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Solid State NMR: AVANCE Solids User Manual - Pascal-Man

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1. Table 10 1 Acquisition Parameters Parameter Value Comments PULPROG cpspindiff old cpnoesy Pulse program NUC1 6 Set 13C in both F2 and F1 column SW 250 ppm To be optimized O1P 120 ppm To be optimized NUC2 1H For CP decoupling only O2P 3 ppm To be optimized for dec PL1 For 13C contact PL11 For 13C flip pulses PL12 For 1H excitation and decoupling PL14 for n spin rate DARR or 120 Recoupling SPO For H contact using shape SPNAMO ramp 100 or ramp70100 100 For 1H 18C contact CPDPRG2 SPINAL64 At PL12 P1 13C excitation flip pulse P3 1H excitation pulse P15 13C H Contact pulse PCPD2 Decoupling pulse for spinal64 D1 Relaxation delay D8 5 500 msec Depending on sample CNST31 MAS speed Used to calculate d31 rotation period L1 calculated from cnst31 and d8 Number of rotor cycles for mixing time AQ MOD DQD TD F1 512 Number of points User Manual Version 001 BRUKER BIOSPIN 103 261 Proton Driven Spin Diffusion PDSD Table 10 1 Acquisition Parameters SW F1 usually SW MASR if possible NUC1 F1 NUC1 TD F2 128 Number of points NDO 1 Not required in TopSpin 2 1 NS 4 n FnMode TPPI States States TPPI Processing Parameters 10 3 1 Process with xfb Table 10 2 Processing Parameters Parameter Value Comment F2 acquisition Be Hiekkaa Left column SI 1k Number of complex
2. Table 3 1 Summary of Acquisition Parameters for Glycine S N Test Parameter Value Comments PULPROG cp cp av for AV1 and 2 NUC1 13C Nucleus on f1 channel O1P 100 ppm 13C offset NUC2 1H Nucleus on f2 channel D1 4s Recycle delay NS 4 Number of scans SWP 300 ppm Spectral width for Glycine TD 2048 Number of acquired complex points CPDPRG2 SPINAL64 Decoupling scheme f2 channel iH SPNAMO ramp 100 or ramp 70100 100 For ramped CP P15 2 ms Contact pulse f1 and f2 channel PL1 Set for 4 4 5 usec P90 SPO or pl2 AV1 2 Set for 4 4 5 usec P90 2 dB optimize PL12 High power level f2 channel iH excitation and decoupling P3 90 H pulse at PL12 f2 channel PCPD2 or SPINAL64 decoupling pulse P31 AV1 2 O2P 2 5 3 ppm 1H offset optimize in 400 500 Hz steps for maximum signal of aliphatic peak Note that the spectral window swp is set in ppm which makes the acquisition time dependant on the By field at a given td of 2k This is intended and accounts for the linewidths dependence on the Bo field The glycine lines show a broadening proportional to By due to chemical shift dispersion To make S N values more comparable this accounts for shorter T5 at higher field User Manual Version 001 BRUKER BIOSPIN 39 261 Basic Setup Procedures Table 3 2 Processing Parameters for the Glycine S N Test Parameter Value Comment Sl 2 4 k Twofold o
3. Table 7 2 Processing Parameters for FSLG HETCOR on tyrosine HCI Parameter Value Comment F2 direct dim 13C si 2 4 k wdw QSINE SSB 2 30r 5 ph_mod pk F1 indirect 1H si 256 1048 mc2 STATES TPPI wdw QSINE ssb 3 5 ph_mod pk 80 261 BRUKER BIOSPIN User Manual Version 001 FSLG HETCOR Results 7 4 A full plot of a FSLG HETCOR on labeled tyrosine HCl is shown in the next figure hetcor AVIII 600 WB 300 usec contact time F1 ppm 10 10 15 200 150 100 50 F2 ppm Figure 7 4 FSLG Hetcor Spectrum Tyrosine HCI The figure above shows a FSLG Hetcor Spectrum Tyrosine HCI with parameters as shown in Table 7 1 Table 7 2 Full transform with slight resolution enhancement qsine SSB 3 Proton shifts calibrated as 2 5 and 12 most high field low field peak Center ridge at 0 ppm along F1 is spin locked signal which does not follow the FSLG evolution Cnst24 is used to separate the proton spectrum from this ridge The contact time of 300 usec shows many long range cou plings The next figure shows the region of interest excluding the center ridge and the spinning sidebands User Manual Version 001 BRUKER BIOSPIN 81 261 FSLG HETCOR F1 ppm 4 10 12 180 160 140 120 100 80 F2 ppm Figure 7 5 FSLG Hetcor Spectrum Tyrosine HCI The figure above shows a FSLG Hetcor Spectrum Tyrosine HCI with parameters as shown in Table 7 1 Table 7 2 Full transform with sl
4. 125 12 3 Data Acquisition 00 cece cec cece cece eee e eee e cece a eee eem eene 126 12 4 Spectral Processing x seed ehren he 127 12 5 13C 13C Single Quantum Correlation with DQ Mixing 128 12 6 Data Acquisition 129 12 7 Spectral Processing coit nein 130 13 PISEMA M M 131 13 1 Introduction ee eee 131 13 2 Pulse Sequence Diagram cccccceeeeeeneeeeneeeneeeeneeeeneeeeneeenneeeneees 132 13 3 SEUD EE 133 Setup 2D Experiment ernennen 134 13 4 Data ACQUISITION RR ee en 135 13 5 Processing sense entalten 137 14 Relaxation Measurements uuu0 u0000000nnannnn nun ann 141 14 1 Describing Relaxation eesseessseesseeeeeee nennen nennen nnn nnns 141 14 2 T1 Relaxation Measurements sss 142 Experimental Methods sssssssssss nennen 142 The CP Inversion Recovery Experiment zuessseessennnsnnnnenn nenn 143 Data Processing odes ee cent Dae E dex d Den dened eeh deet go 145 The Saturation Recovery Experiment sse 147 T1p Relaxation Measurements sssssssee 148 14 3 Indirect Relaxation Measurements sssssss 149 Indirect Proton T1 Measurements sssssssse 150 15 BASIC MO MAS cq 151 15 1 Introduction sssssssssssssssssee e III III II rre 151 15 2 Pulse SEQUENCES eoe pe dE ana AE MR pat eR TR si ara 151 15 3
5. Parameter Value Comment pulprog wpmig2d AV 3 instruments only topspin 2 1 or later FnMODE STATES TPPI Any other method may be used with appropriate changes in ppg NUC1 NUC2 1H sw swh along F1 td Same as for F2 512 1k Needs to be corrected before transform pulse pro gram calculates approximate values to be set before transform ased Depending on resolution 226 261 BRUKER BIOSPIN User Manual Version 001 Table 22 1 Acquisition Parameters CRAMPS 2D 1 td 128 256 Depending on resolution spnam1 wpmlg1 m5m or m5p as DUMBO may be used with modified timing in 1d spnam2 Igs 2 or Igs 4 if used Set I3 2 or 4 depending on desired sw1 DUMBER 22 with modified timing Table 22 2 Phases RF levels and Timings Phases RF Power Levels Timing do 0 STATES TPPI pl12 set for around 100 kHz p1 around 2 5 usec du CYCLOPS 1230 pl12 p1 dh 10 2 02 sp1 sp2 set for 100 130 kHz RF WPMLG calculated via cnst20 field or pl13 for both set in ppg DUMBO p10 set by xau dumbo 011 02 dto dto 03 CYCLOPS 0 123 Data Processing d8 desired mixing time 50 1000 Hs 22 3 The spectral width in both dimensions assumes the absence of shift scaling In order to ac count for the shift scaling effect of the sequence one has to increase the spectral width by the scaling factor Before do
6. sss meme 107 BRUKER BIOSPIN User Manual Version 001 Figures Figure 10 6 13C DARR of Fully Labelled Ubiquitine Spinning at 13 kHz 108 11SUPER 109 Figure 11 1 Pulse Sequence for 2D CPMAS exchange experiment 110 Figure 11 2 The 123 Icon in the Menu Bar of the Data Windows Acquisition Parameter ag is E 111 Figure 11 3 The Acquisition Parameter Window eda see 112 Figure 11 4 The SUPER Spectrum of Tyrosine HCI After Processing Using xfb 115 Figure 11 5 SUPER spectrum after tilting the spectrum setting 1 alpha 1 116 Figure 11 6 Various Cross Sections from the Upper 2D Experiment 117 12Symmetry Based Recoupling 119 Figure 12 1 C7 SQ DQ Correlation Experiment nenn tesa nenn 121 Figure 12 2 Optimization ofthe RF power level for DQ generation reconversion on gly eme E 123 Figure 12 3 Variation of DQ generation reconversion time on a uniformly 13C labeled peptide fMLF ssssssssssssssssseseeenen emen menn erret nnne nnns 124 Figure 12 4 PC7 Recoupling Efficiency at a Spinning Speed of 13 kHz 125 Figure 12 5 5C14 2d SQ DQ correlation on tyrosine HCl seseeeuessss 128 Figure 12 6 PC7 2d SQ SQ correlation on tyrosine HCl cocooccoccccnccccnnccnnnccnanccnnnnns 130 13PISEMA 131 Figure 13 1 Pisema Pulse Sequence sese 132
7. Figure 6 13 CPMAS Spectrum of Tyrosine HCI at 6 5 kHz CPMAS spectrum of tyrosine HCI at 6 5 kHz sample rotation obtained on a 500 WB spectrom eter using a 4 mm CPMAS double resonance probe The red third spectrum is a CPPI spec trum where we see the CH resonance at 35 ppm with a negative intensity The aromatic CH resonances are clearly suppressed where the C shows a slightly negative intensity The po larization inversion pulse p16 was 40 us long The green second spectrum is a CPPIRCP ex periment with p16 40 us and p17 10 us for better nulling of CH resonances but in this case the aromatic CH resonances gained some intensity back The purple first spectrum is a CP PISPI experiment with a similar performance as for the CPPI spectrum Our experience is that one can adjust p15 1 ms in this spectrum p16 30 us in the purple spectrum and so edit for pure CH resonances for example Such tuning needs to be done of course on a known sample which behaves similarly to the one under investigation for the editing to be conclusive and correct Note For more editing experiments consider the Solid State Attached Proton Test experi ment using the sostapt pulse program name or look at 2D editing sequences based on the FSLG HETCOR experiment User Manual Version 001 BRUKER BIOSPIN 73 261 Basic CP MAS Experiments 74 261 BRUKER BIOSPIN User Manual Version 001 FSLG HETCOR Introduction 7 1 This chapter discusses
8. 44s44ssennennnenne nennen 189 Data Processing 2 2 iode een 191 Double ECP di 193 Pulse Sequence Diagram Double CP DCP sees 194 Spectrometer Setup for DCP sssssssssssssss 194 Setup for DCP 13C channel ococccccnccncnncnncnnnnnconnnononnnnnncnnnnnncnnnns 194 15N Channel Setup ssssssssssssssse eres 196 Setup of the Double CP Experiment ssssssssse 197 Setup of the 2D Double CP Experiment sssesssessssse 201 2D Data Acquisition ssssssssssssssee ens 202 Spectral Processing edd bedreet aoa deht 203 Example Spectra 22440444 44nannsnnnennnnnnnnnnnnnn eee nenne 204 CRAMPS E 207 Homo nuclear Dipolar Interactions oooooccoccncccnccnnccnccnncnncnnnninnnnnnnns 207 Multiple Pulse Sequences ssssssss 207 W PMLG and DUMBO eee men Ihnen nnne 208 Quadrature Detection and Chemical Shift Scaling 208 CRAMPS ID EE 211 Pulse Sequence Diagram of W PMLG or DUMBO osne 211 Pulse Shapes for W PMLG and DUMBO sss 212 Analog and Digital Sampling Modi 0 eeeeseeeeeeeeeeeeeeeeeeeeeneeeea tees 213 Analog Mode Sampling sss 214 Digital Mode Sampling 214 SIUE 215 Parameter Settings for PMLG and DUMBO sse 215 Fine Tuning for Best Resolution ssssssem 217 Fine Tuning for Minimum Carrier Spike sseeeee
9. 88 Cien ge lese M 89 dumbonet e 90 HETCOR with Cross Polarization under LG Offset ooooconcnicnnicnccccccoso 91 RFDR NER 93 idera 94 LE e dE ee Zeg 94 REI UCL mmc 95 Set up 2D Experiment 95 Spectral Processing iu 96 Proton Driven Spin Diffusion PDSD 99 Pulse Sequence Diagram sssssse mee 101 SERIEM een eg ni 101 2D Experiment Setup oocoocoonconccocconcconconcconconcnonccnnnoncnonnnnnnnnninaninns 101 Acquisition Parameters ssssssssssssssss emnes 103 Processing Parameters NR 104 Adjust the Rotational Resonance Condition for DARR RAD 104 Example Spectra i Ret sence ded ead oes in er seas kaa De 106 E 109 OvervieW qc 109 Pulse PEOR cum 110 Experiment Setup u en nn 110 Experiment Setup er nenn 110 Setup 2D Experiment nenne nenn 111 BRUKER BIOSPIN User Manual Version 001 Contents 11 4 Data Acquisition nenne nme nnne nen 113 11 5 Spectral Processing ed dek dE ERR ete AER NEE anna e AE vets EE dre das 114 12 Symmetry Based Recoupling sss 119 12 1 Pulse Sequence Diagram Example C7 sss 121 12 2 Sig me 121 Spectrometer Setup for 13 123 Setup for the Recoupling Experiment sssssssssse 123 Setup of the 2D SQ DQ Correlation Experiment
10. 89 Table 8 5 Acquisition Parameters for DUMBO HETCOR on tyrosine HCl 90 9 RFDR 93 Table 9 1 Acquisition Parameters cccccccecceeeceece cece eeeeece ees eee eee eeaeeseeeaeeeaes 95 Table 9 2 Processing Parameters ssssssssse meme 96 10Proton Driven Spin Diffusion PDSD 99 Table 10 1 Acquisition Parameters sssssssssssss een 103 User Manual Version 001 BRUKER BIOSPIN 249 261 Tables 250 261 Table 10 2 Processing Parameters 44 444444snseenenenennnenennnen ernennen 104 11SUPER 109 Table 11 1 Acquisition Parameters 4444444444404nennnennenennne m 113 Table 11 2 Processing Parameters 444444444 4sn snenenennnnnenne nennen 114 12Symmetry Based Recoupling 119 Table 12 1 Recommended Probe Spin Rates for Different Experiments and Magnetic Field Strengths u a 122 Table 12 2 Acquisition parameters for DQ SQ correlation experiments using symmetry based recoupling sequences uuessessnenseennenenennenennnennn e 126 Table 12 3 Processing parameters for DQ SQ correlation experiments using symmetry based recoupling sequences nn nnennne nen 127 13PISEMA 131 Table 13 1 Acquisition Parameters ocococccoccccccnncconnononcnonononnnononnnononnnnnnnnannnnnannns 136 Table 13 2 Processing Parameters sssssssssssssee nne 137 14Relaxation Measurements 141 Table 14 1 Parameters for the 1D CP Inver
11. COMMENT single pulse exci lc acquisition without decoupling SCLASS Solids IM pseudo YPE direct excitation UBTYPE relaxation measurement WNER Bruker to adjust t 0 for acquisition ee E ere 11 0898 99 D An An An in in in d 9 u ne SS in E e K ze di p3 p112 ph1 fi p15 sp0 ph10 f1 go 2 ph31 wr 0 exit phi 0 2 ph10 1 ph31 0 2 3 Power conversion table probe 4mm triple power conversion table nucleus frequency p90 RF field power remarks us kHz W Figures 1 Introduction 9 2 Test Samples 11 3 Basic Setup Procedures 13 Figure 3 1 Routing for a Simple One Channel Experiment u esseensnenennenn 15 Figure 3 2 Probe Connections to the Preamplifier nennen nenn 17 Figure 3 3 Pop up Window for a New Experiment 18 Figure 3 4 ased Table with Acquisition Parameters for the KBr Experiment 19 Figure 3 5 Graphical Pulse Program Display 20 Figure 3 6 Display Example of a Well tuned Probe eeem 21 Figure 3 7 Display Example of an Off Matched and Off Tuned Probe 22 Figure 3 8 Display Example Where Probe is Tuned to a Different Frequency 22 Figure 3 9 FID and Spectrum of the 79Br Signal of KBr used to Adjust the Magic Angle EE 23 Figure 3 10 Routing for a Double Resonance Experiment using High Power Stage for H ANG ed Ensei Abee ee ee e heet bie 24 Figure 3 11 Routing for a Double Resonance E
12. As mentioned above frequency shifts can also be generated by a phase gradient shape A phase change of 360 per second corresponds to a frequency of 1 Hz as can easily be visual ized The frequency shift which needs to be achieved is RF field 2 Since the pulse duration must achieve a 27 rotation off resonance corresponding to a 293 flip angle on resonance it can easily be calculated that a phase change over 209 during a 293 flip angle pulse is re quired to achieve this 52 261 A9 St ALA LA 10 ot DI Ak IPP Figure 4 5 Shape with Phase Gradients In the figure above Shape with phase gradients for positive and negative offsets and corre sponding phase change stdisp display of Igs 1 shape Amplitude is 10096 throughout Vinogradov et al have published shapes with much fewer steps and different phases The pulse length for the shape does not depend on the number of steps but only on the applied RF field Using the include file Igcalc incl a pulse p5 is calculated from cnst20 RF field in Hz The total shape pulse length must be 2 p5 BRUKER BIOSPIN User Manual Version 001 Decoupling Techniques To use pmlg decoupling save the pulse program falg under a different filename and change calculations and loop as follows define loop counter count calculate number of LG periods according to aq count aq 2 p5 define pulse pmlg pmig 2 p5 sp1 pl13 set shape power to pl13 for L
13. Digital Mode Acquisition 20 9 Most parameters stay the same as adjusted in analog mode Table 20 4 Parameters for Digital Mode Parameter Value Comment pulprog dumbod or wpmlgd AV 3 instruments only digmod digital dspfirm sharp or medium aqmod qsim or dad swh 50000 10000 Depending on spectral range and 01 The correction for the scaling factor must be done after acquisition changing the status param eter swh by typing s swh and dividing the value by the scaling factor about 0 578 for WPMLG 0 47 for wpmlgd2 and 0 5 for DUMBO Some pulse programs are written such as to show the correct sweep width in ased which can then be set appropriately as s swh before transform Examples 20 10 MEG power lavas Hil o u Kd LI LU n Po NN J r T r T T T r 40 42 HE 48 43 80 ER Dom Figure 20 6 Optimizing sp1 for Best Resolution 218 261 BRUKER BIOSPIN User Manual Version 001 CRAMPS 1D optimisation for enst25 minimum conter spike at cngt25 20 degrees phase Mist Anu E r r r T 1 T T 50 100 160 ppm Figure 20 7 Optimizing cnst25 for Minimum Carrier Spike Optimized at 120 C oplumisahon for pte optimum ai 0 6 usec about 35 degrees Mp angle should be 90 magic angla T T 02 04 06 08 WEI ppm Figure 20 8 Optimizing p14 for Minimum Carrier Spike Optimized at 0 6 usec Use
14. STMAS Introduction 17 1 The STMAS experiment for half integer quadrupole nuclei is a 2D experiment to separate anisotropic interactions from isotropic interactions In the NMR of half integer quadrupole nu clei the dominant anisotropic broadening of the central 1 2 1 2 transition CT and sym metric multiple quantum MQ transitions is the 2 order quadrupole interaction which can only partially be averaged by MAS The satellite transitions ST e g the 3 2 gt 1 2 transi tions however are broadened by a 1 order interaction which is several orders of magnitude larger than the 2 order broadening Under MAS the 1 order interaction of the ST can be av eraged but spinning cannot be fast compared to the first order broadening of the order of MHz a large manifold of spinning side bands remains The 2nd order broadening of the CT can only be narrowed by a factor of 3 to 4 so a signal is observed that still reflects this 2 d order broadening The 2D STMAS experiment exploits the fact that the 2 d order broadening of the ST transitions e g 3 2 lt gt 1 2 in a spin 3 2 is related to the 2 d order broadening of the CT by a simple ratio A 2D spectrum is recorded which correlates a single quantum coherence of the satellite transitions usually one of the inner transitions 3 2 lt 1 2 and the 1 2 gt 1 2 single quantum coherence of the central transition The resulting 2D spectrum yields an isotropic pro je
15. Sl 256 Sufficient in most cases WDW no Don t use window function unless F1 FID is truncated PH_mod pk Apply phase correction BC_mod no No DC correction is required after full phase cycle ABSF1 1000 Should be outside the observed spectral width ABSF2 1000 Should be outside the observed spectral width STSR 0 Avoid strip FT STSI 0 Avoid strip FT TDoff 0 Avoid left shifts or right shifts before FT Data obtained with stmasdgfe av can be processed with xfb only if IN6 or IN7 have been set appropriately to run a split t experiment Data acquired with the pulse program stmasdgfz av should be processed with the AU program xfshear For information about this program please refer to chapter 16 In an analogous way to MQMAS spectra the apparent Larmor frequency in the indirect dimension is recalculated by multiplying the real Larmor frequency with the corre sponding value of R p The values for the different spin quantum numbers are summarized in Table 17 7 for experiments using the inner ST 3 2 gt 1 2 R determines the shearing User Manual Version 001 BRUKER BIOSPIN 191 261 STMAS ratio i e the slope in a non sheared spectrum R p is the scaling factor for referencing in the indirect dimension Using this procedure the shift positions in the indirect dimension are identi cal in ppm to all MQMAS experiments and the information obtained is the same Refer to the chapter Basic MQ MAS on page 151 for details
16. lar couplings exist between nuclei of different kind While most dipolar couplings between X range nuclei can be removed by magic angle spinning couplings between 1H 19F and X nuclei cannot easily and efficiently be removed by spinning Decoupling of homo nuclear and hetero nuclear interactions can be obtained by different forms of rf irradiation with or without sample spinning It is possible to suppress homo nuclear couplings without suppressing hetero nuclear couplings Most frequently the nucleus 1H must be decoupled when X nuclei like 13C or 19N are observed since it is abundant and broadens the line shapes of coupled X nuclei strongly Hetero nuclear Decoupling 4 1 CW Decoupling 4 1 1 CW decoupling simply means irradiating the decoupled spins usually protons with RF of con stant amplitude and phase The decoupling program is called cw or cw13 and it uses pl12 or pIT3 respectively The decoupling programs select the power level and p 12 does not need to be specified in the pulse program if it is not used elsewhere In the decoupling program there is also a statement setting the RF carrier frequency according to the parameter cnst21 which is zero on resonance by default In order to optimize decoupling one uses the highest permit ted rf field e g 100 kHz for 4mm probes and optimizes the carrier frequency 02 or 02p using popt The cw decoupling program is written as follows 0 5p pl pl12 reset power level to default decoupl
17. spnam1 dumbo 1 40 Both include one DUMBO cycle p3 2 5 3 usec 90 pulse channel 2 at pl12 p15 50 500us Contact pulse width p10 32 or 24us 32 us for 100 kHz field 24 us for better resolution at high magnetic fields gt 500 MHz pcpd2 2 p3 SPINAL64 TPPM decoupling pulse 90 261 BRUKER BIOSPIN User Manual Version 001 HETCOR with Cross Polarization under LG Offset Table 8 5 Acquisition Parameters for DUMBO HETCOR on tyrosine HCl cpdprg2 SPINAL64 TPPM15 Decoupling sequence F1 1H indirect 10 0 Start value 0 incremented during expt 13 2 4 Multiples of DUMBO periods increment per row in_f1 inO as calculated Set according to value calculated by ased F2 13C acquisition d1 2s Recycle delay sw 310ppm Sweep width direct dimension aq 16 20 msec masr 12000 13000 HETCOR with Cross Polarization under LG Offset 8 3 Usually the cross polarization step is executed a less power than what is used for the initial ex citation pulse and decoupling during observe Therefore a second LG condition must be set for the power level during contact Furthermore the HH condition must be re established since the proton spin lock pulse now must be a square pulse not a ramp The ramp can be trans ferred to the carbon F1 side The following steps are involved 1 The RF field for protons during contact must be measured and adjusted With linearized tra
18. spnamO ramp 100 or similar Shape for contact pulse channel f2 sp1 set to pl13 To match cnst20 spnam1 m5m or m5p Both include one FSLG cycle p3 2 5 3 usec 90 pulse channel 2 at pl12 p15 50 500 usec Contact pulse width pcpd2 2 p3 SPINAL64 TPPM decoupling pulse cpdprg2 SPINAL64 TPPM15 Decoupling sequence F1 1H indirect 10 0 Start value 0 incremented during expt 13 2 4 Multiples of FSLG periods increment per row in_f1 inO as calculated Set according to value calculated by ased F2 13C acquisition d1 2s Recycle delay sw 310ppm sweep width direct dimension aq 16 20 msec 88 261 BRUKER BIOSPIN User Manual Version 001 edumbohet HETCOR with DUMBO PMLG or w PMLG Using Shapes 8 2 3 DUMBO decoupling is as efficient as PMLG decoupling As discussed in chapters 22 and 23 it requires to spin a bit slower up to 12 13 kHz and to place the carrier closer to resonance The library of AU programs in TopSpin includes dumbo which calculates the desired shapes for windowed and windowless DUMBO shapes If the windowless version is desired the e dumbo shape is preferred Typing xau dumbo starts a dialog in which e for e dumbo 22 1 for the number of cycles 64 for the number of steps and 0 for an added angle this value would be added to all phases in the shape The program sets p20 to 32 usec as default This is appro priate for an RF field of 100 kHz Spnam2 is set to ed
19. 168 261 borro ro poo br zm zm eda zm mm mm de wm mm wm e e e bk wm wm d wm i i i J e e wl e m axis CS Figure 15 12 Graphical Interpretation of the Spectrum from Figure 15 10 In the 11 7 T spectrum this gives quadrupole induced shifts 5 gj of 75 ppm and 20 ppm for the two sites respectively At 18 8 T the 5 gj of the lower peak in the 2D spectrum decreases to 30 ppm whereas it cannot be determined graphically anymore for the upper peak since the chemical distribution broadens the peak in the F1 dimension more than the theoretical 3 gis of 5 ppm BRUKER BIOSPIN User Manual Version 001 MQ MAS Sensitivity 1 6 Enhancement The MQMAS experiment on half integer quadrupole nuclei is an extremely insensitive experi ment This is due to the low efficiencies of both the excitation of 3Q coherence and their con version to observable magnetization Several approaches have been taken to enhance the efficiency of the excitation and conversion mainly focusing on the conversion as this is the least efficient step Adiabatic pulses can be used for the conversion instead of a single high power CW pulse and alternative phase cycling schemes have also yielded improvements Im proving the efficiency of the MQ excitation pulse has been tried but no generally applicable scheme exists so far Before describing the optimization procedures some experimental ap proaches used in combination with
20. E OO EE 15 77 EDASP ann e 16 CroM 15 o p eevee 17 B16 O Em 89 experiment b tton iii ada 19 F TOQUEN anne ee A cs 15 Frequency Switched Lee Goldbumg cnn 48 208 Frequency Switched Lee Goldburg Heteronuclear Correlation 75 ESG NET 208 ESLG DecoupliNd 4 ea ee 48 FSEG ET 75 BRUKER BIOSPIN User Manual Version 001 Index G Cal Em 11 AS ated ve tue tee 14 e 11 Ee 11 Good Laboratory Practice ernennen 41 graphical display ertet dece ede edes 19 OUD MER RR T 29 H H3SE One een 12 Feat NA seis EE 34 Hartmann Hahn condition oooooccccnnoccccccnoooncccno nano ncnnnn nan cnn cnn nn enne 33 Heteronuclear Decoupling rssu00ssnnassnnonennnnsnnnnnnnnsnnnnnnnnnnnnnnennnnannnennnnn 45 FAG EE 12 ele TTT 12 ele eere me 35 nell Em 35 Me EE 24 elle reiege Ee len Le TTT 14 Homonuclear decoupling esce aA AARAA 47 Homonuclear dipolar Imterachons nennen 207 HPSHPPR modules 2 Ec tata ent soe ond tipo d eee e Presented 31 HPENA 1H Modules re ee me 31 I prr E 77 INVOTSION Bee TEE 142 irradiation Treguency nn 15 K aviae En 12 A E O 12 LEE 12 13 23 en een 12 MODA EE 12 L Lee Goldburg Condition EE 48 LO Offset 91 LIO E EE 12 Mo E 11 12 local MOONS E een 142 logical channel 15 longitudinal relaxa
21. Integrales Sange Structure Pid U 15N Jebeled K lpf in DMPC Figure 13 5 PISEMA Spectrum of 15N Labeled Kdpf Transmembrane Protein PISEMA spectrum of 15N labeled Kdpf transmembrane protein aligned in DMPC courtesy NHMFL T Cross membrane between glass plates using EFREE 700 MHz probe User Manual Version 001 BRUKER BIOSPIN 139 261 PISEMA 140 261 BRUKER BIOSPIN User Manual Version 001 Relaxation Measurements In NMR experiments one is generally concerned with measuring resonance frequencies and relating these to the local molecular environment To do this the state of the system of spins in the sample must be changed from equilibrium At equilibrium the net magnetization due to the spins is aligned along the magnetic field axis By applying a radio frequency pulse the net mag netization is tilted away from the field axis and the resulting precessing magnetization gener ates the observed signal The pulse has disturbed the system from equilibrium and over time the system will return to its equilibrium state This process is called relaxation This chapter describes experiments used for measuring relaxation rates in solid state NMR A basic description of relaxation is provided in order to define terms and introduce the techniques involved but discussion of the significance and use of relaxation data is outside the scope of this manual Many textbooks provide more detail on the theory of relaxation the classic is A
22. Processing Parameters Parameter Value Comment F1 acquisition 13c Left column SI 4096 Number of points and zero fill WDW QSINE Squared sine bell SSB 2 90 shifted sine bell PH mod pk Phase correction if needed BC mod quad DC offset correction Alpha 1 For shearing the spectrum F2 indirect C Right column SI 128 Zero fill MC2 STATES TPPI WDW QSINE Squared sine bell SSB 2 90 shifted sine bell PH mod pk Phase correction if needed BC mod no Automatic baseline correction 114 261 BRUKER BIOSPIN User Manual Version 001 SUPER arso ey ppm BOCH m Zase DINA rour var 50 xerar ow Ki D E De Tt 0 mers Y 00 2600042 E DR E 0 90100040 pes 220 0 3 400340 mec i 0 060749 one 0 0011 ses am t ES e n 6 a S a tw t 50 ac e at a us a were 6 Seen se wer Y M 300069 ses sum WA uc x Lal 1 0 ums 3 15 o 100 009 65 ure ye oo ma 3 e Fui 1 29 e Ln 125 39064225 Ms CURK 17 e nom spanen Bramaning PATA TUM inten 125 3478472 Max X H Yea PL 180 160 140 120 100 80 60 40 20 ppm Figure 11 4 The SUPER Spectrum of Tyrosine HCI After Processing Using xfb User Manual Version 001 BRUKER BIOSPIN 115 261 SUPER 100 190 180 170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 10 ppm Figure 11 5 SUPER spectrum after tilting the spectrum setting 1 alpha 1 Figure 11 5 SUPER spect
23. more transients per slice may be required The recommend ed value is 4 which increases the number of required transients per experiment to 256 Run 1D experiment and make sure everything is set properly 7 Create a new experiment with either iexpno or edc Change to 2D data set BRUKER BIOSPIN User Manual Version 001 SUPER Setup 2D Experiment 11 3 2 After 1D parameter optimization as previously described type iexpno to create a new data file and switch to the 2D mode using the 123 button Set the appropriate FnMode parameter in eda Pulse program parameters are detailed as follows Figure 11 1 shows the pulse se quence ocPars AcquPars Title Pu gcc a Figure 11 2 The 123 Icon in the Menu Bar of the Data Windows Acquisition Parameter Page The 123 icon in the menu bar of the data windows acquisition parameter page is used to tog gle to the different data acquisition modes 1D 2D and 3D if so desired 9 Make sure the correct nucleus is selected in F1 dimension make sure an appropriate quadrature detection mode is selected in FnMode TPPI STATES TPPI or STATES 10 Choose the appropriate sampling time td7 so that the required resolution FIDRES in the indirect dimension is achieved 11 Set pI11 to give a pulse nutation frequency of12 12 rotation rate see chapter 1 12 Set d4 the z filter delay to about 1 ms integer number of rotor periods if possible 13 Set p2 to be a 180x
24. one may click the default button change to powmod high in the command line if necessary Note the routing is only effective if the parameter pow mod high To change to proton observe click SwitchF1 F2 24 261 BRUKER BIOSPIN User Manual Version 001 Basic Setup Procedures frequency logical channel amplifier preamplifier BFI 600 147 MHz NUCI u SFO 600 147 MHz Fl EZ x x 300 W HPHP X8831P OFSI foo Hz 1H v HPHP 19F 1H BF2 150 907083 MHz NUC HPHP X8831P SFO 2 150 912583 MHz F2 eu S iH 1H 100 W HPHP 19F 1H OFS2 55000 He fisc HPLNA IH BF3 600 47 MHz NACH SFO3 600 147 MHz F3 S6U3 x x 300 W OFS3 oo Hz ort x BF4 600 47 MH NACH SFO4 600 147 MHz F4 S6U4 1H E IH 50 w OFS4 foo Hz off bd i cble wiring settings possible QF routing show receiver routing show BF routing corta available C poser os E Sow Ls tch FIF Switch FVF3 Add a logical charra Remove a logical channel Default Info Porm cem Figure 3 11 Routing for a Double Resonance Experiment Changed for Proton Observation In the figure above channel F2 need not be used The settings for F1 and F2 are interchanged Change rg to 8 16 and d1 to 4 sec Set pl1w to 50W or to 10 dB high power proton transmitter 7 dB 500W proton transmitter 5dB 300W proton transmitter or 4 100W transmitter if the green dot does not appear in the 1H channel in edasp Connect the probe proton channel t
25. possible RF routing show receiver routing show RF routing a cortab avoilabl z 2 chow power at probe in Sove Switch Fl FZ Switch FIJF3 Add a logical channel Remove a loza al channel Default Info Param Close Figure 3 1 Routing for a Simple One Channel Experiment The figure above shows the routing for a simple one channel NMR experiment using the 1000 W output from the high power amplifier In this menu 4 RF channels are available These 4 RF channels can be set up for 4 different frequencies The two left most columns labelled frequency and logical channel define the pre cise irradiation frequency by setting the nucleus and the offset O7 from the basic frequency In User Manual Version 001 BRUKER BIOSPIN 15 261 Basic Setup Procedures 16 261 this example we want to set up for pulsing observe on the nucleus 79Br Selecting 79Br for channel F1 defines the basic frequency BF1 of 79Br in this case on a 600 MHz spectrometer 150 360709 and the adjustable offset 9000 Hz in this case Both values are added to show the actual frequency setting SFO1 The frequency setting is taken from a nucleus table which is calculated for the respective magnetic field Bg The index 1 in O1 BF1 and SFOT refers to the RF channel 1 which is also found in the pulse program where the pulse is defined as p1 f1 Note that this index does not exist for the following columns which represent the hardware components SGU 1 4 refers to
26. saturation ee EE 147 saturation recovery experiment nn 143 SAVE Te e 29 Save Display Regilor to ccoo nn 28 SB ie 43 e lee 209 218 SELTICS ae een andern 63 66 67 Ee TTT 48 SEn VI A 13 cca OS aaa 31 SFO EE 16 SOU NET 16 unu TEE 13 Silicone paste in eic it 11 Silicone rubber TT 11 CipeE 40 SKID Een ee seattle I e eto te cune 29 Skip Optimizat OM ES 29 SM2SN297 TTT 11 SIM CY COMOX EE 11 ep 11 spin nutation frequencies nnn 13 err umen Un 13 CDI 47 SPINAL decoupling riri i e Finn to eren eot gen od Le RE Eee 47 User Manual Version 001 BRUKER BIOSPIN 257 261 Index 258 261 spin lattice relaxation nn spin locking pulse neeese eeeeee spinning Oase spin spin relaxation nen spnamd O Start Optlmize nce tne rnit stop optimization oooooononiccccconccnnnccnncnncnnnnns susceptibility compensated DwitchEIIE 2 A e a TPPM decouplmg Fans LE transverse magnetization TREVSB coe V variable dea variable pulse list nn varmod nenn W PMEG acosada X X Low Pass Elter E n X BB Preamplifier nn XIX decoupling nennen nennen BRUKER BIOSPIN User Manual Version 001 Index Y Y NO3 3 6H20 iieiaei iaaea a aa aa Taane aa a ERa 12 Z ZGopti n DISCO ET 32 ZGOPTN ET 62 a carbon at 43 el EE 36 IV 11 iria bo 12 36 a S
27. the magnetization is inverted by a 180 pulse Then there is a delay during which the magnetization relaxes and a 90 pulse converts the remain ing longitudinal magnetization to transverse magnetization and an FID is recorded The inten sity of a particular signal in the resulting spectrum depends on the initial intensity the relaxation delay and the relaxation time constant T as follows S t S S 0 S exp t T Eq 14 1 where t is the relaxation delay Sg is the maximum signal seen when t is infinite S O is the sig nal measure with no relaxation delay and T4 is the relaxation time constant for the spins giving rise to that signal Measurement of S t for a number of relaxation delays allows determination of T4 The disadvantage of the inversion recovery experiment is that the delay between scans needs to be somewhat longer that the longest T4 of the slowest relaxing spins in the sample If cross polarization from protons is possible the initial inversion pulse can be replaced by a cross po larization step followed by a 90 pulse on the nucleus to be observed Then the required delay between scans dT becomes that for relaxation of the protons In most cases the proton T1 is moderate so inversion recovery Torchia method is the method of choice BRUKER BIOSPIN User Manual Version 001 Relaxation Measurements If the T1 relaxation time is extremely long the saturation recovery experiment is preferred Here the transi
28. 001 BRUKER BIOSPIN 49 261 Decoupling Techniques d 3 FSLG decoupling Kr 2n Af Af ka ee um 1LG cycle 1LG cycle 00221133 00 Ton 02201331 Figure 4 3 FSLG Decoupling Pulse Sequence Diagram Ar Am a2 41 40 39 30 37 36 35 24 33 32 21 30 29 29 27 26 2s ppm Figure 4 4 Adamantane FSLG Decoupled The C H J Couplings Shown 50 261 BRUKER BIOSPIN User Manual Version 001 Decoupling Techniques The figure above shows Homo nuclear proton decoupling on center packed adamantane sam ple rotating at 7 kHz 100 kHz 1H decoupling field Note that good B4 homogeneity is required Use a CRAMPS spinner 12 ul sample volume in a 4mm spinner Setting up the experiment 1 Use center packed adamantane in a CRAMPS rotor unlabeled and a spinning rate of 10 kHz for adamantane 2 Start from a data set with well adjusted HH condition on adamantane 3 Generate a new data set with edc 4 Readjust decoupling power pl12 and p3 pcpd2 for 70 100 kHz RF field determine the pre cise RF field preferably via a 360 proton pulse p3 5 Load the pulse program fqlg This uses frequency shifts with simultaneous phase shifts for FSLG decoupling at pl13 6 Set pl13to achieve the same RF field as measured in step 4 set cnst20 to the value of RF field in Hz The pulse program contains the include file lt Igcalc incl gt which calculates the re quired frequency shifts to either side shown in ased as cnst22 and cnst23
29. 1 Pulse Sequence Diagram ssssse ee 211 Figure 20 2 PMLG Shape for wpmlg sp1 ssssssseH Hee 212 Figure 20 3 Shape for DUMBO sp1 ssssssss HH 213 Figure 20 4 Analog Sampling Scheme 214 BRUKER BIOSPIN User Manual Version 001 Figures Figure 20 5 Digital Sampling Scheme 214 Figure 20 6 Optimizing sp1 for Best Resolution 218 Figure 20 7 Optimizing cnst25 for Minimum Carrier Spike Optimized at 120 C 219 Figure 20 8 Optimizing p14 for Minimum Carrier Spike Optimized at 0 6 usec 219 Figure 20 9 WPMLG CRAMPS After Optimization Digital Acquisition 220 21Modified W PMLG 221 Figure 21 1 Pulse Sequence Diagram sss mee 221 22CRAMPS 2D 225 Figure 22 1 Pulse Sequence Diagram sse ee 226 Figure 22 2 Setup and Test Spectrum of Alpha glycine oocooccccoccccncccnncccnnccnnnncnnnnnn 228 Figure 22 3 Spectrum of Tyrosine hydrochloride 229 Figure 22 4 Expansion of the Essential Part of the Spectrum 230 Figure 22 5 Pulse Sequence Diagram sss 231 Figure 22 6 Glycine Proton Proton DQ SQ Correlation Using WPMLG in Both Direc ue cec TUE 234 Figure 22 7 14 5 kHz W PMLG PC7 DQ SQ Correlation at 600 MHz with Tyrosine Hy dr chl ride pede prt eben elle 235 A Appendix 237 User Manual Version 001 BRUKER BIOSPIN 247 261 Figures 248 261 BRUKER BIOSPIN User Manual Version 001 Tables 1 Introduction 9 2 T
30. 100 Phase alignment of freq offset in SPO Offset frequency for SPO User Manual Version 001 FSLG HETCOR In the figure above the frequency offsets for the FSLG part are shown as cnsi22 cnst23 They are different because cnst24 shifts the center frequency by 2000 Hz Table 7 1 Acquisition Parameters for FSLG HETCOR on tyrosine HCI Parameter Value Comments pulprog Ighetfq FSLG program nuc1 13C olp 100 ppm nuc2 1H cnst20 70 100000 Proton spin nutation frequency with PL13 cnst24 0 2000 Place carrier off during evolution pr Power level channel 1 for contact pulse pl12 Power level channel 2 TPPM SPINAL decoupling pl13 Power level channel 2 FSLG decoupling spo Power level channel 2 for contact pulse spnam0 ramp 100 or simil Shape for contact pulse channel f2 p3 2 5 3 usec 90 pulse channel 2 at pl12 p15 50 500 usec Contact pulse width pcpd2 2 p3 0 2 SPINAL64 TPPM decoupling pulse cpdprg2 SPINAL64 TPPM15 Decoupling sequence F1 1H indirect 10 0 Start value 0 incremented during expt 13 2 4 Multiples of FSLG periods increment per row in_f1 in0 as calculated Set according to value calculated by ased F2 13C acquisition di 2s Recycle delay sw 310ppm Sweep width direct dimension aq 16 20 msec masr 10 15000 Hz At 100 kHz RF 15 kHz is ok User Manual Version 001 BRUKER BIOSPIN 79 261 FSLG HETCOR
31. 16 and 17 for some MQMAS experiments will be performed h1 h3 h2 h4 p1 dO p4 p2 d p4 d7 pl11 pl21 oni pl21 Figure 17 3 Four pulse sequence and coherence transfer pathway Four pulse sequence and coherence transfer pathway for the double quantum filtered STMAS experiment with shifted echo acquisition stmasdgfe av Pulses p1 and P2 are non selective pulses Corresponding power level PL11 should be set to achieve around 100 kHz RF field am plitude P3 and P4 are CT selective pulse 90 and 180 pulses of about 20 and 40 us respec tively corresponding to an RF field amplitude of a few kHz Delay DO is the incremented delay for t4 evolution D6 and D7 can be incremented proportional to DO depending on the spin of the observed nucleus Phase lists are as follows incrementation of the phase of the first pulse is not required because a phase modulated data set is acquired with FnMODE being QF phi 0 180 90 270 ph2 0 4 90 4 180 4 270 4 ph3 0 16 90 16 180 16 270 16 ph4 0 receiver ph3 ph1 ph2 BRUKER BIOSPIN User Manual Version 001 STMAS Data Acquisition 17 4 Before the 2D experiment on your sample of interest can be started some setup steps must be done as described in detail below All setup steps should be done on a sample with a Aknown MAS spectrum b With sufficiently good sensitivity to facilitate the set up and c A 2 order quadrupole interaction of the order of the one expected for your sample of intere
32. 176 002 ppm 18714 Peak 2 at 42 269 pom 64875 2 k 8 g T T r 0 5 10 15 20 s 146 261 Figure 14 2 Relaxation of Alpha carbon Signal in Glycine Start calculation This will perform the fitting procedure for all regions The calculated function is displayed as a red line on the same axes as the data points The plus and minus icons can be used to move through the different regions If you wish to change whether the fit is based on the integral or the intensity select the appropriate radio button and repeat the fit using the icons immediately above The gt gt icon will fit all the peaks the gt icon will fit just the current one Display report This displays a text report of the results of the fit including the details of the fit function and the calculated values of the parameters in the function The experimental and cal culated data points are also displayed Note that the experimental data is normalized such that the most intense point has a value of 1 This report file is also saved in the processed data di BRUKER BIOSPIN User Manual Version 001 Relaxation Measurements rectory when the fit is calculated If fitting of a single peak is performed only this result is writ ten to the report If the fit all peaks option is used all results will be stored The results for glycine at 500 MHz and room temperature should be approximately 18 5s and 6 4s for the carbonyl and alpha carbon signals respectively at
33. 217 Correcting for Actual Spectral Width sesseeeese 217 Digital Mode Acquisition sssssssse mH 218 E nl ITT 218 Modified W PMLG ai 221 Pulse Sequence Diagram for Modified W PMLG ssesess 221 Pulse Shapes for W PMLG nennen 222 ijv pL P e 222 Parameter Settings for PMLG and DUMBO sese 223 Fine Tuning for Best Resolution sessssse 224 BRUKER BIOSPIN User Manual Version 001 21 6 21 7 22 22 1 22 2 22 3 22 4 22 5 22 6 22 7 22 8 A 1 Contents Correcting for Actual Spectral Width 224 Digital Mode Acquisition sssss HH 224 CHAMPS 2D unseren ee Segen 225 Proton Proton Shift Correlation spin diffusion gt 225 Pulse Sequence Diagram essen eene enne 226 Data Processing 2 ee eine 227 E let EEN 228 Proton Proton DQ SQ Correlation sessssensnennnnnnennnen nennen 230 Pulse Sequence Diagram sssssse He 231 Data Processing u ee dure du re du d dE EE RR dE er 233 3CclW aecE 233 Appendix de 237 Form for Laboratory Logbooks ssssesee e 237 FIQUrES eet A RARA EDEA 243 ILI m 249 WOON AA O 253 User Manual Version 001 BRUKER BIOSPIN 7 Contents 8 BRUKER BIOSPIN User Manual Version 0
34. 261 FSLG HETCOR Pulse Sequence Diagram for FSLG HETCOR 7 2 6o du 7 Qio TPPM decoupling 2n 2r Af Af e NM 1LGcycle 1LG cycle WE 13 STATES TPP get Joco 00221133 2G r OG e 02201331 00 0 0 00000 Figure 7 1 The FSLG Hetcor Experiment The FSLG Hetcor experiment consists of 3 basic elements the Homo nuclear decoupling se quence during which the 1H chemical shifts evolve the cross polarization sequence during which the information of the 1H spin magnetization is transferred to the X spins followed by ob servation of the X spins under proton decoupling 76 261 BRUKER BIOSPIN User Manual Version 001 FSLG HETCOR Setting FSLG HETCOR Experiments 7 3 1 This experiment requires a probe of 4mm spinner size or smaller One can run it on a 7 mm probe but the results will not be very convincing 2 Start from a data set with well adjusted cross polarization and proton decoupling at fairly high RF fields Unlike standard multiple pulse decoupling which only works well at very high RF fields FSLG requires only moderately high RF fields Decent performance is achieved at 80 100 kHz proton field At lower magnetic fields 200 300 MHz proton frequen cy lower RF fields are adequate RF fields of 100 kHz and higher perform better at higher magnetic fields 500 MHz and up 3 Insert a suitable test sample spin at a suitable speed We recommend 13C labeled tyrosine hydrochloride since i
35. 4 pulse se quence with z filter Both sequences start with an excitation pulse p1 that creates 3Q coher ence which is allowed to evolve during the evolution period dO In the 3 pulse sequence the subsequent conversion pulse p2 flips magnetization back along the z axis which after a short delay d4 to allow dephasing of undesired coherency is read out with a weak CT selective 90 pulse p3 In the 4 pulse sequence however the conversion pulse p2 changes 3Q coherency to 1Q coherency which then passes through a Z filter of two CT selective 90 pulses in a p3 d4 p3 sequence User Manual Version 001 BRUKER BIOSPIN 151 261 Basic MQ MAS 152 261 Figure 15 1 A 3 Pulse Basic Sequence with Z Filter Three pulse sequence and coherence transfer pathway for the 3Q MAS experiment with z filter mp3qzqf av The ratio for pulses p1 and p2 is approximately 3 The corresponding power lev el pl11 should be set to achieve at least 150 kHz RF field amplitude p3 should be some tens of us corresponding to an RF field amplitude of a few kHz Delays d0 and d4 are the incremented delay for t1 evolution and 20 us for z filter respectively Phase lists are as follows for phase sensitive detection in F1 the phase of the first pulse must be incremented by 30 in States or States TPPI mode phi 0 ph2 0 0 60 60 120 120 180 180 240 240 300 300 ph3 0 180 receiver d ph2 Ir pt d p2 di0 p3 d4 p3 piti pl21 Figure 15
36. 5 5 50 200 Triple mode C N 19Sn 149 1 4 62 5 100 Double mode high range 31P 161 9 3 5 71 4 150 Range switch up double mode User Manual Version 001 BRUKER BIOSPIN 57 261 Practical CP MAS Spectroscopy on Spin 1 2 Nuclei 2 Once these values are measured any HH condition can be calculated Assumed you want to cross polarize 119Sn the sample spins at 12 kHz The contact time is anticipated to be rather long because 1195n atoms are large and far away from protons So the power level for the contact should not be too high Let us set the RF field to 50 kHz for the contact We decide to apply a ramp shape on the proton contact pulse covering the 1 spinning side bands This means that we need to apply a ramp from 38 to 62 kHz RF field plus some safety margin about 35 to 65 kHz RF field on the proton ramp For 1195n we need to apply 50 kHz RF field Since the RF field is proportional to the amplitude in a shape RF voltage output is proportional to shape amplitude value the shape power must range from 65 kHz to 35 kHz from 100 to about 50 amplitude Use calcpowlev to calculate the changes in dB to achieve the calculated RF fields enter reference RF field to calculate required RF field instead of pulse lengths In our case the proton contact pulse power sp0 is calculated at 3 74 dB 65 kHz compared to 100 kHz the power level for 119Sn is calculated at 1 94 dB 50 kHz compared to 62 5 kHz Be sure to add the calculated number for a des
37. 9 FID and Spectrum of the 79Br Signal of KBr used to Adjust the Magic Angle Calibrating 1H Pulses on Adamantane 3 3 Spin the KBr sample down and change to a spinner filled with adamantane Spin at 5 10 kHz Generate a new data set from the KBr data set by typing new Set the instrument routing for 13C observe and 1H decoupling as shown in the following figure User Manual Version 001 BRUKER BIOSPIN 23 261 Basic Setup Procedures frequercy logic al channe amplifier preamp BFI 150 907083 MHz NUCI AA SFO 150 916083 MHz m JL ses S x x 300 W HPH fix orsi 000 0 He HPH BF2 600 147 MH NUC2 7L 1H 1000 HH SFO 600 1465 MHz R sev Y 1H 100 W HPH orsz 6000 H in He BF3 600 147 MH NUCH SFO3 600 147 MHz F3 S6U3 x HL x 300 w orsa fo D Hz ort BF4 600 147 MHz NUCA SFO4 600 147 MHz F4 seus IH 1H 60 W OFS4 lo D Hz BZ cable wiring settings possible RF routing show receiver routing show RF routing ab Lab hd SECHER show power at probe in Lowe Switch F1 F2 Switch FI F3 Add a logical channel Remove a logical channel Default Info Figure 3 10 Routing for a Double Resonance Experiment using High Power Stage for H and X nucleus The figure above shows the routing for a double resonance experiment e g a 13C experiment with 1H decoupling For high power transmitters the parameter powmod must be set to high To check which power mode is selected
