Baseline Correction abs C Plot autoplot LAYOUT HD Hxwp v L Warn if processed data exist O 5 Click on 6 Expand the spectrum from 4 ppm to 0 5 ppm 7 Click on 8 All peaks should be phased for postive absorption 98 B7170_00_01 Figure 5 58 Acquisition finished C data3 0 sel_tocsy 1 pdata 1 T if Spectrum ProcPars AcquPars Title PulseProg Peaks Integrals Sample Structure Plot Fid Actu D Selective experiment Reference spectrum aan ME esie mte mte e temi e tona rel 20 10 5 7 6 Plotting two spectra on to the same page 1 Display the selective TOCSY spectrum 2 Click on AI to enter the Multiple display option 3 Drag the Reference spectrum in to the spectral window Figure 5 59 sel tocsy 1 1 C data3 0 ike RMT ith E EEi2 58 selective exp l1 Ci atas 0 sel tocsy 1 1 ch fatas oL 6 4 2 ppm D 1 0 00 01 99 NOTE To adj
23. comprehensive range or even collaborate with you on new developments Our ongoing efforts and considerable investment in research and development illustrates our long term commitment to technological innovation on behalf of our customers With more than 40 years of experi ence meeting the professional scientific sector s needs across a range of disciplines Bruker BioSpin has built an enviable rapport with the scientific community and various specialist fields through understanding specific demand and providing attentive and responsive service Bruker BioSpin Group info bruker biospin com www bruker biospin com OG Bruker BioSpin B7170
24. resolution in the indirect dimension and therefore is recommended when high over lapped carbon spectra precludes an easy resonance assignment Figure 6 21 hmbegplpndat Gl G2 G3 Figure 6 22 6 3 2 Sample 50mM Gramicidin S in DMSO d6 D 1 0 00 01 121 Figure 6 23 6 3 3 Preparation experiment 1 Run a 1D Proton spectrum of Gramicidin in DMSO d6 following the instructions in 1 D Proton experiment Chapter 3 Figure 6 24 proton gramicidin 1 1 C data3 0 Spectrum ProcPars AcquPars Title PulseProg Peaks Integrals Sample Structure Plot Fid Acqu 1 D Proton experiment 50 mM Gramicidin S in DMSO d6 rel 15 10 5 0 ppm NOTE The reference spectrum is necessary to adjust the spectral limits of the sweep width in the F2 dimension and to use it for the projection The HMBCGP parameter set has a default sweep width in the F1 dimension of 222ppm If a regular carbon spectrum of the same sample is available the F1 sweep width can be limited using the setlimit AU program The default sweep width in F1 is used for the experiment in this chapter 122 B7170_00_01 1 Click on the Start tab in the TopSpin Menu bar Figure 6 25 Process Analyse Publish View o 1 Create Dataset a Find Dataset Open Dataset Paste Dataset E Read Pars Manage Acquire F by clicking on it ium i 3 Enter the following inform
25. 2 5 13C 900 decoupler pulse iii 18 2 5 1 Preparation experiment sise 18 2 5 2 Paramotor Se CUP T ERRES 18 2 5 3 Determine the 13C 900 decoupler pulse 22 2 6 A SD ND ane een es eee D eee des ei 24 2 7 15N 900 COLE PSS ae TT MM 25 2 7 1 Parameter SCOR ERa ane en ont sn tome cs 25 2 1 0 TWO CANNES VS san detre etes 28 24 1 2 Three channel system seen asset esse inessten basses qe teste died closes 28 2 7 2 Determine the 15N 900 decoupler pulse 30 2 8 OS OS sae AE E E EEA 32 3 1 D Proton experiment ecce ccc eheu ioco a oca ooo Eo osa yue ae oue ans cepi D n Usa ye asa E Top acess 33 3 1 v i m 33 3 2 1 D Proton experiment russe cuu eaaet anm unen nau amici eva max rus ee GA wi par 33 3 2 1 OO T u 33 3 2 2 EXDODIme nN SIUD andere ne a een en a RORTRUN DUE 34 3 2 3 ACO ION eeror ee E E 36 3 2 4 Processi Oese r AREE ne een 37 4 2 D gradient experiments cccccscsseesesceseeeesseesenseeeeseeeenseeoessesonseeseeneesons 39 4 1 Igiirerelllei MC a es 39 4 2 2101 tes eee Gene ere eee cn ee 39 4 3 2 D gradient COSY 0 ccccccccccssssccceecesseeceeeceeeseeeeeeeeeeeeeeeeeseeeeeeeeesseseeeeeessaageeeeeeeeas 40 4 3 1 Preparation EX DS TIMENTS ann tonte rs nm oui 40 4 3 2 Setting up the COSY experiment iii 41 4 3 3 PCOS LUS LE PR E A E E E 44 4 3 4 PTOC SS SI eee a 44 4 3 5 audio Y 46
26. Figure 2 10 No acquisition running C data pz nmr pulse_calibration 1 pdata 1 Spectrum ProcPars AcquPars Title PulseProg Peaks integrats Sample Structure Fia Acqu m Proton 90 degree pulse calibration 0 1M 15 Urea and 0 1M 130 CH3OH in DMSO d6 20 In the command line type dpl to save the region to parameter F1P F2P 21 In the command line type wpar H1p90 urea all to store the parameter set for future use 2 3 3 Determine the 1H 90 transmitter pulse 1 In the command line type popt 2 Make the following changes OPTIMIZE Step by step PARAMETER p1 OPTIMUM POSMAX STARTVA 2 NEXP 20 VARMOD LIN INC 2 D 1 0 00 01 13 Figure 2 11 pulse _calibration 1 1 C pz store as 2D data ser file I The AU program specified in AUNM will be executed WDW EM I Perform automatic baseline correction ABSF PH mod pk I Overwrite existing files disable confirmation Message FT mod fsc I Stop sample spinning at the end of optimization mash Run optimization in background Start optimize Skip current show Add Read array file Save Save array file as file as Stop optimization optimization Delete Delete parameter Dataset 3 Click on Save NOTE The ENDVAL parameter has been updated 4 Click on Start optimize Figure 2 12 o EEE ok cae 5 Type y into the poptau window 6 Click on NOTE The parameter optimization start
27. O1 position The power level and pulse width of the excitation pulse have to be known and entered into the Prosol parameters 1 In the command line type wrpa and hit Enter Figure 5 23 Copy data set If NAME ends with top the destination will be a 1 file dataset no expna pracna required Please specify destination MAME sel naesy EXPNO PROCNO 1 DIR C datag 0 2 Change NAME sel noesy B7170 00 01 81 3 Click on 4 In the command line type re and hit Enter Figure 5 24 Options Display data in same window Display data in new window NAME sel noesy EXPNE PROCNO DIR Cor cance Browse Ces Crer 5 Change NAME sel noesy 6 Click on 7 Select the Spectrum tab by clicking on it 8 Expand peak at 4 8ppm Figure 5 25 sel noesy 1 1 C data3 0 Spectrum ProcPars AcquPars Title PulseProg Peaks Integrals Sample Structure Plot Fid Acqu 1 D Selective NOES Yexperiment 30 mg Menthy Anthranilate in DMSO d6 4 7971 ppm 1439 7549 Hz Index 37150 Value 0 4042 rel 4 85 4 80 4 75 ppm 9 Move the cursor line to the center of the peak 10 Write down the cursor offset frequency value displayed in the upper left of the spec trum window e g 1439 75 NOTE To display the cursor information right click inside the spectrum window and select Spectra Display Prferences and enable Cursor infor
28. Process Only F2 Axis xf2 by clicking on it D 1 0 00 01 173 8 2 5 Figure 8 15 t1 exp 1 1 C data3 0 1 Proton T1 experiment Pi 30 mg Menthyl Anthra ilate in DMSO d6 BE in N o N in 2 N o 8 6 4 2 F2 ppm T1 calculation 1 Click on the Analyse tab in the TopSpin Menu bar Figure 8 16 E Publish o Ah Multiplets Line Shapes v lt T1 T2 structures v D Simulate Dosyv 2 Click on dam NOTE The flow buttons change for determine the T1 T2 relaxation times see Figure 8 17 below Start Acquire Analyse View Manage Process Figure 8 17 El Stat Acquire Process Analyse Publish View Manage o O Back Eid Peaks Ranges El Relaxation Fitting Calculation Report NOTE While executing the steps below message windows will pop up Please read each message thoroughly and follow the instructions in it E 3 Click on Extract Slice 174 B7170 00 01 Figure 8 18 d Extract a row from 2d data Fid or Spectrum must be extracted From the 2d relaxation data This row should correspond to an experiment with the maximum ar minimum delay time All further data preparation will be done in respect to this row 4 Click on Figure 8 195 Spectrum slice must be extracted From the 2d relaxation data This Spectrum should correspond to an experiment with the maximum ar minim
29. R90 9080 ic I E E J pivot 7 67 ppm Phase increment 0 025 ph0O 0 00 phl 0 00 18 Adjust the phase using the ids and id buttons B7170 00 01 117 Figure 6 18 Phase 2D shape hsqc exp 1 1 C data3 0 AE 0 1 R 90 90180 c Il il HH ul pivot 7 67 ppm Phase increment 0 025 phO 85 77 phl 6 60 I RON SM A OR B BR cr 562123074130914 9 200 360 1202 Ne rennes ERU EMQUE GSC OS QU KU ROI END 302 4002 E162 SES TE 19 Click on Hi 20 Click on el Figure 6 19 Phase 2D shape hsqc exp 1 1 C data3 0 AS 1 R90 90180 cic 11 ll EHI 4 pivot 130 91 ppm Phase increment 0 025 phO 5 04 phl 7 70 21 Adjust the phase if necessary using the 0 and ids buttons 22 Click on Li 118 B7170 00 01 23 Click on ual Figure 6 20 shape_hsqce_exp 1 1 C data3 0 1 Spectrum ProcPars AcquPars Title PulseProg Peaks Integrals Sample Structure Plot Fid Acqu 2D edited HSQC experiment with adibatic pulses experiment F2 ppm B7170_00_01 119 6 2 Observations 120 B7170 00 01 6 3 2 D Selective HMBC experiment 6 3 1 Introduction The Semi selective 2D HMBC experiment is a simple modification of the 2 D HMBC pulse sequence Figure 4 21 in which one of the two carbon 90 degree pulses is applied selectively on a specified region Figure 4 22 The main purpose is to achieve better
30. Shime Al Prosol v Gain v gt Gow Options v Select db spin by clicking on it Figure 5 47 Turn sample rotation on ro on Change sample rotation rate ro MAS Pneumatic Unit masdisp 18 Select ro off by clicking on it NOTE 1 D selective experiments should be run non spinning 19 Select KV Prosol by clicking on it 94 B7170 00 01 NOTE This will load the pulse width and power levels in to the parameter set 20 Click on the down arrow inside the Options button Figure 5 48 IcanMME Automation icannrnr TopGuide topguide One Click Experiments 21 In the shape tool menu bar click on Figure 5 49 e Shape Files File Options Help Find file names enter any string Class Dim Show Recommended Type SubType Reset Filters Bip720 100 10 1 Bip720 50 20 1 Crp20 1 40 1 Crp32 1 5 20 2 Crp48 1 5 20 2 Crp60_xfilt 2 ICrp amp 0 0 5 20 1 Crp80 0 5 20 1 CrpS8 comp 4 Eburp2tr 1000 G4 256 Gaus1_180i 1000 Gaus1_180r 1000 IGaus1_30 1 000 Gaus1 1000 Gaussramp down 1 Gaussramp up 1 Iburp2 1000 Pc9 4 120 1000 Q3 rna c68c1 1 Q3 surbop 1 Q3Ca CaCO 1000 Q5tr sebop 1 Reburp 1000 sinc1 1000 qua100 1000 squaramp 20 1 Tanhtan 300 50 250 Update info 22 Select Gaus1_180r 1000 23 Click on 24 In the main menu click on Analysis Figure 5 5012 ee B B
31. Title PulseProg Peaks Integrals Sample Structure Fid Acqu solvent suppression experiment 2mM Raffinose in 90 H20 10 D20 05 rel 0 1 0 2 0 3 0 4 0 0 15 10 5 0 ppm NOTE Make sure that the SW is large enough to cover the entire Spectrum accounting for the position of O1 The presaturation is applied on resonance at the O1 position The power level for presaturation has to be known and entered into the Prosol parame ters B7170 00 01 141 7 3 1 D Solvent suppression with Presaturation Presaturation is the most common procedure to minimize and suppress the intense sol vent resonance when H spectra are recorded in protonated solutions This experiment is performed by applying a low power continuous wave irradiation on the selected reso nance during the pre scan delay see Figure 5 2 Figure 7 10 7 3 1 Parameter set up 1 Type wrpa 2 on the command line 2 Type re 2 on the command line 3 Expand the Water signal at 4 8 ppm 4 Click on t Figure 7 11 2 1d solvent suppression 2 1 C iBrukeriTOPSPIN nmrsu solvent suppression experiment 2mM Raffinose in 90 H20 10 D20 4 6916 ppm 2346 86 Hz 500 262347 MHz SET SF01 01 FREQUENCIES FROM CURSOR POSITION Define Left click inside data window 5 0 48 46 44 ppm 5 Move the cursor line to the center of the peak and click the left mouse button 142 B 7170 00 01 Figure 7 12 Defin
32. at 3 352 ppm T1 1 4118 Peak 6 at 1 645 ppm T1 266 r40m Peak 7 at 0 064 pom T1 4135 2m 22 Click on E Report Display Report Figure 8 31 3 Fitting report File Edit Search Dataset C data3 0 t1 exp 1 pdata 1 IHTEHSITY fit I t I 0 P exp t T1 10 points for Peak 1 Peak Point at Results Comp 1 I U 9 6869e 001 P 1 856e 000 Ti 1 427s SD 8 622e 003 7 677 ppm tau ppm integral intensity 10 000m 40 000m 000m 000m 000m 000s 000s 4 000 000s 67 2504e 008 BTI 4565e 008 677 3444e 005 BTI 0229e 008 677 6066e 006 676 48159e 008 67 9172e 008 6TT 6415e 009 BTI 8712e 009 9011e 009 m we we we we ee eV 5827e 007 3222e 007 89856e 007 1122e 007 0265e 007 9657e 006 4446e 007 6781e 007 493Te 007 5935e 007 180 B7170 00 01 8 3 Observations B7170_00_01 181 182 B7170 00 01 Appendix A Warning Signs Figures Tables Glossary References Index D 1 0 00 01 183 184 B7170 00 01 Bruker BioSpin your solution partner Bruker BioSpin provides a world class market leading range of analysis solutions for your life and materials science needs Our solution oriented approach enables us to work closely with you to further establish your specific needs and determine the relevant solution package fromour
33. best fit use the 2 2 g Ts tools 4 Click on the Publish tab in the TopSpin Menu bar Figure 5 20 E Start Acquire Publish View Manage Process Analyse H Copy Print L Plot Layouts c PDF gt E Mail 5 Click on the button to print the active window 78 B7170_00_01 5 4 Observations B7170_00_01 79 5 5 1 D Selective NOESY 5 5 1 Introduction This experiment consist of three parts e Selective excitation of the selected resonance using the SPFGE block Mixing period consisting of the basic 90 1H delay 90 1H block in phase polar ization transfer to other spins via NOE Purging gradients are usually applied during the mixing period in order to remove any residual transverse magnetization Proton detection as usual Figure 5 21 Gl Gl G2 5 5 2 Reference spectrum 1 Run a 1D Proton spectrum following the instructions in 1 D Proton experiment Chapter 3 80 B7170_00_01 Figure 5 22 Acquisition finished C data3 0 proton_exp 1 pdata 1 Spectrum ProcPars AcquPars Title PulseProg Peaks Integrals Sample Structure Plot Fid Acqu rel f Proton experiment 30 mg Menthyl Anthranilate in DMSO d6 15 10 5 0 ppm 5 5 3 Selective excitation region set up 5 5 3 1 Off resonance NOTE This method does not require a large SW The shaped pulse is applied off reso nance not on the
