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AVANCE Solids User Manual - UC Davis Nuclear Magnetic

1. Table 9 1 Acquisition Parameters for pmlg HETCOR on tyrosine HCl 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 I3 2 4 Multiples of FSLG periods increment per row in fl inO 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 15 kHz 15 kHz ok at 100 kHz RF field Table 9 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 002 BRUKER BIOSPIN 131 327 Modifications of FSLG HETCOR w pmighet 9 2 2 If one wants to compare a solids proton spectrum acquired via 13C detection us ing 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 bo
2. Y General PULPROG TD 1594 NS 4 DS n SWH Hz 50000 00 AQ s 0 0159900 RG 128 DW uis 10 000 DE us 10 00 CNST11 1 0000000 CNST20 100000 0000000 CNST24 2000 0000000 D1 s 2 00000000 ind s 0 00005654 in a L3 3 count 64 dwell 5 0 00002654 Y Channel ti olitr us 2 00 NUCA 130 P15 us 300 00 PL1 dB 450 PLTW Wy 104 27635956 SFO1 MHZ 150 9220830 Y Channelt2 biktr2 jus 1 00 enat21 o 000000 enst22 68710 679688 enst23 72710 679698 CPOPRG2 spinal54 NUC2 1H Edit P3 us 2 20 ps ps 8 17 PCPD2 ps 4 20 PL2 dB 7 00 PLOW W 47 41231155 PL12 dB 5 00 PL12w DA 59 68856430 PL13 08 6 00 PL13W W 59 68856430 pulsa jus 134 SFO2 MHz 500 1500000 SPO cB 5 10 SPOW w 56 32986739 SPNAM Ramp 70100 100 SPOALO 0 500 SPOFFSO Hz D 00 Figure 8 3 The ased Display BRUKER BIOSPIN Ce LI 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 far acquisition if digmod base LG RF field as adjusted in Hz used to calculat Offset tor proton evolution under LG usually O Recycle delay In 2 0 976713 4 294 360 ycnst20 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 obser
3. 12 Default 58 ele de cu nn 100 digital Mode sitas ida aida 277 Cs 12 nme 70 d PMMA cm 12 ZEE 56 el CEET 56 Bis 12 103 Ul ER 106 DUMBO D 97 134 272 E Corm EHER 57 121 BW er Teer ne 58 AA EE 57 COG 59 eDUMBO EE 133 experiment b tton u enter les 61 F e ee S eee 57 Frequency Switched Lee Goldburg sese 92 272 Frequency Switched Lee Goldburg Heteronuclear Correlation 119 gejic 272 ESLG DecoupliNd circadiano ee 92 ESEGIPUISO 9 EE 119 BRUKER BIOSPIN User Manual Version 002 Index G GAZ E 11 AS 56 ASI AM eM 11 Ciel M 11 Good Laboratory Practice A 84 elle el 61 e tele EE 71 H H3SE O3 M inet vaeeeesntatse 12 Hartmann Mii aaa pei een 77 Hartmann Habn Condtion sn iain aaan Ban BAAREN ANARA 76 Heteronuclear Decoupling urssuu0ssnnassnnonennnnsnonnnnnnsnnnnnnnnnnnnnnnnnnnnnnenn
4. Table 23 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 kHz RF field p8 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 olp 3 8 To be optimized swh 1e6 2 2 p9 1 0 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 User Manual Version 002 BRUKER BIOSPIN 287 327 Modified W PMLG Table 23 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 111 anavpt 4 2 4 8 16 or 32 o1p 5 To be optimized swh 1e6 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 23 5 Fine tuning is done by optimizing power levels pulse widths and carrier offset as before the carrier spike is gon
5. problem Table 14 1 Recommended Probe Spin Rates for Different Experiments and Magnetic Field Strengths i rotor masr max ell max vgr H max Bg max Sequence meh damer me mes Tee gei POST C7 1 7 7 6000 5000 35 7 15 70 3 5 LG 300 4 15000 9000 63 4 100 2 5 LG 500 3 2 24000 12000 84 3 110 zd LG 600 2 5 35000 14000 100 2 5 130 D LG 800 SPC5 2 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 950 182 327 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 C7 is not recommended due to restricted excitation bandwidth 4 Maximum speed results from max possible RF field 5 Maximum 1C RF fields taken from 1 C RF field specification or 1H RF field specification considering the requirement of an off HH condition 6 Maximum RF field for decoupling 7 LG means cw decoupling with optimized LG offset frequency at the given RF field in order to avoid HH contact BRUKER BIOSPIN User Manual Version 002 8 Symmetry Based Recoupling Maximum magnetic field as pr
6. Figures are calculated for a Larmor frequency of 100 MHz From the isotropic shift and the shift position in the MQ dimension the so called SOQE parameter can be calculated dgis being given by equation 2 Eq 17 4 2 2 SOOE Q 14 7 Le r r 9 Q cl 3 PI bo with Eq 17 5 47 27 1 Y Wd 341 1 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 Qc the second order quadrupole induced upfield shift dgis decreases as the spin increases With dgis always being negative this has a direct influence on the appearance of the sheared 2D spectra Figure 17 10 shows 2D 170 3QMAS spectra at 11 7 T and 18 8 T where the Lar mor frequency of this nucleus is 67 8 and 108 4 MHZ respectively The sample is User Manual Version 002 BRUKER BIOSPIN 227 327 Basic MQ MAS 228 327 sodium metaphosphate NaPO in the glassy state The enrichment of 70 is ap prox 30 to 3396 It contains 2 oxygen positions there are bridging oxygen P O P and non bridging oxygen P O Na ppm m A 150 100 50 0 ppm 140 120 100 80 60 ppm Figure 17 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 Q
7. ed o Figure 4 21 Hartmann Hahn Optimization Profile User Manual Version 002 BRUKER BIOSPIN 77 327 Basic Setup Procedures Fie Edi View Spectrometer s Administrator connected te avi tO Processing Analysis Options Window Help The wiggles besides the signals stem from truncation of the FID after 50 msec ac quisition time To exemplify the existence of several HH conditions on a spinning adamantane samples another HH profile Figure 4 22 is shown where a square proton con tact 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 optimize for the spin rate which is used 2475 6 58 2000 4 A L JT i w UutWB ag TTOiio n nu mekEm amp Gaam amp oalM 7t 24 poptau for pit finished POSMAX al expermen 29 pli 76000 NEXP 31 78 327 on LE a a a Figure 4 22 Hartmann Hahn Optimization Profile Using a Square Proton Contact Pulse The sideband order O at 4 8 dB gives a rather small intensity A ramp sweeping over 3 5 6 5 dB would cover both
8. 2 c cccceeeeeedeeedeeeedeeeeeeeeaeteceeeeaueeeaesecadeseadaeaedeceedaeees 149 12 REDOR 155 Table 12 1 Acquisition Parameters for a 13C observed C N REDOR ooocccccncoccnnnoccnnnnccnnnnnonnnos 158 Table 12 2 Results for the M2 Calculation and the Simulations ssssssesese 167 13 SUPER 169 Table 13 1 Acquisition Parameters sssssssssssssssssse eee emen he ehe ne nene nnns 173 Table 13 2 Processing Parameters esseseiesiesesiessi senda este ened eenedensedeneadenteeeneennees 174 14 Symmetry Based Recoupling 179 Table 14 1 Recommended Probe Spin Rates for Different Experiments and Magnetic Field Sgen rc 182 Table 14 2 Acquisition parameters for DQ SQ correlation experiments using symmetry based re coupling Sequences re nre rre rrr rre nnns 186 Table 14 3 Processing parameters for DQ SQ correlation experiments using symmetry based re coupling Sequences ernennen 188 15 PISEMA 193 Table 15 1 Acquisition Parameters ssssssssssssssssssee eee ehe ene ne ne rennes 197 Table 15 2 Processing Parameters for the Pisema Experiment 198 16 Relaxation Measurements 201 Table 16 1 Parameters for the 1D CP Inversion Recovery Experiment 204 Table 16 2 Parameters for 2D Inversion Recovery Experiment 205 Table 16 3 Processing Parameters for CP T1 Relaxation Experiment 205 Table 16 4 Parameters for the Saturation Recovery Experiment 209
9. semen 120 Setting up FSLG HETOOR sss hen enhn nnn nest nnns enr nennen 121 Results eiserne arena a alien ergehen 125 Modifications of FSLG HETCOR isc 127 Carbon Decoupling During Evolution esssseem Hem 128 HETCOR with DUMBO PMLG or w PMLG Using Shapes sse 129 The Sequence pmlghet sssssssssssssssssssssssse e se hee hen he nhe he rrr eere nnne 129 WM m 132 eine H 133 elle 134 HETCOR with Cross Polarization under LG Offset sss 135 HFOR RA 137 Experiment uo rro pese deans ix tede bea ar The SUE athe ee ETERNI A M D re 138 Sij erc EE 138 Data Acquisition EE 139 Set up 2D Experiment hn nennen nennen nennen nen 139 Spectral Processing rade dnte a nennen canes dates ane arten enden er 141 BRUKER BIOSPIN User Manual Version 002 11 11 1 11 2 11 2 1 11 3 11 3 1 11 4 11 5 12 12 1 12 2 12 2 1 12 2 2 12 3 13 13 1 13 2 13 3 13 3 1 13 3 2 13 4 13 5 14 14 1 14 2 14 2 1 14 2 2 14 2 3 14 3 14 4 14 5 14 6 14 7 15 15 1 15 2 15 3 16 16 1 16 2 16 2 1 16 2 2 16 2 3 16 2 4 Contents Proton Driven Spin Diffusion PDSD ann nn nenne 143 Pulse Sequence Diagram esie det feet cadet lee a Sape canadian depen ee 145 BASIC Setup u a 1
10. I Y j d f j j j j F Y nN 4 j b 4 a E T 1 T J ppm Figure 9 1 Comparison of HETCOR with and without 13C decoupling The figure above shows a comparison of HETCOR with and without C decou pling Natural abundance tyrosine HCl was run with 50 usec contact time Reference 1 A Lesage and L Emsley Through Bond Heteronuclear Single Quantum Correlation Spectroscopy in Solid State NMR and Comparison to Other Through Bond and Through Space Experiments J Magn Res 148 449 454 2001 BRUKER BIOSPIN User Manual Version 002 HETCOR with DUMBO PMLG or w PMLG Using Shapes HETCOR with DUMBO PMLG or w PMLG Using Shapes 9 2 These sequences use phase modulated shapes for homonuclear proton decou pling Apart from some smaller differences the sequences are in complete analo gy to the HETCOR sequence using frequency shifts The only differences between these sequences lie in the length and type of shape used for homonuclear 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 1 D Sakellariou A Lesage P Hodgkinson and L Emsley Homonuclear dipolar decoupling in solid state NMR using continuous phase modulation Chem Phys Lett 319 253 2000 Vinogradov E Madhu P K Vega S High resolu
11. 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 com mand edmac and the filename pisemaft for examples write the following com mands using the text editor xf2 zeroim STT 5 Save and close the edmac editor In the future you can do then the processing by simply typing pisemaft into the command line or even creating your own icon in TOPSPIN for this purpose Table 15 2 Processing Parameters for the Pisema Experiment Parameter Value Comment F2 acquisition 1H kkkkkkkkkkkkkk Left column SI 1k Number of complex points in direct dimension WDW no Apodization in t2 F1 indirect IDN ees Right column Sl 128 Number of complex points in indirect dimension MC2 QF 198 327 BRUKER BIOSPIN User Manual Version 002 PISEMA 180 160 140 120 100 80 so 40 20 ppm A PISEMA Spectrum of 9N Labeled Acetylated Valine B FID in t1 over 3 008 ms 64 Data Points Figure 15 2 PISEMA Spectrum of 15N Labeled Acetylated Valine and FID in t1 over 3 008 ms 64 Data Points User Manual Version 002 BRUKER BIOSPIN 199 327 PISEMA Spectrum ProcPars AcquPars Tie PulseProg Peaks Intecorals Sample Structure Fut
12. SHAPE_BWFAC 0 000000E00 SHAPE_BWFAC50 SHAPE_INTEGFAC 6 534954E 17 SHAPE_MODE 0 NPOINTS 10 XYPOINTS 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 000000E02 325 49 1 000000E02 283 92 1 000000E02 242 35 1 000000E02 200 78 HEND TITLE m3p JCAMP DX 5 00 Bruker JCAMP library DATA TYPE Shape Data ORIGIN Bruker BioSpin GmbH HOWNER2 hf DATE 2005 11 29 THETIME 14 47 39 SHAPE_PARAMETERS MINX 1 000000E02 HMAXX 1 000000E02 MINY 1 125000E01 MAXY 3 487500E02 SHAPE_EXMODE None SHAPE_TOTROT 0 000000E00 SHAPE_TYPE Excitation SHAPE_USER_DEF SHAPE_REPHFAC SHAPE_BWFAC 0 000000E00 SHAPE_BWFAC50 SHAPE_INTEGFAC 6 534954E 17 SHAPE_MODE 0 NPOINTS 6 HHXYPOINTS 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 HEND 286 327 BRUKER BIOSPIN User Manual Version 002 Setup Modified W PMLG 23 3 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 re quired for the wpmlg shapes Parameter Settings for PHLG and DUMBO 23 4
13. U 15N iebeled KJpf in DMPC KN a a 3 tu S H a Figure 15 3 PISEMA Spectrum of 15N Labeled Kdpf Transmembrane Protein PISEMA spectrum of 15N labeled Kdpf transmembrane protein aligned in DMPC courtesy of NHMFL Dr T Cross membrane between glass plates using an EFREE 700 MHz probe 200 327 BRUKER BIOSPIN User Manual Version 002 Relaxation Measurements In NMR experiments one is generally concerned with measuring resonance fre quencies 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 magnetization is tilted away from the field axis and the resulting precessing magnetization generates the ob served 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 Abragam A Abragam Principles of nuclear magnetis
14. 1H 50 W OFS4 po 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 FLFR Add a logical channel Remove a logical channel Default Info 66 327 Figure 4 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 We experiment with 1H decoupling For high power transmitters the parameter pow mod must be set to high To check which power mode is selected 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 powmod high To change to proton observe click SwitchF 1 F2 BRUKER BIOSPIN User Manual Version 002 Basic Setup Procedures frequency logical channel amplifier preamplifier BFI 600 147 MHz NUCI u SFO 600 47 MHz Fl Seul 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 cort
15. Channel F1 is the detection channel by default which is no limitation Detection must usually be routed via the same SGU as the F1 pulses are since that SGU will supply the phase coherent reference signal In some cas es pulsing and detection may use different SGU s but provisions must be made to add the signal up coherently using exactly the same frequency on both channels is usually coherent Routing between SGU s and transmitters can only be selected if cf the config uration routine has found a hardware connection that supports this routing Most routine applications will route correctly if the default button is pressed in the short menu version receiver routing not shown BRUKER BIOSPIN 51 327 General Hardware Setup f an illegal or potentially dangerous routing is selected an error message or a warning will pop up Error messages will not allow selection of this routing On AVIII instruments with SGU 2 one SGU can produce two pulse trains within the same NCO frequency setting range 5 MHz irequency KG channer BF 125 764329 300 SFO 120 786009 aa Y _xmew X900 Ww ors aan BE SM 15H NK SPO SU 157947 Er mn vr DEZ 4570 iw Hw SIP nea soetas saitz wi me tere ser sF03 5067892 hee sous P Bere oraa 16520 itz 1H 900 ore 300 190 mit SFG4 500 150 Miz os foo n Abb wg gue seenrwri Swemrura aroa ogeaicnannei genome siogicaicnannet cet re ea
16. General Hardware Setup 1 X Tuning cap 3 Proton reject filter trap 2 Parallel coil 4 Probe ground Figure 3 38 Parallel Coil to Shift the Tuning Range to Higher Frequency This additional coil is electrically connected in parallel to the detection coil Since it is connected behind the proton trap the proton channel is not influenced but as well the X and Y channel will be affected because only part of the inductance is now filled with sample A parallel coil therefore reduces the RF efficiency quite substantially The losses increase as the inductance size and number of turns of the parallel coil decreases A coil of the same inductance as the detection coil will cost 50 in S N and pulse voltage Usually these coils introduce about 30 loss Adding a Frequency Channel to a Probe WB probes only 3 7 3 48 327 Probes are produced as single channel double channel triple or quadruple probes 1 2 or 3 RF connectors on the probe It is not possible to modify a probe produced as a double channel probe into a triple probe but a triple probe may be used as triple or double probe As multiple tuning will reduce the RF performance of a probe on the other channels if they are part of the same RF network it is better to remove an unused RF channel if this is possible The usual case is triple probes or quadruple probes A triple probe can be tuned to H X and Y where X is the higher frequency Y is the lower freq
17. RF Filters in the RF Pathway Please note in Figure 3 7 Only high power preamps allow decoupling through the preamp X BB HPH 1H bandpass BB HPHPPr 1H HPHPPR 1H reject or 0 31P pass or X bandpass Y BB HPHPPr Figure 3 8 Triple Resonance Experiment without X Y Decoupling Figure 3 8 is a triple resonance experiment without X Y decoupling one band pass will suffice note the preamp configuration It is recommended not to put two preamps of the same kind next to each other in order to avoid incorrect wiring of probe and filters For the X channel only the proton frequency needs to be filtered out if X or Y is not decoupled while Y or X is observed protons are usually decou pled Probe X BB HPHPPr i 1H HPHPPR Y BB HPHPPr Figure 3 9 Triple Resonance Experiment with X Y Decoupling Figure 3 9 is a triple resonance experiment with X Y decoupling two band pass es required Care should be taken that the two bandpass filters mutually exclude the other frequency as efficiently as possible Low pass filters will not allow X or Y observe while Y or X is decoupled User Manual Version 002 BRUKER BIOSPIN 23 327 General Hardware Setup 24 327 Probe X pass 19F 1H stop Figure 3 10 Triple Resonance 1H 19F Experiment Figure 3 10 is a double triple resonance HF experiment with 9F observation and H decoupling or X observation with 9F and 1H decoupling WB probes gt 400 MHz onl
18. show RF rout ing Receiver routing SGUS used for transmit and receive show receiver rout ing Power indication Maximum possible power output as measured show pow er at probe in User Manual Version 002 BRUKER BIOSPIN 53 327 General Hardware Setup requecy mt os nw wu wot wn vat on fo WI wnnue M mos wn te we ie ora po ur 20010 we aros 500 isa we ws fo w Dy 00 190 or frequen oy i 159907083 Mn Fol 150 901083 Mite orsi 160080 ve Sr 400147 MHz VO 400 1475 Me C932 500 n Ss 60512367 une wos 40814107 vs ur 22000 ra Ei emote Mir SFO4 600 147 Miz cr 00 H cate wiring 1 possible RF routbng conval svoi able 54 327 opa Canne wore presngiter mm ow Mac Ge 4 n e nn a T un ra m le s B Fa Zeg e senn zen sgseen ERTTST L lz L ps com Figure 3 43 Pulse on F2 Observe on F1 Routing In the figure above the SGU2 is used for pulsing and the SGU1 is used to receive legal crant ame mar prac ter recever er ha wen A wor nx n f an e x d x 300 W PP EBT gt BECI san n 130 D HHP min up tax i ioo0 eani p MS m un is wow Heep ais N ose MEZ n lin D PPLA Ar m ncs y S mw n o sas x Y xsoow Var n 15H asn Ca Ap BOO MICA Fa an it iMSOW 24 Fa ke E 3 tet5ngt E ih ow tecetear routing Wow RF routing drow poser t posten H Sem Semen rine Ses ELE file a l
19. 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 3C 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 T4p Then the most important information about the sample proton T4 proton T45 can be obtained by looking at the protons in the sample Set up for proton ob servation set swh to 100000 500000 rg to 4 and pulprog cpopt if not found in the library copy the pulse program in the appendix p3 and pl12 for p3 p90 Set spnam0 ramp 100 sp0 power level for HH p15 100 us Do 1 scan and fourier transform phase correct Using popt optimize d1 for maxi mum signal Note CP MAS probes usually have a substantial proton background sig nal 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 polarization contact time On protons we measure the time constant Tue Using popt in the previous setup vary p15 between 100 usec and 10 ms even 20 ms at reduced power if a long T s is expected as the distanc
20. 5 7 9 the required information is asked for by the program in order to calculate the shearing correctly Note that using a user designed 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 ad visable to calculate a baseline correction after F2 Fourier transform Note that the range defined by ABSF1 and ABSF2 is used for this You should make sure that BRUKER BIOSPIN User Manual Version 002 Basic MQ MAS 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 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 cor rects the apparent spectrometer 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 17 1 m 81I 1 amp 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 experiment of an order p 3 The program stores the
21. CNST11 1 0000000 To adjust t 0 for acquisition if digmod base D1 5 D 20000000 Recycle delay Y Channeifi 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 on 02651215 Power level for excitation pulse SFO MHz 150 3690213 Frequency of observe channel Figure 4 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 command line and check powmod by clicking the default button as de scribed above The rf routing for this experiment is shown in figure 1 Next set p1 2 us 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 pl1 and pltw if the transmitter 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 pI1 should be set to 10 in case of a 1000W trans mitter 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 002 BRUKER BIOSPIN 61 327 Basic Setup Procedures Proc ora Acqutors Ttle PulseProg Pecks Integrals Sangle Structure F
22. D4 A very short delay is used here just to allow for amplitude and phase switch ing I4 This loop counter is internally used for checking if the echo or anti echo is cur rently being acquired I5 In the acquisition of echo anti echo 2D spectra signals from the echo and anti echo pathways are stored into consecutive FID s in the serial file In MQMAS ex periments these echos and anti echos behave differently For t4 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 contribution 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 or der to increase the overall S N and save spectrometer time However in the pro cessing of echo anti echo data two consecutive FID s are linearly combined in the following way rel im2 iml re re2 rel iml re2 rel im2 im2 iml Eq 18 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 obscur
23. Figure 23 1 Pulse Sequence Diagram Table 23 1 Phrases RF Levels Timings Phases RF Power Levels Timing di CYCLOPS 1230 pl12 set for around 100 kHz p1 around 2 5 usec 010 02 sp1 set for 100 130 kHz cnst20 WPMLG calculated from cnst20 RF field 31 CYCLOPS 0 123 User Manual Version 002 BRUKER BIOSPIN 285 327 Modified W PMLG Pulse Shapes for W PMLG 23 2 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 rotation which is op posite 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 HOWNER2 hf DATE 2005 11 29 HTIME 14 47 39 SHAPE_PARAMETERS MINX 1 000000E02 HMAXX 1 000000E02 MINY 1 125000E01 MAXY 3 487500E02 SHAPE_EXMODE None SHAPE_TOTROT 0 000000E00 SHAPE_TYPE Excitation SHAPE_USER_DEF SHAPE_REPHFAC
24. Proton high resolution preamps are unsuitable for high power pulses especially for durations required for decoupling High resolution X BB preamplifiers are limit ed to 10 msec pulses at 300 500 watts If the back label does not say 500W it is 300W max 1H 19F high power preamps need not necessarily be bypassed but may gradually deteriorate under many decoupling pulses It is therefore recommended to bypass these for decoupling unless the experiment requires that the preamp remain in line Warning These preamps are not optimized for 19F so 9F decoupling should never be done through this preamp H HPLNA preamplifiers need not be bypassed HPLNA preamplifiers are strictly frequency selective a 9F pulse through a H HPLNA will destroy it RF Filters in the RF Pathway 3 3 RF filters are frequently required if more than one frequency is transmitted to the probe Without filtering the noise and spurious outputs from the transmitter of one chan nel would severely interact with signal detection on another channel One has to keep in mind that pulse voltages are in the order of hundreds of volts but NMR signals are in the order of microvolt In High Resolution where the selection of nu clei to run is rather limited it is possible to apply the necessary filtering inside the preamplifier For solids this is not easily possible due to the wide range of possi ble detection frequencies but also due to the additional dead time t
25. Pulse Sequence Diagram Double CP DCP sssse men 256 Double CP Experiment Setup eene ne hehehe nnns 256 Double CP 2D Experiment Setup ocooococcccnconcconconcncncnnnconcnnnnnnncnnnnnnnnnnnn eher 256 TON Channel Setup dante tone rte nba hae a RO PA n eed iind uade eu e did 258 Setup of the Double CP Experiment sssssssssss me 259 Setup of the 2D Double CP Experiment ssssssssssssssseem e 264 2D Data Acquisition EE 265 Spectral Processing enr nenn be ga ade RR ee Sek va BAR dE Ee cn HER area 266 Example Spectra cccccccecec cece eeceecee eee cece eceeece eee ece ees eI hn nnnm herr e rhe hn hi rre reenn nnns 267 CHAMPS General M 271 Homonuclear Dipolar Interactions 271 Multiple Pulse Sequences nr nennen 271 W PMLG and DUMBO ssssssseneee cence cence nnne rene eects erre nnne Erai nei n nani 272 Quadrature Detection and Chemical Shift Scaling cc eeceeeeeeeeeeeeeee terse teen eeeaeeees 273 CRAMPS 1D 275 Pulse Sequence Diagram of W PMLG or DUMBO sss 275 BRUKER BIOSPIN User Manual Version 002 22 2 22 3 22 3 1 22 3 2 22 4 22 5 22 6 22 7 22 8 22 9 22 10 23 23 1 23 2 23 3 23 4 23 5 23 6 23 7 24 24 1 24 2 24 3 24 4 24 5 24 6 24 7 24 8 A 1 Contents Pulse Shapes for W PMLG and DUMBO eee eren 276 Analog and Digital Sampling Modi 277 Analog Mode Sampling 278 Digital Mo
26. The experiment resembles the PDSD or RFDR experiments see Proton 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 ver 5 Vrotor may be used here User Manual Version 002 BRUKER BIOSPIN 189 327 Symmetry Based Recoupling Data Acquisition 14 6 Parameter Value Comments Pulse program pc7cp2dnoe Any sequence may be used make sure to use the correct timing NUC1 13C 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 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 recon
27. WB Probes DVT Probe Connections for RT and HT Measurements User Manual Version 002 BRUKER BIOSPIN 29 327 General Hardware Setup Standard Bore SB Magnet Probes fe P D e C 6 ST i E y d 4 l w T Af Jd 1 Frame flush for VT 2 Ball joint takes bearing gas from the Quickfit connector at the front into the heater dewar 3 Bearing connector for ambient temperature gas Figure 3 19 SB VTN Probe MAS Connections With the standard bore VTN probe quick fit connectors include Bearing 3 Eject 2 Drive 5 Vertical 7 to the tilt stator for eject Magic Angle 8 to tilt stator into the magic angle Bearing sense to supervise bearing pressure shut down in case of a pressure loss For LT experiments the ball joint at the heater dewar must be opened and the transfer line of the heat exchanger or the cooling unit must be connected 30 327 BRUKER BIOSPIN User Manual Version 002 Additional Connections for VT Operation Figure 3 20 SB DVT probe MAS connections The numbered connectors are the same as the VTN probe Connectors 7 and 8 are not present since the probe does not tilt the stator for eject not required for 2 5 mm probes Additional Connections for VT Operation 3 6 If Variable Temperature experiments must be run there are a few connections to be done which are not required for room temperature experiments First of all there must be a flow of VT contro
28. and the information obtained is the same 02 BRUKER BIOSPIN 253 327 STMAS Refer to the chapter Basic MQ MAS on page 213 for details about the informa tion obtained from such spectra Values of R and R p for the Various Spin Quantum Numbers Ob Table 19 7 tained in the STMAS Experiment Spin R IR pl 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 254 327 BRUKER BIOSPIN User Manual Version 002 Double CP Double Cross Polarization DCP experiments use two consecutive cross polar ization steps Usually the first step transfers from protons to one type of X nucle us to achieve high sensitivity 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 nucleus of higher sensi tivity to gain signal intensity is the standard procedure Detection of the most sensitive nucleus protons is also possible but is difficult if the homonuclear pro ton proton dipolar coupling is strong see CHAMPS General on page 271 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 magnetization is transferred from 15N to 13C the
29. 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 experiment 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 the 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 The modifications according to the chapter Modified W PMLG are implement ed 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 de sired spectral range one can make the total acquired window smaller as well along F2 using digital mode as along F1 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 H H Double Quantum CRAMPS NMR Spectroscopy J Am Chem Soc 126 13230 2004 Proton Proton Shift Correlation spin diffusion 24 1 The standard CRAMPS setup must be executed first see chapter 22 23 Any homonuclear dipolar decoupling scheme may be used
30. deene Lu BEE 213 Pulse SEQUENCES Vida Kae nee anne ar 213 PANAS 215 setting Up the Experiment dida 215 Two Dimensional Data Acquisition sssssssssssss eem 220 Data Processing teres sive E 222 Obtaining Information from Spectra 20 eee eect e eee teers eee eee ee nnnn nennen nenn nnn nenn 225 MQ MAS Sensitivity Enhancement 24002000002000nnn ann nun nen ann nn 231 Split t1 Experiments and Shifted Echo Acquisition u r4ssssssenenennenenennennne nenn 231 Implementation of DFS into MOMAS Experiments sssssss 233 Optimization of the Double Frequency Sweep DFS ss 233 2D Data Acquisition u peret elek 238 Data Processing EEN 240 Fast Amplitude Modulation FAM cccccececceeeceeeceeee eee I ers 242 Soft Pulse Added Mixing SPAM sse mene hene hne eens eens need 242 SIMAS A 245 Experimental Particularities and Prerequisites oocoooccoccncccnccocccnccncccncnnnnnnnnnnnnnnnnnnnnnnnns 245 Pulse Sequences sessi eee AAA dameadneaed awa ee ANA 247 Experiment Setup x date a ke dee eed eed uen deba ae Ned dud eranl ed a Ra 249 Setting Up the Experiment ccccccceccc eee eeec eee eece cece ee me m eI nee ne eren 249 Two Dimensional Data Acquisition sssssssssssssssss e emen nen 251 Data Processing 5 3 i rte if dada ia Tan an gate cup Ed du d dua dann gene 253 18 2017 0 2 E 255
31. dex j P Peak ai 43 269 C Area D A ART d s Currert Peak 20t2 Bnet Report 8 Peak 1 at 176 002 ppm T 1874 Pesk 2 at 42 263 ppm T 64875 g k 8 g T T T T T H 5 10 15 20 s User Manual Version 002 Figure 16 2 Relaxation of Alpha carbon Signal in Glycine Start calculation This will perform the fitting procedure for all regions The calcu lated 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 de tails of the fit function and the calculated values of the parameters in the function The experimental and calculated data points are also displayed Note that the ex perimental data is normalized such that the most intense point has a value of 1 This report file is also saved in the processed data directory when the fit is calcu lated If fitting of a single peak is performed only this result is written 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 approximate ly 18 5s and
32. in tensities automatically or use your favorite deconvolution program for data analysis NM o un Tim u 3 36 34 32 ppm d n D D D y 4 n D Figure 12 2 2D data set after xf2 processing In the figure above the data set contains the alternating S and Sg experiments 160 327 BRUKER BIOSPIN User Manual Version 002 Setup a Anis Options Window Help ES Mois Calibration ca m e Ente Peek Picking pp gt ES gt Integration int gm tees Mutiple Spectrum Display Pr TI T2 Relaxation Line Shape Fitting Baron Wu Simulation e Small Molecules Siruciure Edil view Dosy Proleins Start Amix Viewer Figure 12 3 T1 T2 Relaxation for further Analysis ofthe Data Figure and the Analysis Interface If you are going to use topspin you have to choose T1 T2 Relaxation in the Analy sis Menu see Figure 12 3 to open the graphical interface for the data analysis Figure 12 3 To begin extract the first spectrum by using the extract slice button and select the desired peak by manual peak picking To save the data use the button shown in Figure 12 4 CC water Export regions and biggest peak within region to relaxation module and rei SI Fetapon Window Figure 12 4 Saving Data to Continue to the Relaxation Window Now after switching to the relaxation window topspin will show the parameter window which can also be accessed later b
33. 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 9 3 User Manual Version 002 Usually the cross polarization step is executed at less power than what is used for the initial excitation 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 transferred 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 transmitters 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 calcu
34. 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 decremented by 1 dB up to the same power level used for the excitation pulse e g from 20 dB to 0 dB Initially a sweep of one whole rotor period i e LO 1 can be used The optimization of SP1 can be repeated e g for half a rotor period a quarter of a ro tor 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 9 The AU program specified in AUNM will be executed Perform automatic baseline correction ABSF Overwrite existing files disable confirmation Message T Run optimisation in bockground OPTIMIZE PARAMETER OPTIMUM STARTVAL ENDVAL NEXP VARMOD INC ad spt POSMAX 20 Q o LIN A d 10 POSMAX 2 2 1 LIN null ad spi POSMAX 20 0 0 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 d si POSMAX 20 0 0 LIN d Figure 18 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 I0 is 1 Figure 18 5 shows a popt window with successive optimization of SP1 for sever al fractions of rotor periods 0 Note that the check mark for The AU program specified in AUNM will be exec
35. one should get 40 50 DCP efficiency Figure 20 7 compared to the reference direct 13C CP spectrum If this can not 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 13 1H contact Minimise this loss in the following way User Manual Version 002 BRUKER BIOSPIN 261 327 Double CP 262 327 9 Never use a pulsed proton decoupling schemes during p16 Frequency shifted Lee Goldburg decoupling is no alternative since the signal will broaden and decay with shorter Ta Use cw decoupling during p16 and carefully optimize the decoupling power p 13 for maximum signal A slight offset may be set us ing 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 5096 of a standard 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 TanAmpMod 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 select 400 for the dipolar coupling and 50 for the scaling factor Save the shape as tcn5500 if not already available and selec
