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1. sense 161 20 3 SELECTIVE COSY osobteeseseteesatredresrede inner in tre drerit nemen iret rene eee 162 20 3 1 AcquisifiOna ode nedoenoaecacncieienii eit edi e Pin erect n e rer eet edere itera 163 20 9 2 IPFOCESSIRS 3 doo sebo ptione sebo o obo fe beet bb ondas 164 20 4 SEEECTIVE TOCS Y rai Pa AA O EA trina re ri irre ri trea sens ini 165 20 41 Variable Delay List deatur rediere rediit redet eite petiere dr 166 ZOD A CQUISULON m cates 166 202535 IPrOCESSIRSs ARR A RS RSR A babemus dias 168 21 ICONNMR NMR AUTOMATION sense 170 22 APPENDIX A ARTIFACTS IN 2D NMR EXPERIMENTS sense 174 22 T INTRODUCTION ectetuer de tle tede tetti de det eletti tede dese 174 22 11 Whydo artifacts oCGWre s t het ede deett eed tete fud ota 174 22 2 THE DOUBLE QUANTUM FILTERED COS Y EXPERIMENT essent eene nnne enne nnne 175 22 2 L Rapid Scanning ATlfacts JL hike eda tete diete tede eee to had deoa 175 22 2 2 Overload Of ihe Yeceiver i ii beet ette tied t ette dud dede 177 22 2 3 The diamond pattern iet beth ett eed fot 178 22 3 THE HOMONUCLEAR J RESOLVED EXPERIMENT eese eerte eene nnne enne nn entente nnne nn nennen 179 22 3 1 The Effect of digital resolution and tilting of the spectrum ss 179 22 4 INVERSE EXPERIMENTS honored cette tee edet detect tendere cof ve etes 180 22 4 Incorrect proton pulses nist eddie iie deed edet tede ee theft re 180 6 BRUKER Avan
2. edc new create a new data set experiment number or processing number xau iexpno copy the current experiment number including all parameters to the consecutive experiment number wrpa copy of the current data set including the spectra re move to a specific experiment number within the data set rep move to a processing number within the experiment number browse browse the data set directories search find a specific data set wpar save the current parameters rpar select and read a predefined parameter set Table 7 Acquisition Parameters ns number of scans ds number of dummy scans td Time domain number of acquired data points sw sweep width in ppm aq acquisition time olp transmitter frequency of f1 channel in ppm o2p transmitter frequency of f2 channel in ppm rg receiver gain pulprog definition of the pulse program aunmp definition of the acquisition AU program Table 8 Acquisition and Pre acquisition Commands edhead define the current probehead edprosol define probehead specific pulse lengths and power levels getprosol use probehead specific pulse lengths and power levels in the BRUKER Avance 1D 2D current pulse program xau pulse calculate the power level from pulse lengths and vice versa edsp edasp configure the routing
3. April 1 2003 Bruker AG Fallanden Switzerland Version 030401 Avance 1D 2D BRUKER BRUKER Avance 1D 2D 1 INTRODUCTION rmnnnnenenenenenneneneneneeneneneneneneneneneneeneneneneneneneneneneneneneneneeneeneneneneneneneneneneneneneeeneeenenenenenenenes 9 1 1 AN IMPORTANT NOTE ON POWER LEVELS esses eere nennen innen enne nn enne nn nennen ennt nn nennen 9 L2 NMR SPEGCTROMETER htm ttt i eie etat erts Pipe ete rare deu ee Pr eee ete uec n teen eu 10 1 3 CLASSICAL DESCRIPTION OF NMR ccsccsssesseeseeeseescesseeeseeseeesecssesseeesecsesseesseccesseeeseseseeseeeaeceeeseeesaeenseeeesas 10 1 4 SPIN OPERATORS OF A ONE SPIN S YSTEM cesses nennen enitn enne nn terne enn inns eres nnne nennen inneren 11 BAL Efecto YfSPulsesziuuicb edente eitonidiecte ntis te tant tette deste pedet tore eon ELM uso 12 1 4 2 Effect of Chemical Shift Evolution inner 12 L453 Effectof Scalar Coupling adiacente itae etas ep rm betae pe tege ie ibo ege been oe 13 1 5 SENSITIVITY OF NMR EXPERIMENTS nnn nnana iiia iaei ei eie iie iia iei eiie iii edi iini 14 1 6 USEFUL COUPLING CONSTANTS 5 5 2 P ioa t rented eds ae dads hbo ina hti Ed dtd 14 1 6 1 Coupling Constants egisse eec nn A ENNEA a NEon ENVENON NEATE AEAN tein eee tnis eben bete eene dn 14 1 6 Coupling Constants of Hydrocarbons Jp 15 2 PREPARING FOR ACQUISITION smmnnnnnnnnnnnnnnnnnsnnsnnese 17 2 T SAMPEE PREPARATION 4 eee nee eee he estes ee e
4. 2 6 2 Shimming If the sample has been changed the first step after locking is shimming the magnetic field Enter rsh and select an appropriate shim file from the menu Usually only the Z and Z shims and probably the X and Y must be adjusted while observing the lock signal The best shim values correspond to the highest lock level height of the lock signal in the window For further 28 BRUKER Avance 1D 2D discussion of shimming see Chapter 6 Shim Operation of the BSMS User s Manual If you have a gradient probe you can also use the gradient shimming tool which can be started by the command gradshim For more Information please refer to the gradient shimming installation and users guide which is available online in the XWinNMR help menu 2 6 3 Optimize lock settings optional Once the magnetic field has been locked and shimmed the user may wish to optimize the lock settings as described below It is strongly recommended to follow this procedure before running any experiment requiring optimal stability e g NOE difference experiments After the field is locked and shimmed start the auto power routine from the BSMS keyboard see Chapter 2 Key Description of the BSMS User s Manual For lock solvents with long T relaxation times e g CDCls however auto power may take an unacceptably long time and the lock power should be optimized manually Simply increase the lock power level until the signal begins to oscill
5. I cosB Z sin p If the flip angle B 90 then 90 L HI x y Z We find the expected result that a 90 pulse will generate transverse magnetization The rest of this chapter will be concerned with finding out about the fate of this transverse magnetization in time We introduced tacitly the arrow notation where we find on the left side the system before and on the right side after the specific evolution under the operator noted above the arrow This notation is simple very convenient and not only limited to the description of rf pulses We will discuss this notation in more details in the next section 1 4 2 Effect of Chemical Shift Evolution 12 The so called chemical shift Hamiltonian is given by H 6 where is the chemical shift of the corresponding nucleus in the NMR spectrum 8 where oo is the Larmor frequency of the spin and o the carrier frequency of the interaction frame The calculus rules for the chemical shift evolution are the following L 5 1 t L I 51 cos 8 t I sin S t L Sil cos 6t I sin 6 t The time t is the period during which the Hamiltonian is valid The Hamiltonian of a spin system can change with time for example if the experimental setup prescribes first a rf pulse and then a period of unperturbed evolution For the calculus rules it is mandatory that each Hamiltonian is time independent during the time t What s the general idea The whole NMR e
6. which has four columns one for the shaped pulse index number Index one for the power level Power dB one for the offset frequency Offset Frequency and one for the filename of the shaped pulse Filename The pulse program selzg makes use of shaped pulse 1 only In row 1 set the power level for the shaped pulse to 80 dB This parameter is also known as spi For on resonance selective excitation make sure that the offset frequency is set to 0 Hz Click on the filename box with the right mouse button to call up the menu of possible shape files From this list select gauss1 1k with the left mouse button All other acquisition parameters should be the same as for the reference spectrum in particular td o1 sw and rg Acquire and process a selective one pulse spectrum The spectrum should be processed with the command efp using the same phase settings as for the reference spectrum with hard pulse The N H resonance should appear in the middle of the window and no other peaks should be visible Phase correct the N H resonance at 8 1ppm using the O order correction only Note this value but return to the main menu without storing the phase correction This additional phase correction might to be applied to the shaped pulse only not to the hard pulses used in the pulse programs selco and selmlzf below Type 2 phcor 1 and enter the phase correction value Now if the spectrum is reacquired and processed with efp the peptide N H shou
7. 1 6 Useful Coupling Constants Many NMR constants such as chemical shift ranges sensitivities common NMR solvent properties etc can be found in the Bruker Almanac Here we added the values of some common coupling constants that are used more often as parameters cnst1 cnst5 in some pulse programs 1 6 1 Coupling Constants Jcu As a rule of thumb it is possible to estimate the Jcu coupling constant from the following equation Jon 500 fractional CH s character That is 125Hz lt Jcu lt 250Hz so that Jcu 145Hz is a good approximation in most cases The values of Jcn coupling constants increase with increasing HCaCB angles and with the electronegativity of the Cp substituent They vary between 5 and 50Hz 14 BRUKER Avance 1D 2D 1 6 2 Avance 1D 2D The Jcu coupling constants are mostly positive and are maximal at CCCH angles of 0 and 180 The values for trans couplings are larger as for cis couplings Karplus relation Table 2 Useful CH Coupling Constants Compound T Jcu in Hz Ethane 124 9 Acetonitrile 136 0 Ethene 156 Benzene 159 Dichloromethane 178 0 Chloroform 209 0 Formaldehyde 222 0 System Jcu in Hz C sp C sp H 10 to 6 C sp C sp H 0 to 30 C sp C sp H 7 to 4 C sp C sp H 4 to 14 System 3Jcu in Hz C sp C sp C sp H 0to8 C sp C sp C sp H 0 to 20 C sp C sp C sp H 0 to 20 Jtrans gt Jcis
8. P1 H 90 pulse as determined in Section 4 2 4 P5 H 60 pulse calculated from p6 P6 H 90 pulse as determined in Section 4 2 5 P7 H 180 pulse calculated from p6 P17 2 5m 2 5 msec trim pulse D1 2 relaxation delay should about 1 25 T H D9 80ms TOCSY mixing time L1 30 loop for MLEV cycle p6 64 p5 11 p17 2 mixing time calculated internally F1 Parameters Parameter Value Comments TD 256 number of experiments FnMODE States TPPI NDO 1 one dO period INO t increment equal to 2 DW used in F2 SW sw of the optimized H spectrum cosy 1 1 same as for F2 NUC1 select H frequency for F1 same as for F2 parameters set as shown above is 1 3 hours 9 3 Processing Enter edp and set the processing parameters as shown in Table 42 Table 42 TOCSY Processing Parameters Avance 1D 2D F2 Parameters Parameter Value Comments SI 512 SF spectrum reference frequency H WDW SINE multiply data by phase shifted sine function SSB 2 choose pure cosine wave PH mod pk PKNL TRUE BC mod no F1 Parameters BRUKER 95 Parameter Value Comments Sl 512 SF spectrum reference frequency H WDW SINE multiply data by phase shifted sine function SSB 2 choose pure cosine wave PH_mod pk BC_mod no MC2 States TPPI Enter xfb to multiply the time domain data
9. in0 p2 or p4 in other words that d is at least as long as the maximum evolution time t plus the length of the longest pulse p2 or p4 Table 49 COLOC Acquisition Parameters Parameter Value Comments PULPROG colocaf TD 1k NS 8 the number of scans must be 4 ns DS 16 number of dummy scans PL1 high power level on F1 channel C as determined in Section 6 1 4 PL2 high power level on F2 channel H as determined in Section 4 2 4 PL12 low power level on F2 channel H for CPD as determined in Section 6 2 6 P1 13C 90 pulse as determined in Section 6 1 4 P2 13G 180 pulse calculated from P1 P3 H 90 pulse as determined in Section 4 2 4 P4 H 180 pulse calculated from P3 PCPD2 H 90 pulse for cpd sequence as determined in Section 6 2 6 D1 2 relaxation delay should be 1 5 T C D6 50m Delay for evolution of heteronuclear scalar long range J C H couplings D18 33 3m Delay for evolution of heteronuclear scalar long range J C H couplings CPDPRG2 waltz16 cpd sequence for the H decoupling 116 BRUKER Avance 1D 2D F1 Parameters Parameter Value Comments TD 256 number of experiments FnMODE QF NDO 2 two dO periods per cycle SW sw of the optimized H spectrum xhcorr 1 1 INO t increment calculated from SW above NUC1 selects H frequency for F1 The H and the C reference spectra
10. t cos z J t sin t I cos 6 t cost J 4 sin 6 1 2 1 1 cos t sint J t sin t 2 I I sin 0 t sin J 1 sin 9 5 0 Finally the coupling Hamiltonian is applied 200 BRUKER Avance 1D 2D 23 Jj IH Lt o re g c I cost Ja t 2115 sini J t sinQ t cosi J t sin t A cost J 1 2 1 1 sina J t cos 1 cos t J t sin 6 t 2 1 cos t J 1 1 sin t J t cos 8 sin t Ja t sin 6 t Dd E cos x J t I sina Jo t sin S t sin t J t sin 6 t cos t J t sin 6 t cos t J t sin 6 t 1 cost J t cos 6 t cos t Jiz ti sin t L sin t J t sin 6 sin t J t sin 6 t D sin a J t cos t sin Ja t sin f 2 1 J sin t J t cos 6 t cos Ji f sin t 2 1 L sin t J t sin t cos J t sin 1 2 IL cost J t cos t sin t Jy t sin 6 1 2 I L cos t Jy t sin t sin t J t sin fj The corresponding signal function therefore is Tr F 6 2 cos t J 1 sin t cos t Ji t sin 6 t t i cos t J t cos 5 cost J t sin t sin t Jp t sin 6 t sin J t sin 6 t i sin t J t cos 6 t sin a J t sin t 2n dida miv 22 2 e 2 cos sind 1 e J 2a V 2 2m V2 xh E sint Jyyf sin 1 e 2T 0 4
11. 146 Enter ede and change EXPNO to 2 to create the data set homodec 2 1 Setup the relevant parameters according to the table Table 58 Acquisition Parameters for homo decoupling Parameter Value Comments PULPROG zghd 2 Pulse program for homo decoupling CPDPRG2 hd Decoupling sequence during relaxation PL 24 50 Needs to be optimized for good decoupling DIGMOD For AV instruments digital For D X instruments homodecoupling digital HDDUTY 20 Optimise p124 until the multiplet of interest collapses completely to e g a single line Be careful when increasing the power values below 40dB should be avoided The phase correction values of ahomo decoupled spectrum is different to the reference spectrum and must therefore be adjusted for each irradiated signal BRUKER Avance 1D 2D Avance 1D 2D BRUKER 147 19 T Measurement 19 1 Introduction The spin lattice Ti relaxation time of the various H nuclei of a molecule may be determined by using an inversion recovery experiment The pulse sequence begins with a recycle delay tig that is sufficiently long to ensure that all magnetization returns to equilibrium i e pure z magnetization A 180 pulse is applied for the inversion of the whole magnetization During the recovery delay the magnetization is allowed to recover to a certain amount and the final 90 pulse then converts the residual z magnetization into observable transverse magnetization
12. EXPNO 1 PROCNO 1 Click se to create the data set tocsy 1 1 Enter eda and set the acquisition parameters as shown in Table 41 The parameter 11 determines the number of cycles of the MLEV spinlock sequence and thus determines the length of the mixing period The mixing period typically lasts 20 to 100 msec and so 11 should be chosen so that the quantity p6 64 p5 11 p17 2 is 20 to 100 msec The general rule of thumb is that a mixing time of 1 2JHH or approximately 75 msec should be used The parameter p17 determines the length of the trim pulses at the beginning and end of the mixing period A good value for p17 is 2 5 msec The trim pulses are used to ensure that the final 2D spectrum can be phased Note however that for aqueous samples only the first trim pulse should be used in which case 11 should be adjusted so that p6 64 p5 11 p17 is 20 to 100 msec Table 41 TOCSY Acquisition Parameters F2 Parameters Parameter Value Comments PULPROG mlevph TD 1k NS 8 the number of scans should be 8 n DS 16 number of dummy scans PL1 high power level on F1 channel H as determined in Section 4 2 4 PL10 low power level on F1 channel H for MLEV mixing as determined in Section 4 2 5 BRUKER Avance 1D 2D Type rga to set the receiver gain and zg to acquire the time domain data The approximate experiment time for the TOCSY with the acquisition
13. H signals cover almost the entire spectral width Acquire an optimized spectrum Type xau iexpno increment experiment number to create the data set cosy 2 1 Enter eda and set PARMODE to 2D Click on and ok the message Delete meta ext files The window now switches to a 2D display and the message NEW 2D DATA SET appears Enter eda and set the acquisition parameters as shown in Table 37 The F2 parameters olp and sw should be identical to the values used in the optimized H reference spectrum cosy 1 1 Note that in0 and sw F1 are not independent from each other 82 BRUKER Avance 1D 2D Avance 1D 2D Table 37 COSY Acquisition Parameters F2 Parameters Parameter Value Comments PULPROG cosydf see Figure 21 for pulse sequence diagram TD 1k NS 8 the number of scans should be 4 n DS 16 number of dummy scans PL1 high power level on F1 channel H as determined in Section 4 2 4 P1 H 90 pulse as determined in Section 4 2 4 PO P1 0 5 H 45 pulse DO 3 incremented delay t predefined D1 3 relaxation delay should be about 1 25 T4 1H F1 Parameters Parameter Value Comments TD 256 number of experiments FnMODE QF absolute value mode NDO 1 there is one dO period per cycle INO t increment equal to 2 DW used in F2 SW sw of the optimized H spectrum cosy 1 1 same as for F2 NUC1 select H frequency for F1 same as for
14. References H O Kalinowski S Berger S Braun C NMR Spektroskopie Georg Thieme Verlag Stuttgart New York Coupling Constants of Hydrocarbons Ju Usually Juu coupling constants are negative and vary in a range between 0 5Hz and 15Hz in hydrocarbons JHy coupling constants are mostly positive and usually range from 2 up to 18Hz The Ju coupling is positive or negative with smaller absolute values that range from O to 3Hz The Karplus relation is also valid Jirans gt Jois Table 3 Useful HH Coupling Constants System Juu in Hz HC sp H 12 to 15 HC sp H 0 5 to 3 System 3Juu in Hz HC sp C sp H 2109 HC sp C sp H 4to 10 HC sp C sp H 6to 18 HC sp CHO 1to3 HC sp CHO 2104 System Jun abs value in Hz HC sp C sp C sp H 0 HC sp C sp C sp H 0to3 HC sp C sp C sp H 2103 BRUKER 15 Heteroatoms with considerable or M effect can shift the J values dramatically BRUKER Avance 1D 2D 2 Preparing for Acquisition 2 1 Sample Preparation The sample quality can have a significant impact on the quality of the NMR spectrum The following is a brief list of suggestions to ensure high sample quality e Always use clean and dry sample tubes to avoid contamination of the sample e Always use high quality sample tubes to avoid difficulties with shimming e Filter the sample solution e Always use the same sample volume or solution he
15. Set the C Carrier Frequency First a C observe experiment is recorded to determine the correct C carrier frequency which is o1p here but will be o2p in the inverse calibration experiment Create a new data set starting from a previous C data set e g carbon 1 1 re carbon 1 1 enter ede and change the following parameters NAME testinv EXPNO 1 PROCNO 1 Click on SAVE to create the data set testinv 1 1 Type edsp and set the parameters as follows for observing C and decoupling H NUC1 13C NUC2 1H NUC3 off Click on SAVE to save the spectrometer parameters and return to the main window and enter eda to set the acquisition parameters as shown Table 24 Avance 1D 2D BRUKER 59 60 Table 24 1D C One pulse Acquisition Parameters Parameter Value Comments PULPROG zgdc TD 4k NS 1 DS 0 PL1 high power level on F1 channel C as determined in Section 6 1 4 PL12 low power level on F2 channel H for cpd as determined in Section 6 2 6 P1 3 start with less than a 90 pulse PCPD2 1H 90 pulse for cpd sequence as determined in Section 6 2 6 D1 5 SWH 1000 RG 4k olp 77ppm Chloroform resonance o2p 5 CPDPRG2 waltz16 cpd sequence for the H decoupling Notice that the spectral width swh is much smaller than usually used for C spectra because only the chloroform signal must be recorded and o1p is set to almost the correct frequency in th
16. click on with the left mouse button until only the positive peaks are displayed The region can be expanded with the ty button followed by choosing the desired spectral region with the left mouse button depressed The full spectrum is displayed again by clicking the tai button The optimum may be saved by clicking on Defaot and confirming the appearing questions as follows Change levels y Please enter number of positive levels 6 Display contours n BRUKER Avance 1D 2D 8 2 4 Plotting the Spectrum Read in the plot parameter file standard2D rpar standard2D plot which sets most of the plotting parameters to appropriate values Enter edg to edit the plotting parameters Click the ed next to the parameter EDPROJ1 to enter the F1 projection parameters submenu Edit the parameters as follows PF1DU u PF1USER name of user for file cosy 1 1 PF1NAME COSy PF1EXP 1 PF1PROC 1 Click T to save these changes and return to the edg menu Click the ed next to the parameter EDPROJ2 to enter the F2 projection parameters submenu Edit the parameters as follows PF2DU u PF2USER name of user for file cosy 1 1 PF2NAME COSy PF2EXP 1 PF2PROC 1 Click sw to save these changes and return to the edg menu and again to exit the edg menu Create a title for the spectrum setti and plot the spectrum plot A magnitude COSY spectrum of 50 mM Cyclosporin in C D6 is shown in Figure 20 Figure 22 COSY Spectrum of
17. cos Ji 5 2 I L sinf Jj t cos t 4 cost J 5 2 Il D sini J t sin 4 I cost J t cos t 1 cos J t sin 4 2 1 1 sin t J t cos t 2 1 1 sin J 1 sin t gt o Avance 1D 2D BRUKER 199 The first evolution period is identical to what we know from the 1D example The 2D experiment starts now by applying a second pulse after the first evolution period during t 0 5 I cos x Ji 1 cos 1 1 cost Ji 1 sin O t 2 I D sin t Jj t cos 1 2 IL sin t Jj 1 sin 5 6 We now could simply apply the Hamiltonian again for the evolution during tz and battle through 16 operators with countless coefficients just to realize that in fact very few of the original operators contribute to the observable magnetization It s probably more rewarding however to consider o for a while and try to figure out which operators will evolve into observable magnetization and which will just keep us busy liz is a clear cut case as we can see from our calculus table it will not evolve at all The same is true for hylox The reduced density matrix relevant for the observable magnetization is then o l cos t J t sin 6 t 2 1 sin Jy t sin t Applying the chemical shift part of the Hamiltonian yields o Sete cos 6 1 I sin 1 cosQt Jj t sin S t 2 1 1 cos 1 1 sin t sin t J t sin 4 sin 6
18. minimized by acquiring the preirradiated and the reference data in an interleaved manner Reference D Neuhaus and M P Williamson The Nuclear Overhauser Effect in Structural and Conformational Analysis New York VCH Publishers Inc 1989 The sample used to demonstrate a 1D NOE difference experiment in this chapter is 100 mM Pamoic Acid in DMSO d6 The NOE difference pulse sequence is shown in Figure 48 The pulse sequence begins with the recycle delay time d1 This is followed by the cw irradiation period of total time 14 d20 where d20 is the irradiation time for one particular frequency The pulse program makes use of a frequency list fq21ist to determine the frequencies for cw irradiation The final 90 136 BRUKER Avance 1D 2D pulse p1 creates the observable magnetization and is followed by the acquisition period Several spectra are acquired during an NOE difference experiment and for each spectrum a different q21ist is used For the reference spectrum the cw pulse is applied off resonance and the au program noemult is used to acquire the spectra in an interleaved manner Figure 48 ID NOE Difference Pulse Sequence T 2 L14 d20 pl acq 17 2 Acquisition Insert the sample in the magnet Lock the spectrometer Readjust the Z and Z shims until the lock level is optimized Tune and match the probehead for H observation For best results it is recommended to optimize the lock parameters as describ
19. on page 3 SW 20 start with a large spectral width of 20ppm which will be optimized lateron olp 5 will be optimized lateron Enter rga to perform an automatic receiver gain adjustment then enter zg to acquire the FID and edp to set the processing parameters as shown in Table 15 Table 15 1D H one pulse Processing Parameters Parameter Value Comments SI 2k LB 1Hz PSCAL global Fourier transform the spectrum with the command ef and phase the spectrum according to Chapter 3 8 Type sref to calibrate the spectrum and confirm the message no peak found in sref default calibration done 4 2 2 Optimize the Carrier Frequency and the Spectral Width 40 The carrier position o1p should now be set to the signal used for monitoring the 90 pulse calibration which is the quartet signal of the Ethylbenzene H spectrum Expand the spectrum so that only the quartet at 2 6 ppm is displayed Click on M utilities to enter the calibration submenu Click on 91 with the left mouse button move the cursor to the center of the quartet and click the middle mouse button to assign oip to this frequency Click on reum to exit the calibration submenu and return to the main window Reduce the spectral width by entering swh and change the value to 1000 Hz BRUKER Avance 1D 2D Enter zg to acquire a new FID using the new values for o1p and swh and process the spectrum using the command ef 4
20. use a large spectral width when you enter 50 the registered value is slightly different RG 64 suggested receiver gain NUC1 1H observe nucleus O1P 15 position of the carrier frequency is 15 ppm Avance 1D 2D BRUKER 33 Click on SAVE to save the acquisition parameters and return to the main window Click on DONE to save the changes and return to the eda table As with most acquisition parameters however d1 p1 and p11 can also be edited by typing them in the command line of the main XWIN NMR window As mentioned before most of the acquisition parameters for the current pulse program can also be entered in the ased table 3 6 Acquisition Enter acqu to switch to the acquisition window While it is possible to acquire a spectrum from the main window the buildup of the FID can only be observed in the acquisition window Enter the command rga which performs several acquisitions and sets a suitable value for the receiver gain rg Enter zg which deletes any previous data zero and starts the experiment go The message scan 1 1 indicates that the spectrometer is performing the first scan and that only one scan will be performed If at any time a submenu is entered accidentally click on the em button located on the button bar and then enter acqu to switch back to the acquisition window If at some point the message DATA OUT OF WINDOW appears or if the scaling is unsuitably large or small c
21. whereas the signal arising from protons attached to C is suppressed by phase cycling Thus the 1D HMQC spectrum of the current sample 1096 Chloroform in Acetone d6 yields only signal from the C satellites without the large central peak Figure 13 ID HMQC Pulse Sequence T 2 T 13C p3 p3 Create a new data set starting from testinv 3 1 re testinv 3 1 enter edc and set EXPNO to 4 Click SAVE to create the data set testinv 4 1 Enter eda and set the acquisition parameters as shown in Table 30 Avance 1D 2D BRUKER 65 Table 30 ID HMQC Acquisition Parameters Parameter Value Comments PULPROG inv4ndrdid see Figure 13 for pulse sequence diagram TD 8k NS 16 the number of scans should be 4 n in order for the phase cycling to work properly DS 16 number of dummy scans PL1 high power level on F1 channel H as determined in Section 4 2 4 PL2 high power level on F2 channel C as determined in Section 6 1 4 P1 H 90 pulse as determined in Section 4 2 4 P2 H 180 pulse set to 2 P1 P3 C 90 pulse as determined in Section 6 3 6 D1 20 sec relaxation delay should be 1 5 T H CNST2 214 heteronuclear scalar J C H coupling Hz Enter zg to acquire the FID and edp to set the processing parameters as shown in Table 31 Table 31 ID HMQC Processing Parameters Parameter Value Comments SI 4k WDW EM LB 0 30 Hz PKNL TRUE nec
22. 1 cos t sinf J t sing h ie If we sum up the signal functions of both experiments we find Tr F 6 Tr F 0 5 2 cos t J t sin t cos J t sin t t i cos t J t cos t cost Ji t sin fj sin t J t sin t sin t J t sin fj i sin J 1 cos t sin J t sin j cos t J t cos t cos Ji t cos 6 1j i cos t J t sin r cos n J t cos t sin t J t cos 6 t sin t J t cos t i sin t J t sin t sin t J t cos 6 t i A 2Ti V 4251 2m iv rs 5 COS Jo tn sin 4 Ce 2 2 i 27 iV ME 2m 443 224 Sin Jn sin 4 e 2 e Ew ra 1 27 i v 42254 2qi vi y Fe OSC CRC e 2 e T I 4 24 2a i v y Sin Fin 5 cosQ 4 e F8 a 202 BRUKER Avance 1D 2D Which can also be expressed as TAF 6 Tr F 60 5 cost Jo t cos 1 i cos x J t sin S t 2mi v 42254 24d my 2 e 2 Gin Jj 71 cos 0 1 i sin J fj sin 9 f Ji J 2i v M2 2a 4i v 2 4 v5 2 Jta v5 2 ty e The final result will then lead to 24i 2325 24i 1 25 omit ani 72224 2 e 2 2 ee 1 Tr F 6 Tr F o a e Lee re Rene MIL 4 After Fourier transform we find an all positive diagonal peak multiplet and an anti phase cross peak multiplet of four peaks each Avance 1D 2D BRUKER 203 23 11 Summary and Useful Formulae 23 11 1 Eff
23. 2 3 Define the Phase Correction and the Plot Region The phase correction and the spectral region plotted in the output file must be optimized before the automation program for the pulse calibration is executed Phase correct the spectrum according to Chapter 3 8 in a way that the quartet signal is positive Expand the spectrum so that the quartet covers approximately the central quarter of the screen Click on pij with the left mouse button and hit return for the following three prompts or answer them as follows F1 2 8 ppm F2 2 4 ppm change y scaling on display according to PSCAL y The preparations are now completed and the calibration experiment can be executed as described in the next section 4 2 4 Calibration High Power For the 90 pulse calibration an automation program called paropt is used Since the execution of this automation is time consuming it is not the best choice if the correct pulse times and power levels are already known approximately In such cases the correct values are usually just checked by acquiring 1D spectra with different pulse widths to check for maximal signal The automation program is started by typing xau paropt and answering the appearing questions as follows Enter parameter to modify p1 Enter initial parameter value 2 Enter parameter increment 2 Enter of experiments 16 The spectrometer acquires and processes 16 spectra with incrementing the parameter p1 from 2 usec by 2 usec to a fina
24. 2 4 1 Set the Parameters In XWIN NMR enter edsp and set the following spectrometer parameters NUC1 1H NUC2 OFF NUC3 OFF This automatically sets s o1 to a frequency appropriate for H tuning and matching There is no need to adjust s o1 carefully now Exit edsp by clicking SAVE 2 4 2 Start Wobbling Tuning and matching are carried out simultaneously using XWIN NMR During wobbling a low power signal is transmitted to the probehead This signal is swept over a frequency range determined by the parameter wbsw the default value is 4 MHz centered around the carrier frequency i e sfol sfo2 etc depending on which nucleus is being tuned matched Within the preamplifier High Performance Preamplifier Assembly or HPPR the impedance of the probe over this frequency range is compared to the impedance of a 50 Q resistor The results are shown both on the LED display of the HPPR and in the acquisition submenu of XWIN NMR Both displays show the reflected power of the probehead versus the frequency of the Avance 1D 2D BRUKER 25 signal The user observes either one or both of these displays while tuning and matching the probehead Before starting the wobbling procedure ensure that no acquisition is in progress e g enter stop Enter acqu to switch to the acquisition window of XWIN NMR if it is desired to use this to monitor the tuning and matching Notice that being in the acquisition window slows down the wobbling procedure s
25. 2D data set is used by the T calculation routine which allows the user to determine T for any number of peaks of the 1D spectrum 148 BRUKER Avance 1D 2D Figure 52 Inversion Recovery Pulse Sequence T T 2 19 2 Acquisition Insert the sample in the magnet Lock the spectrometer Readjust the Z and Z shims until the lock level is optimized Tune and match the probehead for H observation Create the data set tidata 1 1 starting out from noedif 1 1 and record a H reference spectrum see Section 17 2 From the data set tidata 1 1 enter xau iexpno to create the data set tidata 2 1 This data set will be used for the inversion recovery experiment Although inversion recovery is not technically a 2D experiment it does generate an array of 1D spectra which are most easily handled as one 2D file Thus t1data 2 1 must be changed into a 2D data set as described below Enter parmode and select 2D The window now switches to a 2D display and the message NEW 2D DATA SET appears 19 2 1 Write the variable delay list The inversion recovery experiment requires a variable delay list to provide all the values of the recovery time vd To create the variable delay list enter edlist A menu of list types appears Select vd from this menu This calls up a menu of existing vdlist filenames and gives the user the option of creating a new file Type new name Type the name t1delay to call up the editor Enter the delays in s as
26. 50 mM Cyclosporin in C6D6 Avance 1D 2D BRUKER 8 3 Double Quantum Filtered DQF COSY The DQF COSY pulse sequence consists of three pulses where the third pulse converts part of the multiple quantum coherence into observable single quantum coherence which is detected during the acquisition period One advantage of the DQF COSY experiment is the phase sensitivity i e the cross peaks can be displayed with pure absorption lineshapes in both the F1 and the F2 dimension In general a phase sensitive spectrum has a higher resolution than an otherwise equivalent magnitude spectrum because the magnitude lineshape is broader than the pure absorption lineshape Another advantage is the partial cancellation of the diagonal peaks in a DQF COSY spectrum Thus the diagonal ridge is much less pronounced in a DQF COSY spectrum than in a normal COSY spectrum A third advantage of the double quantum filter is the elimination of strong signals e g the solvent H which do not experience homonuclear J coupling References M Rance O W Sorensen G Bodenhausen G Wagner H R Ernst and K W thrich Biochem Biophys Hes Commun 117 479 1984 A Derome and M Williamson J Magn Reson 88 117 1990 8 3 1 Pulse Sequence The DQF COSY pulse sequence is shown in Figure 23 The pulse p1 must be set to the appropriate 90 pulse length found in Chapter 4 2 4 Note that the DQF COSY experiment is sensitive to high pulse repetition
27. 9 5 9 0 8 5 8 0 7 5 7 0 6 5 6 0 5 5 5 0 4 5 4 0 3 5 3 0 2 5 2 0 1 5 1 0 0 5 ppm 9 0 8 5 8 0 735 70 65 6 9 5 5 5 0 45 49 33 30 2 5 20 1 5 10 ppm 20 3 Selective COSY Many 2D NMR experiments can be converted to analogous 1D experiments by using Gaussian pulses A 1D sequence is advantageous when a limited amount of information is desired which is often the case for medium sized molecules In such cases the total experiment and data manipulation times are shorter for the 1D experiment than for the 2D experiment The 2D COSY experiment is very effective at indicating coupling except in cases where the H chemical shifts are closely crowded together so that many cross peaks overlap Selective COSY gives the same H coupling information at a time without involving a 2D Fourier transform This is useful for probing regions of the spectrum where the H shifts are densely packed provided that some H resonances are sufficiently well separated that they can be picked out for selective irradiation The selective COSY pulse sequence is shown in Figure 57 It is very similar to the standard COSY sequence shown in Figure 21 except that the first pulse is a frequency selective 90 excitation pulse and the delay between the two pulses d14 is not incremented The duration of this delay is measured from the middle of the Gaussian envelope As with 2D COSY the second or coherence transfer pulse is a hard 90 pulse This pulse creates observable ma