34. data are stored and therefore may show different entries as the one in Figure 4 6 above Click on the down arrow button to browse for a specific directory 4 Click on 5 Click on the Aquire tab in the TopSpin menu bar Figure 6 7 Process Analyse Publish Vie Manage e Start Acquire LL Sample v SELock V Tune db Spin Si Shim al Prosol SetLimits V Gain v D Gow Lososa Options 112 B7170_00_01 6 Select db spins by clicking on it Figure 6 8 Turn sample rotation on ro on Change sample rotation rate ro MAS Pneumatic Unit masdisp 7 Select ro off by clicking on it NOTE 2 D experiments should be run non spinning 8 Select A Prosol by clicking on it LA z NOTE This will load the pulse width and power levels in to the parameter set 9 Select Ei setLimits by clicking on it e O Figure 6 9 s setlimits Close this dialog box after setting frequencies o 1 Open 1D dataset from Browser 2 Zoom into region of interest 3 Click OK to set frequencies and return to original dataset 10 To open the 1D Proton spectrum right click on the dataset name in the browser win dow e g proton exp 1 and select Display or click and hold the left mouse button for dragging the 1D Proton dataset in to the spectrum window 11 Expand the spectrum to display all peaks leaving ca 0 2 ppm of baseli
35. from the null point value by using T1 t In 2 Figure 8 1 tlir 8 2 Proton Inversion Recovery T1 experiment 8 2 1 Sample A sample of 30mg Menthyl Anthranilate in DMSO d6 is used for all experiments in this chapter B7170_00_01 167 8 2 2 Figure 8 2 HA at Bie he qu ee 1 iz dd E wm PH a 41 T TA aS 4 xe Pr eS 12 16 ES P n e EL Me 1 2033 Preparation experiment 1 Run a 1D Proton spectrum following the instructions in 1 D Proton experiment Chapter 3 Figure 8 3 Acquisition finished C data3 0 proton_exp 1 pdata 1 Spectrum ProcPars AcquPars Title PulseProg Peaks integrals Sample Structure Plot Fid Acqu ALES rel f Proton experiment 30 mg Menthy Anthranilate in DMSO d6 ppm NOTE The reference spectrum is necessary to adjust the spectral limits of the sweep width to gain more data points 1 Click on the Start tab in the TopSpin Menu bar 168 B7170_00_01 Figure 8 4 Acquire Process Analyse Publish View 9 Create Dataset Find Dataset C Open Dataset Paste Dataset Read Pars 2 Select reale DASE by clicking on i 3 Enter the following information in to the New window Manage Figure 8 5 Prepare for a new experiment by creating a new data set and initializing its NMR parameters according to the selected experiment type
36. parameter through bond or through space connectivity chemical exchange molecular motion The quan titative measurement of such NMR parameters allows us to obtain valuable information about structural parameters such as dihedral angles intermolecular distances relax ation and exchange rates etc For this reason the development of new and or improved NMR methodologies is a key factor to be considered Since the 90 s when the gradients where introduced as a useful tool to incorporate them in to NMR applications the suite of NMR experiments available to researchers has grown A large percentage of them are using pulse field gradients Gradient enhanced NMR spectroscopy is widely used in liquid state spectroscopy for coherence pathway selection solvent suppression artifact reduction and diffusion weighting and has had a tremendous impact by improving the quality of NMR spectra Thus all advantages offering the incorporation of PFG as a powerful elements into high resolution NMR pulse sequences combined with the advanced software tools available at the present time to acquire and process multidimensional NMR experiments with great simplicity has dramatically changed the concept of routine work in NMR for chem ists 4 2 Sample A sample of 30mg Menthyl Anthranilate in DMSO d6 is used for the experiments in this chapter Figure 4 1 H A at jag Hid a3 su ae 2 1 2 es et 1 4 a cd Bd 3 EN 10 m ss et fii
37. phase routine and reverse the spectrum by clicking on the 180 icon Acquire Process Analyse Publish View Manage o D 1 0 00 01 107 Figure 5 73 Carbon exp 2 1 C data3 0 20 ppm 108 B7170 00 01 6 2 D experiments using shaped pulses 6 1 2 D edited HSQC experiment with Adiabatic pulses 6 1 1 Introduction The HSQC experiment is the method of choice for a very well resolved H C correlation However in contrast to the HMQC this experiment uses 180 pulses which causes problems if the 180 pulses become to long e g TXI probes and have to cover a very wide spectral range This leads to phasing problems for high field instruments above 500 MHz To work around this problem is to apply frequency swept adiabatic 180 pulses which can cover the large C spectral width Figure 5 1 shows the regular edited HSQC sequence and Figure 5 2 the edited HSQC sequence using shaped pulses for all 180 pulses on f2 channel with gradients in back inept Figure 6 1 Treo j pad 4 i g at ae fa TN R l pli2 N optional G G2 Gl hsqcetgpsisp shaped pulses for inversion on f2 hsqcetgpsisp 2 shaped pulses for inversion and refocusing on f2 hsqcetgpsisp2 shaped pulses for inversion on f2 gradients in back INEPT hsqcetgpsisp2 2 shaped pulses for inversion and refocusing on f2 gradients in back INEPT hsqcedetgpsisp hsqcedetgpsisp 2 hsqcedetgpsisp2 hsqcedetgpsisp2 2
38. power levels in to the parameter set E by clicking on it 9 Select S Figure 6 29 HA setlimits Close this dialog box after setting frequencies o 1 Open 1D dataset from Browser 2 Zoom into region of interest 3 Click OK to set frequencies and return to original dataset 10 To open the 1D Proton spectrum right click on the dataset name in the browser win dow e g proton gramicidin and select Display or click and hold the left mouse button for dragging the 1D Proton dataset in to the spectrum window 11 Expand the spectrum to display all peaks leaving ca 0 2 ppm of baseline on either side of the spectrum NOTE The solvent peak may be excluded if it falls outside of the region of interest Dig ital filtering however is only applied in F2 and the solvent peak is folding in F1 124 B7170 00 01 Figure 6 30 proton gramicidin 1 1 C data3 0 Spectrum ProcPars AcquPars Title PulseProg Peaks Integrals Sample Structure Plot Fid Acqu 1 D Proton experiment 3 50 mM Gramicidin S in DMSO d rel 8 6 4 2 ppm 12 Click on to assign the new limit Figure 6 31 1H spectral limits copied for F2 dimension i SW 9 9974 ppm OP 4 999 ppm 13 Click on c NOTE The display changes back to the 2D data set 6 3 4 Acquisition 1 Select Gains by clicking on it 2 Select by clicking on it D 1 0 00 01 125
39. sel_noesy 1 1 C data3 0 1 Select the desired file types of the source parameter set 2 Press OK to copy them to the destination data set Keep the following parameters P 1 01 PLW 1 v 8 Click on 9 Select the Title tab by clicking on it 10 Make the following changes 1 D Selective NOESY experiment 30 mg Menthy Anthranilate in DMSO d6 11 Click on to store the title 12 Select the Spectrum tab by clicking on it 13 Click on the Aquire tab in the TopSpin menu bar Figure 5 30 E Start Acquire Process Analyse Publish View Manage o Samples Lock V Tune v Spin Si Shine l Prosol l Gain D Gow Options v Select db spins by clicking on it 84 B7170_00_01 Figure 5 31 Turn sample rotation on ro on Change sample rotation rate ro MAS Pneumatic Unit masdisp 7 Select ro off by clicking on it NOTE 1 D selective experiments should be run non spinning 8 Select Al Prosol by clicking on it L NOTE This will load the pulse width and power levels in to the parameter set 1 Select the AcquPars tab by clicking on it 2 Make the following changes PULPROG selnogp D8 0 450 DS 8 NS 64 SPNAM2 Gaus1 180r 1000 SPOFF2 value from 5 5 3 step 13 e g 413 68 NOTE The mixing time D8 is dependent on the size of the Molecule and the magnetic strength It can vary from a large Molecule to a small one fr
40. shape in the command line B7170_00_01 163 Figure 7 49 Shaped pulse parameter Index Power dB Offset Freq Hz Phase alignment Filename SP SPOFFS SPOAL SPNAM H gauss 1 7235 0 Gaus1 1000 kag 24 Click on to select SPNAM 1 25 Select the user directory in the Source window 26 Select wetshapel from the list 27 Click on Figure 7 50 Shaped pulse parameter Index Power dE Offset Freq Hz Phase alignment Filename SP SPOFFS SPOAL SPNAM SP1 power level adjusted to account for the number of frequency positions see list below 1 frequency calibrated power level e g 59 9db 2 frequencies calibrated level minus 6 dB e g 53 9db 3 frequencies calibrated level minus 9 5 db e g 50 4db 4 frequencies calibrated level minus 12 db e g 47 9db NOTE In this example the power level SP1 for 2 frequencies is used e g 53 9dB 27 Click on 7 8 6 Running the experiment 1 Type lcwetset in the command line 2 Tune the probe NOTE Step 2 is necessary for tuning the F2 frequency which is used to decouple 13C coupling 3 Select Gains by clicking on it 164 B7170_00_01 NOTE To adjust rg manually click on the down arrow inside the Gain icon 4 Select Ba by clicking on it 7 8 7 Processing 1 Click on the Process tab in the TopSpin Menu bar Figure 7 51 3 Start Acquire Process Analyse Publish View M
41. solvsupr wet 2 1 CBrukeriTOPSPIN nmrsu Los SABA mi wt rn 22m Sinc1 1000 1 Offs 1000 Size of Shape 345 63 Offset 1 Eales Sigar O 20 40 60 80 50 150 250 points 12 In the main menu click on Options and select Define Parameter Table by clicking on it Figure 7 45 Window Help Select associated dataset Define Parameter Table Preferences Remote Connection Administration 162 B7170_00_01 Figure 7 46 4 Define Parameter Table Parameter Table P1 Length of hard pulse J 4 PL1 Power level of hard pulse _ P11 Length of shaped pulse SP1 Power level of shaped pulse SPNAM1 Name of shaped pulse P 13 Length of ref shaped pulse SP3 Power level of ref shaped pulse Name of ref shaped pulse EQILIST Name of frequency list 13 Make the following changes Length of shaped pulse p11 Power Level of shaped pulse SP1 Name of shaped pulse SPNAM1 4 Click on 15 Click on E and select Shape Figure 7 47 wetshape1 Title 90 0 Flip Angle Excitation Type of rotation 18 Type wetshapel in the File Name window 19 Click on Figure 7 48 Destination Dir CABrukeATOPSPINvexpistarhnmnlistswaveluser New Name wetshape1 20 Type wetshapel in the New Name window 21 Click on 22 Click on to close the Shape Tool window 23 Type
42. the destination will be a 1 file dataset no expno pracna required Please specify destination MAME EXPNO PROCNO DIR 2 Change NAME sel cosy 3 Click on 4 In the command line type re and hit Enter Figure 5 6 Options Display data in same window Display data in new window NAME EXPO PROCNO ENSEM 5 Change NAME sel cosy 6 Click on 7 Expand peak at 4 8ppm 8 Click on isl to set the RF from cursor 72 B 7170 00 01 Figure 5 7 selective exp 2 1 C data3 0 1 D Selective experiment Reference spectrum 30 mg Menthy Anthranilate in DMSO d6 4 7909 ppm 1437 87 Hz 300 131438 MHz rel 1 5 SET SF01 01 FREQUENCIES FROM CURSOR POSITION MAS LACET ele Nes aide LI LR ES UCM ne 1 0 0 5 0 0 4 90 4 85 4 80 4 75 4 70 ppm 9 Move the cursor line in to the center of the multiplet 10 Click the left mouse button to set the frequency Figure 5 8 1301 02 03 Define SFO1 0O1 frequencies SFO1 MHz 300 131438 O1 2 3 Hz 1437 87 11 Click on 5 3 4 Setting up the Selective COSY 1 Click on the Start tab in the TopSpin Menu bar Figure 5 9 Acquire Publish o _ Create Dataset Find Dataset J Open Dataset Paste Dataset Read Pars Process Analyse View Manage 2 Select by clicking on it D 1 0 00 01 73 Fi
43. too the top of the magnet 9 Select Aj Samples by clicking on it Figure 5 69 Turn on sample lift air ej Control sample temperature edte 10 Select ij by clicking on it NOTE Wait till the sample is lowered down in to the probe and the lift air is turned off A licking sound may be heard 11 Select HE Lock by clicking on it D 1 0 00 01 105 Figure 5 70 e Solvents table F Solvent Description Acetic Acetone Acetone_Hump C6D6 CD2CI2 CD3CN CDCI3 CDCI3 SENS CH3CN D20 D20 DEE Dioxane DME DMF EtOD acetic acid d4 acetone d6 acetone d6 CHCI3 benzene d6 methylenechloride d2 acetonitrile d3 chloroform d chloroform d ETB HPLC Solvent Acetonitril D20 deuteriumoxide diethylether d10 dioxane d8 dimethylether d6 dimethylformamide d7 ethanol d6 H20 D20 90H20 and 10 D20 E Lock nucieus 2H 12 Select DMSO by clicking on it 13 Select by clicking on it NOTE This performs a atma automatic tuning and requires a probe equipped with a automatic tuning module Other options can be selected by clicking on the down arrow inside the Tune button 15 Select db Spin by clicking on it Figure 5 71 Turn sample rotation off ro off Change sample rotation rate ro MAS Pneumatic Unit masdisp 16 Select ro on by clicking on it NOTE Rotation may be turned off for probes su
44. 00 5 8 OS SOMA E AE E AE A I AA I A T T 102 5 9 1 D Carbon DEPT experiment using a shaped 130 pulse 103 5 9 1 A OOU OR E E E 103 5 9 2 EXEM SEUD PET T 104 5 9 3 PCOUISIION RE shan telwetantberadsauuestaiusinan taaneien hiedstanttandeesais 107 5 9 4 FOC SS SIO nn a aies de ie ee ee re 107 6 2 D experiments using shaped pulses 109 6 1 2 D edited HSQC experiment with Adiabatic pulses eeesessssss 109 6 1 1 OO OR 109 6 1 2 2 D edited HSQC experiment using adiabatic pulses ssssss 110 6 1 3 D cee ee 110 6 1 4 Reference Spectrum six TT M ntn EI RUM ER M brass 111 6 1 5 Setting up the HSQC experiment 112 6 1 6 PCO UNISON rcr 114 6 1 7 PO Ce SIO se nee ae anneau ce en re S 115 6 2 OS 120 6 3 2 D Selective HMBC experiment ss 121 6 3 1 It OCG HOM ds ee cc cn 121 6 3 2 SD a adios 121 6 3 3 Preparation experiment cccccssscccesececsesseceeeeeeceueeecseeeesueessaeeessaseeessuseessageenes 122 6 3 4 PG MNS ONO CE ean 125 6 3 5 PO Ce SSN aes En 126 6 3 6 Optimizing the parameters on the carbonyl region 127 6 3 7 Set up the selective pulse 129 6 3 8 Setting up the acquisition parameters 134 6 3 9 Running the experiment cccccccseceeceeeeecee
45. 1 55 4 6 Observations 56 B7170_00_01 4 7 2 D HMBC experiment The basic 2D HMBC pulse sequence see Figure 3 1 is closely related to the HMQC pulse sequence but incorporating the following modifications An optional low pass J filter consisting of a delay 909 1 3C cluster can be included after the initial 90 1H pulse to minimize direct response The de focusing period is optimized to 1 2 J CH 5 10Hz The refocusing period is usually omitted Proton acquisition is performed without X decoupling Using this experiment qualitative heteronuclear long range connectivity including qua ternary carbons or through heteronuclei can be extracted Figure 4 32 hmbclpndaf The non gradient 2D HMBC spectrum of Menthyl Anthranilate in DMSO d6 is illustrated in Figure 3 32 showing considerable artifacts Additionally a minimum number of 8 scans had to be used for the full phase cycling D 1 0 00 01 57 Figure 4 33 hmbc_exp 2 1 C data3 0 2D gradient HMBC experiment 30 mg Menthy Anthranilate in DMSO d6 CHE dh FT ppm 50 100 d j 1 i 150 6 4 2 F2 ppm The main advantages of using gradients in high resolution NMR experiments include Coherence selection and frequency discrimination in the indirect dimension F1 can achieved with a single scan per T1 increment A reduction in the number of required phase cycle steps for the suppress