36. ooonccconccnonccnncccnncccnnccns 284 23 Modified W PMLG 285 Figure 23 1 Pulse Sequence Diagram ssssssseseee meme mee nenne eere 285 24 CRAMPS 2D 291 Figure 24 1 Pulse Sequence Diagram 4422444 24H440nHaannnaannnnannnnannanannnnnnnnnnnnnnnnannnn anne 292 Figure 24 2 Setup and Test Spectrum of Alpha glycine ssssssss mH 294 User Manual Version 002 BRUKER BIOSPIN 313 327 Figures Figure 24 3 Spectrum of Tyrosine hydrochloride mee 295 Figure 24 4 Expansion of the Essential Part of the Gpectum 296 Figure 24 5 Pulse Sequence Diagram sssssssssssssse enemies 297 Figure 24 6 Glycine Proton Proton DQ SQ Correlation Using WPMLG in Both Directions 300 Figure 24 7 14 5 kHz W PMLG PC7 DQ SQ Correlation at 600 MHz with Tyrosine Hydrochloride 301 A Appendix 303 314 327 BRUKER BIOSPIN User Manual Version 002 Tables 1 Introduction 9 2 Test Samples 11 Table 2 1 Setup Samples for Different NMR Sensitive Nucer e 11 3 General Hardware Setup 15 4 Basic Setup Procedures 55 Table 4 1 Summary of Acquisition Parameters for Glycine S N Test 82 Table 4 2 Processing Parameters for the Glycine GIN Test 83 Table 4 3 Reasonable RF fields for Max 2 Duty Cycle sssssem Hem 85 5 Decoupling Techniques 89 Table 5 1 Acquisition Parameters ier to optet ed per E ocn epe in tret Ed npud poet lieh late 95 Table 5 2 Processing Parameters c0cc ccccc
37. probe Often a compromise must be reached between recording an ideal de cay curve and avoiding the risk of probe damage 5 Change parmode to 2D and set other 2D parameters as for the other relax ation experiments 6 Acquire spectrum with zg 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 T relaxation Indirect Relaxation Measurements 16 3 210 327 If proton relaxation measurements are desired the considerable broadening of the proton resonances seen at even high spinning speeds can make resolution of individual components impossible In such cases indirect observation of proton relaxation by X nucleus observation can be used A typical example would be at tempting to observe the proton relaxation of two components of a mixture or multi phase material In general the proton spins within a single molecule are suffi ciently strongly coupled by the homonuclear dipola
38. same species via homonuclear dipolar coupling or chemical exchange The ex periment resembles the NOESY Nuclear Overhauser Effect SpectroscopY pulse sequence in the liquid state by replacing the initial 90 pulse with a cross polariza tion scheme Since spin diffusion between X nuclei is measured cross peak in tensities depend on the probability of interaction between different sites which is low with low natural abundance of NMR active nuclei Therefore these experi ments usually require enrichment for nuclei like 13C or 15N in order to allow sensi ble measurement times The pulse program cpspindiff allows to run several types of PDSD experiments The CP preparation period excites the X nuclei During the evolution time the X magnetization evolves under the effect of the chemical shift interaction The evo lution time ends with an X 90 pulse that stores the chemical shift information along the z axis and marks the beginning of the mixing time The X spins commu nicate 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 interaction is removed by high power 1H decoupling during the prepara tion and the acquisition time The 1H decoupling is switched off during the mixing to dephase the residual X transverse magnetization Spin diffusion between
39. spnam Figure 18 3 Four Pulse Sequence and Coherence Transfer Pathway for the 3Q MAS Experiment Four pulse sequence and coherence 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 I 3 2 nuclei proportional 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 18 4 Three Pulse Sequence and Coherence Transfer Pathway 234 327 BRUKER BIOSPIN User Manual Version 002 MQ MAS Sensitivity Enhancement Three pulse sequence and coherence transfer pathway for the 3Q MAS experi ment with z filter mp3qdfs av Excitation pulse p1 is the same as for mp3qZfil av P4 is a central transition selective 180 pulse usually 2 p3 Delays DO is the in cremented delay for t4 evolution Delays D10 or D11 must be incremented propor tional to DO Power level and duration of the sweep P2 must be optimized Phas
40. 17 Basic MQ MAS 213 Table 17 1 Some Useful Samples for Half integer Spin Nuclet 216 Table 17 2 Initial Parameters for Setup sssssssssssssssssssee ee mensem ehh 219 Table 17 3 F1 Parameters for 2D Acquisition ssssssssssssssss eene 220 Table 17 4 Processing Parameters for 2DET emm memes 222 Table 17 5 Values of R p for Various Spins and Orderep cnn nnn nn canas 226 Table 17 6 Chemical Shift Ranges for all MQ Experiments for All Spins sssssssss 227 18 MQ MAS Sensitivity Enhancement 231 Table 18 1 Initial Parameters for the DFS Experiment 236 Table 18 2 Parameters for 2D Data Acquisition of 3 pulse Shifted Echo Experiment mp3qdfs av 239 Table 18 3 Parameters for 2D Data Acquisition of 4 pulse Z filtered Experiment mp3qdfsz av 240 Table 18 4 Processing Parameters 24 2444444440444HRHAH Han annannnnnnnnnnnnnnn nennen 241 Table 18 5 Parameters for FAM 0 0ccccecceceeeceeeeeeceeece eee eee cece ee eme he nee rennen erre nnns 242 Table 18 6 Further Parameters for 2D Data Acquisition of SPAM MQMAS Experiment UbRPEMREDIB VAEER Em 243 19 STMAS 245 316 327 BRUKER BIOSPIN User Manual Version 002 Table 19 1 Tables Time deviation of the rotor period for spinning frequency variations of 1 and 10 Hz for various spinning frequencies sssssssssssssse enn 246 Table 19 2 Some Useful Samples for Some Nuclei with Half Integer Spin oo
41. 180 pulse Then there is a delay during which the magnetization relaxes and a 90 pulse converts the remaining longitudinal magnetization to transverse magnetization and an FID is recorded The intensity of a particular signal in the resulting spectrum depends on the initial intensity the relaxation delay and the re laxation time constant T as follows S t S S 0 S expC t T Eq 16 1 BRUKER BIOSPIN User Manual Version 002 Relaxation Measurements where t is the relaxation delay Sg is the maximum signal seen when t is infinite S 0 is the signal 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 than the longest T4 of the slowest relaxing spins in the sample If cross polarization from protons is possible the initial inver sion pulse can be replaced by a cross polarization step followed by a 90 pulse on the nucleus to be observed Then the required delay between scans d1 becomes that for relaxation of the protons n most cases the proton T1 is moderate so in version recovery Torchia method is the method of choice If the T1 relaxation time is extremely long the saturation recovery experiment is preferred Here the transitions are saturated by a rapid sequen
42. 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 User Manual Version 002 BRUKER BIOSPIN 191 327 Symmetry Based Recoupling 192 327 BRUKER BIOSPIN User Manual Version 002 PISEMA 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 heteronuclear dipolar coupling to another spin 1 2 nucleus Most of the applications so far reported have been in the field of structural biology therefore the X nucleus is normally 13C or 15N and the other hetero nucleus H The experiment provides orientation infor mation on the vector connecting the 13C or IDN and the 1H nucleus The achiev able 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 heteronuclear dipolar interaction is sin 0 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 sys tem to the transverse plane of the rotating frame system spin locked 15N spin system i e sin O 0 816 the scaling of the heteronuc
43. BIOSPIN User Manual Version 002 Basic Setup Procedures e irate OPSE 21 014 en PON en Admina samnected be att Jun 5a pw A AA LI LH U zt gr a9 770 runs TMRARARMH AM 124 Grosser Last50 Groups Aaa Fite Edit View Spectrometer Processing Analysis Options Window Help 0 ww 2 det 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 Wimpens2007 L2 witer 2 opttopspin v 20 40 Figure 4 13 Setting the Carrier on Resonance Click on the marked red arrow to set the observe frequency set the position of the cursor line and left click on the o1 button Acquire another spectrum ft and phase Then expand the spectrum around the adamantane proton signal including the spinning sidebands by clicking on the left margin of the region of interest and pull ing the mouse to the right margin of the region of interest as shown in the follow ing figure User Manual Version 002 BRUKER BIOSPIN 69 327 Basic Setup Procedures Spun Prec2wrs MAPA Tete 2 abran Path Irtenrsis Aam le Stmurtium Fir m Prana rot i E telo ge e Qu N 12 Distance 80 70 ppm 3 amp 130 08 Hz Figure 4 14 Expanding the Region of Interest Click right mouse button in the Spectrum window When the Save Display Re gion to menu pops up select Parameters F1 2 and OK or
44. Basic CP MAS Experiments CP 3 kHz sample rotation dipolar dephasing 240 114 327 220 200 180 be r D DI H pol x bs p vine Q m iw n Tw sas 160 140 120 100 80 60 40 20 0 20 ppm Figure 7 10 Glycine 13C CPMAS NQS Experiment with a Dephasing Delay Figure 7 10 is a glycine 6C CPMAS NQS experiment with a dephasing delayd3 40 us so that the total dephasing time is 80 us Spinning sidebands are still visi ble BRUKER BIOSPIN User Manual Version 002 Basic CP MAS Experiments Sobds Manual chapter 12 1 Ca jus 1206 Spectrum ProcPars AcquPars Title PulseProg Peaks Integrals Sample Structure Fig c 4 v Al 4 Figure 7 11 Tyrosine C CPMAS NQS Experiment with TOSS Figure 7 11 is a tyrosine C CPMAS NOS experiment with TOSS using a de phasing delay d3 60 us Spinning sidebands are suppressed for a clean spec trum In this experiment the total dephasing time is 20 us shorter than that used for the CPNQS experiment on glycine in Figure 7 10 User Manual Version 002 BRUKER BIOSPIN 115 327 Basic CP MAS Experiments Spectral Editing Sequences CPPI CPPISPI and CPPIRCP 7 5 116 327 These spectral editing sequences help to distinguish CH CH CH3 and quaterna ry carbons in D spectra Common to all are various polarization and depolariza tion times which properly mixed and combined give a series of spectra which can be added and subtracted in order to o
45. CPDPRG2 SPINAL64 At PL12 P1 13C excitation flip pulse P3 1H excitation pulse P15 13 1H 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 SW F 1 usually SW MASR if possible NUC1 F 1 NUC1 TD F2 128 Number of points NDO 1 Not required in TopSpin 2 1 NS 4 n FnMode TPPI States States TPPI 148 327 BRUKER BIOSPIN User Manual Version 002 Proton Driven Spin Diffusion PDSD Processing Parameters 11 3 1 Process with xfb Table 11 2 Processing Parameters Parameter Value Comment F2 acquisition 13G sen Left column SI 1k Number of complex points in direct dimension WDW QSINE Apodization in t2 SSB 2 3 PH mod pk F1 indirect C BESTEN 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 11 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 11 3 2 Set CPDPRG2to cw 3 Use the au program calcpowlev to calculate the power level required for a pro ton decoupling RF field of n x masr using p3 and pl12 as reference values
46. Em IMAG ice eet one te ERR ERE MM E IUS pimlgliet e isos etd ett di ero a IAS ai ee power conversion factor Power Conversion Table oooonininncnnncnnccc0oo preamp EE preamplifier nenn Profile o crecen Proton Bandpass Filter BRUKER BIOSPIN User Manual Version 002 Index eoe mu MP 99 ee PH 78 99 Proton Proton DQ SQ Correlation eene 296 SET ele OR M 205 TEE ALS uud E 11 nib o oro eoceno 61 UD Pm 11 Q en 12 R RD CIOA c rM 11 RENOS A 11 SIE e D 201 Resonance rennen 69 himc Em 56 A Eu M es 137 RE sOMMICIOM CY C ee 55 al Mm 55 RESPOrOrMANCO escitas adidas 55 RF POWER AP 55 RETQU NO encenar nike 55 S rus M 277 saturation ee EE 208 saturation recovery experiment nennen 203 SAVE 71 Save Display REGION to eine rtt e deer em dada Pe dead 70 SB ie 86 sealing TACION ER 274 282 SELTICS ee ae ende E 107 110 111 Ee TTT 92 A 55 cca OS O 73 de 58 Sici L R 58 unu
47. 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 program can be called with the option lastf1 Before giving some further explanations about the experiment Figure 17 7 shows the 2D Rb 3QMAS spectrum of RbNO WT ut 40 42 44 46 48 50 52 54 56 58 60 62 64 66 68 ppm Figure 17 7 2D 87Rb 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 re solved 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 User Manual Version 002 BRUKER BIOSPIN 223 327 Basic MQ MAS 224 327 Since the quadrupole parameters are usually unknown before performing the ex periment 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 its axis upfield negative value or low field positive value accordingly For data which don t need a shear ing
48. Figure 3 34 illustrates modifiers for probe tuning ranges for 400 MHz and up on ly In 300 MHz and lower probes only a A4 line can be used because a 2 would be too long but here A 4 can be tuned over the full range All these modifications may be available for WB probes In SB probes they are usually built in if necessary and operated by a switch 44 327 BRUKER BIOSPIN User Manual Version 002 Probe Setup Operations Probe Modifiers A line inner conductor 1 2 Rotating switch at A 4 position 3 Switch closed rotating counterclockwise seen from probe lower end Contact springs grounded touch the A line at the A 4 position 4 Switch open rotating clockwise 5 Switch operating rod 6 Tuning capacitor at the end of the A line inner conductor Fine tunes the effective length and there fore resonating frequency of the A line This tunes the proton channel frequency tune Figure 3 35 M4 low range and 2 Mode high range 400 MHz Probe At 400 MHz the wavelength is large so the A 4 point is below the closed section 7 of the A line At higher frequencies the A 4 point may fall within the closed sec tion 7 The proton channel decoupling channel is usually tuned via a so called A line transmission line This is just a coaxial cable or a coaxial conductor with an ar rangement of an outer conductor a tube and an inner conductor a rod The rel ative diameters and distances and also the dielectric in
49. It is al ways a good idea to keep track which nucleus was tuned last so it is clear what di rection to tune to Usually turning the tuning knob counter clockwise looking from below will shift to higher tuning frequency User Manual Version 002 BRUKER BIOSPIN 63 327 Basic Setup Procedures Spactrum Proc Pars AsquPars Title PulseProg Peaks Integrals Sample Structure Fia Ss alo 4000 5000 1000 2000 3000 598 599 500 607 Figure 4 7 Display Example of an Off Matched and Off Tuned Probe Spectrum ProcPars AcquPars Title PulseProg Pecks Integrals Sample Structure Fia zc Vs EE 4000 598 599 600 601 Figure 4 8 Display Example Where Probe is Tuned to a Different Frequency 64 327 BRUKER BIOSPIN User Manual Version 002 Basic Setup Procedures The figure above is an example of where the probe is either tuned to a completely different frequency outside this window or the probe is not connected to the se lected 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 reso nance position When the probe is tuned well as shown in Figure 4 6 start an acquisition by typ ing zg in the command line or clicking on the black triangle upper left side in the acquisition display Do a Fourier transformation and a phase correction by typing ft and phase correct Set offset O1 to the
50. Sections from the Upper 2D Experiment ssessssssessssse 177 14 Symmetry Based Recoupling 179 Figure 14 1 C7 SQ DQ Correlation Experiment 181 Figure 14 2 Optimization of the RF power level for DQ generation reconversion on glycine 184 Figure 14 3 Variation of DQ generation reconversion time on a uniformly 13C labeled peptide fM IR ET 184 Figure 14 4 PC7 Recoupling Efficiency at a Spinning Speed of 13 kHz sees 185 Figure 14 5 SC14 2d SQ DQ correlation on tyrosine HC1 sssssem nennen 189 Figure 14 6 PC7 2d SQ SQ correlation on tyrosine HCl nenn nennen 191 15 PISEMA 193 Figure 15 1 Pisema Pulse Sequence zu z0 4s00 nsuansnnnnsnnnnsn nenn renan nen the NANE TEA NENANA iara TA ei 194 Figure 15 2 PISEMA Spectrum of 15N Labeled Acetylated Valine and FID in t1 over 3 008 ms 64 Data POMS ae bie Sadie Sede ge vane de vee Se ve nel ve Sk Ve a we dled dene dR Vo a ve a vv pe cee ve eae weal Jee ee 199 Figure 15 3 PISEMA Spectrum of 15N Labeled Kdpf Transmembrane Protein 200 16 Relaxation Measurements 201 Figure 16 1 The CPX T1 Pulse Sequence eee ceee ee eeee en eee eee et eee mene mene nen rennes 203 Figure 16 2 Relaxation of Alpha carbon Signal in Glycine sssse nnnnn nn 207 17 Basic MQ MAS 213 Figure 17 1 A 3 Pulse Basic Sequence with Z Filter ssssssssssse mmm 214 Figure 17 2 A 4 Pulse Basic Sequence with Z Filter o
51. W PMLG PC7 DQ SQ Correlation at 600 MHz with Ty rosine Hydrochloride User Manual Version 002 BRUKER BIOSPIN 301 327 CRAMPS 2D 302 327 BRUKER BIOSPIN User Manual Version 002 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 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 002 BRUKER BIOSPIN 303 327 304 327 BRUKER BIOSPIN User Manual Version 002 Operator used from to Probe shim file Bo field RA p90 contact mix else p90 contact decouple mix else p90 contact decouple mix else Sample Experiment stored under filename under filename E 2 Pulse program cpopt cpopt TopSp
52. X nu clei is usually very slow and requires very long mixing times since the dipolar cou pling 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 protons are strongly coupled to each other the flip flop transition rate is high along the X4 H4 H25 X 2 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 decou pled 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 Di polar Assisted Rotational Resonance T Terao et al or RAD Rf Assisted Diffu sion see C R Morcombe et al The setup for all these sequences is rather robust requiring only the 1H to X Hart mann Hahn condition and the X 90 hard pulse to be set For RAD and DARR it is usually sufficient to calculate 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 side bands need to be suppressed de Jong et al or where spin diffusion is enhanced by matching the spin rate with the chemic
53. a good size single crystal 1 Set up for static 15N CP observation on the a glycine powder sample pulse program cp Use a ramp pulse if the HH condition is unknown with power level settings for an approximate 5 usec pulse on both channels 2 Determine the proton 90 degree pulse p3 at the respective power level reset the conditions for a square shape and about 50 kHz on both channels for con tact Load the pulse program cplg With cnst17 0 pl2 pl13 and pl1 all set for 50 kHz reestablish the HH condition 3 To adjust the CP condition under a LG offset load the pulse program cplg Cnst 17 sets the LG offset during the contact the offset frequencies are calcu lated as cnst 18 and cnst19 Start with cnst17 0 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 H and set cnst20 accordingly to e g 50 kHz i e cnst17 50000 0 This would give an offset frequency of approximately 35 kHz cnst19 should show this number in the ased display Then adjust the IDN 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 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 reduces the required
54. a single frequency design there are more degrees of freedom in tuning the cir cuit The frequency range is set by a suitable NMR coil see Figure 3 26 Fine tuning is done by a variable capacitance 1 and a fixed capacitance inside an ex changeable tuning insert 3 The purpose of this setup is to adapt the probe to the desired task as much as possible A wideline probe has to cope with a large range of frequencies and line widths and must provide the shortest possible puls es and highest possible sensitivity at the shortest possible dead time These re quirements cannot be met with one standard setup Principles of setting up such a probe are 1 Select an NMR coil 2 with highest inductance that can still be tuned to the re quired frequency Choose the smallest coil diameter permitted by your sample reduce the sample diameter if appropriate 2 Select the symmetrization insert such that the desired frequency is close to the upper end of the available tuning range User Manual Version 002 BRUKER BIOSPIN 41 327 General Hardware Setup 3 Select the Q value of the insert according to the expected line widths higher Q for line widths up to 100 kHz Please note that multiturn coils especially multi filament coils have an intrinsically low Q 1 Tuning capacitance 2 Exchangeable NMR coil 3 Exchangeable symmetrization and Q reduction Figure 3 33 RF Setup of a Wideline Single Frequency Probe Shifting the Probe Tuning Ra
55. anisotropic broadening of the central 1 2 lt gt 1 2 transition CT and symmetric multiple quantum MQ transitions is the 2 d order quadrupole interaction which can only partially be averaged by MAS The satellite transitions ST e g the 3 2 lt gt 1 2 transitions however are broadened by a 1 order interaction which is several orders of magnitude larger than the 24 order broadening Under MAS the 1 order interaction of the ST can be averaged but since the spinning cannot be fast compared to the first order broadening of the order of MHz a large manifold of spinning side bands re mains The 2 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 2 d order broadening which may be of the order of kHz Lineshapes resulting from nuclei in different en vironments 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 corre lates 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 o
56. as the inver sion recovery data above see Table 16 3 for parameters The only differences are that the fitting function should be satrec rather than expdec and the slice se lected 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 ob tained by inversion recovery T1p Relaxation Measurements 16 2 5 Experiment setup Rotating frame relaxation measurements under a spin locking rf field can be used to probe motions on shorter domestically than T measurements with in verse correlation times of the order of the spin locking rf field strength To measure T4 relaxation after CP a variable length spin locking pulse is applied to the X nucleus The remaining X magnetization decays exponentially to zero as a function of spin lock time The parameters of cross polarization can also be de termined from variable contact time CP experiments the function cpt1rho is pro vided in the relaxation analysis tool for this purpose but here only simple T4 measurements will be discussed It should be noted that the relaxation in a T4 experiment might result from pro cesses other than true Ty relaxation For example in glycine the carbon spins are dipolar coupled to protons 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
57. condition acquire reference spectrum 3 P on ADP ammonium dihydrogen phosphate NH H5 PO4 load a glycine 13C reference spectrum set observe nucleus to P in edasp add 6 dB to p 1 optimize HH condition acquire 2 scans reduce rg appropriately User Manual Version 002 BRUKER BIOSPIN 103 327 Practical CP MAS Spectroscopy on Spin 1 2 Nuclei 104 327 BRUKER BIOSPIN User Manual Version 002 Basic CP MAS Experiments The following experiments can be run by calling a 130 CPMAS standard parame ter data set or data loading the appropriate pulse program and loading the pulse parameters obtained previously during the setup see Basic Setup Proce dures Some attention needs to be paid to special experimental parameters Most of those parameters are explained in the header section of the pulse pro grams The CPPI experiment series in section 7 5 requires measuring the HH match us ing a constant amplitude contact pulse This can be accomplished using a rectan gular shape square 100 or using the pulse program cplg Pulse Calibration with CP 7 1 Pulse calibration for C pulses after cross polarization using a flip back pulse hu 63 heteronuclear decoupling Q2 da Oro 130 Figure 7 1 Pulse Program for CP with Flip back Pulse The experiment can be done directly after the CPMAS setup procedure Loading the pulse program cp90 and setting p 1 pI11 allows one to measure the X nucle us spin nutation frequency at
58. couplings between the spins If the coupled spins are of the same kind it is called homonuclear dipolar cou pling heteronuclear dipolar 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 homonuclear and heteronuclear interactions can be obtained by different forms of rf irradiation with or without sample spinning It is possible to suppress homonuclear couplings without suppressing heteronu clear couplings Most frequently the nucleus 1H must be decoupled when X nu clei like SC or IDN are observed since it is abundant and broadens the line shapes of coupled X nuclei strongly Heteronuclear Decoupling 5 1 CW Decoupling 5 1 1 CW decoupling simply means irradiating the decoupled spins usually protons with RF of constant amplitude and phase The decoupling program is called cw or cw13 and it uses pl12 or pl13 respectively The decoupling programs select the power level and pl12 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 re sonance 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 carr
59. cp Pulse Sequence BRUKER BIOSPIN User Manual Version 002 Basic Setup Procedures The following parameters are set pl12 for the initial 90 degree pulse and the decoupling during acquisition set for a 4 5 usec proton 90 degree pulse as previously determined pl for the carbon contact pulse set for a 4 5 usec carbon pulse as previous ly 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 tak en as microseconds cpdprg2 select cw 01 set between both adamantane peaks 02 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 ampli tude e Becher TOPSPIN 2 1 b 14 on PCHF3 as Administrator connected to avi Ow File Edit View Spectrometer Processing Analysis Options Window Help DSDS a A AA JL Fo do w u RF SO Das Sm 7 meENGGGgGSoM t 1 N Cp 098 Neon marsa Spectrum ProcPars AcquPars Toe PulseProg Peaks integras Sample Structure Fid Acqu Prase Proa y poptau for pit finished POSMAX at experiment 6 pif 4 5000 NEXP 9 A O O S E O O E A
60. d1 05 1s 4 lines User Manual Version 002 BRUKER BIOSPIN 11 327 Test Samples Table 2 1 Setup Samples for Different NMR Sensitive Nuclei 130 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 1096 in natrl abundance 79Br KBr MAS 57 d1 lt 50msec angle setting finely powdered reduced volume Co Co CN g MAS shift thermometer 99Mn KMnO MAS gt 500 kHz pattern 99Nb 207pp PbNO3 MAS shift thermometer 0 753 ppm degr d1 gt 10s Pb p tolyl 4 CP 150 5ms 15s 29sj QgMg CPMAS 50 d1 gt 5s reference sample 12 6 108 ppm DSS TMSS CPMAS 0 reference sample O ppm Se H3Se03 CPMAS 1800 HHsetup 8ms contact d1 gt 10s NH4 2SeO CPMAS 200 3ms d1 gt 4s Wee Cd NO3 9 4H 0 CPMAS 350 15ms contact d1 gt 8s 195Pt K Pt OH g CPMAS 12000 1ms contact d1 gt 4s 199Hg Hg acetate gt CPMAS 2500 5ms contact d1 gt 10s Hexakis dimethyl 2313 30 35ms contact d1 gt 10s sulphoxide Holt trifluorome thansulfonate H d PMMA WL 0 wideline setup d1 5s d PE WL 0 wideline setup d1 0 5s 10s amorphous crystalline d DMSO gt WL 0 exchange expt at 315K 6Li LiCl Li org make sure it is not DU depleted d1 gt 60s 179 D20 0 pulse determination 100scans 0 5s 15N a glycine CP 50 sensitivity 4ms contact 4s labelled for fast setup 35C KCI WL MAS
61. di polar 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 men tioned that there are recoupling sequences that do not generate double quantum coherences DRAWS DRAMA and MELODRAMA Symmetry based recoupling sequences recouple specific spin interactions using cyclic sequences composed of N phase shifted repetitions of either 2x C se quences or x R sequences rotation elements Which interaction s are recou pled 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 ele ments The sequences are denoted as e g CN where N is the number of ele ments in the cycle n is the number of rotor peribds spanned by the N elements and the total phase rotation between the elements is 2x v In the simplest imple mentation of a C sequence the 2r rotation element is simply a 27 pulse but other elements are possible Thus the sequence C71 consists of 7 consecutive 27 pulses with the phase of each pulse shifted by 21 7 from the previous one The whole sequence takes two rotor periods each 2r pulse thus takes 2 7 rotor peri od The spin nutation frequency and sample rotation frequency are thus related by Ver 7 2 Vrotor In practice the original C7 sequence uses an addition
62. example chosen here any of the conditions will provide a good qual ity spectrum and the condition with the biggest enhancement at the least power is to be preferred 1 2 rotor period 1 4 rotor period 1 8 rotor period Pul 20 16 12 8 sp1 dB 2D Data Acquisition i eg ped p IER D A A 0 2016 12 8 4 0 20 16 12 8 4 O 20 16 12 8 4 0 sp1 dB sp dB sp1 dB Figure 18 6 Signal Intensities of 9 Rb in RBNOs Signal intensities of Rb in RbNO as function of duration and RF field amplitude for double frequency sweeps 18 2 2 238 327 After the parameters for the DFS are adjusted the 2D data acquisition can be pre pared In tables 2 and 3 the important parameters are listed for the two pulse se quences 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 FAMODE 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 ap plied 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 nuclei with spin I 3 2 where a split t4 experiment can be per formed in which case IN10 must be s
63. experiments using symmetry based recou pling sequences Parameter Value Comment F1 acquisition 13c 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 90 shifted sine bell Processing parameters for DQ SQ correlation experiments using symmetry based recoupling sequences The AU program xfshear may be used with option rotate and argument 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 en ter the value of sr for F2 and add 2 01 188 327 BRUKER BIOSPIN User Manual Version 002 Symmetry Based Recoupling F1 ppm I 180 o DN L N f p e L o E I CS go u F lt u F ti LS N 5 r H L o LT uU LN a a ae a ae EE TT CC a 200 150 100 50 F2 ppm Figure 14 5 SC14 2d SQ DQ correlation on tyrosine HCl SC14 2d SQ DQ correlation on tyrosine HCI 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 14 5 Symmetry based DQ recoupling sequences may also be used as mixing periods in SQ SQ correlation experiments
64. first pulse must be incremented by 30 in States or States TPPI mode ph1 0 ph2 0 0 60 60 120 120 180 180 240 240 300 300 ph3 0 180 receiver hi ph2 I pt dO p2 dio p3 d4 p3 pitt D I Figure 17 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 sequence Corresponding power level pl11 should be set to achieve at least 150 kHz RF field amplitude p3 should be some tens of us corre BRUKER BIOSPIN User Manual Version 002 Data Acquisition Basic MQ MAS sponding to an RF field amplitude of a few kHz Delays d0 and d4 are the incre mented delay for t1 evolution and 20 us for z filter respectively Delay d10 initially is O and can be incremented proportional to dO in10 in0 7 9 if the observe nu cleus 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 phi 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 howev er itis the basic sequence to improve sensitivity by FAM or DFS The 3 pulse se quence itself ca
65. for 2 Different Types of SB Probes Warning The spin rate monitor cable has a probe side connector that is ex actly the same as the power supply cable for the B TO thermocouple oven used with many high resolution probes If this cable is connected to the spin rate monitor assembly at the probe the latter will be destroyed Make sure the B TO cable and the MAS cable labelled probe at the probe side are labelled such that they cannot be mistaken MAS Tubing Connections 3 5 26 327 For any type of fast spinning probe compressed gas is used to provide the spin ner bearing and drive gas Please refer to the installation or site planning manuals to learn about the gas requirements The most important parameters are Mains pressure should be at least maximum required pressure 1 bar to pro vide pressure regulation range Bearing pressure up to 4 5 bar as of February 2008 Drive pressure up to 4 5 bar as of February 2008 This means that at least 5 5 bars of pressure should be available at the outlet If the pressure droop along the supply tube is substantial the internal pressure may drop below 5 bars then the MAS unit stops to regulate and gives a warning Consequently we recommend a primary inlet pressure of min 6 bar preferably 7 8 bar max 10 bar and a low loss gas line 8mm inner diameter distance x5 meters between the instrument and the gas supply to assure trouble free opera tion even under cond
66. in the eda win dow correspondingly Since dipolar couplings between 1H and 15N can be up to 15 kHz the spectral width including the experiment scaling factor should be min around 20 kHz BRUKER BIOSPIN User Manual Version 002 PISEMA Table 15 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 heteronuclear 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 a left column AQ_MOD qsim TD 512 No of points DW Dwell time in t2 F1 indirect 1H en right column TD 64 Number of points IN_F1 L3 2 p5 SF or Scaling factor for PISEMA 0 82 sin 54 7 deg calcu L3 2 5 p5 SF lated by pulse program User Manual Version 002 BRUKER BIOSPIN 197 327 PISEMA Processing 15 3