28. 90 pulse as determined in Section 4 2 4 D1 3 5 relaxation delay D20 50m 50 msec irradiation time L4 T D20 loop counter to determine overall irradiation time T L4 D20 FQ2LIST noedif 1 frequency list for f2 frequency of selective irradiation The pulse program noemul operates such that o2 is set to the first frequency of the q21ist and the selected multiplet is irradiated with this frequency for a time d20 Then o2 is set to the next frequency if there is one of the fq21list and the selected multiplet is irradiated with this frequency for a time d20 This process continues until the multiplet is irradiated for a total of 14 times 17 2 5 Optimize the irradiation power and duration For the NOE difference experiment the au program noemult will run the pulse program noemul with successive fq2lists However the optimization of irradiation power and duration can be performed for a single resonance Thus the au program is not necessary and the pulse program noemul can be started with the command zg Make sure that q21ist is set to noedif 1 or noedif 2 i e make sure that the cw irradiation will be applied on resonance for one of the multiplets Start the acquisition with zg process the spectrum with ef see the processing parameters listed below in Table 56 and manually phase correct the spectrum Compare this spectrum with the reference spectrum noedif 1 1 by using the dual mode From the current data set noe
29. EROR ROO UO Dre DOG Og OO Oda ERO Dra rr Dre ra org 149 19 2 1 Write the variable delay list eese entente tenete tenente eene nennen nennen 149 19 2 2 Set up the acquisition parameters rennes 150 19 2 3 Acquire the 2D data set eee eese eene eene tenente tenente tenente tenete ente tenete nenne 150 19 3 PROCESSING ocieiesetesaecetecet exert PERI elles Peg ev peg exe e ase Pe ben ed nn lan eye tect ne ete tees ces ie es 151 19 3 1 Write the integral range file and baseline point file 151 19 2 Tr CAEGCULATION ctio etetecodecee enter eerte dece ece tette erecto een ete parete Else a 152 I94 HF Check gCUrvesi eos ie er rr eR DR TD ERR ed ee en 153 194 2 Checknumerical results e ete ree ne ne edd 153 AOA SS SE paramel rgs ie eee e RM UEFA wee TE TENE TEUER UFU PER ERE EUR ERE RE Pee TE TENE TUE EY 154 19 5 CREATE STACKED PLEOT eter deceret eme tere ertet deett en eode 154 20 SELECTIVE EXCITATION sense 158 20 1 INTRODUCTION 5 2 3 orn tr ere tr chee ie ra EO ie E RE D Pe PX GE chee dea hes DE D e Hd ve e hee ee 158 20 2 SELECTIVE PULSE CALIBRATION i css esse 3 ct cre crece rer cose he e ea sees Dee ehe rhe a E b be exo eda Das 158 20 2 Protonweference SDECIIUIN tis cs oai egueo doo tetoot di o dioddieeodimtdi mimi leeiodeqis 159 20 2 2 Selective one pulse sequence ner 159 20 2 3 Define the pulse shape inner 159 20 2 4 Acquire and process the selective one pulse Spectrum 159 20 2 5 Perform the pulse calibration
30. RE YE 137 17 2 3 Select the resonances for irradiation ss 138 17 2 4 Set up the acquisition parameters rene rer 139 17 2 5 Optimize the irradiation power and duration ss 139 17 2 6 Perform the multiple NOE experiment eee seien tenete nnne nennen nennen 140 1 3 PROCESSING conan he OD OO D EGRE E IERI EID 141 17 3 1 Perform the Phase Correction nr 141 17 3 2 Create NOE Difference Spectra ner rene 141 17 3 3 Quantitate the NOE isi eee etit teet Entr e per riesen ee sevo ti Ea E delete Reo Fein 142 18 HOMONUCLEAR DECOUPLING eere eara ra ratas tata tata ta tanen enn tata ta tata tata ta tata tata tenen en e tata tanta tnn nuo 144 18 E INTRODUCTION eite oett e e ederet an ent rie tee eme rats finite 144 15 27 ACQUISITION cieteieceiecece cotecetec ote teret teret EEN tete cederet etpteetde etuer ete decet ln eene edo 145 48 2 h Created new file directory si te eee ee e d eec etta e eed 145 19 2 2 ProtoneferenceSepCITUm s cord sven Wes eee tne tees eae Re AE ERE 145 18 2 3 Selection of irradiation frequency eee e esee tenente tenente tenente tenent nente 145 18 2 4 Setting up the homo decoupling parameters iii 146 19 T MEASUREMENT snnnnnnenennenenennenenenenenenenenenennnnennennennenenennennennnnnnnnnnnnnnnnenennenes 148 19 1 INTRODUCTION 455 4 5 ette Dro re reae a cro P Cod Gd e Od og oa cede aed ar ord 148 19 2 ACQUISITION 4 oo trh co or cr Dro FO PO UE UU E
31. Section 4 2 4 SW 20 start with a large spectral width of 20ppm which will be optimized lateron olp 5 Enter rga zg and edp to perform an automatic receiver gain adjustment acquire an FID and set the processing parameters as shown in Table 21 Table 21 1D H one pulse Processing Parameters Parameter Value Comments SI 2k LB 1 PSCAL global Type the command ef to perform line broadening and Fourier transformation and phase correct the spectrum Type sref to calibrate the spectrum and confirm the message no peak found in sref default calibration done Click the left mouse button with the cursor placed in the spectral field of the main window Move the cursor to the top of the Benzene peak at around 7 3 ppm and note the exact ppm value of the cursor position in the small Info window Click the left mouse button to release the cursor from the spectrum 6 2 4 Set the C Carrier Frequency and the Spectral Width These parameters were already determined for the data set test13c 1 1 To transfer all the parameters from test13c 1 1 to the new data set enter re test13c 1 1 enter edc and change the following parameters NAME testdec EXPNO 2 PROCNO 1 Avance 1D 2D BRUKER 55 56 Click on SAVE to create the data set testdec 2 1 Since now H decoupling is required enter edsp and set NUC2 to 1H so that the spectrometer parameters are as follows NUC1 13C NUC2 1H NUC3
32. and TOCSY experiments as well Figure 63 DQF COSY experiment of pamoic acid The receiver is overloaded and additional t noise appears Avance 1D 2D BRUKER 177 22 2 3 The diamond pattern The so called diamond pattern forms an either quadratic or rectangular arrangement of additional peaks In literature this artifact is explained by errors in the phase of that pulse on which the TPPI phase cycle is done in an homonuclear experiment This is true if e The phase preset times of the spectrometer is set to a values which are too short e The pulse width of the proton pulse is set to a short value We recommend to use proton pulses in the order of e g 1lOusec In addition the phase preset time for the F1 channel of the spectrometer can be edited and increased with the XWIN NMR command edscon A diamond pattern can also be caused by temperature oscillation either of the sample or of the room temperature Note that too high values for the receiver gain also can cause a diamond pattern in homonuclear 2D experiment Figure 64 DQF COSY experiment of pamoic acid Artifacts are aligned on the diamond pattern which is shown in red 178 BRUKER Avance 1D 2D 22 3 The Homonuclear J Resolved Experiment 22 3 1 The Effect of digital resolution and tilting of the spectrum The homonuclear J Resolved experiment requires a tilting of the spectrum which is applied after the 2D Fourier transformation
33. are to set as described in Table 52 Table 52 HMBC Acquisition Parameters F2 Parameters Parameter Value Comments PULPROG hmbclpndaf TD 4k NS 64 the number of scans should be 16 n DS 32 CNST2 145 heteronuclear scalar J C H coupling 145 Hz is a good intermediate value D6 50m delay for evolution of long range couplings 1 7 Jx F2 Parameters Parameter Value Comments TD 256 FnMODE QF BRUKER Avance 1D 2D Enter zg to start the HMBC experiment With the acquisition parameters shown above the approximate experiment time is 13 5 hours Enter edp and set the processing parameters as shown in Table 53 Table 53 HMBC Processing Parameters F2 Parameters Parameter Value Comments SI 2k SF spectrum reference frequency H WDW QSINE sine squared window function SSB 0 pure sine squared wave PH mod no PKNL TRUE necessary when using the digital filter BC mod quad F1 Parameters Parameter Value Comments SI 512 SF spectrum reference frequency C WDW SINE sine window function SSB 0 pure cosine wave PH mod mc MC2 QF Enter xfb to multiply the time domain data by the window functions and to perform the 2D Fourier transformation Adjust the display as described for the HMQC spectrum An HMBC spectrum of 50 mM Cyclosporin in CgD is shown in Figure 41 Figure 41 HMBC spectrum of 50 mM Cyclospori
34. be found in XWinNMRHome exp stan nmr lists pp Update info 2 3 Tuning and Matching the Probe In a probehead there are resonant circuits for each nucleus indicated on the probehead label e g one for H and one for C in a dual H C probehead one for H and one for a wide range of nuclei in BBO or BBI probeheads There is also a resonant circuit for the lock nucleus but the standard user will never need to adjust this so we will ignore it in the following Each of the circuits has a frequency at which it is most sensitive the resonance frequency Once the sample is inserted the probehead should be tuned and matched for these individual frequencies Tuning is the process of adjusting this frequency until it coincides with the frequency of the pulses transmitted to the circuit For example the frequency at which the H resonant circuit is most sensitive must be set to the carrier frequency of the H pulses which is s o1 if the H circuit is connected to the f1 channel s o2 if it is connected to the f2 channel etc Matching is the process of adjusting the impedance of the resonant circuit until it corresponds with the impedance of the transmission line connected to it This impedance is 50 O Correct matching maximizes the power that is transmitted to the coil A probehead is said to be tuned and matched when all of its resonant circuits are tuned and matched Once a probehead has been tuned and matched it is not necessary to retune
35. be discussed The different pulse sequences are quite simple and can be explained as follows The first pulse creates transverse magnetization components which evolve chemical shift and homonuclear J coupling during the evolution period t The second pulse mixes the magnetization components among all the transitions that belong to the same coupled spin systems The final distribution of labeled magnetization components is detected by measuring their precession frequencies during the detection period t The COSY spectrum is processed by a 2D Fourier transform with respect to t and t and the cross peaks indicate which H nuclei are J coupled The sample used to demonstrate magnitude and DQF COSY in this chapter is 50 mM Cyclosporin in benzene d6 8 2 Magnitude 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 The signals acquired with one of these experiments have absorptive and dispersive lineshape 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 spectrum must be displayed i
36. contains all the electronics used for transmission and reception of radio frequency rf pulses through the pre amplifier to the probe 3 The computer from where the operator runs the experiments and processes the acquired NMR data 1 3 Classical Description of NMR 10 A more complete theoretical description of NMR is given in chapter 22 Among the various atomic nuclei about a hundred isotopes possess an intrinsic angular momentum called spin and written AI They also possess a magnetic moment u which is proportional to their angular momentum u yu where y is the gyromagnetic ratio The Larmor theorem states that the motion of a magnetic moment in a magnetic field Bo is a precession around that field where the precession frequency is given by yB Larmor frequency BRUKER Avance 1D 2D By convention the external static field Bo is assumed to be along the z axis and the transmitter receiver coil along either the x or y axis After the sample has been inserted into the magnetic field it shows a magnetization vector M along the z axis In this state no NMR signal is observed as we have no tranverse rotating magnetization By application of an additional rotating magnetic field B4 in the x y plane the orientation of M can be tilted into the x y plane where it precesses around the total magnetic field e g the vector sum of Bo and By Such a rotating magnetic field is obtained by applying rf pulses and the com
37. cycling the first C 90 pulse with respect to the receiver After the delay A of about 60msec the second C 90 pulse creates the desired heteronuclear multiple quantum coherence for long range H C J couplings Phase cycling of the second C 90 pulse removes signals from TH without long range coupling to C The final C 90 pulse after the t evolution period is followed immediately by the detection period t The signal detected during t is phase modulated by the homonuclear H J couplings The 2D spectrum is generated by a Fourier transform with respect to t and to If more than one long range H C connectivity is detected for one particular proton the relative intensities of the corresponding resonances are directly related to the magnitude of the coupling constant Because of phase modulation the spectrum has peaks with a combined absorptive and dispersive lineshape It is not possible to phase correct the spectrum so that the peaks are purely absorptive and so the spectrum must be presented in magnitude mode Avance 1D 2D BRUKER 125 Figure 40 HMBC Pulse Sequence T 2 13C p3 p3 di d2 d6 dO dO 15 2 Acquisition and Processing Follow the instructions given for the HMQC experiment Section 14 since the HMBC is very similar to the HMQC experiment Create the data set hmbc 1 1 and set the parameters as described in Section 14 2 except the pulse program number of scans and the delay d6 which
38. dataset related commands 21 DECP90 acquisition parameters 58 DECP90 processing parameters 59 DECP90 pulse sequence 56 define solvent parameters 22 defplot 106 density matrix 195 DEPT 75 DEPT acquisition parameters 77 DEPT processing parameters 78 DEPT pulse sequence 76 DEPT135 19 DEPT45 19 DEPT90 19 detection operator 203 Double Quantum Filtered COSY 89 DQF COSY 19 89 DQF COSY Pulse Sequence with gradients 93 DQF COSY phase correction 91 DQF COSY Processing Parameters 90 DQF COSY Pulse Sequence 89 DQF COSY with gradients 92 eda 18 edg 18 edhead 21 edlist 154 172 edp 18 edsolv 22 elim 158 eliminate datapoints 158 energy level 195 equilibrium matrix 195 Ethylbenzene 40 Euler relations 211 evolution of spin systems in time 196 experiment number 17 experiment time estimation 22 experiments table of common 19 expno 17 FID and free induction decay 198 Eq list 143 frequency list 143 gated H decoupling 71 Gauss 164 gaussian envelope 168 171 gaussian pulse 168 gradshin 30 graphical display of the current pulse program 21 GRASP DQF COSY acquisition parameters 93 GRASP DQF COSY Pulse Sequence 93 Avance 1D 2D GRASP HMBC 133 GRASP HMBC pulse sequence 135 GRASP HMQC 133 GRASP HMQC pulse sequence 134 GRASP HSQC 133 gyromagnetic ratio 192 Hartmann Hahn condition 102 HCCOLOCSW 20 HCCOSW 20 HETCOR 20 HETCORR 113 hetero
39. difference spectra are created by subtracting the reference spectrum from each of the preirradiated spectra Within the data set of each preirradiated spectrum the second and third data sets are defined by using the edc2 command where the second data set refers to the reference spectrum and the third data set refers to the data set where the difference spectrum is stored Avance 1D 2D BRUKER 141 Display the first preirradiated spectrum re 2 1 Enter edc2 and set EXPNO2 and PROCNO to 4 and 1 respectively reference spectrum Set EXPNO3 and PROCNO3 2 and 2 respectively so that the difference spectrum will be stored with the same experiment number and processing number 2 Click on to the main menu Enter the dual submenu by clicking on a Both the current spectrum and the reference spectrum appear on the screen Click on diff and select Save amp return to subtract the reference spectrum from the current preirradiated spectrum The difference spectrum appears automatically in the window Click rtm to save the results and return to the main menu The message result will be put into DU u USER username NAME noedif EXPNO 2 PROCNO 2 click OK if ok appears Click OK and notice that the current data set is now noedif 2 2 Move to the next preirradiated spectrum re 3 1 and repeat the above procedure set EXPNO2 4 PROCNO2 1 EXPNOS 3 and PROCNO2 2 to store the difference spectrum in no
40. ensure that the system reaches steady state conditions before any spectra are added together Enter zg to acquire the FID and efp to add line broadening Fourier transform and phase correct the data after the acquisition is completed As shown in Figure 5 more peaks are visible now However the signal to noise ratio still is unsatisfactory The C carrier frequency must be adjusted and set to the center of the spectrum To do so click on the button _ utilities to enter the calibration BRUKER Avance 1D 2D submenu and then 21 with the left mouse button to select o1p calibration Tie the cursor to the spectrum move it to the Chloroform peak and press the middle mouse button to set o1p to this frequency Click on Feum Acquire and process another spectrum with this new olp zg efp Figure 4 C spectrum of 1 g cholesterylacetate in CDCI3 C E TOGEROGEIROOG RT ER E T F T0X T GTI 150 100 50 0 50 100 ppm The signal to noise ratio can be improved further by the application of H decoupling as shown in the next section Figure 5 C spectrum of 1 g cholesterylacetate in CDCI3 LE EL EE LL RE DE OT E 0 50 100 ppm Avance 1D 2D BRUKER 47 5 3 One Pulse Experiment with H Decoupling The one pulse sequence with H decoupling i is shown in Figure 6 In addition to the one pulse sequence used before H is decoupled throughout the entire length of the pulse program Figure 6 C One Pulse Sequ
41. for H observation It is recommended to run 2D experiments without sample spinning Record a H reference spectrum to determine the correct values for o1p and sw A H reference spectrum of this sample was already created for the magnitude COSY experiment Section 8 2 2 This spectrum is found in the data set cosy 1 1 The NOESY data set can be created from the data set of any of the previous homonuclear 2D experiments run on this sample For example enter re cosy 2 1 to call up the data set cosy 2 1 Enter edc and change the following parameters NAME noesy EXPNO 1 PROCNO 1 Click to create the data set noesy 1 1 Enter eda and set the acquisition parameters as shown in Table 45 104 BRUKER Avance 1D 2D Table 45 NOESY Acquisition Parameters F2 Parameters Parameter Value Comments PULPROG noesyph TD 1k NS 32 the number of scans must 8 n DS 16 number of dummy scans PL1 high power level on F1 channel H as determined in Section 4 2 4 P1 H 90 pulse as determined in Section 4 2 4 D8 350m Mixing time D1 2 F1 Parameters Parameter Value Comments TD 256 number of experiments FnMODE States TPPI NDO 1 one dO period per cycle INO t increment equal to 2 DW used in F2 SW sw of the optimized H spectrum cosy 1 1 same as for F2 NUC1 select H frequency for F1 same as for F2 11 2 1 Optimize Mixing Time Avance 1D 2D The parameter d8 det
42. if aq mod DQD F1 Parameters Parameter Value Comments SI 1k SF spectrum reference frequency H WDW SINE multiply data by phase shifted sine function SSB 2 choose pure sine wave PH_mod pk determine 0 and 1 order phase correction with phasing subroutine BC_mod no MC2 States TPPI States TPPI results in a forward complex FT Enter x b to perform the 2D Fourier transformation and adjust the displayed spectrum as described in Section 8 2 3 8 3 3 Phase correct the spectrum Avance 1D 2D The phase correction of DQF COSY spectra is best performed while examining the cross peaks rather than the diagonal peaks When the BRUKER spectrum is phased properly the cross peaks will be purely absorptive i e they will not have the slowly decaying wings characteristic of dispersion peaks However since DQF COSY peaks are antiphase i e each multiplet has adjacent positive and negative peaks it is not possible to phase the spectrum so that all peaks are positive Generally a 2D spectrum is first phase corrected in the F2 dimension rows and then in the F1 dimension columns To phase correct the spectrum in F2 three rows each with a cross peak should be selected The cross peak of one row should be to the far left of the spectrum the cross peak of the second row should be close to the middle and the one of the third row should be to the far right of the spectrum Enter the phase correction menu by clicking on
43. in 1D NMR spectra are quite well known many spectroscopists have a very little knowledge about artifacts in 2D NMR spectra Modern NMR spectrometer allow to start even complex 2D NMR experiments like ROESY TOCSY and the inverse experiments HSQC and HMBC with single button push even without the need of any knowledge of the theory behind the experiment We therefore like to give an introduction to common artifacts in 2D NMR spectra allowing a more reliable interpretation of the spectra There are three classes of artifacts First those artifacts which are a result of the spectrometer hardware With modern NMR spectrometer that source can be neglected Second artifacts can be a simple result of the spin system under investigation One example is the J Resolved experiment were second order effects of the scalar coupling introduce additional peaks Finally artifacts can be introduced by missettings of the experiment conditions In this small overview we will focus on artifacts which are generated by missettings of acquisition parameters 174 BRUKER Avance 1D 2D 22 2 The Double Quantum Filtered COSY Experiment 22 2 1 Rapid Scanning Artifacts The T relaxation rate of protons differs from less than 1sec for large molecules to values above 5sec for small organic molecules If the relaxation rate is not taken into account and a standard repetition rate of e g 2sec is used so called multiple quantum diagonales will be observed The artifac
44. level for TOCSY spinlock spl f1 channel shaped pulse for selective excitation or f1 channel shaped pulse for water flipback sp2 f1 channel shaped pulse 180 degree or f2 channel shaped pulse 90 degree on resonance sp7 f2 channel shaped pulse 180 degree off resonance2 or f2 channel shaped pulse 180 degree adiabatic or f1 channel shaped pulse for wet po for different applications i e f1 channel variable flip angle high power pulse in DEPT pl f1 channel 90 degree high power pulse p2 f1 channel 180 degree high power pulse p3 f2 channel 90 degree high power pulse p4 f2 channel 180 degree high power pulse p6 f1 channel 90 degree low power pulse p11 f1 channel 90 degree shaped pulse selective excitation or water flipback watergate or wet p15 f1 channel pulse for ROESY spinlock p16 homospoil gradient pulse p17 fi channel trim pulse at pl10 or pl15 p18 f1 channel shaped pulse off resonance presaturation do incremented delay 2D 3 usec d1 relaxation delay 1 5 T1 d2 1 2J d3 1 3J d4 1 4J d6 delay for evolution of long range couplings d7 delay for inversion recovery d8 NOESY mixing time d9 TOCSY mixing time dil delay for disk I O 30 msec d12 delay for power switching 20 usec d14 delay for evolution after shaped pulse BRUKER Avance 1D 2D d16 delay for homospoil gradient
45. levels by getprosol and type xaua to start the acquisition It is assumed that the sample is shimmed and the probe is matched and tuned for the specific nuclei If you are using the Bruker predefined parameter sets you can always process the data by typing xaup The following list is a short summary of the most commonly used experiments and the corresponding parameter sets The emphasis is on the spectroscopic information that you will get from the experiments rather than on the type of experiment For the experiments in this table it is always recommended to use the gradient version of the experiment if you have the required z gradient hardware These experiments usually require less time than the ones without gradients Table 4 Short List of Typical Experiments Parameter Sets and What They Do 18 BRUKER Avance 1D 2D Atom Group Information 1D Experiments a k a Parameter Set H TH chemical shift and coupling 1D H PROTON C TC chemical shift H decoupled signal D C CI3CPD enhancement integration not possible C TC chemical shift H coupled signal 1D C C13GD enhancement integration not possible CH CH CH PC chemical shift select CH CH and DEPT45 C13DEPT45 CH signals only same phase CH TC chemical shift select CH signals DEPT90 C13DEPT90 only CH CH FC chemical shift select CH and CH DEPT135 C1T3DEPT135 signals only
46. oder Yew Find Peraneters bheb Busy unii Walng fer Joti Ewpt Time 00 00 Drar User ki Ha Saveni Expermert N s Avaktis nmednts te 108 DMSO Wee Avsleble nmedata v es aj DMSO r2 maaa 7 fie z 703 vall e You can also setup several experiments on the same sample Therefore press the Add button before you submit the experiment and you will get a new entry line where you can enter the new experiment for the same sample If you click on the Copy button all the parameters you have entered for one sample will be copied to the next allem lm Submit li Cancel lel Edit Delete 2 The automation is parameter set Sem and ee it is very simple to setup your own experiments for the automation in IconNMR The only requirement is a working parameter set for your experiment with AU programs for the data acquisition and processing These AU programs have to be defined under the aunm and aunmp parameters respectively 172 BRUKER Avance 1D 2D Avance 1D 2D BRUKER 173 22 Appendix A Artifacts in 2D NMR Experiments 22 1 Introduction 22 1 1 Why do artifacts occure In general an artifact simply is an artificial signal in the spectrum It cannot be correlated to the chemical structure and therefore can mislead the chemist who tries to determine a structure We therefore have to have at least a basic background about typical artifacts occuring in NMR spectroscopy While artifacts
47. of the spectrum In such cases click on cursor and define the reference peak by moving the cursor onto the desired peak and clicking with the middle mouse button Once the spectrum is phased correctly click on return to exit the submenu and save the phase corrections by selecting Save amp return The 0 and 15t order phase correction values are stored as processing parameters phcO and phc1 respectively To quit the phase correction submenu without saving the corrections simply click on return and select return In either case the display returns to the main menu and the spectrum appears on the screen Note that once suitable values of phcO and phc1 have been stored it is possible to use them for phase correcting subsequent spectra by typing the command pk In addition the Fourier transformation t and the phase correction pk can be performed within one step using the command p 3 9 Windowing Before the Fourier transformation is performed it is common to apply a window or filter function to the time domain data The main reason for this is the improvement of either signal to noise or resolution Usually for a simple 1D spectrum as described here the signal to noise ratio is improved by multiplying the FID with a simple exponential function achieved by the command em Avance 1D 2D BRUKER 35 36 The decay rate of the exponential function determines the amount of line broadening This rate is determined by the proces
48. of the spectrum by defining the appropriate 1D plot range Move the cursor into the display window and press the left mouse button to tie the cursor to the spectrum Move the cursor to one side of the desired zoom region and click the middle mouse button to define it Move the cursor to the other side of the desired plot region and click the middle mouse button again to zoom into this region To display the whole spectrum push the KM button 3 8 Phase Correction Once the spectrum is Fourier transformed it must be phase corrected Click On phase to enter the phase correction submenu Click on biggest for setting the reference for the 0 order phase correction to the position of the biggest peak in the spectrum and adjusts its phase To adjust the 0 order phase manually place the cursor on PHO and hold down the left mouse button Move the mouse until the reference peak is positive and the baseline on either side is as flat as possible Most likely the peaks on either side of the reference peak are not yet phased correctly and require a 1 order phase correction To adjust the 1 order phase correction place the cursor on PH and hold down the left mouse button and move the mouse until the peaks far from the reference point are also in phase Note that it is advisable to select the reference peak for the 0 order phase correction near one edge of the spectrum However for some samples the biggest peak will be located in the middle
49. off Enter eda and set the acquisition parameters values as shown in Table 22 The parameters olp and swh should be set as used in test13c 1 1 Set o2p to the exact H offset frequency determined in the previous section around 7 3ppm The DECP90 experiment will not work as described below unless both o1p and o2p are set correctly Table 22 DECP90 Acquisition Parameters Parameter Value Comments PULPROG decp90 see Figure 9 for pulse sequence diagram TD 4k NS 1 DS 0 PL1 high power level on F1 channel C as determined in Section 6 1 4 PL2 high power level on F2 channel H as determined in Section 4 2 4 P1 C 90 pulse as determined in Section 6 1 4 P3 3 start with 3us which should correspond to less than a 90 pulse D1 5 interscan delay bs because of long T4 CNST2 160 Hz heteronuclear scalar J C H coupling D2 3 125 msec 1 2J C H calculated automatically from cnst2 above SWH 1000 Hz o1p SC offset as determined in Section 6 1 2 O2p H offset as determined in Section 6 2 3 Enter zg to acquire the FID the receiver gain should already be set appropriately enter edp and verify the processing parameters as shown in Table 23 BRUKER Avance 1D 2D Table 23 DECP90 Processing Parameters Parameter Value Comments SI 2k LB 1Hz PSCAL global Fourier transform the spectrum with line broadening by the command ef M
50. or rematch it after slight adjustments of the carrier frequency since the probehead is generally tuned and matched over a range of a couple of hundred kHz On the other hand large adjustments to the carrier frequency necessary when changing nuclei warrant retuning and rematching of the probehead Thus a broadband probe needs to be retuned and rematched each time the heteronucleus is changed 24 BRUKER Avance 1D 2D If you have an ATM probe enter edsp and set the spectrometer parameters for the channels that should be matched and tuned For 1H on channel F1 and 13C on channel F2 enter the following values NUC1 1H NUC2 13C NUC3 OFF This automatically sets s o1 to a frequency appropriate for H and s o2 to the corresponding C frequency for tuning and matching Exit edsp by clicking SAVE Type atma This will invoke the automatic match and tune program for all nuclei that were selected previously in edsp Therefore it is not necessary to tune and match manually 2 4 Tuning and Matching H non ATM Probes When the NMR experiments to be performed are H homonuclear experiments e g H 1D spectroscopy COSY NOESY or TOCSY only the H circuit of the probehead has to be tuned and matched Make sure that the sample is in the magnet and the probehead is connected for standard H acquisition Note that there is no special configuration for tuning and matching Also it is recommended to tune and match without sample spinning
51. rates i e it is important to choose a long recycle delay time d1 in order to avoid multiple quantum artifacts in the spectrum A suitable value for this sample is d1 3 Sec Figure 23 DQF COSY Pulse Sequence T 2 T 2 T 2 8 3 2 Acquisition and Processing From the data set cosy 2 1 enter edc and change EXPNO to 3 Click se to create the data set cosy 3 1 86 BRUKER Avance 1D 2D Enter eda and change the following acquisition parameters It is recommended to use a larger value of td in both F1 type 1 td 512 and F2 type td 2k and a larger number of scans ns 16 for a DQF COSY experiment than for a magnitude COSY experiment The pulse program must be set by typing pulprog cosydfph and the FnMODE in the F1 parameter list in the eda table must be set to States TPPI Enter zg to acquire the data The approximate experiment time for the DQF COSY using the acquisition parameters above can be estimated by the command expt and should be 5 5 hours Enter edp and set the processing parameters as shown in Table 39 Table 39 DOF COSY Processing Parameters F2 Parameters Parameter Value Comments SI 2k SF spectrum reference frequency H WDW SINE multiply data by phase shifted sine function SSB 2 choose pure sine wave PH mod pk determine 0 and 1 order phase correction with phasing subroutine PKNL TRUE necessary when using the digital filter BC mod no
52. resulting signals are intense for all H other than the one excited by the selective pulse These signals are eliminated by the same phase cycling as is used in 2D COSY however the corresponding signals in the 2D experiment are much weaker and so are more easily eliminated by the phase cycling Figure 57 Selective COSY Pulse Sequence wes T 2 1 2 p11 d14 pi 20 3 1 Acquisition For best results run selective COSY experiments non spinning Insert the Cyclosporin sample see Section 20 2 and starting from the data set selex 2 1 create the data set selco 1 1 and record a reference H spectrum for the selective COSY experiment with olp set to the N H resonance at 8 1ppm Enter eda and set the acquisition parameters as shown in Table 64 Avance 1D 2D BRUKER 163 Table 64 Selective COSY Acquisition Parameters Parameter Value Comments PULPROG selco see Figure 57 for pulse sequence diagram TD 32k NS 64 number of scans must be 8 n DS 16 PL1 high power level on F1 channel H as determined in Section 4 2 4 P1 H 90 pulse as determined in Section 4 2 4 SP1 shaped pulse power level on F1 channel H as determined in Section 20 2 P11 H 90 shaped pulse as determined in Section 20 2 D1 2 D14 35m delay for evolution after shaped pulse p1 1 2 di4 1 2 JHH PHCOR 1 additional phase correction applied to shaped pulse p11 See Section 20 2 Note th
53. set the acquisition parameters as shown in Table 50 Table 50 HMQC with BIRD Acquisition Parameters F2 Parameters Parameter Value Comments PULPROG hmacbiph HMQC with BIRD for HMQC without BIRD choose hmqcph TD 1k NS 8 the number of scans should be 4 DS 16 number of dummy scans PL1 high power level on F1 channel H as determined in Section 4 2 4 PL2 high power level on F2 channel C as determined in Section 6 1 4 PL12 low power level on F2 channel C for CPD as determined in Section 6 3 7 P1 H 90 pulse as determined in Section 4 2 4 P2 H 180 pulse calculated from P1 P3 C 90 pulse as determined in Section 6 1 4 P4 SC 180 pulse calculated from P3 PCPD2 SC 90 pulse for cpd sequence as determined in Section 6 3 7 D1 1 5 relaxation delay should be 1 5 T H CNST2 145 heteronuclear scalar J C H coupling 145 Hz is a good intermediate value D2 3 45 msec 1 2J C H calculated automatically from cnst2 above CPDPRG2 garp cpd sequence for the C decoupling D7 100 msec delay for inversion recovery optimize Avance 1D 2D BRUKER 121 F1 Parameters Parameter Value Comments TD 256 number of experiments FnMODE States TPPI NDO 2 there are two dO periods per cycle INO t increment SW 190 sw of the C spectrum typically 190 ppm NUC1 select C frequency for F1 14 2 1 Optimize d7
54. set xhcorr 1 1 Enter rga to perform an automatic receiver gain adjustment Acquire and process a standard H spectrum Calibrate the spectrum and optimize sw and o1p so that the H signals cover almost the entire spectral width Acquire an optimized spectrum 12 2 2 Carbon Reference Spectrum A H decoupled C reference spectrum to determine the correct carrier frequency olp and spectral width sw values for C Since XHCORR detects only C directly bonded to H a DEPT 45 spectrum is typically used 110 BRUKER Avance 1D 2D as a C reference spectrum Enter re dept 1 1 to call up the data set dept 1 1 enter ede and change the following parameters NAME xhcorr EXPNO 2 PROCNO 1 Click _s to create the data set xhcorr 2 1 Enter rga to perform an automatic receiver gain adjustment Acquire and process a C spectrum Calibrate the spectrum and optimize sw and olp so that the C signals cover almost the entire spectral width Acquire an optimized spectrum 12 2 3 Acquire the 2D Data Set Type xau iexpno increment experiment number to create the data set xhcorr 3 1 Enter eda and set PARMODE to 2D Click on EN and ok the message Delete meta ext files The window now switches to a 2D display and the message NEW 2D DATA SET appears Enter eda and set the acquisition parameters as shown in Table 47 Table 47 XHCORR Acquisition Parameters Parameter Valu