46. 3 Type re on the command line Figure 6 36 Options o Display data in same window Display data in new window L MAME shape hmbc exp EXPNO PROCNO DIR 4 Change the EXPNO to 2 5 Expand the carbonyl region including all cross peaks e g 168 ppm to 178 ppm D 1 0 00 01 127 Figure 6 37 shape hmbc exp 2 1 C data3 0 Spectrum ProcPars AcquPars Title PulseProg Peaks Integrals Sample Structure Plot Fid Acqu 2D selective HMBC experiment E 50 mM Gramicidin S in DMSO d6 i amp col 1 17 ppm 351 03 Hz Index 1809 1811 x row 175 9 ppm 13346 8 Hz Index 157 158 Value 6 031e 04 i E 10 o o 9 o o N 8 6 4 2 F2 ppm Figure 6 38 shape hmbc exp 2 1 C data3 0 Spectrum ProcPars AcquPars Title PulseProg Peaks Integrals Sample Structure Plot Fid Acqu 2D selective HMBC experiment SNE Rm te PARTS 168 F1 ppm 174 172 170 176 8 6 4 2 F2 ppm 6 Write down the expanded F1 sweep width in ppm and Hz e g 10 ppm 750 Hz 7 Write down the center frequency O2 of the expanded F1 sweep width in ppm e g 172 ppm 8 Select the AcquPars tab by clicking on it 128 B7170_00_01 9 Select the pulse program parameters view 10 Write down the value for P3 us e g 10 5 us 11 Write down the value for PL2 dB e g 1
47. 7 33 dB 12 Change the PULPROG to shmbcgpndaf 13 Select the Spectrum tab by clicking on it 6 3 7 Set up the selective pulse 1 Click on the Aquire tab in the TopSpin menu bar Figure 6 39 E Start Acquire Process Analyse Publish View Manage oe peneme Y Sample S amp Lock V Tunev db Spin 1 Shim 4 Prosol SetLimits Gain v BD Gow Options gt 2 Click on the down arrow inside the Options button Figure 6 40 IcanMME Automation icannrnr TopGuide topguide One Click Experiments 3 Select Shape Tool spdisp by clicking on it D 1 0 00 01 129 Figure 6 41 Shapelool shape hmbc exp 2 1 C data3 0 HAT i Pod 9 aes ELLID Gauss rParameters 1000 Size of Shape z asiecive HMBC expb meni eee tette ete pL 50 mM Gramicidin S ig DMSO d6 iii im aeni seu BTR EEES 1 0 Truncation Level F2 ppm 5 200 400 600 800 points 3 In the main menu click on Shapes Figure 6 42 Basic shapes Burp Tk Er UU E Adiabatic Shapes Gauss dies a lids Application i Gaussian Pulse Cascade b Imaging Application H HalfGauss Decoupling Shapes Hermite i SERLICE ot Eee Snob vega 2 ShapFour 4 Select Classical Shapes 5 Select Sinc by clicking on it 6 Make the following changes Size of Shape 256 Numb
48. COSY experiment The standard Bruker parameter set is SELCOGP and includes the pulse sequence selcogp shown in Figure 5 3 It consists of the recycling delay four radio frequency RF pulses and the acquisition time during which the signal is recorded The first RF pulse is a 90 degree pulse followed by a 180 degree shaped pulse a 180 degree hard pulse and finally a 90 degree pulse The delay between the 180 and 90 degree pulse is 1 4 J H H The gradient pulses are applied before and after the shape pulse 70 B 7170 00 01 Figure 5 3 5 3 2 Reference spectrum 1 Run a 1D Proton spectrum following the instructions in 1 D Proton experiment Chapter 3 Figure 5 4 Acquisition finished C data3 0 proton_exp 1 pdata 1 Spectrum ProcPars AcquPars Title PulseProg Peaks Integrals Sample Structure Plot Fid Acqu rel 1 Proton experiment 30 mg Menthyl Anthranilate in DMSO d6 15 10 5 0 ppm 5 3 3 Selective excitation region set Up 5 3 3 1 On resonance NOTE Make sure that the SW is large enough to cover the entire Spectrum accounting D 1 0 00 01 T1 for the position of O1 The shaped pulse is applied on resonance at the O1 position The power level and width of the excitation pulse have to be known and entered into the Prosol parameter table 1 In the command line type wrpa and hit Enter Figure 5 5 amp wrpa Copy data set If NAME ends with top
49. Click OK to set frequencies and return to original dataset 10 To open the 1D Proton spectrum right click on the dataset name in the browser win dow e g proton exp 1 and select Display or click and hold the left mouse button for dragging the 1D Proton dataset in to the spectrum window 11 Expand the spectrum to display all peaks leaving ca 0 5 ppm of baseline on either side of the spectrum NOTE The solvent peak may be excluded if it falls outside of the region of interest 170 B7170_00_01 Figure 8 9 proton exp 1 1 C idata3 0 pem qu ge JA m E spectrum ProcPars AcquPars Title PulseProg Peaks Integrals Sample Structure Plot Fid Acqu 1 Proton experiment 30 mg Menthyl Anthranilate in DMSO d6 rel 10 8 6 4 2 ppm 12 Click on to assign the new limit Figure 8 10 ae H spectral limits copied for F1 and F2 dimensions aW 8 5124 ppm TP 4 244 pnm 13 Click on NOTE The display changes back to the 2D data set 14 Select the AcquPars tab by clicking on it 15 Select the pulse program parameters view 15 Make the following changes D1 15 VDLIST tidelay 16 Click on to right of the VDLIST name box B7170 00 01 17 Figure 8 11 EN tidelay C Bruker E File Edit Search 1 g 3 4 z b B g D b M e Ss O amp amp amp H HC 17 Enter the variable delay values as
50. ER a ug m 12 18 T Ba A D 1 0 00 01 39 4 3 2 D gradient COSY Several simple two pulse programs can be used to record a magnitude mode COSY spectrum e g cosy cosy45 and cosy90 These vary with respect to the angle of the final pulse Any value between 20 and 90 may be chosen for the final pulse angle However a pulse angle of 45 is recommended because this yields the best signal to noise ratio together with a simple cross peak structure in the final spectrum A minimum of 8 scans have to be acquired do to the quadrature phase cycle The signals acquired with one of these experiments have absorptive and dispersive line shape contributions in both F1 and F2 dimensions This means that it is impossible to phase the spectrum with all peaks purely absorptive and as a consequence the spec trum must be displayed in magnitude mode A typical spectral resolution of 3 Hz pt is sufficient for resolving large scalar couplings In order to resolve small J couplings fine digital resolution is required which significantly increases the experimental time In gen eral the DQF COSY experiment is recommended if a higher resolution is desired As well the DQF COSY experiment reduces the intensity of the diagonal allowing for anal yses of peaks close in chemical shift Using pulsed field gradients PFG the coherence pathway selection and the axial peak Suppression can be achieved with only one scan per time increment Thus if enoug
51. Integrals Sample Structure Fia Acqu Proton 90 degree pulse calibration 0 1M 15 Urea and O0 1M 13C CH3OH in DMSO d6 2 5 2 Parameter set up 1 Click on the Aquire tab in the TopSpin menu bar 18 B7170 00 01 Figure 2 17 E Start Acquire Process Analyse Publish View Manage o Li Sample v BE Lock iV Tune v db Spin e Shim PL Prosol i Gain v Po Options 2 In the command line type wrpa and hit Enter Figure 2 18 Copy data set If NAME ends with top the destination will be a 1 file dataset no expno procno required Please specify destination NAME EXPNO PROCNO DIR 3 Change NAME p90 carbon 4 Click on 5 In the command line type re and hit Enter Figure 2 19 Options Display data in same window Q Display data in new window NAME p90 ca rbon EXPNO PROCNO DIR Cor cance Cross 6 Change NAME p90 carbon 7 Click on 8 Expand the region between 3 5ppm and 2 8ppm NOTE Normally a single peak set to on resonance is used to determine the 90 trans mitter pulse For practical reason the Methanol signal region from 3 5ppm to 2 8ppm is used to measure the 13C 90 transmitter pulse since the same signals will also be used in determining the 1H 90 decoupler pulse D 1 0 00 01 19 Figure 2 20 No acquisition runni
52. OPSPINidatainmrsummr 1d_soivent_suppression 3 pdatai1 Integrals Sample solvent suppression experiment 2mM Raffinose in 90 H20 10 D20 Spectrum 3 5 NOTE Figure 7 19 above shows the solvent suppressed 1 D spectrum of the Raffinose sample and Figure 7 20 below shows the 1 D spectrum of the Lysozyme sample Figure 7 21 spectrum ProcPars AcquPars Title PulseProg Peaks Integrals Sample Structure Fia 148 B7170 00 01 7 5 1 D Solvent suppression using the noesy sequence This experiment is performed by using the 1 D version of the noesyphpr sequence applying a low power continuous wave irradiation on the water resonance during the pre scan and the during the mixing time period of the NOESY sequence see Figure 7 21 Figure 7 22 noesyprid di plis I presat Iu 7 5 1 Parameter set up 1 Follow the instructions in paragraphs 6 2 2 through 6 2 6 step 9 in this chapter 2 Select the AcquPars tab by clicking on it 3 Make the following changes PULPROG noesyprid D8 s 0 1 4 Select the Spectrum tab by clicking on it 7 5 2 Acquisition 1 Select Gains by clicking on it NOTE To adjust rg manually click on the down arrow inside the Gain icon 2 Select Boo by clicking on it 7 9 3 Processing 1 Process and phase correct the spectrum D 1 0 00 01 149 Figure 7 23 M 1d solvent s
53. Pars Title PulseProg Peaks Integrals Sample structure Fia Acqu F2 ppm NOTE The cross peaks in the selective HMBC show nice separation do to the increased resolution in F1 compared to the regular HMBC The projections are external high reso lution spectra 136 B7170 00 01 7 1 D Solvent suppression experiments 7 1 Introduction Many experiments on samples dissolved in protonated solution require some method to minimize the strong resonance belonging to the solvent This suppression can be per formed in several ways depending on the number of signals to suppress and depending on which part of the pulse sequence can be modified Solvent suppression can be applied during the relaxation period just prior to the conventional pulse sequence as out lined in Figure 5 1 below This is referred to as Presaturation Figure 7 1 However presaturation can also reduce the signal intensities of exchangeable protons For this reason other schemes as the WATERGATE WET and Excitation Sculpting schemes can be used to overcome this problem and are discussed in this chapter In HPLC NMR applications it is mandatory to suppress multiple solvent resonances The incorporation of specific multiple solvent suppression schemes into pulse sequences is made in analogy with classical methods 7 1 1 Samples 2mM Raffinose in 90 H20 10 D2O 2mM Lysozyme in 90 H20 10 D20 D 1 0 00 01 137 7 2 Preparation exp
54. a Calculate Bloch siegert Shift analyze Osiegerts isian Calculate average Power Level analyze calcpaw Integrate Shape analyze integrs Integrate Adiabatic Shape analyze integradial Integrate and compare to Reference analyze integandcomp Simulation analyze simulate 12 Select Calculate Bandwidth for Excitation by clicking on it Figure 6 48 sinc bandh i 80 0 Total rotation degree rResults B 520 Deltaomega DeltaT factor using trans 7 Tr Calculatar DeltaGmega Hz 7428 3 DeltaT usec update parameters 132 B 7170 00 01 13 Make the following changes DeltaOmega Hz 750 e g SW value in Hz from 4 3 6 step 6 14 Press the Enter key NOTE The value of Delta T usec is being calculated e g 7429 3 usec 15 Write down the Delta T value usec e g 7429 3 16 Click on update parameters Figure 6 49 EM Select dataset for parameter update Target dataset NAME saam aall CO PROCNO 4 DR jpfdaaBO fdata3 0 17 Click on 18 In the main menu click on Figure 6 5012 00 Calculate Bandwidth for Excitation analyze bandw2 Calculate Bandwidth for Inve
55. a3 0 spectrum ProcPars AcquPars Title PulseProg Peaks Integrals Sample Structure Plat Fid Acqu 1 D 13C experiment with 1H decoupling 30 mg Menthyl Anthranilate in DWSO d6 rel 160 140 120 100 80 60 40 20 ppm 17 Click on to assign the new limit D 1 0 00 01 63 Figure 4 45 o 13C spectral limits copied far F1 dimension ov 166 6575 ppm centered at 82 714 pom 18 Click on c 4 7 3 Acquisition Gains by clicking on it 1 Select 2 Select by clicking on it 4 7 4 Processing 1 Click on the Process tab in the TopSpin Menu bar Figure 4 46 E Start Publish Manage A Proc Spectrum v Adjust Phase v A Calib Axis 7 R Pick Peaks j Integrate Advanced m O Figure 4 47 Acquire Process Analyse View s apk2d spectrum has no imaginary part MC2 F1 OF PH mod F1 2mc Could not phase real spectrum NOTE This executes a standard processing program proc2 The message shown in Figure 4 47 pops up in case of a magnitude 2D experiment and the apk2d option is enabled To configure the processing program follow the steps below 64 B7170 00 01 3 Click on the down arrow inside the Proc Spectrum button Figure 4 48 Process F2 F1 Axis xfb Process Only F2 Axis xf2 Process Only F1 Axis xf1 Symmetrize Spectrum sym Start Automatio
56. a3 0 Mouse Sensitivity 1 0 1 98 ppm 592 86 Hz Sum 27 4914 DEFINE REGION MODE Define Drag using left mouse button Return Left click highlighted icon rel 10 8 6 4 2 ppm 176 B7170_00_01 11 Click on Li Figure 8 24 Save Regions To intrng cave amp Show LIST 12 Select Export Region To Relaxation Module by clicking on it 13 Click on Relaxation Relaxation Window Figure 8 25 EN Relaxation parameters General Parameters FID for phase determination 1000 0 Left limit for baseline correction 1000 0 Right limit for baseline correction Number of drift points Convergence limit Number of points First slice Hill Slice increment rFitting Functian uxnmrt1 Function Type Number af components List file name Increment auto ino pick data points T Iteratian control parameters GUESSES ET 14 Click on B7170_00_01 177 Figure 8 26 Relaxation t1_exp 1 1 C data3 0 4 2 1 Fitting type Intensity mo IO HP exp VT T Peak f at 7 677 ppm E Area Current Peak 1of7 Brief Report Data preparation is done 15 Click on LE Fitting Fitting Functions Figure 8 27 Please select the function to which the peak intensities or integrals are to be fi
57. ailable N CI show power at probe in Switch F1 F2 Switch F1 F3 Add a logical channel Remove a logical channel Default 16 Click on Save 17 In the AcquPars make the following change O2 ppm 76 D1 10 CNST2 88 5 P3 6 18 Select AV Prosol by clicking on it to read in the Prosol parameters a 19 Select the ProcPar tab by clicking on it 20 Make the following changes SI 2K 21 Select the Spectrum tab by clicking on it 22 In the command line type wpar N15p90 urea all to store the parameter set for future use D 1 0 00 01 29 2 7 2 Determine the 15N 90 decoupler pulse 1 Select NC rune by clicking on it 2 Select by clicking on it 3 Process and Phase correct the spectrum NOTE Phase the left side signal negative and the right side signal positive Figure 2 39 ME pz Spectrum ProcPars AcquPars Title PulseProg Peaks integrals Sample Structure Fia Acqu f5N 90 degree pulse calibration 0 4M 15 Urea and 0 1M 13C CH3OH in DMSO d6 4 Increase p3 in increments of 1 or 2us execute zg followed by the command efp until the signals go through a null or a phase change This will be the 15N 90 decoupler pulse 30 B7170_00_01 Figure 2 40 pulse_calibration 2 1 C pz 10 x B7170_00_01 31 2 8 Observations 32 B7170 00 01 3 1 D Proton experiment 3 1 Sample A sample of 30mg Menthyl Ant