67. is more difficult to observe a small inten sity change on a strong signal which might be caused by fluctuations than a big ger difference on the low abundance nucleus caused by the high abundance of the coupling partner An example The measurement will be more precise observ ing 136 and defocus with 31P pulses than vice versa because the effect on 315 caused by 1 1 of 19C would be max 1 1 a very small change which would re quire extremely high S N and extremely high stability in signal generation and spin rate BRUKER BIOSPIN User Manual Version 002 Pulse Sequence Setup Pulse Sequence 12 1 SPI Number of 0 1 2 Rotorperiods 6713 4501011010 62200112233 m 0 64700110011 4 02132031 Figure 12 1 REDOR Pulse Sequence 12 2 The example of setup given here is based on a biological sample but of course the procedure will not change if you are going to analyze different samples with different combinations of coupled spins Sample fully labelled 15N 13C3 Glycine diluted in natural abundance glycine to 10 Dilution will reduce long range dipolar interactions strongly and lead to a well defined direct interaction between 1 N and C so that a single frequency dipolar modulation is obtained A triple resonance probe with the X channel tuned to 13C and the Y channel tuned to IDN is required Set 13C observe with cross po larization from protons It is recommended that separate preamps are used fo
68. is terminated with the second non selective pulse P2 p4 p2 d4 pl11 pl21 plt1 pl21 Figure 19 2 Four pulse sequence and coherence transfer pathway for the double quantum filtered STMAS experiment with z filter stmasdgfz av Pulses pl and P2 are non selective pulses The corresponding power level PL 11 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 us respectively corre sponding to an RF field amplitude of a few kHz Delays DO and D4 are the incre mented delay for t4 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 in cremented by 90 in States or States TPPI mode User Manual Version 002 BRUKER BIOSPIN 247 327 STMAS 248 327 h1 ph1 02 ph2 0022 ph320000111122223333 ph4 0 8 1 8 2 8 3 8 4 8 receiver 202202002022020021331311313313113 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
69. look like liquids spectra the conditions 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 break through 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 proportional to pulse power and duration Always observe the limits for duty cycle and maximum pulse power Please refer to the probe specifications for more information Never set acqui sition times longer than required Spinners and turbine must be kept extremely clean Any dirt especially oil sweat from fingers water will decrease the breakthrough voltage d
70. mm las dia bd 55 Silicone paste inei e da 11 Silicone rubber TT 11 Cipp eE 83 SKID setenta toot Eta oM ee me D dad 71 Skip Optimizat OM e 71 O une E RA AUN anne OR Van 11 SMV TONE EE 11 SNO2 TEE 11 spin nutation frequencies nn 55 erue eS 55 DI 91 SPINAL decoupling scera ee tlie eo rebns il Lee nu dde 91 User Manual Version 002 BRUKER BIOSPIN 323 327 Index 324 327 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 uo TPPM decoupling eisieinossira aiaa Fans LE transverse magnetization TREVSB coe V variable dea variable pulse list nn varmod ssssssssseeeeener eene W PMLE O acosada X X Low Pass Elter E n X BB Preamplifier nn XIX decoupling esscr BRUKER BIOSPIN User Manual Version 002 Index Y MUTO da ORE 13 Z ZGopti n DIAC ET 75 LOOP Noia 106 aecarbon at Ee EE 80 Sg Ee 12 79 ele LEE 79 User Manual Version 002 BRUKER BIOSPIN 325 327 Index 326 327 BRUKER BIOSPIN User Manual Version 002 End of Document User Manual Version 002 BRUKER BIOSPIN 327 327 Bruker BioSpin your
71. occurs rapidly contact times should be kept short in order to avoid long range transfer leading to unspecific cross peak pat terns since the magnetization then has time to flow from any proton to any X nu cleus 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 proton spins A third modifi cation of the basic sequence uses phase modulated pulses instead of frequency shifts These three modifications to the basic sequence are described in Modifi cations of FSLG HETCOR References 1 H J M deGroot H F rster and B J van Rossum Method of Improving the Resolution in Two Di mensional Heteronuclear Correlation Spectra of Solid State NMR United States Patent No 5 926 023 Jul 20 1999 2 B J van Rossum H F rster and H J M deGroot High field and high speed CP MAS 13C NMR Het eronuclear dipolar correlation spectroscopy of solids with frequency switched Lee Goldburg homonu clear decoupling J Magn Reson A 120 516 519 1997 3 B J van Rossum Structure refinement of photosynthetic components with multidimensional MAS NMR dipolar correlation spectroscopy Thesis University of Leiden Holland 2000 4 B J van Rossum C P deGroot V Ladizhansky S Vega and H J M deGroot A M
72. 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 nucleus SFO MHz 500 0510001 Frequency of 2nd nucleus BF2 MHz 500 0500000 Basic frequency of 2nd nucleus Y Nucleus 3 E ci ET E gt Figure 13 3 The Acquisition Parameter Window eda 172 327 BRUKER BIOSPIN User Manual Version 002 Data Acquisition Table 13 1 Acquisition Parameters SUPER 13 4 Sample Tyrosine HCI Experiment time several hours 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
73. ooooccocccnccnnccnccnnconcnnncnnnnnncnnnnnnnns 22 Figure 3 8 Triple Resonance Experiment without X Y Decoupling coocooicocnnnccncccocccncccnccnnicnnnnns 23 Figure 3 9 Triple Resonance Experiment with X Y Decoupimg 23 Figure 3 10 Triple Resonance 1H 19F Experiment ssssssssssssss nenne nennen 24 Figure 3 11 19F 1H Combiner Filter Set sourina an E EEEE mnes nnne 24 Figure 3 12 Quadruple Resonance HFXY Experiment WB probes gt 400 MHz only 25 Figure 3 13 PICS Probe Connector and Spin Rate Monitor Cable on a WB Probe 25 Figure 3 14 Spin Rate Monitor Cable Connector for 2 Different Types of SB Probes 26 Figure 3 15 WB DVT Probe MAS Tubing Connections me 28 Figure 3 16 VTN Probe MAS Tubing Connections Note WVT Probes are VTN Type Probes 28 Figure 3 17 WB Probes Eject Insert Connechons He 29 Figure 3 18 WB Probes DVT Probe Connections for RT and HT Measurements 29 Figure 3 19 SB VTN Probe MAS Connections ssssssssss meme rre 30 Figure 3 20 SB DVT probe MAS connections meme rennen 31 Figure 3 21 WB Probe MAS VTN and WVT and DVT Probe Connections cooccoccccccnccncccnccnccnncnns 32 Figure 3 22 WB Probe MAS DVT CGonneciions mm mehreren 33 Figure 3 23 SB Probe MAS VIN ih ii kirishi QUA EU A TUE ne 34 Figure 3 24 SB Probe MAS DVT Connections sssssss memes 34 Figure 3 25 WB Wideline or PE Prob6S dii
74. oooooccocccocccnccnnccncconccnnnonconcnonconcnoncnnncnnnnnncnnnnnnninaninannnnnns 96 Pulse Program for Hahn Echo Sequence eee 98 6 Practical CP MAS Spectroscopy on Spin 1 2 Nuclei 99 7 Basic CP MAS Experiments 105 Pulse Program for CP with Flip back Pulse m 105 Pulse Program for CP TOSS iiec abere aL ente do RD eR ot DA Ue ad pee Dese DR ew Eer a 107 BRUKER BIOSPIN User Manual Version 002 310 327 Figures Figure 7 3 Comparison of a CPTOSS and CPMAS Experiment 108 Figure 7 4 CPTOSS243 Experiment on Tyrosine HCI at 6 5 kHz sssse 109 Figure 7 5 CPTOSS Experiment on Tyrosine HCl arpGbktz 110 Figure 7 6 Pulse Program for SELTICS 2 20 002200 ENER icis RR scene dare dat rnt nennen e sehn D nnn a 111 Figure 7 7 SELTICS at 6 5 kHz Sample Rotation on Tyrosine HCl seen nenn 111 Figure 7 8 Cholesterylacetate Spectrum Using Sideband Suppression 112 Figure 7 9 Block Diagram of the Non quaternary Suppression Experiment 113 Figure 7 10 Glycine 13C CPMAS NQS Experiment with a Dephasing Delay 114 Figure 7 11 Tyrosine 13C CPMAS NOS Experiment with TOP 115 Figure 7 12 Block Diagram of the CPPI Experiment 116 Figure 7 13 CPMAS Spectrum of Tyrosine HCl atpGbkHz mee 117 8 FSLG HETCOR 119 Figure 8 1 The FSLG Hetcor Experiment ooocooccconcnncncncccnnccnnconancnnnnnnnn cnn rra rare 120 Figure 8 2 The 12 icon and the ased icon in eda ooccocccnccncccnccnccnnconcnn
75. operator is responsi ble for proper wiring cabling and tubing Since mistakes especially in connection with compressed gas tubing may cause rather expensive repairs it is recom mended to check carefully before an experiment is started The following operations will be described and illustrated with suitable images for WB and SB probes where non trivial differences exist Connections to the Preamplifier on page 15 RF Connections Between Preamplifier and Probe on page 20 RF Filters in the RF Pathway on page 21 Connections for Probe Identification and Spin Detection on page 25 MAS Tubing Connections on page 26 Additional Connections for VT Operation on page 31 Probe Setup Operations Probe Modifiers on page 41 Mounting the Probe in the Magnet Shim Stack on page 50 The edasp Display for a System with two Receiver Channels on page 54 Connections to the Preamplifier 3 1 For solids and liquids there should normally be different sets of preamplifiers Liq uids preamplifiers HPPR High Performance Preamplifiers are not suitable for some of the requirements of solid state NMR Where CP MAS applications are the only solids applications it is however possible to use liquids preamplifiers for X observation Solids preamplifiers HPHPPr High Power High Performance Pre amplifiers are definitely required if high power 2 1 kW is used liquids preamplifi ers take max 500W for X frequencies 50W for proton and fluorine f
76. p C7 sequence is therefore 0 18 dB to higher attenuation than what is required for a 2 5 usec pulse User Manual Version 002 BRUKER BIOSPIN 297 327 CRAMPS 2D Table 24 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 appro 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 24 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 51 DQ selection 298 327 BRUKER BIOSPIN User Manual Version 002 Data Processing Ta
77. parameter window eda in order to get the spectrometer router display Alternatively click on the routing icon in eda frequency logical channel amplifier preamplifier PPI 150 360709 MHz SFO1 150 369709 MHz HPHP X 88315 OFS1 9000 0 Hz HPHP 19F 1H 31 BF2 600 147 MH HPHP XER SFO2 600 147 MHz HPHP 19F 1H OFS2 oo Hr HPLNA 1H BF3 600147 MHz SFO3 600 47 MHz OFS3 0 0 Hr BF4 600147 Mr NUC SFO4 600 147 MH Es seus 1H 1H 50 W OFS4 Jon Hz off cable wiring possible RF routing cortab available settings show receiver routing show RF routing 7 chow power at probe in Seve Switch Fl FZ Switch FI F3 Add a logical channel Remove a logical channel Default Info Param Close User Manual Version 002 Figure 4 1 Routing for a Simple One Channel Experiment The figure above shows the routing for a simple one channel NMR experiment us ing 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 BRUKER BIOSPIN 57 327 Basic Setup Procedures 58 327 channel define the precise irradiation frequency by setting the nucleus and the off set O1 from the basic frequency In this example we want to set up for pulsing ob serve on the nucleus Br Selecting 79Br for channel F1 defines the basic frequency BF1 of Br in this case on a 600 MHz sp
78. 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 10000 15000 Hz At 100 kHz RF 15 kHz is ok User Manual Version 002 BRUKER BIOSPIN 123 327 FSLG HETCOR Table 8 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 124 327 BRUKER BIOSPIN User Manual Version 002 FSLG HETCOR Results 8 3 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 8 4 FSLG Hetcor Spectrum Tyrosine HCI The figure above shows a FSLG Hetcor Spectrum Tyrosine HCI with parameters as shown in Table 8 1 Table 8 2 Full transform with slight resolution enhance ment 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
79. pling 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 frequency 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 Ja beled tyrosine hydrochloride since it has a wide spread 2 5 12ppm of proton shifts a short proton T4 a well resolved 18C spectrum with quite many lines and it is readily available The unlabeled sample can also be used but re quires a few more scans 8 32 4 Optimize the spin rate such that no overlap occurs between center and side bands especially with the labeled sample in order to avoid rotational reso nance broadening Re optimize decoupling and HH condition Check the proton RF field via the proton 90 pulse p3 Set pl13 pl12 set cnsi20 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 Acqu Phase Print nis W ES OV d Installed probe 4 mm MAS BB 1H H12083 0005 F
80. proton RF power by about 70 as compared to the power level for a resonant HH match For the new nutation frequency B1 field for LG condition Bie A sin Q l Bion CH 0 82 By CH the offset frequency for the Lee Goldburg condition is fic cos 0 S Bion res ee 0 578 Bion res CH with the inverse of a 360 pulse Phe Dr User Manual Version 002 BRUKER BIOSPIN 195 327 PISEMA 196 327 Instead of raising the power level for 15N the power level for H is reduced by about 1 7 dB Then the new 2r pulse in the tilted frame is sin 0 0 67B lon _ res Tons Bion res In our example of a contact power level of 50 kHz on 15N one would then calcu late for cnst17 40 807 0 giving an offset frequency of 28855 Hz for the LG fre quency which is calculated automatically 5 n order to verify all calculated power levels and offset frequencies optimize for the appropriate power level using the pulse program cplg 6 Change to the desired PISEMA sample Since a powder contains all possible spin pair orientations the measurement of an oriented sample is much recom mended because it is not only much faster but also allows to judge the perfor mance much better N acetly valine can be rather easily grown to sizeable single crystals and has all properties for a good setup sample decent proton T1 well defined sites and therefore narrow resonances For a good crystal the residual proton line width w
81. quires transfer of the energy to the outside of the spinner which takes sec onds EIS probes eliminate these problems to a large extent 9 The basic double CP experiment can be extended into many different varia tions One example 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 homonuclear recoupling step HORROR DREAM or other Like wise 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 applica tions support for appropriate pulse programs BRUKER BIOSPIN User Manual Version 002 2D Data Acquisition Sample 15N 13C Double CP 20 3 labeled histidine peptide or protein Spinning speed 10 15 kHz depending on 6 spectral parameters rotational resonance must be avoided Experiment time 30 min several hours Acquisition Parameters Table 20 2 Recommended Parameters for the DCP 2D Setup Parameter Value Comments Pulse program doubcp Pulse program nuc1 13C Nucleus on f1 channel o1p 100 ppm 13C offset nuc2 1H Nucleus on f2 channel 02p 2 4 ppm 1H offset optimize nuc3 15N Nucleus on f3 channe
82. reference 130 CP MAS spectrum of the labelled glycine sample using 16 scans This reference spectrum will serve to measure the efficiency of the DCP magnetization transfer To set up the conditions for the N to C transfer one must define 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 excita tion 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 selectively Specific CP An RF field of 35 kHz is a decent compro mise So using the cp90 pulse program and moving the carrier close to the C peak determine a power level p T1 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 other 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 19C channel is set 20 2 2 258 327 8 Create a new experiment using edc Setup
83. refocusing pulses 0 is chosen to a value in a way that there is a noticeable de crease in dephasing experiment normally this is between 0 1 15 and then the refocusing p pulse is optimized for minimum signal intensity in the dephasing ex periment Table 12 1 Acquisition Parameters for a 19C observed C N REDOR Parameter Value Comments pulse program cpredori pulse program nuc1 16 nucleus for f1 channel nuc2 1H nucleus for f2 channel nuc3 15N Nucleus for f3 channel p3 according to specs 1 2 pulse on f2 channel p15 2000 Contact time between f1 and f2 pcpd2 about 2 p3 pulse length for decoupling sequence p2 according to specs 6 10 usec z pulse on f1 p12 about 10 usec 1 pulse on f3 cnst31 masr MAS spinning rate 10 1 starting value for the desired evolution time value must be odd d1 4s recycle delay pl1 for HH condition f1 power level for contact pulse pl11 according to specs f1 power level for x pulse sp0 for HH condition power level for 1H ramp pl2 not used 158 327 BRUKER BIOSPIN User Manual Version 002 Setup Table 12 1 Acquisition Parameters for a 13C observed C N REDOR pli2 adequate power level 1H decoupling pl3 adequate power level for f3 x pulse pl22 120 power level in Sg experiment for the recoupling pulses spnamO ramp ramp file name for CP td f1 32 256 depending on coupling aq 20 40 msec acquisition ti
84. 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 breakthrough limits Note the optimum power levels sp0 and pl1 and contact time p15 Setup of the Double CP Experiment 20 2 3 1 User Manual Version 002 Read the reference carbon data set and generate a new data set using edc or iexpno Select the pulse program doubcp Set the optimum 15N cp parameters as found in the previous step set sp0 p15 and pl3 for proton to 15N cp Set o3 close to the DN peak position Now we have to select the appropriate parameters for the nitrogen to carbon magnetization transfer In the standard doubop pulse program p16 is used as the second contact time and the shapes sp7 and a square pulse at pl5 are specified for the C and 15N contact so p16 sp1 C power level and pl5 5N 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 Using a square pulse on the IDN channel is preferred but the se quence can be rewritten to use a 13c square pulse and have the shape on the 15N channel BRUKER BIOSPIN 259 327 Double CP Table 20 1 Recommended Parameters for the DCP Setup Parameter Value Comments P
85. 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
86. strengths care must be taken over the length of the spin lock pulse If apparently non exponential decay is observed this may result from such alternative relaxation processes Sample Glycine Spinning speed 10 kHz Time 20 minutes User Manual Version 002 BRUKER BIOSPIN 209 327 Relaxation Measurements Data Processing 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 indepen dently of the field strength for the cross polarization In principle the strength of this field can be set to any value within 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 power as pl11 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 Re member 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
87. the motion For efficient relaxation via a particular energy level transition fields fluctu ating with an inverse correlation time close to the frequency of the transition are required Longitudinal relaxation occurs via transitions on a single spin and thus requires fields fluctuating with inverse correlation times near to the Larmor fre quency 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 T4 relaxation involves transitions at the nutation fre quency of the spin locking pulse which can be chosen by the experimenter Mea surement of these relaxation rates can therefore provide information about local motions on a range of time scales T1 Relaxation Measurements 16 2 Longitudinal relaxation can be measured using a number of methods which method is appropriate 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 general 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 16 2 1 202 327 The inversion recovery method is the originally proposed method for measuring T4 values The experiment proceeds as follows firstly the magnetization is invert ed by a
88. the HH contact power Of course the experiment al lows nutation frequencies to be measured at other power levels as well The typical nutation pattern has a cosine form so a 90 degree pulse gives null signal Use glycine spinning at N kHz as before When using POPT for such measure ments the optimization type is ZERO so that the program looks for a zero cross ing at the automatic data evaluation To get nutation patterns without phase User Manual Version 002 BRUKER BIOSPIN 105 327 Basic CP MAS Experiments distortions 90 pulses should always be executed close to the observed reso nance Larger offsets give different shorter p90 values and phase distortions for pulse lengths close to 180 and multiples thereof Table 7 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 ppm Spectral width for Glycine 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 experi ment see next chapter Total Sideband Suppression TOSS 7 2 106 327 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 sequence with four specially spaced 180 pulses As is the case for all extra pulses on the X channel in
89. the high proton frequency the A 4 length shortens the A 4 point 1 moves inside the trans mission line outer conductor 2 The screw 3 is used to set M4 screw in and M 2 mode screw out 4 proton tuning capacitor BRUKER BIOSPIN User Manual Version 002 Probe Setup Operations Probe Modifiers 1 X tuning variable capacitance 4 Low range extension with parallel capacitance 2 Y tuning capacitance 5 Frame flush air outlet to protect sensitive ca 3 Proton reject filter pacitors Figure 3 37 Without with Parallel Capacitance to Shift the Tuning Range to Lower Frequency Adding a parallel capacitance does not decrease the efficiency of a circuit How ever a certain circuit has the highest possible efficiency if it is tuned with maxi mum inductance and minimum capacitance Maximum inductance can usually not be achieved due to spacial restrictions in the stator MAS probes or due to losses in Q if the coil becomes too large increase in resistance Furthermore capaci tances are tunable inductances usually are not so a wide tuning range can only be achieved via exchangeable inductances and or capacitance with a wide tuning range Capacitances may look different than the one shown in the picture Fre quently two larger capacitances are used in series to form a smaller capacitance withstanding higher voltage A parallel capacitance lowers and narrows the tuning range User Manual Version 002 BRUKER BIOSPIN 47 327
90. the proper routing by going into edasp Click the switch F1 F3 button to get 15N on channel 1 Figure 20 3 Routing table for triple resonance setup change for IDN pulse param eter measurement and CPMAS optimization 10 If available set pl 1 and spO for a proton N HH condition in triple mode On a 11 labelled 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 essen tial to optimize the first proton to nitrogen HH contact This is not as trivial as BRUKER BIOSPIN User Manual Version 002 Double CP one might think since the transfer efficiency depends strongly on the timing and RF fields of the HH match The proton Tue 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 polar ized IDN spectrum to the directly observed spectrum using hpdec and 90 de gree pulses at 4 sec repetition one can measure the enhancement factor rather easily Without optimization the enhancement 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
91. the results of the fitting with the solids line shape analysis package included in TopSpin The spectra used for that have been extracted from rows of the 2D spectrum shown in Figure 17 10 BRUKER BIOSPIN User Manual Version 002 Basic MQ MAS 8 g d 3 A E g 3 4 Y f N lagen nene A vo E AED RA TE B Figure 17 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 Alexan drine Flambard LCPS Univ de Lille Spectra that are sheared can be evaluated graphically as follows as shown in Fiqure 17 12 In addition to the red isotropic chemical shift axis indicated as axis CS with the slope A8 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 retaining the same slope so that it intersects a spectral line in its centre of gravity Through the inter section point of the Qis axis with the CS axis a third line can be drawn parallel to the F2 axis This is the dotted black line in Figure 17 12 The shift value that is read from the F1 axis at this position is the isotropic chemical shift of that
92. type dpl in the com mand line x Options Parameters F1 2 e g used by restore display dpl C Parameters ABSFI 2 e g used by absf apkf C Parometers STSR STSI used by strip ft C Parometers SIGFL2 signal region used by sino C Parameters NOISFL2 noise region used by sino C A text file for use with other programs Figure 4 15 Save Display Region to Menu 70 327 BRUKER BIOSPIN User Manual Version 002 Basic Setup Procedures Start parameter optimization by typing popt in the command line The popt win dow will appear euamh 17 1 CTWLKer 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 O Overvrite existing files disable confirmation Message FT mode fqc O stop samale spinning at tne end of op m zanon masn O Run optimizacion in backgrcund OPTMIE GROUP PARAMET OPTMUM STARTAL ENWAL NDP VARMOD INC Stepy step C pi POSMAX los oc 20 JUN E Skip current optimiz S omprotocoh Add parameter t Step ostimization J Delete paraneter Cisplay Dataset Figure 4 16 The popt Window Use optimize step by step parameter p7 to optimize parameter p1 optimum posmax to find the highest signal intensity 90 degree pulse for the given value of pliw or pli and varmod in to use linear increments for optimization The value for group is not used for optimizing only one paramete
93. with 22 ms DARR mixing period for Ca Cx spin diffusion on GB1 protein run using an EFRFE_Probe DARR transfer from Cy to Cg or C generates positive cross peaks HORROR or DREAM transfer generates negative cross peaks See the chapter on spin diffu sion experiments for more information about DARR or PDSD 268 327 BRUKER BIOSPIN User Manual Version 002 Double CP F1 ppm 40 120 rg r perpe P n Am a y paa 150 100 50 H F2 ppm Figure 20 10 NC C correlation experiment with 4 2 ms SPC5 DQ mixing period for C4C spin diffusion on GB1 protein run using an EFFFF Probe at 14 kHz sample rotation and 100 kHz decoupling See the chapter on recoupling experiments for the SPC5 setup and for more infor mation about DQ recoupling sequences Note the inverse phase of the cross peaks generated by the DQ mixing step User Manual Version 002 BRUKER BIOSPIN 269 327 Double CP 270 327 BRUKER BIOSPIN User Manual Version 002 CRAMPS General 2 1 CRAMPS is an acronym standing for Combined Rotation And Multiple Pulse NMR Spectroscopy 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 homonuclear dipolar interactions between the abundant spins mostly protons and chemical shift anisotropy simultaneously through the combination of multip
94. 0 5 AIPO 14 11B 3 2 160 46 5 H3BO3 87Rb 3 2 163 61 0 5 RbNO3 99NIp 9 2 122 25 1 LiNbOs 1 In MHz at 11 7 T i e 500 13 MHz proton frequency 72 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 3 Recycle delays at 11 7 T longer delays may be required at higher fields 216 327 Fiqure 17 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 visi ble which is used as an indication that it is not excited BRUKER BIOSPIN User Manual Version 002 Basic MQ MAS 2000 1500 1000 500 0 500 1000 1500 2000 ppm e e poe p 1 yo ro e 2000 1500 1000 500 0 500 1000 1500 2000 ppm Figure 17 3 Comparison of 87Rb MAS spectra of RbNOS excited with selective and non selective pulses The lower trace is a spectrum excited with a 1 us non selective pulse correspond ing 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 17 4 shows the nuta
95. 0 pulse determ 100 scans 335 KS MAS 0 100 scans in a gt 500 MHz instr 14N NH4CI MAS WL 0 100 scans narrow line 29Mg SEH Anatas MAS 39K KCI MAS WL 0 100 scans 12 327 BRUKER BIOSPIN User Manual Version 002 Test Samples Table 2 1 Setup Samples for Different NMR Sensitive Nuclei 109Ag AgNO MAS 1scan 500s finely powdered AgSO3CH3 CPMAS 70 50 ms contact 10 s repetition 1 scan 89y Y NO3 3 6H20 CPMAS 50 10ms contact d1 gt 10s Literature J M Hook P A W Dean and L C M van Gorkom Magnetic Resonance in Chemistry 33 77 1995 User Manual Version 002 BRUKER BIOSPIN 13 327 Test Samples 14 327 BRUKER BIOSPIN User Manual Version 002 General Hardware Setup Avance instruments are constructed in a way to minimize the requirements to re connect or readjust hardware for different experiments Probe changes are how ever sometimes necessary and require some manual operations This chapter deals with connections that need to be done by the operator and also with other manipulations that are required to set up the instrument in an optimum way Since the RF pathways are under software control up to the preamplifier and un der operator control between preamplifier and probe both setups are considered separately All remaining connections heater cable thermocouple gas flow spin rate cable PICS cable can in no way be under software control so the
96. 0303 0303 1010 1010 3 0123 0321 40 0 rec 0220 0220 1331 1331 Figure 10 1 RFDR Pulse Sequence for 2D CPMAS Exchange Experiment Set up 10 2 Experiment Setup 138 327 Sample C fully labelled histidine Experiment time Less than 1 hour First setup the 1H 13C cross polarization and the Hartmann Hahn match accord ing to the procedures described in Basic Setup Procedures on page 55 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 de coupling 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 achiev able so it should be set as high as possible using a LG offset During the mixing period cpds3 cwlg as shown in Figure 10 1 the Gullion compensated echo sequence used for the mixing period is a XY 8 phase cycling fg XYXY YXYX Consequently the number of rotor periods for the mixing time L1 must be a multiple of 8 BRUKER BIOSPIN User Manual Version 002 RFDR Data Acquisition 10 3 Sample 1C fully labelled histidine Experiment time Several hours Set up 2D Experiment 10 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 appropri ate FnMode parameter in eda Pulse progr
97. 18 2001 User Manual Version 002 BRUKER BIOSPIN 91 327 Decoupling Techniques Homonuclear Decoupling 5 2 Homonuclear decoupling refers to methods which decouple dipolar interactions between like spins Those are only prominent between abundant spins like 1H 19F and 1P and potentially some others This interaction cannot easily be spun out in most cases and renders NMR parameters like chemical shifts of the homo nuclear coupled spins or heteronuclear couplings and J couplings to other X nu clei unobservable Multiple Pulse NMR Observing Chemical Shifts of Homonuclear Coupled Nuclei 5 2 1 Reference Multiple pulse NMR methods are covered in the chapters about CRAMPS of this manual collection 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 pulse sequence In this case the dipolar couplings between those spins are suppressed Short observation windows be tween pulses allow observation of the signal from the decoupled nuclei 1 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 5 2 2 92 327 Multiple Pulse Decoupling Observing dipolar couplings and j couplings to homo nuclear coupled nuclei Homonuclear couplings between abundant spins usually protons sup
98. 2 Figure 4 13 Figure 4 14 Figure 4 15 Figure 4 16 Figure 4 17 Figure 4 18 Figure 4 19 Figure 4 20 Figure 4 21 Figure 4 22 Figure 4 23 Figure 4 24 Figure 4 25 Figure 4 26 Figure 5 1 Figure 5 2 Figure 5 3 Figure 5 4 Figure 5 5 Figure 5 6 Figure 7 1 Figure 7 2 Mounting a Triple Insert Into a Triple Probe nennen 49 Example of a 600 WB NMR Instrument Gite 51 Short Display Pulse Routing Only for C N H DCP or REDOR Experiment observing 13C above ht AE 52 Long Display Pulse and Receiver Roupng nennen nn 53 Pulse on F2 Observe on F1 Routing 54 The edasp Display for a System with two Receiver Channels ssesssss 54 4 Basic Setup Procedures 55 Routing for a Simple One Channel Experiment 57 Probe Connections to the bPreamplfter este eeeeeeeeeaeeseeeaeeas 59 Pop up Window for a New Experiment 60 ased Table with Acquisition Parameters for the KBr Experiment 61 Graphical Pulse Program Display sssssss memes 62 Display Example of a Well tuned Probe e 63 Display Example of an Off Matched and Off Tuned PDrobe ees 64 Display Example Where Probe is Tuned to a Different Frequency ssss 64 FID and Spectrum of the 79Br Signal of KBr used to Adjust the Magic Angle 65 Routing for a Double Resonance Experiment using High Power Stage for H and X nu CCU ada tt EaU ANAA 66 Routing for a Double Resonance Experiment Changed fo
99. 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 States TPPI phase sensitive detection 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 draw backs 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 ex periment is performed The factors R p are listed in Table 17 5 The shift posi tions 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 cas es some enhancement of resolution can provide additional information Table 17 5 Values of R p for Various Spins and Orders p Spin R p 3 IR 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 17 226 327 The spectral width in the MQ dimension of the sheared spectrum is given by spin ning 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 th
100. 293 ExampleS cessent MR ME Ge 294 Proton Proton DQ SQ Correlation ssssssssssee emen nenne 296 Pulse Sequence Diagram ssssssssssseeeeene ene hee eene nn rhe nnne nne nnne 297 Data Processing uat eee re aee db nn dre ER e dE Pe d eve E vate Fo en d en Rea d ba 299 2c E 299 PDDONGIX dime 303 Form for Laboratory Logbooks ssssssssssssssssse ee eene hehehe nenne 303 ghi P 309 TAS e 315 lao aum EE 319 User Manual Version 002 BRUKER BIOSPIN 7 Contents 8 BRUKER BIOSPIN User Manual Version 002 Introduction This manual is intended to help the users set up a variety of different experiments that are nowadays more or less standard in solid state NMR Previously the manuals described the hardware in some detail and also basic setup procedures 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 trans mitters with higher power and preamps and probes that take this power but for the purposes of experimental setup detailed knowledge is not required since the setup does not generally depend on the details of the hardwa