55. spectrometer parameters and return to the main window Set o2p to the o1p value of the C determined in Section 6 3 3 Enter eda and set the acquisition parameters as shown in Table 28 Table 28 DECP90 Acquisition Parameters Parameter Value Comments PULPROG decp90 see Figure 11 for pulse sequence diagram TD 8k NS 1 DS 0 PL1 high power level on F1 channel H as determined in Section 4 2 4 PL2 high power level on F2 channel C as determined in Section 6 1 4 P1 H 90 pulse as determined in Section 4 2 4 P3 0 for the phase correction do not use p3 D1 5 relaxation delay should be 1 5 T H CNST2 214 heteronuclear scalar J C H coupling Hz D2 3 34ms 1 2J C H calculated automatically from cnst2 above SWH 1000 Hz RG use value from testinv 2 1 olp 7 24ppm H offset frequency of chloroform peak as determined in Section 6 3 4 02p 77ppm SC offset frequency of chloroform peak as determined in Section 6 3 3 Enter zg to acquire the FID and enter edp to set the processing parameters as shown in Table 29 Table 29 DECP90 Processing Parameters Avance 1D 2D Parameter Value Comments SI 4k LB 0 3 Hz PSCAL global BRUKER 63 6 3 6 6 3 7 64 Fourier transform the spectrum ef Expand the spectrum to display the region between 8 5 to 7 5 ppm in the region of the chloroform peak with it s two C s
56. the NOE effect is suppressed for this experiment and thus the acquired spectrum can be integrated Note that there is a build up of the NOE effect during the acquisition period when decoupling is active In order to suppress this NOE effect the relaxation delay must be 10 times the T relaxation time for C The pulse sequences for the gated decoupling and the inverse gated decoupling experiments are shown in Figure 15 and Figure 16 respectively However before acquiring a H decoupled C spectrum the frequencies of the cholesterylacetate H signals must be determined See Section 5 3 for the determination of the exact H carrier frequency As a rule of thumb 5 ppm is a safe frequency to select for H decoupling when no optimized H spectrum is available Figure 15 C Pulse Sequence with Gated 1H Decoupling T 2 13C 1H decoupling Avance 1D 2D BRUKER 69 70 Return to the carbon 3 1 data set by entering re carbon 3 1 Enter ede and set EXPNO to 4 Click on swe to create the data set carbon 4 1 for the H gated decoupled C spectrum Enter edsp and set NUC2 to 1H Set o1p to the value determined from the H spectrum or to 5 ppm Enter eda and set the acquisition parameters as shown in Table 32 Table 32 C Acquisition Parameters with Gated and Inverse Gated 1H Decoupling Parameter Value Comments PULPROG zggd zggd for gated decoupling zgig zgig for inverse gated
57. tuned and matched as described above Alternatively if the user already has a data set in which NUC1 1H and NUC2 OFF there is no need to redo edsp for the current data set The user may simply read in the H data set and then type wobb Once the probehead is tuned and matched for C and H exit the wobble routine by typing stop Click on reum to exit the acquisition window and return to the main window Avance 1D 2D BRUKER 27 2 6 Locking and Shimming Before running an NMR experiment it is necessary to lock and shim the magnetic field 2 6 1 Locking To display the lock signal enter Lockdisp This opens a window in which the lock trace appears The most convenient way to lock is to use the XWIN NMR command lock To start the lock in procedure enter lock and select the appropriate solvent from the menu Alternatively enter the solvent name together with the lock command e g lock cdc13 During lock in several parameters such as the lock power the field value and the frequency shift for the solvent are set according to the values in the lock table This table can be edited using the command edlock Note that the lock power listed in this table is the level used once lock in has been achieved The field shift mode is then selected and autolock is activated Once lock in is achieved the lock gain is set so that the lock signal is visible in the lock window At this point the message lock finished appears in the st
58. usec to 40 usec The procedure outlined below uses the paropt routine to determine the corresponding power level However the power level can be estimated roughly by using a rule of thumb The pulse length doubles for an additional 6 dB increase of the power level For example the 90 pulse length p1 was determined 8 usec for p11 0 dB Thus the p1 16 usec for p11 6 GB or the p1 32 usec for p11 12 dB 42 BRUKER Avance 1D 2D For performing the exact determination of the low power pulse return to the file test1h 1 1 re 1 1 Enter p1 and change the value to 35 usec type xau paropt and answer the questions as follows Enter parameter to modify pl1 Enter initial parameter value 0 Enter parameter increment 1 Enter of experiments 16 Again the 16 spectra will be displayed in the file test1h 1 999 and at the end of the experiment the message paropt finished and a value for p11 is displayed This value corresponds to the H transmitter power level for a 90 pulse length of 35 usec Write down this value and follow the procedure described below to obtain a more accurate 90 pulse measurement Return to testih 1 1 re 1 1 type p1 and change the value to 140 usec 360 pulse Acquire and process a spectrum zg e p by using the power level p11 determined by paropt above Change p11 slightly until the quartet undergoes a zero crossing indicating the accurate 360 pulse Divide this 360 pulse time by four to get the
59. without sample spinning Record a H reference spectrum to determine the correct values for olp and sw A H reference spectrum of this sample was already created for the magnitude COSY experiment Section 8 2 2 This spectrum is found in the data set cosy 1 1 The ROESY data set can be created from the data set of any of the previous homonuclear 2D experiments run on this sample For example enter re cosy 2 1 to call up the data set cosy 2 1 Enter edc and change the following parameters NAME roesy EXPNO 1 PROCNO 1 Click se to create the data set roesy 1 1 Enter eda and set the acquisition parameters as shown in Table 43 The pulse p15 at p111 sets the length of the cw spinlock pulse The value listed in Table 43 is appropriate for this sample For other samples with different relaxation properties optimal results may be achieved with slightly different values The typical range for p15 is from 50 to 300 msec A good rule of thumb is that p15 for the ROESY experiment of a molecule should be about the same as d8 for the NOESY experiment of that molecule Table 43 ROESY Acquisition Parameters F2 Parameters Parameter Value Comments PULPROG roesyph TD 1k NS 32 the number of scans must 8 n DS 16 number of dummy scans PL1 high power level on F1 channel H as determined in Section 4 2 4 PL11 low power level on F1 channel H for spinlock as determined in Section 4 2 6 P1 H 90 pu
60. 180 degree pulse in the middle of the evolution period spin echo sequence During the evolution period the different components of the carbon multiplets precess at their individual frequencies During the half of the evolution period the decoupler is OFF to introduce J modulation in the spectrum The length of the evolution period controls the amplitude of the carbon signal Normally the evolution period is set to 1 Jcu then the CH and CH3 groups appear as positive peaks while those from CH2 and quarternary carbons are negative Compared to the DEPT experiment all carbon nuclei are visible in one spectrum Figure 19 APT Pulse Sequence x T HG CPD CPD pl p2 d1 d20 d20 acq References D W Brown T T Nakashima and D L Rabenstein J Magn Res 45 302 1981 S L Patt and N Shoorly J Magn Reson 46 535 1982 A M Torres T T Nakashima and R E D McClung J Magn Reson 101 285 1993 7 3 1 Acquisition and Processing Insert the sample in the magnet Lock the spectrometer Readjust the Z and Z shims until the lock level is optimized Tune and match the probehead for 18C observation and H decoupling 7 3 2 Reference Spectra Since APT is a C observe experiment with H decoupling a reference H spectrum of the sample must be recorded to determine the correct o2p for H Avance 1D 2D BRUKER 77 decoupling Then a H decoupled C spectrum must be recorded to determine the correct olp and sw for the AP
61. 22 2T iv 22 1 2 2 1 iv 2T iN 4 C08 Ju e Vim 27 v e 2n ivn 2n ivn 2 2 Te e TN niv sna Jo t ME ER fe 1 2q ivola 2n dea 2q 472 27 dex is e 2 e 2 _ diee 2 2m i V 2 2m i V ys e i Qn de ya Qn i241 Qn ra Qn dies Sia cm e 2 e 2 e 2 2 2n iV 74245 2n ivn 2 e 2 This signal function is not quiet what we expected first the signals are mirrored in the ti dimension and second the cross peak is in the imaginary part of the spectrum What is the problem Avance 1D 2D BRUKER 201 To be able to distinguish between positive and negative signals we need both the sine and the cosine modulation This is true for the te domain in the above signal function but not for the t part where we only have the sine modulation of the chemical shift What can be done In case of the two pulse COSY fortunately this is quite simple we repeat the experiment but apply the second pulse now around the x axis instead of the y axis After a lot of painstaking manipulations we finally find for the sequence 90 t 90 t2 0 7 l co Jo t cog t cok Jo 4 cos 1 I cost J t sin6 t cost J t cosd 1 1 sint J t cosd t sinf Jh cos 1 L sinf Jgh sin t sint J t cos t Eus compared with the sequence 90 t 90 t 0 I cost J 1 sin6 t cost Jat sing 1 1 cost Jiz h cos t cost J t sint L sinf J ph sin t sinf J t sin6 1 L sinft J
62. 4 2 6 Calibration Low Power for ROESY Spinlock rennes 43 5 BASIC PC ACQUISITION AND PROCESSING see 45 S INTRODUGTION 4 etienne nn ne in OV EO d ed da D e CHO 45 Avance 1D 2D BRUKER Dou ced SG M IEAMIINIEL DUE MUI E NM IUE 45 Iiz Prepare a NEW Data Set esses isos cess e piece tu e nn a quate ataca nent Reve Re nue 45 5 2 ONE PULSE EXPERIMENT WITHOUT IH DECOUPLING oeste retrasa Ra LB be CHER CU RD 45 5 3 ONE PULSE EXPERIMENT WITH IH DECOUPLING 2 32e nacio nmemdona ien a e DR RR Fee een int 48 6 PULSE CALIBRATION CARBON seeeeeeeenenennnnnnnnnnnnes 51 6 1 CARBON OBSERVE 902 BULSE 2 5 ettet ette e e eate PEEL e ETIN irai e N ai eirt en ee NEUE tenue 51 OlT Preparationisscuxuse ee ete e et eee cede ee RR Reden ee rte dene de Rude edes 51 6 1 2 Optimize the Carrier Frequency and the Spectral Width ss 52 6 1 3 Define the Phase Correction and the Plot Region ee eese 52 6 4 Calibration High Power eter ete ege e ne eH eer Cete dite as 52 6 2 PROTON DECOUPLING 90 PULSE DURING P ACQUISITION ses ecetetl e os tes teneo eet leet pede ete tee decode oe 54 6 2 T Samples ette eee essen es dece t sites 54 6 2 2 Pulse S quence oce ee oerte tel Ee i Pe entere Dee ee o Rees 54 6 2 3 Set mie H Carrier Frequefey s eov oe eda nation ieee iota deris MAN erie 54 6 2 4 Set the C Carrier Frequency and the Spectral Width nee 55 6 2 5 Calibration High PoWer e e e e ARS LR RE A Pep beau
63. 60ppm For HMBC NUC1 select C frequency for F1 Gradient Parameters for the gp syntax Parameter Value Comments P16 1 5ms Length of gradient pulses D16 150u Gradient recovery delay gpzi 50 For HMQC and HMBC 80 For HSQC gpz2 30 For HMQC and HMBC 20 1 For HSQC gpz3 40 1 For HMQC and HMBC gpnami SINE 100 gradient shape gpnam2 SINE 100 gradient shape gpnam3 SINE 100 gradient shape With the acquisition parameters shown above the approximate experiment times are 0 3h for HMQC and HSQC and 1 2 hours for HMBC Process the data according to Sections 14 3 and 15 2 respectively except that for the HSQC the parameter MC2 must be set to echo antiecho Figures 43 45 show GRASP HMQC GRASP HMBC and GRASP HSQC spectra of Cyclosporin in benzene Avance 1D 2D BRUKER 133 Figure 45 H C GRASP HMQC experiment of 50mM Cyclosporin in C6D6 Figure 46 H C GRASP HMBC experiment of 50mM Cyclosporin in CDs 134 BRUKER Avance 1D 2D ppm 20 40 60 80 100 120 140 160 he te T sa v 180 85 80 75 70 65 60 55 50 45 40 35 30 25 20 15 10 ppm 75 7 0 6 5 6 0 55 50 43 49 33 3 0 2 5 2 0 15 10 ppm Avance 1D 2D BRUKER 135 17 1D NOE Difference 17 1 Introduction The Nuclear Overhauser Effect NOE is a net change of the signal intensity from one spin due to the relaxation of a saturated spin that is dipole dipole coupled to the first spin NOE s develop due to through space rather than through bond interactions
64. 90 pulse length Note that the parameters used by the TOCSY sequence are p6 for the 90 pulse length and p110 for the power level rather than p1 and p11 4 2 6 Calibration Low Power for ROESY Spinlock The power level required for the cw spinlock pulse used with ROESY experiments corresponds to a 90 pulse length of 100 usec to 120 usec As described for the 90 pulse determination of the MLEV pulse above in Chapter 4 2 5 the power level can again be estimated using the rule of thumb or measured using the paropt automation When using paropt return to the file testih 1 1 re 1 1 enter p1 and change the value to 110 usec and type xau paropt Answer the questions as follows Enter parameter to modify pl1 Enter initial parameter value 10 Enter parameter increment 1 Enter of experiments 16 The results are displayed in the file testth 1 999 and at the end of the experiment the message paropt finished and a value for pli corresponding to the H transmitter power level for a 90 pulse length of 110 usec are displayed Follow the same procedure as described in Chapters 4 2 4 and 4 2 5 for a more accurate determination of the power level Note that since ROESY uses cw spinlock only the power level determination is important here but not the actual 90 pulse length The power level parameter used with the ROESY sequence is p111 rather than p11 Avance 1D 2D BRUKER 43 44 BRUKER Avance 1D 2D 5 Basic C acq
65. CESSING sieste teet iret etes Rd cepe Dee Ee EA CE E ceo E Ne swe PER EL ER HERE UR De ge e re eR EO Roe nont 9 4 PHASE CORRECTION si 95 PLOT THE SPECTRUM a a fie E ies eee a E EEEN 10 1 INTRODUCTION 4 5rd re reb decet ah ite Gh Pea dea eh fele ee rc et eade e p DR eeu 99 10 2 ACQO ISITION arte cr rr re chere trt e Gh ch ha tes eta eae be ee rete cie a a a aed 100 10 3 PROCESSING A 10 4 PHASE CORRECTION AND PLOTTING cscssccscssssessecsscessecesescessecssecseessecesecssesesecsseceeseceseceeesseeeseceeeeeseeeneneees 102 iB P INTRODUCTION TP M seis 11 2 ACQUISITION AND PROCESSING 11 2 1 Optimize Mixing Time z 112 2 Acquire the 2D data Set 32e tede d RUE Rede dg EE NS dei IB NEN PROCESSING eme M Etes 11 4 PHASE CORRECTION AND PLOTTING esee eene nnne enne nne nn nen tn enne nn nennen sinn s enne nennen ennt 107 12 1 INTRODUCTION 12 2 ACQUISITION 5 dnd eh d eae E Pe a e ee ORE ERE Ro REO d CHER RE 12 2 1 Proton Reference Spectrum liked te e D ate dee a d eade ate e dese 110 12 2 2 Carbon Reference Spectrum sense 110 12 2 3 Acquire the 2D DataSet isset hits net tete di deed eed 111 12 3 PROCESSING eccentric terere tft hdd de der edet cft tee ee cde echo 112 12 4 PEOTTING THE SPECTRUM ect fregit eee ce eee tede eee teet 113 T3 L INTRODUCTION nina nn A ee needed 115 13 2 ACQUISITION AND PROCESSING esee eene nenn
66. COSY Acquisition Parameters F2 Parameters Parameter Value Comments PULPROG cosygpmfph TD 2K NS 4 DS 16 PL1 high power level on F1 channel H as determined in Section 4 2 4 P1 H 90 pulse as determined in Section 4 2 4 DO 3u incremented delay ti predefined D1 3 relaxation delay should be about 1 25 T H 90 BRUKER Avance 1D 2D Enter zg to start the DQF COSY experiment With the acquisition parameters Gradient Parameters for the gp syntax Parameter Value Comments P16 1 5m Length of gradient pulses D16 150u Gradient recovery delay gpzi 10 of the maximum gradient amplitude gpz2 20 of the maximum gradient amplitude 20 for double quantum selection 30 for triple quantum selection gpnami SINE 100 Gradient shape gpnam2 SINE 100 Gradient shape F1 Parameters Parameter Value Comments TD 512 number of experiments FnMODE TPPI NDO 1 there is one dO period per cycle INO t increment equal to 2 DW used in F2 SW sw of the optimized H spectrum cosy 1 1 same as for F2 NUC1 select H frequency for F1 same as for F2 shown above the approximate experiment time is 1h Enter edp and set the processing parameters as shown in Table 39 for the conventional DQF COSY except that the F1 parameter MC2 must be set to TPPI instead of States TPPI Enter xfb to multiply the time domain data by the
67. ECTROMETER AND ACQUISITION PARAMETERS cssccsssssesseceseeseeceseceeeseeesecseeseensesaceesecessesseeeseeneesseenesnnees 32 3 3 CREATE A NEW FILE DIRECTORY FOR THE DATA SET eene nennen entente nennen 32 3 4 SET UP THE SPECTROMETER PARAMETERS sees ennt te ennt nn serere nne e reet nenne teens nennen 32 3 5 SET UP THE ACQUISITION PARAMETERG ccsscsssessesseeseenseceseeesecseeeseeesseceeseensecacesseeeseseesseseseeeeeseeaseeseeeeaeeages 33 3 6 ACQUISITION duc hire fee d etre endete ce ec seeded eet tee ea recede te toledo rede de reser ied eden defe ied 34 341 Mie ides T 34 3 8 PHASE CORRECTION ies recense ml te bete ee lege et repe eee ee teuer eges 35 3 9 WINDOWING ae hil decret rete ee dames teens dede tune dec ee A dite red E sales ed te teen mettons tend dec ae 35 SIBI Uer PH 37 4 PULSE CALIBRATION PROTONS ennnnnnnnnnsnnnnnnese 39 4 F INTRODUCTION io awem estende nee eon d Da e da do veo E AR ees E e TORRE 39 4 2 PROTON OBSERVE 90 PULSE eese e e a x e Y REG e Fab Ga e e d ERR ED Re Ea ecu a 39 4 2 he Preparation isse uie be eec titt e EE E TERRE NEED TERI ETE EGRE ETE YU S 39 4 2 20 Optimize the Carrier Frequency and the Spectral Width ss 40 4 2 3 Define the Phase Correction and the Plot Region rennes 41 4 2 4 Calibration High Power 4 2 5 Calibration Low Power for MLEV Pulse Train TOQCSY sse 42
68. F2 The receiver gain is already set correctly Enter zg to acquire the data which requires about 1 4 hours This can be estimated previously by entering expt into the command line 8 2 3 Processing of the 2D COSY Spectrum Enter edp and set the processing parameters as shown in Table 38 Table 38 COSY Processing Parameters BRUKER 84 F2 Parameters Parameter Value Comments Sl 512 SF spectrum reference frequency H WDW SINE multiply data by phase shifted sine function SSB 0 choose pure sine wave PH_mod no this is a magnitude spectrum PKNL TRUE necessary when using the digital filter BC_mod quad F1 Parameters Parameter Value Comments Sl 512 SF spectrum reference frequency H WDW SINE multiply data by phase shifted sine function SSB 0 choose pure sine wave PH_mod mc this is a magnitude spectrum BC_mod no MC2 QF determines type of FT in F1 QF results ina forward quadrature complex FT Enter x b to perform the 2D Fourier transformation For the magnitude COSY sine type window functions are selected to suppress the diagonal peaks relative to the cross peaks Such a window function is also resolution enhancing which is appropriate for a magnitude mode 2D spectrum Adjust the threshold level by placing the cursor on the button holding down the left mouse button and moving the mouse up and down Since this is a magnitude spectrum
69. Griffey and B L Hawkins J Magn Reson 55 301 1983 A Bax and S Subramanian J Magn Reson 67 565 1986 The sample used to demonstrate HMQC in this chapter is 50 mM Cyclosporin in CDe This is the same sample that was used to demonstrate COSY NOESY ROESY and TOCSY The HMQC pulse sequence is shown in Figure 37 which should be used on samples consisting of proteins and other macromolecules The first H pulse creates transverse magnetization some of which evolves into anti phase magnetization at the end of the first 1 2JxH delay This anti phase magnetization is converted into multiple quantum coherence by the 1 2 x pulse and evolves chemical shift during ti In analogy with XHCORR a delay 1 2Jxp is inserted between the final 90 pulse after t and the start of the acquisition so that C decoupling can be used during acquisition Without this delay the H magnetization components would be anti phase at the start of the acquisition and so C decoupling would result in mutual cancellation of the H signals Note that since it is the longitudinal H magnetization present before the first 1 2 u pulse that is converted into heteronuclear multiple quantum coherence itis the H T which determines the appropriate recycle delay Thus it is possible to use a shorter recycle delay for HMQC than for XHCORR For small molecules it is useful to use a BIRD preparation period in conjunction with the HMQC experiment Figur
70. In order to get well resolved multiplets along the F1 dimension of that experiment a high digital resolution is required in the F1 dimension It might be necessary to do a zero filling in the F1 dimension of a factor 16 32 Figure 65 Homonuclear J Resolved experiment of pamoic acid The processing size was set to 2048 points for an experiment were 64 points were collected in the F1 dimension red trace The spectrum obtained with a processing size of 256 points is shown in green O 65 Se gt without tilting with tiIting too low digital resolution Avance 1D 2D BRUKER 179 22 4 Inverse Experiments 22 4 1 Incorrect proton pulses 180 A common artifact of the inverse experiments HMQC and HMBC is caused by an incorrect proton pulse The 180 proton pulse in those experiments is used to refocus the chemical shift evolution of protons If the proton pulses are set incorrectly the chemical shift of protons during the t1 evolution period is not refocussed As a result additional peaks will show up along the F1 dimension Those artifacts can easily assigned as their distance from the correct correlation signal increased with the distance to the centre of the spectrum which is O1P Figure 66 HMQC experiment with pamoic acid Top spectrum is the reference the spectrum on the bottom shows artifacts along the FI dimension due to incorrect proton pulses BRUKER Avance 1D 2D 22 4 2 Rapid scanning artifacts The HSQC
71. LIST z name of vdlist used for z filter PHCOR 1 phase correction applied to shaped pulse P11 Perform a routine acquisition with zg BRUKER 167 20 4 3 Processing Enter edp and set the processing parameters as shown in Table 67 Table 67 Selective TOCSY Processing Parameters Parameter Value Comments SI 16k WDW EM LB 0 30 PKNL TRUE necessary when using the digital filter Add line broadening and Fourier transform the time domain signal with the command e Manually phase correct the spectrum using the 0 order phase correction The resulting spectrum should look like that in Figure 60 Figure 60 Selective TOCSY Spectrum of 5 0mM Cyclosporin in C D 9 5 9 0 8 5 8 0 7 5 7 0 6 5 6 0 5 5 5 0 4 5 4 0 3 5 3 0 2 5 2 0 1 5 1 0 0 5 ppm 168 BRUKER Avance 1D 2D Avance 1D 2D BRUKER 169 21 IconNMR NMR Automation IconNMR is the Bruker NMR Automation Software tool It offers a quick and simple approach to NMR also for unexperienced users via the consequent use of predefined parameter sets To start IConNMR type iconnmr in the XWinNMR command line and click on the Automation button ICON NMR on N162 You need an IconNMR user account in order to run NMR experiments under the IconNMR control Such an account can be created activated and administrated in the Configuration window of IconNMR under User Manager For a detailed description of the IconNMR configuration and a
72. REE SPIN SYSTEM esse 198 23 10 THE COS Y EXPERIMENT isa orere eer ereraa paer Seer SR Te lle 199 23 11 SUMMARY AND USEFUL FORMULAE eret enne nennen nennen nnns asorr a aeae siie ireren eoor 204 23 11 1 Effects on Spins in the Product Operator Formalism eese 204 23 112 Mathematical Relations de te teet n teet te ti ete RS 205 Avance 1D 2D BRUKER BRUKER Avance 1D 2D 1 Introduction This manual gives an introduction into basic one and two dimensional nuclear magnetic resonance NMR spectroscopy After a short introduction the acquisition of basic 1D H and C NMR spectra is described in the Chapters 3 to 7 Homonuclear 2D H H correlation spectra are described in Chapter 8 COSY 9 TOCSY 10 ROESY and 11 NOESY Heteronuclear 2D C H correlation experiments are described in Chapter 12 XHCORR 13 COLOC 14 HMQC and 15 HMBC The Chapter 16 contains the description of inverse 2D C H correlation experiments using pulsed field gradients and some special NMR experiments are described in chapters 17 to 20 A brief introduction to NMR automation with the IconNMR program is given in chapter 21 1 1 An Important Note on Power Levels Several times throughout this manual the user is asked to set the power levels p11 p13 etc to the high power level for the corresponding channel f1 or f2 In order to avoid damaging the probehead or other hardware components the user is adv
73. ROTON TEST neri ertet eret ettet de ede eee Heec dede tede eerte trece eed rede reta TT Z3 JACQUISINON aNd Processing tud teret ere rete certe eee se ee eds 77 7 3 2 EREFETENCES peera o oo eee e erdt e E EE UE ee EU SNS 7 3 59 Create a New Data Set LS A VSDECIRUIm ACQUISIO oec t e eed oe MESSE eter RER D I e eee ne 73 97 Processing of theSpectrulnz s sire e ER tete ie teet re wa es 79 7 3 6 Plotthe Spectra et tbe te ex ARE tee oue ee rte ns 79 e L Y 81 8 L INTRODUCTION TEN 81 82 MAGNITUDE COSY Sn in ee nee ode tote eese ene tete notte 81 G2 Pulse S quence sn es e De epe tene e ee che Heind 82 8 2 2 Acquisition of the 2D COSY Spectrum nee 62 8 2 3 Processing of the 2D COSY Spectrum rennes 63 O24 Plottngthe SDeCtEUm 2 e eerte e ere este tee lette en eet open trees 85 8 3 DOUBLE QUANTUM FILTERED DQF COSY nee 86 Orde s Pulse Sequente sete EL eese et eis BIRR SD dte e 8 3 2 Acquisition and Processing 8 3 3 Phase correct the spectrum 6 34 PlottlesSpectirulmis so tee cte m een e ee ite ee EE 8 4 DOUBLE QUANTUM FILTERED COS Y USING PULSED FIELD GRADIENTS GRASP DQF COS Y 89 SA Pulse Sequence iri od eo ee Ne ee ee Peli es ee te dte e aly Le e SERERE 89 4 BRUKER Avance 1D 2D 0 42 cAcquisition and Processilg au iter IER ete deo ERRORES NE Re EE NE nina 90 9 INTRODUCTION 9 2 ACQUISITION ER T 9 3 PRO
74. ST2 145 heteronuclear scalar J C H coupling 145 Hz is a good intermediate value D2 3 45 msec 1 25 C H calculated automatically from cnst2 above CPDPRG2 waltz16 cpd sequence for the H decoupling Acquire a DEPT 45 spectrum by either selecting the pulse program dept45 type pulprog dept45 or set pO to the length of a 45 pulse pulse type pO and enter the value of 0 5 p1 at the prompt Enter zg to acquire the data the receiver gain should already be set correctly if this data set was created from carbon 3 1 BRUKER Avance 1D 2D 7 2 5 Processing of the Spectrum Enter edp and set the processing parameters as shown in Table 34 Table 34 DEPT Processing Parameters Parameter Value Comments SI 16k WDW EM exponential multiplication LB 2 2 Hz line broadening PKNL TRUE necessary when using the digital filter Add line broadening and Fourier transform the time domain data with the command ef Manually phase correct the spectrum so that all peaks are positive The signals in this spectrum arise from the C nuclei in CH CH and CHs groups 7 2 6 Other spectra To obtain a DEPT 90 spectrum create the data set dept 2 1 and either select the pulse program dept90 type pulprog dept90 or set pO to the length of a 90 pulse type pO and enter the value of p1 at the prompt Acquire zg and process efp the data Only signals from CH groups are visible in this experim
75. T experiments However both steps were already carried out in Section 5 3 a H decoupled C reference spectrum of this sample can be found in carbon 3 1 7 3 3 Create a New Data Set Enter re carbon 3 1 to call up the reference spectrum Enter ede and change the following parameters NAME apt EXPNO 1 PROCNO 1 Click w to create the data set apt 1 1 7 3 4 Spectrum Acquisition Enter eda and set the acquisition parameters as shown in Table 35 Table 35 APT Acquisition Parameters 78 Parameter Value Comments PULPROG jmod spin echo experiment TD 32k NS 4 the number of scans must be 4 ns DS 4 number of dummy scans PL1 high power level on F1 channel C as determined in Section 6 1 4 PL2 high power level on F2 channel H as determined in Section 4 2 4 PL12 low power level on F2 channel H for CPD as determined in Section 6 2 6 P1 C pulse as 90 determined in Section 6 1 4 P2 C 180 pulse calculated from P1 PCPD2 H 90 pulse for cpd sequence as determined in Section 6 2 6 D1 2 relaxation delay should be 1 5 T C CNST2 140 heteronuclear scalar J C H coupling 140 Hz is a good intermediate value CNST11 1 X XH2 positive XH XH3 negative 2 only X D20 7 14 msec 1 1J 76 H calculated automatically from cnst2 cnst11 above CPDPRG2 waltz16 cpd sequence for the H decoupling BRUKER Avance 1D 2D 7 3 5 Processin
76. Three Spin System Our spin system shall include three spins of the same type all three being coupled with each other and two coupling constants should be identical H o 61 6 I 27 Jo gm 27 Ja LL 27 JA LB with J122J13 J The full set of spin operators includes 4 64 elements and we certainly don t want to mess with that many operators The first simplification consists in only considering one spin e g instead of using the full equilibrium density matrix we use only a reduced form In this way we will obtain the signal originating from that particular spin only Most of the time this is absolutely sufficient In our example we will look at spin 1 only 90 gt O aq h Co I 1x Again the Hamiltonian is split it into chemical shift terms and scalar coupling terms which are the applied subsequently But this time we will only keep the terms including ly knowing that the other terms will not have any effect On Go 9l 2 7 Jy TL 2T Jy Tubs Applying this reduced Hamiltonian to oo yields 0 7 l cos t J t cos t Ji t cos 0 t I cos t J t cost J 1 sin t 21 1 sin t Ji 1 cos t Ji 1 cos 0 t J t sin O t 2I L cos t Jj t sint J t cost f 21 J cosa Jg t sini J t sin 6 t 47 J 1 sin Ja t sina J t cos6 t 41 1 J sin t J t sin a Jj t sin f 21 1 sin t J 1 cos t 198 BRUKER Avance 1D 2D The c
77. User kti Holder Type Statues Dak vi Auth NNNM By clicking on the Par button in the setup window 2 red circle in the image above you can modify the parameters that are defined for this user in the IconNMR user specific configuration To submit the experiment press the submit button in the automation window This will either start the sample changer automation or you will be prompted to insert the respective sample manually depending on your configuration Avance 1D 2D BRUKER 171 According to your settings in the IconNMR software configuration IconNMR will then perform the automatic tuning and matching routine lock the spectrometer on the selected solvent shim the sample run the data acquisition and do the spectrum processing automatically It will also generate a plot of the spectrum Certain experiments also require a so called preparation experiment These are for instance 2D experiments where a 1D proton experiment is run as a preparation experiment for the sweep width optimization in the direct dimension These experiments are called composite experiments If you select one of those the preparation experiment will automatically be set up to be run before the 2D experiment In the example below a HSQC experiment was selected and the proton experiment was automatically set up in front Uz Bruker 35 prog curdir changer inmechanger Feb18 2002 1350 kti e xj Ble Run
78. al NMR experiment consisted of applying a variable field B to the sample for the observation of the absorption spectrum so called continuous wave method Double resonance or homonuclear decoupling is the term used for experiments in which a field B2 in addition to B is applied to the sample On modern FT spectrometers equipped with a single coil probe head this decoupling requires a special mode in order to apply the decoupling energy B2 during the acquisition The so called hd mode is applying the energy in pulsed mode within the duty cycle of the dwell time and the preamplifier is switched off during the decoupler pulses The underlying principles of homonuclear decoupling can be illustrated by considering e g the molecule ethyl benzene and coupling pattern of the ethyl group Irradiation of the methylene group will result in the collapse of the methyl group to a singulet and vice versa Figure 50 Figure 51 Spectra of ethyl benzene without and with homo decoupling 144 BRUKER Avance 1D 2D Suitable samples to setup homo decoupling are the proton sensitivity sample 0 1 ethyl benzene in CDCl3 or 100mM pamoic acid in DMSO de 18 2 Acquisition Insert the sample in the magnet Lock the spectrometer Readjust the Z and Z shims until the lock level is optimized Tune and match the probehead for H observation This experiment can be run with sample rotation 18 2 1 Create a new file directory Enter re proton 1 1 to call u
79. and thus contain information on the distances between spins The rate or efficiency of the NOE buildup depends on the rate or efficiency of the dipole dipole relaxation which itself depends on the strength and frequency of the fluctuating fields These fluctuating fields depend on the distance between the nuclei involved the tumbling rate of the molecule and the characteristics of the nuclei themselves The presence of paramagnetic molecules e g metal ions rust or dissolved oxygen distorts any NOE experiment since they dominate T relaxation processes In an NOE difference experiment a H resonance is selectively preirradiated until saturation is achieved During the preirradiation period NOE buildup occurs at other H nuclei close in space A 7 2 pulse then creates observable magnetization which is detected during the acquisition period The experiment is repeated using different preirradiation frequencies including one which is off resonance The latter is used to obtain a reference or control spectrum The final spectra are displayed as the difference between a spectrum recorded with on resonance preirradiation and the reference spectrum Very small phase or frequency shifts between two spectra will give rise to imperfect signal subtraction To minimize subtraction artifacts an efficient signal averaging and maximal acceptable line broadening should be used Other artifacts from temperature instability or magnetic field drift may be
80. anually phase correct the spectrum so that the left peak is positive and the right peak is negative and store the correction The spectrum is already calibrated if the current data set was created from test13c 1 1 Since paropt is not used here neither the phase correction nor the plot region have to be defined 6 2 5 Calibration High Power The H 90 decoupling pulse length should be close to the H 90 observe pulse length for the same power level Set p3 to the value found in Section 4 2 4 and acquire and process a spectrum zg efp If the H 90 pulse p3 is less than 90 the left peak will be positive and the right peak negative If the pulse angle is between 90 and 270 the phase of the two peaks will be opposite If the p3 pulse length corresponds to a 90 pulse the signals show a zero crossing as shown in Figure 10 Avance 1D 2D BRUKER Figure 10 1H Decoupling 90 Pulse Calibration p3 gt 90 p3 90 p3 90 133 131 129 127 ppm 6 2 6 Calibration Low Power for WALTZ 16 Decoupling The WALTZ 16 composite pulse decoupling cpd sequence requires a H 90 decoupling pulse length of 80 to 100 usec Adjust p12 and p3 to determine the 90 pulse length in this range Make use of the rule of thumb 90 pulse length approximately doubles for an additional 6 dB increase in attenuation Note that the parameters used by cpd sequences are pepd2 for the 90 pulse length and p112 for the decoupler power leve