58. als Sample solvent suppression experiment 2mM Raffinose in 90 H20 10 D20 Re Tee eee O OO Pee ee ee ee eee eee eee eee eee eee eee eee eee eer ere eer eee 3 5 NOTE Figure 7 16 above shows the solvent suppressed 1 D spectrum of the Raffinose sample and Figure 7 17 below shows the 1 D spectrum of the Lysozyme sample Figure 7 18 Spectrum ProcPars AcquPars rite PulseProg Peaks integras Sample Structure Fia acau 146 B7170_00_01 7 4 1 D Solvent suppression with Presaturation and Composite Pulses This experiment is performed by applying a low power continuous wave irradiation on the water resonance during the pre scan period followed by a rapid succession of four 90 degree pulses to further reduce the residual hump of the water signal see Figure 7 18 Figure 7 19 pl9 min l 7 4 1 Parameter set up 1 Follow the instructions in paragraphs 6 2 2 through 6 2 6 step 9 in this chapter 2 Select the AcquPars tab by clicking on it 3 Make the following changes PULPROG zgcppr 4 Select the Spectrum tab by clicking on it 7 4 2 Acquisition 1 Select Gains by clicking on it NOTE To adjust rg manually click on the down arrow inside the Gain icon 2 Select Boo by clicking on it 7 4 3 Processing 1 Process and phase correct the spectrum D 1 0 00 01 147 Figure 7 20 M No acquisition running C Bruker T
59. ample rotation rate ro MAS Pneumatic Unit masdisp 7 Select ro off by clicking on it NOTE Solvent suppression experiments should be run non spinning 17 Select amp Shims by clicking on it NOTE This executes the command topshim To select other options click on the down arrow inside the Shim button 18 Select A Prosol by clicking on it NOTE This will load the pulse width and power levels in to the parameter set 7 2 1 Acquisition 1 Select Gains by clicking on it NOTE To adjust rg manually click on the down arrow inside the Gain icon 2 Select Bo by clicking on it 7 2 2 Processing 1 Click on the Process tab in the TopSpin Menu bar 140 B7170 00 01 Figure 7 8 E Start Process Analyse Manage oe A Proc Spectrum Adjust Phase A Calib Axis v 5 Pick Peaks v f Integrate Y Advanced v NOTE This executes a processing program including commands such as an exponen tial window function em Fourier transformation ft an automatic phase correction apk and a baseline correction abs Other options are available by clicking on the down arrow inside the Proc Spectrum button Acquire Publish View Figure 7 9 2 No acquisition running C Bruker TOPSPIN datainmrsuinmr td solvent suppression 1 pdata 1 Le ES Spectrum ProcPars AcquPars
60. anage oe Proc Spectrum v w Adjust Phase v _A Calib Axis d 9 Pick Peaks v J Integrate Advanced NOTE This executes a processing program including commands such as an exponen tial window function em Fourier transformation ft an automatic phase correction apk and a baseline correction abs Other options are available by clicking on the down arrow inside the Proc Spectrum button Figure 7 52 i Acquisition finished 1d_solvsupr_wet 2 1 C Bruker TOPSPIN nmrsu CES Spectrum ProcPars AcquPars Title PuseProg Peaks integrals sample Structure Fid Acqu rel 12 14 10 5 4 3 ppm D 1 0 00 01 165 166 B7170 00 01 8 1T1 experiment 8 1 Introduction The inversion recovery experiment allows to measure longitudinal or spin lattice T1 relaxation times of any nucleus The basic pulse sequence consists of a 180 pulse inverts the magnetization to the z axis During the following delay relaxation along the longitudial plane takes place Mag netization comes back to the original equilibrium z magnetization A 90 pulse creates transverse magnetization The experiment is repeated for a series of delay values taken from a variable delay list A 1D spectrum is obtained for each value od vd and stored ina 2 D data set The relaxation time di must be set to 5 T1 A rough estimation of the T1 value can be calculated
61. and line type efp D 1 0 00 01 15 14 Change p1 slightly and repeat steps 12 and 13 until the signals undergoes a zero crossing as expected for an exact 360 degree pulse NOTE The signals are negative for a pulse angle slightly less then 360 degree and pos itive when the pulse angle is slightly more then 360 degree 15 Simply divide the determined 360 degree pulse value by 4 This will be the exact 90 degree pulse length for the proton transmitter on the current probe 16 B7170_00_01 2 4 Observations B7170 00 01 17 2 5 13C 90 decoupler pulse Figure 2 15 fl channel 30 f channel p3 The pulse program used in this procedure is the decp90 sequence shown in Figure 2 13 The sequence consists of two channels f1 1 and f2 S where in this case f1 is set for 1H and f2 to 13C Channel f1 shows a recycle delay d1 followed by a 90 pulse and a delay d2 1 2J44 for the creation of antiphase manetization A 13C pulse on channel f2 is been executed after the delay d2 and then the 1H signal is detected When the 13C pulse is exactly 909 the 1H signals will go through a null The Methanol signal region from 3 5ppm to 2 8ppm is used for this experiment 2 5 1 Preparation experiment 1 Run a 1D Proton spectrum of Urea Methanol in DMSO d6 following the instructions in 1 D Proton experiment Chapter 3 Figure 2 16 No acqu 1 Spectrum ProcPars AcquPars Title PulseProg Peaks
62. ation in to the New window Figure 6 26 Prepare for a new experiment by creating a new data set and initializing its NMR parameters according to the selected experiment type For multi receiver experiments several datasets are created Please define the number of receivers in the box below NAME shape_hmbc_exp EXPNO PROCNO DIR C data3 0 Solvent DMSO Experiment Dirs CABruker TopSping 0 b 44 exp staninmripar Experiment HMBCGP TITLE 2D selective HMBC experiment 50 mM Gramicidin S in DMSO d6 Show new dataset in new window M Receivers 1 2 16 NOTE The directory DIR is specific to how the data are stored and therefore may show different entries as the one in Figure 4 26 above Click on the down arrow button to browse for a specific directory 4 Click on 5 Click on the Aquire tab in the TopSpin menu bar Figure 6 27 E Start Acquire Process Analyse Publish View Manage oe Sample PE Lock Vv Tune v Jb Spin St Shim v il Prosol SetLimits k Gain v D Gow 6 Select db spins by clicking on it Options B7170_00_01 123 Figure 6 28 Turn sample rotation on ro on Change sample rotation rate ro MAS Pneumatic Unit masdisp 7 Select ro off by clicking on it NOTE 2 D experiments should be run non spinning 8 Select A Prosol by clicking on it ES Fi NOTE This will load the pulse width and
63. ch as BBI TXI TBI and for small sample probes 17 Select amp Shims by clicking on it NOTE This executes the command topshim To select other options click on the down arrow inside the Shim button 18 Select fl Prosol by clicking on it i j 106 B7170 00 01 NOTE This will load the pulse width and power levels in to the parameter set 5 9 3 Acquisition 1 Select Gains by clicking on it NOTE To adjust rg manually click on the down arrow inside the Gain icon 2 Select Boo by clicking on it NOTE Other options are available by clicking on the down arrow inside the Go button 5 9 4 Processing 1 Click on the Process tab in the TopSpin Menu bar Figure 5 72 Start Vi JL Proc Spectrum v Adjust Phase v A Calib Axis v M Pick Peaks v J Integrate Advanced v 2 Click on Jk Pros Spectrum NOTE This executes a processing program including commands such as an exponen tial window function em Fourier transformation ft an automatic phase correction apk and a baseline correction abs Other options are available by clicking on the down arrow inside the Proc Spectrum button Do to the fact that a DEPT135 spectrum con tains negative and positive peaks there is the possibility of getting phase results that are 180 degrees off In this case click on the Adjust Phase button to enter the manual
64. e 86 B7170_00_01 Figure 5 35 Acquisition finished C data3 0 sel_noesy 1 pdata 1 Spectrum ProcPars AcquPars Title PulseProg Peaks Integrals Sample Structure Plot Fia Acqui 1 D Selective NOESYexperiment JU LAE TENN NII 3 cava Rea wt eiat ied sc soto iet ete ttd e astu tot rel 20 20 15 10 5 0 ppm 5 5 7 Plotting two spectra on to the same page 1 Display the selective NOESY spectrum 2 Click on AI to enter the Multiple display option 3 Drag the Reference spectrum in to the spectral window Figure 5 36 sel noesy 1 1 C data3 0 AS RO PtH CEE 25759512585 A ZE 4 rel selective_exp Ea etse io s ioi Ke sel noesy 1 1 d datas 0L 8 6 4 2 ppm D 1 0 00 01 87 NOTE To adjust the spectra for best fit use the 2 2 g Ts tools 4 Click on the Publish tab in the TopSpin Menu bar Figure 5 37 E Stat Acquire Process Analyse Publish View Manage o Bi Copy Printw L J Plot Layout S PDF v E Mail 5 Click on the button to print the active window 88 B7170 00 01 5 6 Observations B7170_00_01 89 5 7 1 D selective gradient TOCSY e This experiment consist of three parts e Selective excitation of the selected resonance using the SPFGE block e Mixing period to achieve in phase polarization transfer to other spins This is usu ally achieved by ap
65. e AcquPars tab by clicking on it 2 Select all parameters view 3 Make the following changes TD F1 64 NS 32 134 B7170 00 01 SW ppm F1 value from 4 3 6 step 6 e g 10 O2P ppm value from in 4 3 6 step 7 e g 172 4 Select the pulse program parameters display 5 Make the following changes P13 us value from in 4 3 7 step 14 e g 7429 3 SP14 dB value from in 4 3 6 step 11 Value from 4 3 7 step 22 e g 24 6501 SPNAM14 Sinc3 256 6 3 9 Running the experiment 1 Select the Spectrum tab by clicking on it 2 Select Bo by clicking on it 6 3 10 Processing 1 Click on the Process tab in the TopSpin Menu bar Figure 6 52 3 Start Analyse Publish View Manage oe Proc Spectrum v v Adjust Phase v Calib Axis 7 tit Pick Peaks v Integrate Advanced m O Figure 6 53 Acquire Process Spectrum has no imaginary part MC2 F1 QF PH mod F1 2mc Could not phase real spectrum NOTE This executes a standard processing program proc2 The message shown in Figure 6 53 pops up in case of a magnitude 2D experiment and the apk2d option is enabled To configure the processing program follow the steps below To avoid the mes sage shown in Figure 6 53 the option Auto Phasing apk2d may be disabled for mag nitude like 2D experiment D 1 0 00 01 135 Figure 6 54 hmbc 400 7 1 C pz Spectrum ProcPars Acqu
66. e 1H signals will go through a null The Urea signal region from 5 6ppm to 5 1ppm is used for this experiment If your system is equipped with a 3rd channel for 15N observation you can still follow the same instructions in this chapter with the exceptions of using the pulse sequence decp90f3 shown in Figure 2 23 and the routing which is illustrated in the section Param eter set up 2 4 2 Figure 2 29 and Figure 2 31 Figure 2 28 decp 0fs i fl channel TA Hi f3 channel g pel 2 7 1 Parameter set up 1 Click on the Aquire tab in the TopSpin menu bar Figure 2 29 Start Acquire Process Analyse Publish View Manage o Y Sample vx HE Lock V Tune v 4b Spin v et Shim FA Prosol V Gain gt Go Options 2 In the command line type wrpa and hit Enter D 1 0 00 01 25 Figure 2 30 Copy data set If NAME ends with top the destination will be a 1 file dataset no expno procno required Please specify destination 3 Change NAME p90 nitrogen 4 Click on ok 5 Inthe com Figure 2 31 Display data in same window Display data in new window 6 Change NAME p90_ nitrogen 7 Click on OK 8 Expand the region between 5 6ppm and 5 1ppm 26 B7170 00 01 Figure 2 32 pulse_calibration 1 1 C pz Spectrum ProcPars AcquPars Title PulseProg Peaks integrals Sample Structure Fia acqu 5N 90 degree pulse calibratio
67. e SFOT1 01 frequencies SFO1 MHz 500 262345 O1 2 3 Hz 2345 29 5 Click on 7 Select the AcquPars tab by clicking on it 8 Make the following changes PULPROG zgpr TD 16k NS 8 DS 4 SW ppm 10 for the Raffinose sample SW ppn 14 for the Lysozyme sample D1 s 2 9 Select the ProcPar tab by clicking on it 10 Make the following changes SI 8k 11 Select the Spectrum tab by clicking on it 7 3 2 Fine tuning 1 Select Gain by clicking on it NOTE To adjust rg manually click on the down arrow inside the Gain icon 2 Click on the down arrow inside he B Gos button D 1 0 00 01 143 Figure 7 13 Transfer Fid To Disk trj Estimate Exp Time expt Optimize Acquisition Params papt Start Automation AU program aua 3 Select Real Time Go setup gs by clicking on it 4 Click on be 5 Select the Offset tab Figure 7 14 2 GS 1d solvent suppression 2 1 C BrukeriTOPSPIN nmrsu ele ea Spectrum ProcPars AcquPars Title PulseProg Peaks Integrals Sample Structure Fid Acqu Pulse Frequency Pulse Power NL EP vH CorPhase Delay Offset Receiver Gain solvent suppression experiment ES 01 2mM Raffinose in 9096 H20 10 D20 sensitivity x o 1 E 8 adjust max 2395 29 m 8 o Offset 8 O1 Hz D o e eo eo N 8 m
68. e number of receivers in the box below NAME iproton exp EXPNO PROCNO DIR v Solvent DMSO v Experiment Dirs C BrukenTopSpin3 0 b 40 exp stan nmrpar v Experiment PROTON v TITLE 1D Proton experiment 30 mg Menthyl Anthranilate in DMSO d6 C Show new dataset in new window M Receivers 1 2 16 NOTE The directory DIR is specific to how the data are stored and therefore may show different entries as the one in Figure 3 4 above Click on the down arrow button to browse for a specific directory 4 Click on 5 Click on the Aquire tab in the TopSpin menu bar Figure 3 5 3 Start Acquire Process Analyse Publish View Manage Sample v Lock W Tune v IE Spin ei Shim fl Prosol Le Gains D Gow Options 6 Select Aj Sample by clicking on it 34 B7170_00_01 Figure 3 6 Turn off sample lift air ij Control sample temperature edte 7 Select ej by clicking on it NOTE Wait till the sample lift air is turned on and remove any sample which may have been in the magnet 8 Place the sample on too the top of the magnet 9 Select Aj Samples by clicking on it Figure 3 7 Turn on sample lift air ej Control sample temperature edte 10 Select ij by clicking on it NOTE Wait till the sample is lowered down in to the probe and the lift air is turned off A licking sound may be heard