101. 45 2D Experiment Setup 146 ee UI Ee 147 Processing Parameters cient bio A ir gau ae deu quu v Sav Zen eat ado 149 Adjust the Rotational Resonance Condition for DARR RAD sssss 149 Example Spectra orrena nand eaa eaa ea ee hee he nhe he nhe hne hs nsi ri hn nennen 151 REDOR me EERE 155 UE I EsI Die 157 SELUP idein dae A A A uet e te dd 157 Data Acquisition sss ee I eI en ne men nene rer ee ne ren re nen nennen 159 Data cerro 160 Final Remarks 167 SUPER e 169 OVEIVIOW 169 Pulse Program 170 2D Experiment Setup 170 Experiment Ssetup 2255400224412 at Sean sabe ae SERE An LUE ah Phe SEDE a es ne ERE EAE Sen dd 170 Setup 2D Experiment nn 171 Data eI MMC 173 spectral ProCeSSINg i con isedet beds een do Eed di an A 174 Symmetry Based Recoupling leer eee ann nn ann nnnn anne 179 Pulse Sequence Diagram Example CT 181 TEE 181 Spectrometer Setup for 13 183 Setup for the Recoupling Experiment ssssssssssss nennen nenne nennen nenn 183 Setup of the 2D SQ DQ Correlation Experiment sssssssssss nennen 185 R I Ree UELL 186 Spectral Processing te ala nt a
102. 5 General Remarks bit etr e ei ie ee dte Haba dern 56 Setting the Magic Angle on KBr rerne r eeart ETE aeara EATER ERRAKETA emen nennen nen nene nenne 57 RF Routing oie do et a A A Bee dee 57 Setting Acquisition Parameters sssssssssssssn eene eene ehe nene nennen nnns 59 Calibrating 1H Pulses on Adamantane sssssssssssse meme nennen 65 Calibrating 13C Pulses on Adamantane and Shimming the Probe en 73 Calibrating Chemical Shifts on AdaMantane nenn 75 Setting Up for Cross Polarization on Adamantane 76 Cross Polarization Setup and Optimization for a Real Solid Glycine 79 Some Practical Hints for CPMAS Spectroscopy ssssssss e 85 Field Setting and Shift Calibration 87 Etera S Sege ne nee 88 Decoupling Techniques u iin uoti AA ds 89 Heteronuclear Decoupling 2 2 5 rn un ng 89 User Manual Version 002 BRUKER BIOSPIN 3 Contents 5 1 1 5 1 2 5 1 3 5 1 4 5 1 5 5 1 6 5 2 5 2 1 5 2 2 5 3 6 1 6 2 6 3 6 4 6 5 7 1 7 2 7 3 7 4 7 5 8 1 8 2 8 3 9 1 9 2 9 2 1 9 2 2 9 2 3 9 2 4 9 3 10 10 1 10 2 10 3 10 3 1 10 4 CW Decoupling 89 TPPM Decoupling u na ee nn nn diia 90 SPINAL Decoupling ssssssse HH meme hen eren emen mene men rennes 91 Swept Frequency TPPM nennen itn hi hh etienne nnns 91 KIX Decoupling Em 91 Pi P lse Dec upl
103. 6 4s for the carbonyl and alpha carbon signals respectively at other field strengths the numbers will be somewhat different If the signals are really un dergoing mono exponential relaxation the curve should be a good fit to the mea sured data BRUKER BIOSPIN 207 327 Relaxation Measurements The Saturation Recovery Experiment 16 2 4 Experiment setup For samples where cross polarization is not possible the inversion recovery ex periment would be very time consuming as the recycle delay d1 would need to be approximately 3x the longest T4 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 forc ing the system into saturation at the beginning of each scan Sample Glycine Spinning speed 10 kHz Time 20 minutes 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 sample this can be left blank to turn off the decoupling Set p1 and pl1 to the measured carbon 90 degree pulse parameters as used in the CP T1 experiment or see chapter Basic Setup Procedures Set d1 to a rela tively long value for the preparation experiments Set the number of pulses in the saturation train 20 to zero and acquire a spec trum This will give an idea of the amount of signal and thus
104. 89 24 CRAMPS 2D 291 Table 24 1 Acquisition Parameters cece eeceeceece eee eee eeeeeseesseesesaeeeaeeeeeeneeees 292 Table 24 2 Phases RF levels and TiMiNgS cooocooccccconcconccncconccnnconcncnconcnoncnnnnonnnnnnnnnnnaninanonannns 293 Table 24 3 Processing Parameters cccccceccececeeeece essen ence eeseeeeeeeeecue ANEREN TEATAN ETERNE EErEE 293 Table 24 4 Acquisition Parameters cece cece eece eee cece eseeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeaes 298 Table 24 5 Phases RF Levels and Timing 298 Table 24 6 Processing ParamMeters cccccnccetiscenetectetenteteneseatecsaneaseesandcaabedebhedanedeabeeeenseeneues 299 A Appendix 303 User Manual Version 002 BRUKER BIOSPIN 317 327 Tables 318 327 BRUKER BIOSPIN User Manual Version 002 Index Symbols O A ee Ee 12 d elei e uie eor te aet eere casio reete Peor beet 11 Numerics 136 Matchirig BOX n T nter tnde te doeet nen ete 59 1H HP Preamplifier Aa aaa ra a pa aa oa in be AaS 59 Te ET 65 QO 0 U 12 HP 55 A Adamantane ito ER Rubin 11 12 PES 103 el e M 13 AIPO 14 EE 11 analog ue 277 DUC se EE 12 AS ale e Un Le TTT 11 ASO M A A N RE 60 AU program DUMBO enn 97 B BOEl RE 82 background sigrial cs odo s or uei Mu 100 background suppressiOH a nasend te Poche tree etra ipea 100 e GETT 279 Basic Setup Procedures roe a e a a e aa 55 EE 93 BRA H 58 B 92 BN ten diete ett e m
105. 9 BIETET 10 Salety ISSUCS D M 10 Contact for Additional Technical Assistance sssssssssse mn 10 Test Dg aee nennen 11 General Hardware Setup ee ERR EEER EEN EE REENEN REESEN RENE 15 Connections to the Preamplifier ssssssssssssssssssssese me emen nnn 15 RF Connections Between Preamplifier and Probe c cccceccceeceeeceeeeeeeeeeeeeeeeeeeeaes 20 RF Filters in the RF Pathway ssssssss meme hehe mre ren rennes 21 Connections for Probe Identification and Spin Detection sssessessssssss 25 MAS T bing CONNECHIONS c resort de hotte rende a a Tae ee ENNER dE ERE aae idee 26 Connections i ganda lle geb ea Ia dile dan Peete eb riy bien oe Muay ba del 27 Wide Bore WB Magnet Probes sss He eren rennes 28 Standard Bore SB Magnet Probes 30 Additional Connections for VT Operation ssssssssssssssss mee 31 Probe Setup Operations Probe Modifiers o ooccocccnconocnconocnonnoconnnonennnnnononnnnnnnnnanonos 41 Setting the Frequency Range of a Wideline single frequency Probe 41 Shifting the Probe Tuning Range 42 Adding a Frequency Channel to a Probe WB probes only sseseeee 48 Mounting the Probe in the Magnet Shim Stack sssssssse nenn nennen 50 EDASP Display Software Controlled Routing sss 51 Basic Setup Procedures iine riot tee 5
106. AcquPars Tite Pulsc rog Peaia integrats Sample Structure Fid Acqu Phase Print poptau for pi Imisheu POSMAX af experiment 12 p 6 0000 NEXP 20 6 00 usec 72 327 13142 d SARNA MA MMM 4 6 9 us ec Figure 4 17 The popt Display after Proton p1 Optimization The figure above shows the popt display after proton p1 optimization the biggest signal is obtained at 6 usec in this case Once you have obtained a 90 degree pulse for a given power setting you can cal culate power levels for different rf fields using the AU program calcpowlev Type xau calcpowlev into the command line and follow the instructions 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 Set the new pl1 as calculated and check whether 2 4 5 usec for pT will give a close to zero signal This is a safe power level for all probes for pulses up to 100 msec length BRUKER BIOSPIN User Manual Version 002 Basic Setup Procedures Calibrating 13C Pulses on Adamantane and Shimming the Probe 4 4 A high power decoupling experiment on 13C of adamantane is used to measure Re pulse parameters d NOTE For experiments where long decoupling pulses on protons are executed the proton preamplifier must be bypassed i e the transmi
107. Based Recoupling Table 14 2 Acquisition parameters for DQ SQ correlation experiments using symmetry based recou pling sequences 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 1H 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 18 CP Step e g ramp 80 10096 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 see para 17 Sweep width indirect dimension above NDO 1 STATES TPPI not required In TS 2 1 User Manual Version 002 BRUKER BIOSPIN 187 327 Symmetry Based Recoupling Spectral Processing Processing Parameters 14 4 Table 14 3 Processing parameters for DQ SQ correlation
108. Be 1 3 ds heteronuclear decoupling 2 2 Prec Figure 7 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 002 Basic CP MAS Experiments Solis Manual chapter2 13 1 C Jos 1206 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 Solids Marual_cheptere 14 1 Can 3951205 Solids Marval chaprerz 13 TE 73021208 alo ll a SS 100 50 9 Figure 7 13 CPMAS Spectrum of Tyrosine HCl at 6 5 kHz CPMAS spectrum of tyrosine HCl at 6 5 kHz sample rotation obtained on a 500 WB spectrometer using a 4 mm CPMAS double resonance probe The red third spectrum is a CPPI spectrum where we see the CH resonance at 35 ppm with a negative intensity The aromatic CH resonances are clearly suppressed where the C4 shows a slightly negative intensity The polarization inversion pulse p16 was 40 us long The green second spectrum is a CPPIRCP experiment 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 CPPISPI 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
109. Bruker BioSpin Cas a al Ge S 3 M 4 56 e Solid State NMR AVANCE Solids User Manual Version 002 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 Benevelli Peter Gierth and Sebastian Wegner Desktop Published by Stanley J Niles May 25 2009 Bruker Biospin GmbH Rheinstetten Germany P N Z31848 DWG Nr Z4D10641 002 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 76287 Rheinstetten Germany Phone 49 72151610 FAX 49 721 5171 01 E mail solids bruker de service bruker de Internet www bruker com Contents 3 1 3 2 3 3 3 4 3 5 3 5 1 3 6 3 7 3 7 1 3 7 2 3 7 3 3 8 3 9 4 1 4 2 4 2 1 4 2 2 4 3 4 4 4 5 4 6 4 7 4 8 4 9 4 10 5 1 GONTENIS nt EE EE 3 let E
110. C CPSPINDIFF of fully labeled tyrosine HCl spinning at 22 kHz 4 6 msec mix Upper PDSD lower DARR User Manual Version 002 BRUKER BIOSPIN 151 327 Proton Driven Spin Diffusion PDSD row 820 from spindiff 10 1 C Bruken TOPSPIN manual F spintire 13 520 Cs Breuer TOFSP2 nenas MY 7 Scale 0 7387 T Bsinsitt 12 Sep Cs Beukec TORSPIa ganz scale 0 8651 f spindif 11 520 C BrukeriTOPSPIN wall 8 Scale 1 101 d N spindiff 30 520 C Beukari TOPSPIN mal an Pa e A A JAN ec XJ v T 10 160 140 120 100 80 ppm Figure 11 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 carbon 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 152 327 BRUKER BIOSPIN User Manual Version 002 Proton Driven Spin Diffusion PDSD efree 4mm rOO SB DARR C C correlation d mser mix 100 rotor penods EW 20 F1 ppm 10 10 X BE hu O A T T T T T T T T I T T T T T T 150 100 50 F2 ppm Figure 11 6 13C DARR of Fully Labelled Ubiquitine Spinning at 13 kHz User Manual Version 002 BRUKER BIOSPIN 153 327 Proton Driven Spin Diffusion PDSD 154 327 BRUKER BIOSPIN User Manual Version 002 REDOR
111. CPMAS experiments with the excep tion 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 ap propriate for lower spinning speeds TOSS B for higher spinning speeds is se lected by setting ZGOPTNS to Dtossb The maximum spinning speed is either determined by common sense if all sidebands are spun out TOSS is not need ed 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 002 Basic CP MAS Experiments 3 heteronuclear decouplin 1H 2 bs ds d 6s Prec Figure 7 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 Progress in Nuclear Magnetic Resonance Spectroscopy 35 1999 203 266 The SELTICS sequence 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
112. EMA References 1 C H Wu A Ramamoorthy and S J Opella High Resolution Heteronuclear 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 heteronuclear 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 15 1 a i 3 44 3 2 ds TPPM decoupling 2 63 7 6 Prec b TPPM decoupling 06 6 Pre Figure 15 1 Pisema Pulse Sequence 194 327 BRUKER BIOSPIN User Manual Version 002 Setup PISEMA Figure 15 1 Pisema pulse sequence a straight PISEMA b clean PISEMA variation for further suppression of phase glitches Ramamoorthy et al Solid State NMR 4 15 2 Sample 15N labelled a glycine powder for power level determination 15N la belled acetylated glycine or acetylated valine or leucine for running the PISEMA experiment preferably as single crystal Setup time 0 5 h on labelled glycine Experiment time 15h on a labelled powder sample 1 2 h on
113. F1 indirect C Right column TD 32 64 Number of real points FnMode TPPI STATES or STATES TPPI User Manual Version 002 BRUKER BIOSPIN 173 327 SUPER Spectral Processing 13 5 Table 13 2 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 174 327 BRUKER BIOSPIN User Manual Version 002 SUPER arso M 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 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 PTT Praeeaxing PATA TUM inten 128 3478472 Max X H Yea PL 180 160 140 120 100 80 60 40 20 ppm Figure 13 4 The SUPER Spectrum of Tyrosine HCI After
114. FSLG evolution Cnst24 is used to separate the proton spectrum from this ridge The contact time of 300 usec shows many long range couplings The next figure shows the region of interest excluding the center ridge and the spin ning sidebands User Manual Version 002 BRUKER BIOSPIN 125 327 FSLG HETCOR F1 ppm 4 10 12 180 160 140 120 100 80 F2 ppm Figure 8 5 FSLG Hetcor Spectrum Tyrosine HCI The figure above shows a FSLG Hetcor Spectrum Tyrosine HCI with parameters as shown in Table 8 1 Table 8 2 Full transform with slight resolution enhance ment qsine SSB 3 Proton shifts calibrated as 2 5 and 12 most high field low field peak Expansion plot 126 327 BRUKER BIOSPIN User Manual Version 002 Modifications of FSLG HETCOR The basic HETCOR sequence can be improved in several respects The protons which are observed are all coupled to 13C carbons since we observe these So the proton shifts evolve also under the residual dipolar coupling and the J cou pling 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 observation HETCOR with the proton spectrum obtained by CRAMPS techniques see chapters 22 23 and 24 observing the protons directly Usually these experiments use phase modulated shapes PMLG or DUMBO In order to make both ex
115. Fig ure 4 14 Figure 4 15 for the peak at 43 ppm and start popt optimizing 02 for maximum signal 2000 Hz around the current position in steps of 500 Hz The following result will be obtained forse rt 14 TY File Edi View Spectromeler Processing Analysis 2 50536m0m63 54 17 u B r aQ TTOLisx mH ZREMPCEMARAQH HOH GH oz e wy ta Spechum Preis Ar Pan Tee Pusero Peas negras Sarge Zut Mid Acqu Phase Print HA poptau toro mister POSGMAX at experiment 5 02 4000 0000 NEXP 9 I eden page ome t nd d Figure 4 24 Optimization of the Decoupler Offset o2 at Moderate Power Using cw Decoupling Since the proton spectrum of glycine extends around 5 ppm the optimum decou pler offset will be obtained at higher frequency than the adamantane proton peak around 1 2 ppm Decoupling 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 corre sponding to a 2 7 usec proton 90 degree pulse This can be obtained 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 cal cpowlev to calculate the required power level p 12 and set p3 to twice the ex pected proton pulse width Check with 4 scans whether a close to zero signal is obtained Compared to 4 5
116. MAS experiment with the same number of scans using dual display The intensity ratio of the aliphatic resonance of the CPMAS compared to the one obtained with the DCP experiment gives the DCP yield see Figure 20 7 Ek n t tj L lE E Eil 7 e tse A E D A a O a VE RE ee probe 20 1 Cridato 00 3020107 cala 0 2026 For art 22 1 Cridate700 32920107 Figure 20 7 Double CP yield measured by comparing CPMAS and DCP ampli tudes of the high field resonance Note that the C4 carbon receives very little magnetization under these conditions the transfer is rather selective User Manual Version 002 BRUKER BIOSPIN 263 327 Double CP The setup for DCP can be rather much sped up and simplified by a python pro gram named dcpset py This program will ask for the 90 pulse widths and associ ated power levels and for the spin rate and calculate the appropriate power levels for the HH condition for all pulses except 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 level script language Setup of the 2D Double CP Experiment 20 2 4 264 327 1 Load a suitable sample spin it up set the desired temperature and match and tune the probe As a simple setup sample full 13C 15N histidine may be used d1 10s 2 4 scans p15 1msec p16 3msec A labeled o
117. Processing Using xfb User Manual Version 002 BRUKER BIOSPIN 175 327 SUPER 100 190 180 170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 10 ppm Figure 13 5 SUPER spectrum after tilting the spectrum setting 1 alpha 1 The figure above is a SUPER spectrum after tilting the spectrum setting 1 alpha 1 and using the command ptilt1 repeatedly until the CSA lines are within the spectral range 176 327 BRUKER BIOSPIN User Manual Version 002 SUPER Figure 13 6 Various Cross Sections from the Upper 2D Experiment The figure above illustrates various cross sections from the upper 2D experiment from which CSA parameters can be determined User Manual Version 002 BRUKER BIOSPIN 177 327 SUPER 178 327 BRUKER BIOSPIN User Manual Version 002 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 aniso tropic interactions like e g dipolar coupling in order to regain specific informa tion 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 sequences see M Hohwy et al 1998 et al and A Brinkmann et al 2000 In these se quences double quantum coherence are excited via the dipolar homonuclear
118. R 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 cor rectly calibrated if the magnetic field Bg 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 recorded in the lab notebook One can also use a spinner filled with H2O to set the field position more precisely Do not spin the sample and make sure the cap is well fitted Set o7p to 4 85 ppm Set for proton observe as described above for adamantane and use gs for con tinuous pulsing and FID display Change the field value in bsmsdisp until the FID is exactly on resonance Then all spectra taken should be correctly referenced with sr 0 For more information on correct field setting and shift calibration see Field Setting and Shift Calibration on page 87 For all these experiments 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 spectra at different fields One can set the sweep am plitude to O in order to avoid suc
119. Refer to chapter Basic Setup Procedures on page 55 for more informa tion User Manual Version 002 BRUKER BIOSPIN 149 327 Proton Driven Spin Diffusion PDSD pectrum Procpars Acquears Tite Puleerog Peak integrais Sample Structure Fit Acqu Figure 11 3 POPT Result for the cw Decoupling Power Variation The figure above shows the POPT result for the cw decoupling power vari ation from about 50 kHz RF field to about 5 kHz RF field spinning the ada mantane sample at 13 kHz The minima at 14 5 and 20 5 dB indicate the n 2and n 1 RR conditions 26 and 13 kHz RF field 4 Vary the decoupler power level p112 used with cw decoupling as indicated in Fiqure 11 3 from a power level value pl12 1 dB below the calculated n 1 condition to 1 dB above the calculated n 2 condition Bandwidth consider ations favor the n 2 condition sample heating 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 pl14 recoupling power for DARR or RAD 6 Using DARR or RAD shorter mixing times are possible 150 327 BRUKER BIOSPIN User Manual Version 002 Example Spectra LLL dL Proton Driven Spin Diffusion PDSD 160 440 120 400 e x m au MI 160 140 120 100 80 60 F2 ppm TT T 77 71 7777 140 120 T T 160 140 120 190 80 ep Ei ppm 160 Figure 11 4 13
120. Rotational Echo Double Resonance NMR Conc Magn Reson 10 277 289 1998 T Gullion Measurement of dipolar interactions between spin 1 2 and quadrupolar nuclei by rotational echo adiabatic passage double resonance NMR Chem Phys Lett 246 325 330 1995 E R H van Eck R Janssen W E J R Maas and W Veeman A novel application of nuclear spin echo double resonance to alumino phosphates and alumino silicates Chem Phys Lett 174 428 432 1990 K T Mueller Analytic solutions for the Time Evolution of Dipolar Dephasing NMR Signals Journal of magnetic resonance A 113 81 93 1995 T Echelmeyer L van W llen and S Wegner A new application for an old concept Constant time CT REDOR for an accurate determination of second moments in multiple spin systems with strong heteronuclear dipolar couplings Solid state nuclear magnetic resonance 34 14 19 2008 M Bak J T Rasmussen N C Nielsen Journal of magnetic resonance 147 296 330 2000 G L Perlovich I K Hansen and A Bauer Brandl The polymorphism of glycine Journal of Thermal Analysis and Calorimetry 66 699 715 2001 M Bertmer H Eckert Dephasing of spin echoes by multiple heteronuclear dipolar interactions in rota tional echo double resonance NMR experiments Solid State Nuclear Magnetic Resonance 15 139 152 1999 BRUKER BIOSPIN User Manual Version 002 SUPEH Overview 13 1 Separation of Undistorted Chemical Shift Anisotrop
121. Rotational Echo DOuble Resonance is an experiment based on the heteronuclear dipolar coupling between the observed nuclei The REDOR sequence investi gates this coupling under high resolution MAS conditions Dipolar couplings between decoupled spin and S observed spins are spun out under MAS if there are no strong homonuclear interactions and if the hetero nuclear coupling is not too big In the case of couplings between most hetero nu clei like 13C 15N and 295i this is usually the case small couplings of a few kHz and dilute spins if the coupled nucleus is 31p 19F or even 1H the coupling may not easily be spun out and the standard REDOR sequence may not be applicable in these cases The REDOR sequence reintroduces the heteronuclear dipolar coupling between the spin S and by applying p pulses every half of a rotor period on the second channel I while the S channel is observed A p pulse at half a rotor period will re focus the dipolar interaction averaged by spinning and dephase the magnetiza tion leading to an attenuation of the observed signal Evaluation requires the acquisition of 2 data sets one with refocusing pulse the other one without so that the natural dephasing can be subtracted out from the dipolar dephasing due to the refocusing pulse Reference experiment without refocusing pulse and de phased experiment with refocusing pulse are subtracted and evaluated Refer ence experiment and dephased experiment
122. SLG decoupling at pI13 6 Set pl13 to 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 lg calc incl which calculates the required frequency shifts to either side shown in ased as cnst22 and cnst23 Cnst24 provides 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 5 1 and Table A spectrum like in fig 4 should be obtained If the splitting is worse optimize with pl13 and cnst24 Usually somewhat less power than calculated is required The FSLG decoupling scheme is also implemented as cpd program cwlgs The include file Igcalc 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 5 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 ramp70100 100 For ramped CP pl12 p3 set for p3 90 SpO 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 User Manual Version 002 BRUKER BIOSPIN 95 327 Decoupling Techniques T
123. Sample Structure Fidi Phase Calibrate Baseline Figure 5 1 Optimization of TPPM Decoupling on Glycine at Natural Abundance The figure above shows optimization of TPPM decoupling on glycine at natural abundance C CPMAS at 5 kHz spin rate Each block represents a 2 degree in crement of the phase toggle and the variation in each block stems from incremen tation of the pulse width in 0 2 uis increments Optimum decoupling was found with a 4 5 us pulse at a 16 phase toggle It is obvious that more than one near opti mum combinations of phase toggle and pulse length exist Reference 1 A E Bennett C M Rienstra M Auger K V Lakshmi and R G Griffin Heteronuclear decoupling in ro tating solids J Chem Phys 103 16 6951 6958 1995 90 327 BRUKER BIOSPIN User Manual Version 002 SPINAL Decoupling Decoupling Techniques 5 1 3 Reference SPINAL provides adequate decoupling bandwidth even for high field 2400 MHz instruments at an RF level of 80 kHz or higher SPINAL 64 64 phase permutati ons outperforms TPPM and may be used as standard decoupling sequence SPI NAL 64 can be optimized in the same way as TPPM by incrementing pcpd2 p31 the phase shifts are fixed The decoupling pulse is an approximate 180 pulse 1 B M Fung A K Khitrin K Ermolaev J Magn Reson 142 97 101 2000 Swept Frequency TPPM 5 1 4 Reference This decoupling method combines TPPM and a frequ
124. Setup Procedures Literature 4 10 Shift referencing 1 3 4 5 R K Harris E D Becker S M Cabral de Menezes R Goodfellow and P Granger NMR Nomencla ture Nuclear Spin Properties and conventions for Chemical shifts Pure Appl Chem Vol 73 1795 1818 2001 W L Earl and D L VanderHart Measurement of 13C 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 html Cross polarization 1 88 327 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 Spinning NMR J Magn Reson A 110 219 227 1994 B H Meier Cross Polarization under fast magic angle spinning thermodynamical considerations Chem Phys Lett 188 201 207 1992 K Schmidt Rohr and H W Spiess Multidimensional Solid State NMR and Polymers Academic Press 1994 S Hediger B H Meier R R Ernst Adiabatic passage Hartmann Hahn cross polarization in NMR un der magic angle sample spinning Chem Phys Lett 240 449 456 1995 BRUKER BIOSPIN User Manual Version 002 Decoupling Techniques Line shapes in solids are often broadened by dipolar
125. Table 7 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 002 BRUKER BIOSPIN 107 327 Basic CP MAS Experiments class AVANCE 10 1 C josf005 lt Rosi L E E ES 95 35 6 A class AVANCE 7 Seale 6 02 Class AVANCE 108 327 L 3026905 Figure 7 3 Comparison of a CPTOSS and CPMAS Experiment Fiqure 7 3 compares a CPTOSS experiment lower spectrum to a CPMAS ex periment 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 per fect suppression 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 suppres sion using the pulse program cptoss243 where the extension av is added in case of the AV2 console Figure 7 4 shows the advantage of the well compensat ed TOSS sequence with its 243 phase cycle steps over the above 4 pulse se quence 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 s
126. Using Shapes edumbohet 9 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 Faster spinning is possible with higher power pulses and shorter pulse widths 24 usec or 16 usec The library of AU programs in TopSpin includes dumbo which calculates the desired shapes for windowed and window less 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 appropriate for an RF field of 100 kHz Spnam2 is set to edumbo22 1 0 Table 9 4 Acquisition Parameters for e DUMBO HETCOR on tyrosine HCl Parameter Value Comments pulprog edumbohet windowless dumbo shape nuc1 13C o1p 100 ppm nuc2 1H 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 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 spn
127. WH masr Equals spinning frequency for rotor synchronization from this IN 010 is calculated correctly if ND 010 is already set NUC1 Select the same nucleus as for F2 so that transmitter fre quency offset is correctly set important for referencing D10 0 Used in mp3qZfil av only IN10 in0 7 9 Used in mp3gzfil av for nuclei with spin I 3 2 only so that no shearing FT is required 220 327 Note the difference in increment handling in Topspin 2 1 and higher BRUKER BIOSPIN User Manual Version 002 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 processing If the pulse program mp3qZfil av is used no shearing is required in case of nuclei with spin I 3 2 if in 10 is set correctly in which case a split t4 experi ment is performed td determines the number of FID s to be accumulated in the in direct dimension This value is determined by the line width and resolution that can be expected in the indirect MQ dimension F1 and which depend on the pro perties of the sample In crystalline material fairly narrow peaks can be expected so a maximum acquisition time in F1 of 2 to 5 ms is expected In disordered mate rials 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 fd 2 i
128. XY 8 echo se quence in order to achieve efficient recovery of single spin magnetization and to generate an effective dipolar recoupling Hamiltonian during the mixing period The critical experimental point is to avoid H X recoupling induced by interference between the H 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 1 T Gullion D B Baker and M S Conradi New compensated Carr Purcell sequences J Magn Reson 89 479 484 1990 2 A E Bennett J H Ok R G Griffin and S Vega Chemical shift correlation spectroscopy in rotating solids Radio frequency driven dipolar recoupling and longitudinal exchange J Chem Phys 96 8624 8627 1992 3 A E Bennett C M Rienstra J M Griffith s W Zhen P T Lansbury and R G Griffin Homonuclear radio frequency driven recoupling in rotating solids J Chem Phys 108 9463 9479 1998 4 B Heise J Leppert O Ohlenschl ger M G rlach and R Ramachandran Chemical shift correlation via RFDR elimination of resonance offset effects J Biomol NMR 24 237 243 2002 User Manual Version 002 BRUKER BIOSPIN 137 327 RFDR Experiment 10 1 Spinal64 Lee Goldburg Spinal64 decoupling decoupling decoupling t N TR t 471 4 0101 1010 bo 71 de
129. a available C poser os i J Switch EE Add a logical charra Remove a logical channel Default Info Panes Close Figure 4 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 d7 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 chan nel to the proton preamp A proton band pass filter must be inserted between pre amp 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 120 Alternatively you can switch to the lower frequency channel within wobb high by clicking on the fre quency 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 002 BRUKER BIOSPIN 67 327 Basic Setup Procedures protons of adamantane rei 15 10 so 40 20 o 20 T Figure 4 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 68 327 BRUKER
130. able 5 1 Acquisition Parameters cnst24 0 To be optimized cnst21 0 Reset proton frequency to SFO2 Table 5 2 Processing Parameters Parameter Value Comment SI 2 td Adequate 4fold zero filling WDW no No apodization PH mod pk Phase correction if needed BC mod quad DC offset correction 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 visualized The frequency shift which needs to be achieved is RF field 2 Since the pulse duration must achieve a 2 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 required to achieve this 1 1 1 1 1 1 1 2 4 1 4 1 e i 1 wo ot ZG ove Save E n vs he Figure 5 5 Shape with Phase Gradients In the figure above Shape with phase gradients for positive and negative offsets and corresponding phase change stdisp display of Igs 1 shape Amplitude is 10096 throughout 96 327 BRUKER BIOSPIN User Manual Version 002 Decoupling Techniques 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 i
131. al Version 002 Basic MQ MAS Obtaining Information from Spectra 17 5 The referencing procedure in xfshear defines the axis in the MQ dimension such that Eq 17 2 Ovo Oso m 17 The value of ogi is given by Eq 17 3 A 02 195 41QI DY 3 In Eq 17 3 I is the spin quantum number Q the quadrupolar coupling con stant g the Larmor frequency and h the asymmetry parameter This makes yg u ag which causes the MQ positions to be field dependent An interesting be havior results as one compares spectra at different fields Plots of the function mg over og are shown in Figure 17 9 for an arbitrary sample 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 op posite case of identical quadrupole couplings separation increases as the field is increased plot B In cases where a difference in isotropic chemical shift jso ex ists and the sites have different quadrupole couplings the relative positions de pend 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 Bo is altered plot C D Figure 17 9 Calculated Shift Positions yg Calculated shift positions yco as function of the static ma
132. al filter applied Figure 22 5 Digital Sampling Scheme 278 327 BRUKER BIOSPIN User Manual Version 002 Setup CRAMPS 1D 22 4 At frequencies of 400 MHz and higher double or triple resonance CP MAS probes may be used on the proton channel at lower fields a CRAMPS probe is re quired 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 imped ance matching between probe and transmitter is important in order to optimise the effect of the pulses on the spins If the 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 precisely set which can easily be achieved with KBr on a double resonance probe or on BaClO3 H20 looking at the proton signal much like one does on the Br 79 resonance Shimming will also be important since protons are observed and on some sam ples good resolution is expected Looking at the protons in adamantane find th
133. al n phase alternation for every second pulse so that 14 pulses are executed during 2 rotor periods requiring Vpr 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 times are required 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 in formation 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 diffi cult to meet It has also been shown that at very high spin rates gt 16 kHz decou pling 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 homonuclear couplings are suppressed User Manual Version 002 BRUKER BIOSPIN 179 327 Symmetry Based Recoupling 180 327 Another important parameter to observe is the required excitation bandwidth of these sequences Naturally going to higher magnetic fields the higher chemical shift spread requires higher RF fields for the recoupled X nuclei requiring ev
134. al shift difference between the sites to be correlated M Ernst et al PDSD is typically applied to high abundance nuclei or labeled materials to detect through space proximity between spins This exper iment has been often used on proteins as an alternative to Radio Frequency DRiven spin diffusion see REDR on page 137 RFDR provides similar infor mation to PDSD but with a different mixing period Here the term frequency driv User Manual Version 002 BRUKER BIOSPIN 143 327 Proton Driven Spin Diffusion PDSD 144 327 1 10 en 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 requiring long spin diffusion times can be observed However the information from this experiment may be ambigu ous because a rather non selective transfer within the proton spin system is uti lized Nevertheless even complex molecules like proteins can be surprisingly well char acterized 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 s