81. asurement Return to the data set test13c 1 1 by entering re 1 1 Type p1 and enter a value corresponding to a 360 pulse i e four times the 90 value determined by paropt above Acquire and process another spectrum zg efp Change p1 slightly acquire and process a spectrum again until the doublet shows a zero crossing indicating the 360 pulse The 360 pulse time divided by 4 is the exact 90 pulse length for the C transmitter for the power level p11 Note that the probe may arc if the 90 pulse length is less than 5 usec for 5 mm probes and less than 10 usec for 10 mm probes In this case the p11 must be set to a higher value increase the attenuation on the transmitter and the corresponding 90 pulse length must be determined again Figure 8 Paropt Results for C 90 Pulse Calibration p1 180 p1 270 W Lund M p1 90 Avance 1D 2D BRUKER 53 6 2 Proton Decoupling 90 Pulse During C Acquisition Ideally this procedure is carried out immediately following the C observe pulse calibration described above since the magnet is then locked and shimmed and the probehead is tuned and matched for both C and H 6 2 1 Sample For the H decoupling pulse calibration the sample must yield a C signal of reasonable intensity with a detectable scalar C H coupling Therefore 80 Benzene in Acetone d6 is a good choice 6 2 2 Pulse Sequence The pulse sequence
82. at in this pulse sequence the delay d14 is to ensure that the magnetization is antiphase when the second pulse is applied This is accomplished by choosing d14 such that p11 2 d14 1 2Jun Perform a routine acquisition with zg The approximate experiment time for Selective COSY with the parameters set as shown above is 4 minutes 20 3 2 Processing Enter edp and set the processing parameters as shown in Table 65 Table 65 Selective COSY Processing Parameters Parameter Value Comments SI 16k WDW EM LB 0 30 PKNL TRUE necessary when using the digital filter Add line broadening and Fourier transform the time domain signal with the command ef Manually phase correct the spectrum The resulting spectrum should look like that inFigure 58 164 BRUKER Avance 1D 2D Figure 58 Selective COSY Spectrum of 50 mM Cyclosporin in C6D6 p 9 5 90 8 5 8 0 7 5 7 0 6 5 6 0 5 5 5 0 4 5 4 0 3 5 3 0 2 5 2 0 1 5 1 0 0 5 ppm 20 4 Selective TOCSY Selective TOCSY gives the same H coupling information as 2D TOCSY without a 2D Fourier transformation The selective TOCSY pulse sequence is shown in Figure 59 It is very similar to the standard TOCSY sequence shown in Figure 27 except that the first pulse is a low power shaped pulse the following delay d14 is not incremented and the spin lock period is followed by a z filter As for the COSY the selective TOCSY sequence begins with a 90 frequency selective excitat
83. ate i e until saturation and then reduce the power level slightly approximately 3 dB For example if the lock signal begins to oscillate at a power of 15 dB the optimal magnetic field stability can be expected when a level of approximately 18 dB or even 20 dB is used The field stability will be significantly worse if a power level of say 35 dB is used instead When the lock power is optimized start the auto phase routine and finally the auto gain routine Take note of the gain value determined by auto gain Using this value select the appropriate values for the loop filter loop gain and loop time as shown below in Table 12 Avance 1D 2D BRUKER 30 Table 12 Lock Parameters BSMS Firmware Version 980930 Lock RX Gain Loop Filter Loop Gain Loop Time after auto gain Hz dB sec dB 120 20 17 9 0 681 30 14 3 0 589 110 50 9 4 0 464 70 6 6 0 384 100 3 7 0 306 160 0 3 0 220 250 3 9 0 158 400 7 1 0 111 90 600 9 9 0 083 1000 13 2 0 059 1500 15 2 0 047 2000 16 8 0 041 So for example if auto gain determines a lock gain of 100 dB the user should set the loop filter to 160 Hz the loop gain to 0 3 dB and the loop time to 0 220 sec see Chapter 4 Menu Description of the BSMS User s Manual for how to set these parameters from the BSMS keyboard BRUKER Avance 1D 2D 3 Basic H Acquisition and Processing 3 1 Intr
84. atellites Correct the phase in a way that the left satellite is positive and the right satellite is negative Calibration High Power Set p3 to the value obtained with the direct method in Section 6 1 Acquire and process another spectrum zg efp If the pulse is less than 90 the left satellite will remain positive and the right satellite negative If the pulse angle is between 90 and 270 the two satellite signals will show opposite phase If the p3 pulse corresponds to 90 the satellites show a zero crossing Figure 12 Figure 12 C Decouple 90 Pulse Calibration Results on the chloroform sample p3 90 p3 90 fe p3 90 T I T T T T T I T I T T T T I T T 8 5 8 3 8 1 7 9 7 7 ppm Calibration Low Power for GARP Decoupling The GARP composite pulse decoupling cpd sequence requires a 90 decoupling pulse length of 60 to 70 usec Adjust p12 and p3 to determine the combination that yields a 90 pulse length in this range Use the rule of thumb The pulse length doubles for a 6 dB increase in attenuation BRUKER Avance 1D 2D Note that the parameters used by cpd sequences are pcpd2 for the 90 pulse length and p112 for the decoupler power level rather than p3 and p12 as used here 6 4 1D Inverse Test Sequence The 1D HMQC pulse sequence shown in Figure 13 is used to check the parameters for inverse experiments This experiment detects only signals of protons directly attached to C nuclei
85. atus line at the bottom of the window The lock in procedure outlined above sets the frequency shift to the exact frequency shift value for the given solvent as listed in the edlock table It also sets the field value to the value listed in the edlock table and then adjusts it slightly to achieve lock in the absolute frequency corresponding to a given ppm value no longer depends on the lock solvent Following this lock in procedure the solvent parameter in the eda table is set automatically which is important if you wish to use the automatic calibration command sref see Spectrum Calibration and Optimization The lock phase adjustment by monitoring the sweep wiggles i e while the field is not locked but is being swept is recommended each time the probehead is changed because autolock may fail If the original phase is reasonably close to the correct value lock in can be achieved and the phase can be adjusted using autophase Note that the lock phase for each probehead is stored in the edlock table In some cases the lock power level listed in the edlock table is set too high leading to a saturation of the lock signal Usually lock in can be achieved but the signal oscillates due to saturation A quick fix is simply to reduce the lock power manually after lock in However it is better to change the power level in the edlock table Note that the appropriate lock power level depends on the lock solvent the field value and the probehead
86. bitals around both nuclei The scalar coupling is expressed in Hz and noted as J The operator expression for the scalar coupling is 27 Jp IL The above Hamiltonian expresses the scalar coupling between spin 1 and spin 2 with a coupling constant Ji2 The evolution Hamiltonian for this spin system is then H 6 I 5 L 27 Jy 1 1 To calculate the effect of this Hamiltonian it is divided into 3 parts 8 I 9 I 27 Jy 1 1 which are applied in sequence where this sequence is arbitrary After a 90 pulse has been applied to the two spins we first calculate the two chemical shift terms 0 l L I cos6 r I sin 1 hs 7 cos n Z sin t Z cos t Z sin t 0 The next step will be to calculate the evolution under the scalar coupling 23 8 2 Evolution under Weak Coupling To apply the last part of the Hamiltonian we need some new calculus rules The scalar coupling term can be evaluated with a simple set of rules Ti 2n Jy ly Dt I I 22412 57 cos J t 21 1 Sina sr L m cos t t 21 1 sin U t 21 1 we 521 1 cos st I sin jr 2 1 1 et 521 1 cos yt I sin f 2 I Petites 2 I From the above equation we immediately recognize a very important fact the scalar coupling can generate observable operators from non observable ones through free evolution This is the reason why we can not neglect the non observable operators until we apply the detection op
87. by the window functions and to perform the 2D Fourier transformation The threshold level can be adjusted by placing the cursor on the j button holding down the middle mouse button and moving the mouse back and forth The optimum may be saved by typing defplot and answering the questions which appear 9 4 Phase Correction To simplify the phasing of the 2D TOCSY spectrum it helps to first phase the second row Enter rser 2 to transfer the second row to the 1D data set TEMP 1 1 Enter sinm to apply the sine bell windowing function and enter ft to Fourier transform the data Manually phase correct the spectrum as any 1D spectrum except that when you are finished click etm and select Save as 2D amp return to save the corrections phcO and phc1 to the 2D data file tocsy 1 1 Click Z to return to the 2D data set tocsy 1 1 Now enter xfb to Fourier transform the TOCSY spectrum again this time applying the appropriate phase correction to F2 The spectrum should now require additional phase correction only in F1 and this can be accomplished in the 2D phasing subroutine Click on phase to enter the phase correction submenu Click on cei with the left mouse button to tie the cursor to the 2D spectrum appearing in the upper left hand corner of the display Move the mouse until the vertical cross hair is aligned with a column towards one end of the spectrum Once the desired column is selected move it to window 1 appearing in the up
88. ce 1D 2D Rapid scanning arijacts aio na ede oa ERE EH Sass ea 181 22 5 THE TOCS Y EXPERIMENT te nn en RU HO nn re E PLEBE UAR ie 182 22 5 1 Sample heating due to the spin lock sequence ss 182 22 5 2 Solvent suppression and trim pulses iii 183 23 APPENDIX B THEORETICAL BACKGROUND OF NMR snnnesese 186 23 1 INTRODUCTION eterne me deese esiste tees EEEO 186 23 2 CLASSICAL DESCRIPTION OF NMR r araea ae eE E ee EE Ee eia aE eS r E aae aSa eSEE EEE 186 23 3 SPIN OPERATORS OF A ONE SPIN SYSTEM csccsssssssseessceseesseecceesecnsecsceesessecseesseceeeccseeeseseaeseeeeseeaeseeeeeeenaes 188 23 4 THE THERMAL EQUILIBRIUM STATE cceccsssesssesseesesseessceeseceseesceessceesaceesecnseecaeeesecesecseessecseseesseeeseeenseeeeages 188 23 5 BEFECEOERE PULSES oiim AT tn deer tn nr beet eel io pe PR scenes 189 23 6 THE HAMILTONIAN EVOLUTION OF SPIN SYSTEMS IN TIME esee nennen nennen nnns 190 23 6 1 Effect of Chemical Shift Evolution eee esee tentent tenente nete ens 191 23 7 OBSERVABLE SIGNALS AND OBSERVABLE OPERATORS een 192 23 8 OBSERVING TWO AND MORE SPIN SYSTEMG ccsccsssescesecsseescecesecsseeseeeseceeescssecceeseseeseceeeeseeeseseeeeeseaeesees 194 23 83 Effect Of Scalar Coupling eat AM all weet e eee eaten ge etel 195 23 8 2 Evolution under Weak Coupling sienne 196 23 8 3 The Signal Function of a Coupled Spectrum siennes 197 23 9 SIMPLIFICATION SCHEMES ON A TH
89. ces soft pulses which selectively excite only one multiplet of a H 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 that the transmitter offset frequency of the selective pulse must be set to the frequency 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 non spinning References C J Bauer R Freeman T Frenkiel J Keeler and A J Shaka J Magn Reson 58 442 1984 H Kessler H Oschkinat C Griesinger and W Bermel J Magn Reson 70 106 1986 L Emsley and G Bodenhausen J Magn Reson 82 211 1989 The sample used to demonstrate selective pulse experiments in this chapter is 50 mM Cycl
90. contours n 12 4 Plotting the Spectrum Read in the plot parameter file standard2D by entering rpar standard2D plot to set most of the plotting parameters to values which are appropriate for this 2D spectrum Enter edg to edit the plotting parameters Click the ed next to the parameter EDPROJ1 to enter the Fi projection parameters submenu Edit the parameters from PF1DU to PF1PROC as follows PF1DU u PF1USER name of user for file xhcorr 1 1 PF1NAME xhcorr PF1EXP 1 PF1PROC 1 Click _s to save these changes and return to the edg menu Avance 1D 2D BRUKER 113 Click the ed next to the parameter EDPROJ2 to enter the F2 projection parameters submenu Edit the parameters from PF2DU to PF2PROC as follows PF2DU u PF2USER name of user for file dept 4 1 PF2NAME xhcorr PF2EXP 2 PF2PROC 1 Click twice _ to save these changes and return to main menu Create a title for the spectrum setti and plot the spectrum plot An XHCORR spectrum of 1 g Cholesterylacetate in CDCls is shown in Figure 34 Figure 34 XHCORR Spectrum of 1g Cholesterylacetate in CDCL LaL ppm pen renner Co eo 114 BRUKER Avance 1D 2D 13 COLOC 13 1 Introduction COLOC COrrelation spectroscopy via LOng range Coupling is a 2D heteronuclear correlation technique very similar to the XHCORR experiment described in the previous Section 12 However unlike XHCORR COLOC makes use also of small long range heteronuclear J couplin
91. cquire the 2D data set Enter zg to acquire the time domain data The approximate experiment time for NOESY with the acquisition parameters set as shown above is 5 8 hours 11 3 Processing 106 Enter edp and set the processing parameters as shown in Table 46 Table 46 NOESY Processing Parameters F2 Parameters Parameter Value Comments SI 512 SF spectrum reference frequency H WDW SINE multiply data by phase shifted sine function SSB 2 choose pure cosine wave PH mod pk PKNL TRUE BC mod no F1 Parameters Parameter Value Comments SI 512 SF spectrum reference frequency H WDW SINE multiply data by phase shifted sine function SSB 2 choose pure cosine wave PH mod pk BC mod no MC2 States TPPI BRUKER Avance 1D 2D Enter x b to multiply the time domain data by the window functions and to perform the 2D Fourier transformation The threshold level can be adjusted by placing the cursor on the button holding down the middle mouse button and moving the mouse back and forth The optimum may be saved by typing defplot and answering the questions which appear 11 4 Phase Correction and Plotting For the phase correction procedure and the plotting procedure please follow the instructions given for the TOCSY spectrum in Sections 9 4 and 9 5 respectively Note that for the NOESY spectrum recorded here the first serial file should be chosen for the F2 p
92. d One of the lists must contain a frequency well off resonance for generating the reference spectrum Here we will create lists with frequencies for the resonances at 4 8 ppm and 8 5 ppm and one with the off resonance frequency 2 ppm The frequency lists are defined using the trist routine which is found in the submenu utilities Note that if it is necessary to expand the spectrum in order to accurately define the irradiation points this must be done before entering the routine Display the peak at 4 8 ppm and enter the __ trist routine and answer the questions as shown below Please enter type of list f1 f2 f3 fi Please enter name of f1 list noedif 1 Write name of f1 list to acqu parameters n The following option appears if a f1 frequency list with the same name already exists Frequency list exists append a overwrite o or quit q Answer a if you wish to add new frequencies to the existing list o if you wish to overwrite the existing list or q if you wish to quit the routine and keep the old list Once the questions have been answered move the mouse until the cursor is tied to the spectrum Click on the peak at 4 8 ppm with the middle mouse button Finish the list by clicking the left mouse button Remember that for a given list multiple irradiation points should all be part of the same multiplet A separate frequency list should be generated for each multiplet irradiated Note The type of list
93. decoupling TD 32k NS 16 DS 4 PL1 high power level on F1 channel C as determined in Section 6 1 4 PL2 120 no high power pulses on F2 channel H PL12 low power level on F2 channel H for CPD as determined in Section 6 2 6 D1 2 2s for gated decoupling 60 60s for inverse gated decoupling P1 C 90 pulse as determined in Section 6 1 4 SW 250 250 ppm RG 8k or use rga o1p 120 frequency of the C carrier 120 ppm O2p frequency of the H carrier see text PCPD2 H 90 pulse for cpd sequence as determined in Section 6 2 6 CPDPRG2 waltz16 H decoupling sequence Enter rga to start the automatic receiver gain adjustment and zg to acquire the FID Enter si and set it to 32k set 1b to 3 and Fourier transform the data using ef Phase correct the spectrum and store the correction The resulting spectrum is noisy compared to the regularly decoupled C spectrum The most intense peak arises from the Chloroform solvent which actually is a triplet BRUKER Avance 1D 2D Figure 16 C Spectrum of 1g Cholesterylacetate in CDCI3 using a Gated Decoupling and 7 1 1 Plotting 1D C Spectra 1D C spectra are most easily plotted using the standard plot parameter file which sets most of the plotting parameters to appropriate values Type rpar and select standard1D from the list of parameter file names Select plot Avance 1D 2D BRUKER 71 from the menu of parameter file types Eq
94. dening factor for em aunmp definition of the processing AU program Table 10 Processing Commands Avance 1D 2D edp edit all processing parameters dpp display all status parameters for processing ft Fourier transform the current data em apply exponential window function ef combined command of t and em phase set the phase correction defined by phcO and phc1 apk Automatically phase correct the spectrum abs Automatically baseline correct and integrate the spectrum efp combined command of t em and phase BRUKER 21 22 sr spectral referencing sref Automatically calibrate the spectrum edc2 select a second and a third data processing number dual invoke the dual display edo select an output device edg edit all graphics and plotting parameters view plot preview xwinplot Start the plot program Table 11 Pulse Program Specific Parameters pli f1 channel power level for pulse default p12 f2 channel power level for pulse default p19 f1 channel power level for presaturation p110 f1 channel power level for TOCSY spinlock plii f1 channel power level for ROESY spinlock p112 f2 channel power level for CPD BB decoupling pl14 f2 channel power level for cw saturation pli5 f2 channel power
95. dif 2 1 enter edc2 to define the Avance 1D 2D BRUKER 139 second data set to be shown in the dual display Set EXPNO2 to 1 and PROCNO to 1 and click s Click 4a to enter the dual display mode Both the spectra from noedif 1 1 and noedif 2 1 appear in the window When the comparison of the spectra is finished click to return to the main 1D processing window Ideally the target resonance is completely saturated by the selective irradiation while all other signals are unaffected by the irradiating field In practice the chemical shift difference between signals is often too small so that neighboring resonances may be saturated as well It is almost always preferable to use low power and hence selective irradiation rather than unwanted saturation of nearby resonances However partial saturation of a multiplet leads to selective population transfer which may obscure NOE effects To avoid this the individual components of a target multiplet are irradiated in an interleaved manner during the preirradiation period before each scan If needed adjust p114 to change the power level of the cw irradiation Note that the total cw irradiation time 14 d20 should be approximately equal to T of the irradiated peak but with the au program noemult it is necessary to use the same total irradiation time for each peak irradiated Thus the irradiation time should be chosen based on the longest Ti Here a total irradiation time of 2 5
96. difference is in the order of 6 5 10 Following the convention of the static field being aligned with the z axis of the reference frame the equilibrium magnetization is also called M 188 BRUKER Avance 1D 2D In the spin operator formalism this result has to be derived from statistical quantum mechanics considerations using ensemble averages and population probabilities For us it will be good enough to know the following result Cu Oeq iS the equilibrium density matrix The density matrix represents the state of the spin system under investigation and is represented as a linear combination of the basis spin operators For a one spin system in thermal equilibrium the coefficients of all but the lz basis operator vanish To understand what happens during an NMR experiment we will have to evaluate the changes in the density matrix during the experiment starting from the equilibrium matrix These changes are also referred to as evolution of the system There are two basic type of evolutions under the effect of an external perturbation e g a RF pulse or the unperturbed evolution which will eventually bring the system back to the thermal equilibrium 23 5 Effect of RF Pulses Let us first consider the evolution under an RF pulse In modern spectrometers pulses are only applied in the x y or transverse plane Pulses in between the x and y axis are calculated by a combination of a rotation around the z axis followed by an x or y
97. dministration please refer to the IconNMR software manual After the login into the automation window the following setup window will appear Here you can setup your experiments 170 BRUKER Avance 1D 2D Uz Bruker 35 prog curdir changer inmechanger Feh18 2003 125 7 ktt j loj x Dh Run Heller Yew Find Persneters Heb CBE Hates Type Stans Disk Mame No Sobert Experiment Par Orig A b1 Available D2 Avaleble b 3 Available Da Available valable Avwaleble Avarlebk Buy Waling for ob EwptTme 0010 Oratie kii E EC EC EC E eE aee el o 1 m zii Date Tima Holdall Nama Mo Esperiment amp TM Rod Look Shm Acqactior If you run IconNMR with a sample changer you can setup several experiments for different sample holders at once If you do not have a sample changer just setup the desired experiments on the first virtual holder and you will be prompted for the sample insert In order to set up the experiments just double click on the line with the respective holder and choose an experiment name accept the suggested experiment number select your solvent and select the experiment that you want to run Then press the green Go button to start the automation run C Bruker 35 prog curdirschanger inenrchamger Feb13 2003 1306 kti dll x Ble Run Hade en Find Parameters Heb es i5 mca Buyuni Wating forlob a dom tum ar 1 Espl lime 0000 Care
98. e 38 The basic idea of this preparation period is to saturate all H not directly attached to a C nucleus Avance 1D 2D BRUKER 119 HMQC is a phase sensitive experiment and after a 2D Fourier transform with respect to t and t the 2D spectrum can be phased so that all peaks are purely absorptive Figure 37 HMQC Pulse Sequence T 2 T idi d2 d2 Figure 38 HMQC with BIRD Pulse Sequence 13C Pee see EE ONE p4 p3 p3 d d2 d a7 a2 do do d2 14 2 Acquisition Insert the sample in the magnet Lock the spectrometer Readjust the Z and Z shims until the lock level is optimized Tune and match the probehead for 1H observation and C decoupling It is recommended to run 2D experiments without sample spinning As for the XHCORR experiment both H and C reference spectra of this sample must be recorded see Sections 12 2 1 and 12 2 2 for the 120 BRUKER Avance 1D 2D corresponding instructions Use wrpa to store the reference spectra as data sets hmqc 1 1 for the H spectrum and hmac 2 1 for the C spectrum Enter re hmqc 1 1 to return to the optimized H spectrum Create the data set hmqc 3 1 by using edc Enter edsp and set NUC2 to 13C Set o2p to the value found for the optimized C spectrum in hmqc 2 1 Enter eda and set PARMODE 2D Click on _ and ok the message Delete meta ext files The window now switches to a 2D display and the message NEW 2D DATA SET appears Enter eda and
99. e Comments PULPROG hxcodf TD 1k NS 8 the number of scans must be 4 ns DS 16 number of dummy scans PL1 high power level on F1 channel C as determined in Section 6 1 4 PL2 high power level on F2 channel H as determined in Section 4 2 4 PL12 low power level on F2 channel H for CPD as determined in Section 6 2 6 P1 13G 90 pulse as determined in Section 6 1 4 P2 13G 180 pulse calculated from P1 P3 H 90 pulse as determined in Section 4 2 4 PCPD2 H 90 pulse for cpd sequence as determined in Section 6 2 6 D1 2 relaxation delay should be 1 5 T C CNST2 145 heteronuclear scalar J C H coupling 145 Hz is a good intermediate value D2 3 45 msec 1 25 C H calculated automatically from cnst2 above Avance 1D 2D BRUKER 111 CNST11 3 used to calculate d3 3 for all multiplicities D3 2 30 msec calculated automatically from cnst11 above CPDPRG2 waltz16 cpd sequence for the H decoupling F1 Parameters Parameter Value Comments TD 256 number of experiments FnMODE QF NDO 2 two dO periods per cycle SW sw of the optimized H spectrum xhcorr 1 1 INO t increment calculated from SW above NUC1 selects H frequency for F1 Since this data set was created from the DEPT 45 reference spectrum the receiver gain is already set correctly Enter zg to acquire the spectrum the approximate experiment time for XHCORR with the acquisition parameters se
100. e eda edp or edg window for acquisition processing or plotting parameters respectively Since these panels contain all possible parameters and are rather large it is often more convenient to use somewhat more reduced parameter editor interfaces The ased command opens the panel for the acquisition parameters that are of importance only for the selected pulse program Here the parameters are also commented on 2 2 1 Predefined Parameter Sets The XWinNMR philosophy is to work with predefined parameter sets that are suitable for most of the NMR tasks and experiments you are facing These parameter sets include the pulse program acquisition and processing AU programs as well as all other necessary parameters except spectrometer specific values for pulse lengths and power levels These standard parameter sets usually have the same base name as the corresponding pulse program Each parameter set can be called up into a dataset of your choice by the command rpar You can modify the parameters and save the new parameter set by the command wpar Bruker predefined parameter sets are written in capital letters and we recommend that you do not change them but rather create new ones that you can use just as well Therefore the most simple way to run a certain experiment is to create a new dataset with a specific name using the command edc Then you would read the corresponding parameter set by rpar i e rpar PROTON all set the pulse lengths and power
101. e evaluated with a simple set of rules I 27 J5 L l t I lz gt Iz 2051 1 1 t I Wet gt I cos aJ t 21 1 sin aJ t I Beet oT cos aJ t 21 L sin aJ t 2 1 L See 2 TL cos aJ t 1 sin aJ t 2 I l ee 5 21 1 cos aJ t 1 sin aJ t 21 lij t 2 lily ey 21 L which can then be applied to the various terms of o above Avance 1D 2D BRUKER 13 o tet I cos a t 2I L sin aJ t cos t I cos 1J 0 2 I L sin 1J t sin 9 t L cos aJ t 2 IL sin aJ t cos 6 t L cos 7J 0 2 I sin aJ t sin 6 t 6 References O W S rensen G W Eich M H Levitt G Bodenhausen R R Ernst Progres in NMR Spectroscopy 16 163 1983 1 5 Sensitivity of NMR Experiments The sensitivity of NMR experiments is given by the signal to noise ratio S IN NY excl y B y ns T S N signal to noise ratio N number of spins in the system sample concentration Yexc gyromagnetic ratio of the excited nucleus Yoet gyromagnetic ratio of the detected nucleus ns number of scans Bo external magnetic field T transverse relaxation time determines the line width T sample temperature Comment here we can already see that it might be useful for a better signal to noise ratio to excite one kind of nuclei and detect another kind with a better gyromagnetic ratio in the same experiment This is done in inverse experiments which are described in sections 14 to 16
102. ects on Spins in the Product Operator Formalism Effect of pulses on magnetization I I cos B 1 sin B I Py cos D 7 sin D 1 1 I I I 5 I cos I sin p L 1I cos p Z sin p If the flip angle B 2 90 then I p L I Effect of chemical shift on magnetization I I 5 1 cos t I sin 8 1 I gt cos 6 f I sin t Effect of scalar coupling on magnetization 27 Ji tiat 12 11722 gt I I L et ji cost ut 2 LL sin 1 I J n falto I cos xJ f 2 1 1 sin tJ pt 2 1 1 W442 2 7 1 cost 1 sin 1 2 1 L 2 521 1 Cos of 1 sin pt 2x Jj lI t tS 2 IL EL 21 1 204 BRUKER Avance 1D 2D 23 11 2 Mathematical Relations The Euler relations where used extensively in the previous paragraphs e cos t i sin t e cos f i sin t l cos t ee gi sin t m i gi Frequently used simplifications in 2D i cosQ sing i cosa i sina i cosQ i sind io i e ip 1 i O t i O t ip COS e x e e 1 i B a i B a t eit BE l rat cc sina eP zc 6 e Au d ae eben agi BNP References O W S rensen G W Eich M H Levitt G Bodenhausen R R Ernst Progres in NMR Spectroscopy 16 163 1983 Avance 1D 2D BRUKER 205 PC acquisition and processing 46 PC NMR with H decoupling 49 PC NMR without decoupling 47 1D HMQC acquis
103. ed NOE difference experiments should be run without sample spinning The parameters and spectra shown below are from a 300 MHz spectrometer The signal enhancements for Pamoic Acid in DMSO d6 at other field strengths will be different than those shown here If an NOE response is difficult to obtain it may be necessary to change the sample temperature or solvent In particular for this sample at 400 MHz it is recommended to use a temperature of 40 C 17 2 1 Create a new file directory Enter re proton 1 1 to call up the data set proton 1 1 Enter ede and create the data set noediff 1 1 Enter edasp and set both NUC1 and NUC2 to 1H The f2 channel is used for cw irradiation during the NOE experiment 17 2 2 Proton reference spectrum Enter rga to perform an automatic receiver gain adjustment Acquire and process a standard H spectrum as described in Chapter 3 Calibrate the Avance 1D 2D BRUKER 137 spectrum and optimize sw and o1p Keep in mind that the control spectrum should be irradiated well off resonance in this case 2 ppm Acquire and process an optimized spectrum 17 2 3 Select the resonances for irradiation The frequencies used by the f2 channel during the preirradiation periods of the NOE experiment must be written to the corresponding fq21ist A separate list must be created for each resonance to be irradiated where a given list may contain several frequencies if irradiation of a resonance at several points is neede
104. edif 3 2 Two NOE difference spectra with cw irradiation on resonance at 8 5 ppm and 4 8 ppm and the reference spectrum with cw irradiation off resonance at 2 ppm of 100 mM Pamoic Acid in DMSO d6 are shown in Figure 49 In both the difference spectra with cw irradiation at 8 5 ppm and 4 8 ppm the large negative peak is the irradiated resonance and the small positive doublet is the NOE Note that these spectra were recorded on a DPX300 at 298 K Experiments recorded at 500 MHz and 298 K will have negative NOE peaks while those recorded at 400 MHz and 298 K may show no NOE peaks at all 17 3 3 Quantitate the NOE To quantitate an observed NOE the integrated intensity of the NOE peak in the difference spectrum is compared with the integrated intensity of the peak that was irradiated However this latter intensity should be measured in the reference spectrum Thus it is necessary to integrate peaks in both the control and the difference spectrum and to use the same normalization constant for the integrals in both spectra see Section 3 10 142 BRUKER Avance 1D 2D Figure 49 NOE Difference Spectra of 100 mg Pamoic Acid in DMSO reference 9 0 8 0 7 0 6 0 5 0 ppm Avance 1D 2D BRUKER 143 18 Homonuclear decoupling 18 1 Introduction The technique of homonuclear decoupling double resonance spin decoupling homo decoupling was established long before routine pulsed FT spectroscopy became popular In those days the usu
105. ee Figure 1 for pulse sequence diagram TD 64 k NS 1 DS 0 D1 2 P1 3 the default unit for pulse lengths is micro seconds entering 3 sets a pulse length of 3 microseconds PL1 high power level on F1 channel C see also Important note on power levels SW 250 SC spectra cover a much broader spectral range than H spectra O1P 100 will be optimized later Enter rga to start the automatic receiver gain adjustment and then zg to acquire the FID Type si and enter a value of 32k Type 1b and enter 3 Enter ef to add line broadening and Fourier transform the data Manually phase correct the spectrum and store the correction Subsequent C spectra can now be processed with the command efp which combines the exponential multiplication Fourier transformation and phase correction using the stored phcO0 and phc1 values The processed spectrum is very noisy and most likely only one single peak is visible arising from the CDCI solvent as shown in Figure 4 Type sref to calibrate the spectrum correctly Note that the sref command works properly only if the parameter solvent is set to the correct solvent in the eda table The signal to noise ratio is improved by acquiring more than one scan Enter edc and set EXPNO to 2 Click on SAVE to create the data set setup13c 2 1 Enter ns number of scans and change the current value to 64 Enter ds dummy scans and change the current value to 4 The four dummy scans
106. een defined the data may be processed and the Ti calculation is carried out using the automation program proc t1 This program first Fourier transforms and phase corrects the rows of the 2D Ti data set It then performs a T calculation on all the peaks indicated by the integral range and baseline point files Start the automation program from the 2D data set by entering xau proc t1 Answer the questions as follows Enter fid no for phase determination 1 Enter left limit for baseline correction 1000 Enter right limit for baseline correction 1000 Enter no of drift points 20 Enter name of baseline point file tibas Enter name of integral range file tireg Enter name of VD list to use tidelay Enter calc type T1 1 T2 2 1 The FID corresponding to the largest value of vd i e full relaxation between the 180 and 90 pulses should be used for the phase determination The values 1000ppm are suggested merely to ensure that the whole spectrum is corrected The automation program applies a baseline correction in F2 abs2 between these two limits and it is important to baseline correct the entire spectral width The number of drift points accounts for the fact that the maximum of a peak selected for a T calculation is usually not at exactly the same position for 152 BRUKER Avance 1D 2D each of the 1D spectra The number of drift points specifies how many digital points the peak maximum may vary This parameter may need some
107. electing three columns rather than rows 8 3 4 Plot the spectrum See the plotting instructions given for the magnitude COSY spectrum in Section 8 2 4 A DQF COSY spectrum of 50 mM cyclosporin in CgD is shown in Figure 24 Figure 24 DQF COSY Spectrum of 50 mM Cyclosporin in C6D6 88 BRUKER Avance 1D 2D 8 4 Double Quantum Filtered COSY using Pulsed Field Gradients GRASP DQF COSY The first high resolution NMR experiments using pulsed field gradients PFG were the COSY experiments mainly to demonstrate that the application of PFGs can replace phase cycling The quality in selecting a desired coherence pathway by PFGs turned out to be more efficient than phase cycling In contrast to phase cycling which requires several scans for each ti increment for coherence selection field gradients allow coherence selection with only a single scan for each t increment There are mainly two common PFG applications with COSY experiments 1 Quadrature detection in the dimension The experiment time for such a COSY is in the order of a few minutes 2 Double quantum filter the quality of the double quantum filter using field gradients is very efficient Therefore solvent signals without homonuclear H coupling like water can be suppressed very efficiently without additional solvent suppression techniques In this chapter we will describe the phase sensitive double quantum filtered COSY experiment and the pulse sequence is shown