69. eee E E E eme nn 75 5 3 7 Plotting two spectra on to the same page cccseececeeeeeseeeeeceeceeeueeeeseeeeeseeseesseeeees 78 5 4 BoI TR MUONS A P EE E 79 5 5 1D SCIE NO 2 niin esere A E ER i 80 5 5 1 WO GIG IOR ee E E E E EEA 80 5 5 2 aici i e x erli PNE 80 5 5 9 Selective excitation region set Up 81 more M O40 erem mm 81 5 5 4 Setting up the Selective NOESY ccccccccccccecesseeeeeeeeeeeeeeeeeeeeeeeeeeeeesaeeeeeeeeseaaees 83 5 5 5 PCOS LUS 85 9 5 6 mise RT en ee ne elite sine ab tte ni 85 5 5 7 Plotting two spectra on to the same page cccseececeseeeceeeeeceeeeeeaeeeeseeeeesseeeessaeeees 87 5 6 OBS NATIONS ee be en an ete seen de tte teens 89 5 7 1 D selective gradient TOCSY accsccecsnsce06ccecierseoncaderssranacedacuanveaunsosenioencaewesanecsegieeeede 90 5 7 1 Selective excitation region set Up 90 OL ON TOSONANOO Sd ae a er ee ed 90 5 7 2 Calculating the selective pulse width and power level 92 5 7 3 Setting up the acquisition parameters 97 4 B7170 00 01 Table of Contents 5 7 4 ive eb i e u Wn T op ne 97 9 4 9 wiee cluoM H 97 5 7 6 Plotting two spectra on to the same page sssseseeeeeeeneen 99 5 7 7 Plotting all 4 experiment on to the same page ccceeeeeceeeeeeeeeeeeeeeeeeseaeeeeeaeees 1
70. eeeceeeeeseeeeeeeeeeseeeeeseeeeesaeeeeseueeesseeeess 135 Di OM cec NETTE en ee D mem 135 7 1 D Solvent suppression experiments 137 7 1 MOOC UO ea ENEE A 137 7 1 1 SPIE enr ces ons EE 137 12 Preparation experiment cccccsssccceescecceseceeseecceeeecseeeeseeeessaseessageeeseeseessaseeess 138 7 2 1 resi Me 140 7 2 2 aioec nemeE s 140 73 1 D Solvent suppression with Presaturation ccccceeeeeneeeeeeeeeeeeeeeeeeeeseeeeeeeees 142 7 3 1 Parameter Set EE R 142 7 3 2 Fine TUMMAG visas snes sxhsteudcesauaneaseinsnd satiiadsiansenieadendanisautteadoetiaatsannianiiWaasdinahaneduanasalonsies 143 7 3 3 eed ze nn a aa en tn ae 145 D 1 0 00 01 5 Table of Contents 7 3 4 FOC 6 SONA ie ee a ae ane dela ee en oo 145 7 4 1 D Solvent suppression with Presaturation and Composite Pulses 147 7 4 1 Parameter set UP cccccccccssecccesscecceeecceesscceueecceueeecsueessuseessseecsaueesseaeesseseeesaaees 147 7 4 2 PCO ION ocuisranbaretdndbntetkev tentem E sect tie en 147 7 4 3 xis 147 7 5 1 D Solvent suppression using the NOESY sequence 149 7 5 1 Parameter Se UD ene E E E E 149 7 5 2 POOU Mo n MEER dt ee 149 7 5 3 OCES SIC er ads du ee et 149 7 6 1 D Solvent suppression with WATERGATE ss 151 7 6 1 Parameter Se
71. eep width in the F1 dimension of 222ppm If a regular carbon spectrum of the same sample is available the F1 sweep width can be limited using the setlimit AU program The steps in 3 7 2 Setting up the HMBC experiment illustrate the limit set ting in both the F2 and F1 dimensions D 1 0 00 01 99 4 7 2 Setting up the HMBC experiment 1 Click on the Start tab in the TopSpin Menu bar Figure 4 36 Acquire Process Analyse Publish View Manage 2 Create Dataset f Find Dataset ii Open Dataset F Paste Dataset Read Pars I 2 Select by clicking on it 3 Enter the following information in to the New window Figure 4 37 Prepare for a new experiment by creating a new data set and initializing its NMR parameters according to the selected experiment type For multi receiver experiments several datasets are created Please define the number of receivers in the box below NAME hmbc exp EXPNO PROCNO DIR v Solvent v Experiment Dirs C Bruker TopSpin3 0 b 44 exp stan nmripar v Experiment HMBCGP v TITLE D gradient HMBC experiment 30 mg Menthyl Anthranilate in DMSO d6 C Show new dataset in new window 4 Receivers 1 2 16 NOTE The directory DIR is specific to how the data are stored and therefore may show different entries as the one in Figure 3 37 above Click on the down arrow button to browse for a specific d
72. elect by clicking on it 4 3 4 Processing 1 Click on the Process tab in the TopSpin Menu bar Figure 4 11 E Start Publish Mange Proc Spectrum v Adjust Phase v Calib Axis v R Pick Peaks v J Integrate v Advanced v m cicking on Figure 4 12 Acquire Process Analyse View Spectrum has no imaginary part MC2 F1 OF PH maod F1 2mc Could not phase real spectrum 44 B7170 00 01 NOTE This executes a standard processing program proc2 The message shown in Figure 4 12 pops up in case of a magnitude 2D experiment and the apk2d option is enabled To configure the processing program follow the steps below 3 Click on the down arrow inside the Proc Spectrum button Figure 4 13 Process F2 F1 Axis xfb Process Only F2 Axis xf2 Process Only F1 Axis xf1 Symmetrize Spectrum sym Start Automation AU Program xaup 3 Select Configure Standard Processing by clicking on it Figure 4 14 ds prac zd Press Execute to process the current dataset Press Save to just change the processing options Changed options will be effective when pressing the one click Proc Spectrum button Fourier Transform xfb s Auto Phasing apk2d x Auto Baseline Correction F2 abs2 s Auto Baseline Correction F1 abs1 Plot autoplot s Warn if processed data exist
73. er of cycle 3 130 B7170_00_01 Figure 6 43 Shapelool shape hmbc exp 2 1 C data3 0 MIE SS MAM i Mold ass E QUE 4 Sinc O Parameters E Qn A mil n PAGOSA expefimenl sss cec ette ettet ttem Fats 20 mM Gramicidin S i DMSCO a6 sm aneren erui mikn 256 Size of Shape 3 0 Number of Cycles F1 ppm 100 150 F2 ppm 20 40 60 80 50 150 250 a 200 400 600 800 points 5 Click on E Figure 6 44 Gradient Fraction 6 Select Shape by clicking on it Figure 6 45 EN Set shape parameters Sines 256 Title 90 0 Flip Angle Excitation Type of rotation 7 Make the following changes Title Sinc3 256 Flip Angle 90 Type of Rotation Exitation D 1 0 00 01 131 8 Click on Figure 6 46 ee Save as Destination Dir C Bruker TopSpin3 0 6 44 expistaninmrlistsiwavewuser Mew Name 9 Make the following changes Destination Dir lt TOPSPIN HOME gt exp stan nmr lists wave user New Name Sinc3 256 10 Click on 11 In the main menu click on Analysis Figure 6 4712 Calculate Bandwidth for Inversion analyze bandw i Calculate Bandwidth far Refocusing My analyze bandwery special Bandwidth Calculations n o Calculate gammaB max analyze calcb1ma om Calculate gammaB imax for Adiabatic shapes analyze calch1adi
74. eriment 1 Click on the Start tab in the TopSpin Menu bar Figure 7 2 Acquire Process Analyse Publish e L Create Dataset a Find Dataset J Open Dataset Paste Dataset E Read Pars View Manage 2 Select by clicking on it is 3 Enter the following information in to the New window Figure 7 1 amp New Ex Prepare for a new experiment by creating a new data set and initializing its NMR parameters according to the selected experiment type For multi receiver experiments several datasets are created Please define the number of receivers in the box below NAME 1d solvent suppression EXPNO Mo PROCNO 4 DIR CABruker TOPSPIN USER nmrsu solvent H20 D20 Experiment Dirs C Bruker TOPSPIN exp stan nmr par Experiment PROTON TITLE p suppression experiment 1 Receivers 1 2 8 NOTE The directory DIR is specific to how the data are stored and therefore may show different entries as the one in Figure 3 4 above Click on the down arrow button to browse for a specific directory 4 Click on 5 Click on the Aquire tab in the TopSpin menu bar Figure 7 3 3 Stat Acquire Process Analyse Publish View Manage o Sample Lock V Tune v IE Spin v amp Shims A Prosol v Gain D Gow Options v 6 Select Aj Sample by clicking on it 138 B7170_00_01 Figure 7 4 Turn off sample lift air ij
75. figure this program or select the right options click on the down arrow inside the Proc Spectrum button Since this is a phase sensitive experiment the phase correction apk2d have to be enabled Acquire Process Analyse View Manage D 1 0 00 01 o3 Figure 4 29 cosymgf exp 1 1 C data3 0 PEE 4 5 5 Spectrum ProcPars AcquPars Title PulseProg Peaks Integrals Sample Structure Plot Fia Acqu 2D gradient DQF COSY experiment E 30 mg Menthyl Anthraniate in DMSO6 c H B N wo 6 4 2 F2 ppm Plotting 1 Use the 4 buttons to adjust for a suitable contour level 2 Click on the Publish tab in the TopSpin Menu bar Figure 4 30 E Process Analyse Publish View Manage o Start ign Copy Printy jPlotLayoute PDF v E Mail Acquire 6 Click on 7 Select the Plot tab by clicking on it 54 B 7170 00 01 Figure 4 31 cosymgf_exp 1 1 C idata3 0 Spectrum ProcPars AcquPars Title PulseProg Peaks Integrals Sample Structure Plot Fid Acqu 0a M Al 2D_hom wp 2D gradient DQF COSY experiment 30 mg Menthyl Anthranilate in DMS0 d De rares x amp t 80 75 70 65 60 55 50 45 40 35 30 25 20 15 10 05 i NOTE If desired any changes can be administered by clicking on the icon to open the Plot Editor 8 Click on the M to plot the spectrum B7170_00_0
76. followed by the 1H signal detection The signal has maximum intensity if p1 is a 90 pulse and 2 Nulls at a B7170_00_01 9 180 and 360 pulse The Methanol signal region from 3 5ppm to 2 8ppm is used for this experiment 2 3 1 Preparation experiment 1 Run a 1D Proton spectrum of Urea Methanol in DMSO d6 following the instructions in 1 D Proton experiment Chapter 3 Figure 2 4 No acquisition running C data pz nmr pulse_calibration 1 pdata 1 Spectrum ProcPars AcquPars Title PulseProg Peaks integrats Sample Structure Fia Acqu Proton 90 degree pulse calibration 0 1M 15 Urea and 0 1M 13C CH3OH in DMSO d6 re i 4 3 ppm 2 3 2 Parameter set up 1 Click on the Aquire tab in the TopSpin menu bar Figure 2 5 E Start Acquire Process Analyse Publish View Manage o JJ Sample v ie Lock V Tune v db spine S Shime fVProso Gainv gt Gow Options v 2 In the command line type wrpa and hit Enter 10 B7170_00_01 Figure 2 6 Copy data set If NAME ends with top the destination will be a 1 file dataset no expno procno required Please specify destination NAME EXPNO PROCNO DIR 3 Change NAME p90 proton 4 Click on 5 In the command line type re and hit Enter Figure 2 7 r Options Display data in same window Q Display data in new window NAME
77. g TopSpin and should be used as a guide through the set up process for some experiments The success of running the experiments in this manual is under the assumption that all parameters have been entered in to the prosol table 1 2 Disclaimer This guide should only be used for its intended purpose as described in this manual Use of the manual for any purpose other than that for which it is intended is taken only at the users own risk and invalidates any and all manufacturer warranties Some parameter values especially power levels suggested in this manual may not be suitable for all systems e g Cryo probes and could cause damage to the unit There fore only persons trained in the operation of the AVANCE systems should operate the unit D 1 0 00 01 7 B7170 00 01 2 Pulse calibration 2 1 Introduction This chapter describes the pulse calibration procedures for determine the 90 transmit ter pulse of the nuclei 1H 13C and 15N gt Warning It is always a good practice to obtain spectra with the power check turned on if your system has been cortabed 2 2 Sample Mixture 0 1M each of 15N enriched Urea Figure 2 1 and 13C enriched Methanol Fig ure 2 2 in DMSO d6 Figure 2 1 15 A 5 Figure 2 2 13CH3 OH 2 5 1H 90 transmitter pulse Figure 2 3 I The pulse program zg is used to determine the 1H 90 transmitter pulse The sequence consists of one channel f1 with a recycle delay d1 a 1H pulse p1
78. gure 5 10 EM Parameter Sets rpar File Options Help Source CABrukerTopSpin3 0 b 44 expistamnmnpar Find file names ystring P re Class Any vi Dim show Recommended SubType Reset Filters SELCOGP SELNOGP SELZG1H D sELU ROESY NOTE Enter SEL in to the Find file names window and hit Enter to display all selec tive parameter sets shown in figure 5 10 3 Select SELCOGP 4 Click on 5 Select the acqu proc and outd parameter options only 6 Click on the down arrow next to the Keep the following parameter window 7 Select P1 O1 PLW1 from the pull down menu Figure 5 11 source Parameter Set C BrukenTopSpin3 D b 44XexpstannmnpanSELNOGP Destination Data Set sel noesy 1 1 C datas 0 1 Select the desired file types of the source parameter set 2 Press OK to copy them to the destination data set acqu prac Keep the following parameters Piovpwi rr 8 Click on 9 Select the Title tab by clicking on it 74 B 7170 00 01 10 Make the following changes 1 D Selective COSY experiment 30 mg Menthy Anthranilate in DMSO d6 11 Click on to store the title 12 Select the Spectrum tab by clicking on it 13 Click on the Aquire tab in the TopSpin menu bar Figure 5 12 E Start Acquire Process Analyse Publish View Manage o Y Sample Lock V Tune v 4b Spine S Shim v EL Prosol V Gain v gt
79. h substance is available a typical gradient COSY experiment with 128 time increments can be recorded in 5 minutes Section 4 2 describes the acquisition and processing of a two dimensional 1H gradient COSY The standard Bruker parameter set is COSYGPSW and includes the pulse sequence cosygpppaf shown in Figure 3 2 It consists of the recycling delay two radio frequency RF pulses separated by the increment delay DO and the acquisition time during which the signal is recorded Both pulses have a 90 degrees angle Two gradient pulses are applied before and after the second pulse in the sequence Purge pulses are applied before di Figure 4 2 cosygpppaf The time intervals depicted in the pulse sequence diagrams are not drawn to scale For example d1 is typically a few seconds while p1 is typically a few microseconds in length 4 3 1 Preparation experiment 1 Run a 1D Proton spectrum following the instructions in 1 D Proton experiment Chapter 3 40 B7170 00 01 Figure 4 3 Acquisition finished C data3 0 proton_exp 1 pdata 1 Spectrum ProcPars AcquPars Title PulseProg Peaks Integrals Sample Structure Plot Fid Acqui T 1 Proton experiment 30 mg Menthvi Anthranilate in DMSO d6 w wo N o 15 10 5 0 ppm 4 3 2 Setting up the COSY experiment 1 Click on the Start tab in the TopSpin Menu bar Figure 4 4 E Publish o Create Dataset
80. hranilate in DMSO d6 is used for the experiment in this chapter Figure 3 1 H H ri Eo ar rar AE 8 OQ Ms ad Ru ed ims SS GESTAM Af Le d 1 E Eo Qu HT n 3 2 1 D Proton experiment 3 2 1 Introduction Section 3 2 describes the acquisition and processing of a one dimensional 1H NMR spectrum using the standard Bruker parameter set PROTON The pulse sequence zg30 Figure 3 2 consists of the recycling delay the radio frequency RF pulse and the acquisition time during which the signal is recorded The pulse angle is shown to be 30 degrees The two parameters D1 and P1 correspond to the length of the recycle delay and the length of the 90 degree RF pulse respectively Figure 3 2 The time intervals depicted in the pulse sequence diagrams are not drawn to scale For D 1 0 00 01 33 example d1 is typically a few seconds while p1 is typically a few microseconds in length 3 2 2 Experiment setup 1 Click on the Start tab in the TopSpin Menu bar Figure 3 3 Acquire Process Analyse Publish View Manage Create Dataset Find Dataset Open Dataset Paste Dataset Read Pars E by clicking on it 3 Enter the following information in to the New window Figure 3 4 Prepare for a new experiment by creating a new data set and initializing its NMR parameters according to the selected experiment type For multi receiver experiments several datasets are created Please define th