38. AVII spectrometers have the extension av pulse programs for the AVIII have no extension Data Acquisition 15 3 Before the 2D experiment on your sample of interest can be started two set up steps must be done as described in detail below All set up steps should be done on a sample with a a known MAS spectrum b with sufficiently good sensitivity to facilitate the set up and c a 2 d order quadrupole interaction in the order of the one expected for your sample of interest In the first step a low power selective pulse must be calibrated in a single pulse experiment With this the MQMAS experiment can be optimized using the 2D pulse sequence for t4 0 Setting Up the Experiment 15 3 1 Sample There are a large number of crystalline compounds which can be used to set up the experiment Please refer to table 1 to select a suitable sample For the general procedure de scribed here the spin of the nucleus is not important of course obtained pulse widths will de pend on the spin I and the Larmor frequency You can use any arbitrary sample showing a considerable broadening by the 2nd order quadrupole interaction to adjust the experiment however reasonable 1D MAS spectra should be obtained quickly for sensitivity reasons The set up must be done in two steps in the first step a central transition selective pulse that merely excites the central transition must be calibrated This pulse must be weak enough so that only this transition is affec
39. CP step e g ramp 80 100 SPO Power level for proton contact pulse 126 261 BRUKER BIOSPIN User Manual Version 001 Symmetry Based Recoupling Table 12 2 Acquisition parameters for DQ SQ correlation experiments using symmetry based recou pling sequences CPDPRG2 SPINAL64 SPINAL64 decoupling CPDPRG1 cwlg To avoid HH contacts during DQ generation reconversion NS 4 32 Number of scans see pulse program phase cycle F2 direct 13C left column TD 1024 or 2048 Number of complex points SW Sweep width direct dimension adjust to experimental require ments F1 indirect 13C right column TD 128 512 Number of experiments in indirect dimension SW see para 17 Sweep width indirect dimension above NDO 1 STATES TPPI not required In TS 2 1 Spectral Processing Processing Parameters 12 4 Table 12 3 Processing parameters for DQ SQ correlation experiments using symmetry based recou pling sequences Parameter Value Comment F1 acquisition 13C left column Sl 2 4 k Number of points and zero fill WDW QSINE Sine bell squared SSB 2 5 Shifted sine bell PH_mod pk Phase correction if needed F2 indirect 1 C right column SI 256 1024 Zero fill MC2 STATES TPPI WDW QSINE Sine bell squared SSB 2 90 shifted sine bell Processing parameters for DQ SQ correlation experiments using symmetry based recoupling sequences The AU program x
40. F1 It is desirable to synchronize sampling along F1 with the rotor spin rate in order to eliminate spinning sidebands fold back onto center band This may however lead to peak fold over since achievable spin rates are usually smaller than the spread of DQ frequencies along F1 This does however not necessarily mean that the spectra are crowded and uninterpretable because frequently the folding does not lead to cross peak overlap Of course the synchronisation to the spin rate need not be used cnst31 can be set equal to the sweep width along F2 which will normally produce spectra free of folding but of course spinning sidebands along F1 will occur and signal intensity will be spread over a larger number of cross peaks An intermediate sampling rate along F1 can be achieved by incrementing the evolution period synchronised to the phase shifted blocks of the sequence one PC7 block being 27 7 T rotor period This will also not generate side bands along F1 but provide a larger sweep width and less fold over M Hong 1999 Fold over can often be tolerated xfshear rotate may be used to shift the spectrum suitably along F1 User Manual Version 001 BRUKER BIOSPIN 125 261 Symmetry Based Recoupling 13 Set the acquisition time along F1 to about 10 msec for a start Lines along the double quan tum dimension may be narrower than along the single quantum dimension so a compro mise between experiment time and digital resolution along F1 must be
41. Figure 13 2 The 123 Icon in the Menu Bar of the Data Windows Acquisition Parameter olm E 134 Figure 13 3 ASED Display for the PISEMA Setup sss HH 135 Figure 13 4 A PISEMA Spectrum of 15N Labeled Acetylated Valine B FID in t1 over 3 008 ms 64 Data Polnts u en 138 Figure 13 5 PISEMA Spectrum of 15N Labeled Kdpf Transmembrane Protein 139 14Relaxation Measurements 141 Figure 14 1 The CPX T1 Pulse Sequence ssse ene 143 Figure 14 2 Relaxation of Alpha carbon Signal in Glycine 146 15Basic MQ MAS 151 Figure 15 1 A 3 Pulse Basic Sequence with Z Filter sseseseeeeee 152 Figure 15 2 A 4 Pulse Basic Sequence with Z Filter seseeeee 152 Figure 15 3 Comparison of 87Rb MAS spectra of RbNO3 excited with selective and non selective pulses ccc cece eee cece cece eee ecee eee eece eee eeseeeseeseesaeeeeeeseeeaes 155 Figure 15 4 Nutation profiles of selective and non selective pulses 156 Figure 15 5 Example for popt Set up for Optimization of p1 and p2 157 Figure 15 6 Signal Intensities of 87Rb Resonances in RONO3 as Function of p1 and p2 EE Ee 158 Figure 15 7 2D 87Rb 3QMAS Spectrum of RbNO3 uussuennsennssnnsnnnnsnnnnnnn anne 161 Figure 15 8 Comparison of Differently Processed 2D 23Na 3Q MAS Spectra of TT NEE 162 Figure 15 9 Calculated Shift Positions dMQ ssssss
42. Hz Add or subtract the spin rate used for instance spinning at 13 yields 74000 or 48000 Hz If a 30 ramp is used and sp0 is set to 74000 with a safety User Manual Version 001 BRUKER BIOSPIN 91 261 Modifications of FSLG HETCOR 92 261 margin of 1000 the HH condition will cover 75000 down to 45000 Hz RF field This in cludes both HH sidebands n 1 4 Optimize the HH condition varying sp0 and p14 phcor1 for maximum signal 5 Run a variation of p15 between 20 and 5000 ms on your sample or tyrosine HCI One should see intensity variations dipolar oscillations which are normally smeared out by ex tensive Homo nuclear dipolar couplings between protons 6 Set up a 2D data set with the pulse program lgcphetfq Fig 3 and fig 4 compare spectra tak en with and without LG offset during cp In th L i e pq 140 126 140 ME Ziel Pal ill IBN INI se Di F pp 10 Mm to a Figure 8 3 HETCOR on tyrosine HCI without left and with LG contact 1msec contact BRUKER BIOSPIN User Manual Version 001 RFDR Radio Frequency Driven Recoupling RFDR with longitudinal magnetization exchange is a Homo nuclear dipolar recoupling experiment This easy setup technique is a zero quantum re coupling sequence that achieves chemical shift correlation under MAS conditions The time dependence of the cross peak amplitudes can be employed to determine inter nuclear distanc e
43. Magn Reson 155 15 28 2002 2 R Tycko G Dabbagh and PA Mirau Determination of Chemical Shift Anisotropy Line shapes in a Two Dimensional Magic Angle Spinning NMR Experiment J Magn Reson 85 265 274 1989 User Manual Version 001 BRUKER BIOSPIN 109 261 SUPER Pulse Program 11 2 Au 3 heteronuclear decoupling H Bo CN ty Nt Figure 11 1 Pulse Sequence for 2D CPMAS exchange experiment Experiment Setup 11 3 Sample Tyrosine HCI natural abundance Experiment time Less than 1 hour Experiment setup 11 3 1 110 261 In order to setup the experiment determine 14 18C and parameters with variable amplitude on H according to Basic Setup Procedures on page 13 Verify the pulse parameters on the 13C channel see Pulse Calibration Techniques with CP on page 66 and calculate the power level required for the recoupling pulses i e fj 12 2 x frot 3 Verify pulse width Calculate power level required for hetero nuclear decoupling during the recoupling pulses pl23 i e 20 30 kHz or gt 25 x frg Low power decoupling during the recoupling pulses is permitted because the 360 degree pulses act like hetero nuclear decoupling pulses pl22 during delays should be high The experiment requires at a minimum accumulation of 64 transients to complete the phase cycle Between 32 and 64 experiments are needed for a 2D data set Depending on the choice for the gamma integral
44. Off Tuned Probe Spectrum ProcPars AcquPars Title PulseProg Peck Integrals Sample Structure Fia 5 Ss dM 4000 598 599 600 601 Figure 3 8 Display Example Where Probe is Tuned to a Different Frequency 22 261 BRUKER BIOSPIN User Manual Version 001 Basic Setup Procedures The figure above is an example of where the probe is either tuned to a completely different fre quency outside this window or the probe is not connected to the selected preamp Check edasp for correct routing Check for correct matching box frequency range Increase WBSW to 50 or 100 and try to find the probe resonance position Next start an acquisition by typing zg in the command line or clicking on the black triangle up per left side in the acquisition display Do a Fourier Transformation and a phase correction by typing ff and phase correct Set offset O1 to the value obtained for the center peak see fig 13 and start xau angle This will allow you to view the fourier transformed spectrum or the FID after ns scans The magic angle is adjusted best when the spikes on the FID or the spinning sidebands in the Spectrum display have maximum size like shown in figure 9 This is most easily seen with the carrier ex actly on resonance and the un shuffled FID display mode The spinning sidebands should have maximum intensity the rotational echoes on the FID should extend out to at least 8msec in the FID display Figure 3
45. RF effi ciency and RF performance Accurate measurement of the pulse lengths and the associated RF power levels is essential for solid state NMR experiments In SSNMR RF field amplitudes are often expressed as spin nutation frequencies instead of 90 pulse widths Spin nutation fre quency ny and 90 pulse width are related through the reciprocal of the 360 pulse duration 4tpgo such that n 1 4t599 7 RF field in Hz with Leon in psec Setting up the magic angle shimming a CPMAS probe setting up cross polarization and mea suring probe sensitivity for 13C will also be explained This is part of probe setup and perfor mance assessment during installation However regularly scheduled performance measurements should be part of the hardware probes and spectrometer maintenance There fore these checks should be performed periodically The checks also need to be performed if an essential piece of hardware has been exchanged In the following we describe all steps which are necessary to assess performance of a CPMAS probe along with all necessary settings Detailed information about TopSpin software com mands is available in the help section within the appropriate chapter Setting up a CPMAS probe from scratch requires the following steps Mount the probe in the magnet and connect the RF connectors of the probe to the appropri ate preamps Connect the spinning gas connectors and the spin rate monitor cable Insert a spinner
46. SA a Figure 3 22 Hartmann Hahn Optimization Profile Using a Square Proton Contact Pulse The sideband order 0 at 4 8 dB gives a rather small intensity A ramp sweeping over 3 5 6 5 dB would cover both most efficient HH conditions Note that increasing the spin rate would shift all maxima except the one at 4 8 dB further out Cross Polarization Setup and Optimization for a Real Solid Glycine 3 7 Adamantane is highly mobile even in the solid state Therefore it behaves differently from a hard solid like glycine For instance it is not sensitive to decoupling mis adjustments and also not sensitive to miss sets of the magic angle It is however extremely sensitive to HH mis adjustment Glycine is therefore used for fine tuning of the decoupling parameters and signal to noise assessment Start with the parameters found for adamantane using a 50 100 ramp ramp 100 and p15 2 msec for contact aq 20 msec Change the sample from adamantane to glycine User Manual Version 001 BRUKER BIOSPIN 35 261 Basic Setup Procedures Since glycine may exist in two different crystal modifications with very different CP parameters and since packing of the spinner determines crucially the achievable S N value it is useful to prepare a reference spinner with pure a glycine finely powdered and densely packed a gly cine is prepared by dissolution of glycine in distilled water and precipitation with acetone quick filtering and careful dry
47. already be set in the standard 1D spec trum Since the experiment is not as dependent on the pulse lengths or the applied RF field amplitude as MQMAS pulse lengths between 1 and 2 us which can be achieved with every probe with 4mm or smaller rotor diameter are sufficient Table 17 3 Initial Parameters for the Set up of stmasdfgz av Parameter Value Comments pulprog stmasdafz av Pulse program NS 16 n For set up the full phase cycle is not so critical DO see text Calculated in pulse program D1 5 T4 Recycle delay use dummy scans if shorter D4 20 us Z filter delay P1 1 5 us Excitation pulse at pl11 P2 1 5 us Conversion pulse at pl11 P3 20 us 90 selective pulse at pl21 taken from previous pulse calibration PL1 120 dB Not used PL11 start with 150 Power level for excitation and conversion pulses to 300 W PL21 Power level for selective pulse approx pl11 30 dB taken from previous pulse calibration 188 261 For PL11 an initial value that corresponds to 150 to 300 W can be used Optimization will be done on the first increment of the 2D sequence which is calculated within the pulse program according to DO 1s L0O CNST31 P1 2 P4 0 3u P2 2 because it is essential that the centres of the pulses PT and P2 are exactly an integer number of rotor periods apart In this formula P1 P2 and P4 are the RF pulses as listed in table 2 CNST31 must be set equal to the spin ning frequency Th
48. also be performed after a probe repair Since a repair may result in a more efficient power conversion start with slightly reduced power settings BRUKER BIOSPIN User Manual Version 001 Basic Setup Procedures SB probes flip the stator vertical for sample eject These probes require some more effort to assure a correct angle setting Remember to always approach the magic angle setting from the same side To check the reproducibility of the magic angle setting take a KBr spectrum stop spinning eject and reinsert the sample take another spectrum into a new data set compare in dual dis play mode If the second spectrum is worse dial less than 1 8 of a turn counterclockwise Take another spectrum compare again A laboratory notebook should be kept with the following entries a suitable form for printout is supplied in the Appendix Name of the shim file and field value for every probe Value of power level in dB and power in watt if available for proton decoupling p 12 pl12W and associated pulse lengths p3 pcpd2 Value of proton contact power level in dB and watt sp0 SpOW e Value of carbon contact power level p 1 pl1W and associated pulse length p1 S N value obtained on glycine SR value for shift calibration line width on a carbon in Hz Literature 3 9 Shift referencing R K Harris E D Becker S M Cabral de Menezes R Goodfellow and P Granger NMR No menclature Nuclear Spin Properties a
49. amplitude This means that quadrature images will always be present if quad detection is used In the case of single detection the signal may be smaller or larger depending on the receiver phase The standard procedure is to use quad detection and suppress the quad images by a suitable phase cycling scheme This is however not as BRUKER BIOSPIN User Manual Version 001 CRAMPS General straightforward as it is with standard excitation observation The quad phase cycling must oc cur in the precession plane so a prepulse is required to tilt the initial magnetization into the di rection of the precession axis Usually a combination of 2 pulses is used for initial excitation 909 y xy T 90 55 adjust Figure 19 1 Difference in Amplitude ofthe Quadrature Channels X and Y The difference in amplitude of the quadrature channels X and Y caused by the tilted preces sion plane Along X the full amplitude is observed along Y only the component in the XY plane is detected As the spins precess around a tilted effective field and not only around the direction of the ex ternal field the precession frequencies are changed which means that the observed chemical shifts are changed As the frequencies are always smaller than in the standard excitation ob servation scheme the chemical shift range appears scaled down The scaling factor depends on the pulse sequence used To achieve a spectrum comparable to spectra acquired conven
50. and R G Griffin Cross Polarization in the tilted frame as signment and spectral simplification in hetero nuclear spin systems Mol Physics 5 1197 1207 1998 User Manual Version 001 BRUKER BIOSPIN 193 261 Double CP Pulse Sequence Diagram Double CP DCP 18 1 1 10 TPPM or SPINAL64 4 Prec 13 C 1SN 2 3 ae L sd p3 pls do p16 aq Aus 1 3 27 0 States TPPI t4 Aus 00001111 brecz0 2203113 22223333 20021331 3 00002222 Figure 18 1 Pulse sequence diagram for 1D t 0 and 2D double CP experiments Spectrometer Setup for DCP 18 2 Setup for DCP C channel 18 2 1 1 Prepare your probe for triple resonance applications H C N 2 Load a sample of glycine 15N and C4 C or only C gt 13C labelled Make sure the sample is a glycine you will get nowhere with y glycine since the proton Tue is very short and CP just does not work with high efficiency Rotate at 11 kHz The sample may be fully labelled or diluted with natural abundance glycine A restricted volume rotor is preferred If a differ ent spin rate is used a different shape must be generated for the second CP step 194 261 BRUKER BIOSPIN User Manual Version 001 Double CP 3 Check the edasp routing and set up 3 RF channels for C H and N such that the lower pow er amplifier 500W or less is used for Gei SN may require more than 500W Set for Tac observation 4 Make sure the preamplifiers in use are set
51. as m Figure 15 6 Signal Intensities of 87Rb Resonances in RbNOS3 as Function of p1 and p2 Each pair of diagrams in A and B shows the signal intensities as function of the excitation pulse p1 and the conversion pulse p2 In A the 3 pulse sequence and in B the 4 pulse sequence was used Note that the signal intensity is much more sensitive to the proper length of the conver sion pulse Maximum intensities were 3 0 us and 1 2 us in A and 3 2 us and 1 0 us in B re spectively This corresponds to approximate RF field amplitudes of 160 kHz Spectra are taken on an AV500WB at a Larmor frequency of 163 6 MHz with a 2 5 mm CP MAS probe spinning at 25 kHz Note the different scaling of the x axes for p1 they range from O to 6 us for p2 from 0 to 2 us Two Dimensional Data Acquisition 15 3 2 Once the pulses are calibrated everything is ready for the 2D data acquisition Create a new data set and change parmode to 2D In the acquisition parameters for the new indirect F1 di mension the following parameters must be set according to the following table Table 15 3 F1 Parameters for 2D Acquisition Parameter Value Comments FnMode States or States TPPI Acquisition mode for 2D TD see text Number of FID s to be acquired ND 010 1 There is only 1 dO delay in the sequence SWH masr Equals spinning frequency for rotor synchronization from this IN 010 is calculated correctly if ND 010 is already set NUC1 Select the s
52. coupling which in turn provides special information Naturally the C N or in general the X Y dipolar coupling is much smaller than any dipolar coupling involving protons For C N it is lt 2 5 kHz This has some experimental consequences 1 There is no need to decouple 15N while observing 13C since the coupling is spun out al ready at moderate spin rates 2 The Hartmann Hahn condition for this cross polarization is extremely sharp and must be adjusted very carefully for every spin rate 3 The magnetization transfer is substantially slower than from protons meaning that contact times are usually longer 4 The transfer occurs unlike CP from protons not out of a bath of abundant spins but be haves especially at high spin rates more like a transfer between spin pairs 5 Labeled samples must be used so that an observable number of coupled spins is present Advanced experimental schemes use tangential pulses to provide adiabatic conditions during the cross polarization S Hediger et al or provide only selective polarization transfer Specific CP Baldus et al References J Schaefer T A Skokut E O Stejskal R A McKay and J E Varner Proc Nat Acad Sci USA 78 5978 1981 J Schaefer E O Stejskal J R Garbow and R A McKay Quantitative Determination of the Concentrations of 13C 15N Chemical Bonds by Double Cross Polarization NMR J Magn Reson 59 150 156 1984 M Baldus A T Petkova J Herzfeld
53. dB The splitting of the two high field lines the protons in the CH gt are in equivalent in the solid state should be below the 50 level Fine Tuning for Minimum Carrier Spike 20 7 The tilt pulse p14 and its phase cnst25 determine the size of the carrier spike Optimise both parameters alternately for minimum spike and make sure the spike does not overlap with a resonance by choosing 01 appropriately N B changing 01 will lead to different values for cnst25 Correcting for Actual Spectral Width 20 8 Since the sampling rate is governed by the multi pulse sequence repetition rate the fore ground parameter swh has no real meaning Once all tuning procedures are done calculate the real spectral width swh according to the formula given in the parameter tables and run a new experiment After FT the spectrum should have an approximately correct spectral width Calibrate the middle position between the two CH peaks to 3 5 ppm the NH3 peak should then be at about 7 5 ppm Since the actual peak positions depend on the probe tuning you will have to recalibrate for your sample using one or more known chemical shifts If the peak sepa ration is incorrect change the status parameter swh by typing s swh and scaling it appropri ately Some pulse programs are written such that upon ased the approximately correct sweep width is shown and can be set as an acquisition parameter User Manual Version 001 BRUKER BIOSPIN 217 261 CRAMPS 1D
54. decoupling 2 2 3 2 93 re Figure 13 1 Pisema Pulse Sequence 13 2 132 261 BRUKER BIOSPIN User Manual Version 001 PISEMA Figure 13 1 Pisema pulse sequence a straight PISEMA b clean PISEMA variation for further suppression of phase glitches Ramamoorthy et al Solid State NMR 4 Setup 13 3 1 Determine HH match of the static sample and the correct 1H offset frequency using the pulse program cplg 2 Then measure the nutation frequency for 1H in order to calculate the FSLG conditions 3 For measuring the LGCP condition create a new experiment and set the cplg pulse pro gram into LGCP by setting the LG flag with ZGOPTNS set to Dlg 4 Two possibilities exist to set the FSLG power levels and offset frequencies a Either use the appropriate offset frequency for the chosen contact power level of 1H and set cnst20 accordingly to e g 50 kHz i e cnst20 50000 0 This would give an offset fre quency of approximately 35 kHz cnst22 should show this number in the ased display Then ad just the 15N power level during the FSLG period to best HH match which is at a power level of appropriately 20 log sin 54 7 1 8 dB higher than for the on resonance contact b If that option is not adequate because of power limitations on 15N one can also leave the on resonance contact levels of IDN and calculate the offset frequency and power level for 1H That would reduce the required power by about 70 as compared to t
55. dimension the so called SOQE param eter can be calculated du being given by equation 2 Eq 15 4 2 2 SOQE Q2 114 L A Q oi 3 i s with Eq 15 5 A1 21 1 Y f 3 41 7 1 3 f I equals 4 16 67 39 2 and 72 for I 3 2 5 2 7 2 and 9 2 respectively So one can see that for a given value of Q the second order quadrupole induced upfield shift du decreases as the spin increases With dgis always being negative this has a direct influence on the appear ance of the sheared 2D spectra Figure 15 10 shows 2D 170 3QMAS spectra at 11 7 T and 18 8 T where the Larmor frequency of this nucleus is 67 8 and 108 4 MHz respectively The sample is sodium metaphosphate NaPO in the glassy state The enrichment of 170 is approx 30 to 33 It contains 2 oxygen positions there are bridging oxygen P O P and non bridging oxygen P O Na User Manual Version 001 BRUKER BIOSPIN 165 261 Basic MQ MAS 166 261 RE EEN REN REN RRE 150 10 50 H ppm 140 120 100 80 60 ppm Figure 15 10 170 MQMAS of NaPO3 at 11 7 T 67 8 MHz on the left and 18 8 T 108 4 MHz on the right The red lines in the spectra indicate the isotropic chemical shift axis Approximate quadrupole parameters of the two sites are Qcc 7 7 MHz h 0 36 diso 125 ppm for the lower peak and Qcc 4 5 MHz h 0 16 diso 85 ppm for the upper peak sample courtesy of Alexan drine Flambard LCPS Univ de Lille The bridging oxy
56. et al or RAD Rf Assisted Diffusion see C R Morcombe et al The setup for all these sequences is rather robust requiring only the 1H to X Hartmann Hahn condition and the X 90 hard pulse to be set For RAD and DARR it is usually sufficient to cal culate an RF power level corresponding to n times the spin rate which is then applied during the mixing period Rotor synchronization of the mixing period is recommended in some cases where cross peaks due to sidebands need to be suppressed de Jong et al or where spin dif fusion is enhanced by matching the spin rate with the chemical shift difference between the sites to be correlated M Ernst et al PDSD is typically applied to high abundance nuclei or la beled materials to detect through space proximity between spins This experiment has been of ten used on proteins as an alternative to Radio Frequency DRiven spin diffusion see RFDR on page 93 RFDR provides similar information to PDSD but with a different mixing period Here the term frequency driven relates to recoupling pulses on the X channel whereas in DARR or RAD the radio frequency that drives the recoupling is the proton RF An important aspect of this experiment is that the mixing time is a simple delay and no pulse or only weak rf irradiation DARR RAD is required Therefore it can be made very long because no technical or experimental problems can arise So the effects even of small dipolar couplings User Manual V
57. first step a central transition selec tive pulse that merely excites the central transition must be calibrated This pulse must be weak enough so that only the central transition is affected and it must be short enough so that the central transitions of all sites in the spectral range are excited These conditions are typical ly fulfilled by a 20 us pulse For the calibration of this pulse a power level around 30 dB with 500 W and 1 kW amplifiers and around 20 dB with 300 W amplifiers should be expected The pulse program zg which uses p1 and pl1 or zgsel av which uses P3 and PL2T can be used For more details please refer to chapter 16 User Manual Version 001 BRUKER BIOSPIN 187 261 STMAS Once the central transition selective 90 pulse is calibrated the STMAS pulse program can be loaded Available pulse programs are stmasdgfz av and stmasdgfe av Both are double quan tum filtered 4 pulse sequences the first with a z filter the second with a shifted echo If this second sequence is to be used a proper setting of the timing for the shifted echo is required to allow collection of the full echo signal This is explained in chapter 17 where a shifted echo can be used in DFS enhanced MQMAS experiments In Table 17 3 and Table 17 4 the starting parameters for the setup of the two sequences are given Typical values for the pulses are entered so one should see some signal for further opti misation Parameters like O1 TD SWH RG should
58. line and check powmod by clicking the default button as described above The rf rout ing for this experiment is shown in figure 1 Next set p1 2 ms ns 8 or 16 The power level at which p1 is executed is pl Having high power transmitters it is important to be aware of the pulse power that is applied With TopSpin 2 0 and later ased shows pl and plw if the trans mitter has been linearized green dot in edasp and the transmitter power has been measured Set the power to about 100W For a non linearized transmitter p11 should be set to 10 in case of a 1000W transmitter and to 4 5 for a 300W or 500W transmitter You can also check durations and power levels in a graphical display by clicking the experiment button in the Pulprog window as it is shown in the figure below User Manual Version 001 BRUKER BIOSPIN 19 261 Basic Setup Procedures Proc ora Acqutors nl PulseProg Pecks Integrals Sangle Structure Fid Kal T Ise IC Bruker TOFSPIN pta vwelists pp single pulse e R BC Bruker TOPSPIN exp stan nmr lists pp seggt Avance II ver Fie Graphical assistant Edit text Options parameters epos ee pl excitatio Expression Y Names F Grid F Blocks p11 power le E Fl ZE 2u COMMENT singi 4CLA3323011ds DIM 1D TYPE direct e 16 SUBTYPE sinpi 14 0UNER Bruker schatll to ad acqt0 lutcnstl pl pli phi go 2 ph3l wr 0 exit phs 0213 ph3l 0 213 4 Ai Figur
59. magnetic field Bo increasing from left to right the y axis dyq increases from bottom to top Plot A is for identical sy plot B for identical quadrupole coupling and In plots C and D shift positions for two sites with large and small dis and large and small ogi and with large and small j and small and large ogi are plotted respectively User Manual Version 001 BRUKER BIOSPIN 163 261 Basic MQ MAS This behavior is independent of the spin quantum number and of the order p of the experiment Higher quantum order experiments are possible for half integer spin quantum numbers gt 3 2 however corresponding pulse programs are not provided in the pulse program library They can easily be derived from the 3Q pulse program by changing the phase cycle In the 3 pulse sequence mp3qzqf e g ph2 should be changed for the 5Q experiment to ph2 0 0 36 36 72 72 108 108 144 144 180 180 216 216 252 252 288 288 324 324 An 18 phase increment of the phase ph1 of the first pulse is required for States or States TPPI phase sensitive acquisition For a full phase cycle a multiple of 20 scans must be used For the 4 pulse sequence mp3qzfil the phases should be changed to ph1 0 36 72 108 144 180 216 252 288 324 ph2 0 40 90 40 180 40 270 40 ph3 0 ph4 0 10 90 10 180 10 270 10 receiver 0 180 5 90 270 5 180 0 5 270 90 5 180 0 5 270 90 5 0 180 5 90 270 5 Again an 18 phase increment of the first pulse for States or State
60. manual should only be used for their intended purpose as described in their respective manual Use of units for any purpose other than that for which they are intended is taken only at the users own risk and invalidates any and all manufacturer warranties Service or maintenance work on the units must be carried out by qualified personnel User Manual Version 001 BRUKER BIOSPIN 9 261 Introduction Only those persons schooled in the operation of the units should operate the units Read the appropriate user manuals before operating any of the units mentioned Pay particular attention to any safety related information Safety Issues 1 2 Please refer to the corresponding user manuals for any hardware mentioned in this manual for relevant safety information Contact for Additional Technical Assistance 1 3 For further technical assistance on the BPSU36 2 unit please do not hesitate to contact your nearest BRUKER dealer or contact us directly at BRUKER BioSpin GMBH am Silberstreifen D 76287 Rheinstetten Germany Phone 49 721 5161 0 FAX 49 721 5171 01 E mail service bruker de Internet www bruker de 10 261 BRUKER BIOSPIN User Manual Version 001 Test Samples Table 2 1 Setup Samples for Different NMR Sensitive Nuclei Nucleus Sample Method O1P Remarks 3H 1H Silicone paste THMAS 0 setup proton channel shim set field Silicone rubber THMAS 0 setup proton channel set fi
61. mp3qspam av sssssssss III III nh Ire herren 181 17STMAS 183 Table 17 1 Time deviation of the rotor period for spinning frequency variations of 1 and 10 Hz for various spinning frequencies eusssssessse 184 Table 17 2 Some Useful Samples for Some Nuclei with Half Integer Spin 187 Table 17 3 Initial Parameters for the Set up of stmasdfqZ av usuuusus 188 BRUKER BIOSPIN User Manual Version 001 Table 17 4 Table 17 5 Tables Initial Parameters for the Set up of stmasdfqe av nenen 189 F1 Parameters for the 2D Data Acquisition cccccceceeeeeeeee eens 190 Table 17 6 Processing Parameters for the 2DFT sssssssssss 191 Table 17 7 Values of R and cR pg for the Various Spin Quantum Numbers Obtained in the STMAS Experiment ssssssssssssss nennen nee 192 18Double CP 193 Table 18 1 Recommended Parameters for the DCP Setup sssssss 197 Table 18 2 Recommended Parameters for the DCP 2D Setup ooccoccoccccccnccncccnccnno 202 Table 18 3 Recommended Processing Parameters for the DCP 2D 203 19CRAMPS General 207 20CRAMPS 1D 211 Table 20 1 Table 20 2 Phases RF Levels Timings cccccccecceeceeeceeeceeceeeaeeseeeseeeeeeeaeees 211 PMLG Analog Mode eee eeeeneese nane nnn tendre nh dne nennen 215 Table 20 3 DUMBO Analog Mode 216 Table 20 4 Param
62. of spinning sidebands Ease of setup therefore depends largely on the availability of a setup sample with decent T4 efficient CP and known chemical shift for referencing Chapter 2 lists some useful setup samples together with known parameters Setup for Standard hetero nuclear Samples 15N 29SI 31P 5 6 58 261 IDN on a glycine calculate HH condition as described above Else Load a glycine 1 C reference spectrum set observe nucleus N15 in edasp add 2 dB to sp0 spnam0 ramp 100 subtract 2 dB from pl more is not required since the transmitter will usually put out 50 more power at 15N frequency set p15 3 ms BRUKER BIOSPIN User Manual Version 001 Practical CP MAS Spectroscopy on Spin 1 2 Nuclei acquire 4 8 scans optimize HH condition acquire reference spectrum with aq 25 35 ms 2 29Si on DSS load a glycine 13C reference spectrum set observe nucleus to Si in edasp add 2 dB to sp0 acquire 4 scans with aq 35 ms optimize HH condition acquire reference spectrum 3 P on ADP ammonium dihydrogen phosphate NH H5 PO load a glycine 13C reference spectrum set observe nucleus to P in edasp add 6 dB to pl1 optimize HH condition acquire 2 scans reduce rg appropriately User Manual Version 001 BRUKER BIOSPIN 59 261 Practical CP MAS Spectroscopy on Spin 1 2 Nuclei 60 261 BRUKER BIOSPIN User Manual Version 001 Basic CP MAS Experiments Introduction 6 1 The fo
63. other field strengths the num bers will be somewhat different If the signals are really undergoing mono exponential relax ation the curve should be a good fit to the measured data The Saturation Recovery Experiment 14 2 4 For samples where cross polarization is not possible the inversion recovery experiment would be very time consuming as the recycle delay d1 would need to be approximately 3x the lon gest T value For glycine at room temperature this would mean a delay of about 60s per scan in addition to the variable relaxation delay The saturation recovery experiment removes the need for long d7 by forcing the system into saturation at the beginning of each scan Sample Glycine Spinning speed 10 kHz Time 20 minutes Experiment setup Start from standard carbon CP parameters and set pulprog to satrect7 Set zgoptns to Ddec to turn on proton decoupling If decoupling is not required on a real sam ple this can be left blank to turn off the decoupling Set pl and pl1 to the measured carbon 90 degree pulse parameters as used in the CP T1 ex periment or see chapter Basic Setup Procedures Set d7 to a relatively long value for the preparation experiments Set the number of pulses in the saturation train 120 to zero and acquire a spectrum This will give an idea of the amount of signal and thus how many scans need to be acquired for each relaxation delay Create a new data set with jexpno and set the saturation
64. parameters 120 5 100 and d20 1 50 ms respectively Acquire a spectrum and verify that saturation is complete there should be no signal at all Setting up the 2D experiment Set the parameters for the 2D acquisition as detailed in Table 14 4 For the variable recovery delay the same values can be used as for the inversion recovery ex periment The recycle delay d7 can be very short but take care not to exceed the duty cycle limits high power pulsing should not exceed 5 of the total scan time In the case of decoupling the ac quisition time comprises most of the pulsing so d1 should be gt 20x aq 1 second is reason able in this case If the experiment is run without decoupling then the saturation period is the only significant period of high power pulsing and d1 can be shorter Acquire the 2D spectrum with zg User Manual Version 001 BRUKER BIOSPIN 147 261 Relaxation Measurements Table 14 4 Parameters for the Saturation Recovery Experiment Parameter Value Comments Parmode 2D Vdlist See text td f1 Number of entries in vd list FnMODE QF This is not a real 2D experiment NS 16 for the glycine sample More scans needed than for the CP experiment due to reduced signal Data Processing The saturation recovery data should be processed in the same way as the inversion recovery data above see Table 14 3 for parameters The only differences are that the fitting function sh
65. points in direct dimension WDW QSINE Apodization in t2 SSB 2 3 PH mod pk F1 indirect 13C PUDE SERIES Right column SI 512 Number of complex points in indirect dimension WDW QSINE Apodization in t1 SSB 2 3 PH mod pk Adjust the Rotational Resonance Condition for DARR RAD 10 4 1 Load the Adamantane sample spin at the same speed as desired for your sample match and tune use a suitable cp setup same as in section 10 3 2 Set CPDPRG2 to cw 3 Use the au program calcpowlev to calculate the power level required for a proton decou pling RF field of n x masr using p3 and pl12 as reference values Refer to chapter Basic Setup Procedures on page 13 for more information 104 261 BRUKER BIOSPIN User Manual Version 001 Proton Driven Spin Diffusion PDSD pectrum ProePars AcquPars Tite PubeProg Peaks integrais sample Structure Fidi Acqu m BND magie calculated ot for Pk 14 9AE utated pl for 135 khe 230 Sete oa culated valua Tos Figure 10 3 POPT Result for the cw Decoupling Power Variation The figure above shows the POPT result for the cw decoupling power variation from about 50 kHz RF field to about 5 kHz RF field spinning the adamantane sample at 13 kHz The minima at 14 5 and 20 5 dB indicate the n 2 and n 1 RR conditions 26 and 13 kHz RF field 4 Vary the decoupler power level p 12 used with cw decoupling as indicated in Figure 10 3 from a power level value p 12 1 dB below the
66. protons attached to the alpha car bon are in equivalent and strongly coupled The cross peaks at 3 and 4 ppm will show at a mix ing time as short as 50 usec the cross peaks to the NH3 protons at 9 ppm require 200 300 usec to show The mixing time here was 500 usec A sequence without carrier spike suppres sion was used here 228 261 BRUKER BIOSPIN User Manual Version 001 CRAMPS 2D m proton spin diffusion E Wide e wpmig2d with center spike suppression i 300 usec mixing N w a 12 10 8 6 4 2 F2 ppm Figure 22 3 Spectrum of Tyrosine hydrochloride The mixing time was 300 usec to show all connectivity Full plot to show that smaller sweep widths can be chosen when the carrier can be conveniently placed within the spectrum User Manual Version 001 BRUKER BIOSPIN 229 261 CRAMPS 2D re proion spin diffusion E hyr HC1 a womig2d with center spike suppression i 300 usec mixing 7 in wo Lo 3 8 7 6 5 4 F2 ppm Figure 22 4 Expansion of the Essential Part of the Spectrum Proton Proton DQ SQ Correlation 22 5 This experiment correlates proton shifts F2 with double quantum frequencies sum of shifts of the correlated sites Double quantum transitions are excited and reconverted by a post C7 or similar sequence 230 261 BRUKER BIOSPIN User Manual Version 001 CRAMPS 2D Pulse Sequence Diagram 22 6 Phases 11 12 22 doo 23 013 ae Pulses and delays taul 3 4 pll p
67. pulse at pl1 for the TOSS sequence 14 Set I5 for the gamma integral typically number of spinning sidebands normally 4 15 Start the experiment User Manual Version 001 BRUKER BIOSPIN 111 261 SUPER Spectrum PracPars AcquPars Title PulseProg Peaks integrals Sample Structure Fid Acqu o n S ev Experiment F2 FI Frequency axis bos Y Experiment Receiver Nucleus PULPROG cpsuper q le Current pulse program Durations AQ_mod DD Acquisition mode Power FnMODE IE Mo States TPP Acquisition mode tor 20 30 Program TD 2048 Size of fid Probe NS 256 Nurnber of scans Usts DS Number of dummy scans Kiel TOO H Loop count for tdD b Y wm Automation p Miscellaneous SWIppmi AESA 333 4823 Spectral width User SWH Hz 37593 984 Spectral width Routing IN 010 Increment for delay DO F1 ND_DI0 1 Number of delays in pulse program for DO F 1 AQ s 0 0272884 Acquisition time FIDRES Hz 18 356438 655 241943 Fid resolution FW Hz 625000 00 Filter width gt Recewer Y Nucleus 1 NUC1 130 130 Observe nucleus O1 Hz 12573 77 12573 77 Transmitter frequency offset OP ppm 100 000 100 000 Transmitter frequency offset SFD1 MHz 125 7502468 125 7502468 Transmitter frequency BF1 MHZ 125 7376730 125 7376730 Basic transmiter frequency Y Nucleus 2 NUC2 1H 2nd nucleus 02 Hz 1000 10 Frequency offset of 2nd nucleus O2P ppm 2 000 Frequency affset af 2nd nucle
68. setup and use of the Frequency Switched Lee Goldburg hetero nucle ar Correlation FSLG HETCOR experiment The FSLG Hetcor experiment correlates TH chemical shifts with X nuclei e g 13C 15N chem ical shifts The experiment provides excellent 1H resolution in the indirect dimension Homo nuclear decoupling in the 1H evolution period is achieved with an FSLG pulse train FSLG per mits relatively high spinning speeds and makes this experiment available for high field sys tems requiring high spinning speeds in order to move spinning sidebands out of the spectral region Decoupling the protons from the coupled X nucleus during evolution is not essential since the high spinning speed already achieves that One can however improve the hetero nu clear decoupling by a rr pulse in the middle of the evolution period see A Lesage et al Mixing is achieved during the cross polarization contact time Since magnetization transfer from protons to X e g 13C occurs rapidly contact times should be kept short in order to avoid long range transfer leading to unspecific cross peak patterns since the magnetization then has time to flow from any proton to any X nucleus A modification of the basic sequence uses cross polarization under a LG frequency offset for the protons In this case the proton magnetization detected by the X nucleus comes from close protons only since the proton spin lock at an LG offset interrupts the communication between the
69. sweep DFS Figure 16 3 shows the 4 pulse sequence with z filter mp3qdfsz av The principle of this sequence is already described in the chapter Basic MQ MAS on page 151 with a CW pulse instead of a DFS for conversion Figure 16 4 shows a 3 pulse sequence with a shifted echo acquisition in a split t4 experiment mp3qdfs av In both sequences the same sweep is used Both sequences start with an excitation pulse p1 that creates 3Q coherence which is allowed to evolve during the evolution period DO The sweep during P2 is used to change non observable 3Q coher ency to observable SQ coherency In the 4 pulse sequence they pass through a z filter by a se quence of CT selective 90 90 pulses P3 D4 P3 In the 3 pulse sequence a delay D6 is introduced so that the obtained signal can first dephase and then be refocused with a 180 CT selective pulse Optimization of the Double Frequency Sweep DFS 16 2 1 At this point it is assumed that the pulses p1 P3 and P4 together with their corresponding power levels PL11 and PL21 are already calibrated as described in the chapter Basic MQ MAS on page 151 Starting from this set up a data set should be created into which either of the two DFS pulse programs is loaded User Manual Version 001 BRUKER BIOSPIN 171 261 MQ MAS Sensitivity Enhancement hi ph2 sp1 spnam Figure 16 3 Four Pulse Sequence and Coherence Transfer Pathway for the 3Q MAS Experi ment Four pulse sequence and co
70. the 2D spectrum shown in Figure 15 10 BRUKER BIOSPIN User Manual Version 001 Basic MQ MAS 8 3 d d A j 4 d 3 E 3 4 y j lagen nene A m po E To wB w w ST Tase TE Sea Figure 15 11 Slices and Simulations of the 18 8 T 170 MQMAS of NaPO3 Fitted parameters are A Qcc 7 7 MHz h 0 36 diso 125 ppm and B Qcc 4 5 MHz h 0 16 diso 87 ppm for the upper peak sample courtesy of Alexandrine Flambard LCPS Univ de Lille Spectra that are sheared can be evaluated graphically as follows as shown in Figure 15 12 In addition to the red isotropic chemical shift axis indicated as axis CS with the slope A6 F2 A8 F1 1 there are two more lines drawn The blue axis indicated as axis Qis is the quadrupole induced shift axis with the slope A8 F2 A8 F1 17 10 This axis is identical for all different spins and all orders p of the MQMAS experiments This axis can be shifted re taining the same slope so that it intersects a spectral line in its centre of gravity Through the intersection point of the Qis axis with the CS axis a third line can be drawn parallel to the F2 ax is This is the dotted black line in Figure 15 12 The shift value that is read from the F1 axis at this position is the isotropic chemical shift of that particular site and the Qis is then given by Eq 15 2 User Manual Version 001 BRUKER BIOSPIN 167 261 Basic MQ MAS
71. the slot position in the AQS rack The lines connecting the software channel F1 to the actual frequency generation and amplification hardware can be drawn ad libitum as long as the required hardware connections are present The connections between transmitter and preamp cannot be routed arbitrarily because every transmitter output is hardwired to a preamplifier so the lines are drawn in green Please note that the nucleus in channel F1 is always the observe nucleus To set up for 79Br observation click on Default and the correct routing will be shown The green dot between SGU1 and amplifier 1 indicates that for this nucleus in this connection the transmitter has been calibrated for amplitude and phase linearity CORTAB For example if you select a nucleus where this has not been calibrated and the green dot is not visible the same power level setting in dB will produce gt 6 dB more power gt 4 fold power which may destroy your probe Calibrate power levels in such a case starting with 10 dB less power higher p n value to prevent destruction of your probe The connections between SGU n and transmitter n can be altered by clicking on either side of the connecting line removes the connection and clicking again on both units you want to connect route In case of high power transmitters you have two power stages which you se lect by clicking on the desired stage High power stages require the parameter powmod to be set to hig
72. these enhancement techniques are introduced Split t Experiments and Shifted Echo Acquisition 16 1 The excitation pulse in the MQMAS experiment creates 3Q coherence that can be refocused into an observable SQ echo by the conversion pulse As the t4 period is incremented in suc cessive slices of the 2D experiment this echo position relative to the conversion pulse changes as a function of the duration of the actual t4 delay If this observable SQ magnetization can be refocused again by a central transition selective 180 pulse a shifted echo acquisition can be implemented The delay between the conversion pulse and the 180 pulse or the delay prior to the start of the acquisition must be incremented proportional to t4 This results in a split t ex periment where the top of the shifted echo appears at a constant position after the final pulse throughout the entire 2D experiment The position of the echo top depends on the spin of the observed nucleus If the delay before the selective 180 pulse is long enough the signal will have decayed and the full build up and decay of the echo can be recorded By this method a phase modulated data set is acquired with a full echo that contains twice the intensity of the simple MQMAS experiment if transverse relaxation can be neglected At the end of the t4 pe riod of the split t4 experiment there is no net evolution under the second order quadrupole broadening This is the case because the evolution o