108. en enne nne enitn eren nni nn sere sense AEE sens innere tenere serere nnn 116 14 1 INTRODUCTION ctt eet tee teen at nel tn ede nie e deep eee 119 14 2 ACQUISITION re aie ree eee ederet eee tee e eee tette tete eee ree de dete 120 14 2 1 Optimize d7 only for HMQC with BIRD tentent nennen eerte 122 14 2 2 Acquire the 2D data S61 ooo e eee entes tu e teet steel 122 14 3 PROCESSING endete erede e eem relient mate ne ree e rete exec eee eee ted aede eg ecole 122 14 4 PHASE CORRECTION scie ette tete d tet tn trs te et nl ete e e euet 123 145 PLOTTING eM 123 15 1 INTRODUCTION a nn mninn SE ee 125 15 2 ACQUISITION AND PROCESSING esses t ce D a ests RH ERE EE Pe E REO DESEE oue 126 16 PROTON CARBON INVERSE SHIFT CORRELATION EXPERIMENTS USING PULSED FIELD I6 1 INTRODUCTION tiet eoe eere ced anakana eee tede eet eee edo atest 129 16 2 GRASP AMQC eere nene e nn e ER EE eg 129 163 GRASE HMBC E26 merce tee te dude deste eder de tede dec E der dune ea 130 16 4 GRASP HSQC ren a d aa aa a d a a a EP OR ESR OE Ula tents 131 16 ACQUISITION AND PROCESSING 3233 62 89 05 28 6509 255 del MM erige LM d E es REI eee tes ne eus 132 LELINPIROD CLEION hee eee eie eee eee edema tec eed ee ede eed de diee eco eee dd 136 APNO e MIBULUM m 137 17 2 I Create a New file directory side tide MAMAN ent eeted tede ode 137 Avance 1D 2D BRUKER 17 2 2 Proton referenc Spectrum eaaa e in tin nan ie REN ER ETE NER SENSU
109. ence with 1H Decoupling T 2 13C 1H decoupling Before acquiring a H decoupled C spectrum the frequency of the 1H signals in must be determined Figure 2 Section 3 9 shows a H Spectrum of cholesterylacetate or look it up with re setuplh 1 1 Most H signals lie in the range of 0 5 to 5 5 ppm An appropriate frequency for H decoupling would therefore be 3 ppm n general 5 ppm is a safe frequency to select for H decoupling if no 1H spectrum is available In case you looked up the H spectrum data set proton 1 1 return to the previous carbon spectrum by entering re setup13c 3 1 Enter edc set EXPNO to 3 and click on SAVE to create the data set setup13c 3 1 Enter eda and set the acquisition parameters as shown in Table 17 48 BRUKER Avance 1D 2D Table 17 C Acquisition Parameters for 1H Decoupled Spectrum Parameter Value Comments PULPROG zgdc see Figure 6 for pulse sequence diagram TD 32k NS 1 DS 0 D1 2 should be 1 5 Ti C P1 3 the default unit for pulse lengths is micro seconds entering 3 sets a pulse length of 3 microseconds PL1 power level for the p1 pulse C See also Important note on power levels Pli2 low power level on F2 channel H as determined in Section 4 2 6 for ROESY spin lock PCPD2 H 90 pulse for cod sequence as determined in Section 4 2 6 SW 350 ppm O1P frequency of C carrier as optimized in Section 5 2 O2P 3
110. ength sp1 must be adjusted so that the pulse is 90 or 270 see below Figure 54 Selective One Pulse Sequence T2 20 2 3 Define the pulse shape Shaped pulses are designed using the shape tool of XWIN NMR version 2 1 and higher Enter stdisp in XWIN NMR Select Gauss from the pull down menu Shapes A small window appears containing default parameters for the shapes pulse Select OK Store the shape by choosing Save As from the File menu and enter the filename gauss1 1k 20 2 4 Acquire and process the selective one pulse spectrum Create the data set selex 2 1 for the 1D selective experiment by typing xau iexpno starting from the data set selex 1 1 Set up the acquisition parameters as shown in Table 63 Avance 1D 2D BRUKER 159 Table 63 Selective One Pulse Acquisition Parameters Parameter Value Comments PULPROG selzg see Figure 54 for pulse sequence diagram TD 8k NS 1 no need for signal averaging yet DS 0 no need for dummy scans yet SP1 80 shaped pulse power level on f1 channel P11 80m 90 shaped pulse on f1 channel D1 10 relaxation delay PLO 120dB Sets power to zero before selective pulse SP edit enter this array to edit power level offset and filename for the shaped pulse In the eda menu enter the power level offset and filename for the shaped pulse by clicking on the edit button next to the parameter SPO7 This calls up the table Power for shaped pulses
111. ent To obtain a DEPT 135 spectrum create the data set dept 3 1 and either select the pulse program dept135 type pulprog dept135 or set pO to the length of a 135 pulse type pO and enter the value of 1 5 p1 at the prompt Acquire zg and process efp the data Only signals from CH and CHs groups are visible in this experiment 7 2 7 Plot the spectra See Section 7 1 1 for instructions on how to plot the acquired spectra DEPT 45 DEPT 90 and DEPT 135 spectra of 1 g Cholesterylacetate in CDCl3 are shown in Figure 18 The DEPT results can be compared with the standard H decoupled C spectrum in carbon 3 1 see Section 5 3 Note that the signals from the quaternary C in the carbon 3 1 do not appear in any of the DEPT spectra From the combination of standard H decoupled and DEPT 45 90 and 135 spectra it is possible to determine which signals arise from primary secondary tertiary and quaternary C s Avance 1D 2D BRUKER 75 76 Figure 18 DEPT Spectra of 1 g Cholesterylacetate in CDCI3 DEPT 45 Arie DEPT 90 d ALI DEPT 135 fo Wainer T I T T T T T T T T T T T T T T T T T T T T T T T T T T T T 120 100 80 60 40 20 0 ppm BRUKER Avance 1D 2D 7 3 APT Attached Proton Test The APT Attached Proton Test is a simple experiment for assigning multiplicities in C NMR spectroscopy The APT pulse sequence is shown in Figure 19 The first 90 degree pulse creates transverse magnetisation followed by a
112. erator signal acquisition Befitted with the above equations we can now evaluate the last part of the Hamiltonian o Att 5 7 cos sp 2 T sin 0 cosQ r I cosQtJ st 2 I I sinu t sin f U cos tJ st 2 T L sinu 0 cosQ rn I cos J t 27 1 sin J t sin f 6 after some rearrangement this leads to 196 BRUKER Avance 1D 2D 6 1 cos t J t cos6 t Jj cos t Ji t sinf 1 L cost Ji t cos t I cost Ji t sin f 2 I sin t Jj t cos 1 2 IL sina Ji t sin t 2 IL sin Jy t cos 1 2 ID sina J 1 sin 9 f cos t J sin t cost Ji t 15 cos t sin t cost J t 2 Hh L cos 0 t 2 11 sin sina J t 2 1 1 cos 0 1 2 TL sin sin t J t Again the final step is the calculation of the expectation value of the detection operator Of course the detection operator also needs to include spin 2 We introduce a detection operator F Poet 1 or more general for a N spin system N F2 In the previous section we were rewriting the density operator in terms of L and L to calculate the detection results While this is very elegant it is also very tedious We know from those equations that F is going to select F in the density operator and we know also that the coefficient of F is obtained by using the coefficients of F zZXlix minus FyzXliy times the complex constant F
113. erence frequency between the two oscillations and is of the order of few 10 kHz The mixing process can be understood as comparing the signal at any time with a rotating reference vector which is exactly what we have done in the interaction frame with a fixed detector on the x or y axis For all practical purposes 1 lo is assumed to be one and the signal function is assumed to F t gre Lo QU2mN ty e Avance 1D 2D BRUKER 193 The radial frequency in radians was replaced by the frequency v in Hz This is the unit in which the spectra are expressed finally The time domain function F tz needs to be Fourier transformed to obtain the spectral function S v F t e ES S 8 pad v Dirac The function S v is zero except for the point v v where it is infinite This is a so called stick spectrum as the intensities are meaningless and only the frequency information is relevant The signal function amp will come up frequently during NMR calculations so that it is worthwhile to remember its Fourier transformation Note that while the time domain function is complex valued the frequency domain function is strictly real Things get more complex once relaxation is taken into account To obtain correct line shape information relaxation becomes vitally important At this point we have successfully evaluated the outcome of a simple NMR experiment consisting of a 90 excitation pulse followed by the detection
114. ermine the correct o2p for H decoupling Then a H decoupled C spectrum must be recorded to determine the correct o1p and sw for the DEPT experiments However both steps were already carried out in Section 5 3 a H decoupled 8C reference spectrum of this sample can be found in carbon 3 1 7 2 3 Create a New Data Set Enter re carbon 3 1 to call up the reference spectrum Enter edc and change the following parameters NAME dept EXPNO 1 PROCNO 1 Click to create the data set dept 1 1 Avance 1D 2D BRUKER 7 2 4 Spectrum Acquisition 74 Enter eda and set the acquisition parameters as shown in Table 33 Table 33 DEPT Acquisition Parameters Parameter Value Comments PULPROG dept or dept45 dept90 dept135 TD 32k NS 4 the number of scans must be 4 ns DS 8 number of dummy scans PL1 high power level on F1 channel C as determined in Section 6 1 4 PL2 high power level on F2 channel H as determined in Section 4 2 4 PL12 low power level on F2 channel H for CPD as determined in Section 6 2 6 PO SC a pulse as 90 determined in Section 6 1 4 a 45 90 135 P1 13G 90 pulse as determined in Section 6 1 4 P2 13G 180 pulse calculated from P1 P3 H 90 pulse as determined in Section 4 2 4 P4 H 180 pulse calculated from P3 PCPD2 H 90 pulse for cpd sequence as determined in Section 6 2 6 D1 2 relaxation delay should be 1 5 T C CN
115. ermines the length of the mixing period during which NOE buildup occurs This should be on the order of Ti The value listed in Table 45 is appropriate for this sample at 300 MHz and room temperature If no appropriate value of d8 is available the following quick and easy procedure can be used Create a 1D data set from the NOESY 2D data set Enter edc set EXPNO to 2 and click _ to create the data set noesy 2 1 Enter eda set PARMODE to 1D click and ok the requests to delete a number of files In eda set PULPROG to zg or enter pulprog zg Set ns to 1 and ds to 0 Use zg and ef to acquire and process a 1D H spectrum Manually phase correct the spectrum and store the correction In eda change PULPROG to the pulse program tiirid or enter pulprog tlirld This is a so called inversion recovery sequence Set d7 to BRUKER 105 approximately 1 msec d7 1m record and process a spectrum using zg and e p The signals should all be negative To set d7 to 1 sec enter d7 1 and record and process another spectrum using zg and efp The signals should all be positive Now find a value for d7 in the range of 300 600ms where all the signals are minimal This length of time is sufficient for NOE buildup in small molecules in order to avoid spin diffusion in macromolecules it may be necessary to use a shorter length of time Return to the NOESY data set by typing re 1 Enter d8 and set this to the value of d7 determined above 11 2 2 A
116. essary when using the digital filter Fourier transform the data e and phase correct the spectrum A 1D HMQC spectrum of Chloroform is shown in Figure 14 Note that the phase cycling is not as perfect to completely suppress the main signal arising from H directly attached to C due to technical limitations 66 BRUKER Avance 1D 2D Figure 14 ID HMQC Spectrum of Chloroform Avance 1D 2D BRUKER 67 68 BRUKER Avance 1D 2D 7 Advanced 1D C Experiments 7 1 Carbon Experiments with Gated H Decoupling In principle the values for scalar couplings between H and C and the signal multiplicity can give additional information for the structure determination The disadvantages of not decoupling H in C spectra are the decreased sensitivity due to the distribution of the signal intensity into the different lines of the multiplet the signal overlap and the missing NOE effect This chapter describes the acquisition and processing of C spectra acquired with commonly used H decoupling techniques called gated decoupling and inverse gated decoupling The gated decoupled C spectrum is recorded with H decoupling during the relaxation delay whereas the decoupling is switched off during C acquisition For the inverse gated decoupling experiment the H decoupling is active during the acquisition whereas it is switched off during the relaxation delay The sensitivity improvement due to
117. et eee re ee Tee Phe iesus eet ve ceu 17 22 BRUKER NMR SOFTWARE 3 endet coe teer eeu ee eec edes e ede e eee eue esee esee ee Pate 17 2 2 Predefined Parameter Sets esee ER REID e ees 18 2 2 20 XWinNMR parameters and commuandis eese esee eset tette tenete tenete netten ente tenente nennen 20 2 2 3 Ghanges for XWinNMR 3 5 eese CERERI ee tte omen AS Les ce 24 2 3 TUNING AND MATCHING THE PROBE ccccsssessesssesseesseesseesecseeesecncesseeesecaeeseeceseceesseeesessaeeseeeneceeeesesesseneesaeeages 24 2 4 TUNING AND MATCHING TH NON ATM PROBES nhe riter eere rie tein tiere 25 2T Sertthe Parameters se eee eub etd tiet ette ede die hebt ee eden 25 242 Stare Wobbling ise di tock cons de tente obe ete ast da 25 24 3 Tune and Match iai e Re de e e RR He Ni eee dee eu ites 26 2 5 TUNING AND MATCHING C NON ATM PROBES sssssesssesssesssesssessscsuesssessecssecssucssscssecssecessseessuessscsssesssessees 27 215 1 Setthe Paramelers is cies de dee dH tl ede te ERN 27 2 25 12 Start Wobbling Tune and Match essent eene nn nennen 27 2 6 LOCKING AND SAMM G i dhvsececeres eco pate n re EP Er ER SEE CAE detect te ERA Ead 28 2 6 1 2 6 2 2 6 3 3 BASIC H ACQUISITION AND PROCESSING 3 L INTRODUCTION e heeft dete teet e nte te t erect ve tec eee ced ed eei eene 31 AGI Samples esc tee dtd dt de eee o eR oed deeds ec nt ed d 31 3 1 2 SPrepaFAllOni s iet teet t p Se c REN deed a an tata op ded OS 3l 3 2 SP
118. eters F2 Parameters Parameter Value Comments SI 8k SF spectrum reference frequency H WDW EM LB 1 PH mod no PKNL TRUE necessary when using the digital filter BC mod quad F1 Parameters Parameter Value Comments SI 16 select a power of two greater than or equal to the number of delays in vdlist BC mod no MC2 QF The spectra will be processed by the automation program proc t1 lf desired however the spectra may be processed manually Simply enter x 2 to multiply the time domain data by the window function and also perform the Fourier transformation in F2 only The 2D data set is displayed automatically 19 3 1 Write the integral range file and baseline point file The automation program proc t1 which will be used to calculate T for the defined peaks requires a predefined integral range file and baseline point file These files must exist before running the automation program From the 2D data set move to a 1D data set containing the row for which vd is a maximum e g the first spectrum here with 10s recovery delay This may be accomplished by entering rser 1 which copies the FID of the first row into the data set TEMP 1 1 Enter e to apply line broadening Manually phase correct the spectrum and store the correction Click integrate to enter the integration mode see Section 3 10 and integrate each peak for which T should be calculated Click the left mouse Ava
119. experiment can show axial peaks as rapid scanning artifacts The axial peaks can be found in the centre of the F1 dimension Increase the repetition delay to avoid these artifacts Figure 67 HMQC experiment with pamoic acid Axial peaks are due to rapid scanning axial peaks Avance 1D 2D BRUKER 181 22 5 The TOCSY experiment 22 5 1 Sample heating due to the spin lock sequence For the spin lock sequence of the TOCSY experiment rather short proton pulses are applied for a duration of 50 100ms This can cause heating of the sample especially if water or salty solutions are used The artefact appears as baseline distortion of the peaks along the F1 dimension The number of dummy scans has to be sufficiently high to avoid artifacts caused by heating In addition the gas flow for temperature regulation can be increased Figure 68 Top A zoom of a TOCSY experiment after Fourier transformation along the acquisition dimension For this experiment the number of dummy scans was insufficiently low therefore a shift of the NMR signal is observed Bottom the right spectrum shown artifacts due to sample heating the left spectrum was recorded with a sufficient number of dummy scans 182 BRUKER Avance 1D 2D 22 5 2 Solvent suppression and trim pulses Commonly the TOCSY experiment contains two trim pulses one located after the t evolution delay and one directly following the spin lock Both trim pulses defocus magnetisation which
120. f1 f2 f3 refers to the directory where the frequency list is stored and not to the spectrometer channel for which the list will be used In any acquisition parameter set it is possible to define eight separate frequency lists q11ist fq2list etc The pulse program noemul uses only one frequency list fq2list Therefore set the parameter fq2list to the appropriate list name noedif 1 within the eda menu The automation program noemult redefines q21ist each time noemul is to be run with a different frequency list Repeat this procedure for the peak at 8 5 ppm and for the off resonance frequency of 2 ppm Write the lists to the files noedif 2 and noedif 3 respectively Note that the automation program noemult requires that all frequency lists have the same base name and increasing extension numbers Click on reum to leave the utilities submenu and return to the main menu 138 BRUKER Avance 1D 2D 17 2 4 Set up the acquisition parameters Enter ede and change EXPNO to 2 to create the data set noedif 2 1 Enter eda and set the acquisition parameters as shown in Table 55 Verify that NUC2 has been set to 1H in edasp Table 55 ID NOE Difference Acquisition Parameters Parameter Value Comments PULPROG noemul TD 64k NS 8 the number of scans must be 8 n DS 2 PL1 high power level on F1 channel H as determined in Section 4 2 4 PL14 70 power level for NOE buildup P1 H
121. frequency C WDW QSINE sine squared window function SSB 2 pure cosine squared wave PH_mod pk apply 0 and 1 order phase correction determined by automation program calcphinv BC mod no MC2 States TPPI Enter xfb to multiply the time domain data by the window functions and to perform the 2D Fourier transformation The threshold level can be adjusted by placing the cursor on the button holding down the middle mouse button and moving the mouse back and forth Both positive and negative peaks can be displayed by clicking on the button The optimum may be saved by typing defplot and answering the questions which appear 14 4 Phase Correction Enter rser 1 to transfer the first row to the 1D data set TEMP 1 1 Enter sinm to apply the sine bell windowing function and enter ft to Fourier transform the data Manually phase correct the spectrum Click rem and select Save as 2D amp return to save the corrections phcO and phc1 to the corresponding F2 parameters in the 2D data file hmqc 3 1 Click 29 with the left mouse button to return to the 2D data set hmqc 3 1 It is convenient to use an automation program to determine the F1 phase correction From the data set hmqc 3 1 simply enter xau calcphinv Note that this automation program is designed specifically for HMQC type experiments Now enter xfb to Fourier transform the HMQC spectrum again using the appropriate phase correction to F1 and F2 The spectr
122. frequency of H carrier CPDPRG2 waltz16 H decoupling sequence Enter zg to acquire an FID Enter e p to perform line broadening Fourier transformation and phase correction A H decoupled C spectrum is shown in Figure 7 Note the improved signal to noise ratio Avance 1D 2D BRUKER 49 50 Figure 7 C spectrum of 1 g cholesterylacetate in CDCI with 1H decoupling 200 150 100 50 0 50 ppm BRUKER Avance 1D 2D 6 Pulse Calibration Carbon 6 1 Carbon Observe 90 Pulse The C observe pulse calibration experiment requires a sample with a strong 13G signal e g 80 Benzene in Acetone d6 If no appropriate sample is available the inverse mode C pulse calibration procedure described in Section 6 3 can be used instead 6 1 1 Preparation Insert the sample and lock the spectrometer Lock Readjust the Z and Z shims until the lock level is maximal use 1ockdisp Tune and match the probehead for C observation and H decoupling see Chapter 2 5 Create a new data set Enter re proton 1 1 enter edc and change the following parameters NAME test13c EXPNO 1 PROCNO 1 Click on SAVE to create the data set test13c 1 1 Enter edsp and set NUC1 to 13C turn off NUC2 and press the DEFAULT button then click on SAVE Enter eda and set the acquisition parameters as shown in Table 18 Table 18 1D H one pulse Acquisition Parameters Parameter Va
123. g of the Spectrum Enter edp and set the processing parameters as shown in Table 36 Table 36 APT Processing Parameters Parameter Value Comments SI 16k WDW EM exponential multiplication LB 2 2 Hz line broadening PKNL TRUE necessary when using the digital filter Add line broadening and Fourier transform the time domain data with the command ef Manually phase correct the spectrum so that CH and CH3 groups are positive and C and CH groups are negative 7 3 6 Plot the spectra See Section 7 1 1 for instructions on how to plot the acquired spectra The APT spectrum of 1 g Cholesterylacetate in CDCI is shown in Figure20 The APT results can be compared with the standard H decoupled C spectrum in carbon 3 1 see Section 5 3 and with the DEPT experiments Note that the signals from the quaternary C is visible in the APT experiment Figure 20 APT Spectrum of 1 g Cholesterylacetate in CDCI3 T T T T T T T T T T T T T T T T T T T 180 170 160 150 140 130 120 110 100 90 80 70 oO 50 40 30 20 10 0 ppm Avance 1D 2D BRUKER 79 80 BRUKER Avance 1D 2D 8 COSY 8 1 Introduction COSY COrrelation SpectroscopY is a homonuclear 2D technique that is used to correlate the chemical shifts of H nuclei which are J coupled to one another In this chapter two types of COSY sequences magnitude COSY and double quantum filtered DQF COSY with and without pulsed field gradients will
124. gnetization from the antiphase coherence present at the end of the fixed delay The acquisition period follows immediately after the second pulse 162 BRUKER Avance 1D 2D The frequency of the selective pulse is set to the chemical shift of a multiplet and the selectivity is chosen so that adjacent multiplets are unperturbed The spectral width is set large enough to cover the entire chemical shift range whatever the transmitter offset The intensity of the transferred signal depends on the magnitude of the appropriate coupling constant and on the length of the fixed delay and varies in a sinusoidal fashion There is a chance that a particular transfer falls accidentally at a null in which case a coupling path would be overlooked This risk can be minimized by selecting the precession interval short compared with the reciprocal of the largest expected coupling constant The lower level of the delay is one half the Gaussian duration needed to get the required selectivity Since the final pulse gives coherence transfer to spins whose couplings are in antiphase to the selectively excited spin 1D selective COSY gives rise to antiphase multiplets which will unavoidably have adjacent positive and negative intensities Thus direct extraction of the coupling constants may be complicated due to annihilation of individual lines within the multiplet Note that the final pulse also converts any longitudinal magnetization into transverse magnetization The
125. gs UxpH n gt 1 for the polarization transfer and detects all C even those which are not directly bonded to H Because of the close similarity to the XHCORR the COLOC experiment is only described in brief here Reference H Kessler C Griesinger J Zarbock and H R Loosli J Magn Reson 57 331 1984 The sample used to demonstrate COLOC in this chapter is 1g Cholesterylacetate in CDCl as already used for the DEPT and XHCORR experiments The COLOC pulse sequence is shown in Figure 35 The evolution time t is incorporated in the polarization transfer period A 1 2 JxH Since the long range heteronuclear coupling constants are small e g Jcu 5 to 20 Hz the time period A is rather long and serious sensitivity losses due to transverse relaxation are inevitable Figure 35 COLOC Pulse Sequence T T 2 Avance 1D 2D BRUKER 115 13 2 Acquisition and Processing Start out from the xhcorr 3 1 data set re xhcorr 3 1 and create the data set coloc 1 1 type ede and change the name to coloc and the experiment number to 1 The acquisition parameters are shown in Table 49 In this pulse sequence the delay time d6 determines the length of the delay for the creation of anti phase magnetization A 1 2 Jci and the time dis determines the length of the refocusing Ai 1 a Jcu where a is generally chosen to be 3 To ensure that the pulses occur during A1 the user must make sure that d6 dO td F1
126. gth p11 of the RF pulse is adjusted to determine the exact conditions for a 90 pulse A common sample used for the H pulse calibration is 0 196 Ethylbenzene in CDCIs Ethylbenzene shows a simple H spectrum with well separated signals which facilitates the selection of a single resonance line However due to the relatively long spin lattice or longitudinal relaxation time T of Ethylbenzene a long recycle delay time must be used 4 2 1 Preparation Insert the sample and lock the spectrometer Lock Readjust the Z and Z shims until the lock level is maximal use 1ockdisp Tune and match the probehead for H observation see Chapter 2 3 First create a new data set Since this will be a H observe experiment it is best to start out from a previous H data set e g proton 1 1 Enter re proton 1 1 then enter edc and change the following parameters NAME testih EXPNO 1 PROCNO 1 Click on SAVE to create the data set test1h 1 1 Enter eda and set the acquisition parameter values as shown in Table 14 Avance 1D 2D BRUKER 39 Table 14 1D H one pulse Acquisition Parameters Parameter Value Comments PULPROG zg see Figure 1 for the pulse sequence diagram TD 4k NS 1 number of scans DS 0 no dummy scans D1 10 interscan delay 10s because of long T P1 3 start with 3us which should correspond to less than a 90 pulse PL1 power level for the p1 pulse see An Important Note on Power Levels
127. has dispersive phase and therefore the trim pulses improve the phase of the signals in the TOCSY experiment One has to take into account that trim pulses act as B gradient pulses This means that the second trim pulse might partially refocus magnetisation which has been defocused by the first trim pulse This effect can be observed in TOCSY experiment with aqueous solutions were a presaturation is used for water suppression The artefact which is caused by the partial refocusing described above results in a poor solvent suppression in the TOCSY spectrum In the FID it appears as an echo signal which is delayed by the length of the spin lock By removing the first spin lock pulse this artefact can be avoided Figure 69 First serial file of a TOCSY experiment Top two trim pulses Bottom one trim pulse gt echo after 100ms z length of spinlock Avance 1D 2D BRUKER 183 Figure 70 Two TOCSY experiments of 2mM sucrose in 90 H2O 10 D20 The spectrum shown on the left side was recorded using one trim pulse the spectrum shown on the right was recorded using two trim pulses and has a poor solvent suppression 184 BRUKER Avance 1D 2D Avance 1D 2D BRUKER 185 23 Appendix B Theoretical Background of NMR 23 1 Introduction In the next few paragraphs an attempt will be made to introduce the spin operator as a handy tool for understanding more or less involved NMR experiments However at the same time we w
128. hase correction type rser 1 instead of rser 2 as for the TOCSY and ROESY spectra Figure 32 NOESY Spectrum of 50 mM Cyclosporin in CgDg Avance 1D 2D BRUKER 107 108 BRUKER Avance 1D 2D 12 XHCORR 12 1 Introduction Heteronuclear X H shift CORRelation spectroscopy is a 2D technique that can be used to determine which H of a molecule are bonded to which C nuclei or other X nuclei Like DEPT XHCORR makes use of the large one bond heteronuclear J coupling JxH for polarization transfer and thus only 13C bonded directly to H s are detected For C and directly attached H JxH 100 to 200 Hz while for more distant H Jx 5 to 20 Hz The final 2D XHCORR spectrum has a projection onto the F2 axis which is the usual H decoupled C spectrum with all quaternary carbons missing and a projection onto the F1 axis which is the normal H spectrum with reduced signal to noise since only H directly attached to C contribute to the signal The XHCORR experiment is not phase sensitive and so the final 2D spectrum must be displayed in magnitude mode Reference A Bax and G A Morris J Magn Heson 42 501 1981 The sample used to demonstrate XHCORR in this chapter is 1g Cholesterylacetate in CDCla which was used to demonstrate DEPT The XHCORR pulse sequence is shown in Figure 33 The short delay between the final C pulse and the start of acquisition is a refocusing period so that the C lines do n
129. have already been recorded for the XHCORR experiment Follow the instructions given there for the acquisition and the processing of the 2D COLOC experiment A COLOC spectrum of 1g Cholesterylacetate in CDCl3 is shown inFigure 36 Avance 1D 2D BRUKER 117 Figure 36 COLOC Spectrum of 1g Cholesterylacetate in CDCI3 ppm 1 0 2 0 3 0 4 0 5 0 160 140 120 100 80 60 40 20 ppm 118 BRUKER Avance 1D 2D 14 HMQC 14 1 Introduction HMQC Heteronuclear Multiple Quantum Correlation spectroscopy is an inverse chemical shift correlation experiment that yields exactly the same information as the XHCORR The advantage of HMQC is that the nucleus with the highest y H is detected and so it is possible to obtain the highest sensitivity The challenge of an inverse chemical shift correlation experiment however is that the large signals from H not coupled directly to a C nucleus must be suppressed in a difference experiment This poses a dynamic range problem the signal of interest is that of H coupled directly to 136 nuclei however the signal detected is dominated by the contribution of TH bonded directly to C nuclei HMQC minimizes this dynamic range problem while optimizing the sensitivity of the experiment The resonance frequency of low y spins can be detected with enhanced sensitivity by the creation and H detection of H C or other X nucleus multiple quantum coherence References A Bax R H
130. he numerical results on the screen set CURPRIN to screen as follows enter edo click the box next to CURPRIN with the left mouse button and enter screen Click _ _ to exit the edo menu The numerical results consist of a table for each selected peak These tables indicate TAU i e vd value CURSOR FREQ PPM INTEGRAL and INTENSITY for each point Below each table is the statement n intensities fit or n areas fit This is an indication of how well the peak picking worked For example if peak picking worked well for the 10 vd values 10 intensities should have been fit for each peak selected If 0 or very few intensities were fit for one or more peaks it is a good idea to redefine the integral range and baseline point files and rerun proc t1 Finally for each selected peak there is a table indicating the T and standard deviation 19 4 3 T parameters If necessary the user may edit a number of parameters used in the Ti calculation routine In the T4 Ta menu type t1 t2 enter edt1 Some appropriate values are indicated in Table 61 Table 61 T Parameters Parameter Value Comments NUMPTS 10 number of delays in vdlist FITTYPE intensity Ti will be calculated from peak intensity rather than integrated area CURSOR 1 start with the first peak chosen CONV B convergence criterion for the fit algorithm DRIFT 20 allowed peak drift for peak picking START 1 starting spectrum for peak
131. he one spin system during detection t2 we get the following expression for our density operator o t 1 cos t I sin f 192 BRUKER Avance 1D 2D After rewriting the equation for l and l 1 1 1 1 1 i pct ue all _ y we can substitute lx and in o t I cos 8 t I sin t 4 I cos 8 d DU I sin 8 t L feos t i sin t 51 leos t i sin 6 t gree L pres When calculating the expectation value of l the observable signal we find Xt Tri o t Tr Ll ie e qoe et TTT peret Trj 0 I The signal function is an oscillation with the frequency and the amplitude Ya lo The amplitude lo is in fact an elegant way to hide a bunch of quantum mechanical constants and it is ignored most of the time Normally one is interested in relative signal intensities rather than in absolute values One might object that 5 is not the Larmor frequency which one might have expected but only the chemical shift relative to the rotation frequency carrier frequency of the interaction frame Remember that also the detection operator is defined in the rotating frame e g is also rotating with the carrier frequency The technical realization of this rotating detector is achieved by mixing the signal from the probe which is in the MHz range with the carrier frequency which is also in the MHz range The mixing process yields the diff
132. her use a high B field or a long pulse duration t Furthermore the gyromagnetic ratio y also strongly influences the behavior of the flip angle This explains the need for specific rf power for different nuclei 23 6 1 Effect of Chemical Shift Evolution So far we discussed the Hamiltonian corresponding to an system under perturbation by an rf pulse and neglecting chemical shift and relaxation at the same time In this simple introduction relaxation will always be neglected The chemical shift Hamiltonian of the unperturbed system will have to describe a precession around the static field We have to remember that for convenience all operations are done in the interaction frame e g that all Larmor frequencies are replaced by the chemical shift or precisely by the difference between the Larmor and the carrier frequency Under this condition the chemical shift Hamiltonian is given by H 6 where is 6 20 0 where oo is the Larmor frequency of the spin and o the carrier frequency of the interaction frame In case that the Larmor frequency is different from the carrier frequency this is a rotation around L in the rotating frame If there is no relaxation shifting the system back to thermal equilibrium this is the expected result The calculus rules for the chemical shift evolution are the following 1 451 I 51 cos t I sin 8 t I hts cos 6 t I sin f The time t is the period during which the Hamiltonian is val
133. id The Hamiltonian of a spin system can change with time for example if the experimental setup prescribes first a rf pulse and then a period of unperturbed evolution For the calculus rules given to be valid it is mandatory that each Hamiltonian is time independent during the time t This means that chemical shift can evolve only in the state of magnetization within the x y plane a k a transversal magnetization Thus the whole experiment is divided into time intervals during which the Hamiltonian can be made time independent by choice of a suitable interaction frame Typical experiments are divided in pulse intervals and free evolution times Avance 1D 2D BRUKER 191 During the pulses the chemical shift and scalar coupling interaction is ignored Only the applied B field is considered This approach is justified for pulses with tpulse lt lt T1 T2 The question now is how to interpret this quantum mechanical result in terms of macroscopic measurements To answer this question we will need to discuss the difference between operators and physical observables This will be the subject of the next paragraph 23 7 Observable Signals and Observable Operators Not all operators correspond to physical forces or fields In fact only a minority gives rise to detectable energy changes of any kind In our particular case only ix ly and are physical observables e g they correspond to physical phenomena which can be measured In a o