81. in 2295 29 Q 2345 29 j o Save Save all Restore Restore all Stop 0 2 04 06 08 1 0 1 2 1 4 s 02 04 06 O8 1 0 12 1 4 s 6 Change the O1 value by clicking just below or above the adjust slider NOTE For smaller changes adjust the sensitivity to smaller values 7 Observe the fid area in the Acquisition information window for a smaller integration value and the FID to become a single line 144 B7170 00 01 Figure 7 15 iia Soe Spectrum ProcPars AcquPars Title PulseProg Peaks Integrals Sample Structure Fid Acqu EC dre ae oS CorPhase Delay Offset Receiver Gain solvent suppression experiment 9 o1 2mM Raffinose in 9096 H20 10 D2O sensitivity CJ 001 9 8 adjust max 2348 23 Offset ie O1 Hz i i D o min 2347 23 234778 o Save Save all Restore Restore all Stop 02 04 06 08 10 12 14 s 02 04 06 08 10 12 14 s 8 Click on 89 Click on Figure 7 16 10 Click on 7 3 3 Acquisition 1 Select Gains by clicking on it NOTE To adjust rg manually click on the down arrow inside the Gain icon 2 Select Ba by clicking on it 7 3 4 Processing 1 Process and phase correct the spectrum D 1 0 00 01 145 Figure 7 17 Spectrum M No acquisition running C Bruker TOPSPIN datainmrsuinmr td solvent suppression 2ipdata 1 2 integr
82. indow D ae EE 4 90 4 85 4 80 4 75 4 70 ppm 9 Move the cursor line in to the center of the multiplet 10 Click the left mouse button to set the frequency D 1 0 00 01 91 Figure 5 42 EM 01 02 03 Define SFO1 01 frequencies SFO1 MHz 300 131438 O1 2 3 Hz 1437 87 11 Click on o 5 7 2 Calculating the selective pulse width and power level NOTE In this example the shaped pulse width and power level are determine using the Calculate Bandwidth option in the shaped tool program Other method of calculating the pulse width and power level can be used 1 Click on rvje to start distance measurement 2 Position the cursor line at the left side of the peak up 1 5 from the baseline 3 Click the left mouse button and drag the cursor line to the right side of the multiplet up 1 5 from the baseline Figure 5 1 sel tocsy 1 1 C idata3 0 Distance measurement left click at first point and move mouse right click to exit Distance 0 0986 ppm 29 6034 Hz Distance 0 0986 ppm 29 6034 Hz 4 80 4 75 470 ppm 4 Write down the value in Hz for the distance between the two cursor lines e g 29 6 92 B 7170 00 01 5 Click on the Start tab in the TopSpin Menu bar Figure 5 43 Analyse Publish View Manage o k Create Dataset L Find Dataset J Open Dataset E Paste Dataset E Read Pars Acquire Process
83. ion of undesired artifacts An important decrease in the total acquisition times for sufficiently concentrated samples The obtaining of higher quality spectra with an important reduction in T1 noise An efficient suppression of undesired signals such as for instance the intense sol vent signal in H2O solution and the 1H 12C 1H 14N magnetization in proton detected heteronuclear experiments at natural abundance In these inverse experi ments the starting BIRD cluster or spin lock pulse are no longer needed A much easier data processing and therefore more accurate spectral analysis A decrease of dynamic range limitation Figure 3 34 shows the gradient HMBC pulse sequence and the instructions below will guide you through the set up of the experiment 58 B 7170 00 01 Figure 4 34 hmbcgplpnaqf Gl G2 G3 4 7 1 Preparation experiment 1 Run a 1D Proton spectrum following the instructions in 1 D Proton experiment Chapter 3 Figure 4 35 Acquisition finished C data3 0 proton_exp 1 pdata 1 Spectrum ProcPars AcquPars Title PulseProg Peaks Integrats Sample Structure Plot Fid Acqu rel f Proton experiment 30 mg Menthyl Anthranilate in DMSO d6 15 10 5 0 ppm NOTE The reference spectrum is necessary to adjust the spectral limits of the sweep width in the F2 dimension and to use it for the projection The HMBCGP parameter set has a default sw
84. irectory 4 Click on 5 Click on the Aquire tab in the TopSpin menu bar Figure 4 38 Start Acquire i Sample v BE Lock V Tunew db Spin amp Shimv EL Prosol SetLimits i Gainv D Gow 6 Select db spins by clicking on it Process Analyse Publish View Manage o Options v 60 B 7170 00 01 Figure 4 39 Turn sample rotation on ro on Change sample rotation rate ro MAS Pneumatic Unit masdisp 7 Select ro off by clicking on it NOTE 2 D experiments should be run non spinning 8 Select Al Prosol by clicking on it NOTE This will load the pulse width and power levels in to the parameter set uem 9 Select Ei SetLimits by clicking on it i i Figure 4 40 HA setlimits Close this dialog box after setting frequencies o 1 Open 1D dataset from Browser 2 Zoom into region of interest 3 Click OK to set frequencies and return to original dataset 10 To open the 1D Proton spectrum right click on the dataset name in the browser win dow e g proton exp 1 and select Display or click and hold the left mouse button for dragging the 1D Proton dataset in to the spectrum window 11 Expand the spectrum to display all peaks leaving ca 0 2 ppm of baseline on either side of the spectrum NOTE The solvent peak may be excluded if it falls outside of the region of interest Dig ital filtering however is only applied i
85. ith excellent selectivity Figure 7 31 pil G21 G22 G23 G24 7 8 1 Sample 2mg Sucrose in Acetonitril and D2O 7 8 2 Preparation experiment 1 Run a 1D Proton spectrum following the instructions in 5 2 Preparation experiment in this Chapter B7170_00_01 155 Figure 7 32 Spectrum ProcPars AcquPars Title PulseProg Peaks Integrals Sample Structure Fid Acqu rel 14 7 8 3 Frequency list set up 1 Type wrpa 2 on the command line 2 Type re 2 on the command line 3 Expand the spectrum to include both peaks for suppression Figure 7 33 4 Click on att 156 B7170_00_01 Figure 7 34 FQILIST wetlist sort frequency list Sort frequencies in descending order O Sort frequencies in ascending order Don t sort frequencies 5 Select FQ1LIST and type a frequency list name e g wetlist1 6 Enable Don t sort frequencies 7 Click on 8 Move the cursor line to the center of the Water peak at 4 7 ppm and click the left mouse button 9 Move the cursor line to the center of the Acetonitril peak at 2 3 ppm and click the left mouse button Figure 7 35 M 1d solvsupr wet 2 1 C BrukeriTOPSPIN nmrsu wa 4 2 031 ppm 1016 006 Hz rei DEFINE LIST OF FREQUENCIES Define Left click inside data window Right click on defined frequency Save Click right mouse button 14 2346 67 Hz 6 8
86. mation in the Spectra 82 B7170_00_01 Display Preferences window 11 Type O1 Figure 5 26 12 Write down the current value e g 1853 43 13 Calculate the difference of step 9 and 11 e g 413 68 14 Click on l NOTE If the signal is down field of O1 a positive value must be entered for spoff If the signal is up field of O1 spoff will have a negative value 5 5 4 Setting up the Selective NOESY 1 Click on the Start tab in the TopSpin Menu bar Figure 5 27 Publish o Create Dataset a Find Dataset Open Dataset r Paste Dataset E Read Pars Process Analyse View Manage Acquire 2 Select Figure 5 28 EM Parameter Sets rpar File Options Help Source C Bruker TopSpin3 0 b 44 exp stan nmripar Find file names string Class Any v Dim Show Recommended subType Reset Filters SELU 1H SELZG1H D 1 0 00 01 83 NOTE Enter SEL in to the Find file names window and hit Enter to display all selec tive parameter sets shown in figure 5 28 3 Select SELNOGP 4 Click on 5 Select the acqu proc and outd parameter options only 6 Click on the down arrow next to the Keep the following parameter window 7 Select P1 O1 PLW1 from the pull down menu Figure 5 29 source Parameter Set C Bruker Topspins 0 b 44 exp staninmripansSELNOGP Destination Data Set
87. ment is an alternative ver sion of the COSY experiment in which a multiple quantum filter is inserted to allow the detection of signals from all coupled spin systems but suppresses signals arising of lower coherence levels Thus a COSY with a double quantum filter 2D COSY DQF experiment experiment efficiently suppress single quantum coherency from singlet uncoupled signals as for instance those of methyl groups or solvents The COSY DQF experiment can be performed in magnitude or phase sensitive modes by selecting the appropriate phase programs and transform algorithm However phase sensitive data is usually recommended In spectrometers equipped with gradient technology gradient based COSY versions are highly recommended The ge 2D COSY MQF experiment allows to obtain a 2D COSY MQF spectrum with a single scan per t1 increment provided that the S N ratio is adequate The main advan tage of such approach is the large reduction in the total acquisition time compared with a conventional phase cycled 2D COSY MFQ experiment Magnitude mode or phase sen sitive data is obtained depending of the selected pulse sequence and acquisition pro cessing procedure The COSY MQF experiment permits to trace out through bond proton proton connectivity via the homo nuclear JE coupling constant Figure 4 18 Figure 4 19 D 1 0 00 01 49 4 5 1 Preparation experiment 4 5 2 1 Run a 1D Proton spectrum following the instructions in 1 D P
88. n 0 1M 15 Urea and 0 1M 13C CH3OH in DMSO d6 9 Click on ie to set the sweep width and the O1 frequency of the displayed region Figure 2 33 amp x New setting of SW SFO1 from current region G SW 0 5000 ppm SWH 150 060 Hz O1 1605 70 Hz SFO1 300 1316057 MHz 10 Click on 11 Select the AcquPars tab by clicking on it 12 Make the following changes PULPROG decp90 TD 4K NS 1 DS 0 13 Click on Edit next to NUC2 in the Nucleus2 section of the AcquPars Figure 2 34 Y Nucleus 2 NUC2 off E dit 2nd nucleus O2 Hz 1853 43 Frequency offset of 2nd nucleus O2P ppm 6 1 75 Frequency offset of 2nd nucleus SFO2 MHz 300 1318534 Frequency of 2nd nucleus BF2 MHz 300 1300000 Basic frequency of 2nd nucleus D 1 0 00 01 27 2 7 1 1 Two channel system 14 Select 15N for NUC2 Figure 2 35 es Edit Spectrometer Parameter frequency logical channel amplifier preamplifier BF1 300 13 MHz NUC1 SFO1 300 131853 MHz 1H 2H 1H OFS1 1653 43 Hz 1H 2H 2H XBB18F 2HS BF2 300 13 MHz SFO2 300 131853 MHZ OFS2 fi 853 43 Hz Cable wiring possible RF routing e cortab available ft probe in Save Switch F 1 F2 Switch F 1 F3 Add a logical channel Remove a logical channel Default Info Param Close 15 Click on Default to set the routing Figure 2 36 le Edit Spectrometer Parameter frequency logical channel amplifier
89. n AU Program xaup 3 Select Configure Standard Processing by clicking on it Figure 4 49 ds prac zd Press Execute to process the current dataset Press Save to just change the processing options Changed options will be effective when pressing the one click Proc Spectrum button Fourier Transform xfb s Auto Phasing apk2d x Auto Baseline Correction F2 abs2 s Auto Baseline Correction F1 abs1 Plot autoplot LAYOUT 2D hom xwp w s Warn if processed data exist NOTE To avoid the message shown in Figure 3 47 the option Auto Phasing apk2d may be disabled for magnitude like 2D experiment D 1 0 00 01 65 4 7 5 Figure 4 50 hmbc exp 1 1 C idata3 0 2D gradient HMBC experiment E 30 mg Menthy Anthranilate in DMSO d6 8 8 8 6 4 2 F2 ppm Plotting 1 Use the 4 buttons to adjust for a suitable contour level 2 Click on the Publish tab in the TopSpin Menu bar Figure 4 51 4 Start Acquire Process Analyse Publish View Manage o Copy amp Print PlotLayoutv D PDF E Mail 6 Click on 7 Select the Plot tab by clicking on it 66 B7170 00 01 Figure 4 52 hmbc_exp 1 1 C data3 0 Spectrum ProcPars AcquPars Title PulseProg Peaks Integrals Sample Structure Plot Fid Acqu O D e M uo inem 2D g
90. n F2 and the solvent peak is folding in F1 D 1 0 00 01 61 Figure 4 41 proton_exp 1 1 C data3 0 Spectrum ProcPars AcquPars Title PuiseProg Peaks Integrals Sample Structure Piot Fid Acqu f Proton experiment 30 mg Menithy Anthranilate in DMSO d6 6 4 2 ppm 12 Click on to assign the new limit Figure 4 42 1H spectral limits capied far F1 and F2 dimensions cav f 9997 nnm TP 4 024 ppm 13 Click on Ci NOTE The display changes back to the 2D data set To set the limits in the F1 dimen sion follow the steps below 14 Select i setLimits by clicking on it 62 B7170 00 01 Figure 4 43 ee setlimits Close this dialog box after setting frequencies o 1 Open 1D dataset from Browser 2 Zoom into region of interest 3 Click OK to set frequencies and return to original dataset 15 To open the 1D C13DEPT spectrum right click on the dataset name in the browser window e g Carbon exp 1 and select Display or click and hold the left mouse button for dragging the 1D C13DEPT dataset in to the spectrum window 16 Expand the spectrum to display all peaks leaving ca 2 ppm of baseline on either side of the spectrum NOTE The solvent peak may be excluded if it falls outside of the region of interest Dig ital filtering however is only applied in F2 and the solvent peak is folding in F1 Figure 4 44 Carbon exp 1 1 C idat
91. n arrow button to browse for a specific directory 4 Click on 5 Click on the Aquire tab in the TopSpin menu bar Figure 4 23 E Start Acquire Process Analyse Publish View Manage oe Y Samples PE Lock V Tune dh Spin 5 Shim v e Prosol SetLimits Gain v Gor 6 Select db spins by clicking on it Figure 4 24 Options Turm sample rotation on ro on Change sample rotation rate ro MAS Pneumatic Unit masdisp 7 Select ro off by clicking on it NOTE 2 D experiments should be run non spinning 8 Select AV Prosol by clicking on it D 1 0 00 01 91 NOTE This will load the pulse width and power levels in to the parameter set 9 Select _ by clicking on it Figure 4 25 ds sethimits Close this dialog box after setting frequencies o 1 Open 1D dataset from Browser 2 Zoom into region of interest 3 Click OK to set frequencies and return to original dataset 10 To open the 1D Proton spectrum right click on the dataset name in the browser win dow e g proton exp 1 and select Display or click and hold the left mouse button for dragging the 1D Proton dataset in to the spectrum window 11 Expand the spectrum to display all peaks leaving ca 0 2 ppm of baseline on either side of the spectrum NOTE The solvent peak may be excluded if it falls outside of the region of interest Dig ital fil
92. ne on either side of the spectrum NOTE The solvent peak may be excluded if it falls outside of the region of interest Dig ital filtering however is only applied in F2 and the solvent peak is folding in F1 B7170 00 01 113 Figure 6 10 proton exp 1 1 C idata3 0 Spectrum ProcPars AcquPars Title PuiseProg Peaks Integrals Sample Structure Piot Fid Acqu f Proton experiment 30 mg Menithy Anthranilate in DMSO d6 6 4 2 ppm 12 Click on to assign the new limit Figure 6 11 1H spectral limits capied far F1 and F2 dimensions cav f 9997 nnm TP 4 024 ppm 13 Click on Ci NOTE The display changes back to the 2D data set 6 1 6 Acquisition 1 Select 2 Select by clicking on it Gains by clicking on it 114 B7170 00 01 6 1 7 Processing 1 Click on the Process tab in the TopSpin Menu bar Figure 6 12 E Start Analyse Publish o Proc Spectrum v w Adjust Phase 7 Calib Axis v t Pick Peaks v J integrate Advanced Acquire Process View Manage NOTE The steps below will guide you through a manually phase correct a phase sensi tive 2 D spectrum 2 In the command line type rser 1 read in the first increment 3 In the command line type qsin executing the window function 4 In the command line type ft 5 Click on 6 Adjust the phase manually NOTE The spectrum