73. through limits Note the optimum power levels sp0 and pl and contact time p75 Setup of the Double CP Experiment 18 2 3 1 Read the reference carbon data set and generate a new data set using edc or iexpno 2 Select the pulse program doubcp1d Set the optimum 15N cp parameters as found in the previous step set sp0 p15 and p 3 for proton to 15N cp Set 03 close to the 15N peak po sition Now we have to select the appropriate parameters for the nitrogen to carbon magnetization transfer In the standard doubcp1d pulse program p16 is used as the second contact time and shapes sp1 and sp2 are specified for the 13C and 5N contact so p16 pl1 Ce power level and pl5 N power level are the relevant parameters The C N contact consists of a square pulse on one channel and a ramp or adiabatic shape on the other channel Setting the square shape on the 15N channel is preferred if the transmitter for 15N does not have ample power and is operated close to the power limits Table 18 1 Recommended Parameters for the DCP Setup Parameter Value Comments Pulse program doubcp1d AVIII Topspin 2 1 only else use doubcp doubcp av NUC1 36 Nucleus on f1 channel O1P 100 ppm 13C offset NUC2 1H Nucleus on f2 channel O2P 2 4 ppm 1H offset optimize NUC3 15N Nucleus on f3 channel O3P 35 glycine 15N offset depending on sample 120 histidine 65 130 protein PL1 Power level for f1 channel N
74. to be preferred 1 1 rotor period 1 2 rotor period 1 4 rotor period 1 8 rotor period 9 We A A ul e ae A E VER OA A eae eae ee 20 16 12 8 4 0 20 16 12 8 4 0 20 16 12 8 4 0 2 16 12 8 4 0 sp1 dB sp1 dB sp dB sp1 dB Figure 16 6 Signal Intensities of Rb in RBNO3 Signal intensities of Rb in RbNOs as function of duration and RF field amplitude for double frequency sweeps 2D Data Acquisition 16 2 2 176 261 After the parameters for the DFS are adjusted the 2D data acquisition can be prepared In ta bles 2 and 3 the important parameters are listed for the two pulse sequences Parameters are listed separately for F1 and for the pulse program relevant parameters which should be set in eda and ased respectively The 3 pulse sequence used in mp3qdfs av creates a phase modulated data set and therefore FnMODE must be QF However since a whole echo acquisition is performed a pure absorption mode spectrum can be obtained Increments for D10 and D11 must be set correctly so that a standard two dimensional FT can be applied Using the 4 pulse sequence mp3qdfsz av FnMode must be States or States TPPI so that the shearing FT can be performed for processing However no shearing is required in case of nu clei with spin I 3 2 where a split t experiment can be performed in which case IN10 must be set correctly For both sequences TD in F1 determines the number of FID s to be accumulated in the indi rect dimension This value i
75. while decoupling protons However observing a nucleus while decoupling it from like spins at the same time is obviously not trivial since the signal cannot be observed under the much higher decoupling RF Observation of the signal and decoupling pulses must therefore be alternately applied Suppression of a Homo nuclear dipolar interaction occurs when the magnetization vector of the coupled spins is rotated around the magic angle This condition can be achieved either by 4 n 2 pulses of suitable phase and spacing multiple pulse methods or by off reso nance irradiation of suitable offset and RF field Lee Goldburg To observe the signal a gap within the pulse sequence must be supplied which is long enough to observe one or several data points while the magnetization vector points along the magic angle This condition obvi ously persists only for a time period short compared to the transverse relaxation of the signal User Manual Version 001 BRUKER BIOSPIN 207 261 CRAMPS General To observe the time dependence of the signal the sequence must be repeated and more data points accumulated until the signal has decayed under the influence of residual broadening Obvious problems of this experiment are the requirement to observe a relatively weak signal shortly after a strong pulse dead time problem and the requirement to time the sequence in such a way that the magnetization vector is accurately aligned with the magic angle requires precise pu
76. 0 Figure 6 4 CPTOSS243 Experiment on Tyrosine HCI at 6 5 kHz Figure 6 4 is a CPTOSS243 experiment on tyrosine HCI at 6 5 kHz sample rotation using a 4 mm CPMAS triple resonance probe at 500 MHz with 243 accumulated transients No spinning sideband residuals can be observed with a noise level below 2 peak to peak compared to the highest peak intensity User Manual Version 001 BRUKER BIOSPIN 65 261 Basic CP MAS Experiments Sehe Manual chnpher2 10 4 jon 1708 Spectrum ProcPars AcquPars Title PulseProg Peaks integrais Sample Structure Fig Phase Callorate Basebne 120 Se 9p CPMAS Tyrosine HC Mast 6503 SELTICS 66 261 Figure 6 5 CPTOSS Experiment on Tyrosine HCI at 6 5 kHz Figure 6 5 is a CPTOSS experiment on tyrosine HCI at 6 5 kHz sample rotation using a 4 mm CPMAS triple resonance probe at 500 MHz with 256 accumulated transients Spinning side band residuals can be observed outside a noise level of approximately 296 peak to peak com pared to the highest intensity The residual sidebands have up to 5 intensity compared to the highest resonance 6 3 Like the TOSS experiment SELTICS Sideband ELimination by Temporary Interruption of the Chemical Shift is an experiment for spinning sideband suppression Pulses on the 1 C chan nel are driven with p111 and pulse times are rotor synchronized For optimum suppression the shortest pulse 1 24 of the sequences where qT is the rotor
77. 001 Basic CP MAS Experiments Sobds Manual chapter 12 1 Ca jos 1206 Spectrum ProcPars AcquPars Title PulseProg Peaks Integrals Sample Structure Fig c 4 v Al 4 Figure 6 11 Tyrosine C CPMAS NOS Experiment with TOSS Figure 6 11 is a tyrosine 13C CPMAS NQS experiment with TOSS using a dephasing delay d3 60 us Spinning sidebands are suppressed for a clean spectrum In this experiment the total dephasing time is 20 us shorter than that used for the CPNQS experiment on glycine in Figure 6 10 User Manual Version 001 BRUKER BIOSPIN 71 261 Basic CP MAS Experiments Spectral Editing Sequences CPPI CPPISPI and CPPIRCP 6 5 These spectral editing sequences help to distinguish CH CH2 CH3 and quaternary carbons in 10 spectra Common to all are various polarization and depolarization times which properly mixed and combined give a series of spectra which can be added and subtracted in order to obtain the various sub spectra All these sequences use constant amplitude CP which should be adjusted for maximum signal intensity For the CPP and CPPISPI sequences the only pa rameter needed in addition to the CP parameters is p16 for which a good starting value is 40 us to give null signal for CH negative signal for CH and positive signal for C and CH3 If necessary this value can be optimized on a sample similar to the sample of interest for better editing p17 for the repolarization step in the CPPIRCP experimen
78. 00E02 325 49 1 000000E02 283 92 1 000000E02 242 35 1 000000E02 200 78 END TITLE m3p JCAMP DX 5 00 Bruker JCAMP library DATA TYPE Shape Data ORIGIN Bruker BioSpin GmbH HHOWNER hf HDATE 2005 11 29 TIME 14 47 39 SHAPE_PARAMETERS MINX 1 000000E02 HMAXX 1 000000E02 MINY 1 125000E01 HMAXY 3 487500E02 SHAPE_EXMODE None H SHAPE_TOTROT 0 000000E00 H SHAPE_TYPE Excitation SHAPE_USER_DEF HSSHAPE_REPHFAC H SHAPE_BWFAC 0 000000E00 HSSHAPE_BWFAC50 5SHAPE_INTEGFAC 6 534954E 17 H SSHAPE_MODE 0 NPOINTS 6 THEXYPOINTS XY XY 1 000000E02 214 64 1 000000E02 283 92 1 000000E02 353 21 1 000000E02 173 2 1 000000E02 103 92 1 000000E02 34 64 END 21 3 Set up Fine tuning is done in the same way as with the original sequence except that the carrier is placed on a convenient position within the spectrum There is no need to minimise the carrier spike it should be all gone Somewhat higher power is required for the wpmlg shapes 222 261 BRUKER BIOSPIN User Manual Version 001 Parameter Settings for PMLG and DUMBO Modified W PMLG Table 21 2 PMLG Analog Mode Parameter Value Comment pulprog wpmlga2 Runs on AV 3 instruments only pli2 for 100 kHz RF field sp1 dto set cnst20 100 000 To be optimized during setup spnam1 m5m or m5p p1 2 5 usec As for 100
79. 01 Introduction This manual is intended to help users set up a variety of different experiments that are to date more or less standard in solid state NMR Previously the manuals described the hardware in some detail and also basic setup proce dures Armed with this knowledge it was assumed the users would be in a position to manage the setup of even complicated experiments themselves In this manual however the hardware is not discussed in detail since there is no longer much hardware which is specific to solid state NMR There are still transmitters with higher power and preamps and probes that take this power but for the purposes of experimental setup de tailed knowledge is not required since the setup does not generally depend on the details of the hardware So this manual is now much more specific to the type of experiment which is to be executed and includes tricks and hints required to set the experiment up properly for best performance If any special hardware or software knowledge is required it is indicated within the experimental section This manual begins with the most frequently used solid state NMR experiments and will be ex tended as time permits and as it is required by new development in NMR The manual is writ ten primarily for Bruker AVANCE III instruments but the experimental part will be identical or similar for AVANCE and AVANCE ll instruments For example pulse programs will have slightly different n
80. 150 180 210 240 270 300 330 ph2 0 ph3 0 12 90 12 180 12 270 12 receiver 0 270 180 90 3 180 90 0 270 3 Initial parameter values are listed in Figure 16 1 A few of these parameters need further ex planation D6 Is calculated as 1s L1 CNST31 P4 2 P2 2 This ensures that the delay from the cen tre of the sweep to the middle of the 180 pulse is an integer multiple of the rotor period L7 must be set so that D6 becomes long enough for a full echo to build up If we assume the FID has decayed after 3 ms spinning frequency is 25 kHz and the 90 pulse is 20 us then L1 should be between 70 and 80 so that D6 is between 2 77 and 3 17 ms P2 Is calculated as 1s CNST31 L0 The duration of the sweep can be adjusted depending on T of the sample The sweep should not be longer than one rotor period for many samples you may find that a quarter of a rotor period or even less is sufficient In ased the values of the two parameters D6 and P2 are greyed because they cannot be set anymore They are calculated in the pulse program from the parameters explained below which must be set accordingly LO Defines the fraction of a rotor period for the duration of the sweep P2 usually between 1 and 8 L1 Defines D6 to be an integer number of rotor periods The sweep will be defined by further parameters CNSTT1 Start frequency in kHz of the sweep the sweep should start slightly off resonance usually 30 to 50 kHz from the resona
81. 2 A 4 Pulse Basic Sequence with Z Filter Four pulse sequence and coherence transfer pathway for the 3Q MAS experiment with z filter mp3gzfil av p1 is the same p2 is usually somewhat shorter than in the three pulse se quence Corresponding power level pl11 should be set to achieve at least 150 kHz RF field am plitude p3 should be some tens of us corresponding to an RF field amplitude of a few kHz Delays dO and d4 are the incremented delay for t1 evolution and 20 us for z filter respectively BRUKER BIOSPIN User Manual Version 001 Basic MQ MAS Delay d10 initially is O and can be incremented proportional to dO in10 in0 7 9 if the observe nucleus has spin I 3 2 Phase lists are as follows for phase sensitive detection in F1 the phase of the first pulse must be incremented by 30 in States or States TPPI mode ph1 0 60 120 180 240 300 ph2 0 24 90 24 180 24 270 24 ph3 0 ph4 0 6 90 6 180 6 270 6 receiver 0 180 3 90 270 3 180 0 3 270 90 3 180 0 3 270 90 3 0 180 3 90 270 3 Of course the sequence with more pulses has slightly inferior sensitivity however it is the ba sic sequence to improve sensitivity by FAM or DFS The 3 pulse sequence itself can be used directly to enhance sensitivity by soft pulse added mixing pulse program mp3qspam av In MQ MAS Sensitivity Enhancement on page 169 some of the sensitivity enhancement techniques will be described Note that pulse programs suitable for AV and
82. 2 7 Processing parameters as above Parameter Value Comment F1 acquisition 6 left column SI 2 4 k Number of points and zero fill WDW QSINE Sine bell squared SSB 2 5 Shifted sine bell PH_mod pk Phase correction if needed F2 indirect 13C right column SI 256 1024 Zero fill MC2 STATES TPPI WDW QSINE Sine bell squared SSB 2 5 90 shifted sine bell NS A uc 8 Ka N E e e P Li i i E Le s Ka T 30 u D DH a 120 T sc 160 Figure 12 6 PC7 2d SQ SQ correlation on tyrosine HCl PC7 2d SQ SQ correlation on tyrosine HCI 56 rotor periods mixing at 13 kHz 2 5 mm probe AV III 700 SB Left 84 rotor periods DQ mixing right 56 rotor periods mixing The 84 periods mixing time show relayed correlations positive blue which are absent at 56 periods mixing except for the SSB cross peaks Direct correlations are negative red 130 261 BRUKER BIOSPIN User Manual Version 001 PISEMA Introduction 13 1 Polarization Inversion Spin Exchange at the Magic Angle is an experiment that correlates the chemical shift of a spin 1 2 X nucleus with the hetero nuclear dipolar coupling to another spin 1 2 nucleus Most ofthe applications so far reported have been in the field of structural biology therefore the X nucleus is normally 13C or 15N and the other heteronucleus 1H The experi ment provides orientation info
83. 5 Se A AVANCE 7 L C 5080905 6 02 class AVANCE 64 261 Figure 6 3 Comparison of a CPTOSS and CPMAS Experiment Figure 6 3 compares a CPTOSS experiment lower spectrum to a CPMAS experiment upper spectrum on tyrosine HCI at 6 kHz sample rotation using a 4 mm CPMAS double resonance probe at 500 MHz with 16 accumulated transients The sequence is not perfectly compensated for experimental artifacts and if perfect suppres sion of SSB is required one can use a 5 pulse sequence with a long phase cycle requiring a minimum of 243 transients for complete artifact suppression using the pulse program cptoss243 where the extension av is added in case of the AV2 console Figure 6 4 shows the advantage of the well compensated TOSS sequence with its 243 phase cycle steps over the above 4 pulse sequence Besides the better compensation the cptoss243 pulse sequence is also shorter and uses only 1 instead of 2 rotor cycles This pulse program can be used with fairly high spinning speeds up to about 12 5 kHz sample rotation depending of course on the width of the employed rt pulses Figure 6 5 shows for a comparison the results obtained with the 4 pulse sequence with 256 scans BRUKER BIOSPIN User Manual Version 001 Basic CP MAS Experiments Salis Momani chapter 5 Spectrum ProcPars AcquPars gel PulseProg Peaks integra Sample Structure Fio Fnase Casprate Baseline E 4130 Setup CPMAS Tyrosine HC JMasr S50
84. 5 usec For 100 kHz RF field p8 1 2usec p14 0 7 usec To be optimized cnst25 140 To be optimized p9 4 2 6 usec To be optimized p10 32 usec or 24 usec Set by xau dumbo d1 4s For a glycine 111 anavpt 4 2 4 8 16 or 32 o1p 5 To be optimized swh 1e 2 2 p9 p10 0 5 To be corrected for proper scaling rg 16 64 td 512 Up to 1024 si 4k digmod analog MASR 10 12 kHz Depending on cycle time 216 261 BRUKER BIOSPIN User Manual Version 001 CRAMPS 1D Fine Tuning for Best Resolution 20 6 For fine tuning the following parameters are important P9 sets the width of the observe window The shorter it is the better the resolution However the natural limit is the size of the sampling period and the dead time of the probe Preamp and receiver play no significant role in the total dead time A CP MAS probe usually has a fairly nar row bandwidth long dead time so p9 3ysec is only possible at frequencies 400 and higher With 111 4 8 p9 can be chosen shorter for better resolution but at the cost of S N Sampling more data points during d9 111 0 1 usec with larger values of 111 will increase S N slightly but requires more time within the window may require a longer p9 and therefore degrade resolu tion Since the decoupling bandwidths are not very large 01 should be close to resonance espe cially for DUMBO For PMLG this is less critical The power level for the shapes should be ad justed in steps of 0 2
85. 64 Number of points NDO 1 INO L3 2 p6 SF or Scaling factor for PISEMA 0 82 sin 54 7 deg calcu L3 2 5 p6 SF lated by pulse program 136 261 BRUKER BIOSPIN User Manual Version 001 PISEMA Processing 13 5 Process the direct dimension with xf2 Accommodate for the cos modulated signal by setting thh imaginary part to zero using the au program zeroim by typing into the command line zeroim 3 Process the indirect dimension with the command xf1 For more automated processing one can write a short macro using the command edmac and the filename 2dft for examples write the following commands using the text editor xf2 zeroim xf1 Save and close the edmac editor 6 In future you can do then the processing by simply typing 2dft into the command line or even creating your own icon in TopSpin for this purpose Table 13 2 Processing Parameters Parameter Value Comment F2 acquisition 1H ee Left column SI 1k Number of complex points in direct dimension WDW no Apodization in t2 F1 indirect IDN ME Right column SI 128 Number of complex points in indirect dimension MC2 QF User Manual Version 001 BRUKER BIOSPIN 137 261 PISEMA B Figure 13 4 A PISEMA Spectrum of 15N Labeled Acetylated Valine B FID in t1 over 3 008 ms 64 Data Points 138 261 BRUKER BIOSPIN User Manual Version 001 PISEMA Spectrum ProcPars AcguPars Tie PulseProg Peso
86. 7 2 Four pulse sequence and coherence transfer pathway for the double quantum fil tered STMAS experiment with z filter stmasdgfz av Pulses p1 and P2 are non selective pulses The corresponding power level PL11 should be set to achieve around 100 kHz RF field amplitude P3 and P4 are CT selective pulse 90 and 180 pulses of about 20 and 40 ys respectively corresponding to an RF field amplitude of a few kHz Delays DO and D4 are the incremented delay for t evolution and 20 us for z filter respec tively Phase lists are as follows for phase sensitive detection in F1 the phase of the first pulse must be incremented by 90 in States or States TPPI mode ph1 02 ph2 0022 ph3 0000111122223333 User Manual Version 001 BRUKER BIOSPIN 185 261 STMAS 186 261 ph4 0 8 1 8 2 8 3 8 4 8 receiver 02202002022020021331311313313113 20020220200202203113133131131331 In the stmasdgfz av pulse sequence this pulse flips the magnetization back along the z axis After a short z filter delay D4 a CT selective 90 pulse P3 creates transverse magnetization In the stmasdgfe av pulse sequence the non selective pulse P2 converts the ST SQ coherence into CT SQ coherence This is allowed to evolve for another delay D6 after which it refocuses into an echo by a CT selective 180 pulse When either the D6 before the 180 pulse or D7 af ter the 180 pulse is incremented proportionally to the t4 period a split t experiment as de scribed in chapters
87. A Lesage P Hodgkinson and L Emsley Homo nuclear dipolar decoupling in solid state NMR using continuous phase modulation Chem Phys Lett 319 253 260 2000 Lyndon Emsley s home page http www ens lyon fr STIM NMR NMR html User Manual Version 001 BRUKER BIOSPIN 53 261 Decoupling Techniques Be Reference 54 261 Transverse Dephasing Optimized Spectroscopy Decoupling optimized under refocused conditions 3 heteronuclear decoupling 2 4 Prec Figure 4 6 Pulse Program for Hahn Echo Sequence Transverse Dephasing Optimized spectroscopy G De Paepe et al 2003 uses a spin echo sequence for optimizing hetero nuclear decoupling The idea behind it is simply the removal of the normally dominant Jo term describing coherent residual line broadening effects in the transverse relaxation rate R A Abragam chapter 8 With the normal CP experiment the ob served line broadening coherence decay time Ts might be caused by other heterogeneous effects such as distribution of chemical shifts or susceptibility effects and not reflect the true Ta coherence lifetime The true Ta achieved through good hetero nuclear decoupling can then be observed with a hahn echo experiment Optimization is done by looking for the maxi mum signal amplitude of the decoupled resonances of interest Be careful not to exceed the maximum decoupling time with high power decoupling G De Paepe N Giraud A Lesage P Hodgkinson A B ckmann a
88. Apart from some smaller differences the sequences are in complete analogy to the HETCOR se quence using frequency shifts The only differences between these sequences lie in the length and type of shape used for Homo nuclear decoupling DUMBO and e DUMBO Emsley et al use principles known from multiple pulse NMR operating on resonance whereas pmlg and w pmlg Vega et al use phase ramps which act like frequency offsets and are therefore derivatives of FSLG References D Sakellariou A Lesage P Hodgkinson and L Emsley Homo nuclear dipolar decoupling in solid state NMR using continuous phase modulation Chem Phys Lett 319 253 2000 Vinogradov E Madhu P K Vega S High resolution proton solid state NMR spectroscopy by phase modulated Lee Goldburg experiment Chem Phys Lett 1999 314 5 6 443 450 E Vinogradov P K Madhu and S Vega Proton Spectroscopy in Solid State NMR with Win dowed Phase Modulated Lee Goldburg Decoupling Sequences Chem Phys Lett 2002 354 193 Leskes Michal Madhu P K Vega Shimon A broad banded z rotation windowed phase modulated Lee Goldburg pulse sequence for 1H spectroscopy in solid state NMR Chem Phys Lett 2007 447 370 374 Leskes Michal Madhu P K Vega Shimon Supercycled Homo nuclear decoupling in solid state NMR towards cleaner 1H spectrum and higher spinning rates J Chem Phys 2007 in press The Sequence pmighet 8 2 1 This sequence uses win
89. Bruker BioSpin in a al Ge S 3 M 4 56 e Solid State NMR AVANCE Solids User Manual Version 001 NMR Spectroscopy think forward The information in this manual may be altered without notice BRUKER BIOSPIN accepts no responsibility for actions taken as a result of use of this manual BRUKER BIOSPIN accepts no liability for any mistakes contained in the manual leading to coincidental damage whether during installation or operation of the instrument Unauthorized reproduction of manual contents without written permission from the publishers or translation into another language either in full or in part is forbidden This manual was written by Hans Foerster Jochem Struppe Stefan Steuernagel Fabien Aussenacc Francesca Benivelli and Peter Gierth Desktop Published by Stanley J Niles November 25 2008 Bruker Biospin GmbH Rheinstetten Germany P N Z31848 DWG Nr Z4D10641 001 For further technical assistance on Solid State NMR please do not hesitate to contact your nearest BRUKER dealer or contact us directly at BRUKER BioSpin GMBH am Silberstreifen D 76237 Rheinstetten Germany Phone 49 721 5161 0 FAX 49 721 5171 01 E mail service bruker de Internet www bruker com Contents 3 1 3 2 3 3 3 4 3 5 3 6 3 7 3 8 3 9 4 1 4 2 5 1 5 2 5 3 eet 3 IntFOOdUCctloll ic a NA iR AAA A 9 Disclaimer c 9 Salety ET 10 Contact for Additional Technical Assistance 10
90. C contact pulse PL3 Power level for IDN channel HN contact PL5 Power level for IDN channel NC contact pulse PL12 Power level decoupling f2 channel and excitation pl13 power level during second contact cw dec cnst24 offset for cw decoupling during p16 P3 Excitation pulse f2 channel PCPD2 Decoupler pulse length f2 channel iH TPPM P15 3 5 msec Contact pulse first contact User Manual Version 001 BRUKER BIOSPIN 197 261 Double CP Table 18 1 Recommended Parameters for the DCP Setup P16 5 12 msec Contact pulse second contact f1 f3 channel D1 5 10s histidine Recycle delay 4s a glycine SPNAMO Ramp for 18 cp step e g ramp 80 100 sp0 Power level for Ramp HN contact pulse 1H spnam1 ramp45 55 Tangential contact pulse ten5500 tcn5500 or on C or square square 100 spnam2 square 100 or Square on N or ramp tangential pulse ramp45 55 tcn5500 CPDPRG2 SPINAL64 SPINAL64 decoupling NS 2 4 or 16 Number of scans 198 261 4 Now we select the shape to use for the C N contact To find the HH contact more rapidly it is a very narrow condition it is recommended to start with a ramp shape In order to find a HH condition independent of the type of shape it is recommended to select shapes which are all centred around 5096 amplitude which allows arbitrary amplitude modulation without changing the HH condition For a start generate a ramp shape from 45 to 55 with 100 sli
91. Cnst24 pro vides an additional overall offset to compensate for phase glitch With proper probe tuning and 500 match cnst24 should be close to zero 7 Set acquisition and processing parameters according to Table 4 1 and Table 4 2 A spectrum like in fig 4 should be obtained If the splitting is worse optimize with p 13 and cnst24 Usually somewhat less power than calculated is required The FSLG decoupling scheme is also implemented as cpd program cwlgs The include file Ig calc incl is also required With cpdprg2 cwlgs the standard cp pulse program can be used The ZGOPTN Dlacq ased should be set in order to allow decoupling times gt 50 ms Table 4 1 Acquisition Parameters Parameter Value Comments pulprog falg AV3 use falg av for AV1 2 d1 4s Recycle delay ns 4 16 Number of scans aq 80 ms Acquisition time spnamO ramp 100 or For ramped CP ramp70100 100 pl12 p3 set for p3 90 Sp pl1 set for cp p15 5 10m pl13 set for 70 100 kHz Optimize for best resolution cnst20 70000 100000 Equals the applied RF field cnst24 0 To be optimized cnst21 0 Reset proton frequency to SFO2 User Manual Version 001 BRUKER BIOSPIN 51 261 Decoupling Techniques Table 4 2 Processing Parameters Parameter Value Comment Sl 2 td Adequate 4fold zero filling WDW no No apodization PH_mod pk Phase correction if needed BC_mod quad DC offset correction
92. Data Acquisition ccc cecc cece cece cece ee cece eee eee s 153 Setting Up the Experiment cccceceecceeeeeeeeeeee tesa eeeeeeeeeaeeeaees 153 Two Dimensional Data Acquisition nme 158 15 4 Data processing uinci icto Eid kind Gee 160 15 5 Obtaining Information from Spectra ccooncconcccnncccnnccnnncinnccinnccinncnnnno 163 16 MQ MAS Sensitivity Enhancement 169 16 1 Split t1 Experiments and Shifted Echo Acquisition 169 16 2 Implementation of DFS into MQMAS experiments 171 Optimization of the Double Frequency Sweep DFS 171 2D Data Acquisition 176 Data Processing teer te as eg 178 User Manual Version 001 BRUKER BIOSPIN 5 Contents 16 3 16 4 17 17 1 17 2 17 3 17 4 17 5 18 18 1 18 2 18 3 18 4 18 5 19 19 1 19 2 19 3 19 4 20 20 1 20 2 20 3 20 4 20 5 20 6 20 7 20 8 20 9 20 10 21 21 1 21 2 21 3 21 4 21 5 Fast Amplitude Modulation FAM 2 c ccecceeeceeeeeeeeeeeeeseeaeeees 180 Soft Pulse Added Mixing SPAM sss 180 RR EE 183 Introduction tem 183 Experimental Particularities and Prerequisites sss 183 Pulse Sequences onr esee na nie A AA AAE ENE NALAN AEE 185 Data Acquisition sss een 187 Set up of the Experiment sssssssssssss 187 Two Dimensional Data Acquisition
93. E P energie 36 User Manual Version 001 BRUKER BIOSPIN 259 261 Index 260 261 BRUKER BIOSPIN User Manual Version 001 End of Document User Manual Version 001 BRUKER BIOSPIN 261 261 Bruker BioSpin your solution partner Bruker BioSpin provides a world class market leading range of analysis solutions for your life and materials science needs Our solution oriented approach enables us to work closely with you to further establish your specific needs and determine the relevant solution package fromour comprehensive range or even collaborate with you on new developments Our ongoing efforts and considerable investment in research and development illustrates our long term commitment to technological innovation on behalf of our customers With more than 40 years of experi ence meeting the professional scientific sector s needs across a range of disciplines Bruker BioSpin has built an enviable rapport with the scientific community and various specialist fields through understanding specific demand and providing attentive and responsive service Bruker BioSpin Group info bruker biospin com www bruker biospin com Bruker BioSpin Z31848
94. F field 3 CYCLOPS 0123 User Manual Version 001 BRUKER BIOSPIN 221 261 Modified W PMLG Pulse Shapes for W PMLG PMLG shapes m3p m3m m5m m5p All these shapes perform similarly M3p and m3m use 6 phases m5p and m5m use 10 phases to generate the phase ramp Obviously 6 phases generate a phase ramp with less resolution but shorter possible duration With the timing resolution available on AV instruments there is no need to prefer the coarse phase ramp The letters m and p refer to the sense of phase rota tion which is opposite between m and p If probe tuning is not perfect m or p may give different results depending on the carrier position The overall added phases of 0 and 180 degrees on consecutive shape pulses are set by the phase program phase list ph10 TITLE m5p JCAMP DX 5 00 Bruker JCAMP library DATA TYPE Shape Data ORIGIN Bruker BioSpin GmbH HHOWNER hf DATE 2005 11 29 TIME 14 47 39 SHAPE_PARAMETERS MINX 1 000000E02 HMAXX 1 000000E02 MINY 1 125000E01 HMAXY 3 487500E02 SHAPE_EXMODE None SHAPE_TOTROT 0 000000E00 SHAPE_TYPE Excitation H SHAPE_USER_DEF SHAPE_REPHFAC H SHAPE_BWFAC 0 000000E00 HSSHAPE_BWFAC50 SHAPE_INTEGFAC 6 534954E 17 SHAPE_MODE 0 NPOINTS 10 THEXYPOINTS XY XY 1 000000E02 20 78 1 000000E02 62 35 1 000000E02 103 92 1 000000E02 145 49 1 000000E02 187 06 1 000000E02 7 06 1 0000
95. G TS2 1 only 3 pmig sp1 ph3 f2 for one full PMLG unit as for Igs 1 shape lo to 3 times count References A Bielecki A C Kolbert and M H Levitt Frequency Switched Pulse Sequences Homo nucle ar Decoupling and Dilute Spin NMR in Solids Chem Phys Lett 155 341 346 1989 A Bielecki A C Kolbert H J M deGroot R G Griffin and MH Levitt Frequency Switched Lee Goldburg Sequences in Solids Advances in Magnetic Resonance 14 111 124 1990 E Vinogradov P K Madhu and S Vega High resolution proton solid state NMR spectroscopy by phase modulated Lee Goldburg experiments Chem Phys Lett 314 443 450 1999 and references cited therein DUMBO DUMBO Decoupling Uses Mind Boggling Optimization is a phase modulation scheme where the phase modulation is described in terms of a Fourier series x p t KS cos no 1 b sin no f n The shape can be created using the AU program DUMBO The DUMBO shape file in the re lease version of TOPSPIN is calculated for 32us pulses To create your own DUMBO shape you can also use the au program dumbo See instructions in the header of the au program for proper use The above pulse program to observe J couplings with DUMBO decoupling would be written as follows define loop counter count calculate number of LG periods according to aq count aq p10 3 p10 sp1 ph3 f2 p10 set by AU program DUMBO n 32 usec lo to 3 times count References D Sakellariou
96. Homo nuclear radio frequency driven recoupling in rotating solids J Chem Phys 108 9463 9479 1998 B Heise J Leppert O Ohlenschl ger M G rlach and R Ramachandran Chemical shift cor relation via RFDR elimination of resonance offset effects J Biomol NMR 24 237 243 2002 User Manual Version 001 BRUKER BIOSPIN 93 261 RFDR Experiment 9 1 Spinal64 Lee Goldburg Spinal64 decoupling decoupling decoupling t N tp t 471 4 0101 1010 bo 71 de 0303 0303 1010 1010 3 0123 0321 40 0 rec 0220 0220 1331 1331 Figure 9 1 RFDR Pulse Sequence for 2D CPMAS Exchange Experiment Set up 9 2 Sample 1C fully labelled histidine Experiment time Less than 1 hour Experiment Setup First setup the 1H 13C cross polarization and the Hartmann Hahn match according to the pro cedures described in Basic Setup Procedures on page 13 An important experimental consideration of the RFDR experiment is that the r f field strengths used in the recoupling channel pI11 and the r f field on the 1H decoupling channel must be sufficiently different ca a factor of 3 to avoid rapid de polarization of the carbon signal during the mixing time This is usually not achievable so it should be set as high as possible using a LG offset During the mixing period cpds3zcwlg as shown in Figure 9 1 the Gullion compensated echo sequence used for the mixing period is a XY 8 phase cycling f4 XYXY
97. LG 500 3 2 24000 12000 84 3 110 E LG 600 2 5 35000 14000 100 2 5 130 I LG 800 SPC5 5 7 6000 5000 25 10 70 3 5 300 4 15000 13000 65 3 85 100 2 5 LG 500 3 2 24000 17000 85 3 none 700 2 5 35000 20000 100 2 5 none 900 SC14 3 5 7 6000 6000 21 12 701 3 5 200 4 15000 15000 52 5 4 75 100 2 5 LG 500 3 2 24000 22000 7713 25 none 700 2 5 35000 28000 100 2 5 none 950 1 C7 is not recommended due to restricted excitation bandwidth 2 SPC5 can be recommended as a standard sequence for 4mm probes and not too high fields 3 SC14 or sequences with similar RF field requirements are recommended for small spinners high fields 4 Maximum speed results from max possible RF field 5 Maximum C RF fields taken from C RF field specification or 1H RF field specification 122 261 considering the requirement of an off HH condition Maximum RF field for decoupling LG means cw decoupling with optimized LG offset frequency at the given RF field in order to avoid HH contact Maximum magnetic field as proton resonance frequency in MHz This results from spin rate requirements for 13C observation to avoid rotary resonance conditions as well as excita tion bandwidth considerations BRUKER BIOSPIN User Manual Version 001 Symmetry Based Recoupling Spectrometer Setup for 13C 12 2 1 1 Load a CPMAS parameter set for 1 C 2 Load a uniformly labeled glycine sample and rotate at the desired rotation rate see ta
98. LG Condition nennen nennen 49 Figure 4 3 FSLG Decoupling Pulse Sequence Diagram ssseese 50 User Manual Version 001 BRUKER BIOSPIN 243 261 Figures 244 261 Figure 4 4 Adamantane FSLG Decoupled The C H J Couplings Shown 50 Figure 4 5 Shape with Phase Gradients nennen nme nenn 52 Figure 4 6 Pulse Program for Hahn Echo Sequence sess 54 5 Practical CP MAS Spectroscopy on Spin 1 2 Nuclei 55 6 Basic CP MAS Experiments 61 Figure 6 1 Pulse Program for CP with Flip back Pulse seseeeeess 61 Figure 6 2 Pulse Program for CPTOSS sssssssesss iania ia NEE 63 Figure 6 3 Comparison of a CPTOSS and CPMAS Experiment 64 Figure 6 4 CPTOSS243 Experiment on Tyrosine HCl at 6 5 KHZ ou eee eee 65 Figure 6 5 CPTOSS Experiment on Tyrosine HCl at 6 5 KHZ ee 66 Figure 6 6 Pulse Program for SELTICS sssssss Hee 67 Figure 6 7 SELTICS at 6 5 kHz Sample Rotation on Tyrosine HCl 67 Figure 6 8 Cholesterylacetate Spectrum Using Sideband Suppression 68 Figure 6 9 Block Diagram of the Non quaternary Suppression Experiment 69 Figure 6 10 Glycine 13C CPMAS NOS Experiment with a Dephasing Delay 70 Figure 6 11 Tyrosine 13C CPMAS NOS Experiment with TOSS sess 71 Figure 6 12 Block Diagram of the CPPI Experiment 72 Figure 6 13 CPMAS Spectr
99. Leet Solvent ww Experiment Use current param Y TEILE ext j Receivers LZ 8 OK Carcel More Info Help Figure 3 3 Pop up Window for a New Experiment Spin the KBr sample moderately 5 2 5mm 10 kHz In order to set up the experiment type ased in the command line to open the table with parameters used for this experiment 18 261 BRUKER BIOSPIN User Manual Version 001 kg 1 1 Joplmewtopspin pkiesis n Basic Setup Procedures 2 Spectrum ProcPars AcquPars Title PulseProg Peaks integrats Sample Structure Fid Acqu Phase Print CARVEVA installed probe 4 mm MASOVT BB 1H H8724 0020 General V General pons ow PULPROG onepulse fe Pulse program for acquisition TD 4096 Time domain size NS 4 Number of scans DS o Number of dummy scans SWH Hz 100000 00 Sweep width in Hz AQ s 0 0205300 Acquisition time RG 64 Receiver gain DW us 5 000 Dweil time DE lus 450 Pre scan delay CNST11 1 0000000 To adjust t 0 for acquisition if digmod base D1 5 0 20000000 Recycle delay Y Channelfi E NUC1 738r fese Nucleus for channel 1 P1 us 2 00 E Excitation pulse length PL1 dB 524 Power level for excitation pulse PLIW W 100 02651215 Power level for excitation pulse SFO MHz 150 3690213 Frequency of observe channel Figure 3 4 ased Table with Acquisition Parameters for the KBr Experiment Then check rf routing by clicking on the Edit button in figure 4 or by typing edasp in the com mand
100. P spectrum If this cannot be achieved even with careful HH matching the following parameters should be checked 8 Re optimize p16 the optimum should be gt 10 msec If the signal gets worse with longer contact time there is a loss due to direct 19C 1H contact Minimise this loss in the following way 9 Never use a pulsed proton decoupling schemes during p16 Frequency shifted Lee Gold burg decoupling is no alternative since the signal will broaden and decay with shorter T5 Use cw decoupling during p76 and carefully optimize the decoupling power pI13 for max imum signal A slight offset may be set using cnst24 10 Instead of a 45 55 ramp a tangential amplitude modulation shape can be used Since this shape provides 100 transfer efficiency on a spin pair system compared to 50 of a stan dard rectangle or ramp shape the DCP efficiency can be increased With such a shape one can get 50 70 DCP efficiency To generate such a shape in stdisp select TanAmp Mod as a shape model select solids notation select 1000 points set the spin rate to half the actual spin rate 5500 set the RF field to the actual RF field used on this channel se lect 400 for the dipolar coupling and 50 for the scaling factor Save the shape as tcn5500 if not already available and select this name for spnam1 or spnam2 The efficiency should be noticeably better Re optimize the first HH contact decoupling and p16 More than 50 should definitely be
101. Solid State NMR of Polymers pp 83 122 Elsevier Science Publisher 1998 4 Matthias Ernst Arno P M Kentgens and Beat H Meier Pure Phase 2D Exchange NMR Spectra un der MAS Journal of Magnetic Resonance 138 66 73 1999 and references cited therein 5 K Takegoshi Shinji Nakamura and TakehikoTerao 13C 1H dipolar assisted rotational resonance in magic angle spinning NMR Chem Phys Lett 2001 344 631 6 F Castellani B van Rossum A Diehl M Schubert K Rehbein and H Oschkinat Structure of a pro tein determined by solid state magic angle spinning NMR spectroscopy Nature 420 98 102 2002 7 C R Morcombe V Gapenenko R A Byrd and K W Zilm Diluting Abundant Spins by Isotope Edited Radio Frequency Field Assisted Diffusion J Am Chem Soc 126 7196 7197 2004 8 S G Zech A G Wand and A E McDermott Protein Structure Determination by High Resolution Solid State NMR Spectroscopy Application to Microcrystalline Ubiquitin J Am Chem Soc 127 8618 8626 2005 9 W Luo X Yao and M Hong Large Structure Rearrangement of Colicin la channel Domain after Mem brane Binding from 2D 136 Spin Diffusion NMR J Am Chem Soc 127 6402 6408 2005 10 A Lange S Luca and M Baldus Structural constraints from proton mediated rare spin correlation spectroscopy J Amer Chem Soc 124 9704 9705 2002 BRUKER BIOSPIN User Manual Version 001 Proton Driven Spin Diffusion PDSD Pulse Sequence D
102. TICS se quence at 5 Hz sample rotation upper spectrum The lower spectrum is the CPMAS spectrum at 5 kHz sample rotation 68 261 BRUKER BIOSPIN User Manual Version 001 Basic CP MAS Experiments Non Quaternary Suppression NQS 6 4 The NQS experiment is a simple spectral editing experiment It relies on the fast dephasing of rare spins coupled to 1H spins through the hetero nuclear dipolar interaction For the dephas ing delay d3 one uses between 30 and 80 us gt 93 heteronuclear decoupling Figure 6 9 Block Diagram of the Non quaternary Suppression Experiment The non quatemary suppression experiment is also called the dipolar dephasing experi ment Use glycine or tyrosine spinning at 11 kHz as before Table 6 3 Acquisition Parameters Parameter Value Comments pulprog cpnqs cptoss_nqs p2 180 pulse on X nucleus pl11 Power level driving P2 on X channel d3 30 80 us Dephasing delay User Manual Version 001 BRUKER BIOSPIN 69 261 Basic CP MAS Experiments CP 3 kHz sample rotation dipolar dephasing pi wat WEG E el Eo ee ee IER Ee et 240 220 200 180 160 140 120 100 80 60 40 20 0 20 ppm Figure 6 10 Glycine 13C CPMAS NQS Experiment with a Dephasing Delay Figure 6 10 is a glycine 130 CPMAS NOS experiment with a dephasing delayd3 40 us so that the total dephasing time is 80 us Spinning sidebands are still visible 70 261 BRUKER BIOSPIN User Manual Version
103. TMAS Spectra of RONOS uuuusssunssssnnnnnnsnnnnnnnnnnnnnnnn nn 190 18Double CP 193 Figure 18 1 Pulse sequence diagram for 1D t120 and 2D double CP experiments aa A AEAEE AE A AAEE AEAEE dere 194 Figure 18 2 The edasp routing tables for H C N double CP eee 195 Figure 18 3 Routing table for triple resonance setup change for 15N pulse parameter measurement and CPMAS optimization 196 Figure 18 4 Shape Tool display with ramp shape from 45 to 55 198 Figure 18 5 Shape Tool display with a tangential shape for adiabatic cross polarization EEUU 199 Figure 18 6 Double CP optimization of PL5 in increments of 0 1 dB 200 Figure 18 7 Double CP yield measured by comparing CPMAS and DCP amplitudes of the high field resonance ssssss Hem 201 Figure 18 8 C N correlation via Double CP in histidine simple setup sample 4mm Tri ple H C N Probe III II 204 Figure 18 9 NCaCx correlation experiment with 22 ms DARR mixing period for Ca Cx spin diffusion on GB1 protein run using an EFREE Probe 205 Figure 18 10 NCaCx correlation experiment with 4 2 ms SPC5 DQ mixing period for CaCx spin diffusion on GB1 protein run using an EFREE Probe at 14 kHz sample rotation and 100 kHz decoupling ssssses 206 19CRAMPS General 207 Figure 19 1 Difference in Amplitude of the Quadrature Channels X and Y 209 20CRAMPS 1D 211 Figure 20
104. Test Samples ans 11 Basic Setup Procedures KEREN SEENEN Ne 13 General Remarks erste EE 14 Setting the Magic Angle on KBr sssseeHH 15 Klee le Lu WEE 15 Setting Acquisition Parameters sss 17 Calibrating 1H Pulses on Adamantane sse 23 Calibrating 13C Pulses on Adamantane and Shimming the Probe 31 Calibrating Chemical Shifts on Adamantane sese 32 Setting Up for Cross Polarization on Adamantane s 33 Cross Polarization Setup and Optimization for a Real Solid Glycine 35 Some Practical Hints for CPMAS Spectroscopy sssssssess 41 Eitetalure ca a e tere eee 43 Decoupling Techniques eee eee eere eren 45 Hetero nuclear Decoupling ssss nennen namen nessa nenn 45 CW eropgeet nen feces tees ee dida 45 TPPEM Decoupling 1 Aint aiid eee aed liessen 46 SPINAL Decou pling u ee ei ik 47 RT Oe BEE 47 Pi Pulse Decouplitg u a ETHER 47 Homo nuclear Decoupling 444 444 snnnenn nenn He 47 Multiple Pulse NMR Observing Chemical Shifts of Homo nuclear Cou pled Nuclel EE 47 Multiple Pulse Decoupling nen 48 BR 24 MREV 8 BLEW 12 sssssssee em 48 ESEG Decoupling 3 2 ete en 48 DUMBO vico een 53 Transverse Dephasing Optimized Spectroscopy 54 Practical CP MAS Spectroscopy on Spin 1 2 Nuclei 55 Introductio
105. YXYX Conse quently the number of rotor periods for the mixing time LT must be a multiple of 8 94 261 BRUKER BIOSPIN User Manual Version 001 Data Acquisition Sample C fully labelled histidine Experiment time Several hours Set up 2D Experiment RFDR 9 3 9 3 1 After 1D parameter optimization as previously described type iexpno to create a new data file and switch to the 2D mode using the 123 button Set the appropriate FnMode parameter in eda Pulse program parameters are indicated below Figure 9 1 shows the pulse sequence ocPars AcquPars Title Pu tHe V d Figure 9 2 The 123 Icon in the Menu Bar of the Data Windows Acquisition Parameter Page The 123 icon in the menu bar of the data windows acquisition parameter page is used to tog gle to the different data acquisition modes 1D 2D and 3D if so desired is used to toggle to the different data acquisition modes 1D 2D and 3D if so desired Table 9 1 Acquisition Parameters Parameter Value Comments pulse program cprfdr av Pulse program nuc1 16 Nucleus on f1 channel olp 100 ppm 13C offset to be optimized nuc2 1H Nucleus on f2 channel 02p 2 3 ppm 1H offset to be optimized pit Power level for contact time on f1 channel pitt Power level for f1 recoupling and excitation DI Power level for contact time on f2 channel pl12 Power level decoupling f2 channel and ex
106. about the information obtained from such spectra Table 17 7 Values of R and R p for the Various Spin Quantum Numbers Obtained in the ST MAS Experiment Spin R R p p 1 3 2 8 9 1 889 5 2 7 24 0 70833 7 2 28 45 0 3777 9 2 55 72 0 263111 192 261 BRUKER BIOSPIN User Manual Version 001 Double CP Double Cross Polarization DCP experiments use two consecutive cross polarization steps Usually the first step transfers from protons to one type of X nucleus to achieve high sensitiv ity the second step transfers to a different Y nucleus in order to probe the dipolar coupling between X and Y The sequence of transfers is in principle arbitrary but usually sensitivity is an issue so transfer from protons to generate a large magnetization and detection on the nucle us of higher sensitivity to gain signal intensity is the standard procedure Detection of the most sensitive nucleus protons is also possible but is difficult if the Homo nuclear proton pro ton dipolar coupling is strong see CRAMPS General on page 207 In this chapter the most popular double CP experiment is described Here the first CP step transfers magnetization from protons to 15N Then in a second cross polarization step magne tization is transferred from 15N to 13C the signal is finally detected on 13C under suitable pro ton decoupling The purpose of this experiment is to gain information about the C N dipolar
107. accumu lated the question is how many anti echos to acquire this depends on the sample In amor phous or disordered materials the FID decays rapidly and so does the anti echo In such a case 4 to 8 anti echos may be sufficient In the case of crystalline materials it takes many more t4 increments before the anti echos decay Hence the number of anti echos should be of the order of half the number of echos It is always better to acquire more anti echos than are really needed because then you can be sure that you acquire a 2D spectrum with a reliable 2D ab sorption line shape Never risk gaining sensitivity or saving experimental time at the expense of quality of lineshapes II6 the value of this loop counter is needed to set the phases of the soft pulses correctly and define what is an echo and what is an anti echo which are different for spin 3 2 and all the other spin quantum numbers Processing of these spectra is done in analogy to spectra obtained with mp3gzqf av However phase correction in the acquisition dimension F2 cannot be determined on the first FID There fore xf2 must be applied first and then F2 phase correction can be determined on either the first slice in case of nuclei with spin 3 2 or the second slice for all other nuclei 2D process ing is then done with the AU program xfshear Alternatively xfshear can be used first with a subsequent 2D interactive phase correction BRUKER BIOSPIN User Manual Version 001
108. addition is the X rr pulse p2 at power level pl At fast spin rates and in fully labeled samples the narrowing effect on the proton spectrum may be small or not noticeable but on samples with natural abundance it may be noticeable At long contact times and transfer from many different protons the line width in the proton spectrum may also be insensitive In fig 1 two columns through the most up field aliphatic peak in tyrosine HCl are shown The rr decoupled trace red is clearly narrower eRe tt Hh EGE 25 595 ee BaAr J column 3069 from tyrosin 5 1 CoBrukenTOPSPIN Ignetfapi tyrorin 999 TCN Erer TOPSPIN ihift 0 1559 ppa 77 5 Ezi tyrorin S 100 C Braker TOPSPIN jobs esl 1 15 rel 10 L 2 T T T T r T r T T r r T T d D 6 4 2 ppm Figure 8 1 Comparison of HETCOR with and without 13C decoupling The figure above shows a comparison of HETCOR with and without C decoupling Natural abundance tyrosine HCl was run with 50 usec contact time Reference A Lesage and L Emsley Through Bond hetero nuclear Single Quantum Correlation Spec troscopy in Solid State NMR and Comparison to Other Through Bond and Through Space Ex periments J Magn Res 148 449 454 2001 84 261 BRUKER BIOSPIN User Manual Version 001 HETCOR with DUMBO PMLG or w PMLG Using Shapes HETCOR with DUMBO PMLG or w PMLG Using Shapes 8 2 These sequences use phase modulated shapes for Homo nuclear proton decoupling