134. ight recommended values 0 6 ml or 4 cm of solution for 5 mm sample tubes 4 0 ml or 4 cm of solution for 10 mm sample tubes This minimizes the shimming that needs to be done between sample changes e Use the depth gauge to position the sample tube in the spinner This is discussed further in Chapter 5 Sample Positioning of the BSMS User s Manual e Check that the sample tube is held tightly in the spinner so that it does not slip during an experiment e Wipe the sample tube clean before inserting it into the magnet e For experiments using sample spinning be sure that the spinner especially the reflectors are clean This is important for maintaining the correct spinning rate 2 2 Bruker NMR software There are three major tasks that are controlled by the NMR software acquisition processing and plotting The XWinNMR program is the user interface for all of these tasks The commands can either be called up by selecting the menu items or by typing the appropriate command in the command line followed by RETURN There are many parameters that are important for each job and they can be accessed and edited by the user These parameters and the measured data as well as the processed spectra are stored in datasets which are specified by names experiment numbers expno and processing numbers procno Avance 1D 2D BRUKER 17 Each parameter can be accessed directly by entering it s name in the command line followed by RETURN or in th
135. ill try to limit the mathematical and purely academic sides of this formalism to an absolute minimum In other words you should not need a degree in mathematical science to be able to use and understand the spin operator formalism as a tool for a better understanding of the experiments covered during this course Also being strictly correct this introduction will try to avoid as many tricky or complicated issues as possible The goal will be to enable the use of the spin operator formalism as a tool and not to give an introduction to quantum mechanics In order to make the first contact with the subject a bit smoother we will introduce the first concepts in analogy to the Bloch equations The Bloch equations are very intuitive and convenient to explain relatively simple 1D experiments But coupled 2 spin systems are already a challenge in this model while a 3 spin system becomes impossible to describe The spin operator formalism for a one spin system is very similar to the Bloch equations and we will use this similarity to ease the first contact However it should be kept in mind that while the Bloch formalism is concerned with macroscopic magnetization only the spin operator formalism describes the full state of the spin system including non observable terms Those non observable terms however ignored in the Bloch equations are the basis of most modern experiments 23 2 Classical Description of NMR Among the various atomic nuclei abo
136. in 35 time units in XWinNMR 24 title for a spectrum 75 TOCSY 19 TOCSY acquisition parameters 97 TOCSY processing parameters 98 TOCSY pulse sequence 96 total correlation spectroscopy 96 transmitter 193 tuning and matching the probe 25 variable delay list 172 vd 172 vdlist 154 weak coupling 202 window function 36 wobbling 22 26 28 wobbling channel selection 21 wpar 18 XHCORR 113 XHCORR acquisition parameters 115 XHCORR processing parameters 116 XHCORR pulse sequence 114 z gradient hardware 19 Avance 1D 2D
137. in Figure 25 8 4 1 Pulse Sequence The GRASP DQF COSY pulse sequence is very similar to the conventional DQF COSY pulse program After the second pulse the spin system exhibits multiple quantum coherence and the application of a PFG G yields complete Avance 1D 2D BRUKER 89 dephasing of all coherences In order to obtain a phase sensitive spectrum later on the effect of chemical shift evolution during G has to be eliminated by a spin echo The third 90 pulse converts part of the multiple quantum coherence into observable single quantum coherence which is rephased by the PFG Ge of proper intensity All the unwanted magnetization stays dephased and can not be observed during the acquisition Only spins J coupled to at least one other spin are detected and solvent signals especially water are suppressed very efficiently The intensity ratio of the PFGs G1 G is 2 1 for a double quantum filter and 3 1 for a triple quantum filter Figure 25 GRASP DQF COSY Pulse Sequence T 2 n 2 T T 2 T Gradient G G 8 4 2 Acquisition and Processing Follow the instructions given in Sections 8 3 2 to 8 3 4 for the conventional DQF COSY and create the data set cosy 4 1 starting out from the DQF COSY data set cosy 3 1 Three parameters related to the PFGs G4 and Ge must be defined The length of the PFG 16 the recovery delay after the PFG 16 and the shape and the intensity of the individual gradients Table 40 GRASP DQF
138. ing the only relevant spectrometer parameters are SFO1 Click on SAVE to save the spectrometer parameters and return to the main window The spectrometer is now prepared to pulse and detect at the H frequency 3 5 Set Up the Acquisition Parameters Enter eda and set the acquisition parameters as shown in Table 13 where only the relevant parameters are listed Note that the parameters d1 p1 and pli are included in the parameter arrays D P and PL in the eda table respectively These parameters can be edited within eda by clicking the Array button next to the corresponding parameter Table 13 Basic H Spectrum Acquisition Parameters Parameter Value Comments PULPROG zg see Figure 1 for the pulse sequence diagram AQ_mod DQD If DQD is not available use qsim TD 32 k 32 k is a standard value for a high resolution 1D spectrum PARMODE 1D One dimensional experiment NS 1 one scan is recorded for parameter optimization DS 0 no dummy scans are recorded D1 2 the default unit for delays is seconds entering 2 sets a delay of 2 seconds click the D Array button P1 3 the default unit for pulse lengths is microseconds entering 3 sets a pulse length of 3 microseconds us click the P Array button PL1 PL1 power level for the p1 pulse see also An Important Note on Power Levels on page 3 click the P Array button SW 50 for the first spectrum of an unknown sample
139. ion pulse This is followed by a fixed delay rather than the variable evolution period of the 2D TOCSY sequence during which in phase coherence is created by evolution due to J coupling The duration of this delay is measured from the middle of the Gaussian envelope Next the coherence transfer occurs during the multiple pulse spin lock period The multiple pulse spin lock sequence most commonly used is MLEV 17 The length of the spin lock period determines how far the spin coupling network will be probed A general rule of thumb is that 1 10 Jun should be allowed for each transfer step and five transfer steps are typically desired for the TOCSY spectrum Immediately after the spin lock period a z filter is introduced which allows easier phase correction of the final spectrum Since the TOCSY correlation peaks arise from magnetization that was in phase during the fixed delay they can be phase corrected to be positive and absorptive Avance 1D 2D BRUKER 165 Figure 59 Selective TOCSY Pulse Sequence tidy l 1 2 p11 p17 p5 p6 p7 p17 p6 p6 di d14 20 4 1 Variable Delay List The z filter in the selective TOCSY experiment requires a variable delay list To create the variable delay list enter edlist A menu of list types appears Select vd from this menu This calls up a menu of existing vdlist filenames and gives the user the option of creating a new file Type new name Simply type the name zf This calls u
140. is data set Enter zg to acquire the FID Enter edp and set the processing parameters as shown in Table 25 Table 25 1D C One pulse Processing Parameters Parameter Value Comments SI 2k LB 1Hz PSCAL global Fourier transform the data e and phase the spectrum manually Type sref to calibrate the spectrum and confirm the message no peak found in sref default calibration done Expand the spectrum until only the chloroform signal at 77 ppm is displayed Enter the calibration submenu by clicking utilities Click on o1 with the left mouse button and move the cursor to the center of the signal Click the middle mouse button to assign o1p to this frequency Click rewm to exit BRUKER Avance 1D 2D 6 3 4 the calibration submenu and return to the main window This o1p value will be the C o2p value for the DECP90 pulse sequence below Set the H Carrier Frequency and the Spectral Width Now a H observe experiment to determine the correct offset for H olp must be recorded Create a new H data set starting from a previous one e g proton 1 1 re proton 1 1 Enter ede and change the following parameters NAME testinv EXPNO 2 PROCNO 1 Click on SAVE to create the data set testinv 2 1 then enter eda and change the acquisition parameters as shown in Table 26 Avance 1D 2D BRUKER 61 6 3 5 62 Table 26 1D H One pulse Acquisition Parameters Pa
141. ised to use only the power levels indicated in Table 1 below if no other information e g final acceptance tests is available Note that these power levels are really attenuation levels and so a higher value corresponds to a lower power Also note that these power levels pertain only to the specific spectrometers and amplifiers listed below which correspond to the AVANCE instruments as of July 2000 It is assumed that no correction tables CORTAB are existing Table 1 Suggested Proton and Carbon High Power Levels for Avance Instruments Nucleus Spectrometer Amplifier Power Level Avance BLA2BB gt 3dB BLARH100 gt 3dB BLAXH300 50 gt OdB Avance DPX BLAXH20 6dB H BLAXH40 3dB BLAXH100 50 gt OdB Avance DRX BLAXH150 50 gt OdB BLAXH300 50 gt OdB Avance DMX BLARH100 gt 3dB Avance 1D 2D BRUKER 9 Nucleus Spectrometer Amplifier Power Level Avance BLA2BB gt 6dB BLAX300 50 gt 6dB BLAX300 gt 6dB BLAX500 gt 9dB Avance DPX BLAXH20 6dB c BLAXH40 6dB BLAXH100 50 gt 3dB Avance DRX BLAXH40 gt 3dB BLAXH150 50 gt OdB BLAXH300 50 gt 6dB Avance DMX BLAX300 gt 6dB BLAX500 gt 9dB 1 2 NMR Spectrometer The NMR spectrometer consists of three major components 1 The superconducting magnet with the probe which contains the sample to be measured 2 The console which
142. ition parameters 68 1D HMQC processing parameters 68 1D HMQC pulse sequence 67 1D proton acquisition and processing 32 H decoupling 90 pulse calibration 60 H decoupling 90 pulse during C acquisition 56 H C chemical shift correlation 19 H H through bond chemical shift correlation 19 90 degree pulse 194 acquisition commands 20 acquisition parameters 21 acquisition parameters with gated and inverse gated decoupling 72 angular momentum 192 antiphase coherences 96 ased 18 atma 26 AU Programs 20 autolock 29 Automation 176 Bo 193 baseline submenu 157 basl 157 baslpnts 157 BIRD parameter optimization 126 BIRD preparation 123 BIRD HMQC 19 Bloch equations 192 193 Boltzman distribution 195 browse the data set directories 21 C13CPD 19 C13DEPT135 19 C13DEPT45 19 C13DEPT90 19 C13GD 19 calcphinv 127 cholesterylacetate 46 Cholesterylacetate 32 classical description 192 cnst 24 COLOC 20 119 COLOC acquisition parameters 120 COLOC pulse sequence 119 configure the routing 21 correlation spectroscopy via long range coupling 119 CORTAB 9 COSY 19 84 205 208 COSY acquisition parameters 86 COSY processing parameters 87 COSY 45 pulse sequence 85 COSY45SW 19 COSYDQFPHSW 19 COS YGPDFPHSW 19 206 BRUKER COSYGPSW 19 coupling constants 14 cross relaxation 107 CURPRIN 159 dit 23 damaging the probehead 9 dataset 17
143. l rather than p3 and p12 as used here 6 3 Carbon Decoupler 90 Pulse Inverse Mode This calibration procedure should yield approximately the same pulse length as for the C observe 90 pulse Section 6 1 4 However the method described here is more convenient because of the more sensitive H detection instead of C In addition the T of H is shorter than for C which allows the selection of shorter interscan delays 6 3 1 Sample For these so called inverse experiments the detected nucleus is H but C satellites must be visible Therefore a convenient sample is the H Lineshape Sample 396 Chloroform in Acetone d6 for frequencies between 300 MHz and 500 MHz and 196 Chloroform in Acetone d6 for frequencies 58 BRUKER Avance 1D 2D gt 600 MHz Here the procedure is described for this readily available standard sample However use the Pulse Calibration sample containing 0 1M 8C methanol and 0 1M N urea in DMSO de if this is available The same DECP90 pulse sequence as used for the H 90 decoupling pulse determination in Section 6 2 is used here except that the H and C channels are interchanged as shown in Figure 11 Figure 11 DECP90 Pulse Sequence T 2 1H tra 1 2J C H acq T 2 136 6 3 2 Preparation 6 3 3 Insert the sample lock the spectrometer readjust the Z and Z shims until the lock level is optimized tune and match the probehead for H observation and C decoupling
144. l value of 32 usec For each of the 16 spectra only the spectral region defined above is plotted and all the spectra are plotted side by side in the file test1h 1 999 as shown in Figure 3 At the end of the experiment the message paropt finished and a value for the parameter p1 is displayed which corresponds to the 90 pulse length of the H transmitter with the power level as defined by p11 Write this value down and follow the procedure described below to obtain a more accurate 90 pulse measurement Return to the data set testih 1 1 by entering re 1 1 Type p1 and enter a value which corresponds to a 360 pulse i e four times the 90 value determined by paropt before Acquire and process a new spectrum by typing zg and efp see Chapter 3 9 respectively Change p1 slightly and acquire and process a spectrum again until the quartet undergoes a zero crossing as expected for an exact 360 pulse Note that the quartet signal is negative for pulse angles slightly less than 360 and positive when the pulse angle is slightly more than 360 Avance 1D 2D BRUKER The 360 pulse length divided by four yields the accurate H 90 transmitter pulse length for the actual power level p11 Figure 3 Paropt Results for 1H 90 Pulse Calibration pi 180 pi 270 p1 90 4 2 5 Calibration Low Power for MLEV Pulse Train TOCSY The H 90 pulse for the MLEV pulse train used during the spinlock period of a TOCSY sequence is between 30
145. ld be phased properly Expand the spectrum so that the N H doublet occupies approximately the center quarter of the window e g so that the region from approximately 9 2 ppm to 8 1 ppm is displayed Save this as a plotting region by clicking on ail with the left hand mouse button and hit return in response to the questions This plotting region will be used by the au program paropt below 160 BRUKER Avance 1D 2D 20 2 5 Perform the pulse calibration The au program paropt may is used to perform an automatic pulse calibration Simply enter xau paropt and answer the questions as follows Enter parameter to modify sp1 Enter initial parameter value 90 Enter parameter increment 2 Enter of experiments 20 At the end of the experiment the message paropt finished and a value for sp1 are displayed This value is the approximate H transmitter power level for a 90 pulse time of 80 msec To obtain a more accurate 90 pulse repeat paropt using a smaller increment for sp1 At this point it may be useful to repeat the above procedure for a range of p11 pulse lengths Paropt results of selective excitation of a N H resonance is shown in Figure 55 A selective one pulse H spectrum of Cyclosporin together with the reference spectrum is shown in Figure 56 Figure 55 Selective One pulse Paropt Results 90 270 Avance 1D 2D BRUKER 161 Figure 56 Selective One pulse Spectrum of 50 mM Cyclosporin in CDs
146. lick on the and KM buttons located on the button bar 3 7 Processing After the FID has been acquired the next step is to process the acquired data The processing parameters are displayed and edited by entering edp First Fourier transformation is performed by entering the command t The number of points used to resolve the resulting spectrum is determined by the processing parameter si size The spectrum consists of si real points and si imaginary points and thus the default setting of si is td 2 where td is the acquisition parameter indicating the number of time domain data points In general td 2 and si are numbers described by powers of 2 2 4 8 16 32 64 128 If si td 2 not all the time domain data is used for the Fourier transformation and if si gt td 2 the time domain data is zero filled with 2 si before the Fourier transformation In 1D spectroscopy it is often recommended to zero fill once i e to set si td Check the value of si Enter si and when prompted enter 32k appropriate for td 32k Enter ft The display automatically switches from the acquisition window to the main window and displays the The FID can still be viewed by returning to the acquisition window If the x axis of the Fourier transformed spectrum is displayed in Hz click on _Hzppm_ to convert into a ppm scale If necessary use the buttons as described above to scale the spectrum 34 BRUKER Avance 1D 2D You can zoom into a part
147. listed below 10 Avance 1D 2D BRUKER 149 Save the file and exit the editor It is recommended to begin and end the list with the longest vd value and to scramble the order of the intermediate values 19 2 2 Set up the acquisition parameters Enter eda and set the acquisition parameters as shown in Table 59 Table 59 Inversion Recovery Acquisition Parameters F2 Parameters Parameter Value Comments PULPROG ttir see Figure 52 for pulse sequence diagram TD 16k NS 8 the number of scans must be 8 n DS 4 number of dummy scans PL1 high power level on F1 channel H as determined in Section 4 2 4 P1 H 90 pulse as determined in Section 4 2 4 P2 H 180 pulse 2 P1 D1 10 10s relaxation delay L4 10 loop counter set to number of entries in vdlist VDLIST tidelay vdlist with various recovery delays F1 Parameters Parameter Value Comments TD 10 number of experiments set to value of L4 19 2 3 Acquire the 2D data set 150 If this data set was created from the receiver gain is already set correctly H reference spectrum t1data 1 1 the Enter zg to acquire the time domain data The approximate experiment time for the inversion recovery experiment with the acquisition parameters set as shown above is 30 minutes BRUKER Avance 1D 2D 19 3 Processing Enter edp and set the processing parameters as shown in Table 60 Table 60 Inversion Recovery Processing Param
148. lse as determined in Section 4 2 4 P15 200m spinlock pulse D1 2 100 BRUKER Avance 1D 2D F1 Parameters Parameter Value Comments TD 256 number of experiments FnMODE States TPPI NDO 1 one dO period per cycle INO t increment equal to 2 DW used in F2 SW sw of the optimized H spectrum cosy 1 1 same as for F2 NUC1 select H frequency for F1 same as for F2 Enter zg to acquire the time domain data The approximate experiment time for ROESY with the acquisition parameters set as shown above is 5 5 hours 10 3 Processing Enter edp and set the processing parameters as shown in Table 44 Table 44 ROESY Processing Parameters Avance 1D 2D F2 Parameters Parameter Value Comments SI 512 SF spectrum reference frequency H WDW SINE multiply data by phase shifted sine function SSB 2 choose pure cosine wave PH mod pk PKNL TRUE BC mod no F1 Parameters Parameter Value Comments SI 512 SF spectrum reference frequency H WDW SINE multiply data by phase shifted sine function SSB 2 choose pure cosine wave PH mod pk BC mod no MC2 States TPPI BRUKER 101 Enter xfb to multiply the time domain data by the window functions and to perform the 2D Fourier transformation The threshold level can be adjusted by placing the cursor on the button holding down the middle mouse button and moving the mouse back a
149. ltiple quantum coherences to differentiate between the different types of C signals Quaternary carbons are missing from DEPT spectra because the large one bond heteronuclear J coupling JxH is used for polarization transfer DEPT may be run with or without H decoupling and it is relatively insensitive to the precise matching of delays with coupling constants and so is much easier to use than the closely related INEPT sequence DEPT on the other hand is more sensitive to pulse imperfections than INEPT The sample used in this chapter is 1 g Cholesterylacetate in CDCla 72 BRUKER Avance 1D 2D The DEPT pulse sequence is shown in Figure 17 The final H pulse with flip angle a selects for the CH3 CH or CH signals This angle is set to 45 in the DEPT 45 sequence which yields spectra with positive CH CH and CH3 signals to 90 in DEPT 90 which yields spectra with only CH signals and to 135 in DEPT 135 which yields spectra with positive CH and CHs signals and negative CH signals Figure 17 DEPT Pulse Sequence T 2 T 13C la di d2 d2 d2 acq 7 2 1 Acquisition and Processing Insert the sample in the magnet Lock the spectrometer Readjust the Z and Z shims until the lock level is optimized Tune and match the probehead for 18C observation and H decoupling 7 2 2 Reference Spectra Since DEPT is a C observe experiment with H decoupling experiment a reference H spectrum of the sample must be recorded to det
150. lue Comments PULPROG zg see Figure 1 for pulse sequence diagram TD 4k NS 1 DS 0 D1 20 interscan delay 20s because of long T4 P1 3 start with 3us which should correspond to less than a 90 pulse PL1 power level for the p1 pulse see An Important Note on Power Levels on page 3 SW 350 start with a large spectral width of 350ppm which will be optimized lateron o1p start with 100ppm it will be optimized later Avance 1D 2D BRUKER 51 Enter rga to perform an automatic receiver gain adjustment enter zg to acquire the FID and edp to set the processing parameters as shown in Table 19 Table 19 1D C one pulse Processing Parameters Parameter Value Comments SI 2k LB 3Hz PSCAL global Add line broadening and Fourier transform the spectrum with the command ef Manually phase correct the spectrum and store the correction Type sref to calibrate the spectrum and confirm the message no peak found in sref default calibration done 6 1 2 Optimize the Carrier Frequency and the Spectral Width The carrier frequency should now be set to the signal used to calibrate the 90 pulse Expand the spectrum until only the doublet at 130 ppm is displayed Enter the calibration submenu by clicking wes Click on o1j with the left mouse button and move the cursor to the center of the doublet Click the middle mouse button to assign o1p to this frequency Click reum to exit the calibration submenu and
151. ly the arrow notation where we find on the left side the system before and on the right side after the specific evolution under the operator noted above the arrow This notation is simple very convenient and not only limited to the description of rf pulses We will discuss this notation in more detail in the next section 23 6 The Hamiltonian Evolution of Spin Systems in Time The arrow notation which was introduced like a deus ex machina in the previous section needs some more explanation First let us introduce a new type of operator the Hamiltonian H Each quantum mechanical system has its associated H which describes the possible changes of energy of the system Once the H is known the evolution of the density matrix of the corresponding system can be described by o t 2 exp i H o 0 expG H r under the condition that H by itself is time independent The above equation in the arrow notation will be T t o 0 20 t In other words the arrow notation is a compact an elegant way of describing the different steps of a time evolution under different Hamiltonians The Hamiltonian corresponding to an rf pulse neglecting relaxation and chemical shift is given by 190 BRUKER Avance 1D 2D which describes a precession around with frequency yB The corresponding flip angle B equals B yB t where t is the duration of the pulse H tey B 1 t BI This result illustrates that for a given flip angle B one can eit
152. n in C6D6 Avance 1D 2D BRUKER 127 ppm 20 49 60 100 120 140 160 180 128 BRUKER Avance 1D 2D 16 Proton Carbon Inverse Shift Correlation Experiments using Pulsed Field Gradients 16 1 Introduction The three most common inverse chemical shift correlation experiments are HSQC HMQC and HMBC which are used to determine which H of a molecule are bonded to which C nuclei or other X nuclei The advantage of inverse experiments over X detection experiments is that with inverse experiments the nucleus with the highest y usually H is detected yielding the highest sensitivity The challenge of an inverse chemical shift correlation experiment however is that the large signals from H not coupled directly to a C nucleus must be suppressed in a difference experiment which poses a dynamic range problem Common techniques for the suppression of H bound to C are the BIRD sequence in HMQC experiments and a trim pulse of 1 2ms during the first INEPT in HSQC experiments However the suppression is still imperfect and usually additional phase cycling is required The introduction of pulsed field gradients in high resolution NMR greatly improved the problem of suppressing signals from H bonded to C The suppression is almost perfect without additional phase cycling In general NMR experiments using PFGs are called GRASP experiments GRASP HSQC GRASP HMQC GRASP HMBC etc Details on the GRASP
153. n 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 general the DQF COSY experiment is recommended if a higher resolution is desired References W P Aue E Bartholdi and R R Ernst J Chem Phys 64 2229 1976 K Nagayama A Kumar K W thrich and R R Ernst J Magn Reson 40 321 1980 Avance 1D 2D BRUKER 81 8 2 1 Pulse Sequence The COSY 45 pulse sequence is shown in Figure 21 The pulse p1 must be set to the appropriate 90 pulse length found in Chapter 4 2 4 Figure 21 COSY 45 Pulse Sequence T 2 T 4 8 2 2 Acquisition of the 2D COSY Spectrum Insert the sample in the magnet Lock the spectrometer Readjust the Z and Z shims until the lock level is optimized Tune and match the probehead for 1H observation It is recommended to run all 2D experiments without sample spinning Record a H reference spectrum to obtain the correct carrier frequency o1p and spectral width sw values Enter re proton 1 1 to call up the data set proton 1 1 enter ede and change the following parameters NAME cosy EXPNO 1 PROCNO 1 Click 1s to create the data set cosy 1 1 Enter rga to perform an automatic receiver gain adjustment Acquire and process a standard H spectrum Calibrate the spectrum and optimize sw and o1p so that the
154. nando ee Rd 6 2 6 Calibration Low Power for WALTZ 16 Decoupling sens 6 3 CARBON DECOUPLER 90 PULSE INVERSE MODE sise 0 3 gt SGM ple co ee tede eei e A A A e see Led p d npe cedri sei bes 6 3 2 Preparation esee tree tete pre eene ee eee rk enel ee eee RU RAR eee aedd 6 3 3 Set the PC Carrier Frequency o acero tdt Ne a tle tede re bo ues se EUN 6 3 4 Set the H Carrier Frequency and the Spectral Width 6 3 5 Preparations for the Inverse Pulse Calibration sn 6 3 0 Calibration High Powers aee diee dei ee deg io cte etre HD Fe Inn it Deere ERE HEC HE tels 6 3 7 Calibration Low Power for GARP Decoupling inner 6 4 TD INVERSE TEST SEQUENCE ro EPOR REPE ENEEERE CE SECHS EREI sacs sashes ODER ARI EG CIO 7 ADVANCED ID C EXPERIMENTS 5i ooponrien euadere tee obi PU e e shine 69 7 1 CARBON EXPERIMENTS WITH GATED H DECOUPLING Re notru setae tertio oet s opuesto ne a 69 7 1 1 Plotting 1D C Spectra RAR MU UT Le 71 FI DI 2 SA MC 72 7251 Acg isiton and Processing Re ep BOR pete tbe AREA ens 73 L22 Reference Speca xc oo ere e erat e OR SA RO tree Eee ARARE a 7 2 59 Create a New Data Set 724 WSBOCIRWIBZACQUISIUDT rec t e ee e e repuesto En Men ee 7 2 9 Processing Of the Spectrum i e eerte t reae ci ee LO e tee ure tee ted 75 72 0 Other SPECT se e e bee ette d ect A eene be ee SIR Lucre 75 42 7 Plobthe spectra sse ie tee e e nf ne tete ede e nega estet Te e Enea estne 75 T APT ATTACHED P
155. nce 1D 2D The entire cycle is repeated until the experiment is finished The number of times this cycle is performed is determined by the value entered for the number of average cycles 17 3 Processing Enter edp and set the processing parameters as shown in Table 56 Table 56 1D NOE Difference Processing Parameters Parameter Value Comments SI 8k WDW EM LB 0 3 PKNL TRUE 17 3 1 Perform the Phase Correction To process the spectra acquired by noemult it is necessary to define the phase correction parameters first Read in the first file re 2 1 Apply the window function and Fourier transformation with the command ef Manually phase correct the spectrum and store the correction see Section 3 8 All spectra are processed identically by entering xau multiefp and answer the questions as follows Enter first expno to process 2 Enter number of expnos 3 Here the first expno to process indicates the spectrum that is already Fourier transformed and phase corrected The au program multiefp reads all processing parameters including 0 and 1 order phase corrections from this data set and uses them to process the following three spectra with the same data set name At this point the data consists of a series of spectra with various saturated resonances and one reference spectrum The procedure for creating the difference spectra is outlined below 17 3 2 Create NOE Difference Spectra The NOE
156. nce 1D 2D BRUKER 131 Figure 44 GRASP HSQC Pulse Sequence T 2 T trim p1 p2 p28 pi p2 pi p2 pi p2 pi p2 acq H ta Ua T nre 148 Jas or t 4 Seu 13C t 2 t 2 p4 p3 p4 p3 p4 p3 p4 di d4 d4 dO do DELTA d24 d24 d4 d4 DELTA1 Gradient A G G2 16 5 Acquisition and Processing The GRASP experiments are best started from a corresponding data set without gradients e g for the GRASP HMQC and GRASP HSQC from the HMQC data set re hmqc 3 1 Section 14 or HMBC Section 15 Table 54 H C HMOC HMBC and HSQC Acquisition Parameters F2 Parameters Parameter Value Comments PULPROG hmqcgpaf For GRASP HMQC hmbcgplpndqf For GRASP HMBC hsqcetgpsi For GRASP HSQC TD 2k NS 2 For GRASP HMQC GRASP HSQC 8 For GRASP HMBC DS 16 number of dummy scans has to be ns 2 n P28 0 5u Trim pulse in HSQC Do not set it to a longer value D4 1 72msec For HSQC only 1 2 J C H 132 BRUKER Avance 1D 2D calculated automatically from cnst2 above D24 0 86msec For HSQC only delay for refocussing Jcu For all multiplicities set the value to 1 8 J C H Only for CH groups set the value to 1 4 C H D4 F1 Parameters Parameter Value Comments TD 256 number of experiments FnMODE QF For GRASP HMQC echo For GRASP HSQC antiecho QF For GRASP HMBC NDO 2 INO t increment SW 190ppm For HSQC and HMQC 2
157. nce 1D 2D BRUKER 151 button to release the cursor from the spectrum Click on and select Save as intrng and return to store the regions and return to the main 1D window Enter wmisc to call up the menu of miscellaneous list types Select intrng to select the integral range file type This calls up the list of possible files Simply type the new name t1reg Now the integral regions selected above are written to the integral range file t1reg Enter bas1 to enter the baseline submenu and from here click on det pts to enter the baseline point subroutine In this subroutine the cursor is tied to the spectrum Use the middle mouse button to select the points for which Ti will be calculated One and only one point must be selected for each integral region defined above Take care to select the point of maximum intensity for each peak region When finished click the left hand mouse button to release the cursor from the spectrum and store the baseline points Next enter wmisc to call up the menu of miscellaneous list types Select baslpnts to select the baseline point file type This calls up the list of possible files Simply type the new name tibas Now the baseline points selected above are written to the baseline point file t1bas Click on return to return to the main 1D window From here click on to return to the 2D data set 19 4 T Calculation Once the T data has been acquired and the integral range and baseline point files have b
158. nd forth The optimum may be saved by typing defplot and answering the questions which appear 10 4 Phase Correction and Plotting For the phase correction procedure and the plotting procedure please follow the instructions given for the TOCSY spectrum in Sections 9 4 and 9 5 respectively Figure 30 ROESY Spectrum of 50 mM Cyclosporin in C6D6 102 BRUKER Avance 1D 2D 11 NOESY 11 1 Introduction NOESY Nuclear Overhauser Effect SpectroscopY is a 2D spectroscopy method whose aim is to identify spins undergoing cross relaxation and to measure the cross relaxation rates Most commonly NOESY is used as a homonuclear H technique In NOESY direct dipolar couplings provide the primary means of cross relaxation and so spins undergoing cross relaxation are those which are close to one another in space Thus the cross peaks of a NOESY spectrum indicate which protons are close to each other in space This can be distinguished from COSY for example which relies on J coupling to provide spin spin correlation and whose cross peaks indicate which H s are close to which other H s through the bonds of the molecule The basic NOESY sequence consists of three 2 2 pulses The first pulse creates transverse spin magnetization This precesses during the evolution time t which is incremented during the course of the 2D experiment The second pulse produces longitudinal magnetization equal to the transverse magnetization component orthog
159. ne spin system obviously only 1 2 E is not a physical observable we neglected this operator already in the beginning But as we will see in paragraph 23 8 1 a two spin system exhibits 16 operators but only 6 of them are physically observable So what are the unobservable operators good for e First they describe quantum mechanical interactions in the system and e second they can evolve into observable magnetization A typical example for this is the scalar coupling which is described in paragraph 23 8 1 How is the FID obtained from these physical observables The trick is to introduce another operator with the same qualities as the physical detector In the spectrometer quadrature detection is used that is we observe the magnetic flux along the x and along the y axis in the rotating frame and combine the results into one complex valued number The corresponding operators are I I i 1 I_ 1 i l In principle we have the choice of selecting either l or l depending how we combine the physical measurements By convention is used as the detection operator It should be noted that l as the detector selects the component of the signal To calculate the physical value of an operator at a given time the trace of this operator is multiplied by the relevant density operator 0 1 0 It is convenient to express o in terms of the operators l and l to evaluate this expression Let s continue with the example of t
160. nuclear X H shift correlation 113 heteronuclear J coupling 113 heteronuclear J couplings 119 Heteronuclear Multiple Bond Correlation 129 heteronuclear multiple quantum coherence 129 Heteronuclear Multiple Quantum Correlation 123 heteronuclear multiple quantum coherence 123 Heteronuclear Single Quantum Correlation 135 HMBC 20 129 HMBC acquisition parameters 130 HMBC processing parameters 131 HMBC pulse sequence 130 HMBCGPLPND 20 HMBCLPND 20 HMQC 19 123 HMQC pulse sequence 124 HMQC with BIRD acquisition parameters 125 HMQC with BIRD processing parameters 126 HMQC with BIRD pulse sequence 124 HMQC HMBC and HSQC acquisition parameters 136 HMQCBI 19 homonuclear Nuclear Overhauser effect 102 HPPR 27 HSQC 19 133 135 HSQC pulse sequence with gradients 136 HSQCGPPH 19 INEPT 135 information on pulse program nomenclature 24 information on pulse program parameters 24 integration of spectra 38 intrng 157 inverse chemical shift correlation 123 133 inverse experiments 133 inverse gated decoupling 71 inversion recovery 153 inversion recovery acquisition parameters 155 inversion recovery processing parameters 156 inversion recovery pulse sequence 154 Karplus relation 15 Larmor frequency 193 Larmor theorem 193 lock parameters 22 lock the spectrometer 29 lockdisp 29 long range H C J couplings 129 long range chemical shift correlation 129 low powe