135. alized signal intensities of about 0 2 0 3 for details see e g reference 10 In the case of very strong dipolar couplings this approach may be restricted to very high MAS spinning speeds because otherwise it will not be possible to get enough data points within the 0 3 area of the curve it here may be useful to use a more efficient REDOR technique for strong dipolar coupled systems like CT RE DOR see e g reference 7 User Manual Version 002 BRUKER BIOSPIN 163 327 REDOR 164 327 ASIS 0 8 0 6 0 4 0 2 0 0 0 000 e Experimental Data Simulation with ideal pulses Simulation with 10deg pulse error 0 002 0 004 0 006 0 008 0 010 0 012 evolution time s Figure 12 7 Experimental data for the glycine 13 15N REDOR Red and green curves are the results of different Simpson simulations Finally the complete REDOR curve can be simulated using the SIMPSON refer ence 8 NMR simulation package The following pages will explain the M ap proach as well as the SIMPSON interpretation of the glycine REDOR data Figure 12 7 shows the experimental data points together with two different SIMPSON sim ulations for details of the geometry and distance information of the labelled 15N 16 spin pair of glycine see reference 9 The red simulation shows the time de pendent evolution assuming ideal p pulse lengths on both the S and channel corresponding to an experiment without any errors on both frequency ch
136. am parameters are indicated below Figure 10 1 shows the pulse sequence ocPars cquPars Title Pu gESVYGg4 Figure 10 2 The 123 Icon in the Menu Bar of the Data Windows Acquisition Pa rameter Page The 123 icon in the menu bar of the data windows acquisition parameter page see Figure 10 2 is used to toggle to the different data acquisition modes 1D 2D and 3D if so desired User Manual Version 002 BRUKER BIOSPIN 139 327 RFDR Table 10 1 Acquisition Parameters Parameter Value Comments pulse program cprfdr av Pulse program nuc1 TE 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 pl2 Power level for contact time on f2 channel pl12 Power level decoupling f2 channel and excitation 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 Su t1 evolution period d1 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 G
137. am2 edumbo22 1 40 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 e DUMBO period increment per row in_f1 in0 as calculated Set according to value calculated by ased User Manual Version 002 BRUKER BIOSPIN 133 327 Modifications of FSLG HETCOR Table 9 4 Acquisition Parameters for e DUMBO HETCOR on tyrosine HCl F2 13C acquisition di 2s Recycle delay sw 310 ppm Sweep width direct dimension aq 16 20 msec masr 12000 13000 dumbohet 9 2 4 This is the windowed version of the previous experiment analogous to wpmlghet 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 experiment 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 resolution 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 experi m
138. ame approach has been used to compare different states of a protein i e bound to a membrane 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 unspecific spin diffusion within the proton spin system is filtered through a double quantum selection Lange et al References 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 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 Matthias Ernst and Beat H Meier Spin Diffusion in Isao Ando and Tetsuo Asakura Eds Solid State NMR of Polymers pp 83 122 Elsevier Science Publisher 1998 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 K Takegoshi Shinji Nakamura and TakehikoTerao 13c 1 dipolar assisted rotational resonance in magic angle spinning NMR Chem Phys Lett 2001 344 631 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 spect
139. ancement 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 5 266 327 BRUKER BIOSPIN User Manual Version 002 Double CP Example Spectra 20 5 13C 15N correlation by double CP 1H gt 15N gt 13C sample histiding U 13C 15N spinning speed 13 kHz 2 4 mm 400 MHZ MB triple resonance probe In H C N mode 1H gt 15N CP at 38 KHz 15N 2 ms ri 15N gt 13C CP ai 35 kHZ for ASN and 22 kHz for 13C 9 ms ramp shape pulse for 1H tangential pulse for 15N e deceupliag SPINAL64 at 100 kHz 2 Gus 34 ms a o A o e T o fei a s o e CN m 200 150 100 50 F2 ppm Figure 20 8 C N correlation via Double CP in histidine simple setup sample 4mm Triple H C N Probe User Manual Version 002 BRUKER BIOSPIN 267 327 Double CP d i N MI AM i A a e See ea VN N Ldba aM Y DI CP NCC 22 ms DARR HE d iz someto solel a amp i ynaisacd pres ffe K i f ann L Di o H lt Ba e EE A i i i ez f a E o t V mL e 8 4 i m Ra e S Ia PL vk tt 4 4 e 4 ep Y y H tl 8 l u 4 D 4 i M N d fr f d 3 98 WM ec Y ail Ge 1 a T d t V V 8 c U i Le J Lu t T T T T T T T r r 7 T Y Y r Y 150 100 50 F2 ppm Figure 20 9 NC C correlation experiment
140. annels for IDN and 13C leading to a slightly too high theoretical REDOR curve compared to the actual experiment The green curve shows the same simulation assuming pulse errors of 10 on both channels corresponding very well with the experi mental data BRUKER BIOSPIN User Manual Version 002 Setup ASIS Experimental Data Simulation with ideal pulses Simulation with 10deg pulse error Simulation with D 850Hz 0 000 0 002 0 004 0 006 evolution time s Figure 12 8 Comparison of Experimental Data to a Simulation with Reduced Di polar Coupling Figure 12 8 shows a zoomed view of the beginning REDOR curve for the glycine sample The third simulated line broken black line shows a SIMPSON simulation with ideal pulses on both channels but varying the dipolar coupling between and S in order to fit to the experiment The simulation using a dipolar coupling con stant of 850 Hz fits the experimental data points very well This coupling can be transformed into a distance between 15N and 13C of about 1 53 A which is com pared to the theoretical value of 1 47 964 Hz an error within 1096 As you can see in an unknown spin system the interpretation by using SIMPSON will always suffer from the fact that a non optimal setup of the experiment will introduce the same error like a reduced dipolar coupling between the analyzed spins these two effects can not be easily separated from each other during the interpretati
141. arameter 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 SI 256 Sufficient in most cases WDW no Don t use window function QSINE Only use if FID in F1 is truncated SSB 2 1 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 User Manual Version 002 Data obtained with mp3gdfsz 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 N10 appropriately The informa tion 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 213 for further details regarding the shearing transformation and the information ob ta
142. ase correction Implementation of DFS into MQMAS Experiments 18 2 Two pulse sequences are available to implement a double frequency sweep DFS Figure 18 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 213 with a CW pulse instead of a DFS for conversion Figure 18 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 ob servable 3Q coherency to observable SQ coherency In the 4 pulse sequence they pass through a z filter by a sequence 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 18 2 1 At this point it is assumed that the pulses p1 P3 and P4 together with their corre sponding power levels PL 11 and PL21 are already calibrated as described in the chapter Basic MQ MAS on page 213 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 002 BRUKER BIOSPIN 233 327 MQ MAS Sensitivity Enhancement hi ph2 sp1
143. at 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 adaman tane previously will look like in Figure 4 23 far from optimum le iiuker IOPSPRN 2 1 b 14 on POF as File Edil View Specirometer Processing Analysis Options Window Help 060594787 4 AAA SIE 5B ap TTD I ie E ZROCMRCEMARARAR AM att A No ACA AECA E Twain AAN AN 1 aen S Spectrum ProcPars Acqu ars Tie PuteProg Peaks integrar Sample Sructure Fid Acqu Phase Print 7 10 o 4 1 4 4 4 4 4 sl 4 4 4 4 4 al 4 4 Mp p 15 109 so o 6 100 ber Figure 4 23 Display Showing a Glycine Taken Under Adamantane Conditions 4 scans User Manual Version 002 BRUKER BIOSPIN 79 327 Basic Setup Procedures The figure above shows a glycine taken under adamantane conditions 4 scans Incorrect carrier setting a carbon at 43 ppm insufficiently decoupled Angle is set correctly because carboxyl peak at 176 03 ppm shows a narrow lorentzian line shape HH condition looks okay Now reset the carrier as shown in Figure 4 13 o1p should be around 100 ppm in the middle of most carbon spectra Acquire a spectrum set the plot limits
144. attenuation and check S N again Re optimize the HH condition observ ing the peak at 176 ppm which is less strongly coupled to protons and therefore exhibits a sharper HH matching condition in steps of 0 3 dB In this case S N im proves 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 1096 improvement in S N Good Laboratory Practice requires that evaluation measurements be taken in suitable periods 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 5 Description of the sample which reference rotor weight of glycine and spin ner 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 acquisition and processing parameters Recalling this data set and acquir ing a new data set should give the same spectrum within 10 of S N BRUKER BIOSPIN User Manual Version 002 Basic Setup Procedures Some Practical Hints for CPMAS Spectroscopy 4 8 Some general recommendations for reasonab
145. ays at 11 7 T longer delays may be required at higher fields User Manual Version 002 BRUKER BIOSPIN 249 327 STMAS As for MQMAS the setup must be done in two steps in the first step a central tran sition selective 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 typically fulfilled by a 20 us pulse For the calibration of this pulse a power level around 30 dB with 500 W and 1 kW amplifi ers and around 20 dB with 300 W amplifiers should be expected The pulse pro gram zg which uses p1 and pIT or zgsel av which uses P3 and PL21 can be used For more details please refer to chapter 16 Once the central transition selective 90 pulse is calibrated the STMAS pulse pro gram can be loaded Available pulse programs are stmasdqfz av and stmas dgfe av Both are double quantum 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 prop er 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 19 3 and Table 19 4 the starting parameters for the setup of the two se quences are given Typical va
146. between usually air in WB probes determine the impedance of the transmission line Since such a line is as well an inductance as a capacitance it is a resonating circuit If the length of the transmission line equals A 4 or A 2 of the RF wave it is a V4 or N2 line Since the upper end of the transmission line inner conductor is connected to the coil high voltage is required there This means that the M4 point has low voltage but high current whereas the A 2 point is at high voltage and low current A short between User Manual Version 002 BRUKER BIOSPIN 45 327 General Hardware Setup inner and outer conductor at the A 4 position enforces a low voltage high current and fixes a certain resonance frequency Some probes are tuned for the proton resonance frequency by a tunable capacitor at the end of the A 2 line 400 MHz and up which changes the effective length of the A 2 line Some probes 400 MHz and below are tuned by shifting the position of the A 4 short to a higher higher frequency shorter length or lower lower frequency longer length position 46 327 Figure 3 36 A A4 only probe left and a A4 2 probe right On the left side of the figure above is a MA only probe 200 300 MHz 400 low range only probes The transmission line is only A 4 proton tuning is done by moving the brass block to ground 1 Proton matching is done with capacitor 2 On the right side of the figure above is a M4 N2 probe 600 MHz Due to
147. ble 24 6 Processing Parameters CRAMPS 2D 24 7 The spectral width in both dimensions assumes the absence of shift scaling In or der to account 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 sw to call the status parameters for both F2 and F1 and replace both val ues by current value gt 0 6 After xfb the relative peak positions will be approxi mately correct but the absolute peak positions must be corrected by calibrating a known peak position to the correct value 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 24 8 These spectra were both taken without the modification according to CHAMPS 2D on page 291 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 002 BRUKER BIOSPIN 299 327 CRAMPS 2D een Ev v 10 9 8 7 6 5 a 3 ppm Figure 24 6 Glycine Proton Proton DQ SQ Correlation Using WPMLG in Both Directions 300 327 BRUKER BIOSPIN User Manual Version 002 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 13 2 H 10 9 8 7 6 5 4 3 2 ppm Figure 24 7 14 5 kHz
148. btain the various sub spectra All these se quences use constant amplitude CP which should be adjusted for maximum sig nal intensity For the CPP and CPPISPI sequences the only parameter 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 ex periment 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 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 H
149. but executes the contact at a proton offset cal culated from the proton RF field during the spin lock contact pulse This modifies the HH condition which must be reestablished using the pulse program Igcp In the following sections the specifics of these modified sequences are dis cussed User Manual Version 002 BRUKER BIOSPIN 127 327 Modifications of FSLG HETCOR 9 1 Carbon Decoupling During Evolution 15 rel 10 128 327 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 addition is the X rr pulse p2 at power level pl1 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 vd Rx t A dHLE EC 75 5 ze 359 5S A 3 M A column 3069 from tyrosin 5 1 CoBrukenTOPSPIN ighettapi ty orin 999 CrnrWeriTCPSEPIK m Shift 0 1559 ppm 77 979 Ez f A eyrarin S 03 C Braker Tepepry EN It hi if fi A f i Ij M II F 4 F 11 i I i j 4 j 1 1 j 8 A IN 1 j A 4 1 f j d
150. but in the following the ex periment is described using windowed pmlg w PMLG The reasons are the fol lowing 1 At fast spin rates over 10 kHz only w PMLG and DUMBO work well The se quence 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 frequency 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 gly cine and generate a 2D data set by clicking on the symbol 1 2 in the headline of the acquisition parameters User Manual Version 002 BRUKER BIOSPIN 291 327 CRAMPS 2D Pulse Sequence Diagram Phases i Di u ds Pulses and delays pi LG p13 d3 d3d3 d Pulse power pl2 pll3 pli2 oli 10 excitation evolution mixing detection Figure 24 1 Pulse Sequence Diagram 24 2 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 principle a windowless sequence can be used as well and should give better resolution along F1 The power level for a windowless se quence is however usually slightly different from the windowed sequence so this needs to be adjusted separately Likew
151. 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 text 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 236 327 BRUKER BIOSPIN User Manual Version 002 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 calcu lates 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 sp1 As a first guess a value
152. can be acquired consecutively or in an interleaved mode so that experimental drifts will not cause large errors Usual ly the experiment is set up as a pseudo 2D experiment where the number of rotor periods with p pulses is increased before detection The experiment can either be used to investigate isolated spin systems or multi speed systems In both cases the time dependent difference of the echo Sg with out the reintroduction of the heteronuclear dipolar coupling and the second echo experiment S with the reintroducing p pulses applied on the channel can be used for calculating the distance information for the two involved spins or the sec ond moment of the spin system respectively In isolated two spin systems the measured REDOR dephasing curve can be used to determine distance information between the two involved spins In case of investigating multispin systems the experimental REDOR dephasing curves can only be used to determine the second moment M5 1 E x Eq 12 1 By the relation of the second moment to the distance by this information in combi nation with theoretical simulations can be used to determine a mean distance be tween the involved spins as well In the case of very strong dipolar couplings the normal REDOR approach for mul tispin systems can not be used without introducing severe errors into the calculat ed second moment With very strong dipolar couplings the signal intensity may be lost after very
153. cc 4 5 MHz h 0 16 diso 85 ppm for the upper peak sample courtesy of Alexandrine Flambard LCPS Univ de Lille The bridging oxygen give rise to the lower peaks in the 2D spectra of Figure 17 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 nega tive quadrupole induced shift is scaled down and subtracted from the isotropic shift to give the MQ shift In the example shown in Figure 17 10 two sites are vis ible 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 respec tively 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 di mension has a much smaller quadrupole coupling which can immediately be rec ognized 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 interac tion at 11 7 T is so much reduced at 18 8 T that the width of the peak is now deter mined by the distribution of chemical shift This is expressed in the fact that the peak is dispersed along the diagonal Fiqure 17 11 shows
154. cdeccceecccneccgeesegeccedeectaeesaerscececeeceenees enn enn nena NENNEN 96 6 Practical CP MAS Spectroscopy on Spin 1 2 Nuclei 99 Table 6 1 Power Conversion Table 101 7 Basic CP MAS Experiments 105 Table 7 1 Acquisition Parameters meme II ee e nen ernennen ernennen 106 Table 7 2 Acquisition Parameters e eem een ee ene rennen rris 107 Table 7 3 Acquisition Parameters cece eeee cents eeeeeseeeceeseeeseeseeseeeeeseesaeeeaes 113 8 FSLG HETCOR 119 Table 8 1 Acquisition Parameters for FSLG HETCOR on tyrosine HCl eseese 123 Table 8 2 Processing Parameters for FSLG HETCOR on tyrosine HCl essees 124 9 Modifications of FSLG HETCOR 127 Table 9 1 Acquisition Parameters for pmlg HETCOR on bwrosine HCH 130 Table 9 2 Processing Parameters for pmlg HETCOR as for FSLG on tyrosine HCI 131 Table 9 3 Acquisition Parameters for wpmlg HETCOR on tyrosine HCl nesese 132 Table 9 4 Acquisition Parameters for e DUMBO HETCOR on tyrosine HCl 133 Table 9 5 Acquisition Parameters for DUMBO HETCOR on tyrosine HC l ssssse 134 10 RFDR 137 Table 10 1 Acquisition Parameters sss eee eme em emnes 140 Table 10 2 Processing PDarameiers eene mee nennen nennen nnns 141 11 Proton Driven Spin Diffusion PDSD 143 User Manual Version 002 BRUKER BIOSPIN 315 327 Tables Table 11 1 Acquisition Parameters ssssssssssssssssss nenne nme nhe ne nennen 148 Table 11 2 Processing Parameters
155. ce of hard pulses such that no signal remains There is then a variable delay during which relax ation 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 T4 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 16 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 approxi mate temperature of 20 C The form of the pulse program is shown in the follow ing figure decouple vd C Figure 16 1 The CPX T1 Pulse Sequence User Manual Version 002 BRUKER BIOSPIN 203 327 Relaxation Measurements Starting from the glycine spectrum create a new data set set parameters accord ing to Table 16 1 and acquire a 1D spectrum The relaxation delay after inver sion is controlled by a variable delay list this can be crea
156. contact your nearest BRUKER dealer or contact us directly at BRUKER BioSpin GMBH am Silberstreifen D 76287 Rheinstetten Germany Phone 49 721 51610 FAX 49 721 5171 01 E mail service bruker de Internet www bruker de BRUKER BIOSPIN User Manual Version 002 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 field 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 106 direct observe 19F CP CP 1 1 1H 13C 19F 13C low sensitivity PTFE 19FMAS 126 direct observe 3He 203 209 31p NH4 H2PO4 HRIPCP 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 65Cu Cu metal powder wideline knight shift 2500ppm Ga Ga505 hahn echo CT 300 kHz wide 129X as hydroquinon CPMAS 0 d1 gt 5s Clathrate gas in air 0 single pulses overnight 1s 23Na NagHPO MQMAS 0 dep on crystal water 2 5 lines Na3P309 MQMAS 51y NH VO 1237 27Al AIPO 14 MQMAS 0
157. d not reflect the true TI coherence lifetime The true T achieved through good heteronuclear decou pling can then be observed with a hahn echo experiment Optimization is done by looking for the maximum signal amplitude of the decoupled resonances of inter est Be careful not to exceed the maximum decoupling time with high power de coupling Reference 1 G De Paepe N Giraud A Lesage P Hodgkinson A B ckmann and L Emsley Transverse De phasing Optimized Solid State NMR Spectroscopy JACS 125 13938 13939 2003 98 327 BRUKER BIOSPIN User Manual Version 002 Practical CP MAS Spectroscopy on Spin 1 2 Nuclei 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 different from 130 Nevertheless sometimes one comes across samples where it is difficult to observe 13C This chapter deals with strate gies to optimize acquisition parameters for 13C and other spin nuclei Possible Difficulties 6 1 Usually 136 spectra are easily acquired Several sample properties may however make observation difficult 1 Low concentration of 19C in the sample 2 Noor too few protons in the sample 3 Long proton T4 4 Long T s 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 cond
158. d 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 SI 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 User Manual Version 0 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 stmasdqfz 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 corresponding value of R p The values for the different spin quantum numbers are summa rized in Table 19 7 for experiments using the inner ST 3 2 gt 1 2 R deter mines the shearing 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 identical in ppm to all MQMAS exper iments
159. d transverse or spin spin relaxation Both the transverse magnetization and the difference between the current and equilibrium z magnetization decay exponentially with time constants denoted T4 for longitudi nal relaxation and T3 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 it is important During cross polarization the mag netization on the dilute spins is increased by transfer from another nucleus but it will also decay since the radio frequency field weak compared 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 con tinued the transverse magnetization will decay exponentially with a time constant denoted T4 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 spontaneous 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 motion and the transition rates depend on the strength and details of the fluctuations of these local fields Since the fluctua tions are random the rate of fluctuation is defined by the correlation time of
160. dditional Connections for VT Operation Figure 3 25 WB Wideline or PE Probes 1 Bell shaped glass dewar around sample chamber 2 Insulating and sealing Al20 felt ring 3 Cover for coil sample compartment fixed with plastic or metal screws Figure 3 26 WB Wideline or PE Probe Connections User Manual Version 002 BRUKER BIOSPIN 35 327 General Hardware Setup Figure 3 27 Low Temperature Heat Exchanger for VTN Probes old style In the figure above is a low temperature heat exchanger for VTN probes old style 1 turn exchanger loop for SB probes 2 turn loop for WB probes A turn loop for DVT probes 36 327 BRUKER BIOSPIN User Manual Version 002 Additional Connections for VT Operation Figure 3 28 Low Temperature Heat Exchanger for DVT Probes The low temperature heat exchanger for DVT probes in the figure above uses the exchanger coil with 6 turns the larger one may be used for high resolution probes To use the spring loaded connection device shown in the close up 1 Compress the spring 2 Move the hollow part over the ball joint 3 Release the spring User Manual Version 002 BRUKER BIOSPIN 37 327 General Hardware Setup A t Her f to probe DVT DN Ze v A A 7 i A K MM nitrogen ustre VT ga flow from B VT 3001 A A EN A X 1 Nitrogen exhaust refill 3 VT Gas flow from B VT 3000 2 Transfer line to probe DVT 4 N2 level control to B VT 3000 te
161. de Sampling eiae ee Le ee Leere iia dere do ee Dre BAR d 278 SELUP rinnen r 279 Parameter Settings for PMLG and DUMBO sss hene 279 Fine Tuning for Best Resolution sssssssssse eene eee enne 281 Fine Tuning for Minimum Carrier Spike sssssse eee 281 Correcting for Actual Spectral Width sss 281 Digital Mode ACQUISITION 12 a eat aa dee Sege See qu ER ER 282 Examples sale ee eb een geing 282 Modified W PMLG EE 285 Pulse Sequence Diagram for Modified W PMLG nenn nenn nennen 285 Pulse Shapes for WePMLG cece eeceece cece eeeeese eee nennen nhe ene ne hne rer nenne 286 SIX 287 Parameter Settings for PMLG and DUMBO sss mener 287 Fine Tuning for Best Resolution cecinere nete teneo tee nennen 288 Correcting for Actual Spectral Width sssssssssssssssss e 288 Digital Mode ACQUISITION A0 ee KREE 440442 00004 00 04 ce ER e da ee an eee d re du ve d wate Ye Le ER ea Ra ca bees 289 CRAMPS 2D 291 Proton Proton Shift Correlation spin diffusion oooooocoonncconicinnccnnncinnccinncnnnncannncannnnannn 291 Pulse Sequence Diagram sssssssssssssse eene ee ehe hene ne nennen nes 292 Data Processing 2 aae dedere doves deh e dez rez re Dee du ove ques AE FPE LR Pe ee ebe Dee d e d va d
162. 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 experiment using the sostapt pulse program name or look at 2D editing se quences based on the FSLG HETCOR experiment User Manual Version 002 BRUKER BIOSPIN 117 327 Basic CP MAS Experiments 118 327 BRUKER BIOSPIN User Manual Version 002 FSLG HETCOR This chapter discusses setup and use of the Frequency Switched Lee Goldburg Heteronuclear Correlation FSLG HETCOR experiment The FSLG Hetcor experiment correlates H chemical shifts with X nuclei e g 13C 15N chemical shifts The experiment provides excellent 1H resolution in the indirect dimension Homonuclear decoupling in the 1H evolution period is achieved with an FSLG pulse train FSLG permits relatively high spinning speeds and makes this experiment available for high field systems requiring high spin ning speeds in order to move spinning sidebands out of the spectral region De coupling 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 heteronuclear 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 136
163. e lists are as follows 2D data are acquired in QF mode which means that no phase incrementation is required ph1 0 30 60 90 120 150 180 210 240 270 300 330 ph2 0 ph3 0 12 90 12 180 12 270712 receiver 0 270 180 90 3 180 90 0 270 3 Initial parameter values are listed in Figure 18 1 A few of these parameters need further explanation D6 Is calculated as 1s L1 CNST31 P4 2 P2 2 This ensures that the delay from the centre 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 L7 should be between 70 and 80 so that D6 is between 2 77 and 3 17 ms P2 Is calculated as 1s CNSTS31 LO 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 param eters 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 param
164. e power level for a 2 5 usec 90 pulse Set the Bo field or o1 to be close to reso nance see chapter Basic Setup Procedures for more details Calibrate the ada mantane proton shift to 1 2 ppm Then load a spinner with a glycine precipitate from cold water with acetone and dry if you are not sure about the composition of your sample A spinner with 50 pl 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 literature Parameter Settings for PMLG and DUMBO 22 5 Table 22 2 PMLG Analog Mode Parameter Value Comment pulprog wpmlga Runs on AV 3 instruments only pli2 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 User Manual Version 002 BRUKER BIOSPIN 279 327 CRAMPS 1D Table 22 2 PMLG Analog Mode 111 anavpt 4 2 4 8 16 or 32 o1p 10 or 1 To be optimized swh 1e 2 2 p9 10 p5 0 6 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 22 3 DUMBO Ana
165. e scheme exists so far Before describing the optimization procedures some exper imental approaches used in combination with these enhancement techniques are introduced Split t Experiments and Shifted Echo Acquisition 18 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 successive slices of the 2D experiment this echo position relative to the conversion pulse changes as a function of the duration of the actual t4 de lay 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 experiment where the top of the shifted echo appears at a constant posi tion 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 period of the split t
166. e spikes at both sides may appear Correcting for Actual Spectral Width 288 327 23 6 The modified sequence has a slightly different scaling factor of 0 47 BRUKER BIOSPIN User Manual Version 002 Digital Mode Acquisition Modified W PMLG Most parameters stay the same as adjusted in analogue mode Table 23 4 Parameters for Digital Mode 23 7 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 o1 The correction for the scaling factor must be done after acquisition changing the status parameter swh by typing s swh and dividing the value by the scaling factor about 0 47 for WPMLG and 0 5 for DUMBO User Manual Version 002 BRUKER BIOSPIN 289 327 Modified W PMLG 290 327 BRUKER BIOSPIN User Manual Version 002 CRAMPS 2D CRAMPS methods allow measurement of chemical shifts in the presence of strong homonuclear dipolar interactions Therefore CRAMPS type sequences can be applied to measure chemical shifts of protons where these sequences work most efficiently and where fast spinning cannot easily be used As an ex ample the proton X heteronuclear chemical shift correlation experiment see chapter 5 uses FSLG to suppress homonuclear dipolar couplings between pro tons to resolve the proton chemical shifts CRAMPS type pulse sequences must
167. e Concen trations of 15C 195N Chemical Bonds by Double Cross Polarization NMR J Magn Reson 59 150 156 1984 3 M Baldus A T Petkova J Herzfeld and R G Griffin Cross Polarization in the tilted frame assign ment and spectral simplification in heteronuclear spin systems Mol Physics 5 1197 1207 1998 User Manual Version 002 BRUKER BIOSPIN 255 327 Double CP Pulse Sequence Diagram Double CP DCP 20 1 i 10 TPPM or SPINAL64 da Qrec 15 2 03 ty m EE p3 pls do pl6 aq Aus 1 3 7 0 States TPPI t4 44 000011141 702203113 22223333 20021331 dees 00002222 Figure 20 1 Pulse sequence diagram for 1D t 0 and 2D double CP experi ments Double CP Experiment Setup 20 2 Double CP 2D Experiment Setup 20 2 1 1 Prepare your probe for triple resonance applications H C N 2 Load a sample of glycine N and C4 C3 or only C 13C labelled Make sure the sample is a glycine you will get nowhere with y glycine since the proton T4p 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 gly cine A restricted volume rotor is preferred If a different spin rate is used a dif ferent shape must be generated for the second CP step 256 327 BRUKER BIOSPIN User Manual Version 002 Double CP 3 Check the edasp routing and set up 3 RF channels for C H and N such that the lower pow
168. e Fd Ao of 2 MAL are Figure 14 3 Variation of DQ generation reconversion time on a uniformly 1 C la beled peptide fMLF 184 327 BRUKER BIOSPIN User Manual Version 002 Symmetry Based Recoupling Both times were incremented in units of 2 rotation periods One can clearly see the different maxima for the C4 the alphatic carbons and the mobile CH groups Spinning speed was 13 kHz T Optimize the cwlg decoupling if needed by variation of enst20 in increments of 5000 and check whether a different offset condition helps improving the signal intensity Run one experiment and compare with the direct CP experiment to measure the DQ recoupling yield reccupling 1 1 C iBruker TUPSPIN sama MY Scale D 6768 reccupling 6 1 C Brukec TUPSPIN EAE r 3 r8 da e H el ber Bed 2 B et O T Y T T T T T 1 T T T 200 150 100 so o ppm Figure 14 4 PC7 Recoupling Efficiency at a Spinning Speed of 13 kHz Figure 14 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 generation reconversion Quite a noticeable loss on the glycine a peak due to insufficient HH suppression is visible Efficiency is 67 on the car boxyl peak AVIII 700 SB Setup of the 2D SQ DQ Correlation Experiment 14 2 3 9 10 11 User Manual Version 002 Running such a correlation e
169. e STMAS spectrum This can easily be understood as the rotational echoes decay much more rapidly when the magic angle is off Experimentally it has been found that the precision for setting the angle must be lt 0 002 The de pendence on the accuracy 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 special goni ometer 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 an gle 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 19 2 Fiqure 19 2 and Figure 19 3 show two of the basic sequences which are 4 pulse sequences with z filter stmasdgfz av and stmasdgfe av Both sequences start with a non selective excitation pulse p7 that creates SQ coherency on the in nermost ST which is allowed to evolve during the evolution period DO Shortly be fore the end of the t4 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
170. e be tween nucleus of interest is long or the mobility is high leading to a small het eronuclear 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 5096 of the initial proton signal amplitude will still give a 2 fold en hancement on C If the proton signal is below 5096 at 1ms spin lock time or even less a full cp enhancement cannot be expected Now we know the minimum relaxation delay and the maximum contact time With these parameters used as d1 and p15 the measurement is just a matter of patience BRUKER BIOSPIN User Manual Version 002 Practical CP MAS Spectroscopy on Spin 1 2 Nuclei Possible Approaches for Non 13C Samples 6 3 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 13C In this case running a PE cp mas spectrum allows setting and determining all proton parameters recycle time contact time from the C 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 effec tive field at the X frequency equals the effective field at proton frequency spin rate Example setting the HH condition for IDN from known