109. ame nucleus as for F2 so that transmitter fre quency offset is correctly set important for referencing D10 0 Used in mp3gzfil av only IN10 in0 7 9 Used in mp3qZfil av for nuclei with spin I 3 2 only so that no shearing FT is required 158 261 1 Note the difference in increment handling in Topspin 2 1 and higher BRUKER BIOSPIN User Manual Version 001 Basic MQ MAS Some further comments and explanations on the parameters listed above FnMode must be States or States TPPI so that the shearing FT can be performed for process ing If the pulse program mp3qzfil avis used no shearing is required in case of nuclei with spin I 3 2 if in10 is set correctly in which case a split t experiment is performed td determines the number of FID s to be accumulated in the indirect dimension This value is determined by the line width and resolution that can be expected in the indirect MQ dimension F1 and which de pend on the properties of the sample In crystalline material fairly narrow peaks can be expect ed so a maximum acquisition time in F1 of 2 to 5 ms is expected In disordered materials where the line width is broader and determined by chemical shift distribution a total acquisition time in F1 of 1 ms may be sufficient The total acquisition time aq in F1 equals td 2 in_010 For rotor synchronized experiments in_010 1 spinning frequency so will typically be between 100 us 10 kHz spinning and 28 5 us 35 kHz spinn
110. ames differing usually in the pulse program name extension Contact your nearest applications scientist if you do not find the experiment pulse program that you are look ing for Users of older instruments DSX DMX DRX should refer to the Solids Users Manual delivered within the Help system at Help gt Other topics gt Solids Users Manual Even though the pulse programs may look similar they will not run on these instruments The first five chapters deal with basic setup procedures subsequent chapters are dedicated to specific types of experiments There may be many different sub experiments within a given type since the same information can often be obtained with pulse sequences differing by sub units only or in using a totally different principle The experiments outlined here are usually the most important ones and or the ones that were common at the time when the manual was writ ten New chapters will be added as the manual consists of largely self contained units rather than being a comprehensive single volume This structure was adopted in order to be more flexible in updating replacing individual chapters So do not be surprised if some chapters are still missing they will be completed in the near future and implemented as they are finished and proofread The individual chapters are written by different people so there will be some differ ences in style and composition Disclaimer 1 1 Any hardware units mentioned in this
111. and cnst20 62500 an offset frequency of 44 2 kHz for the LG condition is calcu lated and consequently an inO of 42 86 us which is already corrected for the scaling factor of 0 82 Data Acquisition 13 4 Sample 1N labeled glycine for power level determination and N labeled acetylated glycine or acetylated valine or leucine for running the PISEMA experiment Experiment time 15h User Manual Version 001 BRUKER BIOSPIN 135 261 PISEMA Table 13 1 Acquisition Parameters Parameter Value Comments PULPROG Pisema pisema Pulse programs clean NUC1 15N SW Reasonable SW in F2 O1P 90 160 ppm For 15N labeled acetylated glycine NUC2 1H O2P to be optimized For 15N labeled acetylated glycine PL1 For 15N contact PL11 Or 15N evolution PL2 For 1H contact and excitation PL12 For 1H hetero nuclear decoupling during t2 PL13 For 1H Evolution under FLSG condition P3 1H excitation pulse P15 15N 1H Contact pulse P6 1H LG 294 degree pulse D3 1 4 us For frequency amp phase setting D X only cnst20 1H spin nutation frequency achieved with PL 13 cnst21 0 Offset from 02 in Hz cnst22 LG frequency in Hz calculated cnst23 LG frequency in Hz calculated L3 1 3 Loop counter for appropriate t1 increment F2 acquisition 15N DIMUS left column AQ MOD qsim TD 512 No of points DW Dwell time in t2 F1 indirect 1H RRE right column TD
112. ar defines the axis in the MQ dimension such that Eq 15 2 10 Ovo S i zu 17 The value of Aus is given by Eq 15 3 De s ar t 1 3 0 ue ii aiQ1 1 In Eg 15 3 is the spin quantum number Q the quadrupolar coupling constant wo the Lar mor frequency and h the asymmetry parameter This makes mo H ec which causes the MQ positions to be field dependent An interesting behavior results as one compares spectra at dif ferent fields Plots of the function yg over ox are shown in Figure 15 9 for an arbitrary sam ple with two sites If the isotropic chemical shifts of the two sites are identical then it is obvious that the separation of the two lines increases as the field decreases plot A In the opposite case of identical quadrupole couplings separation increases as the field is increased plot B In cases where a difference in isotropic chemical shift dj exists and the sites have different quadrupole couplings the relative positions depend on which site has the larger quadrupole coupling The separation of the lines will always increase as the field decreases plots C and D but in some cases a crossover of the shift positions may be observed as the field Bg is al tered plot C D Figure 15 9 Calculated Shift Positions yg Calculated shift positions yg as function of the static magnetic field Bg for two different sites with arbitrary jsy and gjs The x axis in each plot is the static
113. arbon signals will be derived only from directly bonded protons and thus any differ ences in proton relaxation within a molecule could be isolated Such indirect observation can be implemented conveniently for both T4 and T4 relaxation For T4 a proton saturation recovery step is inserted prior to the cross polarization step in a stan dard CP sequence The proton magnetization immediately prior to CP and thus the observed carbon signal depends on the extent of recovery after the saturation so the carbon signal as a function of recovery delay gives the proton T4 value For Tip a variable length proton only spin lock pulse is applied after the 90 degree pulse in the CP experiment The proton magnetization after this pulse and thus the carbon signal after CP depends on the proton Tip relaxation User Manual Version 001 BRUKER BIOSPIN 149 261 Relaxation Measurements Indirect Proton T1 Measurements 14 3 1 Sample Glycine Spinning speed 10 kHz Time 20 minutes Start from standard CP parameters as for the X T4 measurement with CP and set pulprog to cph 1 Set the saturation loop 120 to zero and acquire a spectrum to check signal intensity Signal to noise should be comparable with the standard CP experiment so a similar number of scans to that used for the carbon T experiment should suffice Saturation parameters can be set as for carbon saturation previously 120 5 100 and d20 1 50 ms Acquire a spectrum with these para
114. ared The spectrum on the left was obtained after the first execution of the experiment The spectrum on the right was obtained after several iterations of resetting the angle and rerunning the spec trum From this it is obvious that the spectrum of the sample for the setup must be known be cause otherwise it is impossible to judge whether a shoulder or a splitting is due to an incorrectly set angle or another signal from another site in the sample Figure 17 4 87Rb STMAS Spectra of RbNO3 190 261 BRUKER BIOSPIN User Manual Version 001 STMAS While the left spectrum has been obtained after adjusting the magic angle with KBr the right spectrum can be obtained after several iterations of readjusting the angle and rerunning the 2Dspectrum Data Processing 17 5 Processing parameters should be set according to the table below Table 17 6 Processing Parameters for the 2DFT Parameter Value Comments F2 acquisition dimension SI Usually set to one times zero filling WDW No Don t use window function PH_mod Pk Apply phase correction BC_mod No No DC correction is required after full phase cycle ABSF1 1000 ppm Should be outside the observed spectral width ABSF2 1000 ppm Should be outside the observed spectral width STSR 0 Avoid strip FT STSI 0 Avoid strip FT TDoff 0 Avoid left shifts or right shifts before FT F1 indirect dimension
115. asic Setup Procedures The figure above is an Adamantane 13C FID with 50 msec aq with setsh display showing no shim values N b spinning removes part of the By in homogeneities Probes which do not use susceptibility compensated coil wire can show much shorter T5 and require much more shim ming effort only with older probes up to 400 MHz proton frequency seda Fatz Aca Par 10 PuneProg Pesa meget Saroe Sruwe 1 g Amu rate ame CG rr EF ap gs 987 Patriot ung en degen Set shim values and lock parameters Press ENTER to set new value Setsnm FIT Ka Close Figure 3 19 Adamantane 13C FID with 50 msec aq setsh with Optimized Z Shim Value For optimum shims rarely required set the shims x y 25 xy and x y as well as xz and yz You may need to increase the acquisition time aq to see the effect of increasing resolution Safe the shims using the command wsh followed by a suitable name Note For long acquisition times aq gt 0 05 s the decoupling power level p 72 must be set to 3 dB and d1 must be increased to 6s To allow longer acquisition times than 50 msec the ZGoption Dlacq must be set in ased if the pulse program contains the include file aq prot incl Make sure the Dlacq option is not left set for the following steps Calibrating Chemical Shifts on Adamantane 3 5 32 261 In TOPSPIN as well as in the XWIN NMR 3 5 release the frequency list for NMR nuclei fol lows the IUPAC recommend
116. asr Equals spinning frequency for rotor synchronization from this inO is calculated correctly ND 010 must be set first NUC1 Select the same nucleus as for F2 so that transmitter frequency offset is set the same in both dimensions essential for refer encing Pulse program In ased parameters D10 0 IN10 INO 7 9 For spin I 3 2 so that no shearing FT is required 0 For all other spin I Data Processing 16 2 3 Processing parameters should be set according to Table 16 4 Data obtained with mp3qdfs av can be processed with xfb alone if INTO or IN11 have been set appropriately to run a split t experiment Since a whole echo is accumulated FT along F2 from A gt t gt A is done which ne cessitates a large 1 order phase correction to compensate for the start of the acquisition be fore the echo top This correction can easily be calculated as given in Eq 16 1 and should be stored into the parameter PHC1 This gives an approximate value which can be precisely ad justed in the interactive phase correction routine The phase of the spectrum must be corrected such that there is no signal in the imaginary part as described above 178 261 BRUKER BIOSPIN User Manual Version 001 MQ MAS Sensitivity Enhancement Table 16 4 Processing Parameters Parameter Value Comments F2 acquisition dimension Sl Usually set to one times zero filling WDW no Don t use win
117. at stores the chemical shift information along the z axis and marks the beginning of the mixing time The X spins communicate through chemical exchange or spin diffusion depending on the properties of the material and the duration of the mixing time At the end of the mixing period another X 90 read pulse and the data acquisition follow The usually strong 1H X dipolar in teraction is removed by high power 1H decoupling during the preparation and the acquisition time The H decoupling is switched off during the mixing to dephase the residual X transverse magnetization Spin diffusion between X nuclei is usually very slow and requires very long mix ing times since the dipolar coupling between all X nuclei is small However turning the proton decoupling field off during the mix period allows another process to take place spin diffusion via the proton spin system Since the rare nuclei are strongly coupled to protons and all pro tons are strongly coupled to each other the flip flop transition rate is high along the X4 H H gt X pathway In fact the spin exchange is almost solely due to proton mediated mechanisms except when chemical exchange is present At high spin rates spin diffusion may however still be slow since the X H spins are decoupled A simple procedure to recouple the X H interaction is to irradiate the protons at an RF field of n times the spin rate These modified sequences are DARR Dipolar Assisted Rotational Resonance T Terao
118. ation of the rotational echo is 1 us it will be missed when the de viation is larger which is the case for a 1 Hz deviation at 10 kHz but requires a fluctuation of 10 Hz at 30 kHz spinning Table 17 1 Time deviation of the rotor period for spinning frequency variations of 1 and 10 Hz for various spinning frequencies Fluctuation of ae Ha Deviation from precise rotor Deviation from precise rotor period desired spinning period after 1 rotor period after 100 rotor periods frequency 10 Hz 30 kHz 11 ns 1 1 us 1 Hz 30 kHz 1 1 ns 110 ns 10 Hz 20 kHz 25 ns 2 5 us 1 Hz 20 kHz 2 5 ns 250 ns 10 Hz 10 kHz 100 ns 10 us 1 Hz 10 kHz 10 ns 1 us 184 261 Typically the spinning frequency must be stable within 1 Hz throughout the entire 2D data ac quisition Secondly the accuracy of the magic angle setting is extremely important The side bands resulting from the first order broadening are narrowed from the full first order line width by a factor of 3cos 0 1 hence for a deviation of d from the magic angle the broadening is 3cos sindd9 which close to the magic angle is v2d4 The magnitude of the interaction that must be narrowed in the present case is in the order of MHz so even a small deviation causes BRUKER BIOSPIN User Manual Version 001 STMAS a severe broadening in the STMAS spectrum This can easily be understood as the rotational echoes decay much more rapidly when the magic an
119. ations see R K Harris E D Becker S M Cabral de Menezes R Goodfellow and P Granger NMR Nomenclature Nuclear Spin Properties and conventions for Chemical Shifts Pure Appl Chem Vol 73 1795 1818 2001 for reference Set the 19C low field signal of adamantane to 38 48 ppm This will set the parameter SR which is used to calculate the chemical shift axis and the peak positions in the spectrum Note All data sets generated from this data set will have the peak positions correctly calibrated if the magnetic field By is not changed However you must make sure that the magnetic field is always the same It may change if the magnet has a slight drift or if different shim settings are loaded Therefore the same shim file should be loaded and the field be set to the same value using the BSMSDISP command If the magnet drift is noticeable the calibration should be redone in suitable intervals and the field value re corded in the lab notebook BRUKER BIOSPIN User Manual Version 001 Basic Setup Procedures One can also use a spinner filled with H20 to set the field position more precisely Do not spin the sample and make sure the cap is well fitted Set ofp to 4 85 ppm set for proton observe as described above for adamantane and use gs for continuous pulsing and FID display Change the field value in bsmsdisp until the FID is exactly on resonance Then all spectra tak en should be correctly referenced with sr 0 For all these expe
120. basic double CP experiment can be extended into many different variations One ex ample is the double transfer N C Cg where the second transfer step is made selective so only a carbons are polarized from the nitrogen then magnetization is transferred from the a carbons to the adjacent B carbons This can be done by a simple PDSD or DARR proton spin diffusion step or by a 13C 13C Homo nuclear recoupling step HORROR DREAM or other Likewise the N C C experiment transfers from the a carbons to all X carbons which are in close enough proximity to the a carbons Check with your applications support for appropriate pulse programs 2D Data Acquisition 18 3 Sample 15N 13 labeled histidine peptide or protein Spinning speed 10 15 kHz depending on 13C spectral parameters rotational resonance must be avoided Experiment time 30 min several hours Acquisition Parameters Table 18 2 Recommended Parameters for the DCP 2D Setup Parameter Value Comments Pulse program doubcp2d Pulse program NUC1 13C Nucleus on f1 channel O1P 100 ppm 13C offset NUC2 1H Nucleus on f2 channel O2P 2 4 ppm 1H offset optimize NUC3 15N Nucleus on f3 channel O3P 65 150 ppm 15N offset depending on sample PL1 Power level for f1 channel NC contact pulse PL3 Power level for IDN channel HN contact PL5 Power level for IDN channel NC contact pulse PL12 Power level decoupling f2 channel and exc
121. ble 1 depending on the recoupling experiment planned and the sample under investigation Con sider possible rotational resonance conditions in the sample of interest 3 Tune and match the probe optimize the 13C and 1H pulse parameters for excitation and de coupling 4 Use the cp90 pulse program with p 11 pl to measure the nutation frequency for 13C in order to calculate the recoupling conditions see chapter Basic Setup Procedures Cal culate the power levels required by the spin speed see table 1 using calcpowlev 5 Set pl11 back to 120 dB p1 to zero and run 1 experiment with 16 4 scans as a reference Setup for the Recoupling Experiment 12 2 2 1 Create a new experiment and load the appropriate pulse program spc5cp1d use the same routing Set the appropriate sample rotation rate as required for step 14 set cnst31 equal to the rotation rate 2 Load the power level calculated for the necessary C recoupling B field into p 11 set p1 as determined in step 4 3 Set 0 15 should be but need not be a multiple of 5 for SPC5 or of 7 for PC7 SC14 This determines the DQ build up time DQ generation The reconversion time is usually also controlled by 0 it may however be written such as to be independently controlled by a dif ferent loop counter For glycine about 5msec will be the optimum 4 Set the decoupling program cpdprg1 to cwlg Set pl13 such as to yield the desired decou pler RF field during t
122. bragam A Abragam Principles of nuclear magnetism Oxford Clarendon Press 1961 but simpler descriptions can be found in the books of Slichter and Levitt C P Slichter Principles of magnetic resonance Springer 1996 gd ed M H Levitt Spin dynamics Basics of nuclear magnetic resonance Wiley 2001 Some discussion of T4 relaxation including effects of dipolar coupling to proton spins can be found in D L VanderHart and A N Garroway 130 NMR rotating frame relaxation in a solid with strongly coupled protons polyethylene J Chem Phys 71 2773 2787 1979 Details of the X T4 experiment with CP are in DA Torchia The measurement of proton enhanced 13C T4 values by a method which sup presses artifacts J Magn Reson 30 613 616 1978 The TOPSPIN software includes a tool for processing the data obtained in relaxation measure ments and this will be demonstrated for the different types of relaxation experiment Describing Relaxation 14 1 Relaxation of the net magnetization can be described in terms of two processes After a pulse the state of the system differs from the equilibrium in two ways the z magnetization is not equal to the equilibrium value and the net magnetization in the transverse plane is non zero The return of the z magnetization to equilibrium is termed longitudinal relaxation or spin lattice relaxation and the return of transverse magnetization to zero is termed transverse or spin spin relaxation Bo
123. c mod no Automatic baseline correction 96 261 BRUKER BIOSPIN User Manual Version 001 RFDR Figure 9 3 C Histidine Signal Decay as a Function of the RFDR Mixing Time ppa 20 40 0 30 100 120 140 160 130 160 160 140 120 100 80 60 40 20 ppm Figure 9 4 2D RFDR Spectrum of 13C fully Labelled Histidine RFDR mixing time 1 85 ms User Manual Version 001 BRUKER BIOSPIN 97 261 RFDR 98 261 BRUKER BIOSPIN User Manual Version 001 Proton Driven Spin 1 0 Diffusion PDSD PDSD is a 2D experiment that correlates a spin 1 2 nucleus to another spin of the same spe cies via Homo nuclear dipolar coupling or chemical exchange The experiment resembles the NOESY Nuclear Overhauser Effect SpectroscopY pulse sequence in the liquid state by re placing the initial 90 pulse with a cross polarization scheme Since spin diffusion between X nuclei is measured cross peak intensities depend on the probability of interaction between dif ferent sites which is low with low natural abundance of NMR active nuclei Therefore these experiments usually require enrichment for nuclei like 13 or N in order to allow sensible measurement times The pulse program cpspindiff allows to run several types of PDSD experiments The CP prep aration period excites the X nuclei During the evolution time the X magnetization evolves un der the effect of the chemical shift interaction The evolution time ends with an X 90 pulse th
124. c7cp2dnoe Any sequence may be used make sure to use the correct timing NUC1 6 Nucleus on f1 channel O1P 100 ppm 13C offset NUC2 1H Nucleus on f2 channel O2P 2 3 ppm 1H offset PL1 for gt 50 kHz vge Power level for f1 channel CP and p1 PL11 dep on masr Power level for f1 channel recoupling power PL12 as specified Power level decoupling f2 channel and excitation PL13 p112 optimize or Power level decoupling f2 channel during cw or cwlg decoupling 120 fast spinning P3 Excitation pulse f2 channel PCPD2 Decoupler pulse length f2 channel iH TPPM P15 Contact pulse D1 Recycle delay CNST20 Spin nutation frequency at PL13 for cwlg decoupling LO for 0 5 10msec Use multiples of 5 7 or 16 spc5 pc7 sc14 for full phase cycle mix in ased SPNAMO Ramp for 13 CP step e g ramp 80 100 SPO Power level for proton contact pulse CPDPRG2 SPINAL64 SPINAL64 decoupling CPDPRG1 cwlg To avoid HH contacts during DQ generation reconversion NS 4 32 Number of scans see pulse program phase cycle F2 direct 13C left column TD 1024 or 2048 Number of complex points SW Sweep width direct dimension adjust to experimental require ments F1 indirect 13C right column TD 128 512 Number of experiments in indirect dimension SW usually sw F2 Sweep width indirect dimension NDO 1 STATES TPPI not required in TS 2 1 User Manual Version 001 BRUKER BIOSPIN 129 261 Symmetry Based Recoupling Spectral Processing 1
125. cal shifts in solids Combined multiple pulse NMR and magic angle spinning J Chem Phys 72 vol 1 1980 Homo nuclear Dipolar Interactions 19 1 Multiple Homo nuclear dipolar interactions among spins with a strong magnetig moment and high natu ral abundance mainly 1H or 19F and to a much smaller extent P are usually very large un less averaged by high mobility Especially in the case of protons spin exchange is usually rapid compared to routinely achievable rotation periods meaning that MAS alone cannot sup press the Homo nuclear dipolar broadening Even spin rates in the order of 70 kHz which is no longer a mechanical problem cannot fully average this interaction in rigid solids As chemical shift differences among the coupled nuclei become larger the interaction becomes more heter ogeneous and MAS can suppress it more efficiently This is the reason why fast spinning alone works much better on 9F or 31P than on protons hetero nuclear dipolar coupling such as be tween 13C and 1H can in principle be spun out but only H the Homo nuclear coupling between protons is small or averaged by motion or a suitable pulse sequence CRAMPS sequences therefore play an important role also in experiments where X nuclei are observed Pulse Sequences 19 2 Dealing with a hetero nuclear dipolar coupling is easy continuous high power irradiation of one coupling partner will decouple it from the other nucleus as in the case of 13C observation
126. calculated n 1 condition to 1 dB above the calculated n 2 condition Bandwidth considerations favor the n 2 condition sample heat ing considerations favor the n 1 condition An RF field of 2 x proton chemical shift range is on the safe side 5 Enter the power level determined above as pI14 recoupling power for DARR or RAD 6 Using DARR or RAD shorter mixing times are possible User Manual Version 001 BRUKER BIOSPIN 105 261 Proton Driven Spin Diffusion PDSD Example Spectra 10 5 Ld BE E a a ee amp 5 D a re Le a o o e a L la pe L lt sop t ay esa 3 F Sun a e i ro a gt 8 H 160 140 120 100 80 60 F2 ppm l N IN jl ses e 5 TE ES LS a es p B D s a La ro r Lo ro Tr To ro L s H a LQ ere af _ gt ue a a l Lg L7 ue i Le o le O 8 160 140 120 100 80 60 F2 ppm Figure 10 4 13C CPSPINDIFF of fully labeled tyrosine HCI spinning at 22 kHz 4 6 msec mix Upper PDSD lower DARR 106 261 BRUKER BIOSPIN User Manual Version 001 Proton Driven Spin Diffusion PDSD le row 820 from spindiff 10 1 C Bruker TOPSPIN manual H spindiff 13 52 zuguer TOFSPT namia lll Seale 0 7357 ssintitt 32 SSO Cs BeukecsTOrsrsg nara Le Scale 0 8651 f spindift 11 520 C Bruker TOPSPIN na
127. ces using shape tool stdisp Store the ramp as ramp4555 100 Select this ramp as spnam 1 or spnam2 if the shape should be executed on the Y channel e Y e 2 m DI peresi Figure 18 4 Shape Tool display with ramp shape from 45 to 55 The amplitude factor is 50 corresponding to 50 RF field or a power level change of 6 dB since the amplitude corresponds directly to the pulse voltage BRUKER BIOSPIN User Manual Version 001 Double CP 5 Since the shape is centred around 50 the RF voltage here is down by a factor of 2 6 dB in voltage the power must be increased by 6 dB to get the same RF field as with a 100 square pulse 6 Usually the ramp shape is set on C but it can also be used on 1N Set spnam2 to square 100 pl5 to 46 or 24 kHz RF field on 15N Set spnam1 to ramp4555 100 pl1 to 35 kHz RF field on 19C 6 dB Set pl13 pl12for a start set p16 to 5 msec Optimize the power level pI5 A variation over 1 to 1 dB in steps of 0 2 dB should be ample In order to be sure one can optimize p 7 and pl5 as an array pI1 in steps of 0 5 dB pl5 in steps of 0 2 dB Use a full phase cycle to avoid signal from a direct proton to carbon transfer which is cancelled by the phase cycle Optimize p16 between 5 and 15 msec See fig 6 for an op timization of pI5 19N square pulse power 7 With a ramp shape for the N C transfer one should get 40 50 DCP efficiency fig 7 com pared to the reference direct 136 C
128. cical aa La d a bech Edt Shape Parambeters o D 100 gt PE Figure 20 3 Shape for DUMBO sp1 Analog and Digital Sampling Modi 20 3 AV3 instruments allow different acquisition modi one which resembles the previous mode of analogue filtering in so far as the down conversion is done without simultaneous digital filter ing whereas the digital mode always down converts and filters simultaneously Remember that at a standard sampling rate of 20 MHz the fixed sampling rate of the DRU down conversion must be done to obtain data sets of reasonable sizes The pulse programs dumboa and wpml ga are written for the pseudo analog mode without digital filtering dumbod and wpmlgd are written for the digitally filtered mode User Manual Version 001 BRUKER BIOSPIN 213 261 CRAMPS 1D Analog Mode Sampling 20 3 1 2 complex data points down converted into 1 data point no points sampled in between Figure 20 4 Analog Sampling Scheme Digital Mode Sampling 20 3 2 Continuous sampling throughout the pulse train eere ef e D Ze Ze af B e af digital blanking during pulse and deadtime zeroes are sampled Ka downconversion of decim data points into one digital filter applied Figure 20 5 Digital Sampling Scheme 214 261 BRUKER BIOSPIN User Manual Version 001 CRAMPS 1D 20 4 Setup At frequencies of 400 MHz and higher doubl
129. citation pl13 Power level LG decoupling f2 channel pl 90 excitation pulse on f1 channel p2 180 excitation pulse on f1 channel p3 90 excitation pulse on f2 channel p15 Contact pulse on f1 and f2 channel do 3u t1 evolution period User Manual Version 001 BRUKER BIOSPIN 95 261 RFDR Table 9 1 Acquisition Parameters di Recycle delay cpdprg2 Spinal64 Spinal64 decoupling on f2 channel cpdprg3 cwlg cwlg decoupling on f2 channel ns 16 Number of scans cnst20 100000 Proton rf field in Hz to calculate LG parameters cnst21 0 f2 channel offset cnst24 Additional Lee Goldburg offset cnst31 Spinning speed in Hz 11 for 2 40 msec Number of rotor cycles for mixing time F2 direct 13C Left column td 4k Number of complex points sw 200 ppm Sweep width direct dimension F1 indirect 13C Right column td 256 Number of real points sw Rotor synchronized sweep width ndo 1 Number of inO in pulse program FnMode TPPI or STATES Or STATES TPPI Spectral Processing Table 9 2 Processing Parameters 9 4 Parameter Value Comment F1 acquisition e Left column si 4k Number of points and zero fill ph_mod pk Phase correction if needed bc_mod quad DC offset correction F2 indirect 13C Right column si 256 Zero fill mc2 TPPI or STATES Or STATES TPPI ph mod pk Phase correction if needed b
130. clockwise tunes to higher frequency Do not change the matching adjustment until you have found the current tuning position of the probe else you may loose the probe response totally Do not tune without having the appropriate matching box fitted to the preamp Fake resonances may appear due to filters between probe and preamp because filters are also tuned circuits Remove all filters before tuning over a wide range and fine tune again wbsw lt 10 MHz when the probe is tuned close to the desired frequency Changing the proton tuning will affect the X tuning so always tune the proton channel first then the X channel Setting a probe from high range to low range mode lambda 4 switch will shift the X tuning to lower frequency by many MHZ the proton frequency will only change by a few MHZ An empty probe may tune as much as 10 MHz higher on the proton channel compared to a probe with a spinner in When a probe has not been used over an extended period humidity may collect inside the tur bine causing a few harmless arcs RF breakthrough on the proton channel If the arcing does persist and or gets worse have the probe checked Usually this means that dirt has accumulat ed inside the turbine or on the RF coil Cleaning should be done by a trained person only Regular probe performance checks comprise Checking the magic angle setting KBr Checking the shims Adamantane Checking S N performance on glycine These checks must
131. ction where the 2nd order broadening has disappeared The information content is in full anal ogy to the MQMAS experiment Experimental Particularities and Prerequisites 17 2 In contrast to the MQMAS experiment the first pulse in the STMAS experiment excites single quantum SQ coherency The signal which is thus generated consists of contributions from both the CT and the ST In Figure 17 1 the contribution of the CT shows up in the cosine curve starting at the blue filled rectangle resembling the initial pulse Figure 17 1 Principle of 2D Data Sampling in STMAS Experiments The blue filled rectangle on the left symbolizes the first pulse which starts the evolution peri od t After each revolution of the rotor rotational echo s show up which are indicated by the User Manual Version 001 BRUKER BIOSPIN 183 261 STMAS red filled circles The open rectangles symbolize the second pulse one pulse at the end of each individual t increment They must always occur precisely on top of the rotational echo For each increment the t data acquisition which is not shown here starts after the second pulse In the following discussion we will ignore this part completely It showed up in the original ex periments but can be completely suppressed by a double quantum filter The contribution of the ST rides on top of the CT signal like spikelets Since MAS efficiently averages the 1st or der quadrupole interaction of the ST t
132. dB Not used PL11 start with 300 W Power level for excitation and conversion pulses PL21 Power level for selective pulse approx pl11 30 dB taken from previous pulse calibration Hence it is always better to optimize the pulse lengths p1 and p2 In this case p2 should be optimized before pT because the signal intensity is much more sensitive to this pulse length A suitable set up for the parameter optimization procedure popt is shown in following figure store as 2D dato ser file The AU program specified in AUNM will be executed Por form automatic baseline correction ABSF T Overwrite existing files disable confirmation Message C Run optimisation in background OPTIMIZE To PARAMETER OPTIMUM p2 POSMAX pi POSMAX 05 15 15 45 STARTVAL ENDVAL nexe VARMOD INC LIN LIN oo N Sa Figure 15 5 Example for popt Set up for Optimization of p1 and p2 In the first step p2 is optimized to which the experiment is the more sensitive In the second step p1 is optimized using the optimum value found for p2 in the first step For more details about using the popt procedure to optimize a series of parameters please re fer to the manual Figure 15 6 shows the signal amplitudes as functions of pulse lengths p2 an User Manual d p1 Version 001 BRUKER BIOSPIN 157 261 Basic MQ MAS sinum Hd vnen we nv UH uu a ae t 4 4 3 4 m PT Ap
133. dow function PH_mod pk Apply phase correction BC_mod no No DC correction is required after full phase cycle ABSF1 1000 ppm Should be outside the observed spectral width ABSF2 1000 ppm Should be outside the observed spectral width STSR 0 Avoid strip FT STSI 0 Avoid strip FT TDoff 0 Avoid left shifts or right shifts before FT F1 indirect dimension Sl 256 Sufficient in most cases WDW no Don t use window function QSINE Only use if FID in F1 is truncated SSB 2 n 2 shifted squared sine bell PH_mod pk Apply phase correction PHC1 d6 dw 180 BC_mod no No dc correction is required after full phase cycle ABSF1 1000 Should be outside the observed spectral width ABSF2 1000 Should be outside the observed spectral width STSR 0 Avoid strip FT STSI 0 Avoid strip FT TDoff 0 Avoid left shifts or right shifts before FT Data obtained with mp3qdfsz av can be processed with the AU program xfshear unless in case of nuclei with spin 3 2 where a straight 2D FT can be used if a split t4 experiment has been recorded by setting IN10 appropriately The information obtained from the DFS enhanced spectra is the same as from the standard MQMAS experiments Please refer to the chapter Basic MQ MAS on page 151 for further details regarding the shearing transformation and the information obtained from MQMAS spectra User Manual Version 001 BRUKER BIOSPIN 179 261 MQ MAS Sensitivity En
134. dowless phase ramped shapes One can write these shapes as multi ples of FSLG cycles to manipulate the length of the T4 increment Usually 2 FSLG cycles make sense The pulse program calculates the required shape pulse length from the RF field during the FSLG evolution In pmlghet a shape with 2 cycles is assumed in the calculation The sequence is optimized for a simple twofold linear phase ramp supplied as Igs 2 The car rier may be placed in the middle of the proton spectrum during evolution which may allow using fewer increments and therefore saving time However one should be aware of the presence of proton spinning sidebands along F1 which may inappropriately fold in if the spectrum window along F1 is chosen too small Processing is done in complete analogy to the FSLG experiment as for all following sequenc es User Manual Version 001 BRUKER BIOSPIN 85 261 Modifications of FSLG HETCOR J T M e F E r amp d Le Lu hm IM Lo c pal y i I Le c i L a y i E l 1 rd Lo e I Le L H FR T T T T T T T T T T T T A T T T T T T T T T T T T T a 200 150 100 50 F2 ppm Figure 8 2 HETCOR Using Windowless Phase Ramps The figure above shows the HETCOR using windowless phase ramped shapes for proton Homo nuclear decoupling during evolution The carrier was placed in the middle of the proton spectrum and the usual carrier ridge was suppressed by phase cycling Leskes et al C
135. e Click on the marked red arrow to set the observe frequency set the position of the cursor line and left click on the 01 button Acquire another spectrum ft and phase Then expand the spectrum around the adamantane proton signal including the spinning side bands by clicking on the left margin of the region of interest and pulling the mouse to the right margin of the region of interest as shown in the following figure User Manual Version 001 BRUKER BIOSPIN 27 261 Basic Setup Procedures Spaun Prer2srs MAPA 3l Tet gt habran Parts Irtenrsis Same Siess Fir m Prana arnt E teli dme ete en 12 Distance 80 70 ppm 3 amp 130 08 Hz Figure 3 14 Expanding the Region of Interest Click right mouse button in the Spectrum window When the Save Display Region to menu pops up select Parameters F1 2 and OK or type dplin the command line 3 Save display region to Options Parameters F1 2 e g used by restore display dpl C Parameters ABSF1 2 e g used by absf apkf C Parameters STSR STSI used by strip ft C Parameters SIGFL2 signal region used by sino C Parameters NOISFL2 noise region used by sino C A text file for use with other programs Figure 3 15 Save Display Region to Menu 28 261 BRUKER BIOSPIN User Manual Version 001 Basic Setup Procedures Start parameter optimization by typing popt in the command line The popt window will ap pear e
136. e 3 5 Graphical Pulse Program Display In the figure above the experiment button for opening the graphical display is marked with a red circle Then match and tune the probe for this sample using the command wobb This will start a fre quency sweep over the range of SFO1 WBSW 2 The swept frequency will only be absorbed by the probe at the frequency to which it is tuned 20 261 BRUKER BIOSPIN User Manual Version 001 Basic Setup Procedures Spectrum Proc Pars Acqurars eie Pulse Prog Peaks Integrals Sarete Structure Fid zen Fs 2 en reg 2000 3000 4000 5000 1000 598 599 600 601 Figure 3 6 Display Example of a Well tuned Probe At frequencies where the probe is not matched to 50 Ohms the curve will lift off the zero line If tuned to a frequency within SFO1 WBSW 2 but SFOT the probe response will be off center N B Fake resonances may appear which do not shift with probe tuning It is always a good idea to keep track which nucleus was tuned last so it is clear what direction to tune to Usually turning the tuning knob counter clockwise looking from below will shift to higher tun ing frequency User Manual Version 001 BRUKER BIOSPIN 21 261 Basic Setup Procedures Spectrum Proc Pars AsquPars Title PulseProg Peaks Integrals Sample Structure Fia zc Fs alo y 4000 5000 1000 2000 3000 598 599 600 607 Figure 3 7 Display Example of an Off Matched and
137. e or triple resonance CP MAS probes may be used on the proton channel at lower fields a CRAMPS probe is required due to the increased ring down time at lower field Spinner diameters of 4mm or smaller are preferred since we want to spin over 10 kHz Since only one nucleus is observed no filters are required and should be avoided Good impedance matching between probe and transmitter is important in order to optimise the effect of the pulses on the spins Ifthe RF cable has been too strongly bent or the connectors been twisted the cable may not have 50 Ohms and the result will al ways be bad Likewise if the preamp is burnt it is not possible to get good results The fewer connectors are between probe and preamp the better you can expect the 50 Ohm match to be In edasp set F1 for 1H observation select the high power proton amplifier and high power preamplifier Tune and match as usual It is assumed that the magic angle is pre cisely set which can easily be achieved with KBr on a double resonance probe or on BaClO3 H 0 looking at the proton signal much like one does on the Br resonance Shimming will also be important since protons are observed and on some samples good res olution is expected Looking at the protons in adamantane find the power level for a 2 5 usec 90 pulse Set the By field or 01 to be close to resonance see chapter Basic Setup Procedures for more details Calibrate the adamantane proton shift to 1 2 ppm Then l
138. e previous experiment analogous to wpmighet Run xau dumbo select d for dumbo 1 for 1 cycle O for added angle The calculated shape dumbo_1 0 will be entered as spnam1 p10 will be set to 32 usec The projection of this exper iment can be compared to the result of a direct proton detected CRAMPS experiment using dumbod2 At higher fields p10 may have to be set to 24 rather than 32 usec for better resolu tion The same pulse length p10 the same shape the same window p9 and the same power level should be used in both experiments The resolution along the proton dimension is comparable for all these experiments The FSLG experiment is the most forgiving requiring just the knowledge of the RF power level for decou pling at a certain RF field Setting cnst20 to this RF field 5 or 10 is all that needs to be set if the 19C observe parameters are well adjusted Table 8 5 Acquisition Parameters for DUMBO HETCOR on tyrosine HCI Parameter Value Comments pulprog dumbohet Windowless dumbo shape nuc1 13C olp 100 ppm nuc2 1H cnst24 1000 3000 Place carrier within proton spectrum for evolution pr Power level channel 1 for contact pulse pl12 Power level channel 2 TPPM SPINAL decoupling pl13 Power level channel 2 DUMBO decoupling sp0 Power level channel 2 for contact pulse spnamO ramp 100 or similar Shape for contact pulse channel f2 sp1 set to pl13 100 kHz for default duration 32 ps
139. eaks See the chapter on spin diffusion experiments for more information about DARR or PDSD User Manual Version 001 BRUKER BIOSPIN 205 261 Double CP 206 261 Ft ppm 40 In mm A Am A A La A A MR es TERRA ee AR eee ee E MEA Am T T 150 100 50 H F2 ppm Figure 18 10 NC C correlation experiment with 4 2 ms SPC5 DQ mixing period for C 4C spin diffusion on GB1 protein run using an EFRFE_Probe at 14 kHz sample rotation and 100 kHz decoupling See the chapter on recoupling experiments for the SPC5 setup and for more information about DQ recoupling sequences Note the inverse phase of the cross peaks generated by the DQ mixing step BRUKER BIOSPIN User Manual Version 001 CRAMPS General CRAMPS is an acronym standing for Combined Rotation And Multiple Pulse NMR Spectros copy Multiple Pulse Spectroscopy had long been thought not to work under spinning around the magic angle but in fact it does work as long as the pulse cycle times are substantially shorter than the rotation period CRAMPS suppresses Homo nuclear dipolar interactions between the abundant spins mostly protons and chemical shift anisotropy simultaneously through the combination of multiple pulse techniques and magic angle spinning J couplings and large hetero nuclear dipolar cou plings are not suppressed Reference L M Ryan R E Taylor A J Patt and B C Gerstein An experimental study of resolution of proton chemi
140. efine the RF field at which the transfer is to take place and find the appropriate power levels to achieve these RF fields In order to minimize losses due to insufficient excitation bandwidths and T4 relaxation the contact should be executed at high power However there are limitations in terms of what the probe can take and there are losses due to unwanted HH contact to the proton spin system On the other hand in many samples bio samples the spread of chemical shifts that one wants to cover is not extremely wide or one even wants to execute the transfer se lectively Specific CP An RF field of 35 kHz is a decent compromise So using the cp90 pulse program and moving the carrier close to the C peak determine a power level pI11 which corresponds to 35 kHz RF 7 14 usec 90 pulse However since the sample spins at 11 kHz the HH condition will require 46 or 24 kHz on one nucleus and 35 kHz on the oth er nucleus If you decide to account for the spin rate on the 13C side calculate the required power levels for 24 and 46 kHz 35 kHz RF field 11 kHz spin rate RF field using xau calcpowlev Now the 13C channel is set 15N Channel Setup 18 2 2 196 261 8 Create a new experiment using edc 9 Setup the proper routing by going into edasp Click the switch F1 F3 button to get 15N on channel 1 Figure 18 3 Routing table for triple resonance setup change for 15y pulse parameter mea surement and CPMAS optimi
141. elay sw 310ppm Sweep width direct dimension aq 16 20 msec masr 10 15 kHz 15 kHz ok at 100 kHz RF field Table 8 2 Processing Parameters for pmlg HETCOR as for FSLG on tyrosine HCl Parameter Value Comment F2 direct dim 1 C si 2 4 k wdw QSINE SSB 2 3 or 5 ph_mod pk F1 indirect H si 256 1048 mc2 STATES TPPI wdw QSINE ssb 3 5 ph_mod pk User Manual Version 001 BRUKER BIOSPIN 87 261 Modifications of FSLG HETCOR w pmighet 8 2 2 If one wants to compare a solids proton spectrum acquired via 13C detection using HETCOR and a direct detect proton spectrum using w pmlg it is useful to do this using exactly the same parameters power levels and timings in both experiments If w pmlg is used and opti mized for the direct detect experiment the same parameters will work with w pmlghet provid ed that both experiments are done with the same probe Table 8 3 Acquisition Parameters for womlg HETCOR on tyrosine HCI Parameter Value Comments pulprog wpmlghet Phase ramp allows detection window nuc1 13C olp 100 ppm nuc2 1H cnst20 usually gt 100 kHz As prepared with wpmlgd proton detect exp cnsi24 1000 3000 Place carrier within proton spectrum for evolution pit Power level channel 1 for contact pulse pl12 Power level channel 2 TPPM SPINAL decoupling pl13 Power level channel 2 w PMLG decoupling sp0 Power level channel 2 for contact pulse
142. eld Adamantane THMAS 0 setup proton channel set field shim under CRAMPS conditions Glycine CRAMPS 3 setup CRAMPS Malonic Acid CRAMPS 3 resolution CRAMPS d1 60s 19F PVDF 19FMAS direct observe 9F CP CP 1H 9F 1H 13C 19F 13C low sensitivity PTFE 9FMAS direct observe 3He 203 209 nm 31p NH4 H2PO4 IHR PCP 0 powdered sample piezoelectric 4s Li LiCl MAS GAT Sr Sn cyclohexyl CP 5ms contact d1 gt 10s Sm Sn gt 07 SnO3 MAS VT shift thermometer d1 lt 1s Sm2Sn gt 207 gt 60s SnO temp independent 87Rb RbNO3 RbCIO MQMAS 0 0 5s repetition 1 BN MAS Boric Acid MQMAS gt 5s repetition Ga Ga 03 hahn echo CT 300 kHz wide 65Cu Cu metal powder wideline knight shift 2500ppm 71Ga 129X as hydroquinon CPMAS 0 d1 gt 5s Clathrate gas in air 0 single pulses overnight 1s 23Na Na 5HPO MQMAS 0 dep on crystal water 2 5 lines Na3P309 MQMAS aly NH4VO4 12376 User Manual Version 001 BRUKER BIOSPIN 11 261 Test Samples Table 2 1 Setup Samples for Different NMR Sensitive Nuclei 27 Al AIPO 14 MQMAS O d1 05 1s 4 lines PC Adamantane CP DEC 50 HH setup shim a glycine CP 110 sensitivity decoupling Prep precipitate with acetone from aq solution C N fully labelled for fast setup recoupling REDOR 10 in natrl abundance 79Br KBr MAS d1 lt 50msec angle setting finely powdered reduced volume 59Co Co CN g MAS shift t
143. emented by 1 dB up to the same power level used for the excitation pulse e g from 20 dB to O dB Initially a sweep of one whole rotor period i e LO 1 can be used The optimization of SP7 can be repeated e g for half a rotor period a quarter of a rotor period and so on With a shorter sweep you will find that higher RF power will be needed T store os 2D dato ser file P The AU program specified in AUNM will be executed Perform automatic baseline correction ABSF C Overwrite existing files disable confirmation Message T Run optimisation in bockground OPTIMIZE PARAMETER OPTIMUA STARTVAL ENDVAL NEXP VARMOD INC ad spt POSMAX 20 Q 0 LIN A 10 POSMAX 2 2 1 LIN null ad spi POSMAX 20 0 o LIN 1 P lo POSM AX 4 4 1 LIN null sph POSMAX 20 0 0 LIN 1 a 10 POSMAX 8 8 1 LIN null ad spi POSMAX 20 0 0 LIN d Figure 16 5 Example for popt to Set up for Optimization of DFS Note that the option The AU program specified in AUNM will be executed is checked This ensures that the sweep is recalculated for the variation of I0 and stored in the shape file dfs The initial value of 10 is 1 Figure 16 5 shows a popt window with successive optimization of SP1 for several fractions of rotor periods 0 Note that the check mark for The AU program specified in AUNM will be exe cuted MUST be set in order to force recalculation of the shape for each step of the optimisa tion In this case acquisition is run with t
144. ent on Tec samples requires reasonable enrichment Usually fully enriched samples are used sometimes diluted in natural abundance samples to reduce nonspecific long range inter actions As always rotary resonance conditions overlap of side and center bands should be avoided unless specifically desired Running the experiment on enriched 15N samples is of course possible but one should consid er that most samples will not have nitrogen atoms directly attached to each other so small cou User Manual Version 001 BRUKER BIOSPIN 121 261 Symmetry Based Recoupling plings will prevail requiring long DQ excitation and reconversion times with a nucleus that requires high RF power levels to achieve a certain RF field On the other side the shift range is not large allowing relatively slow spinning Considering a nucleus like 31P there is no need for enrichment but cases with directly bonded 3 P atoms are rare Phosphates are usually easy since the shift range is small couplings are also rather small If however a large shift range possible with 31P needs to be covered there may be a substantial problem Table 12 1 Recommended Probe Spin Rates for Different Experiments and Magnetic Field Strengths a rotor masrmaX rtl max vgr H max Bg max Sequence me dame rae ms ee gei POST C7 7 7 6000 5000 35 7 15 70 3 5 LG 300 4 15000 9000 63 4 100 2 5