161. o if the HPPR LED display will be used to monitor tuning and matching it is best to remain in the main XWIN NMR window and not to switch to the acquisition window Start the frequency sweep by typing wobb The curve that appears in the acquisition window is the reflected power as a function of frequency Unless the probehead is quite far from the correct tuning and matching there will be a noticeable dip in the curve When the H circuit is properly tuned the dip will be in the center of the window denoted by the vertical marker and when the circuit is properly matched the dip will extend all the way down to the x axis Similar information is conveyed by the LED display on the HPPR The horizontal row of LED s indicates tuning and the vertical row matching When the circuit is properly tuned and matched the number of LEDs is minimized This usually means that only green LED are lit in both the horizontal and vertical displays 2 4 3 Tune and Match Adjust the tuning and matching screws labeled T and M at the base of the probehead Note that the screws are color coded and those for the H circuit are usually yellow Also note that the screws have a limited range and attempting to turn them beyond this range will damage the probehead Since there is an interplay between tuning and matching it is generally useful to adjust the T and M screws in an iterative fashion Turn the M screw until the dip is well matched at some frequency the dip e
162. oduction This chapter describes the acquisition and processing of a 1D H NMR spectrum using the simple one pulse NMR experiment shown in Figure 1 The pulse sequence consists of the recycling delay ta the radio frequency RF pulse and the acquisition time during which the signal is recorded The pulse angle is shown to be 7 2 although in practice it is often chosen less The two parameters d1 and p1 correspond to the length of the recycle delay tra and the length of the RF pulse respectively Note that 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 Figure 1 1D H NMR One Pulse Sequence T 2 3 1 1 Sample The sample used for demonstrating the basic 1D H experiment is 100 mg Cholesterylacetate in CDCl3 with 0 5 TMS 3 1 2 Preparation Make sure that you have done the following steps see also Chapter 2 Preparing for Acquisition e Insert a suitable probehead e Read in the corresponding shim file e Insert the sample e Lock the spectrometer Avance 1D 2D BRUKER 31 e Optimize the Z and Z and probably X and Y shims e Tune and match the probehead for H 3 2 Spectrometer and Acquisition Parameters Before the acquisition of a spectrum a new data set must be created All the spectrometer and acquisition parameters are entered within the new data set The spectrometer pa
163. of the spectrometer edcpul open the current pulse program in a text editor window eda edit all acquisition parameters ased as edit the acquisition parameters that are relevant for the current pulse program PPS graphical display of the current pulse program spdisp open the graphical pulse program editor dpa display all status parameters for the acquisition wbchan select the wobbling channel for tuning and matching wobb tuning and matching the probe atma automatic tune and match the ATM probe atmm manually tune and match the ATM probe edsolv define solvent parameters edlock define lock parameters for probhead and solvent lock Automatically lock on solvent parameters defined in edlock lockdisp open the lock display window rsh select and read shim values gradshim start the gradient shimming subprogram wsh save the current shim values edte open the temperature control window edau select or edit AU programs stdisp open the shape tool expt estimate the experiment time rga Automatically adjust the receiver gain zg start acquisition xaua start the acquisition AU program this also starts the acquisition gs Interactive adjustment of acquisition parameters tr data transfer during acquisition halt stop stop the acquisition kill kill a specific process Table 9 Processing Parameters si size of the real spectrum phc0 phcl Parameters for zero order and first order phase corrections lb line broa
164. onal to the pulse direction Thus the basic idea is to produce an initial situation for the mixing period tm Note that for he basic NOESY experiment tmis kept constant throughout the 2D experiment The third pulse creates transverse magnetization from the remaining longitudinal magnetization Acquisition begins immediately following the third pulse and the transverse magnetization is observed as a function of the time te The NOESY spectrum is generated by a 2D Fourier transform with respect to t and te Axial peaks which originate from magnetization that has relaxed during Tm can be removed by the appropriate phase cycling NOESY spectra can be obtained in 2D absorption mode Occasionally COSY type artifacts appear in the NOESY spectrum however these are easy to identify by their anti phase multiplet structure References J Jeener B H Meier P Bachmann R R Ernst J Chem Phys 69 4546 1979 G Wagner and K W thrich J Mol Biol 155 347 1982 The sample used to demonstrate NOESY in the chapter is 50 mM Cyclosporin in CgDe The NOESY pulse sequence is shown in Figure 31 The delay d8 determines the length of the mixing period during which NOE buildup occurs Avance 1D 2D BRUKER 103 Figure 31 NOESY Pulse Sequence T 2 T 2 T 2 11 2 Acquisition and Processing Insert the sample in the magnet Lock the spectrometer Readjust the Z and Z shims until the lock level is optimized Tune and match the probehead
165. only for HMQC with BIRD Set the acquisition parameters as shown above and choose a starting value of 400 msec for d7 Enter acqu to enter the acquisition window Enter gs to start the go setup routine Click the left mouse button to fix the acquisition gs window somewhere on the screen and then click on the box in the upper right hand corner of the window to iconize it While monitoring the intensity of the time domain data adjust the value of d7 simply enter d7 and then a new value at the prompt The optimum value of d7 corresponds to the minimum signal intensity Once the optimum value of d7 is found and stored enter rga to optimize the receiver gain for this minimum signal 14 2 2 Acquire the 2D data set Enter zg to start the HMQC experiment With the acquisition parameters shown above the approximate experiment time is 1 2 hours 14 3 Processing Enter edp and set the processing parameters as shown in Table 51 Table 51 HMQC with BIRD Processing Parameters F2 Parameters Parameter Value Comments SI 1k SF spectrum reference frequency H WDW QSINE sine squared window function SSB 2 pure cosine squared wave PH mod pk apply 0 and 1 order phase correction determined by phase correcting the first row PKNL TRUE necessary when using the digital filter BC mod no BRUKER Avance 1D 2D F1 Parameters Parameter Value Comments Sl 512 SF spectrum reference
166. opposite phase Correlation Information 2D Experiments a k a Parameter Set H H HH nearest neighbor through bond COSY COSYGPSW chemical shift correlation COSY45SW H H H H nearest neighbor through bond DGF COSYGPDFPHSW chemical shift correlation plus coupling COSY COSYDQFPHSW constants H 24H H H total spin system through bond TOCSY MLEVPHSW chemical shift correlation C H Sensitive H C directly bound HSQC HSQCGPPH chemical shift correlation one bond HMQC HMQC lower resolution in C dimension C H Sensitive H C directly bound BIRD HMQCBI chemical shift correlation one bond HMQC lower resolution in C dimension small molecules solemnly select C H not 2C H C H Insensitive H C directly bound HETCOR HCCOSW chemical shift correlation one bond high resolution in C dimension C 24H Sensitive H C long range chemical HMBC HMBCGPLPND shift correlation more than one bond HMBCLPND lower resolution in C dimension C nH Insensitive H C long range chemical COLOC HCCOLOCSW shift correlation one and more bonds high resolution in C dimension HoH HH non bound structural neighbor ROESY ROESYPHSW through space chemical shift correlation small molecules low fields HoH H H non bound structural neighbor NOESY NOESYPHSW through space chemical shift correlation large molecules proteins In most of the 2D parameter sets there is a s
167. optimization It is important to select the number of drift points large enough so that you are always sure to find the peak maximum yet small enough so that the maximum is always of the same peak If the number of drift points is chosen incorrectly peak picking will not work properly and the T curves will not be smooth exponential curves When proc t1 is finished the message T1 result stored in tir appears The pathname of this file is t1data 2 pdata 1 tir i e it is in the same directory as the processed NMR data The peak intensity vs vd time data are also gathered and plotted for each resonance To view these results type t1 t2 to enter the T T routine The first T4 curve appears automatically in the window Enter nxtp to view the Ti curves for successive peaks 19 4 1 Check T curves The T curve for each selected resonance must be verified that the vd values were chosen so that all curves are clearly defined If any T curve is not well defined it is necessary to edit the vdlist tidelay and rerun the experiment so that reliable T measurements for those resonances may be obtained as well Also check all T curves to be smoothly exponential If not the T calculation can be redone with bad points eliminated for the calculation Points may be removed from a curve one at a time by typing elim and then selecting a point with the middle mouse button click the left mouse button to quit without choosing a peak Eliminated point
168. orresponding signal function is Tr 1 6 cost J 1 cos t J t cos t i cos t Ju f cos t J t sin 6 t T 1 Re 2a ET 4 Ja J Ja J 2m iv 822 2m iv 15 202 19109703 e p In the case where J12 J13 J this simplifies to Tr 1 0 ge VIDT Srg T UN vi 5 which is the well known 1 2 1 triplet we expected Note that the Jos is totally irrelevant for the signal of the spin 1 We have now seen 3 examples of a 1D experiment Let us now turn to 2D experiments As an example for all 2D experiments we will study the most fundamental 2D the magnitude COSY experiment 23 10 The COSY Experiment The minimum spin system size for a COSY is a two spin system as we need at least two coupled spins The pulse sequence of a COSY is very simple first we use a 90 excitation pulse from the y direction followed by a free evolution time t and a second 90 pulse around y just before the acquisition time t As you may have noticed the number of operator terms has a tendency to dramatically increase during free evolution periods Therefore we will discuss more simplifications in order to keep the problem within reasonable size For the COSY we use a two spin system with the Hamiltonian H 0 1 40 L 2 Jy IL For the equilibrium density operator again we use the reduced version from the previous section 1 0 Ou Tz 0 1x The evolution of oo under the above Hamiltonian therefore yields 0 gt I
169. osporin in CgDe 20 2 Selective Pulse Calibration Insert the sample in the magnet Lock the spectrometer Readjust the Z and Z shims until the lock level is optimized Tune and match the probehead for H observation Selective experiments are measured without sample spinning Before performing selective excitation experiments it is necessary to calibrate the selective pulse First a H reference spectrum must be recorded and the resonance frequency of the desired resonance is determined second define the shaped pulse and third perform the pulse calibration experiment 158 BRUKER Avance 1D 2D 20 2 1 Proton reference spectrum Acquire and process a standard H spectrum in the data set selex 1 1 Optimize o1p and sw Click on M utilities to enter the calibration submenu Click on Of with the left mouse button to select the o1p calibration Move the cursor to the center of the doublet at 8 1ppm and click the middle mouse button to assign o1p to this frequency Exit the utilities submenu and return to the main window Make sure sw is large enough to include the entire H spectrum with this new olp value Acquire and process a H spectrum 20 2 2 Selective one pulse sequence The pulse sequence used to calibrate the selective pulse is shown in Figure 54 This sequence is identical to the standard one pulse sequence shown in Figure 1 except for the pulse is applied with low power and a shape The pulse length p11 and the pulse str
170. ot have opposite phase and thus do not cancel one another when H decoupling is applied The optimal refocusing time A2 depends on whether the C belongs to a CH CH2 or CHs group Generally a compromise value of A gt 1 3Jx is chosen C couplings during t are removed by adding a C x pulse in the middle of tj so that there is refocusing by the end of t4 To enable maximum polarization transfer a fixed delay A 1 2JxH is added after t This delay allows anti phase magnetization to be re established In this pulse sequence the delay time d2 determines the length of the delay for the creation of anti phase magnetization A 1 2Jx and the time d3 determines the length of the refocusing delay A2 1 oJxH were a is usually chosen to be 3 Avance 1D 2D BRUKER 109 Figure 33 XHCORR Pulse Sequence T T 2 13C di l do dO d2 d3 acq 12 2 Acquisition Insert the sample in the magnet Lock the spectrometer Readjust the Z and Z shims until the lock level is optimized Tune and match the probehead for 136 observation and H decoupling It is recommended to run 2D experiments without sample spinning 12 2 1 Proton Reference Spectrum Record a H reference spectrum to obtain the correct H carrier frequency olp and spectral width sw values Enter re proton 1 1 to call up the data set proton 1 1 enter ede and change the following parameters NAME xhcorr EXPNO 1 PROCNO 1 Click _s to create the data
171. p the data set proton 1 1 Enter ede and create the data set homodec 1 1 Enter edasp and set both NUC1 and NUC2 to 1H The f2 channel is used for cw irradiation during the NOE experiment 18 2 2 Proton reference sepctrum Enter rga to perform an automatic receiver gain adjustment Acquire and process a standard H spectrum as described in Chapter 3 Calibrate the spectrum and optimize sw and o1p Acquire the standard spectrum using the parameters outlined in the table Table 57 Acquisition Parameters for 1H Reference Spectrum Parameter Value Comments PULPROG zg30 One pulse acquisition with 30 flip angle NS 8 number of scans DS 2 number of dummy scans Process the FID with em t and phase correct it 18 2 3 Selection of irradiation frequency The frequency used by the f2 channel for the irradiation of the multiplet can be defined by entering the submenu was and clicking on the button for selecting the o2 frequency jj The cursor is now bound to the spectrum and changed his shape vertical arrow The mouse buttons in this mode do have the functions left return middle define frequency rightzunused Move the cursor to the position of interest and press the middle mouse button to define SFO2 O2 frequency Leave the utilities submenu with the return button The determined value for o2 is now stored in the current data set Avance 1D 2D BRUKER 145 18 2 4 Setting up the homo decoupling parameters
172. p the editor Enter the delays desired some appropriate values are listed below 0 004 0 016 0 010 0 006 0 004 0 010 0 017 0 011 0 018 0 012 When the list is complete save the file and exit the editor 20 4 2 Acquisition From the data set selco 1 1 create the data set seltoc 1 1 and record a H reference spectrum of Cyclosporin exactly as described for the selective COSY in Section 20 3 Enter eda and set the acquisition parameters as shown in Table 66 166 BRUKER Avance 1D 2D Avance 1D 2D Table 66 Selective TOCSY Acquisition Parameters F2 Parameters Parameter Value Comments PULPROG selmizf TD 32k NS 32 the number of scans should be 16 n DS 16 number of dummy scans PL1 high power level on F1 channel H as determined in Section 4 2 4 PL10 low power level on F1 channel H for MLEV mixing as determined in Section 4 2 5 SP1 shaped pulse power level on F1 channel H as determined in Section 20 2 P11 H 90 shaped pulse as determined in Section 20 2 P5 H 60 pulse calculated from p6 P6 H 90 pulse as determined in Section 4 2 5 P7 H 180 pulse calculated from p6 P17 2 5m 2 5 msec trim pulse D1 2 relaxation delay should about 1 25 T4 H D14 delay for evolution after shaped pulse p1 1 2 d14 1 Juy L1 30 loop for MLEV cycle p6 64 p5 11 p17 2 2 mixing time L4 10 number of delays in vdlist VD
173. parameters Click the ed next to the parameter EDPROJ1 to enter the F1 projection parameters submenu Edit the parameters from PF1DU to PF1PROC as follows PF1DU u PF1USER name of user for file cosy 1 1 PF1NAME COSy PF1EXP 1 PF1PROC 1 Click se to save these changes and return to the edg menu Click the ed next to the parameter EDPROJ2 to enter the F2 projection parameters submenu as described for EDPROJ1 above Click to save all the above changes and exit the edg menu Enter setti to open the title file and enter a title To plot the spectrum simply enter plot A TOCSY spectrum of 50 mM Cyclosporin in CDe is shown in Figure 28 Figure 28 TOCSY Spectrum of 50 mM Cyclosporin in C6D6 Avance 1D 2D BRUKER 97 98 BRUKER Avance 1D 2D 10 ROESY 10 1 Introduction ROESY Rotating frame Overhauser Effect SpectroscopY is an experiment in which homonuclear Nuclear Overhauser effects NOEs are measured under spin locked conditions ROESY is especially suited for molecules with motional correlation times te such that ox 1 were is the angular frequency o 3B In such cases the laboratory frame NOE is nearly zero but the rotating frame NOE or ROE is always positive and increases monotonically for increasing values of te In ROESY the mixing time is the spin lock period during which spin exchange occurs among spin locked magnetization components of different nuclei recall that spin exchange in NOESY occu
174. pectral width optimization implemented PULSEPROGRAMSW So if you acquire the corresponding 1D experiments in the previous experiment number the spectral width for the 2D will be optimized according to the 1D information A complete list of parameter sets can be called up by typing rpar without a following name The nomenclature of the parameter sets follows the rules for 1 z gradient hardware required Avance 1D 2D BRUKER the nomenclature of the pulse programs They can be found in the file XWinNMRHome exp stan nmr lists pp Pulprog info However in this manual we focus on the manual setup of the experiments from scratch and the optimization of the corresponding parameters therefore the rpar command will not be used throughout this text 2 2 2 XWinNMR parameters and commands 20 A list of commonly used acquisition and processing commands and parameter names as well as a description of the corresponding command or parameter is given in short in the tables below Table 5 General Commands and AU Programs setres customize the XwinNMR interface edmac edit or create an XWinNMR macro edau edit or create an XWinNMR AU program edpul edit or create an XWinNMR pulse program xau create a file called listall in your home directory with a list of all listall au available AU programs including short descriptions edcpul edit the current pulse program Table 6 Data Set Related Commands
175. per right hand corner of the display see Section 8 3 3 Repeat the above procedure to select two further columns one with a diagonal peak in the middle and one with a peak at the other end of the spectrum Move these columns to window 2 and 3 respectively Now that three columns have been selected the 0 and 1 order phase corrections in F1 are determined manually exactly as for the DQF COSY spectrum see Section 8 3 3 When the phase correction is satisfactory click on and select Save amp return to save the results and confirm the xf1p option to apply this phase correction to the spectrum 96 BRUKER Avance 1D 2D At this point the spectrum should be phased correctly If however the user wishes to make further adjustments the above procedure can be repeated to adjust the F1 phasing To further phase correct the spectrum in F2 repeat the above procedure for rows rather than columns Phase correct as described above and confirm the xf2p option It should be possible to phase correct the spectrum so that all TOCSY peaks are positive 9 5 Plot the Spectrum Set the region the threshold and peak type positive and or negative to be used for plotting the spectrum Make sure the spectrum appears as desired on the screen type defplot and answer the following questions Change levels Please enter number of positive levels Please enter number of negative levels Display contours 2 0920 Enter edg to edit the plotting
176. period and the evaluation of the detection operator L In the next paragraph we are going to extend our example from one to two spins and calculate the outcome of the same experiment 23 8 Observing Two and More Spin Systems A one spin system does indeed not show much complexity So let us then proceed to a two spin system Traditionally there are two notations widely used to distinguish different spins l and lo as indices or and S with different names For protons we use the indices notation and for heteronuclei we use S or S4 As a first two spin system let us consider at a homonuclear system with two H nuclei The number of operators in the basis of a spin system is given by 4N where N is the number of spins in the system Fortunately it is very simple to construct such a basis The basis for two single spins compare section 23 3 are multiplied to yield the needed 16 operators 1 zh Le laz 21 1 Indie Ef OY et turbo LE I 21 5 2L yla I des bd A DAE BOT PE y 2x 2y 2y 2L I Note that 1 2 E and 2 E were consistently omitted from the notation In section 23 7 we discussed observable vs non observable operators In general only single spin operators along x y or z are observable operators and only those along x or y will give rise to a NMR signal In this particular case the operators that relevant for NMR are hx lox hy and lzy 194 BRUKER Avance 1D 2D 23 8 1 Avance 1D 2D BRUKER 195 In a two s
177. picking INC 1 increment for next spectrum used in peak picking NUMTERM 3 number of variables used in fitting routine 19 5 Create a Stacked Plot This section describes a method for obtaining a stacked plot of the 2D data set The plot is created by the au program stack2d which uses the plot parameter set stackplot Note that stackplot is a 1D plot parameter set To create the 1D parameter set first return to the reference spectrum enter re 1 1 and select an appropriate region for plotting Next create the plot parameter set to be used by the au program Enter edg to call up the plot parameter menu Make sure that SPECT is set to YES but that XAXIS YAXIS TITLE INTEGR and PARAM are set to NO Click on the ED which 154 BRUKER Avance 1D 2D appears next to the option EDSPECT to call the submenu Spectrum Plot Parameters The following selected parameter values are suggested for A4 8 5 11 paper Table 62 Spectrum Plot Parameters for Stacked Plot Parameter Value A4 Comments Name SXLLEFT 2 0cm SYLLEFT 1 0cm CX 20 0 cm SHEI 20 0 cm F1P F1 F2P These parameters were set when DP1 was used to define the plot F2 region PPMCM HZCM DHEI 17 5 cm SZERO 2 0 cm CY 6 0 cm Size of each individual spectrum Click SAVE to save these changes and return to the main edg menu There click SAVE to save all changes and exit edg Ne
178. pin system the thermal equilibrium density operator now includes also the second spin and is given by Cu z D h When applying a rf pulse e g a 90 pulse the same rules still apply However the corresponding Hamiltonian has changed to include also the operator from spin 2 H y B 1 1 The above Hamiltonian can be split into two Hamiltonians H y B 4 and H y B being applied one after the other The first acts only on operators of spin 1 and is ignored by all spin 2 operators The second applies accordingly only to spin 2 operators Accordingly a selective rf pulse could be realized by applying e g a pulse with the respective Hamiltonian to achieve a selective pulse on a certain spin This issue will be discussed again when the theory of the inverse experiments is discussed In the arrow notation if not explicitly mentioned otherwise the rf pulse always applies to all spins in the system In our example we apply a 90 pulse to the equilibrium density matrix 6 0 0 1 h The next step will be to evaluate the free evolution during the acquisition time But before we can do so we need to have a look at the corresponding Hamiltonian and there we will find a new phenomenon the scalar coupling Effect of Scalar Coupling Apart from the chemical shift there is a second very import interaction between spins the scalar coupling The scalar depends on the mediation of electrons which are confined in or
179. ple is in the magnet and the probehead is connected for the appropriate experiment Also it is recommended to tune and match without sample spinning 2 5 1 Set the Parameters In XWIN NMR enter edsp and set the following spectrometer parameters NUC1 13C NUC2 OFF NUC3 OFF This automatically sets s o1 to a frequency appropriate for C tuning and matching Exit edsp by clicking SAVE 2 5 2 Start Wobbling Tune and Match Ensure that no acquisition is in progress enter stop Enter acqu to switch to the acquisition window if this will be used to monitor the tuning and matching Start the frequency sweep by typing wobb The curve that appears in the acquisition window is for C Adjust the tuning and matching following the guidelines given above for H Notice that some probeheads e g broadband probeheads have sliding bars instead of screws one set labeled tuning and another labeled matching Set the tuning and matching sliding bars to the values indicated for C on the menu Adjust the tuning and matching bars until the dip is well tuned and matched at some frequency as described above for H Once the C circuit is tuned and matched the C wobbling must be stopped before the H wobbling Exit the wobble routine by typing stop Enter edsp change NUC1 to 1H and exit by clicking SAVE Start the H frequency sweep by typing wobb After a few seconds the H curve appears in the acquisition window and the H circuit can be
180. ponents of M are described by the Bloch equations d M 0 dt d igr Te YyB M d wee M YB Assuming the magnetization at time O to be along the z axis with amplitude Mo we find the following solution to the above equation M t M sin yB t M t M cos yB t The magnetization vector is precessing around the B axis which is aligned with the x axis of the reference system If we choose the time t of suitable duration we obtain TU B YyBt p which is defined as the 90 degree pulse creating maximum y magnetization which in turn yields maximal signal intensity 1 4 Spin Operators of a One Spin System All NMR experiments start from the thermal equilibrium In thermal equilibrium the classical description gives a magnetic moment parallel to the static field Mz In the Spin Operator formalism this is described by Guy l where Ogg is the equilibrium density matrix representing the state of the spin system under investigation Now there are only two basic types of evolutions 1 An external perturbation e g a rf pulse or 2 an unperturbed evolution which will eventually bring the system back to the thermal equilibrium Avance 1D 2D BRUKER 11 1 4 1 Effect of rf Pulses The effect of an rf pulse is that of a rotation along the pulse axes according to the following calculus rules I I cos B I sin B I Bar cos D 7 sin D I f1 I I I I cos I sin p I
181. pulse In the classical description we moved to a rotating coordinate system to describe the effect of the rf pulse In the Spin Operator formalism a similar approach is taken although with a slightly different vocabulary The rotating coordinate system is called rotating frame or interaction frame For the same reason then in the classical approach the rotational axis is chosen along the z axis parallel to the static field Bo and the By field is assumed along the x axis The interaction frame rotates by definition with the frequency of the rf pulse or the reference frequency of the detector which is identical to the former and is called the carrier frequency As a consequence all Larmor frequencies are changed into chemical shift frequencies defined by 0 0 The pulse is assumed to be of very short duration such that chemical shift evolution and relaxation during the pulse can be ignored Then the effect of an rf pulse is that of a rotation along the pulse axes according to the following calculus rules Avance 1D 2D BRUKER 189 I 3 I cosp I sinp I akar cos Z sin D I gt I I posu cos 1 sin p I I cosp 1 sin p If the flip angle B 90 then eee ae L I We find the expected result that a 90 pulse will generate transverse magnetization The rest of this chapter will be concerned with following the fate of this transverse magnetization in time We introduced tacit
182. r pulse calibration 43 magnetic moment 192 193 Avance 1D 2D magnitude COSY 84 maximum power levels 9 mixing period NOESY 107 mixing time optimization NOESY 109 mixing time ROESY 102 MLEV 17 96 MLEVPHSW 19 multiefp 146 NOE 107 141 NOE Difference 141 NOE difference acquisition parameters 144 NOE difference parameter optimization 144 NOE difference processing parameters 146 NOE difference pulse sequence 142 NOE quantitation 147 noemul 143 noemult 144 NOESY 20 107 NOESY acquisition parameters 109 NOESY processing parameters 110 NOESY pulse sequence 108 NOES YPHSW 20 Nuclear Overhauser effect 102 Nuclear Overhauser Effect 107 141 Nuclear Overhauser Effect Spectroscopy 107 observable operators 198 observable signals 198 one spin system 194 p 23 paropt 42 phase correction 1D 36 phase cycling 163 pl 23 population 195 power level 9 predefined parameter set 18 preirradiation 141 probehead 199 proc_t1 156 processing commands 20 processing number 17 processing parameters 22 processing size 35 procno 17 PROTON 19 pulse calibration for protons 40 pulse calibration carbon 53 pulse program display 21 pulse program information 20 pulse program nomenclature 24 pulse program parameters 23 pulse sequence with gated H decoupling 71 read shim values 22 receiver coil 193 receiver gain adjustment auto 22 recovery time value
183. rameter Value Comments PULPROG zg see Figure 1 for pulse sequence diagram TD 8k NS 1 DS 0 PL1 high power level on F1 channel H as determined in Section 4 2 4 P1 3 H pulse less than a 90 pulse D1 5 SW 20 ppm olp 5 Enter rga to perform an automatic receiver gain adjustment zg to acquire the FID and edp to set the processing parameters as shown in Table 27 Table 27 1D H One pulse Processing Parameters Parameter Value Comments SI 4k LB 0 3 Hz PSCAL global Fourier transform the data e and phase correct the spectrum Type sref to calibrate the spectrum and confirm the message no peak found in sref default calibration done Expand the spectrum until only the chloroform signal at 7 2ppm is displayed and set the o1p value exactly on the methanol peak in the utilities menu follow the same procedure as described in Section 4 2 2 The spectral width swh can now be reduced to 1000 Hz Preparations for the Inverse Pulse Calibration The correct H and C frequencies have now been determined Next a DECP90 spectrum is acquired to determine the appropriate phase correction values Create a new data set by entering ede and change EXPNO to 3 Click SAVE to create the data set testinv 3 1 Enable C decoupling by entering edsp and set the following spectrometer parameters BRUKER Avance 1D 2D NUC1 1H NUC2 13C NUC3 off Click on SAVE to save the
184. rameters are responsible for the hardware settings necessary for configuring the spectrometer for a particular experiment The command edsp calls up a window in which the spectrometer parameters for the observe and the decoupler channel s are set The acquisition parameters include all pulse sequence parameters the number of data points number of scans receiver gain and many others These may be displayed and edited by entering eda Notice that the spectrometer parameters are also listed in the eda table It is important to set the spectrometer parameters before setting the acquisition parameters because the values from edsp automatically overwrite the corresponding ones from the eda table 3 3 Create a New File Directory for the Data Set To create a new data set type edc in the command line of the XWIN NMR window This calls up a small window entitled Current Data Parameters Enter a data set name NAME an experiment number EXPNO a processed data number PROCNO the disk unit DU where the data is stored the user id USER and the data type TYPE Change the parameters as follows NAME proton EXPNO 1 PROCNO 1 Click on SAVE This exits edc and creates the data set proton 1 1 The message NEW DATA SET should now appear on the screen 3 4 Set Up the Spectrometer Parameters Enter edsp and set the following spectrometer parameters NUC1 1H NUC2 off NUC3 off 32 BRUKER Avance 1D 2D Since there is no decoupl
185. recovery d17 delay for DANTE pulse train dis delay for evolution of long range couplings d19 delay for binomial water suppression d20 for different applications cnstO for different applications cnstl J HH cnst2 J XH cnst3 J XX cnst4 J YH enst5 J XY cnst11 for multiplicity selection cnst12 for multiplicity selection ve variable loop counter taken from vc list vd variable delay taken from vd list 11 loop for MLEV cycle p6 64 p5 11 p17 2 mixing time 12 loop for GARP cycle 12 31 75 4 p9 gt AQ 13 loop for phase sensitive 2D or 3D using States et al or States TPPI method 13 td1 2 14 for different applications i e noediff Note that the default units for pulses are microseconds u the units for delays are seconds s but one can always enter a value combined with a unit to define a time slot in XWinNMR The nomenclature here is s seconds m milliseconds and u microseconds For example To set the value of d1 to 500m would define d1 to last for half a second The complete information on the nomenclature and default usage of the pulse program parameters can be found in XWinNMRHome exp stan nmr lists pp Param info The nomenclature and description of the standard pulse programs and predefined parameter sets can be found in XWinNMRHome exp stan nmr lists pp Pulprog info Acquisition processing and plotting commands can be given either in the XWinNMR command line or via men
186. return to the main window Reduce the spectral width by entering swh and change the value to 1000 Hz Acquire and Fourier transform another spectrum zg ef 6 1 3 Define the Phase Correction and the Plot Region Now it is necessary to define the phase correction and spectral region that will be plotted in the output file produced by paropt Phase correct the spectrum so that the doublet is positive Expand the spectrum so that the doublet covers approximately the central third of the screen Click on api with the left mouse button and answer the three questions as follows F1 133 ppm F2 127 ppm change y scaling on display according to PSCAL y 6 1 4 Calibration High Power 52 As for the H 90 calibration Chapter 4 2 4 the automation program paropt is used Type xau paropt and answer the questions as follows Enter parameter to modify p1 Enter initial parameter value 2 Enter parameter increment 2 Enter of experiments 16 BRUKER Avance 1D 2D Paropt acquires and processes 16 spectra while incrementing the parameter pi from 2usec to 32usec The result is displayed side by side in test13c 1 999 and should resemble Figure 8 At the end of the experiment the message paropt finished appears and a value for p1 is displayed which corresponds to the 90 pulse length of the C transmitter using the current power level p11 Write this value down and follow the procedure below to obtain a more accurate 90 pulse me