171. e decoupling bandwidths are not very large o1 should be close to reso nance especially for DUMBO For PMLG this is less critical The power level for the shapes should be adjusted in steps of 0 2 dB The splitting of the two high field lines the protons in the CH are in equivalent in the solid state should be be low the 50 level Fine Tuning for Minimum Carrier Spike 22 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 o1 appropriately N B changing 01 will lead to different values for cnst25 Correcting for Actual Spectral Width 22 8 Since the sampling rate is governed by the multi pulse sequence repetition rate the foreground 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 be tween the two CH gt 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 separation is incorrect change the status parameter swh by typing s swh and scaling it appropriately Some pulse pr
172. e glycine sample Sample dependent need to see a reasonable spectrum but must be an even number Data Processing 16 2 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 16 3 lists the relevant parameters No processing is done in the indirect dimen sion 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 frequency di mension does not affect the analysis so exponential multiplication with Ib of the order of the observed line width can be applied to improve the signal to noise ra tio Table 16 3 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 TD F1 Must be 2n but any zeros will be ignored User Manual Version 002 BRUKER BIOSPIN 205 327 Relaxation Measurements 206 327 Once the parameters are set pr
173. e indirect dimension than a 3Q experiment BRUKER BIOSPIN User Manual Version 002 Basic MQ MAS We see that a 5Q experiment has a 5 times smaller range than the 3Q experiment and therefore 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 respectively Table 17 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 Mathemat ically this is solved in the AU program xfshear in such a way that the observe Lar mor frequency is multiplied by the factor R p to redefine an apparent Larmor frequency in the MQ dimension Table 17 6 Chemical Shift Ranges for all MQ Experiments for All Spins I 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
174. e irradiation frequency jumps between RF field sqrt 2 for the duration of two 360 pulses on resonance 7293 pulses at the LG frequency with a 0 180 phase alternation The include file lt Igcalc incl gt calculates all values according to the RF field set in Hz as cnst20 within the pulse program Bz Bor Figure 5 2 Geometry for the FSLG Condition Note that Beg points along the 1 1 1 direction in the 3 dimensional space see Ref erence 2 Note the sign of Bog when calculating the actual direction of the effec tive field A positive Bor and a B4 with phase 0 results in the effective field being in the positive quadrant along the magic 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 to get 2x The relationship describes the rate at which a phase of the rf pulse must be changed in order to achieve a cer tain frequency offset Vinogradov et al describe this approach under the acronym PMLG Phase Modulated Lee Goldburg Used in combination with cp signal generation both methods allow observing pro ton J couplings to the observed X nucleus However only samples with very nar row lines will produce well resolved J couplings as shown below on adamantane Harder sol
175. e observed Multiple Pulse Sequences 21 2 Dealing with a heteronuclear dipolar coupling is easy continuous high power irra diation of one coupling partner will decouple it from the other nucleus as in the case of 13C observation 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 Sup pression of a homonuclear dipolar interaction occurs when the magnetization vec tor of the coupled spins is tilted into 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 resonance irradiation of suitable offset and RF field Lee User Manual Version 002 BRUKER BIOSPIN 271 327 CRAMPS General Goldburg To observe the signal a gap within the pulse sequence must be sup plied which is long enough to observe one or several data points while the mag netization vector points along the magic angle This condition obviously persists only for a time period short compared to the transverse relaxation of the signal 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 require
176. ectrometer 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 ta ble which is calculated for the respective magnetic field Bg The index 1 in Of BF1 and SFO1 refers to the RF channel 1 which is also found in the pulse pro gram 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 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 connec tions 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 pr
177. ed Therefore in case of doubt it is probably the best idea to set L5 TD F1 2 If less anti echos are to be accumulated the question is how many anti echos to acquire this depends on the sample In amorphous 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 absorption 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 mp3qzqf av However phase correction in the acquisition dimension F2 cannot be determined on the first FID Therefore 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 processing is then done with the AU program xfshear Alternatively xfshear can be u
178. egaat channel Been we a ngical channel Dela nta Berar few Ji Figure 3 44 The edasp Display for a System with two Receiver Channels In the figure above the edasp display for a system with two receiver channels set for observe on 19C and 9N while decoupling on protons The same SGU is used for pulse and detection on both receiver channels BRUKER BIOSPIN User Manual Version 002 Basic Setup Procedures This chapter contains information and examples on how to set up basic solid state NMR SSNMR experiments We ll begin with the settings for the RF routing of the spectrometer some basic setup procedures for MAS probes and how to mea sure their radio frequency RF efficiency and RF performance Accurate mea surement of the pulse lengths and the associated RF power levels is essential for solid state NMR experiments In SSNMR RF field amplitudes are often ex pressed as spin nutation frequencies instead of 90 pulse widths Spin nutation frequency n and 90 pulse width are related through the reciprocal of the 360 pulse duration 4f 99 such that Ho isen RF field in Hz with Leon in psec Setting up the magic angle shimming a CPMAS probe setting up cross polariza tion and measuring probe sensitivity for 13 will also be explained This is part of probe setup and performance assessment during installation However regularly scheduled performance measurements should be part of the hardware probes and spectrometer maintena
179. en higher RF fields for protons So the tendency is going to high spin rates also de sired to get rid of spinning sidebands and turning the decoupling off during recou pling which represents a much lower RF load to the probes and increases experimental stability substantially Table 1 shows the sample rotation rate and the required spin nutation frequencies for the X nucleus The spin nutation frequency must be 7 times the sample rota tion rate for C7 5 times the sample rotation rate for SPC5 and 3 5 times the sam ple 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 Especially the choice of the MAS probe is essential to achieve a sensible setup Table 14 1 shows the selection parameters for three standard recoupling sequences References 1 E A Bennett R G Griffin and S Vega Recoupling of homo and heteronuclear dipolar interaction 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 recoupling in the nuclear magnetic resonance of rotat
180. ency variation via pulse length variation to achieve a wider decoupling bandwidth The decoupling efficien Cy is better than TPPM especially at high fields and comparable to if not better than SPINAL 64 The corresponding cpd program is called swftppm 1 R S Thakur N D Kurur and P K Madhu J Magn Res 193 77 2008 XiX Decoupling 5 1 5 Reference XiX decoupling requires high spinning speeds but decouples at a moderate RF level 180 proton pulses are used synchronized to the rotor speed such that re coupling does not occur pcpd2 n 4 rotor periods Usually pcpd2 is selected to be about 1 3 rotor period The decoupler power level must be adjusted to produce a 180 pulse of rotor period 3 1 A Detken E H Hardy M Ernst and B H Meier Chem Phys Lett 356 298 304 2002 Pi Pulse Decoupling Reference 5 1 6 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 pro tons cannot be sufficiently decoupled with this method but it is very suitable to re move couplings to 31B 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 homonuclear decoupled by spinning 19F 1 S F Liu and K Schmidt Rohr Macromolecules 34 8416 84
181. enerate sidebands 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 spec trum suitably along F1 Set the acquisition time along F1 to about 10 msec for a start Lines along the double quantum dimension may be narrower than along the single quantum di mension so a compromise between experiment time and digital resolution along F1 must be found Start the experiment 14 3 Data Acquisition Sample Fully 13C labelled tyrosine HCl or a suitable fully labelled small peptide Spinning speed 5 20 kHz depends on experimental requirements see Table 14 1 Experiment time 1 4 hours Table 14 2 Acquisition parameters for DQ SQ correlation experiments using symmetry based recou pling sequences Parameter Value Comments Pulse program spc5cp2d See Table 14 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 Ec 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 186 327 BRUKER BIOSPIN User Manual Version 002 Symmetry
182. ensitivity 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 affected 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 transition signals must not ex tend 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 us l 1 2 12 5 kHz 1 1 2 This means that wge wg as a prerequi User Manual Version 002 BRUKER BIOSPIN 215 327 Basic MQ MAS site for a CT selective 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 expected The pulse program zg which uses p1 and pl1 or zgsel av which uses P3 and PL21 can be used Table 17 1 Some Useful Samples for Half integer Spin Nuclei Nucleus Spin eae d1 bi 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 27 Al 5 2 130 32 5 YAG 27 Al 5 2 130 32 0 5 Al203 27 Ni 5 2 130 32 0 5 VPI 5 27A 5 2 130 32
183. ente edet vt feet e tendat Se 11 BOM ACIO eoi nee fe eL rA nte dt UE 11 BR 24 ee tete is teet ulus e edd EE de WE e SE 92 272 breakthrougli 2 m IUe o LUPUS gc Uc P Egger 56 bsmisdiS xe tede atem eu oce dide Md 75 bypassing the Dreamp nnne nennen nnne nenne 73 C C24 EE 272 CA CO WIEV 72 Galibrating WEE 75 User Manual Version 002 BRUKER BIOSPIN 319 327 Index 320 327 ecu 69 Cd NO3 2 ABI2Q iia ARTES ARR ees 12 Chemical Shifts oooooooonococcnnncnncnncccccccnnonnoononnnnnonnncnnnnnnnnnnnn nn nn nnnnnnnnnn nana nn EEE nnnnnnnnn 75 Suri ccm 11 193 119710 pU CREME 121 CO CN G a 12 COMPIeSSOSS nn 56 CONCUCHVILY d 100 contact me 76 CORTAB eee 58 velo 81 CPP A aa ai ada 105 116 117 CPPIRCP EE 116 117 CPPISPI e M 116 117 enc 271 Cross polariSation EE 76 cross polarisation sssssssssssssesee nennen enne nene nennen nnns 55 cross polarization under a LG frequency OMS Ot oooocccoccocccnccccnccccconconcononnnnnnnnnnn 119 Cu metal Dowder eene meer tnnt nene n nenne nennen sensns 11 CW decoupliig nee rre cipe tpe dba dea E isses 89 D po EMT 12 OsDMSO2 D
184. ents The FSLG experiment is the most forgiving requiring just the knowledge of the RF power level for decoupling at a certain RF field Setting cnst20 to this RF field 5 or 10 is all that needs to be set if the 13C observe parameters are well adjusted Table 9 5 Acquisition Parameters for DUMBO HETCOR on tyrosine HCI Parameter Value Comments pulprog dumbohet Windowed dumbo shape nuc1 13C olp 100 ppm nuc2 1H cnsi24 1000 3000 Place carrier within proton spectrum for evolution pli 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 spnam1 dumbo 1 40 Both include one DUMBO cycle p3 2 5 3 usec 90 pulse channel 2 at pl12 134 327 BRUKER BIOSPIN User Manual Version 002 HETCOR with Cross Polarization under LG Offset Table 9 5 Acquisition Parameters for DUMBO HETCOR on tyrosine HCI 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 2500 MHz 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 DUMBO periods
185. equirement 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 Magn Reson 155 15 28 2002 2 R Tycko G Dabbagh and P A Mirau Determination of Chemical Shift Anisotropy Lineshapes in a Two Dimensional Magic Angle Spinning NMR Experiment J Magn Reson 85 265 274 1989 User Manual Version 002 BRUKER BIOSPIN 169 327 SUPER Pulse Program 13 2 Au 3 heteronuclear decoupling H Bo ta te GN Ir tizNt Figure 13 1 Pulse Sequence for 2D CPMAS exchange experiment 2D Experiment Setup 13 3 Sample Tyrosine HCI natural abundance Setup time Less than 1 hour Experiment setup 13 3 1 170 327 1 In order to setup the experiment determine 1H 18C and parameters with vari able amplitude on 1H according to Basic Setup Procedures on page 55 2 Verify the pulse parameters on the C channel see Pulse Calibration with CP on page 105 and calculate the power level required for the recoupling pulses i e fr 12 2 x frot 3 Verify pulse width 4 Calculate power level required for heteronuclear decoupling during the recou pling 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 het eronuclea
186. er amplifier 500W or less is used for 1 C UN may require more than 500W Set for 1 C observation 4 Make sure the preamplifiers in use are set up for the appropriate frequencies The following external RF filters are required proton bandpass e bandpass and IDN low pass The channel isolation required between X and Y here 13C and 15N is usually sufficient with a bandpass on one of the channels but a fil ter to remove the proton decoupling RF interference 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 PPP CITI 5 geng 910 dee ege 4 8 TT WI e Li LJ t 7 v k D D joe Send L al Figure 20 2 The edasp routing tables for H C N double CP Three examples are shown Setup with only one X HP preamplifier must be reca bled for 1 C and IDN setup setup with 2 X BB HP preamplifiers and 2 HP trans mitter 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 13 CP operation in triple mode Remember that a double tuned probe has better signal to noise and requires less power on X than a tri ple probe User Manual Version 002 BRUKER BIOSPIN 257 327 Double CP 5N Channel Setup 6 Optimize decoupling and CP condition run a
187. erefore a single frequency irradiation is sufficient for spin 3 2 Higher spins have more satellite transitions and therefore a correspondingly larger num ber of irradiation frequencies are required DFS is here the most convenient solu tion Start values for the parameters determining FAM are listed in Table 18 5 Table 18 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 optimizing PL14 P2 and L2 consecutively This can conve niently be performed with the parameter optimization procedure popt where two or three iterations can automatically be performed Soft Pulse Added Mixing SPAM 18 4 242 327 A simple and ingenious experimental trick can immediately give a signal enhance ment Starting from the standard 3 pulse sequence the phase cycling of reconver sion pulse P2 and the CT selective 90 pulse P3 is eliminated This changes the coherence transfer pathway from 0 gt 3 gt 0 gt 1 to 0 gt 3 5 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 i
188. erent values of Spec trum Reference sr To reduce the requirement of readjustment and to make referencing more reli able the field should be set to be the same for all spectra To keep the field the same 2 parameters must be taken into account 1 The drift of the magnetic field 2 The difference in shims and field between different probes The drift of the magnet should be measured in the following way Insert a sample of D20 and run a proton spectrum just like it was done on ada mantane in Calibrating 1H Pulses on Adamantane on page 65 Without changing any parameter rerun the spectrum every 10 minutes for a full day This can be done with popt storing the data as a 2D experiment or use the pulse program zg in a 2D data set with appropriate settings for d1 and td1 so that the drift of the magnet can be followed by the changes in peak positions of the D20 protons The pattern of changes in the peak position will reveal whether the changes are solely due to magnet drift or whether there are additional disturbanc es to the magnet field A magnet drift will always be constant towards lower fre quency note A freshly charged magnet may also drift to higher frequency but this will change so it makes no sense to account for this initial drift A nonlinear drift pattern indicates temperature changes abrupt changes reveal magnetic dis turbances elevator cars or trucks trains or the keys in your pocket only if your magnet is n
189. erimpose their heteronuclear dipolar couplings to X spins and J couplings to X spins so these distinct couplings are not observable homonuclear decoupling protons while observing X spins makes these couplings observable Any method used in multiple pulse NMR section 5 2 1 may be used to achieve this BR 24 MREV 8 BLEW 12 Used as heteronuclear decoupling methods the window between pulses may be shortened or omitted semi windowless or windowless sequences These se quences 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 supplied as a standard cpd pro gram It consists of a windowless sequence of 90 pulses with suitable phases High RF levels for decoupling provide better resolution FSLG Decoupling The Frequency Switched Lee Goldburg FSLG sequence may be used at spin ra tes up to 15 kHz It is a homonuclear decoupling sequence which rotates the inter action Hamiltonian around an effective field aligned at the magic angle arctan A2 with respect to the Zeeman field in the rotating frame The tilt is achieved by off resonance irradiation at the Lee Goldburg frequency fj according to the Lee Goldburg condition BRUKER BIOSPIN User Manual Version 002 Decoupling Techniques The off resonance condition depends on the RF field of irradiation not very sensi tively however Th
190. ersion 002 CRAMPS 1D auEgEFMAm s re urine dumbo_1 0 p Open i 54 NPONTS None EXMODE 00 TOTROT Pat AG INTEGFAC 2 MODE 20 40 o S Edit Shape Parambelers o asl 2 DN 50 100 dasaalaasadasaalasalasaaLaa 1 3 PE Figure 22 3 Shape for DUMBO sp1 Analog and Digital Sampling Modi 22 3 AV3 instruments allow different acquisition modi one which resembles the previ ous mode of analogue filtering in so far as the down conversion is done without si multaneous digital filtering 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 wpmlga are written for the pseudo analog mode without digital filtering dumbod and wpmlgd are written for the digitally filtered mode User Manual Version 002 BRUKER BIOSPIN 277 327 CRAMPS 1D Analog Mode Sampling 22 3 1 2 complex data points down converted into 1 data point no points sampled in between Figure 22 4 Analog Sampling Scheme Digital Mode Sampling 22 3 2 Continuous sampling throughout the pulse train es 0000000000000 e D Ze Ze af B e af digital blanking during pulse and deadtime zeroes are sampled E Ka downconversion of decim data points into one digit
191. es the DQ build up time DQ generation The reconver sion time is usually also controlled by 0 it may however be written such as to be independently controlled by a different loop counter For glycine about 5msec will be the optimum Set the decoupling program cpdprg1 to cwlg Set pI13 such as to yield the desired decoupler RF field during the DQ generation reconversion or set it to 120 if the spin rate suffices to omit decoupling Set cnst20 corresponding decoupling RF field 5 Optimize p 11 for maximum signal intensity User Manual Version 002 Optimize 0 for optimum signal intensity In a multi site spectrum the optima may differ for different spin pairs BRUKER BIOSPIN 183 327 Symmetry Based Recoupling 20 15 Tre EEE T m RAR Ben Jam ar 5 T Y T Y T T T T d T T T T T T T T T T T 24 26 28 3 0 32 3 4 48 Figure 14 2 Optimization of the RF power level for DQ generation reconversion on glycine In principle both peaks must grow together as one approaches the RF 7 MASR condition but the resonances are differently influenced by non ideal oft H condi tions 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 H be Ede Ve Spectrometer Procamung Amts Open Window Hep J90 6 520 Mw L 4i1fuLl Nu u eQg TTootii B5 TRUACERMRAR EA tii wenn Fan Aa Em Pus agi Pape rte Canis Crat
192. et correctly BRUKER BIOSPIN User Manual Version 002 MQ MAS Sensitivity Enhancement For both sequences TD in F1 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 and which depend on the properties of the sample In crys talline material fairly narrow peaks can be expected so that a maximum acquisi tion time in F1 of 2 to 5 ms is expected In disordered 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 be between 100 us 10 kHz spinning and 28 5 us 35 kHz spinning so only 100 to 250 experiments might be required The rotor synchronization im mediately means that the spectral range in F1 is limited Dependent on chemical shift range spinning frequency and quadrupole interactions the positions of the peaks may fall outside this range In such a case care must be taken when inter preting 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 sp
193. eters CNSTT1 Start frequency in kHz of the sweep the sweep should start slightly off resonance usually 30 to 50 kHz from the resonance 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 approxi mately 1 MHz Therefore it does not make sense to have this value bigger than 1000 User Manual Version 002 BRUKER BIOSPIN 235 327 MQ MAS Sensitivity Enhancement Table 18 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 Aus 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
194. ethod for Measur ing Heteronuclear 1H 1 C Distances in High Speed MAS NMR J Am Chem Soc 122 3465 3472 2000 5 D P Burum and A Bielecki An Improved Experiment for Heteronuclear Correlation 2D NMR in Sol ids J Magn Res 94 645 652 1991 6 A Lesage and L Emsley Through Bond Heteronuclear Single Quantum Correlation Spectroscopy in Solid State NMR and Comparison to Other Through Bond and Through Space Experiments J Magn Res 148 449 454 2001 User Manual Version 002 BRUKER BIOSPIN 119 327 FSLG HETCOR Pulse Sequence Diagram for FSLG HETCOR 8 1 do du 7 Qio TPPM decoupling 2n 2r Af Af n Mr 1LG cycle 1LG cycle jer 13 STATES PPI det Joco de 00221133 u 2G r OG dree 02201331 0 00 00000 Figure 8 1 The FSLG Hetcor Experiment The FSLG Hetcor experiment consists of 3 basic elements the homonuclear de coupling sequence during which the 1H chemical shifts evolve the cross polariza tion sequence during which the information of the 1H spin magnetization is transferred to the X spins followed by observation of the X spins under proton de coupling 120 327 BRUKER BIOSPIN User Manual Version 002 FSLG HETCOR Setting up FSLG HETCOR 8 2 1 This experiment requires a probe of 4 mm 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 decou
195. experiment there is no net evolution under the second or der quadrupole broadening This is the case because the evolution of the MQ co herence 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 relat ed 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 18 1 The FID generated by the initial 90 pulse is not sampled but after it is decayed it is refocused with a 180 User Manual Version 002 BRUKER BIOSPIN 231 327 MQ MAS Sensitivity Enhancement 232 327 pulse into a so called shifted echo meaning that the position of the echo can be shifted by adjusting the delay d6 phi ph2 p3 p4 pl21 d6 Figure 18 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 acqu
196. experiments outlined here are usually the most important ones and or the ones that were common at the time when the manual was written New chapters will be added as the manual consists of largely self contained units rather than being a comprehensive single volume This was done 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 im plemented as they are finished and proofread The individual chapters are written by different people so there will be some differences in style and composition Note Concerning Future TopSpin Release Upon the release of this manual a new TopSpin version was in development In the new version which is scheduled to be released later this year there are fairly big changes that will influence all of the setup routines described in this manual User Manual Version 002 BRUKER BIOSPIN 9 327 Introduction Disclaimer Safety Issues In the future version of TopSpin there will be a different way of setting pulse pow ers There will be a watt scale which refers to the pulse power in watts This al lows you to set pulse powers in a spectroscopically more relevant scale Moreover different transmitters and different routings will not anymore have an in fluence on the pulse power setting since it is referenced to an absolute not rela tive scale This means however that some setup routine
197. f 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 H 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 H excitation and decoupling P3 90 1H 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 82 327 Note that the spectral window swp is set in ppm which makes the acquisition time dependant on the Bo 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 broaden ing proportional to Bg due to chemical shift dispersion To make S N values more comparable this accounts for shorter T at higher field BRUKER BIOSPIN User Manual Version 002 Basic Setup Procedures Table 4 2 Processing Parameters for the Glycine S N Test Parameter Value Comment SI 2 4 k Twofold or fourfold zero filling WDW No No apodization used for S N measure
198. f pulse lengths p2 and pf User Manual Version 002 BRUKER BIOSPIN 219 327 Basic MQ MAS 4 um a E H E 2 E mm 2 E as m ssim Hd vin sn we uv uu a Figure 17 6 Signal Intensities of 87Rb Resonances in RbNO3 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 conversion pulse Maximum intensities were 3 0 us and 1 2 us in A and 3 2 us and 1 0 us in B respectively 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 0 to 6 us for p2 from 0 to 2 us Two Dimensional Data Acquisition 17 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 dimension the following parameters must be set accord ing to the following table Table 17 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 S
199. f the 3Q transition A projec tion of the 2D spectrum perpendicular to this slope yields an isotropic spectrum free from quadrupolar broadening 17 2 Fiqure 17 1 and Fiqure 17 2 show two of the basic sequences a 3 pulse and a 4 pulse sequence with z filter Both sequences start with an excitation pulse p1 that creates 3Q coherence which is allowed to evolve during the evolution period dO In the 3 pulse sequence the subsequent conversion pulse p2 flips magnetiza tion back along the z axis which after a short delay d4 to allow dephasing of un desired 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 002 BRUKER BIOSPIN 213 327 Basic MQ MAS 214 327 Figure 17 1 A 3 Pulse Basic Sequence with Z Filter Three pulse sequence and coherence transfer pathway for the 3Q MAS experi ment with z filter mp3qzqf av The ratio for pulses p1 and p2 is approximately 3 The corresponding power level 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 ampli tude 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
200. f your sample of interest are considered before the experiment is started Table 14 1 illustrates the proper choice of hardware for the observe nucleus C Obviously observation of DQ co herence requires samples with reasonable dipolar couplings and reasonable probability of coupled species So running this experiment on TuS samples re quires reasonable enrichment Usually fully enriched samples are used some times diluted in natural abundance samples to reduce nonspecific long range interactions As always rotary resonance conditions overlap of side and center bands should be avoided unless specifically desired User Manual Version 002 BRUKER BIOSPIN 181 327 Symmetry Based Recoupling Running the experiment on enriched N samples is of course possible but one should consider that most samples will not have nitrogen atoms directly attached to each other so small couplings will prevail requiring long DQ excitation and re conversion 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 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
201. few or only one refocusing pulse so the decay curve cannot be User Manual Version 002 BRUKER BIOSPIN 155 327 REDOR 156 327 measured and the reference experiment does not show a decay independent of the heteronuclear dipolar coupling Faster spinning may solve the problem Alter natively the constant time CT REDOR sequence may be used which can help to enhance the performance in these cases and to reduce the experimental time needed dramatically Here the refocusing pulse is stepped in small time increments from the beginning of the rotor period where it has a small effect on the signal intensity to the middle of the rotor period where it has the maximum effect Finally if one or both of the involved nuclei do have a quadrupolar moment the here used REDOR sequence should not be used but there are several different REDOR like experiments in literature which have an enhanced performance on observing quadrupolar nuclei These are for example the Rotational Echo Adia batic Passage DOuble Resonance REAPDOR or TRAnsfer of Population in DOuble Resonance TRAPDOR sequences Usually the setup is chosen such that the more sensitive nucleus is observed The measured coupling is of course independent from the choice of the nucleus but there may be reasons to consider carefully which nucleus is observed Of course it is tempting to observe the nucleus with higher isotopic concentration but this is usually not recommended since it
202. for 7 2 IN0 72 55 Used for 9 2 In Figure 19 4 two 2D plots of the Rb STMAS experiment on RbNO3 are com pared The spectrum on the left was obtained after the first execution of the exper iment The spectrum on the right was obtained after several iterations of resetting the angle and rerunning the spectrum From this it is obvious that the spectrum of the sample for the setup must be known because 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 LJ EJ amp 3 i Figure 19 4 87Rb STMAS Spectra of RONO3 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 252 327 BRUKER BIOSPIN User Manual Version 002 Data Processing STMAS 19 4 Processing parameters should be set according to the table below Table 19 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 observe
203. g 2 Jamie 7 0 5 0 5 1 M Eq 12 4 The comparison of the second moment directly calculated from the experimental data with the second moment extracted from the best fitting SIMPSON simulation gives you in this example 747 Hz compared to 850 Hz Of course this result can be improved by introducing more data points within the interpretation region of the parabolic fit which can easily be done by running the experiment again with high er spinning speeds Afterwards the data sets can be combined before running the interpretation process 166 327 BRUKER BIOSPIN User Manual Version 002 Final Remarks Table 12 2 Results for the M Calculation and the Simulations Measurement M gt s Dipolar coupling Hz Distance experiment 4 4E6 747 1 6 ideal pulses 7 31 6 3 E6 964 896 1 47 1 5 10 pulse error 7 31 5 7 E6 964 850 1 47 1 53 850 Hz dipolar coupling 5 7 5 0 E6 850 800 1 53 1 56 theoretical reference 9 7 3E6 964 1 47 Final Remarks 12 3 The REDOR sequence is a powerful tool to measure distances in different spin systems But as seen above already a small error during the setup procedure will finally lead to severely stretched distances calculated in the interpretation process of the measured data Although the results using SIMPSON simulations are in a much better agreement with the expected values the simulations cannot compen sate for the errors i
204. ged anemones 188 13C 13C Single Quantum Correlation with DQ Mixing eeem nennen 189 Data Acquisitlon odere eee es ea 190 Spectral Processing 191 PISEMA T 193 Pulse Sequence Diagram circa daa ea Pu PR E Re quera uu const 194 SIUE 195 Processing igi 198 Relaxation Measurement wissiseserscvsesessassnarsenisedusndnnconunsvesanenveusnassens 201 Deseribing Relaxation 24 2 Zen faut ai dee d india iat deet eet deve Y dx a Ru e a ae AREE dA Hrn 201 T1 Relaxation Measurements ene ne hen ne nnn nnns 202 Experimental Methods 202 The CP Inversion Recovery Experiment unsuessssesssnnsnnnsnnnen memes 203 Data Processing ER 205 The Saturation Recovery Experiment u4sss44sssnnnsnnnnnnnnenneen nenn mee mene 208 User Manual Version 002 BRUKER BIOSPIN 5 Contents 16 2 5 16 3 16 3 1 17 17 1 17 2 17 3 17 3 1 17 3 2 17 4 17 5 18 18 1 18 2 18 2 1 18 2 2 18 2 3 18 3 18 4 19 19 1 19 2 19 3 19 3 1 19 3 2 19 4 20 20 1 20 2 20 2 1 20 2 2 20 2 3 20 2 4 20 3 20 4 20 5 21 21 1 21 2 21 3 21 4 22 22 1 T1p Relaxation Measurements ssssssssssssssssse eee hehe he hen enhn nnns 209 Indirect Relaxation Measurements ssssssssssss eere 210 Indirect Proton T1 Measurements 211 Baste MG MAS 213