145. erate RF level 180 proton pulses are used synchronized to the rotor speed such that recoupling does not occur pcpd2 n 4 rotor periods Usually pcpd2 is selected to be about 1 3 rotor period The decou pler power level must be adjusted to produce a 180 pulse of rotor period 3 Reference A Detken E H Hardy M Ernst and B H Meier Chem Phys Lett 356 298 304 2002 Pi Pulse Decoupling 4 1 5 Pi pulse decoupling is a decoupling program for weaker nuclear interactions like J couplings or weak dipolar interactions using rotor synchronized 180 pulses n pulse decoupling uses the xy 16 phase cycle for large bandwidth Abundant protons cannot be sufficiently decoupled with this method but it is very suitable to remove couplings to 31P which is hard to do by cw or tppm since the chemical shift range is wide Likewise it can be used to decouple dilute spins or spins which are homo nuclear decoupled by spinning 9F Reference S F Liu and K Schmidt Rohr Macromolecules 34 8416 8418 2001 Homo nuclear Decoupling 4 2 Homo nuclear decoupling refers to methods which decouple dipolar interactions between like spins Those are only prominent between abundant spins like 1H 19F and p and potentially some others This interaction cannot easily be spun out in most cases and renders NMR pa rameters like chemical shifts of the homo nuclear coupled spins or hetero nuclear couplings and J couplings to other X nuclei unobs
146. erent offset condition helps improving the signal intensity 8 Run one experiment and compare with the direct CP experiment to measure the DQ recou pling yield 124 261 BRUKER BIOSPIN User Manual Version 001 Symmetry Based Recoupling y reccupling 1 1 C Srukec TOPSOIN sar MY Scale 0 6768 p reccupling 6 1 C Brukec TOPSPIN A E L rs TEES P ONES FERIEN ER RL es wi 2 1 um A a E SE T T mE v m T T T T T T 200 150 100 so o ppm Figure 12 4 PC7 Recoupling Efficiency at a Spinning Speed of 13 kHz Figure 12 4 illustrates PC7 recoupling efficiency at a spinning speed of 13 kHz about 100 kHz RF field using a 2 5 mm probe LG decoupling at 125 kHz was used during DQ genera tion reconversion Quite a noticeable loss on the glycine a peak due to insufficient HH sup pression is noticeable Efficiency is 67 on the carboxyl peak AVIII 700 SB Setup of the 2D SQ DQ Correlation Experiment 12 2 3 9 10 11 12 Running such a correlation experiment on glycine makes little sense so insert a sample with more 13C sites fully labelled tyrosine HCl or histidine or any other suitable labelled sample Optimize 0 for the best compromise in signal intensities Generate a new data set set the mode to 2D using the 123 button in eda Load the pulse program spc5cp2d Make sure C is selected as nuc1 in the F1 dimension set FnMode STATES TPPI Set the spectral window along
147. ersion 001 BRUKER BIOSPIN 99 261 Proton Driven Spin Diffusion PDSD requiring long spin diffusion times can be observed However the information from this exper iment may be ambiguous because a rather non selective transfer within the proton spin sys tem is utilized Nevertheless even complex molecules like proteins can be surprisingly well characterized by PDSD experiments with different mixing times The buildup of cross peak intensities can be studied and correlated for instance to the structure of a macromolecule in the solid state The same approach has been used to compare different states of a protein i e bound to a mem brane or free as can be found in the recent literature on solid state NMR applied to protein studies More elaborate derivatives of PDSD are also known in bio molecular NMR where the unspe cific spin diffusion within the proton spin system is filtered through a double quantum selection Lange et al References 100 261 1 N M Szeverenyi M J Sullivan and G E Maciel Observation of Spin Exchange by Two Dimensional Fourier Transform 13C Cross Polarization Magic Angle Spinning J Magn Reson 47 462 475 1982 2 A F de Jong A P M Kentgens and W S Veeman Two Dimensional Exchange NMR in Rotating Solids a technique to study very slow molecular motions Chem Phys Lett 109 4 337 1984 3 Matthias Ernst and Beat H Meier Spin Diffusion in Isao Ando and Tetsuo Asakura Eds
148. ervable Multiple Pulse NMR Observing Chemical Shifts of Homo nuclear Coupled Nuclei 4 2 1 Multiple pulse NMR methods are covered in the chapters about CRAMPS of this manual col lection The principle of those methods CRAMPS if MAS is used to average CSA interactions simultaneously is to set the magnetization of the spins into the magic angle using a suitable User Manual Version 001 BRUKER BIOSPIN 47 261 Decoupling Techniques Reference pulse sequence In this case the dipolar couplings between those spins are suppressed Short observation windows between pulses allow observation of the signal from the decoupled nu clei S Hafner and H W Spiess Multiple Pulse Line Narrowing under Fast Magic Angle Spinning J Magn Reson A 121 160 166 1996 and references therein Multiple Pulse Decoupling 4 2 2 48 261 Multiple Pulse Decoupling Observing Dipolar Couplings and J couplings to Homo nuclear Coupled Nuclei Homo nuclear couplings between abundant spins usually protons superimpose their hetero nuclear dipolar couplings to X spins and J couplings to X spins so these distinct couplings are not observable Homo nuclear decoupling protons while observing X spins makes these couplings observable Any method used in multiple pulse NMR section 4 2 1 may be used to achieve this BR 24 MREV 8 BLEW 12 Used as hetero nuclear decoupling methods the window between pulses may be shortened or omitted semi window
149. es are simpler and shorter requiring fewer adjustments and allowing higher spin rates Both sequences use repetitive shaped pulses with detection in between PMLG uses the prin ciple of a Frequency Switched Lee Goldburg FSLG sequence continuous irradiation with a net RF field along the magic angle where the frequency shifts are replaced by a phase modu lation DUMBO basically works like a windowless MREV type pulse sequence where the indi vidual pulses are replaced by a single pulse with phase modulation References E Vinogradow P K Madhu and S Vega High resolution proton solid state NMR spectrosco py by phase modulated Lee Goldburg experiment Chem Phys Lett 314 443 450 1999 D Sakellariou A Lesage P Hodgkinson and L Emsley Homo nuclear dipolar decoupling in solid state NMR using continuous phase modulation Chem Phys Lett 319 253 2000 Quadrature Detection and Chemical Shift Scaling 19 4 208 261 Under Homo nuclear decoupling the magnetization precesses in the transverse plane of a tilt ed rotating frame whose new z axis is along the direction of the effective field The projection of this plane into the X Y plane is therefore an ellipse and the signal intensities sampled in the two quadrature channels along the x and y direction are different since these are the major and the minor axes of the projected ellipse As can be seen from fig 1 the X and Y observe di rection will see a signal of different
150. es confirmed by the optimi zation procedure Parameters like O1 TD SWH RG should already be set in the standard 1D spectrum For 4 mm probes these pulse lengths are about the limit of what can be achieved for 2 5 mm probes somewhat shorter pulses can be obtained For 3 2 and 5 2 nuclei the ratio of p1 p2 3 For pl11 an initial value that corresponds roughly to 300 W can be used Optimization will be done on the first increment of the 2D sequence i e d0 1 us Two strategies for the optimiza tion procedure can be followed either the pulse lengths p1 and p2 or the power level pI11 can be optimized for maximum signal amplitude However the latter can be disadvantageous be cause a power level above the probe limit might be applied in order to clearly determine the optimum power In the case of 300 W amplifiers the maximum signal amplitude may not be ob tained even at full power with the chosen pulse lengths BRUKER BIOSPIN User Manual Version 001 Basic MQ MAS Table 15 2 Initial Parameters for Setup Parameter Value Comments Pulprog mp3qzqf av or Pulse program mp3qzfil av NS 12 n zaf Full phase cycle is important 96 n zfil DO tu Or longer t4 period D1 5 T4 Recycle delay use dummy scans if shorter D4 20 us Z filter delay P1 3 6 us Excitation pulse at pl11 P2 1 2 us Conversion pulse at pl11 P3 20 us 90 selective pulse at pl21 taken from previous pulse calibration PL1 2120
151. est Samples 11 Table 2 1 Setup Samples for Different NMR Sensitive Nuclei sssss 11 3 Basic Setup Procedures 13 Table 3 1 Summary of Acquisition Parameters for Glycine S N Test 39 Table 3 2 Processing Parameters for the Glycine S N Test a n 40 Table 3 3 Reasonable RF fields for Max 2 Duty Cycle eseese 41 4 Decoupling Techniques 45 Table 4 1 Acquisition Parameters sssssssssssssse ernennen 51 Table 4 2 Processing Parameters essetis tient there nna nnns 52 5 Practical CP MAS Spectroscopy on Spin 1 2 Nuclei 55 Table 5 1 Power Conversion Table sssssssssss eee 57 6 Basic CP MAS Experiments 61 Table 6 1 Acquisition Parameters ssssssssssssse ernennen 62 Table 6 2 Acquisition Parameters sssssssssssse ernennen 63 Table 6 3 Acquisition Parameters ssssssssssssssse nn 69 7 FSLG HETCOR 75 Table 7 1 Acquisition Parameters for FSLG HETCOR on tyrosine HCl 79 Table 7 2 Processing Parameters for FSLG HETCOR on tyrosine HCl 80 8 Modifications of FSLG HETCOR 83 Table 8 1 Acquisition Parameters for pmlg HETCOR on tyrosine HCl 86 Table 8 2 Processing Parameters for pmlg HETCOR as for FSLG on tyrosine HCl TEE 87 Table 8 3 Acquisition Parameters for wpmlg HETCOR on tyrosine HCl 88 Table 8 4 Acquisition Parameters for e DUMBO HETCOR on tyrosine HCl
152. eta ddl dira edidit ius 56 lee GETT 215 Basic Setup Procedures roe a a a e a a e a a 13 EE 49 A 16 BLE ds cad 48 BN et een debet ed 11 BON Acid ooi ne fe E A nte d de 11 BR 24 ee atte i teet Ulo eie to do te bez EE 48 208 breakthrough 2 Ine o LEUTE PUES gcc PESE Mete URINE 14 EE El E xe edet teme oc Eee dide inda 33 bypassing the Dreamp nene emere n nnn nennen 31 C C24 S 208 CA CO WEY 30 Calibrating WEE 32 User Manual Version 001 BRUKER BIOSPIN 253 261 Index 254 261 CANET 27 Cd NO3 2 4A2O 5 iei deena ane einen eee ee 12 Chemical Shifts c ccccccccceceeeeeeeeececeaeceeeeeeeeeeeseesecaeaaecaeeeeeeeeteeeesecscenaeeeeeeees 32 Suri ccm 11 eli pP 77 CO CN G iii da 12 Lomptessore nn 14 CONCUCHVILY tii acia 55 contact me 33 RE 16 e nl re EE 38 CPP ME 61 72 73 egli Ee 72 73 CPPISP NEE 72 73 enc 207 Cross polariSation u n e td en e eae n en 33 cross polarisation sssssssssssssessese nennen entren enne nnne nnns 13 cross polarization under a LG frequency oftset 75 Cu metal Dowder nn nn nnnnnnnnnnnnnrnnnnnnnnnnnrnninnnnn 11 CW decoupling een 45 D D20 cionan EMT 12 ee e KETTER 12 Default aia 16 dielectric lOSS fiat anne eu dann 55 digital Mode sitas dais 213 d TEE 12 OD RE 28 d PMMA cT 12 YAS TT 14 el CEET 14 RTE 12 59 Ei ET 62 DUMBO EE 53 90 208
153. eters for Digital Mode ssss nenn 218 21Modified W PMLG 221 Table 21 1 Phrases RF Levels Timings ssem nennen 221 Table 21 2 Table 21 3 Table 21 4 PMLEG Analog Mode oer ea A e d n 223 DUMBO Analog Mode 223 Parameters for Digital Mode 224 22CRAMPS 2D 225 Table 22 1 Table 22 2 Table 22 3 Table 22 4 Table 22 5 Table 22 6 Acquisition Parameters 42444s44 snesenennenenennenennnennne nenne nnne nn 226 Phases RF levels and Timings nennen 227 Processing Parameters ness secet cansada ead nea Reda na du nd 227 Acquisition Parameters ssssssssssssssss ene 232 Phases RF Levels and Timing c cccceeceeeeeeceeeeeeeeeeeeeeeeeeneneees 232 Processing Parameters 233 A Appendix 237 User Manual Version 001 BRUKER BIOSPIN 251 261 Tables 252 261 BRUKER BIOSPIN User Manual Version 001 Index Symbols O A A ees 12 d elei e EE 11 Numerics 136 Matching Box rnit ii 17 1H HP Sie lu EE 17 Te ET 23 QO 0 U E ae IPIE 13 A Adamantane ite It ette 11 12 ADP zit ee ebe ihn nn Reais Ee had To due 59 el e EE 12 AIP O EE 12 analog ue 213 Anatas iii EE 12 AS ale e Un Le ee 11 ASO iii eine E N RE 18 AU program DUMBO 1 0 0 eect erent etree neste tieeeeeeeiaeeeeeenaeeeseetieeeeeenea 53 B BO field os an a ri ei eee awa 39 backgr und Signalz 25 Ha Se ta Se ee ee 56 background suppression ek sed idee aet P be
154. ext Fraction of rotor period for sweep L1 see text Number of rotor cycles for synchronization used in mp3qdfs av only cnst1 see text Start frequency of sweep in kHz cnst2 see text End frequency of sweep in kHz cnst3 50 in ns timing resolution for sweep cnst31 masr Spinning frequency 174 261 BRUKER BIOSPIN User Manual Version 001 MQ MAS Sensitivity Enhancement CNST3 in ns 50 This is the maximum timing resolution that can be obtained on a shaped pulse with AV or AVII hardware However if cortab is defined for that nucleus 100 ns is the maximum timing resolution possible in a shaped pulse CNST31 Magic angle spinning frequency used for the calculation of the duration of the sweep and the echo delay The calculation of the sweep is done via an AU program called zg_dfs It calculates the sweep according to the parameters given above and stores it as a shape file which is called dfs After the calculation the AU program starts the acquisition In order to ensure that the correct sweep is always used it is advisable to enter the name of this AU program into the parameter aunm and start all acquisitions using the command xaua All that needs to be optimized now is the appropriate RF power level for the sweep defined as parameter sp7 As a first guess a value of 3 dB less RF power than for the excitation pulse should be used i e SP1 PL11 3 dB You may use popt for the optimization where SP1 is decr
155. f the MQ coherence in the first part of t4 is cancelled out by the evolution of the SQ coherence in the part of t4 after the conversion pulse the lengths of the two periods are related by the ratio of the second order broadening of the MQ and SQ coherence The resulting 2D spectrum thus requires no shearing transformation to make f2 the isotropic dimension Whether this experimental trick is useful for your sample of interest can easily be determined by running a simple Hahn echo experiment where the delay r in the 90 1 180 sequence is adjusted so that the FID of the signal is decayed before the 180 pulse is applied This is shown in Figure 16 1 The FID generated by the initial 90 pulse is not sampled but after it is decayed it is refocused with a 180 pulse into a so called shifted echo meaning that the posi tion of the echo can be shifted by adjusting the delay d6 User Manual Version 001 BRUKER BIOSPIN 169 261 MQ MAS Sensitivity Enhancement 170 261 phi ph2 p3 p4 pl21 d6 Figure 16 1 Hahn Echo Pulse Sequence and Coherence Transfer Pathway After the initial 90 pulse p3 the magnetization dephases and is refocused by the 180 pulse p4 If the r delay d6 is long enough a full echo can be acquired If data acquisition starts immediately after the second pulse the whole echo will be acquired The integrated intensity of the echo will be almost twice the intensity of the single FID it is just T relaxation during r tha
156. found 14 Start the experiment Data Acquisition 12 3 Sample Fully 13C labelled tyrosine HCl or a suitable fully labelled small peptide Spinning speed 5 20 kHz depends on experimental requirements see Table 12 1 Experiment time 1 4 hours Table 12 2 Acquisition parameters for DQ SQ correlation experiments using symmetry based recou pling sequences Parameter Value Comments Pulse program spc5cp2d See Table 12 1 for hints which sequence to prefer spc5cp2dlsw Rule of thumb high field fast spinning sc14 sc14cp2d low field slow spinning pc7 Intermediate spc5 r14cp2d N b sc14 usually has low DQ yield 3596 but that may not mat pc7cp2d ter NUC1 13G Nucleus on f1 channel O1P 100 ppm 13C offset NUC2 1H Nucleus on f2 channel O2P 2 3 ppm 1H offset PL1 for gt 50 kHz vrF Power level for f1 channel CP and p1 PL11 dep on masr Power level for f1 channel recoupling power PL12 as specified Power level decoupling f2 channel and excitation PL13 pl12 optimize or Power level decoupling f2 channel during cw or cwlg decoupling 120 fast spinning P3 Excitation pulse f2 channel PCPD2 Decoupler pulse length f2 channel iH TPPM P15 Contact pulse first contact D1 Recycle delay CNST20 Spin nutation frequency at PL13 for cwlg decoupling LO for 0 5 10 msec Use multiples of 5 7 or 16 spc5 pc7 sc14 for full phase cycle mix in ased SPNAMO Ramp for 1
157. frequen cy offsets When the experiment setup for the 1D experiment has been executed no further setup is required for the 2D experiment Start from the 1D experiment on your sample the recommended setup sample is glycine and generate a 2D data set by clicking on the sym bol 1 2 in the headline of the acquisition parameters User Manual Version 001 BRUKER BIOSPIN 225 261 CRAMPS 2D Pulse Sequence Diagram Phases Di u ds Pulses and delays pl LG p13 d3 d3d3 d Pulse power pl2 pll3 pli2 oli 22 2 excitation evolution 10 mixing detection Figure 22 1 Pulse Sequence Diagram This sequence is written in such a way that the windowed PMLG unit is used both for detection and for the shift evolution along F1 This was done to minimize the setup requirements In prin ciple a windowless sequence can be used as well and should give better resolution along F1 The power level for a windowless sequence is however usually slightly different from the win dowed sequence so this needs to be adjusted separately Likewise decoupling during t1 could be implemented using real frequency shifts as in the HETCOR sequence see Decoupling Techniques on page 45 If a windowless sequence is incorporated the windows d3 must of course be removed A simple windowless FSLG unit can be used with a shape like Igs 2 or Igs 4 having duration of twice or 4 times the length of the w pmlg pulse Table 22 1 Acquisition Parameters
158. fshear may be used with option rotate and argument 6 ppm to shift the spectrum suitably along F1 Setting 1 sr 2 sr 01 will set the referencing along F1 correctly just type sr and in f1 enter the value of sr for F2 and add 2 01 User Manual Version 001 BRUKER BIOSPIN 127 261 Symmetry Based Recoupling LAN LI Fi ppm I 180 o LN E _ f p E L e We rs go u e TT F lt ti 8 N lt D r L Po o A T1 1T 1T 1T 1171737 1711717 7711717 717171 200 150 100 50 F2 ppm Figure 12 5 SC14 2d SQ DQ correlation on tyrosine HCI SC14 2d SQ DQ correlation on tyrosine HCl 56 rotor periods mixing at 26 kHz 2 5 mm probe AV III 700 SB With the sampling window along F1 spin rate only the a B correlation is folded 13C 13C Single Quantum Correlation with DQ Mixing 12 5 Symmetry based DQ recoupling sequences may also be used as mixing periods in SQ SQ cor relation experiments The experiment resembles the PDSD or RFDR experiments see Pro ton Driven Spin Diffusion PDSD and RFDR as a NOESY type correlation will be generated Similarly the MELODRAMA see Bennett et al Dusold et al sequence with vgr 5 Vrotor May be used here 128 261 BRUKER BIOSPIN User Manual Version 001 Data Acquisition Symmetry Based Recoupling 12 6 Parameter Value Comments Pulse program p
159. g Peaks integrais Sample Structure Fid Acqui Prase Pring 15 1 td y poptau for pit finished POSMAX at experiment 6 pif 4 5000 NEXP 9 34 261 ne o gt a Figure 3 21 Hartmann Hahn Optimization Profile The wiggles besides the signals stem from truncation of the FID after 50 msec acquisition time BRUKER BIOSPIN User Manual Version 001 Basic Setup Procedures To exemplify the existence of several HH conditions on a spinning adamantane samples an other HH profile Figure 3 22 is shown where a square proton contact pulse is used There are several maxima corresponding to matches on the sideband orders n 2 n 2 n 1 n 0 and n 1 The largest intensity is seen for n 1 the intensities are very sensitive to the RF level which is varied in 0 2 dB steps Using a ramp makes the experiment much more stable and more quantitative Problems may arise if the proton T4 is short since usually longer contact times must be employed It makes therefore sense to use a flatter ramp 70 100 and opti mize for the spin rate which is used 1 5 14 on POHE J as Administrator connected Te ae Fille Edt View Spectrometer Processing Analysis Options Window Help J amp 765870 4 M A L ELA AA n P anetsooeop o aW e sz att A Spectrum ProcPars AcquPars Tie PusePreg Peaks integras Sample Sinxture Ma Acqu Prise Pere Y popotau for pit finished POSMAX al expenmen 29 pli 76000 NEXP 31 1
160. gen give rise to the lower peaks in the 2D spectra of Figure 15 10 the non bridging ones give rise to the upper peak An additional red line is drawn into the spectrum which represents the diagonal meaning d F2 d F1 One can see that all line positions must be below this diagonal because the negative quadrupole induced shift is scaled down and sub tracted from the isotropic shift to give the MQ shift In the example shown in Figure 15 10 two sites are visible with distinct differences in their spectroscopic parameters In the sheared spectra we find the lower peak at 170 ppm 11 7 T and 140 ppm 18 8 T respectively in the 3Q dimension This peak is dispersed parallel to the F2 axis which means that its line width is mainly due to second order quadrupole broadening The upper peak at 100 ppm 11 7 T and approx 90 ppm 18 8 T in the 3Q dimension has a much smaller quadrupole coupling which can immediately be recognized from the fact that the peak is much closer to the diagonal It is very nice example where the second order broadening which is still the dominant interaction at 11 7 T is so much reduced at 18 8 T that the width of the peak is now determined by the distri bution of chemical shift This is expressed in the fact that the peak is dispersed along the diag onal Figure 15 11 shows the results of the fitting with the solids line shape analysis package includ ed in TopSpin The spectra used for that have been extracted from rows of
161. gle is off Experimentally it has been found that the precision for setting the angle must be lt 0 002 The dependence on the accura cy is so important that the experiment itself must be used to find the most precise magic angle setting It is obvious that this can only be done on a well known sample as we will see later in the chapter In order to achieve the necessary precision for the adjustability of the angle a spe cial goniometer screw with an adapted gear transmission ratio is provided for the magic angle setting knob as an upgrade for Bruker WB MAS probes Once the best angle setting is found it is advisable to leave the probe in the magnet Sample changes however on the WB probes doesn t change the setting noticeably Pulse Sequences 17 3 Figure 17 2 and Figure 17 3 show two of the basic sequences which are 4 pulse sequences with z filter stmasdqfz av and stmasdqfe av Both sequences start with a non selective exci tation pulse p1 that creates SQ coherency on the innermost ST which is allowed to evolve dur ing the evolution period DO Shortly before the end of the t period there is a selective 180 pulse P4 This provides a double quantum filter by which magnetization of the CT transition is eliminated which will otherwise give a strong diagonal signal from a CT gt CT coherence transfer pathway The t4 period is terminated with the second non selective pulse P2 h1 h3 p2 d4 p3 pl11 pl21 pl11 pl21 Figure 1
162. gnal 2000 Hz around the current position in steps of 500 Hz The following result will be obtained 36 261 BRUKER BIOSPIN User Manual Version 001 Basic Setup Procedures File Edi View Speciromeler Processing Analysis Options Window Help MELO LEERSE E SILA TILA ASES ob TREMP 10220 W esee lt via foedum ProePaet Adan TOS Pusero Peas ig en Samos Gtuctue Fid Acqu Phase Print 7 ES poptau foro fmstoc POGMAX at experiment 5 07 4000 0000 NEXP 9 E 3 S a S f E 2000 2000 4000 d Hertz Figure 3 24 Optimization of the Decoupler Offset 02 at Moderate Power Using cw Decoupling Since the proton spectrum of glycine extends around 5 ppm the optimum decoupler offset will be obtained at higher frequency than the adamantane proton peak around 1 2 ppm Decou pling is still inefficient since cw decoupling is used which does not cover the whole proton shift range Also decoupling power is too low with a proton pulse of 4 5 usec Glycine requires about 90 kHz of decoupling RF corresponding to a 2 7 usec proton 90 degree pulse This can be ob tained with probes of 4mm spinner diameter and smaller 2 5 3 2 mm For a 7 mm probe 3 5 4usec can be expected at proton frequencies below 500 at 500 MHz Use calcpowlev to calculate the required power level p 12 and set p3 to twice the expected proton pulse width Check with 4 scans whether a close to zero signal is obtained Compared to 4 5 usec a 2 7 usec pul
163. h Selecting a path which is not fully routed will generate an error message To leave the display click on Save Make sure your probe X channel is connected to the se lected preamplifier the sequence of preamplifiers in edasp represents the physical position of the preamplifier in the stack If the preamp is a high power type make sure the correct match ing box is inserted into the preamp for 500 800 MHz systems it would be labelled for the fre quency range 120 205 MHz Connections are shown in the figure below BRUKER BIOSPIN User Manual Version 001 Basic Setup Procedures 1 X Low Pass Filter 5 13C Matching Box 2 Proton Bandpass Filter 6 X Probe Connector 3 X BB Preamplifier 7 1H Probe Connector 4 1H HP Preamplifier Figure 3 2 Probe Connections to the Preamplifier The figure above shows the probe connections to the preamplifier with appropriate filters placed on a table for better illustration Setting Acquisition Parameters 3 2 2 Create a new data set for the experiment by typing edc in the command line User Manual Version 001 BRUKER BIOSPIN 17 261 Basic Setup Procedures B New x Prepare for a new experiment by creating a new data set and initializing its NAAR parameters according to the selected experiment type For multi receiver experiments several datosets are created Please define the number of receivers in the box below NAME Pea EXPNO j PROCNO ho DIR le Bruker TOPSPIN USER
164. h that no RR condition occurs and sidebands do not overlap with peaks if possible set the sweep width in F1 7 swh equal to the spin rate or such that sidebands folding in along F1 do not interfere Make sure the correct nucleus is selected in the F1 dimension make sure to choose an ap propriate quadrature detection mode in FnMode usually STATES TPPI Choose the appropriate sampling time fd so that the required resolution FIDRES in the indirect dimension is achieved Set the desired mixing time as d8 The required multiple of spin periods from cnst37 is calculated as 7 the real mixing time my deviate by fractions of a rotor period The required mixing time may vary widely depending on the sample properties from a few milliseconds to hundreds of milliseconds if long distance correlations in a mobile sample need to be ob served Note that longer mixing times will result in S N deterioration as the mixing time ap proaches the T of the observed nuclei test 51 1 C Bruker TOPSPIN dents Jax Spectrum ProcPars AcquPars Title PulseProg Peaks Integrals Sample Structure Fid vi n s kg aA y EI Installed probe 4 mm MASDYT BE 1H H8724 0020 Expenment EZ F1 Frequency 305 W Experiment Receiver MEUS PULPROG Een zi E Current pulse program Durations AQ mod pap Acquisition mode Power FnMODE States TPP zl Acquisition mode for 20 3D etc Program TD 128 512 Sue of fid Probe NS la Number of sca
165. hancement Fast Amplitude Modulation FAM 16 3 It must be mentioned at this point that similar approaches have been made where the frequen cy of irradiation is established by a fast modulation of the amplitude of the pulses This is real ized by a repetitive train of either pulse delay pulse delay or delay pulse pulse delay Pulses and delays in these trains are of the same length The phases of the pulses are alter nating x and x which creates a fast cosine type amplitude modulation The frequency of this amplitude modulation appears to the spin system as an irradiated frequency Two pulse programs are available mp3qfamz av and mp3qfam av They correspond to the pulse sequences depicted in Figure 16 3 and Figure 16 4 but the shaped pulse realizing the DFS is replaced with a sequence D2 P2 P2 D2 embedded in a loop repeated by loop counter L2 and with power level PL14 for the pulses These sequences are only useful for spin 3 2 nuclei There are also sequences for higher spins which are not included in the pulse program library In those cases it is recommended to use DFS The difference between FAM and DFS can be understood in such a way that FAM establishes the irradiation of a single distinct fre quency whereas DFS continuously irradiates sweeps over a range of frequencies These fre quencies must lie in the range of the satellite transitions therefore a single frequency irradiation is sufficient for spin 3 2 Higher spi
166. hat do not gener ate double quantum coherence DRAWS DRAMA and MELODRAMA Symmetry based recoupling sequences recouple specific spin interactions using cyclic se quences composed of N phase shifted repetitions of either 2x C sequences or x R sequenc es rotation elements Which interaction s are recoupled by a given sequence is determined by the relationship between the sample rotation rate the spin rotation rate and the rate of phase shift between the elements The sequences are denoted as e g where N is the number of elements in the cycle n is the number of rotor periods spanned by the N elements and the total phase rotation between the elements is 2x v In the simplest implementation of a C se quence the 2r rotation element is simply a 2x pulse but other elements are possible Thus the sequence C71 consists of 7 consecutive 27 pulses with the phase of each pulse shifted by 27 7 from the previous one The whole sequence takes two rotor periods each 2x pulse thus takes 2 7 rotor period The spin nutation frequency and sample rotation frequency are thus related by vpe 7 2 Vrotor In practice the original C7 sequence uses an additional n phase alternation for every second pulse so that 14 pulses are executed during 2 rotor periods re quiring vr 7 vrotor For all C and R sequences the spin nutation frequency must be accurately matched to the sample rotation rate Since X X dipolar couplings are usually small long mixing
167. he DQ generation reconversion or set it to 120 if the spin rate suffices to omit decoupling Set cnst20 corresponding decoupling RF field 5 Optimize pl11 for maximum signal intensity 6 Optimize 0 for optimum signal intensity In a multi site spectrum the optima may differ for different spin pairs HUET T T T r T T T T T T T T 2 8 3 0 32 3 4 de Figure 12 2 Optimization of the RF power level for DQ generation reconversion on glycine User Manual Version 001 BRUKER BIOSPIN 123 261 Symmetry Based Recoupling In principle both peaks must grow together as one approaches the RF 7 MASR condition but the resonances are differently influenced by non ideal off HH conditions The glycine a peak is usually hard to get off HH so it is frequently too small Optimise the LG decoupling condition on the glycine a peak step 12 4 Eje Ede View Spectrometer prscateng Arab Otem Window Heb Ja 328 nm wi AAL rs iz Yu 9 YTm sm TREAT ERRAR RAR CE Hr wets T mam kafa fs P Pape tage Lampen Chat T us laz ee Figure 12 3 Variation of DQ generation reconversion time on a uniformly 1 C labeled peptide fMLF Both times were incremented in units of 2 rotation periods One can clearly see the different maxima for the C the alphatic carbons and the mobile CH3 groups Spinning speed was 13 kHz 7 Optimize the cwlg decoupling if needed by variation of cnsi20 in increments of 5000 and check whether a diff
168. he command xaua which ensures that the correct sweep is stored in the shape file dfs In the above example optimization proceeds such that in the first run SP1 is varied from 20 to 0 in 21 steps Then LO is set to 2 and SP1 is again varied from 20 to 0 Then 0 is set to 4 and so on Figure 16 6 shows results of the variation of the RF power level of sweeps with different durations Experiments have been run at 20 kHz and opti mization procedures for 1 0 5 0 25 and 0 125 rotor periods corresponding to 50 25 12 5 and 6 25 us respectively have been run One can see that as the duration of the sweep is re duced the required RF field amplitude is higher This is true when the spinning frequency is kept constant and the sweep is a smaller fraction of a rotor period and when the sweep is kept at e g one rotor period and the spinning frequency is increased and hence the rotor period de creased On real life samples the differences between signal intensities at one rotor period compared to e g Y rotor periods will be more pronounced than on a crystalline model com pound Since the spinning frequency is usually determined by the spectrum itself the only de User Manual Version 001 BRUKER BIOSPIN 175 261 MQ MAS Sensitivity Enhancement gree of freedom is the amplitude of the sweep In the example chosen here any of the conditions will provide a good quality spectrum and the condition with the biggest enhance ment at the least power is
169. he corresponding MHz broad signal is now narrowed into a huge number of spinning side bands These coherency originating from the ST dephase rap idly and refocus into rotational echoes with each rotor cycle A pulse precisely on top of such a rotational echo can transfer the SQ coherency from the ST to SQ coherency of the CT the sig nal from which can then be acquired under standard MAS conditions The evolution in the indi rect dimension is achieved in such a way that the delay between the two pulses which is the evolution period t4 is incremented by integer multiples of the rotor period Two extremely important points must be considered for the experimental realization of the ST MAS experiment Firstly the spinning frequency must be kept absolutely constant The dura tion of the rotational echoes in the STMAS experiment is determined by the width of the satellite transition giving a length of e g 1 us for a satellite transition of 1 MHz width If the ro tor period varies from that specified in the parameters the calculated delay in the pulse pro gram is incorrect and the pulse misses the echo top so less or no signal intensity is obtained Table 17 1 summarizes the time deviation that occurs when the spinning frequency fluctuates by 1 Hz and 10 Hz at various desired spinning frequencies One can see that when the t increment accumulates to as much as 100 rotor periods it is possible to miss an echo com pletely For example if the dur
170. he dipolar coupling between the correlated sites 2 Proton proton DQ SQ correlation similar to the high resolution INADEQUATE correlates proton chemical shifts with DQ frequencies of dipolar coupled sites References 1 P Caravetti P Neuenschwander R R Ernst Macromolecules 18 119 1985 2 S P Brown A Lesage B Elena and L Emsley Probing Proton Proton Proximities in the Solid State High Resolution Two Dimensional 1H H Double Quantum CRAMPS NMR Spec troscopy J Am Chem Soc 126 13230 2004 The modifications according to the chapter Modified W PMLG are implemented in order to remove the carrier spike Without a carrier spike 2D experiments are much easier and faster to set up Being able to set the carrier close to the desired spectral range one can make the total acquired window smaller as well along F2 using digital mode as along F1 Proton Proton Shift Correlation spin diffusion 22 1 The standard CRAMPS setup must be executed first see chapter 20 21 Any Homo nuclear dipolar decoupling scheme may be used but in the following the experiment is described using windowed pmlg w PMLG The reasons are the following 1 At fast spin rates over 10 kHz only w PMLG and DUMBO work well The sequence can easily be modified to use DUMBO replacing the w PMLG shapes by DUMBO shapes and modifying the shape timing accordingly 2 W PMLG is easy to set up since it is rather insensitive to power level missets and
171. he one obtained with the DCP experiment gives the DCP yield see Figure 18 7 200 261 BRUKER BIOSPIN User Manual Version 001 Double CP ie noe t he E Ej 5 es 5 8 A E 09801 0 for suste 20 1 Ciidata700 3020207 Peale 0 7000 Figure 18 7 Double CP yield measured by comparing CPMAS and DCP amplitudes of the high field resonance Note that the C4 carbon receives very little magnetization under these conditions the transfer is rather selective The setup for DCP can be rather much sped up and simplified by a python program named dcpset py This program will ask for the 90 pulse widths and associated power levels and for the spin rate and calculate the appropriate power levels for the HH condition for all pulses ex cept for the proton channel Contact solids bruker de to receive this python program and some instructions for use This program serves as an example on how the experiment setup can be controlled by the high lev el script language Setup of the 2D Double CP Experiment 18 2 4 1 Load a suitable sample spin it up set the desired temperature and match and tune the probe As a simple setup sample full C 19N histidine may be used d1 10s 2 4 scans p15 1msec p16 3msec A labeled oligopeptide or small protein will of course provide a more interesting spectrum With proteins good results should only be expected if the prep aration is micro crystalline In such a case water salt and c
172. he power level for on reso nance HH match For the new nutation frequency B1 field for LG condition Bio CH sin 0 Bion H 0 82 li Bion S CH the offset frequency for the Lee Goldburg condition is fic cos 0 T Phocas CH 0 578 Boss CH with the inverse of a 360 pulse Instead of raising the power level for Dior js 1 7 15N the power level for 1H is reduced by about 1 7 dB Then the new 27 pulse in the tilted frame is sin 0 0 67 B lon _ res 1 Tous B lon res In our example of a contact power level of 50 kHz on 15N one would then calculate for cnst20 40 807 0 giving an offset frequency of 28855 Hz for the LG frequency which is calculat ed automatically 5 n order to verify all calculated power levels and offset frequencies optimize for the appro priate power level using the pulse program cplg 6 If the sample does tune and match very differently than the setup sample check the HH conditions briefly and verify that the found parameters are valid Correct power levels or pulse parameters if needed This is especially important for saline lipid water mixtures 7 Create a new experiment and setup a 2D data set as described in the previous chapter User Manual Version 001 BRUKER BIOSPIN 133 261 PISEMA Setup 2D Experiment 13 3 1 134 261 After 1D parameter optimization as previously described type iexpno to create a new data file and switch to the 2D mode using
173. hem Phys Lett This allows reduced measurement times Pmlghet wpmlghet dumbohet and ed umbohet should give rather similar spectra Table 8 1 Acquisition Parameters for pmlg HETCOR on tyrosine HCl Parameter Value Comments pulprog pmlghet Using phase ramps nuc1 13C olp 100 ppm nuc2 1H cnst20 80 100000 Proton spin nutation frequency with PL13 cnst24 1000 3000 Place carrier within proton spectrum for evolution pr Power level channel 1 for contact pulse pl12 Power level channel 2 TPPM SPINAL decoupling pl13 Power level channel 2 PMLG decoupling 86 261 BRUKER BIOSPIN User Manual Version 001 HETCOR with DUMBO PMLG or w PMLG Using Shapes Table 8 1 Acquisition Parameters for pmlg HETCOR on tyrosine HCI sp0 Power level channel 2 for contact pulse spnam0 ramp 100 or similar Shape for contact pulse channel f2 sp1 set to pl13 To match cnst20 spnam1 Igs 2 To match ppg calculation of inO in f1 p3 2 5 3 usec 90 pulse channel 2 at pl12 p15 50 500us Contact pulse width pcpd2 2 p3 SPINAL64 TPPM decoupling pulse cpdprg2 SPINAL64 TPPM15 Decoupling sequence F1 1H indirect 10 0 Start value 0 incremented during expt 13 2 4 Multiples of FSLG periods increment per row in_fl in0 as calculated Set according to value calculated by ased F2 13C acquisition di 2s Recycle d
174. herence transfer pathway for the 3Q MAS experiment with z filter mp3dfsz av and double frequency sweep DFS Excitation pulse p1 and selective pulses P3 are the same as for mp3qzfil av Delays DO and D4 are the incremented delay for t4 evolution and 20 us for z filter respectively Delay D10 can be incremented for spin 3 2 nuclei propor tional to DO Power level and duration of the sweep P2 must be optimized Phase lists are as follows for phase sensitive detection in F1 the phase of the first pulse must be incremented by 30 in States or States TPPI mode ph1 0 60 120 180 240 300 ph2 0 24 90 24 180 24 270 24 ph3 0 ph4 0 6 90 6 180 6 270 6 receiver 0 180 3 90 270 3 180 0 3 270 90 3 180 0 3 270 90 3 0 180 3 90 270 3 hi ph2 spi Figure 16 4 Three Pulse Sequence and Coherence Transfer Pathway 172 261 BRUKER BIOSPIN User Manual Version 001 MQ MAS Sensitivity Enhancement Three pulse sequence and coherence transfer pathway for the 3Q MAS experiment with z filter mp3qdfs av Excitation pulse p1 is the same as for mp3qzfil av P4 is a central transition se lective 180 pulse usually 2 p3 Delays DO is the incremented delay for t4 evolution Delays D10 or D11 must be incremented proportional to DO Power level and duration of the sweep P2 must be optimized Phase lists are as follows 2D data are acquired in QF mode which means that no phase incrementation is required ph1 0 30 60 90 120
175. hermometer 55Mn KMnO MAS gt 500 MHz pattern 93Nb 207pp PbNOz MAS shift thermometer 0 753 ppm degr d1 gt 10s Pb p tolyl 4 CP 150 5ms 15s 295 QgMg CPMAS 50 d1 gt 5s reference sample 12 6 108 ppm DSS TMSS CPMAS 0 reference sample O ppm Se H3Se03 CPMAS 1800 HH setup 8ms contact d1 gt 10s NH4 2Se04 CPMAS 200 3ms d1 gt 4s ca Cd NO3 4H50 CPMAS 350 15ms contact d1 gt 8s 195Pt K2Pt OH 6 CPMAS a 1ms contact d1 gt 4s 12000 19 Hg Hg acetate CPMAS 2500 5ms contact d1 gt 10s 2H d PMMA WL 0 wideline setup d1 5s d PE WL 0 wideline setup d1 0 5s 10s amorphous crystalline d DMSO WL 0 exchange expt at 315K e LiCl Li org make sure it is not 9Li depleted d1 gt 60s 170 D20 0 pulse determination 100scans 0 5s 15N a glycine CP 50 sensitivity 4ms contact 4s labelled for fast setup ol KCI WL MAS O pulse determ 100 scans sis KoS MAS 0 100 scans in a gt 500 MHz instr 14N NH4CI MAS WL 0 100 scans narrow line 25Mg eem Anatas MAS 39K KCI MAS WL 0 100 scans 109 y AgNO3 MAS 1scan 500s finely powdered Say Y NO3 3 6H20 CPMAS 50 10ms contact d1 gt 10s 12 261 BRUKER BIOSPIN User Manual Version 001 Basic Setup Procedures This chapter contains information and examples on how to set up basic solid state NMR SSN MR experiments We ll begin with the settings for the RF routing of the spectrometer some basic setup procedures for MAS probes and how to measure their radio frequency
176. iagram 10 1 oy O10 1H SPINAL 64 SPINAL64 decoupling decoupling cw DARR pl14 02 bs s prec A ti mixing t2 63 00000000 1 Qrec 02201331 Figure 10 1 CPSPINDIFF Pulse Sequence Basic Setup 10 2 1 On a standard sample i e glycine determine HH match and decoupling parameters 2 Check the X 90 hard pulse with cp90 on the standard sample 3 Ifthe real sample does tune and match very differently than the setup sample verify the HH conditions briefly and eventually the X 90 hard pulse 4 Set the spin rate as high as possible make sure to avoid rotational resonance conditions overlap between center bands and sidebands recheck the HH condition Set cnst31 spin rate 5 Optimize contact time o1 and o2 on a 13C 1D CP experiment if necessary 6 Create a new experiment with either iexpno or edc 7 Change to a 2D data set 2D Experiment Setup 10 2 1 8 Type expno to create a new data file and switch to the 2D mode using the 123 button Load the pulse program cpspindiff User Manual Version 001 BRUKER BIOSPIN 101 261 Proton Driven Spin Diffusion PDSD 11 12 13 Recheck pulse widths and power levels using ased Go into eda and set parameters for sampling in the indirect dimension the spectral width 7 swh Note that in TopSpin 2 1 or later the parameter N F1 replaces the parameters inO and nd0 Usually 1 swh equals swh Choose a suitable spin rate suc
177. ic Angle on KBr 3 2 For all following steps generate new data sets with appropriate names using the edc com mand to record all individual setup steps RF Routing 3 2 1 The spectrometer usually has 2 or more RF generation units SGU s transmitters and pream plifiers In order to connect the appropriate SGU to the appropriate transmitter and the trans mitter to the associated preamp where the probe channels are connected there are several routing possibilities In order to minimise errors in hardware connections the routing is under software control where possible Where cable connections need to be done manually the soft ware does not allow a change These connections are made during instrument installation Enter the edasp command or click the Edit button in the nucleus section in the acquisition parameter window eda in order to get the spectrometer router display Alternatively click on the routing icon in eda frequency logical channel amplifier preamplifier BFI 150 360709 MHz NUCI ae SFO1 150 369709 me pn L su x E xow momo vegan OFS1 9000 0 Hz 792e HPHP 19F IH Hane xena 1H 1000 BF2 600 147 MH NUC P a SFO2 600 147 MHz F sour 1H Y 1H 100 W HPHP 19F 1H OFS2 oo Hr ES v HPLNA IH Bf3 600 147 MH NACH SFO3 600 147 MHz F3 Seu3 X HL x 300 W OFS3 10 0 Hz lors v BF4 600147 Miro NUCH SFO4 600 147 MHz F4 GET 1H 1H 50 W OFS4 0 0 Hz ES cable wiring settings
178. ight resolution enhancement qsine SSB 3 Proton shifts calibrated as 2 5 and 12 most high field low field peak Expansion plot 82 261 BRUKER BIOSPIN User Manual Version 001 Modifications of FSLG HETCOR The basic HETCOR sequence can be improved in several respects The protons which are ob served are all coupled to 13 carbons since we observe these So the proton shifts evolve also under the residual dipolar coupling and the J coupling to 13C This can be refocused bya 136 Tr pulse in the middle of the proton evolution The pulse program Ighetfqpi will serve this purpose Furthermore it may be desirable to compare the proton shift spectrum obtained with X obser vation HETCOR with the proton spectrum obtained by CRAMPS techniques see chapters 22 23 and 24 observing the protons directly Usually these experiments use phase modulat ed shapes PMLG or DUMBO In order to make both experiments comparable it is favored to use the same type of proton shift evolution in both sequences The pulse programs which use phase gradient shapes to achieve Homo nuclear proton dipolar decoupling are pmighet and wpmlghet If DUMBO decoupling is desired the pulse programs are dumbohet or edumbohet These pulse programs use either windowless pulse trains or windowed pulse trains which can be timed in exact analogy to the CRAMPS type sequences wpmlg 2 and dumbo 2 These se quences also suppress the center ridge efficiently so that the carrier freq
179. igned pulse program that contains e g a string 5q but performs a 3Q experiment and vice versa will yield erroneous shearing When started the AU program prompts for Apply ABS2 and F1 shift in ppm It is advisable to calculate a baseline correc tion after F2 Fourier transform Note that the range defined by ABSF1 and ABSF2 is used for this You should make sure that the limits are at least as large as the spectral width to allow baseline correction of the whole spectrum The F1 shift in ppm allows shifting the spectrum BRUKER BIOSPIN User Manual Version 001 Basic MQ MAS including its axis in the vertical direction for cases where peaks are folded due to a limited spectral window in a rotor synchronized experiment For the first processing both prompts are typically returned At the end of the processing the AU program corrects the apparent spec trometer frequency of the indirect dimension by a factor R p where R is defined in equation 1 and p is the order of the experiment e g 3 for 3QMAS Eq 15 1 p SIG 1 8 5m 5 181 I 1 3 5 This ratio is calculated from the spin quantum number of the nucleus and the magnetic spin quantum number m which is determined by the experiment e g 3 2 in case of a 3Q experi ment of an order p 3 The program stores the F1 shift that was calculated and will prompt for it when data are processed next time If the same F1 shift should be applied as before the AU pr