93. ng C data pz nmr pulse_calibration 1 pdata 1 Spectrum ProcPars AcquPars Title PulseProg Peaks integrals Sample Structure Fia Acqu Proton 90 degree pulse calibration 0 1M 15 Urea and 0 1M 13C CH3OH in DMSO d6 9 Click on de to set the sweep width and the O1 frequency of the displayed region Figure 2 21 amp x New setting of SW SFO1 from current region G SW 0 7000 ppm SWH 210 080 Hz O1 945 40 Hz SFO1 300 1309454 MHZ 10 Click on 11 Select the AcquPars tab by clicking on it 12 Make the following changes PULPROG decp90 TD 4K NS 1 DS 0 13 Click on Edit next to NUC2 in the Nucleus2 section of the AcquPars Figure 2 22 Y Nucleus 2 NUC2 off E dit 2nd nucleus O2 Hz 1853 43 Frequency offset of 2nd nucleus O2P ppm 6 1 75 Frequency offset of 2nd nucleus SFO2 MHz 300 1318534 Frequency of 2nd nucleus BF2 MHz 300 1300000 Basic frequency of 2nd nucleus 20 B 7170 00 01 14 Select 13C for NUC2 Figure 2 23 ica Edit Spectrometer Parameter frequency BF1 300 13 MHz sFO1 300 130945 MHz OFS1 945 4 Hz BF2 300 13 MHZ SFO2 300 13 MHZ OFS2 0 0 Hz cable wiring possible RF routing cortab available preamplifier 1H 2H 1H 1H 2H 2H XBB19F 2HS logical channel amplifier NUC1 F1 SGU1 1H 3 ft probe in Save Switch F1 F2 Switch F1 F3 Add a logical channel Rem
94. nsform ft Auto Phasing apk Set Spectrum Reference sref Auto Baseline Correction abs C Plot autoplot LAYOUT 1D Hxwp x L Warn if processed data exist O 5 Click on 76 B 7170 00 01 Figure 5 17 Acquisition finished C data3 0 sel_cosy 1 pdata 1 Spectrum ProcPars AcquPars Title PulseProg Peaks Integrals Sample Structure Plot Fid Acqu ll IL L i AL 1 D Selective experiment Reference spectrum AN cit e Y 10 5 0 ppm 6 Expand the spectrum from 4 ppm to 0 5 ppm 7 Click on 8 Adjust the 0 order phase on the peak at 2 0 ppm to display a antiphase pattern Figure 5 18 Acquisition finished C data3 0 sel_cosy 1 pdata 1 A 0 1 R 90 90190 2 gt NEY 4 pivot 1 99 ppm Phase increment 0 025 phO 32 32 phl 0 00 1 D Selective experiment Reference spectrum rel 10 e 10 10 15 3 5 3 0 2 5 2 0 1 5 1 0 ppm 9 Click on Lii to store the phase value D 1 0 00 01 17 5 3 7 Plotting two spectra on to the same page 1 Display the selective COSY spectrum 2 Click on A to enter the Multiple display option 3 Drag the Reference spectrum in to the spectral window Figure 5 19 sel cosy 1 1 C data3 0 HEE RMT l E E Ei As Sets b AA TE sel cosy C data3 0 a 8 6 4 2 ppm NOTE To adjust the spectra for
95. om 100 ms to 800 ms 5 5 5 Acquisition 1 Select Boos by clicking on it 5 5 6 Processing 1 Click on the Process tab in the TopSpin Menu bar Figure 5 32 E Start Publish View Manage 2 JL Proc Spectrum 7 Adjust Phase x A Calib Axis v 912 Pick Peaks Integrate Advanced v 2 Click on the down arrow inside the Proc Spectrum button Acquire Process Analyse D 1 0 00 01 85 Figure 5 33 Window Multiplication wm Fourier Transform ft Start Automation AU Program xaup 3 Select Configure Standard Processing by clicking on it 4 Deselect the following options Auto Phasing apk Set Spectrum Reference sref Auto Baseline correction abs Warn if Processed data exist Figure 5 34 e proc 1 d Press Execute to process the current dataset Press Save to just change the processing options Changed options will be effective when pressing the one click Proc Spectrum button Exponential Multiply em LB HJ 0 1 Fourier Transform ft Auto Phasing apk Set Spectrum Reference sref CO Auto Baseline Correction abs C Plot autoplot LAYOUT 4 AD H Xwp v L Warn if processed data exist E 5 Click on 6 Expand the spectrum from 4 ppm to 0 5 ppm 7 Click on 8 To asure the correct phasing of the NOE peaks phase the signal at 4 8 ppm negativ
96. ove a logical channel Default Info Param Close 15 Click on Default to set the routing Figure 2 24 EM Edit Spectrometer Parameter frequency BF1 300 13 MHz SFO1 300 130945 MHz OFS1 945 4 Hz BF2 75467749 MHz SFO2 75 475295 MHz OFS2 7546 3 Hz cable wiring possible RF routing e cortab available logical channel amplifier preamplifier NUC1 Et SGU1 1H 2H 1H 1H 1H 2H 2H NUC2 XBB18F 2HS F2 SGU2 13C m settings I show receiver routing TM show RF routing I show power at probe in Info Param Clase Save Switch F 1 F2 Switch F 1 F3 Add a logical channel Remove a logical channel 16 Click on Cave 17 In the AcquPars make the following change O2 ppm 49 D1 10 CNST2 130 P3 3 18 Select 19 Select the ProcPar tab by clicking on it by clicking on it to read in the Prosol parameters 20 Make the following changes SI 2K 21 Select the Spectrum tab by clicking on it D 1 0 00 01 21 22 In the command line type wpar C13p90 urea all to store the parameter set for future use 2 5 3 Determine the 13C 90 decoupler pulse 1 Select V Tune amp by clicking on it 2 Select by clicking on it 3 Process and Phase correct the spectrum NOTE Phase the left doublet negative and the right doublet positive The Water peak at 3 3 ppm can be ignored and does not have to be in phase
97. phs 6 2 2 through 6 2 6 step 9 2 Select the AcquPars tab by clicking on it 3 Make the following changes PULPROG zgesgp P12 us 2000 SP1 dB calculate using the AU program pulse and subtract 6dB since this is a 180 pulse e g 44 5 SPNAM1 Squai00 1000 GPZ1 31 GPZ2 96 11 4 Select the Spectrum tab by clicking on it 7 7 2 Acquisition 1 Select T Gains by clicking on it NOTE To adjust rg manually click on the down arrow inside the Gain icon 2 Select Boo by clicking on it 7 7 3 Processing 1 Process and phase correct the spectrum D 1 0 00 01 153 Figure 7 29 M No acquisition running C Bruker TOPSPIN datainmrsu nmr 1d_solvent_suppression 5ipdata 1 solvent suppression se 2mM Ramnose in 90 H20 10 Spectrum rei 3 5 NOTE Figure 7 28 above shows the solvent suppressed 1 D spectrum of the Raffinose sample and Figure 7 29 below shows the 1 D spectrum of the Lysozyme sample Figure 7 30 Spectrum ProcPars AcquPars Tite PulseProg Peaks integrats Sample Structure Fia 154 B7170 00 01 7 8 1 D Solvent suppression with WET This pulse sequence uses a shaped selective pulse and pulse field gradients to sup press one or more solvent signals The option of carbon decoupling is available for sup pression of solvent signals with large C13 satellites It provides very efficient suppression w
98. plying some isotropic mixing sequence like MLEV WALTZ or DIPSI pulse trains This in phase transfer avoids possible cancellation when the coupling is poorly resolved Proton detection as usual Figure 5 38 5 7 1 Selective excitation region set up 5 7 1 1 On resonance NOTE Make sure that the SW is large enough to cover the entire Spectrum accounting for the position of O1 The shaped pulse is applied on resonance at the O1 position The power level and width of the excitation pulse have to be known and entered into the Prosol parameter table 1 In the command line type wrpa and hit Enter Figure 5 39 amp wrpa Copy data set If NAME ends with top the destination will be a 1 file dataset no expno pracna required Please specify destination NAME sel tocsy EXPNO 1 PROCNO DIR C datas 0 90 B7170_00_01 2 Change NAME sel tocsy 3 Click on 4 In the command line type re and hit Enter Figure 5 40 Options Display data in same window Display data in new window PROCNO 5 Change NAME sel tocsy 6 Click on 7 Expand peak at 4 8ppm 8 Click on iS to set the RF from cursor Figure 5 41 selective exp 2 1 C data3 0 1 D Selective experiment Reference spectrum 30 mg Menthy Anthranilate in DVISO d6 4 7909 ppm 1437 87 Hz 300 131438 MHz sET SF01 01 FREQUENCIES FROM CURSOR POSITION Define Left click inside data w
99. preamplifier BF1 300 13 MHz NUC1 SFO1 300 131606 MHz F1 SGU1 1H 2H 1H OFS1 1605 7 Hz 1H 1H 2H 2H BF2 30411909 MHz NUC2 XBB19F 2HS SFO2 30 419455 MHz F2 SGU2 OFS2 7546 3 Hz 15N cable wiring settings possible RF routing I show receiver routing TM show RF routing corab available Save Switch F1 F2 Switch F1 F3 Add a logical channel Remove a logical channel l j into Param Close 2 7 1 2 Three channel system 14 Select 15N for NUC2 28 B7170_00_01 Figure 2 37 s Edit Spectrometer Parameter frequency logical channel amplifier preamplifier BF1 399 87 NUC 1 SFO1 399 872469 SGU1 X 150 vv 1H 2H 1H OFS1 2469 36 1H 2H 2H SFO2 399 872469 scu2 OFS2 2469 36 X 300 W BF3 399 87 SFO3 399 872469 s SGUS 2H 20 W OFS3 2469 36 cable wiring possible RF routing cortab available v 15 Clickon Default to set the routing Figure 2 38 s Edit Spectrometer Parameter frequency logical channel amplifier preamplifier BF1 399 87 NUC1 SFO1 399 872469 sour J X 150 W 1H 2H 1H OFS1 2469 4 aH 2 1H 2H 2H BF2 399 87 NUC2 SFO2 399 87 OFS2 0 0 of ne Heow 60 vv XBB19F 2HS SGU2 xaoow BFS 40516475 NUCS SFO3 40 525975 OFS3 7499 8 15N v sous 2H20w 20 wv cable wiring rsettings possible RF routing C show receiver routing CI show RF routing e cortab av
100. pul offs Single Sine Modulation manipul sinm2 Single Cosine Modulation manipul cosm2 Modulation acc to Freq Sweep gt Power of Amplitude manipul power Scale Amplitude manipul scale Add constant Phase manipul addphase Time Reversal manipul trev Calc Shape from Excitation Region manipul region Add Shapes manipul addshapes Expand Shape manipul expand 6 Enable Beginning at Phase 0 ly gt 1z 7 Enable Reference O1from current Data Set 8 Enable Frequencies taken from Frequency List 9 Change Parameters Length of pulse usec 10000 Name of Frequency List wetlist1 Figure 7 42 amp Manipulate command offs Alignment Beginning at Phase D hv Izl O Phase 0 at Middle of Shape ly gt ly 15 17 Ending at Phase Iz hl Reference Frequency O Na Reference Frequency specified Reference 01 from current Data Set O Reference First Frequency in List rGptians Frequencies taken from Frequency List With additional Phase Setting With additional Scaling Parameters 10000 Length of pulse usec wetlist1 Mame of Frequency List 10 Click on B7170 00 01 161 Figure 7 43 amp Manipulate command offs Reference 2001 D Reference Frequency Frequency List Diff to Reference 2346 67 345 63 1160 10 840 94 11 Click on Figure 7 44 M ShapeToo 1d
101. radient HMBC experiment 30 mg Menthyl Anthranilate in DMS0 d nai Fix o 75 70 65 60 55 50 45 40 35 30 25 20 15 10 O5 Dy NOTE If desired any changes can be administered by clicking on the icon to open the Plot Editor 8 Click on the M to plot the spectrum B7170 00 01 67 4 8 Observations 68 B7170 00 01 5 1 D experiments using shaped pulses 5 1 Introduction Selective homonuclear 1D experiments usually start from the selective H excitation of a given resonance followed by a mixing process When PFG s are available the SPFGE scheme is highly recommended as a selective excitation scheme The SPFGE or Single Pulsed Field Gradient Echo scheme is a single echo experiment in which the central selective 180 degree pulse is flanked by two gradient pulses It is used for efficient selective excitation purposes Figure 5 1 Select ve 8L Selective 1D experiments can be easily derived by adding the corresponding mixing pro cess between the SPFGE block and the acquisition period NOTE To run this experiment the instrument has to be equipped with the hardware to do Shaped Pulses and Gradients Three different ways to run this experiment are dis cussed in this chapter and can also be applied to other selective experiments such as SELCOSY SELROESY and SELTOCSY 5 2 Sample A sample of 30mg Menthyl Anthranilate in DMSO d6 is used for all experiments in this chapter D 1 0 00 01 69
102. roton experiment Chapter 3 Figure 4 20 Acquisition finished C data3 0 proton_exp 1 pdata 1 Spectrum ProcPars AcquPars Title PulseProg Peaks Integrals Sample Structure Plot Fid Acqu f Proton experiment 30 mg Menthy Anthranilate in DMSO d6 15 10 5 0 ppm Setting up the MFQ COSY experiment 1 Click on the Start tab in the TopSpin Menu bar Figure 4 21 Publish o 5 Create Dataset a Find Dataset y Open Dataset J Paste Dataset E Read Pars 2 Selec reale Dalasel by clicking on i 3 Enter the following information in to the New window Acquire Process Analyse View Manage 50 B 7170 00 01 Figure 4 22 Prepare for a new experiment by creating a new data set and initializing its NMR parameters according to the selected experiment type For multi receiver experiments several datasets are created Please define the number of receivers in the box below NAME cosymqf exp EXPNO PROCNO DIR C data3 0 Solvent Experiment Dirs C Bruker TopSpin3 0 b 44 exp stan nmripar Experiment COSYGPDFPHSW TITLE D gradient DQF COSY experiment 30 mg Menthyl Anthranilate in DMSO d6 Show new dataset in new window a Receivers 1 2 16 NOTE The directory DIR is specific to how the data are stored and therefore may show different entries as the one in Figure 3 22 above Click on the dow
103. rsion analyze bancdwzi Calculate Bandwidth for Refocusing My analyze bandwery special Bandwidth Calculations d Calculate qammaB1max analyze calch1rma Calculate qammab1max for Adiabatic shapes analyze calcbladia Calculate Bloch Siegert Shift analyze bsieqerts t I Calculate average Power Level analyze calcpas Integrate Adiabatic Shape analyze integradia Integrate and compare to Reference analyze integandcamp Simulation analyze simulate 19 Select Integrate Shape analyse integr3 by clicking on it D 1 0 00 01 133 Figure 6 51 Since r integra f 7429 3 Length of pulse usec 30 Total rotation degree Iu 10 5 80 deg hard pulse usec rResults 0 17752 integ Ratio comp to square on res 15 01507 Corresponding difference dE 41 98010 Change of power level dE update parameters 20 Make the following changes Length of pulse usec value from in 4 3 7 step 14 e g 7429 3 21 Press the Enter key Total rotation degree 90 22 Press the Enter key 90 deg Hard pulse usec value from in 4 3 6 step 8 e g 10 5 21 Press the Enter key 22 Write down the change of power level dB value e g 41 9801 23 Click on to close the Shape Tool window 6 3 8 Setting up the acquisition parameters 1 Select th