205. gnetic field Bg for two dif ferent sites with arbitrary jso and Aus The x axis in each plot is the static magnet ic field Bg increasing from left to right the y axis yyg increases from bottom to top Plot A is for identical plot B for identical quadrupole coupling and In plots C User Manual Version 002 BRUKER BIOSPIN 225 327 Basic MQ MAS and D shift positions for two sites with large and small d sy and large and small jjs and with large and small is and small and large Au are plotted respectively 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 pro vided 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 20 ph4 0 10 90 10 180 10 270 10 receiver 0 180 5 90 270 5 180 0 5
206. h an accidental error condition User Manual Version 002 BRUKER BIOSPIN 75 327 Basic Setup Procedures Setting Up for Cross Polarization on Adamantane 4 6 Graphical asestart Edit text Y Expression i F2 F1 76 327 Cross polarization is used to enhance the signals of X nuclei like C The strong proton polarization 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 condition Hartmann Hahn condition is met the transfer of proton magnetization to carbon is optimum Since the pro ton 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 sideband 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 136 under proton decoupling 1 4 Load the pulse program cp in eda or typing pulprog cp The pulse sequence is depicted in the following figure EB CADOCUME 1 ADMINI TALOCALS 1 Temp SPDISP_50494 tmp cpedit hf Options fee ee eee el Names F Values M Grid P3 phi 1 3 2 Su P15 po pue 0 ramp 100 Figure 4 20 A
207. hannel BF 00 NUC1 SFO 00 F1 OFS1 oo Hz HPHP XBB31P 1100 HPLNA 1H BF2 00 NUC2 SFO2 00 F2 SGU2 1H 1H 100 W 2H OFS2 oo HPHP XBB31P BF3 00 NUC3 SFO3 0 0 F3 SGu3 x 500 W HPHP 19F 1H V show receiver routing F add eretic Figure 3 3 The edasp setpreamp Display Note for Figure 3 3 Transmitter to preamplifier wiring must reflect hardware con nections 18 327 BRUKER BIOSPIN User Manual Version 002 Connections to the Preamplifier 1 RF signal out to receiver 6 RS 485 control connection and DCin 2 Lock signal out to lock receiver 7 Additional DC supply for gt 3 preamps 3 Tune RF in from SGU 2 aux out 8 High voltage DC for HPLNA preamp 4 PICS probe ID cable to probe 9 Additional controls for multi receiver 5 ATMA and AUX connectors Figure 3 4 Additional Connections to the Preamplifier Stack User Manual Version 002 BRUKER BIOSPIN 19 327 General Hardware Setup 1 Pulse from TX 4 Signal from probe 2 Pulse pathway to probe 5 Signal to preamplifier 3 To probe Figure 3 5 Matching Box Setup for High Power X BB Preamplifiers The frequency of the observed nucleus must be within the bandwidth of the matching box the matching box contains a low pass filter to suppress frequencies above the X nucleus frequency range 1H 19F and a passive diode multiplexer which directs the RF pulse into the probe and the NMR signal into the preamplifi er High res
208. hat filters may cause So all filtering is done with external filters If a single channel NMR experi ment is run no filters are required Usually one filter per RF channel is required Both filters should mutually exclude the frequency of the other channel s Usual attenuations of the frequency to be suppressed should be around 80 dB in special cases when both frequencies User Manual Version 002 BRUKER BIOSPIN 21 327 General Hardware Setup amp gt 22 327 are rather close gt 140 dB may be necessary as in the case 14 19 More than 90 dB is usually hard to achieve with one filter Using external filters has three principal safety aspects 1 Make sure you do not pulse into a filter with a frequency that this filter does not pass 2 Make sure the pulse power you apply does not exceed the power rating of this filter Most modern Bruker filters will survive 1 kW pulses of 5msec but older fil ters or non Bruker filters may not 3 Remember that filters may attenuate the pulse RF voltage by as much as 1 5 dB about 20 The following figures illustrate the most common filter combinations Probe 1H reject or 0 31P pass or X bandpass Figure 3 6 Standard Double Resonance CP Experiment Bypassing the Proton Preamp Probe IH HPHPPR X BB HPHPPr 1H reject or 0 31P pass or X bandpass Figure 3 7 Standard CP Experiment Proton Preamp in Line BRUKER BIOSPIN User Manual Version 002
209. he 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 pro jected ellipse As can be seen from fig 1 the X and Y observe direction will see a signal of different 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 straightforward as it is with standard excitation observation The quad phase cycling must occur in the precession plane so a prepulse is required to tilt the initial magnetization into the direction of the preces sion axis Usually a combination of 2 pulses is used for initial excitation 90 y x y 90 55 adjust Figure 21 1 Difference in Amplitude of the Quadrature Channels X and Y The difference in amplitude of the quadrature channels X and Y caused by the tilted precession 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 direc tion of the external field the precession frequencies are changed which means that the observed chemical shifts are changed As t
210. he frequencies are always smaller than in the standard excitation observation scheme the chemical shift User Manual Version 002 BRUKER BIOSPIN 273 327 CRAMPS General range appears scaled down The scaling factor depends on the pulse sequence used To achieve a spectrum comparable to spectra acquired conventionally 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 scal ing factor in order 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 ac count will be given in the practical chapter 274 327 BRUKER BIOSPIN User Manual Version 002 CRAMPS 1D As outlined above many sequences are available to achieve homonuclear dipolar decoupling 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 22 1 Phases 164 1010 10 10 Figure 22 1 Pu
211. hee hene rene rne 152 Figure 11 6 13C DARR of Fully Labelled Ubiquitine Spinning at 13 KHZ 153 12 REDOR 155 Figure 12 1 REDOR Pulse Sequence sient etui to da kd dan AE ETERRA 157 Figure 12 2 2D data set after xf2 processing sssssssssssssssee eere 160 Figure 12 3 T1 T2 Relaxation for further Analysis of the Data Figure and the Analysis Interface 161 Figure 12 4 Saving Data to Continue to the Relaxation WiNd0W sss 161 Figure 12 5 Setting the Correct Analysis Parameter 162 Figure 12 6 Plot of the Normalized Signal Intensity Versus the Evolution Time 163 Figure 12 7 Experimental data for the glycine 13C 15N REDOR sese 164 Figure 12 8 Comparison of Experimental Data to a Simulation with Reduced Dipolar Coupling 165 Figure 12 9 Experimental data with the corresponding M2 parabolic analysis 166 User Manual Version 002 BRUKER BIOSPIN 311 327 Figures 13 SUPER 169 Figure 13 1 Pulse Sequence for 2D CPMAS exchange evperiment 170 Figure 13 2 The 123 Icon in the Menu Bar of the Data Windows Acquisition Parameter Page 171 Figure 13 3 The Acquisition Parameter Window eda sss 172 Figure 13 4 The SUPER Spectrum of Tyrosine HCI After Processing Using xfb 175 Figure 13 5 SUPER spectrum after tilting the spectrum setting 1 alpha 1 176 Figure 13 6 Various Cross
212. how many scans need to be acquired for each relaxation delay Create a new data set with iexpno and set the saturation 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 208 327 Set the parameters for the 2D acquisition as detailed in Table 16 4 For the variable recovery delay the same values can be used as for the inversion recovery experiment 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 acquisition time comprises most of the pulsing so d1 should be 220x aq 1 second is reasonable in this case If the experiment is run without de coupling then the saturation period is the only significant period of high power pulsing and dT can be shorter Acquire the 2D spectrum with zg BRUKER BIOSPIN User Manual Version 002 Relaxation Measurements Table 16 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
213. id Kal T Ise ICA Bruker TOFSPIN pta ml pp single pulse e R BC Bruker TOPSPIN exp stan nmr ists pp seggt Avance II ver Fie Graphical assistant Edit text Options parameters epos ee spl excitatio Expression Y Names l Grid f Blocks pll power le ZE 2u 4COMMENT sings Fl 74CLASS Solids 4DIM 1D TYPE direct e 16 SUBTYPE sinpi 14 0UNER Bruker schatll to ad acqt0 lutcnstl pl pli phi go 2 ph3l vr 0 exit phs 0213 ph3l 0 213 4 Ai Figure 4 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 frequency sweep over the range of SFO1 WBSW 2 The swept fre quency will only be absorbed by the probe at the frequency to which it is tuned 62 327 BRUKER BIOSPIN User Manual Version 002 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 4 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 SFO7 the probe response will be off center N B Fake resonances may appear which do not shift with probe tuning
214. ids require careful adjustment 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 cou pling in addition to CSA sidebands User Manual Version 002 BRUKER BIOSPIN 93 327 Decoupling Techniques dn 93 FSLG decoupling Af Af 1LGcycle 1LG cycle amp 00221133 0 L se 02201331 Figure 5 3 FSLG Decoupling Pulse Sequence Diagram Figure 5 4 Adamantane FSLG decoupled showing the downscaled C H J couplings 94 327 BRUKER BIOSPIN User Manual Version 002 Decoupling Techniques The figure above shows homonuclear proton decoupling on center packed ada mantane sample 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 p 12 and p3 pcpd2 for 70 100 kHz RF field de termine the precise RF field preferably via a 360 proton pulse p3 5 Load the pulse program fqlg This uses frequency shifts with simultaneous phase shifts for F
215. ier frequency o2 or o2p using popt The cw decoupling program is written as follows 0 5p pl pl12 reset power level to default decoupling 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 becomes more evident since the separation due to the mag netic field increases CW decoupling requires fairly high decoupling power to be efficient User Manual Version 002 BRUKER BIOSPIN 89 327 Decoupling Techniques TPPM Decoupling 5 1 2 TPPM decoupling surpasses the traditional cw decoupling The decoupling pro grams tppm15 and tppm20 use a 15 and 20 degree phase shift between the two pulses respectively Both operate at power level p 12 The cpd program tppm13 uses 15 degree phase shift as tppm15 but operates at power level p 73 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 cou pled 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 pcpd2 available in TS2 0 and higher mm OreePars AcquPars Tite Det Pesis rtegals
216. igh 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 13 4D CP experiment if necessary 6 Create a new experiment with either iexpno or edc 7 Change to a 2D data set User Manual Version 002 BRUKER BIOSPIN 145 327 Proton Driven Spin Diffusion PDSD 2D Experiment Setup 11 2 1 146 327 8 c 11 12 13 Type iexpno to create a new data file and switch to the 2D mode using the 123 button Load the pulse program cpspindiff Recheck pulse widths and power levels using ased Go into eda and set parameters for sampling in the indirect dimension the spectral width 1 swh Note that in TopSpin 2 1 or later the parameter N F1 re places the parameters inO and od Usually 1 swh equals swh Choose a suit able spin rate such that no RR condition occurs and sidebands do not overlap with peaks if possible set the sweep width in F1 1 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 appropriate quadrature detection mode in FnMode usually STATES TPPI Choose the appropriate sampling time td so that the required resolution EL DRES in the indirect dimension is achieved Set the desired mixing time as d8 The required multiple of sp
217. igure 8 2 The 12 icon and the ased icon in eda 6 Performing ased will show all parameters which are essential for the acquisi tion 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 pro gram between ze and exit In this sequence the proton chemical shift evolu tion is influenced by the RF field cnst20 under which the shifts evolve and the type of homonuclear 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 IN F1 in eda A button in ased This will set the sweep width along F1 Note that the time increment here is gener ated 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 sampling 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 002 BRUKER BIOSPIN 121 327 FSLG HETCOR OAM CY d installec prove 4 mm MAS DECH H13762 0001 General Channel f1 Channel f2 122 327
218. ii increase as one goes to nuclei with higher atomic mass In short HH condi tions may be very sharp T s may be long but proton T4 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 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 Heteronuclear Samples 15N 29SI 31P 6 5 102 327 1 15N on a glycine calculate HH condition as described above Else Load a glycine C reference spectrum set observe nucleus N15 in edasp add 2 dB to sp0 spnam0 ramp 100 BRUKER BIOSPIN User Manual Version 002 Practical CP MAS Spectroscopy on Spin 1 2 Nuclei subtract 2 dB from pl1 more is not required since the transmitter will usually put out 5096 more power at 15N frequency set p15 3 ms acquire 4 8 scans optimize HH condition acquire reference spectrum with aq 25 35 ms 2 Sion 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
219. ill reflect the quality of the setup Beware of crystal twinning The orientation of the sample should be selected carefully 7 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 impor tant for saline lipid water mixtures 8 Depending on the decision taken in 4 set a either PL13 PL2 case 4a and set PL11 to the optimized value higher pow er i e a value of about 1 8 dB below pl1 b or set PL11 PL1 case 4b and PL13 to the obtained value in the cplg experi ment i e about 1 8 dB higher than PL2 which is less RF power 9 Depending on the orientation of the single crystal O1 and O2 need to be re optimized since the peak positions will change Select an orientation that shows the most low field IDN peak positions because that will usually corre spond to the biggest 1H 15N dipolar couplings 10 Reoptimise power levels for the HH contact 11 Create a new experiment and setup a 2D data set using the 1 2 3 icon Load the pisema pulse program Go into the eda window 12 Make sure the correct nucleus is selected in the F1 dimension 13 In order to set the t4 increment go into the ased window clicking the pulse symbol and choose 13 to be 1 2 or 3 This sets the t4 increment and the pa rameter inO is updated Use the calculated value and set inf
220. in 2 1 single pulse excitation acquisition without decoupling a 4 P 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 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 CUR E ere 11 ae an 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 General Hardware Setup 15 Figure 3 1 All Connections to the Back of the Preamplifier m 16 Figure 3 2 Transmitter Cables only Wired to Back of the Preamplfier 17 Figure 3 3 The edasp setpreamp Display 18 Figure 3 4 Additional Connections to the Preamplifier Stack sssee 19 Figure 3 5 Matching Box Setup for High Power X BB Preamplifiers esee 20 Figure 3 6 Standard Double Resonance CP Experiment Bypassing the Proton Preamp 22 Figure 3 7 Standard CP Experiment Proton Preamp in Line
221. in periods from cnst31 is calculated as l1 the real mixing time may deviate by fractions of a rotor period The required mixing time may vary widely depending on the sam ple properties from a few milliseconds to hundreds of milliseconds if long dis tance correlations in a mobile sample need to be observed Note that longer mixing times will result in S N deterioration as the mixing time approaches the T4 of the observed nuclei BRUKER BIOSPIN User Manual Version 002 Proton Driven Spin Diffusion PDSD test 51 1 C BrukeriTOPSPIN denis Spectrum ProcPars AcquPars Title PulsePrag Peaks integrals Sample Structure Fid o n s W als Wa installed probe 4 mm MASDYT BE 1H HO724 0020 Expenment n F1 Frequency as W Experiment Receiver Weier PULPROG cpspindimt Current pulse program ren AQ_mod pap Acquisition mode Power FnMODE States TPPI D Acquisition mode for 20 3D etc Program TD 128 i2 Seeoffid Probe NS a Number of scans Lists DS b 1 Number of dumrriy scans Wobole TDD fi Loop count tor too rade width Automation MENGE ERAS Sw ppm 250 9772 ae 1443 Spectral width Fees SWH Hz 7878789 9001348 Spectral width Routing IN F ys i632 Increment for delay AG s o 0017396 b 0196903 Acquisition time FIDRES Hz 295 928040 25 393251 Fid resolution Fw Hz 250000 00 Fiter woth gt Receiver Y Nucleus 1 NUC 1 rac Observe nucle us O1 Hz Gesi 18108 85 Transmitter frequency offset O1P pp
222. ine the 90 degree carbon pulse p1 using popt Recalculate ol for a 4 5 usec carbon pulse using calcpow lev 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 4 18 and Figure 4 19 show the adamantane 13C FID without shims with z shim adjusted and the corresponding setsh displays User Manual Version 002 BRUKER BIOSPIN 73 327 Basic Setup Procedures EE A serch Setshim Set shim values and lock parameters Press ENTER to set new value slelelelololololololololololololoim Figure 4 18 Adamantane 1 C FID with 50 msec aq setsh Display The figure above is an Adamantane C 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 shimming effort only with older probes up to 400 MHz proton frequency Soedus Eat Aca Par IER PuneProg Pera Fa Saroe Suwe nd Amu sae ame Ss IA Setsnim Set shim values and lock parameters Press ENTER to set new value 5000 0 D D 0 o D D 0 0 D n D D n o Close Figure 4 19 Adamantane 1 C FID with 50 msec aq setsh with Optimized Z Shim Value 74 327 BRUKER BIOSPIN User Manual Version 002 Basic Setup Procedures For optimum shims rarely required se
223. ined from MQMAS spectra BRUKER BIOSPIN 244 327 MQ MAS Sensitivity Enhancement Fast Amplitude Modulation FAM 18 3 It must be mentioned at this point that similar approaches have been made where the frequency of irradiation is established by a fast modulation of the amplitude of the pulses This is realized 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 alternating x and x which creates a fast cosine type amplitude modulation The frequency of this amplitude modulation ap pears to the spin system as an irradiated frequency Two pulse programs are available mp3gfamz av and mp3qfam av They corre spond to the pulse sequences depicted in Figure 18 3 and Figure 18 4 but the shaped pulse realizing the DFS is replaced with a sequence D2 P2 P2 D2 em bedded in a loop repeated by loop counter L2 and with power level PL 14 for the pulses These sequences are only useful for spin 3 2 nuclei There are also se quences 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 frequency whereas DFS continuously irradiates sweeps over a range of frequencies These frequencies must lie in the range of the satellite tran sitions th
224. ing 1 1 iic ne ea eu and 91 Homonuclear Decoupling Iit eftt ine Hesse din 92 Multiple Pulse NMR Observing Chemical Shifts of Homonuclear Coupled Nuclei 92 Multiple Pulse Decoupling ccccccceccecceeeceeeceeceeeceeeeeece esse aA a aa aaa 92 BR 24 MREV 8 BLEW 12 ooo cece ec cccecee ce eeeeee ee eeee eee eeeee ae teeee aaa eeesaaeeeesaaeeeeaeaeeeeesaeeeeeaa 92 FSLG Decoupling u oed deb dese tached aves DR Ep dE pede tdeo tle ici 92 DUMBO 97 Transverse Dephasing Optimized Spectroscopy sssssssssse 98 Practical CP MAS Spectroscopy on Spin 1 2 Nuclei 99 Possible Difficulties iie ecd eee ea ge Cope ode DI d uode id 99 Possible Approaches for 13C Samples ssssssssssssssssssen enn 99 Possible Approaches for Non 13C Samples ssssssssss nn nenn 101 Hints Tricks Caveats for Multi nuclear CP MAS Spectroscopy 102 Setup for Standard Heteronuclear Samples 15N 29SI 21 102 Basic CP MAS Experiments nenn 105 Pulse Calibration with CP u Br tette Raub ena Ree e D np DR en a e a do RR Lu RR 105 Total Sideband Suppression TOSS sss emen 106 SELVICS EL 110 Non Quaternary Suppression NQS sssssssssee emen 113 Spectral Editing Sequences CPPI CPPISPI and CPPIRCP sese 116 FSLOG HETUCOMH E 119 Pulse Sequence Diagram for FSLG HETCOR
225. ing 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 homonuclear recoupling in rotating solids Application to double quantum spectroscopy J Chem Phys 110 7983 1999 M Hong Solid State Dipolar INADEQUATE NMR Spectroscopy with a Large Double Quantum Spec tral Width J Magn Reson 136 86 91 1999 A Brinkmann M Ed n and M H Levitt Synchronous helical pulse sequences in magic angle spin ning 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 homonuclear dipolar recoupling in rotating solids J Chem Phys 117 4974 2002 C E Hughes S Luca and M Baldus RF driven polarization transfer without heteronuclear decou pling in rotating solids Chem Phys Letters 385 435 440 2004 BRUKER BIOSPIN User Manual Version 002 Symmetry Based Recoupling Pulse Sequence Diagram Example C7 14 1 Qi 10 1 H CW or CW LG decoupling TPPM dec b2 4 0161 13 014 ds Orec eee chee t gem RF 2 rotor periods 14 2x pulses 1 phase rotation about 360 57 1230 11 127 9 180 13 14 7 90 QD phase o 9 180 80 doo 0 123 bez 0321 Ld Figure 14 1 C7 SQ DQ Correlation Experiment Setup 14 2 As mentioned before it is essential that the parameters o
226. inning side bands in the indirect dimension extend over a very wide range which cannot be truncated by e g filtering Therefore rotor syn chronization together with States or States TPPI phase sensitive acquisition helps to fold spinning sidebands from outside back onto centre bands or other side bands Table 18 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 NUC1 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 IN0 19 12 For spin 5 2 1N0 101 45 For spin 7 2 IN0 91 36 For spin 9 2 User Manual Version 002 BRUKER BIOSPIN 239 327 MQ MAS Sensitivity Enhancement Table 18 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 Acquis
227. ion EL DRES in the indirect dimension is achieved 11 Set pl1 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 p2to be a 180x pulse at pl1 for the TOSS sequence 14 Set 15 for the gamma integral typically number of spinning sidebands nor mally 4 15 Start the experiment User Manual Version 002 BRUKER BIOSPIN 171 327 SUPER Spectrum PracPars AcquPars Title PulseProg Peaks integrals Sample Structure Fid Acqu o n S W m 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 IDE 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 DD F 1 AQ 5 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
228. ired The integrated intensity of the echo will be almost twice the intensity of the single FID it is just T2 relaxation during r that leads to attenuation In MAS ex periments it is advisable to synchronize 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 18 2 because after a normal FT the signal looks quite unconventional To obtain the usual spectrum a magnitude cal culation can be done on 1D spectra with the loss of phase information Alterna tively and in particular in 2D spectra it is possible to apply a large 1 order phase correction phc1 to compensate for the time delay before the echo top The value of this is d6 hel 22 4808 P d w Eq 18 1 This value can be entered into the processing parameters and a phase correction pk can be performed After this the 0 order phase correction still needs to be ad justed 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 002 MQ MAS Sensitivity Enhancement FT PK p EE WER ENEE Lee 0 5 ms 10 40 60 ppm 40 60 ppm Figure 18 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 cor rect first order ph
229. ise decoupling during t1 could be imple mented using real frequency shifts as in the HETCOR sequence see Decou pling Techniques on page 89 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 24 1 Acquisition Parameters Parameter Value Comment pulprog wpmlg2d 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 292 327 BRUKER BIOSPIN User Manual Version 002 CRAMPS 2D Table 24 1 Acquisition Parameters sw swh along F1 Same as for F2 Needs to be corrected before transform pulse pro gram calculates approximate values to be set before transform ased td 512 1k Depending on resolution 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 322 or 4 depending on desired sw1 DUMBER 22 with modified timing Table 24 2 Phases RF levels and Timings Phases RF Power Levels Timing du 0 STATES TPPI CYCLOPS 1230 pl12 pl12 set for around 100 kHz p1 around 2 5 usec p1 05 2 10 2 02 sp1 sp2 set for 100 130 kHz RF WPMLG calculated via cnst20 field or p
230. isition Parameters 4 2 2 Create a new data set for the experiment by typing edc in the command line User Manual Version 002 BRUKER BIOSPIN 59 327 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 Leet Solvent ww Experiment Use current param Y TEILE ext j Receivers LZ 8 OK Cancel More Info Help Figure 4 3 Pop up Window for a New Experiment Spin the KBr sample moderately 5 2 5 mm 10 kHz In order to set up the ex periment type ased in the command line to open the table with parameters used for this experiment 60 327 BRUKER BIOSPIN User Manual Version 002 Basic Setup Procedures 2 kg 1 1 opthewtopspin pkiesis n Spectrum ProcPars AcquPars Title PulseProg Peaks integrats Sample Structure Fid Acqu Phase Print e A Ze NM EST A Installed probe 4 mm MASDVT 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 j 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 ys A 50 Pre scan delay
231. ition 8 Unknown relaxation properties proton T4 Tip T s Possible Approaches for 13C Samples 6 2 1 Collect as much information about the sample as possible Do not accept samples for measurement 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 nucleus of in terest mobility rigid environment expect long T4 and repetition delay proximity to protons can one use cross polarization User Manual Version 002 BRUKER BIOSPIN 99 327 Practical CP MAS Spectroscopy on Spin 1 2 Nuclei 100 327 conductivity dielectric loss expect tuning and RF heating problems if sample is dielectrically lossy or even conductive Collect information about the sample first by running an easy nucleus Feasibility of cross polarization parameters is the required key information be cause it decides the steps to follow If the sample information which you have collected shows that a SC 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 p15 to 1ms wait 1 min do one scan There should be a visible signal From there on optimize the required repetition rate d1 contact time p75 number of scans ns
232. ition mode for 2D States TPPI TD see text Number of FID s to be acquired SWH masr 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 N0 7 9 For spin I 3 2 so that no shearing FT is required 0 For all other spin I Data Processing 18 2 3 240 327 Processing parameters should be set according to Table 18 4 Data obtained with mp3qdfs av can be processed with xfb alone if IN10 or INT 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 necessitates a large 1 order phase cor rection to compensate for the start of the acquisition before the echo top This cor rection can easily be calculated as given in Eg 18 1 and should be stored into the parameter PHC1 This gives an approximate value which can be precisely adjusted 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 BRUKER BIOSPIN User Manual Version 002 Table 18 4 Processing Parameters MQ MAS Sensitivity Enhancement P
233. itions of high gas throughput The maximum throughput de pends on the experimental conditions and the probe type BRUKER BIOSPIN User Manual Version 002 MAS Tubing Connections The following gas requirements exist 1 2 At room temperature or higher dew point min 30 C compressed air will do At temperatures 200 C or higher suitable probe required nitrogen is re quired to prevent coil oxidation At temperatures between room temperature and 50 C using a B CUX cryo cooler with 80 C exchanger temperature nitrogen or compressed air with a dew point x 100 C At temperatures below 50 C using liquid nitrogen and heat exchanger boil off nitrogen with a dew point 196 C Any compressed gas used in NMR probes must be free of any liquid droplets or of oil from compressor lubrication Especially oil even smallest amounts will lead to probe arcing and or spinning problems and potentially expensive repair Using boil off nitrogen should be carefully considered since it is by far the most reliable stable and trouble free source of compressed gas to be used at any temperature Connections 3 5 1 MAS tubing connections are quite different between different types of probes for stationary non spinning probes only frame flush and VT gas are required 1 WB probes VTN WVT and DVT probes VTN VT normal range WVT VT wide range These probes have a diameter of 72 mm and are l
234. l 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 excitation p3 Excitation pulse f2 channel pcpd2 Decoupler pulse length f2 channel WEN 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 10 s for histidine Recycle delay optimize on 1d spnamO Ramp for 18 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 User Manual Version 002 BRUKER BIOSPIN 265 327 Double CP Table 20 2 Recommended Parameters for the DCP 2D Setup Sw 200 ppm Sweep width direct dimension adjust to experimental require ments F1 indirect N right column td 128 512 Number of real points sw 100 150 ppm Sweep width indirect dimension Spectral Processing Processing parameters 20 4 Table 20 3 Recommended Processing Parameters for the DCP 2D Parameter Value Comment F1 acquisition 6 left column si 2 4k FT size wdw QSINE Squared sine bell ssb 2 5 Shifted square sine bell gt 2 res enh
235. l gas For MAS probes this flow can be the bearing gas VTN or it can be separate DVT In any case the VT control gas will flow through a dewar which contains a heater There will be at least one thermocouple which senses the temperature as close as possible to the sample Several requirements must be fulfilled to obtain precise and stable temperature readings as close as possible to the real sample temperature This will however be part of a different chapter Some connections are required to control the temperature of the probe sample others are necessary to protect the probe and the magnet from illegal tempera tures MAS probes are usually not as well insulated as for instance a wideline or PE stationary probe is Therefore the probe outer shell warms cools down during the experiment The heat transfer between heater and probe electronics probe environment must be kept at a safe level User Manual Version 002 BRUKER BIOSPIN 31 327 General Hardware Setup 32 327 A 1 Probe heater connector 3 TC connector s Safety precautions involve flushing the probe frame this serves to keep the tuning elements at decent temperatures Furthermore the magnet must be kept at legal temperatures to prevent freezing of O rings or excessive ex pansion of the inner bore tube The shim stack must be kept at tempera tures below 70 C else the shim coils might be damaged With MAS probes it must be made sure that no wet air is sucked in
236. l13 for both set in ppg DUMBO p10 set by xau dumbo 11 202 dto dto 4 CYCLOPS 0 1 23 d8 desired mixing time 50 1000 us Data Processing 24 3 The spectral width in both dimensions assumes the absence of shift scaling In or der to account 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 sw to call the status parameters for both F2 and F1 and replace both val ues by lt current value gt 0 6 After xfb the relative peak positions will be approxi mately correct but the absolute peak positions must be corrected 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 24 3 Processing Parameters Parameter Value Comment mc2 STATES TPPI wdw QSINE Slight moderate resolution enhancement is usually required User Manual Version 002 BRUKER BIOSPIN 293 327 CRAMPS 2D Table 24 3 Processing Parameters ssb 3or5 si 2k 4k 1si 512 1k Examples 24 4 ppm JIM 91 LU Hi ppm 10 o gt gt 4 3 20 10 o ppm i Glycine proton proton shift 74 correlation using pmlg for homonuclear decoupling in both dimensio
237. late 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 calculating the required RF field for the HH match can be done in the following way BRUKER BIOSPIN 135 327 Modifications of FSLG HETCOR 136 327 140 Load the pulse program Igcp into a standard CP MAS data set with all parame ters set and optimized Set p14 to about 54 flip angle Use tyrosine HCI or the sample of interest From p3 calculate the power level p 2 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 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 margin of 1000 the HH condition will cover 75000 down to 45000 Hz RF field This includes both HH sidebands n 1 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 Mni uM rm hi wi HCI One should see intensity variations dipolar oscillations
238. le RF fields used in WB probes Table 4 3 Reasonable RF fields for Max 296 Duty Cycle Decoupling power over 50 ms 200ms Prope 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 24 130 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 6 71 kHz 3 5 us probe 15 kHz max sample rotation 62 kHz 4 us 4 mm CPMAS triple resonance probe 130 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 re 55 kHz 4 5 us 7 kHz sample rotation 50 kHz 5 us Note Higher RF power levels should only be applied if necessary and within specifications For special probes max allowed RF fields may be lower Check with your Bruker BioSpin applications support if in doubt In order to have quantitative information about the precision of your magic angle one may measu
239. le pulse techniques and magic angle spinning J couplings and large heteronuclear dipolar couplings are not suppressed Reference 1 L M Ryan R E Taylor A J Patt and B C Gerstein An experimental study of resolution of proton chemical shifts in solids Combined multiple pulse NMR and magic angle spinning J Chem Phys 72 vol 1 1980 Homonuclear Dipolar Interactions 21 1 Homonuclear dipolar interactions among spins with a strong magnetic moment and high natural abundance mainly 1H or 19F and to a much smaller extent P are usually very large unless 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 suppress the homonuclear dipolar broadening Even spin rates in the order of 70 kHz which is no longer a mechani cal problem cannot fully average this interaction in rigid solids As chemical shift differences among the coupled nuclei become larger the interaction becomes more heterogeneous and MAS can suppress it more efficiently This is the reason why fast spinning alone works much better on 19F or 31P than on protons hetero nuclear dipolar coupling such as between 13C and 1H can in principle be spun out but only if the homonuclear 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 ar