180. ing so only 10 to 40 experiments in amor phous samples but 50 to 200 experiments in crystalline samples might be required The rotor synchronization means that the spectral range in F1 is limited Depending on the chemical shift range spinning frequency and quadrupole interactions the positions of the peaks may fall out side this range In such a case care must be taken when interpreting the spectrum Acquisition with half rotor synchronization to double the spectral window in F1 may help However in this situation one set of spinning sidebands appears and it must be avoided that the spinning side bands of one peak fall on top of other peaks Some sort of rotor synchronization is always rec ommended because spinning side bands in the indirect dimension extend over a very wide range which cannot be truncated by e g filtering Therefore rotor synchronization together with States or States TPPI phase sensitive acquisition helps to fold spinning sidebands from outside back onto centre bands or other side bands User Manual Version 001 BRUKER BIOSPIN 159 261 Basic MQ MAS Data processing 15 4 Processing parameters should be set according to the following table Table 15 4 Processing Parameters for 2D FT Parameter Value Comments F2 acquisition dimension SI Usually set to one times zero filling WDW no Don t use window function PH_mod pk Apply phase correction BC_mod no No DC cor
181. ing in a desiccator Drying is important because wet glycine may readily transform especially when kept warm into y glycine a glycine has two carbons with shifts of 176 03 and 43 5 ppm y glycine shows resonances somewhat shifted to higher field sharper lines longer proton T4 and shorter proton T4 which results in longer experiment time and less signal to noise Spin the glycine sample at 5 kHz 7mm spinner or 10 kHz smaller spinners 4 3 2 or 2 5 tune and match the probe The glycine cp mas 13C spectrum taken under the same conditions as adamantane previously will look like in Figure 3 23 far from optimum e DT Elle Edil View Specirometer Processing Analysis Options Window Help QGCcS 9 6 m8 o4 A 17b u 8 apg TTO tie El nans 14484820 AN gt ee att A ho ACT ARCE opt Tas wo span E AYRUP nmi GANA paura Figure 3 23 Display Showing a Glycine Taken Under Adamantane Conditions 4 scans The figure above shows a glycine taken under adamantane conditions 4 scans Incorrect car rier setting a carbon at 43 ppm insufficiently decoupled Angle is set correctly because car boxyl peak at 176 03 ppm shows a narrow lorentzian line shape HH condition looks okay Now reset the carrier as shown in Figure 3 13 o1p should be around 100 ppm in the middle of most carbon spectra Acquire a spectrum set the plot limits Figure 3 14 Figure 3 15 for the peak at 43 ppm and start popt optimizing 02 for maximum si
182. ing power level 1 100up 0 fq cnst21 reset decoupling carrier frequency to o2 cnst21 jump to 1 repeat until decoupler is switched off by do in the main ppg CW decoupling suffers from the fact that protons have different chemical shifts so irradiating at a single frequency does not decouple all protons evenly At higher magnetic fields this be comes more evident since the separation due to the magnetic field increases CW decoupling requires fairly high decoupling power to be efficient User Manual Version 001 BRUKER BIOSPIN 45 261 Decoupling Techniques TPPM Decoupling 4 1 2 TPPM decoupling surpasses the traditional cw decoupling The decoupling programs tppm15 and tppm20 use a 15 and 20 degree phase shift between the two pulses respectively Both op erate at power level p 12 The cpd program tppm13 uses 15 degree phase shift as tppm15 but operates at power level p 13 In order to optimize the decoupling one optimizes pcpd2 AV3 or p31 AV1 2 with popt and the carrier frequency by varying 02 or o2p Strongly proton coupled 13C resonances narrow substantially especially at high magnetic fields gt 300 MHz The figure below shows an arrayed optimization using popt for the TPPM phase tilt and pepd2 available in TS2 0 and higher 75 Tite Det Pazi Mtega Sample Structure Fidi Shae Cabri Baseline Figure 4 1 Optimization of TPPM Decoupling on Glycine at Natural Abundance The figure above
183. ing the 2D fourier transformation type s sw to call the status parame ters for both F2 and F1 and replace both values by current value gt 0 6 After xfb the relative peak positions will be approximately correct but the absolute peak positions must be correct ed by calibrating a known peak position to the correct value The pulse program is written such that the correctly scaled sweep widths are calculated and indicated upon ased These values are set as status parameters before transform as indicated above Table 22 3 Processing Parameters Parameter Value Comment mc2 STATES TPPI wdw QSINE Slight moderate resolution enhancement is usually required ssb 3or5 si 2k 4k 1 si 512 1k User Manual Version 001 BRUKER BIOSPIN 227 261 CRAMPS 2D Examples glycine proto proton shift correlation using ug for homonuclear decoupling in bot x mixing time shows all possible corri plot of ROI only plot of full spectrum including carrie a n Le uw F alasasasasalasasasasala EI pS Ki K D H D D D D D D D HE te 22 4 ppm J LIA 0 P ol ow 5 10 je e ee e ep 20 D I vv vw AS id penne 3 20 10 0 ppm f l N u Be e y ppm Figure 22 2 Setup and Test Spectrum of Alpha glycine The figure above shoes the setup and test spectrum of alpha glycine N B glycine samples containing gamma glycine will show additional peaks The
184. ion is defined by the correlation time of the motion For efficient relaxation via a particular energy level transition fields fluctuat ing with an inverse correlation time close to the frequency of the transition are required Longi tudinal relaxation occurs via transitions on a single spin and thus requires fields fluctuating with inverse correlation times near to the Larmor frequency Transverse relaxation occurs also via flip flop transitions of pairs of spins which have energies close to zero and so local fields fluctuating very slowly will cause transverse relaxation T relaxation involves transitions at the nutation frequency of the spin locking pulse which can be chosen by the experimenter Measurement of these relaxation rates can therefore provide information about local motions on a range of time scales T1 Relaxation Measurements 14 2 Longitudinal relaxation can be measured using a number of methods which method is appro priate depends on the sample involved Here the experiments are demonstrated on glycine which has a very simple spectrum and will give results using all the methods discussed In gen eral the only setup required is to calibrate pulses for the nucleus under observation and to have some idea of the relaxation time constants involved Experimental Methods 14 2 1 142 261 The inversion recovery method is the originally proposed method for measuring T4 values The experiment proceeds as follows firstly
185. ired RF field lower than the reference field subtract the number if the desired RF field is higher If such a table is not available but an oscilloscope is one can measure the RF voltage for the X contact pulse of the known 130 HH condition calculate the pp voltage for the un known HH condition from the NMR frequencies of the two nuclei and set this voltage for the unknown HH condition Hints Tricks Caveats for Multi nuclear CP MAS Spectroscopy 5 5 1 Since T4 relaxation tends to be slow in solids direct observation of hetero nuclei is usually time consuming so CP is widely used because the proton T4 is usually bearable However CP can only be used if the hetero nucleus is coupled to protons or whatever nucleus the magnetization is drained from Whereas 13C and 9N usually bear directly bonded protons this is not the case for many other spin hetero nuclei So the magnetization must come from more remote substitutes More remote they may also be because atomic radii increase as one goes to nuclei with higher atomic mass In short HH conditions may be very sharp Ti s may be long but proton T may still be short Chemical shift ranges and chemical shift anisotropies increase with nuclei of higher order number and number of electrons in the outer shell Therefore one may be confronted with two problems to find the signal somewhere within the possible chemical shift range to find the signal within a forest
186. is means that the first increment can last between 100 us 10 kHz spinning and 28 5 us 35 kHz spinning Since P4 is the 180 selective pulse which can be as long as 40 us LO must be set large enough to avoid the situation where the calculated d0 is negative Optimization of the pulses PT and P2can be done using popt in full analogy to the optimization of the pulses in MQMAS BRUKER BIOSPIN User Manual Version 001 STMAS Table 17 4 Initial Parameters for the Set up of stmasdfqe av Parameter Value Comments pulprog stmasdafe av Pulse program NS 16 n For set up the full phase cycle is not so critical DO See text Calculated in pulse program D1 5 T4 Recycle delay use dummy scans if shorter D6 See text D7 See text P1 1 5 us Excitation pulse at pl11 P2 1 5 us Conversion pulse at pl11 P3 20 us 90 selective pulse at pl21 taken from previous pulse cali bration PL1 120 dB Not used PL11 Start with 150 to 300 W Power level for excitation and conversion pulses PL21 Power level for selective pulse approx pl11 30 dB taken from previous pulse calibration Two Dimensional Data Acquisition 17 4 2 Once the pulses are calibrated the 2D data acquisition can be used to find the correct and pre cise magic angle setting Create a new data set and change parmode to 2D The acquisition parameters for the new indirect F1 dimension must be set according to Table 17 5 Similar co
187. itation P3 Excitation pulse f2 channel 202 261 BRUKER BIOSPIN User Manual Version 001 Double CP Table 18 2 Recommended Parameters for the DCP 2D Setup PCPD2 Decoupler pulse length f2 channel H TPPM P15 1 5 msec First contact optimize on 15N cp spectrum P16 3 10 msec Second contact f1 f3 channel optimize on 1D dcp spectrum D1 5 10s for histidine Recycle delay optimize on 1d SPNAMO Ramp for 18t CP step e g ramp 80 100 SPO Power level for Ramp HN contact pulse 1H SPNAM1 tcn5500 Tangential or ramp contact pulse spnam2 square 100 Shape on 15N channel CPDPRG2 SPINAL64 SPINAL64 decoupling NS 2 or 16 Number of scans F2 direct 13C left column TD 2k Number of complex points SW 200 ppm Sweep width direct dimension adjust to experimental requirements F1 indirect IDN right column TD 128 512 Number of real points SW 100 150ppm Sweep width indirect dimension Spectral Processing Processing parameters 18 4 Table 18 3 Recommended Processing Parameters for the DCP 2D Parameter Value Comment F1 acquisition 13c left column SI 2 4k FT size WDW QSINE Squared sine bell SSB 2 5 Shifted square sine bell gt 2 res enhancement PH_mod pk Phase correction if needed F2 indirect IDN right column SI 512 1024 Zero fill MC2 STATES TPPI WDW QSINE Squared sine bell SSB 2 5 Use
188. ization will stop without evaluation of the data You can also type kill in the command line and click on the bar with poptau exe to stop optimization This will work like stop optimization Popt will generate a data set where the selected expansion part of the spectrum is concate nated for all different parameter values in this case for p7 It will have a procno around 999 To achieve this processing parameters are changed appropriately Fourier transforming a nor mal FID in such a window will generate an incorrect spectrum window User Manual Version 001 BRUKER BIOSPIN 29 261 Basic Setup Procedures Therefore Never start an acquisition in such a window first read in the procno where popt was started using the rep n command where n is the source procno No acaso runna ope on sarita manva matt town Triebe CARTE Zrd 0099 Somlrum ProcPars AcquPars Tite Pulse rog Peaks integrats Sample Structure Fid Acqu Phase Print postau for pi teistie POSMAX at experiment 12 pt 6 0000 NEXP 20 2 4 6 9 us ec Figure 3 17 The popt Display after Proton p1 Optimization The figure above shows the popt display after proton p1 optimization the biggest signal is ob tained at 6 usec in this case Once you have obtained a 90 degree pulse for a given power setting you can calculate power levels for different rf fields using the AU program calcpowlev Type xau calcpowlev into the command line and follow the inst
189. kHz RF field ps 1 2 usec p9 4 2 6 usec To be optimized p5 not used calculated from cnst20 To be optimized d1 4s For a glycine 111 anavpt 4 2 4 8 16 or 32 o1p 3 8 To be optimized swh 1e6 2 2 p9 10 p5 0 47 To be corrected for proper scaling rg 16 64 td 512 Up to 1024 si 4k digmod analog MASR 12 15 kHz Depending on cycle time Table 21 3 DUMBO Analog Mode Parameter Value Comment pulprog dumboa2 Runs on AV 3 instruments only pli2 For 100 kHz RF field sp1 up to 130 kHz To be optimized during setup spnam1 dumbo1 64 Set by xau dumbo p1 2 5 usec For 100 kHz RF field p8 1 2usec p9 4 2 6 usec To be optimized p10 32 usec or 24 usec Set by xau dumbo d1 4s For a glycine User Manual Version 001 BRUKER BIOSPIN 223 261 Modified W PMLG Table 21 3 DUMBO Analog Mode 111 anavpt 4 2 4 8 16 or 32 o1p 5 To be optimized swh 1e 2 2 p9 p10 0 5 To be corrected for proper scaling rg 16 64 td 512 Up to 1024 si 4k digmod analog MASR 10 12 kHz Depending on cycle time Fine Tuning for Best Resolution 21 5 Fine tuning is done by optimizing power levels pulse widths and carrier offset as before the carrier spike is gone spikes at both sides may appear Correcting for Actual Spectral Width 21 6 The modified sequence has a slightly different scaling factor of 0 47 Digital Mode Acquisition 21 7 Most parameters stay the same as adjusted in analogue m
190. lds a 4 5 usec proton pulse Set pl such that in ased the power displayed is 200W for 86 7mm probe 150W 4mm probe or 80W 2 5 mm probe If the green dot is not visible in edasp for the 13C channel set pl1 to 12 dB 1 kW transmitter 9 dB 500W transmitter or 7 dB 300W transmitter for any probe Make sure the proton channel is tuned wobb high and the carbon channel is also tuned wobb With d1 4s rg 256 swh 100000 td 4k 02 set to be on resonance on the adamantane pro tons as found above accumulate 4 scans Set the carrier frequency between both adamantane 56 peaks Reduce the spectral width swh to 50 kHz set aq 50 msec Acquire 2 4 scans and define the plot limits as shown in Figure 3 14 for the larger of the two peaks Define the plot limits and determine the 90 degree carbon pulse p7 using popt Recal culate pl1 for a 4 5 usec carbon pulse using calcpowlev Pulse continuously using gs and shim the z gradient for highest FID integral The gradient settings can be conveniently changed in the setsh display Figure 3 18 and Fig ure 3 19 show the adamantane C FID without shims with z shim adjusted and the corre sponding setsh displays SGT Setshim Set shim values and lock parameters Press ENTER to set new value b D 0 0 0 0 0 0 0 5 ck olojoloalolo Figure 3 18 Adamantane 19C FID with 50 msec aq setsh Display User Manual Version 001 BRUKER BIOSPIN 31 261 B
191. lective pulse this is not the case User Manual Version 001 BRUKER BIOSPIN 155 261 Basic MQ MAS 05 10 15 20 25 30 35 ppm 20 40 60 80 ppm 156 261 Figure 15 4 Nutation profiles of selective and non selective pulses Left diagram shows signal intensity of 87 Rb resonances in RbNO3 as a function of a non selec tive pulse at approx 150 W RF power the right diagram shows the signal intensity as function of a selective pulse at less than approx 0 5 W Spectra are taken on AV500WB at a Larmor frequency of 163 6 MHz with 2 5 mm CP MAS probe spinning at 25 kHz Note the different scaling of x axis which is displayed as ppm but corresponds to the used pulse lengths in us apart from the sign Once the central transition selective 90 pulse is calibrated the parameters can be copied to a new data set with jexpno and the MQMAS pulse program can be loaded Available pulse pro grams are mp3qzqf and mp3qzfil The first is a 3 pulse sequence the second a 4 pulse se quence The sequence with fewer pulses will be slightly more sensitive whilst the 4 pulse sequence can be used as an initial set up for experiments with sensitivity enhancement meth ods like DFS or FAM see MQ MAS Sensitivity Enhancement on page 169 describing sensitivity enhancement methods In Table 15 2 the starting parameters for the set up are displayed This table gives typical val ues for the pulses and powers that should be close to the final valu
192. less or windowless sequences These sequences work well but have rather long cycle times and are therefore not suitable for fast spinning samples Else they work in a similar fashion as the sequences covered in the following BLEW 12 decoupling is sup plied as a standard cpd program It consists of a windowless sequence of 90 pulses with suit able phases High RF levels for decoupling provide better resolution FSLG Decoupling The Frequency Switched Lee Goldburg FSLG sequence may be used at spin rates up to 15 kHz It is a Homo nuclear decoupling sequence which rotates the interaction Hamiltonian around an effective field aligned at the magic angle arctan 2 with respect to the Zeeman field in the rotating frame The tilt is achieved by off resonance irradiation at the Lee Goldburg frequency f G according to the Lee Goldburg condition fLG 2f with f 1 41 p being the nutation frequency of the magnetization in the rotating field under on resonance conditions t p is the 90 pulse width In the rotating frame the frequency switching induces 2p rotations in op posite directions in the tilted rotating frame Such rotations can be achieved by irradiation peri ods at the Lee Goldburg frequency f c of duration t_g sin 54 7 f1 2 3 f with the rf carrier jumping between the two frequencies he and he with a simultaneous x phase shift BRUKER BIOSPIN User Manual Version 001 Decoupling Techniques Figure 4 2 Geometry forthe FSLG C
193. llowing experiments can be run by calling a C CPMAS standard parameter data set or data loading the appropriate pulse program and loading the pulse parameters obtained previ ously during the setup see Basic Setup Procedures Some attention needs to be paid to special experimental parameters Most of those parameters are explained in the header sec tion of the pulse programs The CPPI experiment series in 6 5 requires measuring the HH match using a constant ampli tude contact pulse This can be accomplished using a rectangular shape square 100 or using the pulse program cplg Pulse Calibration with CP 6 1 1 Pulse calibration for C pulses after cross polarization using a flip back pulse hu 63 heteronuclear decoupling Q2 94 Ore 36 Figure 6 1 Pulse Program for CP with Flip back Pulse The experiment can be done directly after the CPMAS setup procedure Loading the pulse pro gram cp90 and setting p 1 pI11 allows one to measure the X nucleus spin nutation frequency at the HH contact power Of course the experiment allows nutation frequencies to be mea sured at other power levels as well The typical nutation pattern has a cosine form so a 90 de gree pulse gives null signal Use glycine spinning at N kHz as before When using POPT for such measurements the optimization type is ZERO so that the program looks for a zero User Manual Version 001 BRUKER BIOSPIN 61 261 Basic CP MAS Experiments crossing at the auto
194. lse lengths and phases and it requires RF fields strong compared to the interaction and shift distribution Many sequences have been devised after the original WHH 4 or WaHu Ha sequence which yield better results due to better error compensation MREV 8 BR 24 C 24 TREV 8 MSHOT Modern hardware has made the setup and application of these se quences a lot easier since pulse phase and amplitude errors are negligible higher magnetic fields have led to better chemical shift dispersion and also to shorter dead times The resolu tion achieved with long highly compensated sequences like BR 24 is very good but their ap plicability at limited spin rates because of the need for the cycle time to be short with respect to the rotor period often presents a problem References S Hafner and H W Spiess Multiple Pulse Line Narrowing under Fast Magic Angle Spinning J Magn Reson A 121 160 166 1996 and references therein M Hohwy J T Rasmussen P V Bower H J Jakobsen and N C Nielsen 1H Chemical Shielding Anisotropies from Polycrystalline Powders Using MSHOT 3 Based CRAMPS J Magn Res 133 2 374 1998 and references cited therein W PMLG and DUMBO 19 3 W PMLG and DUMBO are shorter sequences which avoid turning high power pulses rapidly on and off which is what most multiple pulse sequences do This avoids undesired phase glitches Also they use higher duty cycles during the decoupling period As a result the se quenc
195. lve and the type of Homo nuclear decoupling sequence FSLG in this case which scales chemical shifts by about 0 578 in this case In order to obtain proton chemical shifts at the standard scale both parameters are taken into account and an increment along F1 is calculated which yields correct chemical shifts for protons Transfer this increment to N F1 in eda A button in ased This will set the sweep width along F1 Note that the time incre ment here is generated by a loop counter counting the periods of FSLG The loop counter I3 is used to multiply this increment Usually 13 is set to 2 4 in order to reduce the F1 sam pling width to a reasonable value Cnst24 is usually set to 1000 2000 in order to move the spectrum away from the center ridge in F1 User Manual Version 001 BRUKER BIOSPIN 7T 261 FSLG HETCOR o AM EDY d installec prove 4 mm MAS DECH H13762 0001 78 261 General Y General Channel f1 PULPROG Channel f2 TD 1594 NS 4 DS n SWH Hz 50000 00 AQ s o 0169900 RG 126 DW ps 10 000 DE sl 10 00 CNST11 1 0000000 CNST20 100000 0000000 CNST24 2000 0000000 D1 s 2 00000000 Ind ls 0 00005654 in n L3 3 count 64 dwell 5 0 00005654 Y Channel ti dIktr us 2 00 NUCA 130 P15 us 300 00 PL1 dB 4 50 PLTW Ww 104 27635956 SFO1 MHz 150 9220830 Y Channelt2 biktr2 sl 1 00 cnst21 a 000000 enst22 68710679688 enst23 72710 679698 CPOPRG2 spinal d NUC2 1H Edit P3 us 2 20 p
196. ma g d Scale 1 101 g spinditt 10 520 CiM rukertTOPZPIN namali i 5 m en A n A BAS La i r D 1 o ra j j m La m L Lo L U A p ER M ium nee ee _ _ FF o D j 160 140 12 100 80 ppm Figure 10 5 Comparison of DARR PDSD The figure above is a comparison of DARR PDSD with 4 6 and 20 msec mixing time sample tyrosine HCI spinning at 22 kHz Traces through peak at 115 ppm most high field aromatic car bon Traces from below DARR at 4 6 msec mix PDSD at 4 6 msec mix DARR at 20 msec mix and PDSD at 20 msec mix Note that some cross peak intensities differ substantially User Manual Version 001 BRUKER BIOSPIN 107 261 Proton Driven Spin Diffusion PDSD O d a Aca N Wid V i L efree 4mm rOO SB DARR C C correlation 8 msec mix 100 rotor periods d 20 F1 ppm 10 10 A Mm rk A O T T T T T T T T T T T T T 100 50 F2 ppm T T T T 150 Figure 10 6 13C DARR of Fully Labelled Ubiquitine Spinning at 13 kHz 108 261 BRUKER BIOSPIN User Manual Version 001 SUPER Overview 11 1 Separation of Undistorted Chemical Shift Anisotropy Powder patterns by Effortless Recoupling SUPER correlates CSA powder patterns in the F1 dimension with the isotropic chemical shift in the F2 dimension The SUPER experiment is based on Tycko s CS CSA correlation exper iment but provides better compensati
197. matic data evaluation To get nutation patterns without phase distortions 90 pulses should always be executed close to the observed resonance Larger offsets give different shorter p90 values and phase distortions for pulse lengths close to 180 and multi ples thereof Table 6 1 Acquisition Parameters Parameter Value Comments pulprog cp90 AVIII cp90 av for older instruments nuc1 13C Nucleus on f1 channel nuc2 1H Nucleus on f2 channel sw 300 Spectral width for Glycine ppm olp 45 Close to C a td 2048 Number of points sampled Fine adjustment of the x pulse on 13C can also be done using the TOSS experiment see next chapter Total Sideband Suppression TOSS 6 2 62 261 The TOSS sequences permit complete suppression of spinning sidebands SSB in CPMAS experiments The TOSS sequence consists of the basic CP sequence plus a 2 rotor period se quence with four specially spaced 180 pulses As is the case for all extra pulses on the X channel in CPMAS experiments with the exception of symmetry based sequences see further below these 180 pulses are set with pl11 This experiment can be optimized for minimum spinning sideband intensity either by variation of the 180 pulse width or the associated power level pl11 Two variations of the TOSS sequence exists the default is TOSS A which is appropriate for lower spinning speeds TOSS B for higher spinning speeds is selected by setting ZGOPTNS to D
198. ment i e about 1 8 dB higher than PL2 which is less RF power Start the experiment BRUKER BIOSPIN User Manual Version 001 A Channel f2 PULPROG TD NS os SVH Hz AQ s RG DW us DE ps CNST11 CNST20 CNST24 Di 5 ind 5 0 La count Y Channel 11 NUCI P14 lus PL dB PL11 dB PL11W DN PLIW il SFO1 MHz Y Channel f2 enst21 cn322 enst29 CPDPRG2 NUC 37878 79 0 0270836 32 13200 1 00 1 DO0000D 62500 0000000 1 0000000 5 00000000 0 00004286 32 e 2000 00 20 12000 0 00000000 158 49931895 125 7502468 0 000000 44195 171875 44193 171875 spinal64 1H Eot RE Le Figure 13 3 ASED Display for the PISEMA Setup PISEMA Pulse program tor acquisitan Time domain size Number of scans Number of dummy scans Sweep wicth in Hz Acquisition time Receiver gain Dwell time Pre scan delay To adjust tO for acquistion if digmod base LG RF field as adjusted In Hz used ta cacuat Offset far proton evolution under LG usualy D Recycle delay h0z2 p5 3 0 82 LO 0 For gwel in 11 22 pS 3 0 62 Count t01 2 Nucleus for channel 1 Cortact pulse For X cortact puse For X cortact during SEMA For x contact during SEMA For X contact puse Frequency of observe channel Cnst21 0 Cnst22zcnst20 sqrt 2 cnst24 Cnst23 cnst20 sqrt 2 cnst24 Cw pms spinale4 Nucleus for channel 2 With L3 2
199. meters and verify that there is again no signal Make a new data set with jexpno and set parameters for 2D acquisition as for the previous experiments D1 can be short with the same proviso about duty cycle as the X saturation re covery experiment A reasonable set of delays for the vdlist would be 10 ms 22 ms 45 ms 100 ms 220 ms 450 ms 1 s 2 2 s 4 5 s 10 s Data processing 150 261 The data should be processed in the same way as for the X saturation recovery experiment Both the carbonyl and alpha carbon peaks derive their carbon polarization from the same pro ton spins and so analysis of the two peaks should give the same result If you have a sample containing some gamma glycine gives peaks at slightly lower shifts than the more common al pha glycine form this should show different T1 values for the two sets of peaks At 500 MHz the proton relaxation time should be approximately 520 ms at room temperature BRUKER BIOSPIN User Manual Version 001 Basic MQ MAS Introduction 15 1 The MQMAS experiment for half integer quadrupole nuclei is a 2D experiment to separate anisotropic interactions from isotropic interactions In the NMR of half integer quadrupole nu clei the dominant anisotropic broadening of the central 1 2 lt gt 1 2 transition CT and sym metric multiple quantum MQ transitions is the 2nd order quadrupole interaction which can only partially be averaged by MAS The satellite transitions ST e g
200. mig pli oui 34 Pulse power p17 pli2 sp2 pl2 pl p112 sp1 mm Biel EE DQ excitation evolution DQ reconversion detection PC7 pmlg DUMBO PC Gef aq cb Figure 22 5 Pulse Sequence Diagram When applied to X nuclei like 13C the RF field during this sequence must be carefully matched to the 7 fold spin rate since the dipolar couplings are small and care must be taken that the excitation bandwidth of the sequence chosen covers the whole shift range of the X nucleus In the case of protons this is rather forgiving since the shift range to be covered is small and the required power levels are easily achieved for protons Usually it is enough to calculate the re quired power level from the spin rate and the known proton 90 degree pulse using the au pro gram calcpowlev Assume the spin rate is 14000 Hz and post C7 is used The required RF field is then 7 14000 98000 Hz The known proton 90 degree pulse is 2 5 usec 1 4 2 5e 6 100000 Hz Type calcpowlev and enter 100000 return then enter 98000 return The output will be change power level by 0 18 dB The power level for the p C7 sequence is therefore 0 18 dB to higher attenuation than what is required for a 2 5 usec pulse User Manual Version 001 BRUKER BIOSPIN 231 261 CRAMPS 2D Table 22 4 Acquisition Parameters Parameter Value Comment pulprog wpmlgdqsq AV 3 instruments only topspin 2 1 or later FnMODE STATES TPPI Any other method may be used with a
201. n 4 itu nene e as IU 55 Possible Difficulties ooo een 55 Possible Approaches for 13C Samples c ccceeeeeeeeeeeeeeeeeeeeeees 55 User Manual Version 001 BRUKER BIOSPIN 3 Contents 5 4 5 5 5 6 6 1 6 2 6 3 6 4 6 5 7 1 7 2 7 3 7 4 8 1 8 2 8 3 9 1 9 2 9 3 9 4 10 10 1 10 2 10 3 10 4 10 5 11 11 1 11 2 11 3 Possible Approaches for non 13C Samples cccccceceeeeeeeeeeeeeees 57 Hints Tricks Caveats for Multi nuclear CP MAS Spectroscopy 58 Setup for Standard hetero nuclear Samples 15N 29SI 31P 58 Basic CP MAS Experiments eese een 61 IMTROGDUGCHON erm 61 Pulse Calibration with CP oocooonoccoccccccnccnnnonannnnnnnnnnnnnnnnnnnnnnn narra 61 Total Sideband Suppression TOSS sss 62 SELTIGS en IM III IINE EM 66 Non Quaternary Suppression NQS sss 69 Spectral Editing Sequences CPPI CPPISPI and CPPIRCP 72 ei e E 75 Introduction heii ibe ai ea hee a ae adele 75 Pulse Sequence Diagram for FSLG HETCOR sese 76 Setting FSLG HETCOR Experiments e 77 E EE EN Modifications of FSLG HETCOR ener 83 Carbon Decoupling During Evolution se 84 HETCOR with DUMBO PMLG or w PMLG Using Shapes 85 The Sequence pmlghet ccceeeeeeceeeeeeceeeee eee eeeeeeeeeeeeeeeeeeees 85 UE E c
202. nce of the central transition CNST2 End frequency in KHz of the sweep the sweep should cover the satellite transition but this is often broader than the band width of the probe of approximately 1 MHz Therefore it does not make sense to have this value bigger than 1000 User Manual Version 001 BRUKER BIOSPIN 173 261 MQ MAS Sensitivity Enhancement Table 16 1 Initial Parameters for the DFS Experiment Parameter Value Comments pulprog mp3qdfs av or Pulse program mp3qdfsz av NS 48 n dfs Full phase cycle is important 96 n dfsz DO 3 us Or longer t4 period D1 5 T Recycle delay use dummy scans if shorter If d1 is too short artefacts in the 2D spectrum may show up D4 20 us Z filter delay mp3qdfsz av only D6 see text Calculated in pulse program mp3qdfs av only D10 4u D11 0 Used in mp3qdfs av only P1 lt 3 6 us Excitation pulse at pl11 P2 see text Calculated in pulse program P3 20 us 90 selective pulse at pl21 used in mp3qdfsz av only P4 40 us 180 selective pulse at pl21 used in mp3qdfs av only PL1 120 dB Not used PL11 Power level for excitation pulse use value from standard MQMAS optimization PL21 Power level for selective pulse approx pl11 30 dB taken from previ ous pulse calibration spnam1 dfs Set by AU program zg_dfs sp1 to be optimized Power level for dfs aunm zg_dfs AU program to calculate sweep LO see t
203. nd L Emsley Transverse Dephasing Optimized Solid State NMR Spectroscopy JACS 125 13938 13939 2003 BRUKER BIOSPIN User Manual Version 001 Practical CP MAS Spectroscopy on Spin 1 2 Nuclei Introduction 5 1 Once good setup parameters have been obtained to observe 13C and get good S N on glycine it should be easy to also observe 13C CP MAS spectra on other samples and on nuclei differ ent from 13C Nevertheless sometimes one comes across samples where it is difficult to ob serve 13C This chapter deals with strategies to optimize acquisition parameters for 13C and other spin nuclei Possible Difficulties 5 2 Usually 13C spectra are easily acquired Several sample properties may however make obser vation difficult Low concentration of 19C in the sample No or too few protons in the sample Long proton T4 A O N gt Long Ts 5 Short proton Tue If a nucleus different from 13C should be observed there are additional potential difficulties 6 Unknown chemical shift 7 Unknown Hartmann Hahn condition 8 Unknown relaxation properties proton T4 T45 Tel Possible Approaches for 13C Samples 5 3 1 Collect as much information about the sample as possible Do not accept samples for mea surement with unknown composition Request information about possible hazards upon a rotor explosion concentration of the nucleus to be measured structural information about the molecular environment of the nucle
204. nd conventions for Chemical shifts Pure Appl Chem Vol 73 1795 1818 2001 W L Earl and D L VanderHart Measurement of 19C Chemical Shifts in Solids J Magn Res 48 35 54 1982 C R Morcombe and K W Zilm J Magn Reson 162 p479 486 2003 IUPAC recommendation Harris et al http sunsite informatik rwth aachen de iupac reports provisional abstract01 harris_310801 ht ml Cross polarization D Michel and F Engelke Cross Polarization Relaxation Times and Spin Diffusion in Rotating Solids NMR Basic Principles and Progress 32 71 125 1994 G Metz X Wu and S O Smith Ramped amplitude Cross Polarization in Magic Angle Spin ning NMR J Magn Reson A 110 219 227 1994 B H Meier Cross Polarization under fast magic angle spinning thermodynamical consider ations Chem Phys Lett 188 201 207 1992 K Schmidt Rohr and H W Spiess Multidimensional Solid State NMR and Polymers Academ ic Press 1994 S Hediger B H Meier R R Ernst Adiabatic passage Hartmann Hahn cross polarization in NMR under magic angle sample spinning Chem Phys Lett 240 449 456 1995 User Manual Version 001 BRUKER BIOSPIN 43 261 Basic Setup Procedures 44 261 BRUKER BIOSPIN User Manual Version 001 Decoupling Techniques Line shapes in solids are often broadened by dipolar couplings between the spins If the cou pled spins are of the same kind it is called homo nuclear dipolar coupling Hetero nuclear dipo
205. need to see a reasonable spectrum but must be an even number 144 261 BRUKER BIOSPIN User Manual Version 001 Relaxation Measurements Data Processing 14 2 3 Table 14 3 Once the pseudo 2D data has been recorded the processing parameters must be set and checked before it can be evaluated using the T4 T relaxation tool Table 14 3 lists the rele vant parameters No processing is done in the indirect dimension the relaxation dimension but the size must still be set to a power of two for TOPSPIN to create a processed data file The size should be next power of two larger than the number of relaxation delays used The zero points appended are ignored by the relaxation analysis In principle the line shape in the fre quency dimension does not affect the analysis so exponential multiplication with Ib of the or der of the observed line width can be applied to improve the signal to noise ratio Processing Parameters for CP T1 Relaxation Experiment Parameter Value Comment F2 acquisition dimension SI TD Zero fill LB Matched to line width WDW EM Ft_mod FQC ABSF1 1000 Limits for baseline correction ABSF2 1000 Should cover entire F2 width F1 relaxation dimension SI Smallest power of 2 greater than Must be 2n but any zeros will be TD F1 ignored Once the parameters are set process the data with xf2 to execute a Fourier transform in the f2 dimension o
206. nly The phase can be adjusted from within the relaxation analysis tool but baseline correction should be carried out with abs2 Start the relaxation analysis guide with the command t1guide The sequence of icons guides you through the analysis as follows Extract slice The first spectrum row should be selected for phase correction as this contains maximum signal The spectrum should then be phased to give positive peaks Define ranges Here you must define integral regions containing the peaks of interest The fit ting routine can either use the integral of the signal or the intensity in which case the maxi mum signal in each integral region is used Regions can be defined via the cursor or between specified limits via a dialogue box The integral regions need to be saved to a special file by clicking the disk icon towards the left of the integral window not the standard save integrals button on the right and selecting export regions to relaxation module and ret Relaxation window Here the intensity or area values from the first integral region are dis played The icons at the top of this window allow you to move between the integral regions ex clude points from the calculation display the data on a variety of axes and start the fit for the displayed region or all regions Figure 14 1 shows the decay of the a carbon signal of glycine as a function of relaxation delay along with the fit and calculated relaxation parameters Note tha
207. ns Lists DS lo Number of dumery scans Wobole TDD h Loop count for too RR Y width Automaton has Ber Miscellaneous Hi Irpmi 260 9772 86 1443 Spectral width ieee SWH Hz 37878 780 13001 345 Spectral width Routing IN_F us ze 92 Increment for delay AQ s 0 0017396 o c 196903 Acquisition time FIDRES Hz 295 928040 las 393251 Fid resoluton Fw bi 250000 op Fiter woth gt Receiver Y Nucleus 1 102 261 NUC 1 186 Edit tT3C Observe nucleus O1 Hz 18108 85 18108 85 Transmitter frequency offset O1P ppm 120 000 120 000 Transmitter frequency offset SFO1 MHz 150 9251918 150 9251918 Transmitter frequency BF 1 MHz 180 9070830 180 9070830 Basic transmitter frequency Y Nucleus 2 NUCI 1H Edit 2nd nucleus 02 Hz 1500 00 Frequency offset of 2nd nucleus 02P aprn Dao 5 Frequency offset of 2nd nucleus SFO2 MHz 600 1485000 Frequency of 2nd nucleus BF2 MHz 500 1470000 Basic frequency of 2nd nucleus zj Figure 10 2 The Acquisition Parameter Window eda BRUKER BIOSPIN User Manual Version 001 Proton Driven Spin Diffusion PDSD 14 Set p 14 if DARR RAD is desired else make sure p 14 120 dB For DARR RAD calculate the required power level using calcpowlev or use the setup procedure shown in 11 7 15 Start the experiment Acquisition Parameters Sample Experiment time 90 min 20 min 10 3 13C labelled histidine labelled tyrosine HCl
208. ns have more satellite transitions and there fore a correspondingly larger number of irradiation frequencies are required DFS is here the most convenient solution Start values for the parameters determining FAM are listed in Table 16 5 Table 16 5 Parameters for FAM Parameter Value Comments PL14 PL11 3 dB Less RF power than for a CW pulse is sufficient P2 0 8 us D2 P2 Calculated in the pulse program L2 2 All other parameters are in full analogy to the other MQMAS pulse programs in particular for z filtering and creating the shifted echo What is left is to find the best conditions for FAM optimiz ing PL14 P2 and L2 consecutively This can conveniently be performed with the parameter optimization procedure popt where two or three iterations can automatically be performed Soft Pulse Added Mixing SPAM 16 4 180 261 A simple and ingenious experimental trick can immediately give a signal enhancement Start ing from the standard 3 pulse sequence the phase cycling of reconversion pulse P2 and the CT selective 90 pulse P3 is eliminated This changes the coherence transfer pathway from 0 gt H gt 0 gt 1 to 0 gt 3 1 0 1 gt 1 and 0 gt 3 gt 1 0 1 gt 1 It has been shown that this leads to a substantial gain in sensitivity However it requires that data are acquired in echo anti echo mode in order to store the two different coherence transfer pathways in consec utive FID s in
209. nsiderations for the maximum t4 period determined by the number of FID s to be ac quired and the t4 increment can be made as for MQMAS Because the shift range in ppm is twice a big as in 3QMAS a larger increment can be used to give an equivalent shift range the increment being calculated from the spinning speed Since the magic angle is probably not yet perfect 32 to 64 FID s will be sufficient initially Processing parameters are described in the next section User Manual Version 001 BRUKER BIOSPIN 189 261 STMAS Table 17 5 F1 Parameters for the 2D Data Acquisition Parameter Value Comments F1 parameters In eda FnMode States TPPI 2D acquisition mode for stmasdqfz av or States QF 2D acquisition mode for stmasdafe av TD see text Number of FID s to be acquired ND 010 1 There is only 1 DO delay in the sequence SWH masr Equals spinning frequency for rotor synchronization from this INO is calculated correctly if ND_010 is already set NUC1 Select the same nucleus as for F2 so that transmitter frequency offset is correctly set important for referencing Pulse program In ased parameters D6 0 Used in stmasdafe av only ING N0 8 9 Used in stmasdafe av for 3 2 D7 0 Used in stmasdafe av only IN7 N0 7 24 Used in stmasdgfe av for 5 2 IN0 28 45 Used for 7 2 IN0 72 55 Used for 9 2 In Figure 17 4 two 2D plots of the 87Rb STMAS experiment on RbNO are comp
210. nsmitters the required power level can be calculated from a known reference pulse width 2 The LG frequency offset must be calculated from the RF field RF sqrt 2 3 The HH condition must be reestablished varying the F1 13C RF field As the effective field required to match for both nuclei at the HH condition is the vector sum of RF field and frequency offset a higher power pulse is required on F1 with increasing offset So it is recommended not to set the proton power higher than to 50 kHz RF field For the setup the pulse program lgcp is used It contains an include file Igcalc2 incl which will calculate the LG offset from a given RF field specified as cnst17 It will set the calculated offset as cnst19 during contact In ased it will calculate also cnst16 which shows the effective field under cnst17 RF field and cnst18 cnst19 offset The X contact pulse is executed as ramp shape Any standard ramp is possible but a flat ramp 70 100 or 90 100 is preferred Usually cal culating the required RF field for the HH match can be done in the following way 1 Load the pulse program Igcp into a standard CP MAS data set with all parameters set and optimized Set p14 to about 54 flip angle Use tyrosine HCI or the sample of interest 2 From p3 calculate the power level plI2 for 50 kHz RF field using calcpowlev 3 Enter cnst17 50000 type ased Read the value of cnst16 effective field under cnst18 cnst19 offset about 61000
211. nt can not be expected Now we know the minimum relaxation delay and the maximum contact time With these pa rameters used as d1 and p15 the measurement is just a matter of patience BRUKER BIOSPIN User Manual Version 001 Possible Practical CP MAS Spectroscopy on Spin 1 2 Nuclei Approaches for non 13C Samples 5 4 If an arbitrary X nucleus of spin is under investigation quadrupolar spins must be treated separately the strategy follows the one described above if the sample contains the protons bound to 1 C In this case running a BE cp mas spectrum allows setting and determining all proton parameters recycle time contact time from the E setup To run the X nucleus cross polarized from protons one just needs to set the HH condition from the known proton RF field the spin rate and the transmitter power at the NMR Frequency of the X nucleus such that the effective field at the X frequency equals the effective field at proton frequency spin rate Example setting the HH condition for 15N from known parameters for 13C CP MAS The gyro magnetic ration of 15N is lower by a factor of 2 5 compared to carbon proton frequency 400 MHz 13C frequency 100 MHz 5N frequency 40 MHz The probe efficiency is about the same for 1 C and N but not H so one needs about 2 5 times higher RF voltage for the 15N contact pulse than for the 13C contact pulse if the spin rate and the proton RF field are the same This is equivalent t
212. o 2 5 6 25 times the power in watts So if ased shows pl1W 150W for a well optimized 13C CP setup 15N will require 6 25 150 W 938 W This is far above specs so the same proton contact power level cannot be used it needs to be lowered The maximum allowed power for a contact pulse on 15N is 500W This means that the proton contact power should be lowered by approximately a factor of sqrt 938 500 1 37 Precalcu lating power levels like this will get the parameters close enough to see a cp signal on a good test sample so further optimization is possible See Test Samples for suitable test samples The most efficient way of precalculating power levels for multi nuclear spectroscopy is the fol lowing 1 Determine the power conversion factor for some nuclei of interest on a suitable test sample from the low end to the high end of the probe tuning range This means measuring a pre cise 360 pulse make sure it is 360 not 180 or 540 and the associated power level Make a table in your lab notebook as follows see Appendix Table 5 1 Power Conversion Table Probe Amm Triple Power Nucleus Frequency P90 us Rf field Khz W or dB Remarks 1H 400 13 2 5 100 100 Low range 19F 376 3 Not available 15N 40 5 6 5 38 6 300 Probe in double mode 15N 40 5 6 5 38 6 500 Probe in triple mode C N 2981 79 5 6 41 7 300 Double mode low range 130 400 5 4 62 5 150 Double mode low range 13C 100
213. o the proton preamp A proton band pass filter must be inserted between preamp and probe Tune the proton channel of the probe using the command wobb high This means that the highest frequency is tuned first Stop and type wobb again Then adjust the tuning of the X channel to 13C Alternatively you can switch to the lower frequency channel within wobb high by clicking on the frequency table symbol in the wobb display or by pressing the second touch button on the preamp cover module twice Then acquire ns 2 scans on the protons of adamantane User Manual Version 001 BRUKER BIOSPIN 25 261 Basic Setup Procedures protons of adamantane rei 15 10 so 40 20 o 20 T Figure 3 12 Proton Spectrum of Adamantane at Moderate Spin Speed Set the carrier frequency O1 on top of the biggest peak using the encircled button in TopSpin 26 261 BRUKER BIOSPIN User Manual Version 001 Basic Setup Procedures Cra HORSE 2 1 0614 en PON en Admina samnected be rn 2 om a 5a nn u A AA LI LH U FB gt a9 770 runs TMRARARMH AM 124 Grosser Last50 Groups Aaa Filo Edit View Spectrometer Processing Analysis Options Window Help 0 ww O der06 ME ousreceve 2 entemcooung Res O gaser SET 701 01 FREQUENCIES FROM CURSOR POLTTION Define Left clich inside data window vi wo5o0 C3 wi mann remen a A CQ Wimpens2007 L2 wither 2 opttopspin x 20 0 Figure 3 13 Setting the Carrier on Resonanc
214. oad a spinner with a glycine precipitate from cold water with acetone and dry if you are not sure about the com position of your sample A spinner with 50 ul or less sample volume is preferred since high H4 homogeneity is desired although it is by far less important than is commonly stated in the liter ature 20 5 Parameter Settings for PHLG and DUMBO Table 20 2 PMLG Analog Mode Parameter Value Comment pulprog wpmlga Runs on AV 3 instruments only pl12 for 100 kHz RF field sp1 dto set cnst20 100 000 To be optimized during setup spnam1 wpmlg1 m5m or m5p p1 2 5 usec As for 100 kHz RF field p8 1 2usec p14 0 7 usec To be optimized cnst25 140 To be optimized p9 4 2 6 usec To be optimized p5 1 5 usec or calculated from cnst20 To be optimized d1 4s For a glycine 111 anavpt 4 2 4 8 16 or 32 o1p 10 or 1 To be optimized User Manual Version 001 BRUKER BIOSPIN 215 261 CRAMPS 1D Table 20 2 PMLG Analog Mode swh 1e8 2 2 p9 10 p5 0 6 To be corrected for proper scaling rg 16 64 td 512 Up to 1024 si Ak digmod analog MASR 12 15 kHz Depending on cycle time Table 20 3 DUMBO Analog Mode Parameter Value Comment pulprog dumboa Runs on AV 3 instruments only pl12 for 100 kHz RF field sp1 up to 130 kHz To be optimized during setup spnam1 dumbo1 64 Set by xau dumbo p1 2
215. obtained Shape lool HAY pe 14 1 Cadets jew dO CET IKEA TangAmplitudeMod 4 Amplitude Parameters Size of Shape deta MAS dipolar coupling RF field at match Ampl Scaling Factor Use general notation Figure 18 5 Shape Tool display with a tangential shape for adiabatic cross polarization User Manual Version 001 BRUKER BIOSPIN 199 261 Double CP The amplitude factor is 50 corresponding to 50 RF field or a power level change of 6 dB since the amplitude corresponds directly to pulse voltage 11 Optimize the DCP condition on the rectangular pulse using 0 1 dB steps over a range of 1 dB around the optimum found with the ramp Moor probe 22 393 CAsataran jn50507 Spectrum ProcPars ArquPars Title PulseProg Peaks tegal Sample Structure Fid Acqu o k 12 o 4 6 8 A Ba a ee Dr DDr Tan a Hr Be LE Te Dr 2 T T Eu 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 T 3 2 1 0 E 2 ppm Figure 18 6 Double CP optimization of PL5 in increments of 0 1 dB Note how narrow the optimum DCP conditions are However with diligent preparation one should be very close to the optimum with the first try 12 Run an experiment with 16 scans and compare the signal amplitude with the signal intensity of the 5C CPMAS experiment with the same number of scans using dual display The in tensity ratio of the aliphatic resonance of the CPMAS compared to t