104. s The spectrometer acquires and processes 20 spectra with incrementing the parameter p1 from 2 us by 2 us to a final value of 40 us For each of the 20 spectra only the spectral region defined above is plotted and all the spectra are plotted side by side in the file pulse calibration 2 999 as shown in Figure 2 13 14 B7170 00 01 Figure 2 13 No acquisition running C data pz nmr pulse_calibration 1 pdata 999 Spectrum ProcPars AcquPars Tite PulseProg Peaks Integrals Sample Structure Fia Acqu poptau for of finished POSMA at experiment 6 p1f1 120000 NEXP 20 7 Select the Title tab by clicking on it Figure 2 14 Ho acquisition running C idata pzinmripulse_calibration 1 pdata Spectrum Procpars AcquPars Title PulseProg Peaks E H o E paptau for p1 finished FOSMAx at experiment p1 12 0000 NEXP 20 NOTE The POSMAX value of p1 is displayed in the title window which is the 90 degree pulse along with the experiment number and the NEXP value Write this value down To obtain a more accurate 90 degree pulse measurement follow the steps below 8 Close the popt setup window 9 In the command line type re 2 1 10 In the command line type p1 11 Enter the value which corresponds to a 360 degree pulse the second zero crossing in the popt spectrum which should be approximately 4 times the POSTMAX value 12 Select Boo by clicking onit 13 In the comm
105. s tab by clicking on it 3 Make the following change PULPROG p3919gp D19 s 0 00015 1 2 d where d distance to next null in Hz GPZ1 20 4 Select the Spectrum tab by clicking on it 7 6 2 Acquisition 1 Select Gains by clicking on it NOTE To adjust rg manually click on the down arrow inside the Gain icon 2 Select Bo by clicking on it B7170 00 01 151 7 6 3 Processing 1 Process and phase correct the spectrum Figure 7 26 t No acquisition running C Bruker TOPSPINidatainmrsuinmri1d_solvent_suppression 4ipdata 1 gt ler ex Spectrum ProcPars AcquPars Tite PulseProg Peaks Integrals Sample Structure Fid Acqu solvent suppression experiment T 2mM Rafihose in 90 H20 10 D20 wo N o 5 5 5 0 4 5 4 0 3 5 ppm NOTE Figure 7 25 above shows the solvent suppressed 1 D spectrum of the Raffinose sample and Figure 7 26 below shows the 1 D spectrum of the Lysozyme sample Figure 7 27 M 1d solvent suppression 10 1 C Bruker TOPSPIN nmrsu Sc Spectrum ProcPars AcquPars Title PulseProg Peaks Integrals Sample Structure Fid Acqu Lu N o 10 8 6 4 2 0 ppm 152 B7170 00 01 7 7 1 D Solvent suppression with excitation sculpting Figure 7 28 pi spi G amp Gl GlG2 Gz 7 7 1 Parameter se up 1 Follow the instructions in the paragra
106. shown in Figure 8 11 above 18 Click on File and select Save by clicking on it 19 Click on File and select Close by clicking on it 20 Select the Spectrum tab by clicking on it 8 2 3 Acquisition Gains by clicking on it by clicking on it 1 Select 2 Select 8 2 4 Processing 1 Click on the Process tab in the TopSpin Menu bar Figure 8 12 Hj Start Publish o Proc Spectrum v Adjust Phase v A Calib Axis v 3 Pick Peaks ff Integrate Advanced Acquire Process Analyse View Manage 1 In the command line type rser 10 2 In the command line type ef 3 Click on amp 4 Adjust the phase manually 172 B7170 00 01 Figure 8 13 TEMP 1 1 C data3 0 14 0 1 R 90 0800 I RE 4 pivot 0 88 ppm Phase increment 0 025 phO 82 25 phl 11 20 fid 10 from t1_exp 1 1 C datas 0 rel 12 10 8 6 4 rs ppm 5 Click on Li to store the 2 D phase values 6 Click on m NOTE The spectrum will go back to the unphased view since the phase correction val ues where stored only for the 2 D spectrum 7 Click on LI going back to the 2 D spectrum display 8 Click on the down arrow inside the button Figure 8 14 Configure Standard Processing broc4d Process F2 F1 Axis Gb Process Only F1 Axis xf1 aymimetrize Spectrum sym otat Automation AU Program xaup 9 Select
107. t UD dant mec een een eu nr 151 7 6 2 OCTO a de a te 151 7 6 3 xisec cio 152 7 7 1 D Solvent suppression with excitation sculpting 153 1511 PAG MOI XU 153 1 1 2 A COUI OR E autaen 153 1 1 9 EOS SA ae a ee ce ao demo nina 153 7 8 1 D Solvent suppression with WET ssssesessseeeeeeeeennn nnn 155 7 8 1 scc aes 155 7 8 2 Preparation experiment eeeeesseseeseeeeee eene nennen nnn nnn nnne nnn nnn 155 7 8 3 Frequency list set Up PR SN E m 156 7 8 4 Setting up the acquisition parameters 158 7 8 5 DPBIECLIV DUISES SOL UD 159 7 8 6 Running the experiment iii 164 7 8 7 OS SSS aca Secreta noi 165 SE 1 e iR ERE 167 8 1 Inifeeloi H 167 8 2 Proton Inversion Recovery T1 experiment 167 8 2 1 IMDS ITEE E A E O T E EA E 167 8 2 2 Preparation CXC NING Me oct 168 8 2 3 PAC CUIISIUION RETO TT m 172 8 2 4 FOC CS aM nn TT le D te ace ne T 172 8 2 5 LR CANCUN QUO mc M e 174 8 3 OS ONS east ea ee d aie 181 PAO CUI qe 183 6 B7170_00_01 1 Introduction 1 1 General This manual was written for AVANCE systems runnin
108. ter the following information in to the New window Figure 5 66 Prepare for a new experiment by creating a new data set and initializing its NMR parameters according to the selected experiment type For multi receiver experiments several datasets are created Please define the number of receivers in the box below NAME deptsp EXPNO 1 PROCNO 1 DIR CAdata3 0 v solvent DMSO v Experiment Dirs CABruker TopSpin3 0 b 44 exp staninmrpar de Experiment C13DEPT135 v TITLE 1 D Selective 13C DEPT135 experiment 30 mg Menthyl Anthranilate in DMSO d6 CI Show new dataset in new window 1 Receivers 1 2 16 NOTE The directory DIR is specific to how the data are stored and therefore may show different entries as the one in Figure 3 4 above Click on the down arrow button to browse for a specific directory 104 B7170_00_01 4 Click on 5 Click on the Aquire tab in the TopSpin menu bar Figure 5 67 3 Start Acquire Process Analyse Publish View Manage o Sample v Lock V Tune v db Spin gt Si Shim le Prosol i Gain D Gow Options v 6 Select Aj Sample by clicking on it Figure 5 68 Turn off sample lift air ij Control sample temperature edte 7 Select ej by clicking on it NOTE Wait till the sample lift air is turned on and remove any sample which may have been in the magnet 8 Place the sample on
109. tering however is only applied in F2 and the solvent peak is folding in F1 Figure 4 26 proton_exp 1 1 C data3 0 TE Spectrum ProcPars AcquPars Title PulseProg Peaks Integrals Sample Structure Plot Fid Acqui f Proton experiment 30 mg Menthy Anthranilate in DMSO d6 4 2 ppm 52 B 7170 00 01 12 Click on to assign the new limit Figure 4 27 1H Spectral limits copied for F1 and F2 dimensions SW 7 8997 ppm RC ES pum 13 Click on arr NOTE The display changes back to the 2D data set 4 5 3 Acquisition NOTE The first increment of the DQF COSY experiment has a low signals to noise ratio and the signals grow as the experiment is progressing It is therefore not advisable to use the automatic receiver gain adjustment rga since it adjusts the receiver gain on the first increment In this case an AU program au_zgcosy is available Executing this AU program changes the pulse program to zg and performs a rga and then changes back again to cosygpmfph and then starts the acquisition 1 Type au_zgcosy on the command line 4 5 4 Processing 1 Click on the Process tab in the TopSpin Menu bar Figure 4 28 E Start Publish o JL Proc Spectrum di Adjust Phase v A Calib Axis 912 Pick Peaks v f Integrate Advanced m cicking ont NOTE This executes a standard processing program proc2 To con
110. ters 11 Select the Spectrum tab by clicking on it 7 8 5 Selective pulses set up NOTE One shaped pulse is created and can be tailored to select for a single or multiple resonances 1 In the main menu click on Spectrometer and select Shape Tool or type stdisp in the command line 2 In the shape tool menu bar click on and select Shape D 1 0 00 01 159 Figure 7 39 d Shape Files File Options Help Source C Bruker TOPSPIN exp stan nmnlistswave M Search in names sen J Bip720 100 10 1 Bip720 50 20 1 Crp20 1 40 1 Crp32 1 5 20 2 Crp42 1 5 20 2 Crp80 0 5 20 1 Crp amp ocomp4 Eburp2 1000 JEburp2tr 1000 63 256 G4 266 Q o4tr25b6 Gausii1000 Gaust_180i 1000 Gausi_180r 1000 Gaus 270 1000 Gausi 901000 Mp Jpc3 4 1201000 Pe 4 90100 3 1000 O3Ca CaCO000 Q3 ma c68c1 1 Q5 1000 stron Reburp 1000 Rsnob 1000 Seduce 100 ISIN Squaramp 20 Tanhtan 300 50 250 Updaeifo o 0 0000000 MELOMANI NPOINTS Universal EXMODE 90 0 TOTROT 1 606000 BWFAC 0 566900 INTEGFAC MODE Edit Shape Parameters points 5 In the main menu click on Manipulate and select Phase Modulation acc to Offset Freq by clicking on it 160 B7170 00 01 Figure 7 41 Manipulate Options Window Help Phase Modulation acc to Offset Freq mani
111. tted depending on the experiment which produced the relaxation data Settings dialog provides all possibilities for Relaxation analysis adjustment 16 Read the message and then click on 17 Click on 18 Click on Calculation Start Calculating 178 B7170 00 01 Figure 8 28 Click on one of the following 2 icons to start the calculation look at the status line or the icon tool tip to locate the correct icon Do fitting for the given function for all peaks regions Do fitting for the given function for the current peak region Click an the PreviousNext icon to navigate peaks integrals YOU may now start calculation for it 19 Click on 20 Select Area for Fitting Type Figure 8 29 al xl Relaxationt1 exp 2 1 C pz 6e0606 No ks is a 07 co rFitting type T4 C ntensity Area NMR Relaxation Guide r Current Integral d of 4 Brief Report Region from 8 660 to 8 P 1 832e 000 0 9 969e 001 Peaks Ranges 4 ii 1 2435 1 SD 3 934e 003 Relaxation Window Extract Slice we Print Export 21 In the T1 data display window click on to calculate all regions D 1 0 00 01 179 Figure 8 30 Erief Report Peak 1 at 7 677 ppm TT 1 4275 Peak 2 at 7 234 ppm Tl 1 3125 Peak 3 at 6 653 ppm T1 23f f 55m Peak 4 at 4 784 ppm I1 641 00m Peak 5
112. um delay time All further data preparation will be done in respect to this Spectrum Slice Number ao 5 Select Slice Number 10 6 Click on Figure 8 20 TEMP 1 1 C data3 0 Spectrum ProcPars AcquPars Title PulseProg Peaks Integrals Sample Structure Plot Fia Acqul rel row 10 from t1 exp 1 1 C data3 0 12 10 8 6 4 2 ppm 7 Click on D 1 0 00 01 175 Figure 8 21 ee Define Peaks and or Intervals fou choose manual integration module you can define integration regions 4 Most intensive peaks within the intervals will be found automatically I you choose manual peak picking module you can define peaks without intervals or peaks within integration regions Manual Integration Manual Peak Picking 8 Click on Manual Integration Figure 8 22 Prepare relaxation data Integration module provides the integration regions definition When you export intervals to relaxation module the mast intensive peaks within the regions will be selected automatically Peaks can be positive and negative To import intervals from 2d data set click Exp button in relaxation module C Do not show this message again 9 Click on 10 Define the regions by clicking the left mouse button and the use of the cursor lines Figure 8 23 TEMP 1 1 C data3 0 ap Teu o fb t imag 2 28 X amp T XE row 10 fromt1 exp 1 1 C3dat
113. uppression 14 1 C Bruker TOPSPIN nmrsu Spectrum ProcPars AcquPars Tite PulseProg Peaks integrals Sample Structure Fia Acqu 3 5 NOTE Figure 7 22 above shows the solvent suppressed 1 D spectrum of the Raffinose sample and Figure 7 23 below shows the 1 D spectrum of the Lysozyme sample Figure 7 24 Spectrum ProcPars AcquPars Tite PulseProg Peaks integrals Sample Structure 150 B7170 00 01 7 6 1 D Solvent suppression with WATERGATE The WATERGATE WATER suppression by GrAdient Tailored Excitation technique which uses pulsed field gradients is claimed to be independent of line shape yielding better suppression compared with other methods Exchangeable protons are not affected and there is no phase jump at the water resonance although signals very close to the water resonance are also suppressed The sequence is in principle a spin echo experiment in which the 180 degree pulse is embedded between two pulsed field gradients After excitation by the first pulse p1 the field gradient G1 dephases all coherence The selective inversion element consists of a symmetrical 3 9 19 pulse sequence 3a t 9a t 19a t 19a t 9a t 3a with 26a 180 degree Figure 7 24 Additional suppression appears at different sidebands 1 t Figure 7 25 p3919gp 7 6 1 Parameter set up 1 Follow the instructions in the paragraphs 6 2 2 through 6 2 6 step 9 2 Select the AcquPar
114. ust the spectra for best fit use the 2 2 g Ts tools 4 Click on the Publish tab in the TopSpin Menu bar Figure 5 60 Publish View Manage C Copy Printe Plot Layout v PDF 1 E Mail 5 Click on the button to print the active window 5 7 7 Plotting all 4 experiment on to the same page Acquire Process Analyse 1 Display the selective NOESY spectrum 2 Click on AI to enter the Multiple display option 3 Drag the selective COSY spectrum in to the spectral window 4 Drag the selective TOCSY spectrum in to the spectral window 5 Drag the Reference spectrum in to the spectral window Figure 5 61 sel noesy 1 1 C idata3 0 ESRT ith E E Ei 25 59 AZ DJ dl sel cosy I L C data3 0L Scale 3 1936 sel no sy loud c vallas ol DS STE Er 5 4 3 2 1 ppm NOTE To adjust the spectra for best fit use the 2 2 g Ts tools 6 Click on the Publish tab in the TopSpin Menu bar 100 B7170_00_01 Figure 5 62 El Start Acquire Process Analyse Publish View Manage o 7 Click on the button to print the active window B7170 00 01 101 5 8 Observations 102 B7170_00_01 5 9 1 D Carbon DEPT experiment using a shaped 13C pulse 5 9 1 Introduction The basic DEPT pulse sequence consists of the following steps e Relaxation period di to achieve a pre equilibrium state e 90 1H pulse p1 to create transverse 1H magnetization ly
115. will have positive and negative peaks showing the CH and CH3 as positive where the CH2 will be negative To assure the right phase correct the Aromatic peaks 7 9 ppm positive Figure 6 13 TEMP 1 1 C data3 0 40 1 R 90 90180 2 c NE I pivot 7 69 ppm Phase increment 0 025 phO 28 39 phl 10 40 from shape hsqc exp 1 1 C data3 o rel 10 10 6 4 2 ppm 7 Click on Li to store the 2 D phase values 8 Click on man B7170_00_01 115 NOTE The spectrum will go back to the unphased view since the phase correction val ues where stored only for the 2 D spectrum 9 Click on LI going back to the 2 D spectrum display 10 Type xfb fourier transform the 2 D spectrum 11 Click on 12 Select the peak at 7 7ppm 130 9ppm 13 Click the left mouse button Figure 6 14 Remove Row Col li Remove All es eres ee Seg ee ee ee 14 Select Add Figure 6 15 shape hsqc exp 1 1 C data3 0 1 MER cI 4 Click the right mouse button to select the peaks for phase correction 6 4 2 F2 ppm 15 Repeat steps 13 and 14 for the peaks at 4 8ppm 73 2ppm and 0 76ppm 16 8ppm 116 B7170 00 01 Figure 6 16 shape hsqgc exp 1 1 C data3 0 1 MO amp cI amp 4 Click the right mouse button to s lect the peaks for phase correction 6 4 2 F2 ppm 17 Click on B Figure 6 17 Phase 2D shape hsqc exp 1 1 C data3 0 Als 0 1

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