187. rs while magnetization is aligned along the z axis Different spectral density functions are relevant for ROESY than for NOESY and these cause the ROE to be positive for all values of Tc ROESY spectra can be obtained in 2D absorption mode This is also useful for the identification of certain artifacts Spurious cross peaks both COSY type and TOCSY type can be observed due to coherence transfer between scalar coupled spins COSY type artifacts anti phase arise when the mixing pulse transfers anti phase magnetization from one spin to another TOCSY type artifacts which have the same phase as the diagonal peaks while ROESY cross peaks have opposite phase arise when the Hartmann Hahn condition is met e g when spins A and B have opposite but equal offsets from the transmitter frequency or when they have nearly identical chemical shifts In general to minimize these artifacts it is suggested to limit the strength of the spin locking field Reference A Bax and D G Davis J Magn Reson 63 207 1985 The sample used to demonstrate ROESY in this chapter is 50mM Cyclosporin in CgDe The ROESY pulse sequence is shown in Figure 29 Figure 29 ROESY Pulse Sequence T 2 Avance 1D 2D BRUKER 99 10 2 Acquisition Insert the sample in the magnet Lock the spectrometer Readjust the Z and Z shims until the lock level is optimized Tune and match the probehead for H observation It is recommended to run 2D experiments
188. s that 1 10 Jun should be allowed for each transfer step and five transfer steps are typically desired for the TOCSY spectrum The sample used to demonstrate TOCSY in this chapter is 50 mM Cyclosporin in CgDe References L Braunschweiler and R R Ernst J Magn Reson 53 521 1983 A Bax and D G Davis J Magn Reson 65 355 1985 The TOCSY pulse sequence is shown in Figure 27 The pulse p1 must be set to the appropriate 90 time found Section 4 2 4 and the MLEV 17 sequence used during the spinlock period requires the calibrated 90 time p6 as determined in Section 4 2 5 Figure 27 TOCSY Pulse Sequence T 2 1H MLEV 17 p11 p17 p5 p6 p7 p17 d1 do Avance 1D 2D BRUKER 9 2 Acquisition 94 Insert the sample in the magnet Lock the spectrometer Readjust the Z and Z shims until the lock level is optimized Tune and match the probehead for H observation It is recommended to run 2D experiments without sample spinning Record a H reference spectrum to determine the correct values for o1p and sw A H reference spectrum of this sample was already created for the magnitude COSY experiment Section 8 2 2 This spectrum is found in the data set cosy 1 1 The TOCSY data set can be created from the data set of any of the previous homonuclear 2D experiments run on this sample For example enter re cosy 2 1 to call up the data set cosy 2 1 Enter edc and change the following parameters NAME tocsy
189. s list 154 relaxation 153 restore eliminated datapoints 158 RF pulses 193 BRUKER 207 RF Pulses 195 ROE 102 ROESY 20 102 ROESY acquisition parameters 103 ROESY processing parameters 105 ROESY pulse sequence 102 ROESYPHSW 20 rotating coordinate system 195 rotating frame 195 Rotating frame Overhauser Effect SpectroscopY 102 rpar 18 rstp 158 save shim values 22 search for a specific dataset 21 selco 165 selective COSY 168 selective COSY acquisition parameters 170 selective COSY pulse sequence 169 selective excitation 163 selective irradiation 168 selective one pulse sequence 164 selective pulse 163 selective pulse calibration 163 selective TOCSY 171 selective TOCSY acquisition parameters 173 selective TOCSY processing parameters 174 selective TOCSY pulse sequence 172 selmlzf 165 selzg 165 setti 75 shape tool 22 shaped pulses 164 Shapes 164 shimming the sample 29 single quantum magnetization 135 solvent parameters 22 sp 23 spin 192 spin lattice relaxation 153 spin lock 102 208 BRUKER spinning 85 stacked plot 159 stackpar 160 stackplot 160 stdisp 164 stop the acquisition 22 T1 calculation AU program 156 T curves 158 T measurement 153 T parameters 159 T relaxation 153 tlbas 157 tidelay 154 158 tireg 157 temperature control window 22 The Hamiltonian 196 thermal equilibrium 194 time doma
190. s may be restored by entering rstp this restores all eliminated points to all T curves Once the bad points have been removed from a curve enter ct1 to begin the T calculation for that resonance Enter nxtp to call up the next curve remove the bad points enter ct1 to calculate T for that peak and so on Alternatively remove the unwanted points from all curves and then enter dati to begin the Ti calculation for all selected peaks Note that unless CURPRIN is changed before using ct1 or dati to recalculate Ti the numerical results from proc t1 will be overwritten as discussed below If there are too many bad points for a given T curve to be a reliable fit proc t1 should be rerun It may be necessary to use a different number of drift points or to redefine the integral range and baseline point files 19 4 2 Check numerical results The numerical results generated by the T calculation routine may be stored in a file displayed on the monitor or sent directly to the printer The automation program proc t1 automatically stores the results in the file ttr in the processed data subdirectory After running proc t1 enter edo to call up the plotter options menu and note that CURPRIN is set to tir Each time a T calculation is run with CURPRIN set to tir this file is overwritten However before using ct1 or dat1 the user also has the option to set CURPRIN to screen or to the appropriate printer Avance 1D 2D BRUKER 153 To display t
191. sec is used which is longer than the T of the peak at 8 5 ppm 17 2 6 Perform the multiple NOE experiment To start the NOE difference experiment type xau noemult and answer the questions as follows base name of all frequency lists noedif of frequency lists 3 of cycles through each list L4 of average cycles 8 The number of frequency lists is the number of fq2lists written above and it will be the number of spectra acquired The number of cycles through each list is the loop counter 14 The number of average cycles controls the total number of scans for each frequency list For each frequency list and hence for each spectrum the total number of scans is ns where ns should be as small as possible e g 8 and then the signal to noise ratio is improved by increasing the number of average cycles to e g 10 The au program automatically starts the pulse program noemul using the acquisition parameters defined in the current data set noedif 2 1 and the o2 frequencies defined in the first fq2list noedif 1 Next an experiment is performed using the o2 frequencies defined in the second fq2list noedif 2 and the results are written to the next data set noedif 3 1 and so on Note that new data sets created by noemult have the same name as the original data set but increasing EXPNO Here the spectrum irradiated at 4 5 ppm is noedif 2 1 that at 8 5 ppm is noedif 3 1 and that at 2 ppm is noedif 4 1 140 BRUKER Ava
192. sing parameter 1b in Hz Enter 1b and set the value to 0 3 which corresponds to an appropriate line broadening for high resolution H spectra Enter em to perform the multiplication and then enter p to Fourier transform and phase correct the filtered data You can also use the combined command efp which performs the windowing Fourier transformation and the phasing with the previously determined phase correction The final spectrum should look like the one shown in Figure 2 Figure 2 H 1D Spectrum of 100 mg Cholesterylacetate in CDCI3 9 0 8 0 7 0 6 0 5 0 4 0 3 0 2 0 1 0 0 0 ppm BRUKER Avance 1D 2D 3 10 Integration To quantitatively analyze an observed signal the integrated intensity of the peaks are compared within each other Click integrate to enter the integration submenu To integrate a peak first move the cursor into the spectral window and click the left mouse button Next click the middle mouse button once at each end of the range of interest the integral appears automatically Click the left mouse button again to release the cursor from the spectrum An asterisk or a vertical arrow appears next to the right end of the integral if not select the integral with the left mouse button Correct the baseline of the integral with the slope bias buttons Integrate the other areas or peaks in the same way For the calibration select an integral asterisk arrow and click on calibrate Enter 100
193. t as shown above is 2 5 hours 12 3 Processing Enter edp and set the processing parameters as shown in Table 48 Table 48 XHCORR Processing Parameters F2 Parameters Parameter Value Comments SI 1k SF spectrum reference frequency C WDW EM LB 3 a value of 2 5 Hz is appropriate PH mod no this is a magnitude spectrum PKNL TRUE necessary when using the digital filter BC mod quad 112 BRUKER Avance 1D 2D F1 Parameters Parameter Value Comments Sl 512 SF spectrum reference frequency H WDW QSINE multiply data by squared sine function SSB 1 choose pure sine bell PH_mod mc this is a magnitude spectrum BC mod no MC2 QF Enter x b to perform the 2D Fourier transformation The threshold level can be adjusted by placing the cursor on the button holding down the left mouse button and moving the mouse up and down The sewer button is used to set the number of levels The user can choose to display positive peaks only negative peaks only or both positive and negative peaks by clicking on with the left mouse button Since this is a magnitude spectrum only positive peaks need to be displayed Since this is a magnitude spectrum no phase adjustment can be made When the spectrum appears as desired on the screen click permet and answer the following questions Change levels y Please enter number of positive levels 6 Display
194. tating coordinate system is chosen to rotate at the same frequency than B1 thus making both Bo and B time independent in this reference system The Bloch equations in this coordinate system are then d r r re M lyB u M lyB 0 yB M z d r re M YB w is the rotational frequency of the coordinate system The relaxation during the rf pulse is neglected as the pulse is assumed to be very short compared with the relaxation time By assuming an effective magnetic field O Boy By Jf 0 y we recognize the Bloch equation from the static coordinate system The magnetization precessess in the rotating frame around Ber instead of Bo By choosing o to be Q YyD Ber vanishes and the Bloch equation simplifies to d y C O dt d M yB M dt y Y 1 z d ao M IB Avance 1D 2D BRUKER 187 Assuming the magnetization at time O to be along the z axis with amplitude Mo we find the following solution to the above equation system M t M sin yB M t M cos yB t This means that the magnetization vector is precessing around Ber Bi e g the magnetization is rotating around the B axis which is aligned with the x axis of the reference system If we choose the time t of suitable duration we obtain TU B yB 2 which is defined as the 90 degree pulse As we can see the 90 creates a maximum of y magnetization which in turn yields a maximal signal intensity This results will no
195. technique are covered by the GRASP course and will not be discussed here 16 2 GRASP HMQC See Section 14 for information about the HMQC experiment The GRASP HMQC pulse sequence is shown in Figure 42 The PFGs in the experiment are used for coherence selection and the quadrature detection in the o1 dimension The two gradients applied during t gradients Gy and Gp dephase all H magnetization while the third gradient G3 rephases the magnetization of interest The gradient ratio Gi Ga Gs for a GRASP HMQC is 2 1 for C and 5 1 for N This version of the GRASP HMQC experiment is not phase sensitive Avance 1D 2D BRUKER 129 Figure 42 GRASP HMQC Pulse Sequence T 2 T p3 p3 di d2 dO dO Gradient G G G Note that all correlation peaks of any HMQC experiment are splitted along the w dimension due to evolution of the homonuclear H H coupling which cannot be refocussed by the 180 H pulse applied during ti Therefore the resolution of the HMQC experiment along the dimension is limited For a better resolution in the o dimension the HSQC experiments must be done References A Bax R H Griffey and B L Hawkins J Magn Reson 55 301 1983 A Bax and S Subramanian J Magn Reson 67 565 1986 16 3 GRASP HMBC See Section 15 for information about the HMBC experiment The GRASP HMBC pulse sequence is shown in Figure 43 Identical gradient ratios are used for the GRASP HMBC as for
196. the GRASP HMQC see Section 16 2 130 BRUKER Avance 1D 2D Figure 43 GRASP HMBC pulse sequence T 2 T p3 p3 p3 d1 d2 d6 do do Gradient G G G 16 4 GRASP HSQC HSQC Heteronuclear Single Quantum Correlation yields the same spectrum as HMQC but is based on single quantum NMR In the HSQC sequence the pulse scheme prior the t evolution period represents a so called INEPT sequence and creates transverse single quantum magnetization on the X nucleus e g C or N which evolves X chemical shift during ti The G4 gradient dephases all the transverse magnetization This gradient is located in a spin echo in order to refocus chemical shift evolution during the gradient Then a second INEPT segment transfers the magnetization to H where it is detected after it has been rephased by a second gradient Go The field gradients in this version of a GRASP HSQC experiment are solely used for the coherence selection The gradient ratio G G2 for a GRASP HSQC is 4 1 for C and 10 1 for PN This version of the GRASP HMQC experiment is phase sensitive Compared to the HMQC experiment no line broadening along the o dimension appears as only C single quantum magnetization is present during the t1 evolution period References A Bax R H Griffey and B L Hawkins J Magn Reson 55 301 1983 A Bax and S Subramanian J Magn Reson 67 565 1986 Pulse sequence for GRASP HSQC is shown in Figure 44 Ava
197. the _ phase button Select one row by clicking on rw with the left mouse button to tie the cursor to the 2D spectrum appearing in the upper left corner of the display Move the mouse until the horizontal cross hair is aligned with a row that has a cross peak Select the row by clicking the middle mouse button If the selected row does not intersect the most intense portion of the cross peak click 1 with the left mouse button until it does Once the desired row is selected click on ses 1 with the left mouse button to move the row to window 1 appearing in the upper right hand corner of the display Repeat the selection of rows described above for a row with a cross peak in the middle and another row with a cross peak at the right edge of the spectrum and move them to window 2 and 3 respectively Now that three rows have been selected the 0 and 1 order phase corrections in F2 are determined by hand exactly as described for the 1D spectrum in Section 3 8 Click on the 11 orthe 1 button to tie the cursor to the biggest peak of the row in window 1 Phase Correct this row using the 0 order phase correction Em button P Correct the 1 order phase correction for the other two rows using the button and observe the rows in window 2 and 3 respectively Save the phase correction by returning to the main window select Save amp return at the prompt To phase correct the spectrum in F1 repeat the above procedure by s
198. to calibrate this integral to 10096 Upon return select Save amp store intrng to save the integral and normalization constant and return to the main 1D processing window It is also possible to compare integral values of spectra located in different data sets Integrate both spectra and calibrate the integral s in one of them e g to 100 as described above Enter the integration mode in the second spectrum select the corresponding integral asterisk arrow and click on the lastsca button to display the integral value compared to the calibrated 10096 of the other signal Avance 1D 2D BRUKER 37 38 BRUKER Avance 1D 2D 4 Pulse Calibration Protons 4 1 Introduction This chapter describes pulse calibration procedures for H and C It is assumed that the user is already familiar with acquisition and processing of simple 1D NMR spectra Appendix A Data Sets and Selected Parameters which lists all data sets generated throughout this course and Appendix B Pulse Calibration Results which provides all the pulse lengths and power levels determined during this course maybe useful in this context 4 2 Proton Observe 90 Pulse For the calibration of a H 90 pulse on the observe channel F1 the one pulse sequence described in Chapter 3 is used The carrier frequency olp is set onto the resonance frequency of a peak in the H spectrum of an appropriate sample This peak is monitored while the length p1 and or the stren
199. ts are additional peaks which can be placed on those additional diagonales The multiple quantum diagonales can be quite easily found in the spectrum They have either twice three times four times the slope of the regular diagonal Figure 61 Reference spectrum DQF COSY experiment of pamoic acid recorded with a repetition rate of 5s No artifacts are visible Avance 1D 2D BRUKER 175 Figure 62 DQF COSY experiment of pamoic acid recorded with a repetition rate of 1s Artifacts appear on the double quantum diagonal Double quantum diagonal 176 BRUKER Avance 1D 2D 22 2 2 Overload of the receiver With the introduction of field gradients in high resolution NMR spectroscopy the spectral quality of 2D experiments was improved dramatically With field gradients just the magnetisation of interest can be selected For the gradient assisted DQF COSY experiment the intensity of the first t FID is zero as no magnetisation transfer can occur at that state As a consequence the automated receiver gain adjustment will fail and the receiver gain will be set to a too large value With the receiver overloaded the phase cycling of the receiver will not work properly anymore leading to errors like quadrature artifacts along the F1 dimension e g a diamond pattern In addition the baseline will be distorted and high t noise can appear Please note that care has to be taken for the automated adjustment of the receiver gain for ROESY
200. u selection Examples are zg which starts the acquisition t which performs a Fourier transformation on the current data or apk which invokes the automatic phase correction Another possibility to manage different task in XWinNMR are AU programs They handle many routine jobs an can be selected or edited by the edau command AU programs have to be compiled before first usage Compile and start AU Programs by entering xau followed by the program name XWinNMR also offers extensive online documentation which can be accessed via the help menu in the XWinNMR menu bar Avance 1D 2D BRUKER 23 2 2 3 Changes for XWinNMR 3 5 XWinNMR version 3 5 is shipped with new systems now There are some new commands and the handling of some pulse programs have changed from the software version 3 1 e n XWinNMR 3 5 the names of pulse program and parameter files have been adjusted to the general NMR nomenclature For recording HSQC HMQC and HMBC spectra pulse program and parameter files starting with the 4 letter code hsqc hmqc and hmbc respectively have to be given in the pulprog line in the eda table e A new parameter TDO is now available in the eda table This parameter brings about a storage of your 1D data after recording ns TDO scans This is especially useful for very long 1D experiments For more information on general changes please refer to the release letter of your software packet Information for pulse program specific changes can
201. uisition and processing 5 1 Introduction This chapter describes the acquisition and processing of a C spectrum acquired with a one pulse sequence with and without H decoupling 5 1 1 Sample Since NMR is much less sensitive to C nuclei than to H it is advisable to replace the 100 mg sample of cholesterylacetate used in chapter 3 Basic H Acquisition and Processing with a 1g sample of cholesterylacetate in CDCl 5 1 2 Prepare a New Data Set Create a new data set starting from setup1h 1 1 created in the last chapter Enter edc and change the following parameters NAME setup13c EXPNO 1 PROCNO 1 Click on SAVE to create the data set setup13c 1 1 Enter edsp and set the following spectrometer parameters NUC1 13C NUC2 1H Click on SAVE The spectrometer is now ready to pulse and detect at the C frequency and to pulse and decouple at the H frequency Lock the spectrometer Lock cdc13 adjust the Z and Z shims until the lock level is optimized use 1ockdisp tune and match the probehead for 136 and H 5 2 One Pulse Experiment without H Decoupling The one pulse sequence without decoupling is identical to the one used in Chapter 3 Figure 1 except that the RF pulse is applied at the C frequency Enter eda and set the acquisition parameters values as shown in Table 16 Avance 1D 2D BRUKER 45 46 Table 16 C Basic Acquisition Parameters Parameter Value Comments PULPROG zg s
202. uivalently simply enter rpar standardlD plot To select the spectral region full or expanded to be plotted make sure the spectrum appears on the screen as desired and then type defplot Hit return in response to the following three questions F1 lt return gt F2 lt return gt Change y scaling on display according to PSCAL return Unless special precautions are taken to deal with the long C T relaxation times and potential NOE build up during H decoupling the integrated intensities in 1D C NMR spectra do not reflect the correct numbers of different types of C nuclei in a given molecule Thus standard C spectra are usually not integrated and the integrals are therefore not plotted Type edg and click the button next to the parameter INTEGR so that it toggles to no Click EE to exit the edg menu Finally create a title for the spectrum by entering setti and write a title Save the file and simply enter plot provided the correct plotter is selected in edo to plot the spectrum 7 2 DEPT DEPT Distortionless Enhancement by Polarization Transfer is a polarization transfer technique used for the observation of nuclei with a small gyromagnetic ratio which are J coupled to H most commonly C DEPT is a spectral editing sequence that is it can be used to generate separate C subspectra for methyl CH3 methylene CH2 and methine CH signals DEPT makes use of the generation and manipulation of mu
203. um should be phased correctly and all peaks should be positive Further adjustments can be made in the 2D phase subroutine as described in previous chapters 14 5 Plotting Follow the instructions given for the previous experiments i e XHCORR An HMQC spectrum of 50 mM Cyclosporin in CDs is shown in Figure 39 Avance 1D 2D BRUKER 123 Figure 39 HMQC Spectrum of 50 mM Cyclosporin in CDs ppm 19 20 30 49 om 50 2e Xe 4 to stop que Gp ae 60 dm 70 um 80 30 RL DS ES D A M S 73 70 6 5 6 0 535 5 0 4 5 49 33 3 0 2 5 2 0 15 1 0 ppm 124 BRUKER Avance 1D 2D 15 HMBC 15 1 Introduction HMBC Heteronuclear Multiple Bond Correlation spectroscopy is a modified version of HMQC suitable for determining long range H C connectivities Since it is a long range chemical shift correlation experiment HMBC provides basically the same information as COLOC but it has a higher sensitivity since it is an inverse experiment Reference A Bax and M F Summers J Am Chem Soc 108 2093 1986 The sample used to demonstrate HMBC in this chapter is 50 mM cyclosporin in C D6 as was used to demonstrate HMQC The HMBC pulse sequence is shown in Figure 40 The first C 90 pulse which is applied 1 2 JxH after the first H 90 pulse serves as a low pass J filter to suppress one bond correlations in the 2D spectrum by creating H 13C heteronuclear multiple quantum coherence This unwanted coherence is removed by phase
204. urthermore we elegantly disposed of the Yalo factors in a constant which here we can replaced by Fo or simply be omitted altogether While neither being elegant nor exactly correct we can obtain very useful results much faster then going through all the details 23 8 3 The Signal Function of a Coupled Spectrum By omitting Fo we obtain the following signal function for our coupled two spin system Tr F 0 2 cos r i sin r cos r Jp f cos 1 i sin 6 f cos t J 1 ijt i t cos t J t e cos t J t e zt pg ty t 4 utet ander ge 2 2a 2a 2 2i 2a iV y e e e te To get the results above we made extensive use of the Euler relation The form of the signal function should look familiar it describes a frequency spectrum with four signals at the frequencies v1 J12 2 v1 J12 2 v2 J12 2 and Avance 1D 2D BRUKER 197 Vo J12 2 We recognize a spectrum with two doublet signals each doublet having two lines of equal intensity that are separated by J 2 Hz At this point we are able two handle a two spin system in a 1D experiment Most of the calculations using the spin operator formalism will never include a spin system larger then two as the number of operators quickly become too cumbersome to handle Nevertheless let us take a look at a simple 3 spin system in order to introduce some important simplification schemes for handling such large system 23 9 Simplification Schemes on A
205. used is called DECP90 and it is shown in Figure 9 The sequence consists of a recycle delay tra d1 followed by a 90 C pulse p1 a delay 1 2J 8C H 42 enst2 for the evolution of antiphase C H magnetization a H pulse p3 and the C detection period acq aq For the calibration of the 90 H decoupling pulse the length and or the strength of the H pulse p3 and or the power level p12 is adjusted No signal is acquired for an exact H 90 pulse p3 since only undetectable multiple quantum coherence C magnetization is present Figure 9 DECP90 Pulse Sequence T 2 13C du 1 2J C H acq di pi d2 p3 6 2 3 Setthe H Carrier Frequency The first spectrum will be a H observe experiment to determine the correct frequency for the DECP90 H decoupling pulse Create a new data set starting from e g proton 1 1 re proton 1 1 enter edc and change the following parameters NAME testdec EXPNO 1 PROCNO 1 Click on SAVE to create the data set testdec 1 1 and enter eda to set the acquisition parameters as shown in Table 20 54 BRUKER Avance 1D 2D Table 20 1D H one pulse Acquisition Parameters Parameter Value Comments PULPROG zg see Figure 1 for the pulse sequence diagram TD 4k NS 1 DS 0 D1 10 interscan delay 10s because of long T P1 3 start with 3us which should correspond to less than a 90 pulse PL1 high power level on F1 channel H as determined in
206. ut a hundred isotopes possess an intrinsic angular momentum called spin and written AI They also possess a magnetic moment u which is proportional to their angular momentum u oy where y is the gyromagnetic ratio The Larmor theorem states that the motion of a magnetic moment m where M represents the bulk magnetic moment of a collection of identical nuclei in 186 BRUKER Avance 1D 2D a magnetic field Bo is a precession around that field The precession frequency is given by yB Larmor frequency By convention the external static field Bo is assumed to be along the z axis and the transmitter receiver coil along either the x or y axis After the sample has reached its thermal equilibrium in this context the equilibrium magnetic polarization the system shows a magnetization vector m along the z axis In this state no NMR signal is observed as we have no transverse rotating magnetization By application of an additional rotating magnetic field B4 in the x y plane the orientation of can be tilted into the x y plane as the precession of Mm is always around the total magnetic field e g the vector sum of Bo and Bi A rotating magnetic field is obtained by using RF pulses To describe the motion of M in the presence of the rotating B4 it is convenient to use a rotating coordinate system instead of a static one By convention B is assumed to be along the x axis of a coordinate system rotating around the z axis The ro
207. w be presented in the quantum mechanical notation 23 3 Spin Operators of a One Spin System In the spin operator formalism the state of a spin is represented by a linear combination of four operators l ly lz and 2 E The first three can be understood as M M and M respectively also this is not strictly correct as M refers to a macroscopic magnetization while refers to a single spin For all practical purposes this detail can be neglected The fourth operator 1 2 E or unity operator is added for reasons of mathematical consistency and is usually omitted in the notation We will also follow this convention and omit Ve E The operators form a basis in the so called Liouville space which is the mathematical frame work in which the spin system is described But we don t need to worry about this for the moment 23 4 The Thermal Equilibrium State All NMR experiments start from the thermal equilibrium In thermal equilibrium the classical description gives rise to a magnetic moment parallel to the static field This is due to the fact that the energy level for spins in a parallel orientation with the external field is slightly lower than the one for the antiparallel spins According to Boltzman the lower energy level will have a higher population than the high energy level the difference being proportional to the energy difference The energy difference between these two Zeeman levels being very small the resulting population
208. which is detected during the acquisition period Note that for a very short recovery delay time the pulse sequence is equivalent to a 270 pulse and the detected signal has full negative intensity if the delay is very long full T relaxation occurs between the 180 and 90 pulses and the detected signal has full positive intensity T can be determined by repeating the experiment with different recovery delay values The resulting curve is an exponential with rate 1 T Note that for some intermediate value of the recovery delay the peak intensity is zero and T1 tnu In 2 The procedure described in this chapter is for determining H T values A similar procedure may be used for measuring C T values However for measuring C Tis it is important to use inverse gated H decoupling to improve the spectral signal to noise ratio without selectively enhancing peak intensities through NOE effects It is also important to use a sufficiently long recycle delay recall that C T4 can be much longer than H T The sample used to demonstrate a T experiment in this chapter is 100 mM Pamoic Acid in DMSO d6 The inversion recovery pulse sequence is shown in Figure 52 The 180 pulse p2 is followed by the recovery delay vd The value of vd is determined by the delays contained in the appropriate vdlist and is varied over the course of the experiment A 1D spectrum is obtained for each value of vd and the results are stored in a 2D data set The
209. window functions and to perform the 2D Fourier transformation adjust the threshold level set the phase correction and plot the spectrum A GRASP DQF COSY spectrum of 50 mM Cyclosporin in CDs is shown in Figure 26 Avance 1D 2D BRUKER 91 92 Figure 26 GRASP DQF COSY experiment of 50mM Cyclosporin in C6D6 BRUKER Avance 1D 2D 9 TOCSY 9 1 Introduction TOCSY TOtal Correlation SpectroscopY provides a different mechanism of coherence transfer than COSY for 2D correlation spectroscopy in liquids In TOCSY cross peaks are generated between all members of a coupled spin network An advantage is that pure absorption mode spectra with positive intensity peaks are created In traditional COSY cross peaks have zero integrated intensity and the coherence transfer is restricted to directly spin coupled nuclei In TOCSY oscillatory exchange is established which proceeds through the entire coupling network so that there can be net magnetization transfer from one spin to another even without direct coupling The isotropic mixing which occurs during the spin lock period of the TOCSY sequence exchanges all in phase as well as antiphase coherences The coherence transfer period of the TOCSY sequence occurs during a multiple pulse spin lock period The multiple pulse spin lock sequence most commonly used is MLEV 17 The length of the spin lock period determines how far the spin coupling network will be probed A general rule of thumb i
210. xperiment is divided into time intervals during which the Hamiltonian can be made time independent by BRUKER Avance 1D 2D choice of a suitable interaction frame Typical experiments are divided in pulse intervals and free evolution times During the pulses the chemical shift and scalar coupling interaction is ignored Only the applied B field is considered This approach is justified for pulses with toulse T1 T2 1 4 3 Effect of Scalar Coupling Apart from the chemical shift there is a second very import interaction between spins the scalar coupling The scalar depends on the mediation of electrons which are confined in orbitals around both nuclei The scalar coupling is expressed in Hz and noted as J The operator expression for the scalar coupling is 2xJ IL The above Hamiltonian expresses the scalar coupling between spin 1 and spin 2 with a coupling constant Ji2 The evolution Hamiltonian for this spin system is then H 6 1 6 L 21 LL To calculate the effect of this Hamiltonian it is divided into 3 parts 9 I 5 L 27 J IL which are applied in sequence where this sequence is arbitrary After a 90 pulse has been applied to the two spins we first calculate the two chemical shift terms Ox h I cos 8 t 1 sin t L tx _ J cos 8 t I sin t L cos 6 t L sin 6 t o The next step will be to compute the evolution under the scalar coupling The scalar coupling term can b
211. xt save these parameters as the plot parameter file by entering wpar stackpar plot Avance 1D 2D BRUKER 155 156 Enter NAME Enter EXPNO Enter PROCNO Enter USER Enter DISK Repeat dialog r or continue c Enter first row to plot Enter row increment Enter number of rows Enter row for scaling Enter x increment cm Enter y increment cm BRUKER Return to the 2D processing menu enter re 2 1 and start the stacked plot automation program by entering xau stack2d Answer the questions as tidata 2 1 user name The resulting stacked plot is sent to the plotter specified by the parameter CURPLOT To check or change this parameter enter edo to call up the output device parameter menu and click on the box next to CURPLOT to open the menu of plotter options Select one of these with the left hand mouse button and exit the edo menu A stacked plot of the results of the inversion recovery sequence run on 100 mM Pamoic Acid in DMSO d6 is shown in Figure 53 Avance 1D 2D Figure 53 Inversion Recovery Spectra of 100 mM Pamoic Acid in DMSO d6 vd 10s IL AUX I AUC A AA AA JUL AA AAA A MA JN Uh A wW vd 0 01 s LR Y XY vY Y Avance 1D 2D 20 Selective Excitation 20 1 Introduction The hard pulses used in all the experiments from the previous chapters are used to uniformly excite the entire spectral width This chapter introdu
212. xtends to the x axis and the number of LEDs lit in the vertical HPPR display is minimized Most likely this will not be the desired frequency Adjust the T screw slightly to move the dip toward the center of the window or equivalently to reduce the number of LEDs lit in the horizontal HPPR display Rematch the dip by adjusting the M screw again Note that it is possible to run out of range on the M screw If this happens return M to the middle of its range adjust T to get a well matched dip at some frequency and walk the dip towards the correct frequency as described above As mentioned above ideal tuning and matching is when the dip is centered in the window and extends to y 0 the x axis on the acquisition window or equivalently when the number of LED s lit on the preamplifier is minimized in both the vertical and horizontal display When the H circuit is tuned and matched exit the wobble routine by typing stop Click on return to exit the acquisition window and return to the main window 26 BRUKER Avance 1D 2D 2 5 Tuning and Matching C non ATM Probes Since most C experiments make use of H decoupling besides C the H should be tuned and matched as well When tuning and matching a probehead with multiple resonant circuits it is best to tune and match the lowest frequency circuit first Thus when tuning and matching a probehead for both H and C first do the C and then the H adjustments Make sure that the sam

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