216. ode Table 21 4 Parameters for Digital Mode Parameter Value Comment pulprog dumbod2 or wpmlgd2 AV 3 instruments only digmod digital dspfirm sharp or medium aqmod qsim or dqd swh 50000 10000 Depending on spectral range and 01 The correction for the scaling factor must be done after acquisition changing the status param eter swh by typing s swh and dividing the value by the scaling factor about 0 47 for WPMLG and 0 5 for DUMBO 224 261 BRUKER BIOSPIN User Manual Version 001 CRAMPS 2D CRAMPS methods allow measurement of chemical shifts in the presence of strong Homo nu clear dipolar interactions Therefore CRAMPS type sequences can be applied to measure chemical shifts of protons where these sequences work most efficiently and where fast spin ning cannot easily be used As an example the proton X hetero nuclear chemical shift corre lation experiment see chapter 5 uses FSLG to suppress Homo nuclear dipolar couplings between protons to resolve the proton chemical shifts CRAMPS type pulse sequences must be used in both dimensions if proton chemical shifts are to be correlated Two types of proton proton correlation experiment will be described here 1 Proton proton shift correlation via spin diffusion similar to the high resolution NOESY ex periment In this case the dipolar coupling between protons acts during the mixing period The size of the off diagonal cross peaks indicates the size of t
217. ogram can be called with the option lastf1 Before giving some further explanations about the experiment Figure 15 7 shows the 2D Rb 3QMAS spectrum of RbNO3 M ra ww 40 42 44 46 48 50 52 54 56 58 60 62 64 66 68 ppm Figure 15 7 2D Rb 3QMAS Spectrum of RbNO3 Top and left projections are the summations over the signal ranges The spectrum included in the 2D map is a cross section through the resolved peak resonating at approximately 53 ppm Note that at 11 7 T two of the three sites cannot be resolved in the 2D spectrum The spectral range shown in F1 corresponds to the spinning frequency Spectra are taken on AV500WB at a Larmor frequency of 163 6 MHz with a 2 5 mm CP MAS probe spinning at 25 kHz Since the quadrupole parameters are usually unknown before performing the experiment the positions of the peaks in the indirect dimension cannot be predicted Therefore it may happen that a peak is positioned at the border of the spectral range in the F1 dimension or even folded When using xfshear the prompt F1 shift in ppm can be used to shift the spectrum including User Manual Version 001 BRUKER BIOSPIN 161 261 Basic MQ MAS 162 261 its axis upfield negative value or low field positive value accordingly For data which don t need a shearing transformation the ppm axis in F1 can be correctly calibrated by running the AU program xfshear with the option rotate It will calibrate the F1 axis and
218. on T4 proton T can be ob tained by looking at the protons in the sample Set up for proton observation set swh to 100000 500000 rg to 4 and pulprog cpopt if not found in the library copy the pulse pro gram in the appendix p3 and pl12 for p3 p90 Set spnam0 ramp 100 sp0 power lev el for HH p15 100 us Do 1 scan and fourier transform phase correct Using popt optimize d1 for maximum signal Note CP MAS probes usually have a substantial proton background signal Do not be misled by this it will not behave like a regular signal it will grow steadily with longer pulses it will not show spinning sidebands it will cancel when a background suppression pulse program like aring is used with a full phase cycle Knowing the required relaxation delay the following step is to determine the cross polariza tion contact time On protons we measure the time constant Tue Using popt in the previ ous setup vary p15 between 100 usec and 10 ms even 20 ms at reduced power if a long Tis is expected as the distance between nucleus of interest is long or the mobility is high leading to a small hetero nuclear dipolar coupling between nucleus of interest and protons This measurement will tell you how long the contact time p15 may be A value of p15 giving 50 of the initial proton signal amplitude will still give a 2 fold enhancement on 13C if the proton signal is below 50 at 1ms spin lock time or even less a full cp enhanceme
219. on for experimental imperfections such as B in homoge neities and pulse imperfections Also both experiments produce scaled powder patterns in F1 and the scaling factor is more favorable in SUPER than the factor 0 39 in Tycko s version As a consequence the SUPER experiment does not require high spinning speeds to fit the F1 line shape into the rotor synchronized spectral window or very strong 13C pulses SUPER has several advantages First of all it covers a large bandwidth for the isotropic chem ical shift Secondly no requirements exist for 1H decoupling during the recoupling pulses be cause it uses 360 pulses instead of the 180x pulses in Tycko s experiment Exact 360 pulses automatically decouple the hetero nuclear dipolar interaction so that no or only weak 1H de coupling is required during the recoupling pulses The scaling factor is normally 0 155 so that a spectral width over 40 kHz can be achieved in the indirect dimension As a consequence mod erate spinning speeds of up to 6 5 kHz can be chosen so that experiments can be performed without serious problems on high field instruments The limiting factor in the choice of the spin ning speed is the rotor synchronization requirement of the recoupling 360 pulses Ver 12 12V References 1 S F Liu J D Mao and K Schmidt Rohr A Robust Technique for Two Dimensional Separation of Un distorted Chemical Shift Anisotropy Powder Patterns in Magic Angle Spinning NMR J
220. on rate and the required spin nutation frequencies for the X nucleus The spin nutation frequency must be 7 times the sample rotation rate for C7 5 times the sample rotation rate for SPC5 and 3 5 times the sample rotation rate for SC14 Be careful to obey the maximum allowed spin nutation frequencies for the hardware in use It is essential that all these parameters are considered carefully in context with the properties of your sample before the experiment is started so that the appropriate hardware is used Espe cially the choice of the MAS probe is essential for achieving a sensible setup Table 12 1 shows the selection parameters for three standard recoupling sequences References 120 261 EA Bennett R G Griffin and S Vega Recoupling of homo and hetero nuclear dipolar inter action in rotating solids NMR Basic Principles and Progress 33 3 77 1994 S Dusold and A Sebald Dipolar Recoupling under Magic Angle Spinning Conditions Annual Reports on NMR Spectroscopy 41 185 264 2000 M Hohwy H J Jakobsen M Eden M H Levitt and N C Nielsen Broadband dipolar recou pling in the nuclear magnetic resonance of rotating solids A compensated C7 pulse sequence J Chem Phys 108 2686 1998 M Hohwy C M Rienstra C P Jaroniec and R G Griffin Fivefold symmetric Homo nuclear re coupling in rotating solids Application to double quantum spectroscopy J Chem Phys 110 7983 1999 M Hong Solid State Dipola
221. ondition Note that Beg points along the 1 1 1 direction in the 3 dimensional space see Reference 2 Note the sign of Bog when calculating the actual direction of the effective field A positive Bog and a B with phase 0 results in the effective field being in the positive quadrant along the mag ic angle in the X Z plane of the rotating frame Two methods are available to achieve such a frequency switch experimentally One method is simultaneous switching of frequencies and phases The other method uses phase modulation Frequency time and phase relate to each other as derivative of phase and time as 0 t to get2nf 00 01 The relationship describes the rate at which a phase of the rf pulse must be changed in order to achieve a certain frequency offset Vinogradov et al describe this ap proach under the acronym PMLG Phase Modulated Lee Goldburg Used in combination with cp signal generation both methods allow observing proton J cou plings to the observed X nucleus However only samples with very narrow lines will produce well resolved J couplings as shown below on adamantane Harder solids require careful ad justment and fairly high power levels to show barely resolved couplings since the linewidths achieved are broader than what can be achieved with standard decoupling sequences like tppm Since the hetero nuclear X H coupling remains there may be spinning sidebands from this coupling in addition to CSA sidebands User Manual Version
222. or special probes max allowed RF fields may be lower Check with your Bruker BioSpin ap plications support if in doubt In order to have quantitative information about the precision of your magic angle one may measure the line width of the KBr central peak and compare it with the line width of the 5th spinning sideband If the linewidths compare within 8 then the MA setting is acceptable The line width comparison is conveniently achieved with the command peakw expanding the display first around the center line typing peakw and then repeating this with the 5th sideband to either side Most cp mas probes are tunable over a large range of X frequencies It can sometimes be fair ly difficult to retune a probe to an arbitrary frequency within the tuning range NEVER just load a nucleus and blindly tune and match the probe using a small wobble width wbsw of 10 MHz or less Instead either note the current tuning position of the probe into the lab notebook and start retuning to the new nucleus frequency from this frequency on following the probe re sponse over the whole frequency range using a large wbsw of 50 MHZ Alternately check the micrometer setting of the X tuning adjustment and conclude from that to which nucleus the probe is tuned Make a list of micrometer settings for the most frequently measured nuclei Remember which way to turn the tuning knob to tune to higher and lower frequencies On most probes turning the adjustment counter
223. or synchronization together with States or States TPPI phase sensi tive acquisition helps to fold spinning sidebands from outside back onto centre bands or other side bands Table 16 2 Parameters for 2D Data Acquisition of 3 pulse Shifted Echo Experiment mp3qdfs av Parameter Value Comments pulprog mp3qdfs av F1 parameters In eda FnMode QF Acquisition mode for 2D TD see text Number of FID s to be acquired ND_010 1 There is only one dO delay in the sequence SWH masr Equals spinning frequency for rotor synchronization from this inO is calculated correctly if ND 010 must be set first NUCA Select the same nucleus as for F2 so that transmitter frequency off set is set the same in both dimensions essential for referencing pulse program In ased parameters D10 0 IN10 IN0 7 9 For spin 3 2 0 for all other spin l D11 0 IN11 0 For spin 3 2 1N0 19 12 For spin 5 2 1N0 101 45 For spin 7 2 1N0 91 36 For spin 9 2 User Manual Version 001 BRUKER BIOSPIN 177 261 MQ MAS Sensitivity Enhancement Table 16 3 Parameters for 2D Data Acquisition of 4 pulse Z filtered Experiment mp3qdfsz av Parameter Value Comments pulprog mp3qdfsz av F1 parameters In eda FnMode States or Acquisition mode for 2D States TPPI TD see text Number of FID s to be acquired ND_010 1 There is only one dO delay in the sequence SWH m
224. ould be satrec rather than expdec and the slice selected for processing should be the last one signal is maximum at long recovery times The calculated relaxation time constants should be the same as those obtained by inversion recovery T1p Relaxation Measurements 14 2 5 Rotating frame relaxation measurements under a spin locking rf field can be used to probe motions on shorter domestically than T4 measurements with inverse correlation times of the order of the spin locking rf field strength To measure Ty relaxation after CP a variable length spin locking pulse is applied to the X nu cleus The remaining X magnetization decays exponentially to zero as a function of spin lock time The parameters of cross polarization can also be determined from variable contact time CP experiments the function cpt1rho is provided in the relaxation analysis tool for this pur pose but here only simple T4 measurements will be discussed It should be noted that the relaxation in a T4 experiment might result from processes other than true T4 relaxation For example in glycine the carbon spins are dipolar coupled to pro tons and there is a possible fast relaxation pathway via the protons which is not T4 relaxation This is inhibited by having a high spin lock field strength but at large field strengths care must be taken over the length of the spin lock pulse If apparently non exponential decay is ob served this may result from such alternati
225. ounter is internally used for checking if the echo or anti echo is currently being acquired 15 In the acquisition of echo anti echo 2D spectra signals from the echo and anti echo path ways are stored into consecutive FID s in the serial file In MQMAS experiments these echos and anti echos behave differently For t 0 both signals have their echo top immediately after the selective 90 pulse As t4 is incremented the top of the echo appears at later point in time whereas the top of the anti echo appears at an earlier point in time It means that the contribu tion of the anti echo becomes less and less until finally the signal fades out completely and only noise is sampled It can be advantageous to terminate the acquisition of this noise in order to increase the overall S N and save spectrometer time However in the processing of echo anti echo data two consecutive FID s are linearly combined in the following way rel im2 iml re2 re2 rel iml re2 rel im2 im2 iml Eq 16 2 Where re and im refer to the real and imaginary points of FID s 1 and 2 Hence acquiring a smaller number of anti echos than echos leads to the usual truncation effects wiggles in the spectrum Furthermore since both signals contribute to the phase information care must be taken that the pure absorption line shape of the 2D peaks is not obscured Therefore in case of doubt it is probably the best idea to set L5 TD F1 2 If less anti echos are to be
226. perform the 2D FT Figure 15 8 compares the same 2D 3Q MAS spectrum processed with no shift and an addi tional shift of 5 ppm respectively We see that without the additional shift the uppermost peak is at the border of the spectral range and the projection shows that the edge of this peak reen ters into the spectral range from the opposite side In summary the AU program xfshear can be called with the following options lastf1 Use the F1 shift value from last processing abs Do abs2 after F2 Fourier transform of data noabs Don t do abs2 after F2 Fourier transform of data rotate don t calculate shearing only use F1 shift to rotate spectrum along F1 axis ratio Use different value for ratio R value can either be entered or passed A A l E dw S MM MM A y e ppm t pr Ip ppm EEN CS ai 10 _ 15 B md 5 1 10 E D 2 f E j 40 20 D 20 40 60 ppm 40 20 0 20 40 60 ppm Figure 15 8 Comparison of Differently Processed 2D 23Na 3Q MAS Spectra of Na4P207 The left spectrum was processed with an additional F1 shift of O ppm the right spectrum with 5 ppm Spectra are taken on an AV500WB at a Larmor frequency of 132 3 MHz with a 4 mm CP MAS probe spinning at 10 kHz Note that the F1 range equals the spinning frequency of 10 kHz in both cases BRUKER BIOSPIN User Manual Version 001 Basic MQ MAS Obtaining Information from Spectra 15 5 The referencing procedure in xfshe
227. period should be a 7 2 pulse or stronger Choose pI11 accordingly Unlike TOSS SELTICS is only 0 5 rotor periods long BRUKER BIOSPIN User Manual Version 001 Basic CP MAS Experiments heteronuclear decouplin 93 H 2 bs e 798 Prec 1 12 1 12 1 112 G 1 24 t 24 Figure 6 6 Pulse Program for SELTICS In Figure 6 6 one can see that the SELTICS experiment takes only rotor period compared to the 2 rotor periods required in the TOSS experiment Use glycine or tyrosine HCl at reasonable spinning speed PrarcFars AcquPars Tale PuseProg Peaks Inteprais Sample Smacrure Mic Phase Calbrate Baseline 137 Satun COMAS Tyrosine HOY Mast 6509 Figure 6 7 SELTICS at 6 5 kHz Sample Rotation on Tyrosine HCI User Manual Version 001 BRUKER BIOSPIN 67 261 Basic CP MAS Experiments In Figure 6 7 the amplitude of the spinning sidebands are reduced to more than 10 com pared to the original spectrum without sideband suppression 256 transients were recorded Das c_experiments 6 1 Cudata5Utkubisdb jas O Ble ra eL E E Ei 73 os 9 5 A E jesic_experiarnts 6 Cr darssMrbrxnb jos 605 ME ZEN CN GNE NES XV Bsasc_experzuents 6 l Ci darsSmorbrrbb jos0P05 MEC Dec D Au ei E ANS uL d Sa a i Ba es ET a ol ol oe 200 180 100 Figure 6 8 Cholesterylacetate Spectrum Using Sideband Suppression Figure 6 8 is a cholesterylacetate spectrum using sideband suppression with the SEL
228. power level of the contact needs to be increased Set spnam0 ramp70100 100 Set sp0 and pl1 to about 2 dB less attenuation and check S N again Re opti mize the HH condition observing the peak at 176 ppm which is less strongly coupled to pro tons and therefore exhibits a sharper HH matching condition in steps of 0 3 dB In this case S N improves to 100 1 Then optimize the decoupling pulse pcpd2 in steps of 0 2 usec observing the peak at 43 ppm which is more sensitive to decoupling mis sets Here this led to another 10 improvement in S N Good Laboratory Practice requires that evaluation measurements be taken in suitable peri ods Store the optimized glycine spectrum together with the following important information 1 Value of field setting 2 Name of the shim file 3 Name of the operator 4 Probe setup triple mode or double mode high range or low range setting WB probes only name or part number of the probe al Description of the sample which reference rotor weight of glycine and spinner 6 Any additional comments for instance the reading of the micrometer setting for the X tuning adjustment not available on all probes Write this information into the title file so it is stored with the data set as well as all other acqui sition and processing parameters Recalling this data set and acquiring a new data set should give the same spectrum within 10 of S N Some Practical Hints for CPMAS Spectrosco
229. ppro priate changes in ppg NUC1 NUC2 1H sw swh along F1 same as for F2 Needs to be corrected before transform pulse program calculates approximate val ues upon ased td 512 1k Depending on resolution 1 td 128 256 Depending on resolution cnst31 spin rate 10 15 000 Depending on available RF field I1 number of pc7 cycles 2 7 depending on dipolar coupling spnam1 m5m or m5p as in 1d setup DUMBO may be used with modified timing spnam2 Igs 2 or Igs 4 if used Set I3 2 or 4 depending on desired sw1 DUMBER 22 with modified timing Table 22 5 Phases RF Levels and Timing Phases Rf Power Levels Timing 11 12 POST C7 0 3 14 pl7 set for RF 7 spin rate tau1 3 4 calculated from cnst31 11 12 incremented for DQ evol 11 12 incremented for DQ select di CYCLOPS pl12 p1 025 3 pl12 p11 45 93 1 pl12 p11 10 0 sp1 set for 100 130 kHz RF WPMLG calculated via cnst20 field DUMBO p10 set by xau dumbo 031 DQ selection 232 261 BRUKER BIOSPIN User Manual Version 001 Data Processing CRAMPS 2D 22 7 The spectral width in both dimensions assumes the absence of shift scaling In order to ac count for the shift scaling effect of the sequence one has to increase the spectral width by the scaling factor Before doing the 2D fourier transformation type s swto call the status parame ters for both F2 and F1 and replace both values by lt cu
230. proton spins A third modification of the ba sic sequence uses phase modulated pulses instead of frequency shifts These three modifica tions to the basic sequence are described in Modifications of FSLG HETCOR References H J M deGroot H F rster and B J van Rossum Method of Improving the Resolution in Two Dimensional hetero nuclear Correlation Spectra of Solid State NMR United States Patent No 5 926 023 Jul 20 1999 B J van Rossum H F rster and H J M deGroot High field and high speed CP MAS 13C NMR hetero nuclear dipolar correlation spectroscopy of solids with frequency switched Lee Goldburg Homo nuclear decoupling J Magn Reson A 120 516 519 1997 B J van Rossum Structure refinement of photosynthetic components with multidimensional MAS NMR dipolar correlation spectroscopy Thesis University of Leiden Holland 2000 B J van Rossum C P deGroot V Ladizhansky S Vega and H J M deGroot A Method for Measuring hetero nuclear 1H 13C Distances in High Speed MAS NMR J Am Chem Soc 122 3465 3472 2000 D P Burum and A Bielecki An Improved Experiment for hetero nuclear Correlation 2D NMR in Solids J Magn Res 94 645 652 1991 A Lesage and L Emsley Through Bond hetero nuclear Single Quantum Correlation Spec troscopy in Solid State NMR and Comparison to Other Through Bond and Through Space Ex periments J Magn Res 148 449 454 2001 User Manual Version 001 BRUKER BIOSPIN 75
231. py Some general recommendations for reasonable RF fields used in WB probes 3 8 Table 3 3 Reasonable RF fields for Max 2 Duty Cycle Decoupling power over 50 ms 200ms Prone Nucleus 500ms Contact pulse up to 10 ms 2 5mm CPMAS double resonance 35 1H 115 kHz 2 2us 90 pulse 75 kHz 40 kHz kHz max sample rotation 71 kHz 3 5 us contact 2 5mm CPMAS double resonance 35 6 83 kHz 3 us 90 pulse kHz max sample rotation 71 kHz 3 5 us 3 2mm CPMAS double resonance 24 1H 110 kHz 2 3 us 60 kHz 35 kHz kHz max sample rotation 68 kHz 3 7 us 3 2mm CPMAS double resonance 24 e 78 kHz 3 2 us kHz max sample rotation 68 kHz 3 7 us 4 mm CPMAS double resonance 1H 92 5 kHz 2 7us 90 50 kHz 30 kHz probe 15 kHz max sample rotation 62 kHz 4 us 4 mm CPMAS double resonance 18 71 kHz 3 5 us probe 15 kHz max sample rotation 62 kHz 4 us User Manual Version 001 BRUKER BIOSPIN 41 261 Basic Setup Procedures Table 3 3 Reasonable RF fields for Max 2 Duty Cycle 4 mm CPMAS triple resonance probe 18 66 kHz 3 8 us 15 kHz max sample rotation 50 kHz 5 us 7mm CPMAS double resonance probe 1H 70 kHz 3 6 us 90 pulse 35 kHz 20 kHz 7 kHz sample rotation 50 kHz 5 us 7mm CPMAS double resonance probe Tee 55 kHz 4 5 us 7 kHz sample rotation 50 kHz 5 us 42 261 Note Higher RF power levels should only be applied if necessary and within specifications F
232. r INADEQUATE NMR Spectroscopy with a Large Double Quan tum Spectral Width J Magn Reson 136 86 91 1999 A Brinkmann M Ed n and M H Levitt Synchronous helical pulse sequences in magic angle spinning nuclear magnetic resonance Double quantum recoupling of multiple spin systems J Chem Phys 112 8539 2000 M Hohwy C M Rienstra and R G Griffin Band selective Homo nuclear dipolar recoupling in rotating solids J Chem Phys 117 4974 2002 C E Hughes S Luca and M Baldus RF driven polarization transfer without hetero nuclear decoupling in rotating solids Chem Phys Letters 385 435 440 2004 BRUKER BIOSPIN User Manual Version 001 Symmetry Based Recoupling Pulse Sequence Diagram Example C7 12 1 Qi 10 1 H CW or CW LG decoupling TPPM dec b2 du O11 O12 13 014 ds Orec dk chee t gem RF 2 rotor periods 14 2x pulses 1 phase rotation about 360 57 1230 11 127 9 180 13 14 90 QD phase 0 9 180 80 doo 0 123 dec 0321 td Figure 12 1 C7 SQ DQ Correlation Experiment Setup 12 2 As mentioned before it is essential that the parameters of your sample of interest are consid ered before the experiment is started Table 12 1 illustrates the proper choice of hardware for the observe nucleus 1 C Obviously observation of DQ coherence requires samples with rea sonable dipolar couplings and reasonable probability of coupled species So running this ex perim
233. r Manual Version 001 BRUKER BIOSPIN 203 261 Double CP Example Spectra 18 5 13C 15N correlation by double CP 1H gt 15N gt 13C E sample histiding U 13C 15N spinning speed 13 kHz g 4 mm 400 MHZ MB triple resonance probe In H C N mode 1H gt 15N CP at 38 KHz 15N 2 ms e 15N gt 13C CP ai 35 kHz for ASN and 22 kHz Tor ARC 9 ms ramp shape pulse for 1H tangential pulse for 15N o decoupling SPINALG4 at 100 kHz 26 us 34 ms a Q A o e Td o Lo T a s o E CN m 200 150 100 50 F2 ppm Figure 18 8 C N correlation via Double CP in histidine simple setup sample 4mm Triple H C N Probe 204 261 BRUKER BIOSPIN User Manual Version 001 Double CP A 4 A l f f NE V NN uf D MY 4 UAM P rE la OP 90 kHz TEPM 12 hm iv 6 Ww 21580 preamplifier fi WEE EST E X U i L e I 2 i E a app i RE qtr f mer 4 i I L A 1 e of tt Ki v4 gt D c d Li T b l I d a tht e 1 0 D VW u UN g t AE A ne H i 1 I i Bac Y Y VW a sn E eg d 4 Lj 2 lt M i he a i T 1 1 e e t t D t t 1 r Y T Y 150 100 60 F2 ppm Figure 18 9 NCC correlation experiment with 22 ms DARR mixing period for Ca Cx spin dif fusion on GB1 protein run using an EFRFF_Probe DARR transfer from Ca to Cg or C generates positive cross peaks HORROR or DREAM transfer generates negative cross p
234. r Manual Version 001 BRUKER BIOSPIN 219 261 CRAMPS 1D ema CRAMPES z womlg CRAMPS 19lycine itus Is what should be achieved with Oftfe omon e d U U I D I J j f j Fi fy b j 1 TA gt j tf 1 j V 1 d d Qi ei lt WA 4 T T T T T T T T T T T T T 16 n 6 E 2 o ppm Figure 20 9 WPMLG CRAMPS After Optimization Digital Acquisition 220 261 BRUKER BIOSPIN User Manual Version 001 Modified W PMLG 2 1 Recently a modified version of WPMLG was published by Leskes et al which suppresses the carrier spike completely and therefore allows placing the carrier frequency 01 o1p arbitrarily This is achieved by a 180 degree phase alternation between consecutive WPMLG pulses The magic angle tilt pulse is then not required anymore This reduces setup time and enhances ex perimental possibilities significantly Reference M Leskes P K Madhu and S Vega A broad banded z rotation windowed phase modulated Lee Goldburg pulse sequence for 1H spectroscopy in solid state NMR Chem Phys Lett 447 370 2007 Pulse Sequence Diagram for Modified W PMLG 21 1 Phases 1 010010 O10 10 Figure 21 1 Pulse Sequence Diagram Table 21 1 Phrases RF Levels Timings Phases RF Power Levels Timing di CYCLOPS 1230 pl12 set for around 100 kHz p1 around 2 5 usec 019202 sp1 set for 100 130 kHz cnst20 WPMLG calculated from cnst20 R
235. r fourfold zero filling WDW No No apodization used for S N measurement In this case PH_mod Pk Phase correction if needed BC_mod Quad DC offset correction on FID FT_mod Fqc Note No line broadening is applied since the acquisition time is set appropriately File Edi View Spectrometer Processing Analysis Options Window Help NETO ATEN ani rex snot Brae rTryryot iia mH rnane 100220 aM gt att A Sominum Pramas A quOMS Tele Ppgucg Peaks rien Sarga Structure Mid Acqu Phase Print 15 o a SEE H 200 200 160 106 ze o 60 ppm 4 4 Figure 3 26 Glycine Spectrum with Spinal64 Decoupling at 93 kHz RF field Here the line width of the line at 43 ppm is fitted to be about 50 Hz Correspondingly the inten sity is much higher The sinocal routine calculates 80 1 S N using 250 to 50 ppm spectrum range 50 to 40 signal range and 10 ppm noise range On this triple probe more than 100 1 is expected What else needs to be optimized Two more parameters are essential 1 The power level at HH contact 2 The decoupling pulse pcpd2 40 261 BRUKER BIOSPIN User Manual Version 001 Basic Setup Procedures The spectrum of Figure 3 26 was taken at contact power levels as set for adamantane Fur thermore a 50 ramp was used which has a rather low average RF level corresponding to about 5 7usec in this case 25 less than 4 5 usec This does not spin lock the protons well enough So the
236. re fore folding of peaks will always occur even at fast spinning For even higher quantum orders the shift ranges are 7 and 30 times smaller for 7Q and 9Q than for the 3Q experiment respec tively Table 15 6 summarizes ppm ranges for the maximum spinning frequencies of 2 5 3 2 and 4 mm probes respectively A Larmor frequency of 100 MHz is assumed One can see that the ranges become less than the typical chemical shift range for many nuclei The expression R p acts like a scaling factor that scales the frequency scale directly Mathematically this is BRUKER BIOSPIN User Manual Version 001 Basic MQ MAS solved in the AU program xfshear in such a way that the observe Larmor frequency is multi plied by the factor AR p A to redefine an apparent Larmor frequency in the MQ dimension Table 15 6 Chemical Shift Ranges for all MQ Experiments for All Spins Spin and MQ 15 kHz 25 kHz 35 kHz Experiment 4 mm probe 3 2 mm probe 2 5 mm probe 3 2 39 6 ppm 66 0 ppm 92 4 ppm 5 2 3Q 105 6 ppm 176 0 ppm 246 4 ppm 5 2 5Q 26 1 ppm 35 2 ppm 49 3 ppm 7 2 3Q 197 4 ppm 329 0 ppm 460 6 ppm 7 2 5Q 39 5 ppm 65 8 ppm 92 1 ppm 7 2 TQ 14 1 ppm 23 5 ppm 32 9 ppm 9 2 3Q 319 2 ppm 532 0 ppm 744 8 ppm 9 2 5Q 63 2 ppm 160 4 ppm 144 9 ppm 9 2 7Q 45 6 ppm 76 0 ppm 106 4 ppm 9 2 9Q 10 6 ppm 17 7 ppm 24 8 ppm Figures are calculated for a Larmor frequency of 100 MHz From the isotropic shift and the shift position in the MQ
237. reakthrough limit must be applied and that currents of far more than 20 A occur It is therefore essential that Power levels for pulses must be carefully considered before they are applied Always start at very moderate power levels with an unknown probe find the associated RF field or pulse length and then work your way towards specified values The same applies for pulse lengths especially decoupling periods since the power dissipation inside the probe is pro portional to pulse power and duration Always observe the limits for duty cycle and maxi mum pulse power Please refer to the probe specifications for more information Never set acquisition times longer than required Spinners and turbine must be kept extremely clean Any dirt especially oil sweat from fin gers water will decrease the breakthrough voltage dramatically Make sure the spinner is always clean wipe before inserting touch the drive cap only and the spinning gas supply is carefully checked to provide oil free and dry dew point below 0 C spinning gas Com pressors and dryers must be checked and maintained on a regular basis Any dirt inside the turbine will eventually cause expensive repairs The following setup steps need only be executed upon installation or after a probe repair The test spectrum on glycine should be repeated in regular intervals to assure probe performance BRUKER BIOSPIN User Manual Version 001 Basic Setup Procedures Setting the Mag
238. rection is required after full phase cycle ABSF1 1000 ppm Should be outside the observed spectral width ABSF2 1000 ppm Should be outside the observed spectral width STSR 0 Avoid strip FT STSI 0 Avoid strip FT TDoff 0 Avoid left shifts or right shifts before FT F1 indirect dimension Sl 256 Sufficient in most cases WDW no Don t use window function unless F1 FID is truncated PH_mod pk Apply phase correction BC_mod no No DC correction is required after full phase cycle ABSF1 1000 Should be outside the observed spectral width ABSF2 1000 Should be outside the observed spectral width STSR 0 Avoid strip FT STSI 0 Avoid strip FT TDoff 0 Avoid left shifts or right shifts before FT 160 261 Data obtained with mp3gzfil av from nuclei with spin 3 2 can be processed with xfb if INTO has been set appropriately to run a split t experiment If this is not the case data can be sheared in order to align the anisotropic axis along the F2 axis This is done with the AU pro gram xfshear The AU program checks the nucleus to determine the spin quantum number checks the name of the pulse program and decides what type of experiment has been per formed In case the nucleus is unknown to the program or the pulse program has a name that does not contain a string nq nor nQ with n 3 5 7 9 the required information is asked for by the program in order to calculate the shearing correctly Note that using a user des
239. resonance offset and an incorrect H carrier frequency may cause some intensity loss and a zero frequency contribution Diligent adjustment of the LG condition and the rf carrier is critical for accurate measurement of the dipolar coupling as the splitting increases quadratically with increasing proton frequency offset Simulations of the spin dynamics show that the hetero nuclear term in the Hamiltonian leads to a complicated spectrum for small hetero nuclear dipolar couplings usually introduced by re mote protons see Z Gan s paper for more information User Manual Version 001 BRUKER BIOSPIN 131 261 PISEMA References 1 C H Wu A Ramamoorthy and S J Opella High Resolution hetero nuclear Dipolar Solid State NMR Spectroscopy J Magn Reson A 109 270 272 1994 2 A Ramamoorthy C H Wu and S J Opella Experimental Aspects of Multidimensional Solid State NMR Correlation Spectroscopy J Magn Reson 140 131 140 1999 3 A Ramamoorthy and S J Opella Two dimensional chemical shift hetero nuclear dipolar coupling spectra obtained with polarization inversion spin exchange at the magic angle sample spinning PISE MAMAS Solid State NMR 4 387 392 1995 4 Zhehong Gan Spin Dynamics of Polarization Inversion Spin Exchange at the Magic Angle in Multiple Spin Systems J Magn Reson 143 136 143 2000 Pulse Sequence Diagram a 9 3 440 3 2 ds TPPM decoupling d dd 6 s Ae b TPPM
240. riments the field sweep must be off When the BSMS unit is turned off and on again the sweep will always be on Running spectra with the sweep on will superimpose spec tra at different fields One can set the sweep amplitude to 0 in order to avoid such an accidental error condition Setting Up for Cross Polarization on Adamantane 3 6 Cross polarization is used to enhance the signals of X nuclei like 13C The strong proton polar ization is transferred cross polarized to the X nuclei coupled to the protons via strong dipolar couplings To achieve this the protons and the X nuclei must nutate at the same frequency This frequency is the RF field applied to both nuclei at the same time contact time If this con dition Hartmann Hahn condition is met the transfer of proton magnetization to carbon is opti mum Since the proton signal of adamantane is resolved into spinning sidebands even at slow spin rates this Hartmann Hahn condition can be set to match for every proton spinning side band Using a ramp for the proton contact pulse the Hartmann Hahn match is swept over these possible match conditions and becomes insensitive to miss sets and different spin rates Start from the data set used for observing C under proton decoupling 1 4 Load the pulse program cp in eda or typing pulprog cp The pulse sequence is depicted in the following fig ure EB CADOCUME 1 ADMINI TALOCALS 1 Temp SPDISP_50494 tmp cpedit hf Graphics assistar Edtte
241. rmation on the vector connecting the 13C or 15N and the 1H nu cleus The achievable high resolution of the CS as well as the dipole coupling makes the experiment well suited for 3D NMR experiments on aligned systems or single crystals Unlike normal FSLG experiments where the dipolar and CS interactions are scaled by cos 0 577 the scaling factor for the hetero nuclear dipolar interaction is sin O 0 816 because the coupling takes place in the transverse plane of the rotating frame the spin locked state The projection is from the tilted frame locked 1H spin system to the transverse plane of the rotating frame system spin locked 15N spin system Through the combination of spin exchange dipolar flip flop term and the Homo nuclear de coupling using FSLG PISEMA achieves a line width that is an order of magnitude better than its predecessor the separated local field experiment The central line in the dipolar dimension can among other things be caused by a proton fre quency offset introducing a constant term in the time domain signal That offset frequency also makes the splitting larger See additional test procedures in A Ramaamoorthy et al Experi mental Aspects of Multidimensional Solid State Correlation Spectroscopy PISEMA is not very sensitive to the exact Hartmann Hahn condition A mismatch has only little effect on the dipolar coupling The scaling factor in the indirect dimension depends of the 1H
242. rrent value gt 0 6 After xfb the relative peak positions will be approximately correct but the absolute peak positions must be correct ed by calibrating a known peak position to the correct value Table 22 6 Processing Parameters Parameter Value Comment mc2 STATES TPPI Or whatever used wdw QSINE Slight moderate resolution enhancement is usually required ssb 3or5 si 2k 4k 1 si 512 1k Examples 22 8 These spectra were both taken without the modification according to CRAMPS 2D on page 225 so the offset is placed to the down field side and the spectrum width was chosen larger than necessary The small plots show the full spectrum User Manual Version 001 BRUKER BIOSPIN 233 261 CRAMPS 2D tring sping in both de gem atten 10 9 8 7 6 5 4 3 ppm Figure 22 6 Glycine Proton Proton DQ SQ Correlation Using WPMLG in Both Directions 234 261 BRUKER BIOSPIN User Manual Version 001 CRAMPS 2D 14 5 kHz W PMLG PC7 DQ SQ correlation at 600 MHz with ppm h VU tyrosin hydrochloride em 107 40 30 20 10 ppm 1312 11109 8 7 6 5 4 3 2 ppm Figure 22 7 14 5 kHz W PMLG PC7 DQ SQ Correlation at 600 MHz with Tyrosine Hydrochlo ride User Manual Version 001 BRUKER BIOSPIN 235 261 CRAMPS 2D 236 261 BRUKER BIOSPIN User Manual Version 001 Appendix Form for Laboratory Logbooks A 1 The form on the following page may be printed and filled out by every user using the instrument to
243. ructions in the popup window Calculate the power level in Watts or dB required to achieve a 4 5 usec proton 90 degree pulse In this case 6 usec were obtained The command calcpowlev calculates a power level 2 5 dB higher than used above to achieve 4 5 usec pulse length Check whether 2 4 5 usec for p1 will give a close to zero signal This is a safe power level for all probes for pulses up to 100 msec length 30 261 BRUKER BIOSPIN User Manual Version 001 Basic Setup Procedures Calibrating 13C Pulses on Adamantane and Shimming the Probe 3 4 A high power decoupling experiment on 13C of adamantane is used to measure 13C pulse pa rameters NOTE For experiments where long decoupling pulses on protons are executed the pro ton preamplifier must be bypassed e the transmitter should be wired to the probe di rectly via the proton bandpass filter without going through the preamp if a high power proton preamplifier is not available For HP HPPR modules H F this is not absolutely necessary but recommended For HPLNA 1H modules it is not required to bypass Note that when bypassing the preamp which attenuates by about 1 dB the proton power lev els should be corrected by adding 1 dB to the pl values Type edasp in the command line You should get a display like in Figure 3 11 Click on SwitchF1 F2 to set for C observation with proton decoupling Load the pulse program hpdec Set cpdprg2 cw Set pl12to the power level that yie
244. rum after tilting the spectrum setting 1 alpha 1 and using the command ptilt1 repeatedly until the CSA lines are within the spectral range 116 261 BRUKER BIOSPIN User Manual Version 001 SUPER Figure 11 6 Various Cross Sections from the Upper 2D Experiment Figure 11 6 illustrates various cross sections from the upper 2D experiment from which CSA parameters can be determined User Manual Version 001 BRUKER BIOSPIN 117 261 SUPER 118 261 BRUKER BIOSPIN User Manual Version 001 Symmetry Based Recoupling Sample rotation averages most anisotropic interactions and therefore removes the information available from them Therefore selective recoupling of anisotropic interactions is desired for structural analysis re coupling reintroduction of anisotropic interactions like e g dipolar cou pling in order to regain specific information The topic has been thoroughly reviewed by E A Bennett et al and by S Dusold et al One strategy is the use of symmetry based recoupling se quences see M Hohwy et al 1998 et al and A Brinkmann et al 2000 In these sequences double quantum coherence are excited via the dipolar Homo nuclear dipolar coupling Single quantum coherence are suppressed by phase cycling The size of the dipolar coupling can be determined from the build up rate of DQ signal intensity measured after reconversion into SQ coherence It should also be mentioned that there are recoupling sequences t
245. ryo protectant glycol glycerol will very likely be present This means that the probe proton channel will be detuned to low er frequency and tuning may be difficult if not impossible at high proton frequencies and salt contents In such cases E probes are recommended 2 Run standard 1D cp 13C and 15N experiments determine the required offsets for all fre quencies and the required sampling windows 3 Re optimize the H N and N C HH conditions 4 Generate a new data set and switch to 2D data mode using the 123 icon in eda 5 In eda set the pulse program to doubcp2d Set FnMode as desired usually STATES TP PI User Manual Version 001 BRUKER BIOSPIN 201 261 Double CP Make sure the correct nucleus SN is selected in the F1 dimension Set the sampling windows for both dimensions from the previously acquired 1D spectra Both acquisition times in F2 and F1 should be considered with care since the decoupler is on at high power during both periods Especially for biological samples where the RF heat ing may be high and the samples are temperature sensitive it is essential not to use overly long acquisition times and high duty cycles Remember that the heating effect is generated inside the sample where the temperature increases within milliseconds whereas cooling re quires transfer of the energy to the outside of the spinner which takes seconds Eee probes eliminate these problems to a large extent The
246. s With short dipolar recoupling times only spins in close spatial proximity lead to cross peak facilitating assignment of 13C resonances in uniformly labelled peptides for instance RFDR may also be used in order to correlate chemical shifts and crystallographic sites on materials samples The Homo nuclear dipolar recoupling is implemented via the application of rotor synchronised 180 degree pulses one inversion pulse per rotor period The phases of the 180 degree puls es are cycled with Gullion s compensated XY 8 echo sequence in order to achieve efficient re covery of single spin magnetization and to generate an effective dipolar recoupling Hamiltonian during the mixing period The critical experimental point is to avoid 1H X recoupling induced by interference between the TH decoupling rf field and 13C rf recoupling field This effect can be removed using a 1H decoupling rf field 3 times as strong as the 13C rf field used for recoupling or by using Lee Goldburg 1H decoupling during the mixing period References T Gullion D B Baker and M S Conradi New compensated Carr Purcell sequences J Magn Reson 89 479 484 1990 A E Bennett J H Ok R G Griffin and S Vega Chemical shift correlation spectroscopy in ro tating solids Radio frequency driven dipolar recoupling and longitudinal exchange J Chem Phys 96 8624 8627 1992 A E Bennett C M Rienstra J M Griffith s W Zhen P T Lansbury and R G Griffin
247. s ps 8 17 PCPD2 ps 4 20 PL2 dB 7 00 PLOW W 4741231155 PL12 08 6 00 PL12w DA 59 68856430 PL13 08 5 00 PLISWY DA 59 58856430 pulsa us 1 34 SFO2 MHz 500 1500000 SPO GB 5 10 SPOW w 50 32980739 SPNAM Ramp 70100 100 SPOALO 0 500 SPOFFSO Hz 0 00 Figure 7 3 The ased Display Ce LI BRUKER BIOSPIN Ighetfg LE Pulse program for acquisition Time domain size Number of scans Number af dummy scans Sweep width in Hz Acquisition time Receiver gain Owell time Pre scan delay To adjust t 0 for acquisition if digmod base LG RF field as adjusted in Hz used to calculat Offset tor proton evolution under LG usually O Recycle delay INO 0 570713 4 294 360Venst20 LO 0 For dwell inti 4 p5 I3 0 278 Countz tdi1 2 Dwellf1 inO BiKt 1 2u Nucleus far channel 1 Contact pulse short 30 200 us For X contact pulse For X contact pulse Frequency of observe channel Elktr2 1u Cnst21 0 Cnst22 cnst2 sqrt 2 4enst24 Cnst23z cn3620 3q t 2 cnst24 Topm 15 SPINALSA Nucleus for channel 2 90 degree 1H pulse excitaton PS 294 360y cnst20 y 1e5 Pulse length in decoupling sequence 120dB not used 120dB not used For decoupling and excitation 1H For decoupling and excitation 1H For homonuclear decoupling For homonuclear decoupling Pul amp 4 n37547 900 Frequency or observe channel Proton power level during contact Proton power level during contact Shape for contact pulse ramp
248. s TPPI phase sensitive de tection in F1 is needed Thus a full phase cycle can be performed with a multiple of 160 scans The usefulness of such a 5Q experiment is limited and there are several drawbacks Firstly the sensitivity is much inferior to the 3Q experiment because of the lower transition probability and a less efficient excitation Secondly the shift range in ppm in the indirect dimension is much smaller when a rotor synchronized experiment is performed The factors R p are listed in Table 15 5 The shift positions in the MQ dimension in a sheared spectrum are the same for all orders p and therefore no additional information can be expected However the observed line widths are slightly reduced in the higher order experiments so in special cases some en hancement of resolution can provide additional information Table 15 5 Values of R p for Various Spins I and Orders p Spin I R p 3 R p p 3 IR p p 5 IR p p 7 IR p p 9 3 2 7 9 3 78 5 2 19 12 1 42 7 08 7 2 101 45 0 76 3 78 10 58 9 2 91 36 0 47 2 36 6 61 14 17 164 261 The spectral width in the MQ dimension of the sheared spectrum is given by spinning speed R p in a rotor synchronized experiment A 5Q experiment e g gives a 7 08 1 42 5 times smaller spectral range in the indirect dimension than a 3Q experiment We see that a 5Q experiment has a 5 times smaller range than the 3Q experiment and the
249. s determined by the line width and resolution that can be expected and which depend on the properties of the sample In crystalline material fairly narrow peaks can be expected so that a maximum acquisition time in F1 of 2 to 5 ms is expected In disor dered material where the line width is broader and determined by distribution a total acquisition time in F1 up to may be 1 ms may be sufficient The total acquisition time aq in F1 equals TD 2 IN 010 For rotor synchronized experiments IN 010 1 spinning frequency so will typically BRUKER BIOSPIN User Manual Version 001 MQ MAS Sensitivity Enhancement be between 100 us 10 kHz spinning and 28 5 us 35 kHz spinning so only 100 to 250 exper iments might be required The rotor synchronization immediately means that the spectral range in F1 is limited Dependent on chemical shift range spinning frequency and quadrupole inter actions the positions of the peaks may fall outside this range In such a case care must be tak en when interpreting the spectrum Acquisition with half rotor synchronization to double the spectral window in F1 may help However in this situation one set of spinning sidebands ap pears and it must be avoided that the spinning side bands of one peak fall on top of other peaks Some sort of rotor synchronization is always recommended because spinning side bands in the indirect dimension extend over a very wide range which cannot be truncated by e g filtering Therefore rot
250. sample should be dried at 70 C for a couple of hours before packing the rotor in order to eliminate crystal water completely Kai Recycle delays at 11 7 T longer delays may be required at higher fields 154 261 BRUKER BIOSPIN User Manual Version 001 Basic MQ MAS Figure 15 3 shows a comparison of a spectrum excited by a short non selective pulse with a spectrum that has been obtained by a weak selective pulse Note that in the latter the spinning sidebands from the satellite transition are no longer visible which is used as an indication that it is not excited 2000 1500 1000 500 0 500 1000 1500 2000 ppm of gt IS ER mm 20 A0 40 80 ppm od p EES x ET m F T RAA ih 3 2000 1500 1000 500 0 500 1000 1500 2000 ppm Figure 15 3 Comparison of 87Rb MAS spectra of RbNOS excited with selective and non se lective pulses The lower trace is a spectrum excited with a 1 uis non selective pulse corresponding to a small flip angle Above is a spectrum excited with a 20 us selective 90 pulse Note that in the latter no spinning side bands from the satellite transition are observed Spectra are taken on AV500WB at a Larmor frequency of 163 6 MHz with a 2 5 mm CP MAS probe spinning at 25 kHz Figure 15 4 shows the nutation profiles of a non selective and a selective pulse respectively Note that for the selective pulse a fairly precise 180 pulse of a length of 2 tgy can be deter mined whereas for a non se
251. se HH 163 Figure 15 10 170 MQMAS of NaPO3 at 11 7 T 67 8 MHz on the left and 18 8 T 108 4 MHZ onthe right 42 dE po raped ebe 166 User Manual Version 001 BRUKER BIOSPIN 245 261 Figures 246 261 Figure 15 11 Slices and Simulations of the 18 8 T 170 MQMAS of NaPOS 167 Figure 15 12 Graphical Interpretation of the Spectrum from Figure 15 10 168 16MQ MAS Sensitivity Enhancement 169 Figure 16 1 Hahn Echo Pulse Sequence and Coherence Transfer Pathway 170 Figure 16 2 Processing of Hahn Echo Left is the Shifted Echo 171 Figure 16 3 Four Pulse Sequence and Coherence Transfer Pathway for the 30 MAS Experiment E 172 Figure 16 4 Three Pulse Sequence and Coherence Transfer Pathway 172 Figure 16 5 Example for popt to Set up for Optimization of DFS sssss 175 Figure 16 6 Signal Intensities of 87Rb in RbNO3 sssssse nennen 176 Figure 16 7 Pulse Sequence and Coherence Transfer Pathways for SPAM 3QMAS tad 181 17STMAS 183 Figure 17 1 Principle of 2D Data Sampling in STMAS Experiments 183 Figure 17 2 Four pulse sequence and coherence transfer pathway for the double quan tum filtered STMAS experiment with z filter stmasdqfz av 185 Figure 17 3 Four pulse sequence and coherence transfer pathway 186 Figure 17 4 87Rb S
252. se requires about 4 5 dB more power corresponding to almost 4 times more power With p3 properly set a spectrum like in Figure 3 25 should be obtained with about 93 kHz de coupling RF field User Manual Version 001 BRUKER BIOSPIN 37 261 Basic Setup Procedures File Edi View Spectrometer Processing Analysis Options Window Help DEM PSHRwwAAAL SEX AICA AA B TRANS TBRARRAR SA atta Seecrum Grofen AcauPars Tae PuseProg Peake riega Simple Suche Fic Acq Phase Pret 514 Figure 3 25 Glycine with cw Decoupling at 90 kHz RF Field In the spectrum above a lorentzian deconvolution Analysis menu shows a line width of 71 Hz for the peak at 43 ppm The line width achievable under optimum decoupling conditions varies with the magnetic field At fields below 9 4 Tesla 400 MHz this line is substantially broadened by second order quadrupolar interaction to 14N At fields above 9 4 Tesla 500 MHz and higher the residual line width is mostly determined by chemical shift dispersion and insuf ficient decoupling Here less than 60 Hz at 600 MHz are expected More efficient decoupling schemes must be applied especially at higher magnetic fields A more efficient decoupling scheme is spinal64 Select cpdprg2 spinal64 set pcpd2 to proton 180 degree pulse 0 2 usec for a start A glycine spectrum as shown in Figure 3 26 is obtained 38 261 BRUKER BIOSPIN User Manual Version 001 Basic Setup Procedures
253. set other 2D parameters as for the other relaxation experi ments 6 Acquire spectrum with zg Data Processing The data can be processed in the same way as the other relaxation experiments The slice with shortest spin lock time contains most signal so this slice should be used for processing The fitting function should be set to expdec and vplist should be selected as the list file name in the fitting function dialogue At 500 MHz with a 60 kHz spin lock field the T4 values should be approximately 400 ms and 48 ms for the carbonyl and alpha carbons respectively The data for the alpha carbon does not give a perfect fit to a single exponential but this may result from the relatively low spin lock field allowing non T4 relaxation Indirect Relaxation Measurements 14 3 If proton relaxation measurements are desired the considerable broadening of the proton res onances seen at even high spinning speeds can make resolution of individual components im possible In such cases indirect observation of proton relaxation by X nucleus observation can be used A typical example would be attempting to observe the proton relaxation of two compo nents of a mixture or multi phase material In general the proton spins within a single molecule are sufficiently strongly coupled by the Homo nuclear dipolar coupling that different relaxation is not seen for the different sites If the experiments are set up with short contact times the in dividual c
254. shows optimization of TPPM decoupling on glycine at natural abundance 13C CPMAS at 5 kHz spin rate Each block represents a 2 degree increment of the phase tog gle and the variation in each block stems from incrementation of the pulse width in 0 2 us incre ments Optimum decoupling was found with a 4 5 us pulse at a 16 phase toggle It is obvious that more than one near optimum combinations of phase toggle and pulse length exist Reference A E Bennett C M Rienstra M Auger K V Lakshmi and R G Griffin Hetero nuclear decou pling in rotating solids J Chem Phys 103 16 6951 6958 1995 46 261 BRUKER BIOSPIN User Manual Version 001 Decoupling Techniques SPINAL Decoupling 4 1 3 SPINAL decoupling is a super cycled TPPM decoupling sequence SPINAL16 provides ade quate low power B1 field around 50 kHz decoupling for static samples exceeding decoupling performance of higher power cw decoupling and provides an adequate decoupling bandwidth at ny of approximately 50 kHz For rotating samples SPINAL64 and SPINAL128 exceed TPPM15 decoupling at high decou pling fields gt 80 kHz SPINAL64 can be optimized in the same way as TPPM by incrementing pcpd2 p37 the phase shifts are fixed The decoupling pulse is an approximate 180 pulse Reference B M Fung A K Khitrin K Ermolaev J Magn Reson 142 97 101 2000 XiX Decoupling 4 1 4 XiX decoupling requires high spinning speeds but decouples at a mod
255. sion Recovery Experiment 143 Table 14 2 Parameters for 2D Inversion Recovery Experiment 144 Table 14 3 Processing Parameters for CP T1 Relaxation Experiment 145 Table 14 4 Parameters for the Saturation Recovery Experiment 148 15Basic MQ MAS 151 Table 15 1 Some Useful Samples for Half integer Spin Nuclei 154 Table 15 2 Initial Parameters for Setup ooocooccoccccccncconcccnconcnoncnncnnnconcncnninannnninannnns 157 Table 15 3 F1 Parameters for 2D Acquisition oooooncocccocccoccnccnccnncnnnnnnnnnnnnnnnnnnnnno 158 Table 15 4 Processing Parameters for 2D FT sss 160 Table 15 5 Values of R p for Various Spins and Orders p 164 Table 15 6 Chemical Shift Ranges for all MQ Experiments for All Spins 165 16MQ MAS Sensitivity Enhancement 169 Table 16 1 Initial Parameters for the DFS Experiment ssesessesesssse 174 Table 16 2 Parameters for 2D Data Acquisition of 3 pulse Shifted Echo Experiment Lu RD DIS ui co 177 Table 16 3 Parameters for 2D Data Acquisition of 4 pulse Z filtered Experiment ul e LC naeh anne 178 Table 16 4 Processing Parameters nenn nennen nnnn nn 179 Table 16 5 Parameters for FAM 2 cccceeeceeeceeceeeeeeeeeee enne 180 Table 16 6 Further Parameters for 2D Data Acquisition of SPAM MQMAS Experiment
256. st In a first step a low power selective pulse must be calibrated in a single pulse experiment Af ter this the STMAS experiment can be optimized using the 2D pulse sequence for the first t4 in crement Set up of the Experiment 17 4 1 Sample There are a large number of crystalline compounds that can be used to set up the ex periment Please refer to Table 17 2 to select a suitable sample For the general procedure described here the spin of the nucleus is not important of course the obtained pulse widths will depend on the spin I and the Larmor frequency For the STMAS experiment in contrast to MQMAS it is advisable to use a well known sample for the setup because the accuracy of the magic angle setting is extremely critical Table 17 2 Some Useful Samples for Some Nuclei with Half Integer Spin Nucleus Spin ae d1 s Sample Comments 170 5 2 67 78 2 NaPO gt 10 enriched 11B 3 2 160 42 gt 5 H3BO3 23Na 3 2 132 29 10 Na HPO 2 7A 5 2 130 32 5 YAG 87Rb 3 2 163 61 0 5 RbNO3 93Nb 9 2 122 25 1 LiNbO 1 In MHz at 11 7 T i e 500 13 MHz proton frequency 2 Alternatively Na gt HPO 2H50 can be used For anhydrous Na HPO the sample should be dried at 70 C for a couple of hours before packing the rotor in order to eliminate crystal water completely KW Recycle delays at 11 7 T longer delays may be required at higher fields As for MQMAS the setup must be done in two steps in the
257. st set as the parameter vdlist Table 14 1 Parameters for the 1D CP Inversion Recovery Experiment Parameter Value Comments Pulprog cpxt1 Vdlist See text Relaxation delays after inversion pulse Short value to set spectrum phase correctly User Manual Version 001 BRUKER BIOSPIN 143 261 Relaxation Measurements Table 14 1 Parameters for the 1D CP Inversion Recovery Experiment d1 3s Needs only to be 3x proton T1 pl11 Measured X pulse power p1 Measured 90 X pulse length at pl1 Ns 2 Should be enough to see a reasonable spec trum Table 14 2 This pulse program uses the method of Torchia in which the phase of the contact pulse and the receiver is inverted in alternate scans In the first scan the first 90 pulse creates mag netization and in the second scan it creates z The phase cycling of the receiver means that the difference between the two scans is recorded For short relaxation delays neither relaxes significantly and so the maximum signal is recorded At longer relaxation delays both the z magnetization which is larger than the equilibrium value as it is created by CP and the z magnetization relax and the recorded signal decays exponentially as a function of the relax ation delay At long times both have relaxed back to equilibrium and the two scans yield a zero signal The resulting spectrum should be phased to give posi
258. t 1970 sp1 set for 100 130 kHz WPMLG p5 1 2 1 5 usec or calculated from cnst20 RF field DUMBO p10 set by xau dumbo Q4 CYCLOPS 0123 User Manual Version 001 BRUKER BIOSPIN 211 261 CRAMPS 1D Pulse Shapes for W PMLG and DUMBO 20 2 Both shapes are purely phase modulated pulses their amplitudes are constant throughout The PMLG shape is a standard shape delivered with the software wpmlg1 m5m m5p DUMBO shapes are generated using the standard AU program dumbo Calling dumbo with xau dumbo will ask for the slice length of the shape usually 1 usec the number of slices usually 32 generate the shape and load the name of this shape into the current parameter set it will also set the length of the shaped pulse p70 to 32 usec N B at magnetic fields higher than 500 MHz it is recommended to replace the standard 32 usec timing by 24 usec timing and increasing the power level accordingly since this has been found to give better re sults G4Brumereucne wpmigt open E H NPONTS EXMOOEC 00 TOTROT 0 000000 BWFAL 0 000000 INTE GF AL 0 MODE 20 40 en S Edt Shape Paramteters 1 po 50 100 ETT suerte ERBRRETEREETORUNER Figure 20 2 PMLG Shape for wpmlg sp1 212 261 BRUKER BIOSPIN User Manual Version 001 CRAMPS 1D 3 UBA mM swine dumbo_1 0 e Open f m NPOMTS None EXMODE 00 TOTROT BAFA INTEGFAC 0 MODE 20 40 o S Lassslrar iararlirpiiccinl
259. t is about 10 20 us For this experiment to succeed reliably one should use moderate spinning speeds around up to 10 kHz At slow rotation rates no advantage was found in measuring the exact HH match Running the experiment at constant amplitude CP optimized for maximum signal proved to be sufficient References for these experiments 72 261 1 X Wu K Zilm Complete Spectral Editing in CPMAS NMR J Magn Reson A 102 205 213 1993 2 X Wu K Zilm Methylene Only Subspectrum in CPMAS NMR J Magn Reson A 104 119 122 1993 3 X Wu S T Burns K Zilm Spectral Editing in CPMAS NMR Generating Subspectra Based on Proton Multiplicities J Magn Reson A 111 29 36 1994 4 R Sangill N Rastrup Andersen H Bildsoe H J Jakobsen and N C Nielsen Optimized Spectral Editing of 13C MAS NMR Spectra of Rigid Solids Using Cross Polarization Methods J Magn Reson A 107 67 78 1994 3 ds heteronuclear decoupling 2 2 Prec Be Figure 6 12 Block Diagram of the CPPI Experiment Typical pulse widths for the second part of the CP pulse with the phase inversion are 40 us BRUKER BIOSPIN User Manual Version 001 Basic CP MAS Experiments Solis Manual chapter2 13 1 C Jos1206 Me Rar et LJE E El 5 5 05 ls AA X MJ Solids Marual chapter2 16 1 Ch josl2065 re VER a A Ale Ge m Solids Marual_cheptere 14 1 Can 3951205 Solids Marval chaprerz 13 TE 73021208 alo ll a SS 100 50 9
260. t any peaks with integrals or intensities too close to zero will be omitted from the analysis by User Manual Version 001 BRUKER BIOSPIN 145 261 Relaxation Measurements E intersty C Area Currert Peak 2 at 2 the software if you see less points in the relaxation window than were actually recorded this may be because they have insufficient intensity Fitting function Here the parameters of the fitting calculation are set The general parameters should be determined automatically but ensure that the limits for baseline correction are set to cover the whole spectrum The fitting function depends on the experiment but in this case the signals decay exponentially so the function expdec should be chosen The list filename should be vd this will take the specified vdlist from the data set Note that when the experi ment is run the selected vdlist is written into the acquisition data directory as the file vdlist so it is always available even if the source list is edited The fitting program can calculate multi exponential fits but data with very good signal to noise is required for this to be accurate Un less there is obvious overlap of peaks the assumption is usually that each peak corresponds to a single nuclear site and thus a single T4 value bio u gil Tg 1 3 ire ussqun ia 0608 1 Mio ae aot Oe Fitting type gegen Kn as T4 r N ele 7 3 Peak Mi W Bret Raport 8 Peak 1 2t
261. t has a wide spread 2 5 12ppm of proton shifts a short proton T4 a well resolved 1SC spectrum with quite many lines and it is readily available The unlabeled sample can also be used but requires a few more scans 8 32 4 Optimize the spin rate such that no overlap occurs between center and sidebands espe cially with the labeled sample in order to avoid rotational resonance broadening Re opti mize decoupling and HH condition Check the proton RF field via the proton 90 pulse p3 Set pl13 pl12 set cnst20 to RF field in Hz as calculated from p3 5 Generate a new data set with edc new Set pulprog Ighetfq and change to a 2D parameter set using the 123 button in eda Set FnMode to STATES TPPI Type ased or click the pulse symbol in eda Spectrum ProcPars ACQUPars Title PulseProg Peaks Integrals Sample Structure Fid Ae qu Phase Print nis W ES OV d Installed probe 4 mm MAS 8B 1H H12083 0005 Figure 7 2 The 12 icon and the ased icon in eda 6 Performing ased will show all parameters which are essential for the acquisition not all available parameters In addition it performs calculations which are specified in the pulse program Note that all parameters which are calculated are not editable and will show only if explicitly used during the main pulse program between ze and exit In this sequence the proton chemical shift evolution is influenced by the RF field cnst20 under which the shifts evo
262. t leads to attenuation In MAS experiments it is advisable to synchro nize the echo with the sample rotation i e make r an integer multiple of rotor periods For FT of the shifted echo FID there is a slight inconvenience as shown in Figure 16 2 because after a normal FT the signal looks quite unconventional To obtain the usual spectrum a magnitude calculation can be done on 1D spectra with the loss of phase information Alternatively and in particular in 2D spectra it is possible to apply a large 1 order phase correction phc1 to com pensate for the time delay before the echo top The value of this is d6 here qug P d w Eq 16 1 This value can be entered into the processing parameters and a phase correction pk can be performed After this the O order phase correction still needs to be adjusted interactively The best method is to phase the spectrum to give minimum signal intensity and add or subtract 90 to the obtained value click 90 or 90 in the TopSpin phasing interface BRUKER BIOSPIN User Manual Version 001 MQ MAS Sensitivity Enhancement FT PK VEER ENEE t VR Eege EIL 0 9 ms 10 40 60 ppm 40 60 ppm Figure 16 2 Processing of Hahn Echo Left is the Shifted Echo The middle shows the spectrum after FT On the right is the spectrum with the correct first or der phase correction Implementation of DFS into MQMAS experiments 16 2 Two pulse sequences are available to implement a double frequency
263. ted and it must be short enough so that the central transitions of all sites in the spectral range are excited As an example the sinc shape excitation profile of a 20 us pulse has its zero crossings at 1 20 us 25 kHz which means that the central transi tion signals must not extend beyond this range otherwise severe line shape distortions will be observed On the other hand the corresponding RF field amplitude of a 20 us 90 pulse will be 1 80 ys 1 1 2 12 5 kHz 1 1 2 This means that waF lt lt wg as a prerequisite for a CT selec tive pulse is most likely to be fulfilled For the calibration of this pulse a power level around 30 dB with 500 W and 1 kW amplifiers and around 20 dB with 300 W amplifiers should be expect ed The pulse program zg which uses p1 and ol or zgsel av which uses P3 and PL21 can be used User Manual Version 001 BRUKER BIOSPIN 153 261 Basic MQ MAS Table 15 1 Some Useful Samples for Half integer Spin Nuclei Nucleus Spin zn d1 sT Sample Comments 179 5 2 67 78 2 NaPOz gt 10 enriched 11B 3 2 160 42 gt 5 H3BO3 23Na 3 2 132 29 10 Na HPO 2 27 Al 5 2 130 32 5 YAG 27 Al 5 2 130 32 0 5 Al203 27 Al 5 2 130 32 0 5 VPI 5 27 Al 5 2 130 32 0 5 AIPO 14 11B 3 2 160 46 5 H3BO3 87Rb 3 2 163 61 0 5 RbNO3 93Nb 9 2 122 25 1 LiNbO 1 In MHz at 11 7 T i e 500 13 MHz proton frequency 2 Alternatively Na gt HPO 2H50 can be used For anhydrous Na HPO the
264. th the transverse magnetization and the difference between the current and equi librium z magnetization decay exponentially with time constants denoted T for longitudinal re laxation and T for transverse relaxation Relaxation also occurs while radio frequency pulses are being applied to the system Normally this is ignored but in the case of spin locking pulses itis important During cross polarization the magnetization on the dilute spins is increased by User Manual Version 001 BRUKER BIOSPIN 141 261 Relaxation Measurements transfer from another nucleus but it will also decay since the radio frequency field weak com pared to the static field Bo is insufficient to maintain the resulting transverse magnetization If the pulse on the excitation nucleus is stopped and only that on the detection nucleus contin ued the transverse magnetization will decay exponentially with a time constant denoted Ty This rate of decay will be strongly affected by the amplitude of the spin locking pulse Both of these processes occur via spin energy level transitions It turns out that the spontane ous transition rate is very low and thus relaxation is dominated by stimulated transitions Such transitions are stimulated by local magnetic fields which fluctuate due to local molecular mo tion and the transition rates depend on the strength and details of the fluctuations of these lo cal fields Since the fluctuations are random the rate of fluctuat
265. the 123 button Set in eda the appropriate FnMode param eter Pulse program parameters are listed in Table 13 1 in eda set ocPars AcquPars Title Pu EVA Figure 13 2 The 123 Icon in the Menu Bar of the Data Windows Acquisition Parameter Page The 123 icon in the menu bar of the data windows acquisition parameter page is used to tog gle to the different data acquisition modes 1D 2D and 3D if so desired 8 Go into eda and set parameters for sampling in the indirect dimension the spectral width 10 11 12 13 In order to set the t4 increment go into the ased window and choose 3 to be 1 2 or 3 This sets the t4 increment and the parameter inO is updated To get the calculated parameter inO such that one can inspect the appropriate spectral width in F1 which should be no less than 20 kHz type inO and enter the value given behind inO in the ased window Figure 13 3 Make sure the correct nucleus is selected in Me dimension Choose the appropriate sampling time TDT so that the required resolution FIDRES in the indirect dimension is achieved Depending on the decision above whether the rf power on the X or on the 1H channel are changed during the SEMA part of the experiment set a either PL13 PL2 case 4b and set PL11 to the optimized value higher power i e a value of about 1 8 dB below pl1 b or set PL11 PL1 case 4a above and PL13 to the obtained value in the cplg experi
266. the 3 2 lt gt 1 2 transi tions however are broadened by a 1 order interaction which is several orders of magnitude larger than the 2 d order broadening Under MAS the 1 order interaction of the ST can be av eraged but since the spinning cannot be fast compared to the first order broadening of the or der of MHz a large manifold of spinning side bands remains The 24 order broadening of the CT can only be narrowed by a factor of 3 to 4 by MAS so a signal is observed that still reflects this 24 order broadening which may be of the order of kHz Lineshapes resulting from nuclei in different environments are thus likely to be unresolved in a simple 1D spectrum The 2D MQMAS experiment exploits the fact that the 2nd order broadening of the symmetric MQ transitions e g 3 2 3 2 in a spin 3 2 is related to the 2nd order broadening of the CT by a simple ratio A 2D spectrum is recorded which correlates e g a 3 2 3 2 3Q coherence involving the satellite transitions and the 1 2 1 2 single quantum coherence of the central transition This spectrum shows a ridge line shape for each site with slope given by the ratio of the second order broadening of the two transitions 7 9 in the case of the 3Q transition A pro jection of the 2D spectrum perpendicular to this slope yields an isotropic spectrum free from quadrupolar broadening Pulse sequences 15 2 Figure 15 1 and Figure 15 2 show two of the basic sequences a 3 pulse and a
267. the serial file BRUKER BIOSPIN User Manual Version 001 MQ MAS Sensitivity Enhancement Figure 16 7 Pulse Sequence and Coherence Transfer Pathways for SPAM 3QMAS It is extremely convenient that the setup of the pulse lengths and power levels can be done with the pulse program mp3qzqf av The setup procedure is exactly the same as described for this experiment in the chapter Basic MQ MAS on page 151 Before the start of the 2D data acquisition all that needs to be set is the pulse program mp3qspam av and a small number of other parameters These are listed in Table 16 6 Table 16 6 Further Parameters for 2D Data Acquisition of SPAM MQMAS Experiment mp3qspam av Parameter Value Comments pulprog mp3qspam av Further F1 parameters In eda FnMode Echo Anti echo Acquisition mode for 2D Further pulse program In ased parameters D4 0 5 us Not 20 us like in mp3qzqf av 14 1 Set by the pulse program internally used counter 15 see text Number of anti echos to be acquired 0 gt L5 gt td F1 2 16 3 For spin I 3 2 1 For all other spins User Manual Version 001 BRUKER BIOSPIN 181 261 MQ MAS Sensitivity Enhancement 182 261 FNMODE Even though this parameter is not evaluated by the pulse program it will be used by the processing AU program xfshear D4 A very short delay is used here just to allow for amplitude and phase switching 14 This loop c
268. times are re quired to reintroduce the dipolar coupling When 1H decoupling is required it is important to avoid any transfer of magnetization to or from the proton spin system HH condition which would destroy the desired information This means that the effective fields on X and H must be very different However proton decoupling must still be efficient as well It has been shown that the two RF fields should differ by a factor of 3 which in practice is extremely difficult to meet It has also been shown that at very high spin rates gt 16 kHz decoupling is not necessary at all A possible trick is also to use off resonant LG decoupling during the recoupling sequence This enhances the effective proton field vector sum of RF field and offset and sharpens the HH condition since the Homo nuclear couplings are suppressed Another important parameter to observe is the required excitation bandwidth of these sequenc es Naturally going to higher magnetic fields the higher chemical shift spread requires higher RF fields for the recoupled X nuclei requiring even higher RF fields for protons So the tenden cy is going to high spin rates also desired to get rid of spinning sidebands and turning the de coupling off during recoupling which represents a much lower RF load to the probes and increases experimental stability substantially User Manual Version 001 BRUKER BIOSPIN 119 261 Symmetry Based Recoupling Table 1 shows the sample rotati
269. tion cocida nn ea ale 141 User Manual Version 001 BRUKER BIOSPIN 255 261 Index 256 261 M magic angle nn Malonic Acid cert tens matching bon monoexponential oooooococccccccccococconcncconnnnnn Multiple Pulse Decoupling Multiple pulse NMR sssssssss N le observe nucleus occoccccccncoconconnoncnncnnncnnnnnnnns offset Ola anne nun oil free gas cooooonnocccccnnnocincconncannnccnanan cnn ele EE optimum POSMAX coccccccccocccconcnnncnononcnnnnnannnnns P PDNOS vaticina ias dcs een IMAG E pimlgliet 2 tette seid ero a IAS ai ee power conversion factor Power Conversion Table preamp EE preamplifier nenn Profile o crecen Proton Bandpass Filter BRUKER BIOSPIN User Manual Version 001 Index GESOT ek a ce da aay ceases E a aar 55 ee ee Heu eed ieee ee 35 55 Proton Proton DQ SQ Correlation nennen 230 SET ele OR EE 145 PI E ee ee 11 O 19 a EI 11 Q en 12 R iso c D 11 RENOS A 11 le P M M 141 TA 27 RE VONAG EEN 14 A 93 e A ee euch 13 a Mm 13 immer e nu Le 13 RF POWER u nenne ara A 13 RETQU NO EE 13 S rus M 213
270. tionally the shift range must be scaled up again by this scaling factor i e the spectral window given by the repetition rate of the pulse sequence must be multiplied by this scaling factor in or der to place the resonances correctly This scaling factor can be calculated from the tilt angle but is also slightly dependant on the offset and RF field Since the correct chemical shifts are usually unknown one must be aware of the fact that the shifts may not be as precise as they are in high resolution liquids experiments An example of shift calibration taking the scaling factor into account will be given in the practical chapter User Manual Version 001 BRUKER BIOSPIN 209 261 CRAMPS General 210 261 BRUKER BIOSPIN User Manual Version 001 CRAMPS 1D As outlined above many sequences are available to achieve Homo nuclear dipolar decou pling We want to concentrate on those that allow fast spin rates and are easy to set up The performance of DUMBO and W PMLG is very similar The pulse sequence is also very similar on AV3 instruments just different shapes and different timings are loaded Pulse Sequence Diagram of W PMLG or DUMBO Phases 104 010910 10 10 20 1 Figure 20 1 Pulse Sequence Diagram Table 20 1 Phases RF Levels Timings Phases RF Power Levels Timing bj CYCLOPS 1230 pl12 set for around 100 kHz p1 around 2 5 usec Q4 0 cnst25 adjust ditto p4 about 45 degrees adjus
271. tions are saturated by a rapid sequence of hard pulses such that no signal re mains There is then a variable delay during which relaxation occurs and then a 90 read out pulse If the relaxation delay is very short no signal is seen and at long relaxation times the maximum signal is seen The advantage is that the saturation time required does not need to be many times the longest Ty value The state of the system at the start of the experiment is forced by the saturation pulses so a long recycle delay is not required The CP Inversion Recovery Experiment 14 2 2 Sample Glycine Spinning speed 10 kHz Experiment time 20 minutes Before starting the experiment the spectrometer should be set up as described in the basic setup procedures chapter including measurement of the carbon pulse lengths and the CP spectrum of glycine should be acquired for reference Since relaxation times are necessarily temperature dependent control of the sample temperature is desirable The data shown here were all acquired at an approximate temperature of 20 C The form of the pulse program is shown in the following figure decouple hy H vd C Figure 14 1 The CPX T1 Pulse Sequence Starting from the glycine spectrum create a new data set set parameters according to Table 14 1 and acquire a 1D spectrum The relaxation delay after inversion is controlled by a vari able delay list this can be created using edlist and the name of the li
272. tive peaks given the very short recov ery delay no appreciable relaxation will have occurred Now we can set parameters for the 2D acquisition as in Figure 14 1 Since this is a pseudo 2D experiment the only relevant param eter in F1 is the number of points which should be the number of entries in the vd list The most important setting is the range of relaxation delays set in the vd list Ideally the list should run from times short enough for no appreciable relaxation to occur up to a few times the lon gest T value Of course the accurate relaxation time constants are not known in advance but order of magnitude estimates can be obtained by running the 2D experiment with a small num ber of relaxation delays and a small number of scans per slice The relaxation delays should be approximately equally spaced in log delay in order that decays with all time constants in the range are equally well characterized Data can always be improved either by increasing the number of relaxation delays sampled or by averaging more FIDs at each relaxation delay For the glycine sample a suitable list of times would be 100ms 220ms 450ms 1s 2 2s 4 5s 10s 22s 45s Parameters for 2D Inversion Recovery Experiment Parameter Value Comments Parmode 2D Vdlist See text td f1 Number of entries in vd list FnMODE QF This is not a real 2D experiment NS 4 for the glycine sample Sample dependent
273. tossb The maximum spinning speed is either determined by common sense if all side bands are spun out TOSS is not needed low field instruments or by the shortest delay which is d26 in both cases For TOSS B d26 0 0773s cnst31 p2 with cnst31 the rotation rate in Hz and p2 the 180 pulse width in us For TOSS A d26 0 0412s cnst31 p2 so the maximum spinning rate is lower BRUKER BIOSPIN User Manual Version 001 Basic CP MAS Experiments 3 heteronuclear decouplin 1H 2 bs 6 d 6s Prec Figure 6 2 Pulse Program for CPTOSS If the timing becomes a problem alternative TOSS schemes need to be found see e g O N Antzutkin Sideband manipulation in magic angle spinning nuclear magnetic resonance Prog ress in Nuclear Magnetic Resonance Spectroscopy 35 1999 203 266 The SELTICS se quence is an alternative Set up the experiment using glycine or tyrosine HCl at a moderate spinning speed Get a good CPMAS spectrum first then run a TOSS spectrum Table 6 2 Acquisition Parameters Parameter Value Comments pulprog cptoss cptoss243 p2 180 pulse on X nucleus pl11 Power level driving P2 on X channel cnst31 Spinning speed in Hz e g 5 kHz the entry would be 5000 zgoptns Dtossb Tossb if needed because of high spinning speed or long p2 User Manual Version 001 BRUKER BIOSPIN 63 261 Basic CP MAS Experiments class AVANCE 10 1 C j080005 PE class_ scale X 1 L E E E 5
274. trace eventual problems and provide information for the next us er Another copy may be printed for every user s own laboratory notebook One form per probe used should be filled out The following form serves as an example Operator used from to 10 12 07 10 00h 13 12 07 18 00h Probe shim file Bprfield Ge tr tripled hf es C N H 360 14 Experiment T us plin dB watt ak SNL T CP S N test F1 p90 C contact mix else p90 contact decouple mix else p90 contact decouple mix else stored under filename glycine 4 opt topspin reference 1 1 comments spinning at 10 kHz ok mains pressure at 6 bars linewidth a C 50 Hz O2 1500 spinal decoupling User Manual Version 001 BRUKER BIOSPIN 237 261 238 261 BRUKER BIOSPIN User Manual Version 001 Operator usedfrom Im Probe shim file Bo field RA p90 contact mix else p90 contact decouple mix else p90 contact decouple mix else Sample Experiment J stored under filename under filename E 2 Pulse program cpopt cpopt TopSpin 2 1 single pulse excitation acquisition without decoupling a 4 fu pa 30 M om fu K ai KA excitation pulse length as in cp me me me IO Ud decoupling excitation power level for H Ww P bo usual shape for cp usual shape power for cp contac mae ae in IO 2 d fu B c in IO Ca
275. uamh 17 CATWLKer TOPSPIN setup O store as 2D data ser file O Tre AU program specified In AUNM wil be executed WDW no O Perform automatic haseline correction ARSF PH_mnri nk 3 Overwrite existing files Jisable confirmation Message FT mode fqc O stop samale spinning at tne end of op m zanon masn O Run optimizacion in backgrcund OPTRAZE GROUP PARAMET OPTMUM STARTVAL ENIVAL NEP VARMOD INC Stepoystep C p1 lPOSMAX 05 oc 20 ILN 15 Skip current optimiz S210 protocol Add parameter Y d Step oxtimization Delete paraneter Cisplay Dataset Figure 3 16 The popt Window Use optimize step by step parameter p1 to optimize parameter p1 optimum posmax to find the highest signal intensity 90 degree pulse for the given value of pl1w or pI1 and varmod in to use linear increments for optimization The value for group is not used for optimizing only one parameter and the number of experiments nexp is set automatically when clicking on the save button Then save table by clicking on the save button and click on start optimize to start optimization procedure The parameter value obtained by the program is written into the parameter set of the actual ex periment at the end of the optimization In order to stop the execution of popt use the skip or stop optimization buttons Skip optimi zation will evaluate the obtained data as if popt had finished regularly and writes the parameter into the parameter set Stop optim
276. uency need not be shifted out of the proton range during evolution cnst24 0 In contrary it is possible to shift the carrier to the proton shift range center A third modification addresses the problem of poor discrimination between sites which are strongly and weakly coupled to protons In the standard sequence this is solely achieved set ting contact times short Of course this reduces cross peaks from remote couplings more than it reduces cross peaks from directly bonded protons However the remote couplings are al ways present through the Homo nuclear coupling between all protons These couplings can however be suppressed by executing the contact with a Lee Goldburg proton offset Then the protons are Homo nuclear decoupled and the transfer from protons to X only follows the hete ro nuclear dipolar coupling between those The pulse program Ighetfqlgcp works completely in analogy to Ighetfq but executes the contact at a proton offset calculated from the proton RF field during the spin lock contact pulse This modifies the HH condition which must be reestab lished using the pulse program Igcp In the following sections the specifics of these modified sequences are discussed User Manual Version 001 BRUKER BIOSPIN 83 261 Modifications of FSLG HETCOR Carbon Decoupling During Evolution 8 1 The only difference between Ighetfq and Ighetfqpi is the decoupling rr pulse at the center of the evolution period All what needs to be set in
277. um of Tyrosine HCl at 6 5 kHz essessssss 73 7 FSLG HETCOR 75 Figure 7 1 The FSLG Hetcor Experiment 76 Figure 7 2 The 12 icon and the ased icon in eda nenne 77 Figure 7 3 The HEI EE 78 Figure 7 4 FSLG Hetcor Spectrum Tyrosine HCI eee eae 81 Figure 7 5 FSLG Hetcor Spectrum Tyrosine HCI eee eee 82 8 Modifications of FSLG HETCOR 83 Figure 8 1 Comparison of HETCOR with and without 13C decoupling 84 Figure 8 2 HETCOR Using Windowless Phase Ramps en nnnnnn 86 Figure 8 3 HETCOR on tyrosine HCI without left and with LG contact 1msec con TACL EE 92 9 RFDR 93 Figure 9 1 RFDR Pulse Sequence for 2D CPMAS Exchange Experiment 94 Figure 9 2 The 123 Icon in the Menu Bar of the Data Windows Acquisition Parameter Page E EE E A E T E EEN leans 95 Figure 9 3 13C Histidine Signal Decay as a Function of the RFDR Mixing Time 97 Figure 9 4 2D RFDR Spectrum of 13C fully Labelled Histidine RFDR mixing time 1 85 AUEREN 97 10Proton Driven Spin Diffusion PDSD 99 Figure 10 1 CPSPINDIFF Pulse Sequence nennen 101 Figure 10 2 The Acquisition Parameter Window eda ssseee 102 Figure 10 3 POPT Result for the cw Decoupling Power Variation 105 Figure 10 4 13C CPSPINDIFF of fully labeled tyrosine HCl spinning at 22 kHz 4 6 msec mix Upper PDSD lower DARR sss 106 Figure 10 5 Comparison of DARR PDSD
278. umbo22_1 0 Table 8 4 Acquisition Parameters for e DUMBO HETCOR on tyrosine HCI Parameter Value Comments pulprog edumbohet windowless dumbo shape nuc1 13C olp 100 ppm nuc2 1H cnst24 1000 3000 Place carrier within proton spectrum for evolution pit Power level channel 1 for contact pulse pl12 Power level channel 2 TPPM SPINAL decoupling pl13 for 100 kHz Power level channel 2 DUMBO decoupling sp0 Power level channel 2 for contact pulse spnam0 ramp 100 or similar Shape for contact pulse channel f2 sp2 set to pl13 100 kHz for default duration 32 us spnam2 edumbo22 1 0 Both include one e DUMBO cycle p3 2 5 3 usec 90 pulse channel 2 at pl12 p15 50 500us Contact pulse width p20 32 us 32 us for 100 kHz field set by xau dumbo pcpd2 2 p3 SPINAL64 TPPM decoupling pulse CPDPRG2 SPINAL64 TPPM15 Decoupling sequence F1 1H indirect 10 0 Start value 0 incremented during expt 13 2 4 Multiples of ee DUMBO period increment per row in_f1 inO as calculated Set according to value calculated by ased F2 13C acquisition d1 User Manual Version 001 2s BRUKER BIOSPIN Recycle delay 89 261 Modifications of FSLG HETCOR Table 8 4 Acquisition Parameters for e DUMBO HETCOR on tyrosine HCI sw 310 ppm Sweep width direct dimension aq 16 20 msec masr 12000 13000 dumbohet 8 2 4 This is the windowed version of th
279. up for the appropriate frequencies The following external RF filters are required proton bandpass 13C bandpass and 15N low pass The channel isolation required between X and Y here C and 9N is usually sufficient with a bandpass on one of the channels but a filter to remove the proton decoupling RF interfer ence is required for X and Y This means that one of the band pass filters on X or Y may be replaced by a proton reject X low pass filter If the channel isolation between X and Y is not adequate the probe cannot be tuned d A e ne r x LJ A H x i mg D D Figure 18 2 The edasp routing tables for H CN double CP Three examples are shown Setup with only one X HP preamplifier must be recabled for 130 and IDN setup setup with 2 X BB HP preamplifiers and 2 HP transmitter and setup with one HP transmitter and one 500W transmitter The higher frequency nucleus is set for the lower power amplifier 5 Set up for standard 13C CP operation in triple mode Remember that a double tuned probe has better signal to noise and requires less power on X than a triple probe 6 Optimize decoupling and CP condition run a reference 130 CP MAS spectrum of the la belled glycine sample using 16 scans This reference spectrum will serve to measure the efficiency of the DCP magnetization transfer User Manual Version 001 BRUKER BIOSPIN 195 261 Double CP 7 To set up the conditions for the N to C transfer one must d
280. us SFO MHz 500 0510001 Frequency of 2nd nucleus BF2 MHz 500 0500000 Basic frequency of 2nd nucleus Y Nucleus 3 ER r gege gt Figure 11 3 The Acquisition Parameter Window eda 112 261 BRUKER BIOSPIN User Manual Version 001 Data Acquisition Sample Tyrosine HCI SUPER Experiment time several hours Table 11 1 Acquisition Parameters Parameter Value Comments Pulse program cpsuper Pulse program NUC1 13 Nucleus on f1 channel O1P 100 ppm 13C offset NUC2 1H Nucleus on f2 channel O2P 0 ppm 1H offset can be optimized for best decoupling PL1 Power level for f1 channel PL11 Power level for f1 recoupling P2 180 pulse on F1 during TOSS with PL 1 PL2 Power level for f2 channel PL12 Power level decoupling f2 channel and excitation P3 Excitation pulse f2 channel P15 Contact pulse first contact CPDPRG2 TPPM or SPINAL64 NS 64 n 15 Number of scans CNST31 Spinning speed in Hz L5 L5 cnst31 counter for increment in t1 and number of gamma inte gral typically number of SSB s F2 direct 13C left column TD 2048 Number of complex points SW 300 ppm Sweep width direct dimension F1 indirect C Right column TD 32 64 Number of real points FnMode TPPI STATES or STATES TPPI User Manual Version 001 BRUKER BIOSPIN 113 261 SUPER Spectral Processing Table 11 2
281. us of interest mobility rigid environment expect long T4 and repetition delay proximity to protons can one use cross polarization conductivity dielectric loss expect tuning and RF heating problems if sample is dielectri cally lossy or even conductive User Manual Version 001 BRUKER BIOSPIN 55 261 Practical CP MAS Spectroscopy on Spin 1 2 Nuclei 56 261 2 Collect information about the sample first by running an easy nucleus Feasibility of cross polarization parameters is the required key information because it de cides the steps to follow If the sample information which you have collected shows that a 13 CP MAS experiment should be feasible sample contains more than 20 protonated carbons load a reference cross polarization data set S N test spectrum of glycine spin the sample at the same spin rate set contact time p75 to 1ms wait 1 min do one scan There should be a visible sig nal From there on optimize the required repetition rate d1 contact time p15 number of scans ns spin rate masr and Hartmann Hahn adjustment until the signal is optimum In very few cases the decoupler offset 02 may require readjustment If no C signal is found the reasons may be incorrect setup recheck reference sample concentration lower than expected unusual relaxation properties long Tj s long proton T4 short proton Tp Then the most important information about the sample prot
282. ve relaxation processes Experiment setup 148 261 Sample Glycine Spinning speed 10 kHz Time 20 minutes 1 Start from standard CP parameters The only additional calibration required is the carbon RF field strength of the spin lock pulse This can be set independently of the field strength for the cross polarization In principle the strength of this field can be set to any value with BRUKER BIOSPIN User Manual Version 001 Relaxation Measurements in probe limits to probe motions on a range of time scales However only at relatively large field strengths is true T4 relaxation the only significant relaxation pathway 2 Set pulprog to cp90 and measure the required power level for a 70 kHz RF field 3 57 us 90 degree pulse 3 Make a new data set with iexpno change pulprog to cpxtirho and set this measured pow er as pI11 4 Set up a variable pulse list for the incrementation of the spin lock with the command edlist vp Check that this list is set as the parameter vplist Remember that this is a high power pulse so the duration should not be too long For the glycine sample a possible set of times would be 1ms 2ms 5ms 10ms 15ms 20ms 25ms 30ms 40ms 50ms This will not allow the signals to decay completely so is not ideal but should not place undue stress on the probe Often a compromise must be reached between recording an ideal decay curve and avoiding the risk of probe damage 5 Change parmode to 2D and
283. with finely ground KBr and spin at 5 kHz It is assumed that these operations are known If not please refer to the following sources Probe manual MAS II pneumatic unit manual in TopSpin help SBMAS manual in TopSpin help Solids manual in TopSpin help User Manual Version 001 BRUKER BIOSPIN 13 261 Basic Setup Procedures This chapter will include Setting the Magic Angle on KBr Calibrating 1H Pulses on Adamantane Calibrating 13C Pulses on Adamantane and Shimming the Probe Calibrating Chemical Shifts on Adamantane Setting Up for Cross Polarization on Adamantane Cross Polarization Setup and Optimization for a Real Solid Glycine Some Practical Hints for CPMAS Spectroscopy Literature General Remarks 3 1 14 261 Despite the fact that most spectra taken on a CP MAS probe look like liquids spectra the con ditions under which they are taken must account for the presence of strong interactions This basically means that fast spinning and high power pulses are applied Fast spinning requires a high precision mechanical system to allow spinning near the speed of sound This requires careful operation of the spinning devices Please read the probe manual carefully High power decoupling in solids requires 20 fold RF fields compared to liquids spectroscopy since we are dealing with gt 20 kHz dipolar couplings rather than maximum 200 Hz J couplings This means that RF voltages near the b
284. xperiment Changed for Proton Obser EC EE 25 Figure 3 12 Proton Spectrum of Adamantane at Moderate Spin Speed 26 Figure 3 13 Setting the Carrier on Resonance nenn nennen 27 Figure 3 14 Expanding the Region of Interest sssessse nn 28 Figure 3 15 Save Display Region to Menu nn 28 Figure 3 16 The popt Window ssssssssse IH nennen ernennen 29 Figure 3 17 The popt Display after Proton p1 Optimization ssssssssssss 30 Figure 3 18 Adamantane 13C FID with 50 msec aq setsh Display 31 Figure 3 19 Adamantane 13C FID with 50 msec aq setsh with Optimized Z Shim Value Oa aR 32 Figure 3 20 A cp Pulse Sequence sssssssssssss e 33 Figure 3 21 Hartmann Hahn Optimization Profile 34 Figure 3 22 Hartmann Hahn Optimization Profile Using a Square Proton Contact Pulse geil batad bavi E hid tad tbe o rt o E eg 35 Figure 3 23 Display Showing a Glycine Taken Under Adamantane Conditions 4 scans M EE eaves enka 36 Figure 3 24 Optimization of the Decoupler Offset o2 at Moderate Power Using cw De Coupling EE 37 Figure 3 25 Glycine with cw Decoupling at 90 kHz RF Field essc 38 Figure 3 26 Glycine Spectrum with Spinal64 Decoupling at 93 kHz RF field 40 4 Decoupling Techniques 45 Figure 4 1 Optimization of TPPM Decoupling on Glycine at Natural Abundance 46 Figure 4 2 Geometry for the FS
285. xt Options see PoR Cte a lv Expression Y Wames F Values M Grid F2 P3 phi 1 3 2 5u PiS sp0 ph10 0 i dB ramp 100 PAS 2m Di 6 im besch Figure 3 20 A cp Pulse Sequence F1 User Manual Version 001 BRUKER BIOSPIN 33 261 Basic Setup Procedures de IS The following parameters are set pI12 for the initial 90 degree pulse and the decoupling during acquisition set for a 4 5 usec proton 90 degree pulse as previously determined pl1 for the carbon contact pulse set for a 4 5 usec carbon pulse as previously determined p3 4 5 usec spnamo set to ramp 100 to sweep the proton contact RF field from 50 to 100 sp0 set to pl12 3 dB to account for the lower average RF over the ramp p15 2 5 msec after the value specify m to make it milliseconds else it is taken as micro seconds cpdprg2 select cw 01 set between both adamantane peaks o2 set to be on resonance on adamantane protons Acquire 2 or 4 scans then set plot limits for both peaks and optimise p3 2 usec and pl1 2 dB for best signal Fig 21 shows a Hartmann Hahn match optimization over 4 dB using a ramp contact pulse going from 50 to 100 amplitude TOPSPIN 2 1 b 14 on PCHE J as Administrator connected to avi600 File Edit View Spectrometer Processing Analysis Options Window Help Q 9 0 9 6 i E324 AAN JL Ti UF PP e w TFT SOA A m s run ENG a man OB o ak att A Spectrum ProcPars AcquPars Twe PulsePro
286. zation 10 If available set pl1 and spO for a proton 19N HH condition in triple mode On a labelled 11 sample even the previous settings for C should give a signal which allows optimizing the HH condition When the HH condition is optimized find the power level to achieve a 35 kHz RF field 7 14 usec 90 pulse carrier close to the 15N resonance It is essential to optimize the first pro ton to nitrogen HH contact This is not as trivial as one might think since the transfer effi ciency depends strongly on the timing and RF fields of the HH match The proton T4 of the glycine NH protons from which the nitrogen is polarized is fairly short so the polarization transfer is not efficient On a fully labelled sample a maximum enhancement factor of 8 3 is possible 5 protons transfer to one nitrogen Comparing the cross polarized 15N spectrum to the directly observed spectrum using hpdec and 90 degree pulses at 4 sec repetition BRUKER BIOSPIN User Manual Version 001 Double CP one can measure the enhancement factor rather easily Without optimization the enhance ment factor may be as low a 5fold It should be at least 6 5fold more than 7 5fold is hard to achieve To achieve a good result the HH RF fields should be set as high as possible with a contact time of 4 msec higher proton RF fields yield a longer proton T1p and allow longer contact times Of course the RF field is limited by transmitter power and probe break

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