240. lear dipolar coupling strength Through the combination of spin exchange dipolar flip flop term and the homo nuclear decoupling 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 be caused among other things by a proton frequency offset introducing a constant term in the time domain signal That offset frequency makes also the splitting larger See additional test proce dures in the paper about experimental 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 di mension depends of the 1H resonance offset and a wrong 1H carrier frequency can cause besides a wrong scaling factor some intensity loss and as mentioned above 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 quadratic with increasing proton frequency offset Simulations of the spin dynamics show that the heteronuclear term in the Hamilto nian leads to a complicated spectrum for small heteronuclear dipolar couplings usually introduced by remote protons see Z Gan s paper for more information User Manual Version 002 BRUKER BIOSPIN 193 327 PIS
241. ligopeptide or small protein will of course provide a more interesting spectrum With proteins good results should only be expected if the preparation is micro crystalline In such a case water salt and cryo protectant glycol glycerol will very likely be pres ent This means that the probe proton channel will be detuned to lower fre quency and tuning may be difficult if not impossible at high proton frequencies and salt contents In such cases Ef probes are recommended 2 Run standard 1D cp 136 and 15N experiments determine the required offsets for all frequencies 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 doubcp Set FnMode as desired usually STATES TPPI 6 Make sure the correct nucleus SN is selected in the F1 dimension 7 Set the sampling windows for both dimensions from the previously acquired 1D spectra 8 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 heating 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
242. llows see Appendix Table 6 1 Power Conversion Table Probe 4mm Triple Nucleus Frequency P90 us Rf field Khz Power 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 130 400 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 002 BRUKER BIOSPIN 101 327 Practical CP MAS Spectroscopy on Spin 1 2 Nuclei 2 Once these values are measured any HH condition can be calculated As sumed you want to cross polarize 19Sp the sample spins at 12 kHz The con tact 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 sidebands 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 119Sn we need to apply 50 kHz RF field Since the RF field is proportional to the am plitude in a shape RF voltage output is proportional to shape amplitude val ue the shape power must ra
243. log Mode Parameter Value Comment pulprog dumboa 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 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 280 327 BRUKER BIOSPIN User Manual Version 002 CRAMPS 1D Fine Tuning for Best Resolution 22 6 For fine tuning the following parameters are important P9 sets the width of the observe window The shorter it is the better the resolu tion 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 narrow bandwidth long dead time so p9 lt 3usec is only possible at frequencies 400 and higher With 11 4 8 p9 can be chosen shorter for better resolution but at the cost of S N Sampling more data points during d9 11 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 resolution Since th
244. lse Sequence Diagram Table 22 1 Phases RF Levels Timings Phases RF Power Levels Timing di CYCLOPS 1230 pl12 set for around 100 kHz p1 around 2 5 usec da 0 cnst25 adjust ditto p4 about 45 degrees adjust 10 0 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 Q5 CYCLOPS 0123 User Manual Version 002 BRUKER BIOSPIN 275 327 CRAMPS 1D Pulse Shapes for W PMLG and DUMBO 22 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 p10 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 results aulgFgo m V ucne wpmigi Open 00 0 000000 0 000000 0 NPONTS Ev Ce TOTROT Bwrac INTEGRAL MODE Edt Shape Parameters 276 327 aw L Figure 22 2 PMLG Shape for wpmlg sp1 BRUKER BIOSPIN User Manual V
245. lues for the pulses are entered so one should see some signal for further optimization Parameters like O1 TD SWH RG should al ready be set in the standard 1D spectrum Since the experiment is not as depen dent 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 19 3 Initial Parameters for the Set up of stmasdfgz av Parameter Value Comments pulprog stmasdqfz 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 250 327 For PL11 an initial value that corresponds to 150 to 300 W can be used Optimiza tion will be done on the first increment of the 2D sequence which is calculated within the pulse program according to DO 1s L0 CNST31 P1 2 P4 0 3y P2 2 BRUKER BIOSPIN User Manual Version 002 STMAS because it is essential that the centres of the p
246. m 20000 120 000 Transmitter frequency offset SFO1 MHz 150 9251918 150 9251918 Transmitter frequency BF1 MHz 150 9070830 150 9070830 Basic transmitter frequency Y Nucleus 2 NUCI 1H Edit 2nd nucleus 02 Hz 1500 00 Frequency offset of 2nd nucleus 02P pprn 2 426 Frequency offset of 2nd nucleus SFO2 MHz 600 1485000 Frequency of 2nd nucleus BF2 MHZ 500 1470000 Basic frequency of 2nd nucleus ni Figure 11 2 The Acquisition Parameter Window eda 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 pro cedure shown in 11 7 15 Start the experiment Acquisition Parameters 11 3 Sample 13C labelled histidine labelled tyrosine HCl Experiment time 90 min 20 min User Manual Version 002 BRUKER BIOSPIN 147 327 Proton Driven Spin Diffusion PDSD Table 11 1 Acquisition Parameters Parameter Value Comments PULPROG cpspindiff old cpnoesy Pulse program NUC1 Se Set 1 C 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 H excitation and decoupling PL14 for n spin rate DARR or 120 Recoupling SPO For 1H contact using shape SPNAMO ramp 100 or ramp70100 100 For 1H 19C contact
247. m 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 13C 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 D A Torchia The measurement of proton enhanced 13c T4 values by a method which suppresses artifacts J Magn Reson 30 613 616 1978 The TOPSPIN software includes a tool for processing the data obtained in relax ation measurements and this will be demonstrated for the different types of relax ation experiment Describing Relaxation 16 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 trans User Manual Version 002 BRUKER BIOSPIN 201 327 Relaxation Measurements verse magnetization to zero is terme
248. me in f2 ns 8 number of scans per experiment fnmode QF phase correction mode in the f1 dimension rg 16 64 receiver gain level digmod baseopt or digital digitizing mode Data Acquisition 12 2 1 Setup of the 2D data set After the optimization as described above type iexpno to create a new data set afterwards switch to 2D data mode by using the 123 button Set the time domain for the F1 Dimension according to the maximum desired evolution time of the final REDOR curve To calculate the value for td1 you have to keep in mind that the REDOR Program is organized in multiples of two rotation periods compare with the pulse program scheme E g for a given MAS rate of 10 kHz and a desired overall evolution time of 10 ms you have to set td1 to a value of 200 This will re cord 100 sets of Sp and S experiments with an maximum evolution time in the last two data rows of the desired 10 ms The pulse program library supplies the follow ing REDOR sequences cpredor standard REDOR for dipolar couplings gt 500 Hz incrementing in 2 rotor period intervals Two data sets are required with pl3 set for pulsing REDOR ex periment or no pulse 2120 dB reference experiment cpredori stores these data sets in the 2D data frame by interleaving scans of the So and S experiments for the same evolution period see Figure 12 2 cpredorxy8 increments in units of 16 rotor periods for small couplings Here the XY 8 scheme is
249. ment 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 Eile Edi View Spectrometer Processing Analysis Options Window Help JB DSB HUN 424 17 z ut B aQg TTUO is 7nunmekwAaGaugmeoeaM f 1i Serum Probar AcQuPan Tele PumeProg Peaks Megan Saepe Structure Mid Acqu Phase Print Figure 4 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 Correspond ingly the intensity 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 opti mized Two more parameters are essential 1 The power level at HH contact 2 The decoupling pulse pcpd2 User Manual Version 002 BRUKER BIOSPIN 83 327 Basic Setup Procedures 84 327 The spectrum of Figure 4 26 was taken at contact power levels as set for ada mantane Furthermore 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 power level of the contact needs to be increased Set spnam0 ramp70100 100 Set sp0 and pl1 to about 2 dB less
250. ment 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 pulse 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 WaHuHa 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 sequences a lot easier since pulse phase and amplitude errors are negligible higher magnetic fields have led to better chemical shift dis persion and also to shorter dead times The resolution achieved with long highly compensated sequences like BR 24 is very good but their applicability 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 1 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 2 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 21 3 W PMLG and DUMBO are shorter seque
251. most efficient HH conditions Note that increas ing the spin rate would shift all maxima except the one at 4 8 dB further out BRUKER BIOSPIN User Manual Version 002 Basic Setup Procedures Cross Polarization Setup and Optimization for a Real Solid Glycine 4 7 Adamantane is highly mobile even in the solid state Therefore it behaves differ ently 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 how ever extremely sensitive to HH misadjustment 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 Since glycine may exist in two different crystal modifications with very different CP parameters and since packing of the spinner determines crucially the achiev able S N value it is useful to prepare a reference spinner with pure a glycine fine ly powdered and densely packed a glycine is prepared by dissolution of glycine in distilled water and precipitation with acetone quick filtering and careful drying in a desiccator Drying is important because wet glycine may readily transform espe cially when kept warm into y glycine a glycine has two carbons with shifts of 176 03 and 43 5 ppm y glycine shows resonances somewh
252. mperature controller Figure 3 29 Low Temperature Liquid N Dewar with DVT Probe Heat Exchanger 38 327 BRUKER BIOSPIN User Manual Version 002 Additional Connections for VT Operation Q flush he rulation then les 1 Gas in 4 Spin rate 7 Transfer line 2 Frame flush 5 Heater 8 Read and regulation thermocouples 3 Shims 6 Magnet bore 9 Support for transfer line Figure 3 30 Bottom view of Low Temperature DVT Probe Heat Exchanger User Manual Version 002 BRUKER BIOSPIN 39 327 General Hardware Setup EMIT ech Fee eee ex nd tray fu read lation then 1 B CU X heat exchanger and transfer line 4 Read and regulation thermocouples 2 Bypass 5 PICS probe identification 3 Heater 6 Bearing drive Figure 3 31 Low Temperature Setup with B CU X or B CU 05 In the figure above is the low temperature setup with a B CU X or B CU 05 for DVT probes only shown from the probe magnet side 40 327 BRUKER BIOSPIN User Manual Version 002 Probe Setup Operations Probe Modifiers heater in out VT gas in unused from B VT 3000 p p9 I gg to bypass a when heater contra _ to B 10 Y 1 To bypass when heater is off 3 Control to B VT 3000 2 VT gas in from B VT 3000 4 Heater in out unused Figure 3 32 Low temperature setup with B CU X Probe Setup Operations Probe Modifiers 3 7 Setting the Frequency Range of a Wideline single frequency Probe 3 7 1 In
253. n 010 For rotor synchronized experiments in 010 1 spinning frequency so will typical ly be between 100 us 10 kHz spinning and 28 5 us 35 kHz spinning so only 10 to 40 experiments in amorphous 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 outside 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 recommended 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 002 BRUKER BIOSPIN 221 327 Basic MQ MAS Data processing 17 4 Processing parameters should be set according to the following table Table 17 4 Processing Parameters for 2D FT Parameter Value Comments F2 acqui
254. n be used directly to enhance sensitivity by soft pulse added mixing pulse program mp3qspam av In MQ MAS Sensitivity Enhancement on page 231 some of the sensitivity enhancement techniques will be described Note that pulse programs suitable for AV and AVII spectrometers have the exten sion av pulse programs for the AVIII have no extension 17 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 ex periment can be optimized using the 2D pulse sequence for t4 0 Setting Up the Experiment 17 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 described here the spin of the nucleus is not important of course obtained pulse widths will depend 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 s
255. n order to store the two different coherence transfer pathways in consecu tive FID s in the serial file BRUKER BIOSPIN User Manual Version 002 MQ MAS Sensitivity Enhancement Figure 18 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 213 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 list ed in Table 18 6 Table 18 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 002 BRUKER BIOSPIN 243 327 MQ MAS Sensitivity Enhancement 244 327 FNMODE Even though this parameter is not evaluated by the pulse program it will be used by the processing AU program xfshear
256. n the very short recovery delay no appreciable relaxation will have occurred Now we can set parameters for the 2D acquisition as in Figure 16 1 Since this is a pseudo 2D experiment the only relevant parameter 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 longest T4 value Of course the accurate relaxation time constants are not known in ad vance but order of magnitude estimates can be obtained by running the 2D ex periment with a small number of relaxation delays and a small number of scans per slice The relaxation delays should be approximately equally spaced in log de lay in order that decays with all time constants in the range are equally well char acterized 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 BRUKER BIOSPIN User Manual Version 002 Relaxation Measurements Table 16 2 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 th
257. nce Therefore these checks should be performed pe riodically 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 infor mation about TopSpin software commands is available in the help section within the appropriate chapter Setting up a CPMAS probe from scratch requires the following steps 1 Mount the probe in the magnet and connect the RF connectors of the probe to the appropriate preamps 2 Connect the spinning gas connectors and the spin rate monitor cable 3 Insert a spinner 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 User Manual Version 002 BRUKER BIOSPIN 55 327 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 4 1 Despite the fact that most spectra taken on a CP MAS probe
258. nce Transfer Patbway 234 312 327 BRUKER BIOSPIN User Manual Version 002 Figures Figure 18 5 Example for popt to Set up for Optimization of DFS ooooccocicoccccccnconnconcnnncnnnnnnnnnnnos 237 Figure 18 6 Signal Intensities of 87Rb in RbNOS coccccocccnccccnccccncnnnnnnnn nano memes 238 Figure 18 7 Pulse Sequence and Coherence Transfer Pathways for SPAM 3QMAS 243 19 STMAS 245 Figure 19 1 Principle of 2D Data Sampling in STMAS Experiments sssssesss 245 Figure 19 2 Four pulse sequence and coherence transfer pathway for the double quantum filtered STMAS experiment with z filter stmasdqfz av ssssss nenn 247 Figure 19 3 Four pulse sequence and coherence transfer pathway oooooonoccocccnccccoccncncnncnnancnanono 248 Figure 19 4 87Rb STMAS Spectra of RDNOZ3 mme menm nennen rhe 252 20 Double CP 255 Figure 20 1 Pulse sequence diagram for 1D t1 0 and 2D double CP experiments 256 Figure 20 2 The edasp routing tables for H C N double CP 0 eee enters terres 257 Figure 20 3 Routing table for triple resonance setup change for 15N pulse parameter measurement and CPMAS optimization nennen 258 Figure 20 4 Shape Tool display with ramp shape from 45 to bbneo 261 Figure 20 5 Shape Tool display with a tangential shape for adiabatic cross polarization 262 Figure 20 6 Double CP optimization of PL5 in increments of 0 1 dB oononncnicnnnoccncnnocc
259. nces 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 de coupling period As a result the sequences are simpler and shorter requiring few er adjustments and allowing higher spin rates Both sequences use repetitive shaped pulses with detection in between PMLG uses the principle of a Frequency Switched Lee Goldburg FSLG sequence con tinuous irradiation with a net RF field along the magic angle where the frequency shifts are replaced by a phase modulation DUMBO basically works like a window less MREV type pulse sequence where the individual pulses are replaced by a single pulse with phase modulation References 1 E Vinogradow P K Madhu and S Vega High resolution proton solid state NMR spectroscopy by phase modulated Lee Goldburg experiment Chem Phys Lett 314 443 450 1999 2 D Sakellariou A Lesage P Hodgkinson and L Emsley Homonuclear dipolar decoupling in solid state NMR using continuous phase modulation Chem Phys Lett 319 253 2000 272 327 BRUKER BIOSPIN User Manual Version 002 CRAMPS General Quadrature Detection and Chemical Shift Scaling 21 4 Under homonuclear decoupling the magnetization precesses in the transverse plane of a tilted rotating frame whose new z axis is along the direction of the effec tive field The projection of this plane into t
260. ncl a pulse p5 is cal culated from cnst20 RF field in Hz The total shape pulse length must be 2 p5 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 LG TS2 1 only 3 pmlg sp1 ph3 f2 for one full PMLG unit as for Igs 1 shape lo to 3 times count References 1 A Bielecki A C Kolbert and M H Levitt Frequency Switched Pulse Sequences Homonuclear De coupling and Dilute Spin NMR in Solids Chem Phys Lett 155 341 346 1989 2 A Bielecki A C Kolbert H J M deGroot R G Griffin and M H Levitt Frequency Switched Lee Gold burg Sequences in Solids Advances in Magnetic Resonance 14 111 124 1990 3 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 AU Ya cos no f 4 b sin nof ax n 0 The shape can be created using the AU program DUMBO The DUMBO shape file in the release version of TOPSPIN is calculated for 32 us pulses To create your own DUMBO shape yo
261. nconnnnnnnnannnnnnannnnnnnnns 121 Figure 8 3 he ased Display near e aee nalen ee A Ud Re eae 122 Figure 8 4 FSLG Hetcor Spectrum Tyrosine HCl sssssssssese eene 125 Figure 8 5 FSLG Hetcor Spectrum Tyrosine HCl oocooocccoccccnccccnnccnnoconncnnnnccnnnconannonnncnnncconancnanins 126 9 Modifications of FSLG HETCOR 127 Figure 9 1 Comparison of HETCOR with and without 13C decoupling cccooccoccccncccconccnnaccnncnns 128 Figure 9 2 HETCOR Using Windowless Phase HRamps sss 130 Figure 9 3 HETCOR on tyrosine HCI without left and with LG contact 1msec contact 136 10 RFDR 137 Figure 10 1 RFDR Pulse Sequence for 2D CPMAS Exchange Experiment 138 Figure 10 2 The 123 Icon in the Menu Bar of the Data Windows Acquisition Parameter Page 139 Figure 10 3 13C Histidine Signal Decay as a Function of the RFDR Mixing Time 141 Figure 10 4 2D RFDR Spectrum of 13C fully Labelled Histidine RFDR mixing time 1 85 ms 142 11 Proton Driven Spin Diffusion PDSD 143 Figure 11 1 CPSPINDIFF Pulse Sequence 00 cee eee teres mehreren 145 Figure 11 2 The Acquisition Parameter Window eda sssssee HH 147 Figure 11 3 POPT Result for the cw Decoupling Power Variation 150 Figure 11 4 13C CPSPINDIFF of fully labeled tyrosine HCl spinning at 22 kHz 4 6 msec mix Up per PDSD lower DARR esee nenne ne nemen sisse ns se rasan essa nnns 151 Figure 11 5 Comparison of DARR PDSD ene Hee
262. ncy range shift shown in Figure 3 34 As the triple tuning insert will act as a load to the whole circuit the frequency range will shift a bit to lower frequencies 2 Both frequencies X and Y should be within the same basic probe tuning range high or low A 2 high range or N4 low range mode 1 X Tuning capacitor 3 X Y trap stops X frequency into 2 Y Tuning capacitor Y channel but not Y into X channel Figure 3 39 Mounting a Triple Insert Into a Triple Probe User Manual Version 002 BRUKER BIOSPIN 49 327 General Hardware Setup These inserts can be made to optimize the X or the Y channel They should be mounted in exactly the position as indicated on the information sheet included with the probe Reversing the trap may mess up tuning Observe probe manual instructions For low range below T nuclei the probe must be in A 4 mode Combinations of low high range nuclei are difficult impossible and always lossy Mounting the Probe in the Magnet Shim Stack 3 8 50 327 Usually the service engineer installing the magnet and the rack has considered the local restrictions and placed the system such that all operations which may be required for the proper probe installation are conveniently possible This refers to the ease of access of the magnet bore from below and above to mount the probe and the sample insert devices and also to the possibility to place VT equipment liquid N dewar B CU 05 or B CU X in a co
263. nge 3 7 2 42 327 Most probes cover a fairly wide frequency range Changing the frequency range of a probe requires either a change in the inductance or capacitance of the circuit The inductance of a circuit is hard to change unless a coil is mechanically length ened or shortened Most probes are tuned over a certain range by variation of a capacitance The frequency range is then determined by the minimum and maxi mum capacitance that can be set In order to make the inductance as high as pos sible since the signal from the oscillating magnetization is detected in the inductive part one usually chooses a capacitance with very small minimum ca pacitance which again means a not large enough maximum capacitance So in most probes additional tuning components must be inserted removed to achieve the full tuning range The highest signal to noise is always reached with maximum inductance and minimum capacitance i e at the high end of the tuning frequency achieved with maximum inductance BRUKER BIOSPIN User Manual Version 002 Probe Setup Operations Probe Modifiers In a wideline probe the NMR coil is easily replaced So with a few coils of different inductance one can extend the tuning range determined by the tuning capacitor inside the probe This cannot easily be done in a MAS probe for two reasons 1 2 The coil must be carefully aligned such that it does not touch the spinning rotor There are two frequency ranges to be
264. nge from 65 kHz to 35 kHz from 100 to about 5096 amplitude Use calcpowlev to calculate the changes in dB to achieve the calculated RF fields enter reference RF field to calculate required RF field in stead of pulse lengths In our case the proton contact pulse power spO is cal culated at 3 74 dB 65 kHz compared to 100 kHz the power level for 1195n is calculated at 1 94 dB 50 kHz compared to 62 5 kHz Be sure to add the calculated number for a desired RF field lower than the reference field sub tract 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 13C HH condition calculate the pp voltage for the unknown 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 6 4 1 Since T4 relaxation tends to be slow in solids direct observation of hetero nu clei 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 cou pled to protons or whatever nucleus the magnetization is drained from Whereas 13C and 15N usually bear directly bonded protons this is not the case for many other spin 72 hetero nuclei So the magnetization must come from more remote substituents More remote they may also be because atomic rad
265. nnnn 89 ue aaa 12 Hgtacetale 2 etr tee HE Eb re Horae Doa FR bet ead 12 ele eere m 78 HELL profile Em 78 A 66 Highipower decouplirig nti pre nen 56 Homonuclear decoupling sarasini arnai annia nA ANAA aA 92 Homonuclear dipolar Imterachons 271 HPSHPPR modules 2 se arena 73 APENA IHA Baue IEN 73 I METER 121 INVEPSION MOCOVETY Mm 202 irradiation frequency enne enne nennen enn 58 K KPOP 2 4 12 Mu l 12 ABP accede LP 12 55 65 en TIT 12 KMNO A 12 L Lee Goldburg Condition EE 92 LO rr 135 LN ONG c 12 A AAE E NA E A E E 11 12 local MOONS vivio ee 202 logical channel 57 longitudinal relaxation n ne anna aan 201 User Manual Version 002 BRUKER BIOSPIN 321 327 Index 322 327 M magic angle coococcconncocococononaconcno conan nnnr oran nn Malonic Acid cert tens matching bon monoexponential oooooococccccccccococconcncconnnnnn Multiple Pulse Decoupling Multiple pulse NMR sssssssss N NOS gadaa observe nuceus occoccccccncoconconnoncnncnnncnnnnnnnns offset Ola anne nun oil free gaS cooooonnocccccnnnocincconncannnccnanan cnn ele EE optimum POSMAX coccccccccocccconcnnncnononcnnnnnannnnns P PDNOS vaticina ias PMG
266. nnnnninnnnnns 263 Figure 20 7 Double CP yield measured by comparing CPMAS and DCP amplitudes of the high field Lena 263 Figure 20 8 C N correlation via Double CP in histidine simple setup sample 4mm Triple H C N Probe E nee 267 Figure 20 9 NCaCx correlation experiment with 22 ms DARR mixing period for Ca Cx spin diffusion on GB1 protein run using an EFEREE Probe ee 268 Figure 20 10 NCaCx correlation experiment with 4 2 ms SPC5 DQ mixing period for CaCx spin dif fusion on GB1 protein run using an EFREE Probe at 14 kHz sample rotation and 100 kHz decoupling ecc 269 21 CRAMPS General 271 Figure 21 1 Difference in Amplitude of the Quadrature Channels X and ve 273 22 CRAMPS 1D 275 Figure 22 1 Pulse Sequence Diagram 2 4422444444H440H Hann ENNER ENER ENEE REENEN anne nnn nnns 275 Figure 22 2 PMLG Shape for wpmlg ep 276 Figure 22 3 Shape for DUMBO ep 277 Figure 22 4 Analog Sampling Scheme z 244444444240H400Rn Hann Hann nannanannaannn nennen seen inna 278 Figure 22 5 Digital Sampling Gcheme meme mene mh herren renes 278 Figure 22 6 Optimizing sp1 for Best Resolution ssssssssssese mH 282 Figure 22 7 Optimizing cnst25 for Minimum Carrier Spike Optimized at 120 C 283 Figure 22 8 Optimizing p14 for Minimum Carrier Spike Optimized at Opuserc 283 Figure 22 9 WPMLG CRAMPS After Optimization Digital Acquisition
267. ns All 91 correlations show with 500 10 psec mixing time u Small plot full spectrum large 124 plot ROI only 10 9 8 7 6 5 4 3 ppm Figure 24 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 protons at tached to the alpha carbon are in equivalent and strongly coupled The cross peaks at 3 and 4 ppm will show at a mixing 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 suppression was used here 294 327 BRUKER BIOSPIN User Manual Version 002 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 24 3 Spectrum of Tyrosine hydrochloride The mixing time was 300 usec to show all connectivities Full plot to show that smaller sweep widths can be chosen when the carrier can be conveniently placed within the spectrum User Manual Version 002 BRUKER BIOSPIN 295 327 CRAMPS 2D re proion spin diffusion E hyr HC1I a womig2d with center spike suppression i 300 usec mixing be in w Lo 3 8 7 6 5 4 F2 ppm Figure 24 4 Expansion of the Essential Part of the Spectrum Proton Proton DQ SQ Correlation 24 5 This experiment correlates prot
268. ntroduced by a faulty setup Additionally it is not always possib le to use the simulations for the interpretation of experimental data e g in the case of multispin systems or amorphous systems it may not possible to get reli able input data for the simulations setup In any case in order to be sure of the correct setup of the experiment it is abso lutely necessary to proof the experimental setup on a known spin system like the glycine in order to check the robustness of the overall sequence setup After this validation and calibration process the sequence can then be used to determine distances or M values for unknown samples by using the calibrated experimental setup Experiments on unknown samples should be measured as close to the cal ibration run as possible to minimize the influence of experimental fluctuations pressure changes and consecutive spin rate changes temperature changes and consecutive pulse power changes and the like Of course a qualitative comparison within a set of samples is always possible with the same set of experimental parameters without doing a full calibration run User Manual Version 002 BRUKER BIOSPIN 167 327 REDOR 168 327 References 1 10 T Gullion J Schaefer Rotational echo double resonance NMR J Magn Reson 81 196 200 1989 T Gullion J Schaefer Measurement of Heteronuclear Dipolar Couplings by MAS NMR Adv Magn Reson 13 58 83 1989 T Gullion Introduction to
269. nvenient location that allows access without restricting standard operations Depending on the type of probe VT control gas enters the probe from the side or from behind So it must be possible to attach the heat exchanger transfer line from the appropriate side Furthermore the weight of the transfer line must be relieved from the probe dewar ball joint so there should be appropriate fixation points for the transfer line It is important that the transfer line enters the ball joint as straight as possible as a ball joint will cut off the flow when strongly tilted to an angle It should also be possible to reach the probe tuning elements since they need to be operated frequently as easily as possible from the operators chair Also when tuning the probe the video screen and the preamplifier display should be easily visible BRUKER BIOSPIN User Manual Version 002 EDASP Display Software Controlled Routing Figure 3 40 Example of a 600 WB NMR Instrument Site The example of a 600 WB NMR instrument site in the figure above provides easy access to the probe from either side EDASP Display Software Controlled Routing 3 9 The menu edasp shows all relevant RF routing and allows the routing which are under software control to be changed The following restrictions apply User Manual Version 002 Connections between transmitter and preamplifier cannot be changed the command edasp setpreamp with NMR Super user permissions does that
270. obe Calibrate power lev els 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 select by clicking on the desired stage High power stages require the parameter powmod to be set to high 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 selected 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 matching box is inserted into the preamp for 500 800 MHz systems it would be labelled for the frequency range 120 205 MHz Con nections are shown in the figure below BRUKER BIOSPIN User Manual Version 002 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 4 2 Probe Connections to the Preamplifier The figure above shows the probe connections to the preamplifier with appropri ate filters placed on a table for better illustration Setting Acqu
271. ocess the data with xf2 to execute a Fourier transform in the f2 dimension only The phase can be adjusted from within the re laxation analysis tool but baseline correction should be carried out with abs2 Start the relaxation analysis guide with the command tfguide 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 posi tive peaks Define ranges Here you must define integral regions containing the peaks of in terest The fitting routine can either use the integral of the signal or the intensity in which case the maximum 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 re gion are displayed The icons at the top of this window allow you to move between the integral regions exclude points from the calculation display the data on a va riety of axes and start the fit for the displayed region or all regions Figure 16 1 shows the decay of the a carbon signal of glycine as a function of
272. ograms are written such that upon ased the approximately correct sweep width is shown and can be set as an acquisi tion parameter User Manual Version 002 BRUKER BIOSPIN 281 327 CRAMPS 1D Digital Mode Acquisition 22 9 Most parameters stay the same as adjusted in analog mode Table 22 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 dqd swh 50000 10000 Depending on spectral range and o1 The correction for the scaling factor must be done after acquisition changing the status parameter 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 22 10 S oimtmisalion for WPMLO power level optimum sf 2 6 5 dE A j d if N HU di J J i alu Roo EL ww Loa E m eT 40 E K 46 43 e ER geet Figure 22 6 Optimizing sp1 for Best Resolution 282 327 BRUKER BIOSPIN User Manual Version 002 CRAMPS 1D optimisation for enst25 minimum conter spike at cngt25 20 degrees phase Mist An e e T T r T 1 T T 50 100 160 ppm Figure 22 7 Optimizing cnst25 for Minimum Carrier Spike O
273. oherence of the central transition The resulting 2D spectrum yields an isotropic projection where the 2 order broadening has disappeared The information content is in full anal ogy to the MQMAS experiment Experimental Particularities and Prerequisites 19 1 In contrast to the MQMAS experiment the first pulse in the STMAS experiment ex cites single quantum SQ coherency The signal which is thus generated consists of contributions from both the CT and the ST In Figure 19 1 the contribution of the CT shows up in the cosine curve starting at the blue filled rectangle resem bling the initial pulse Figure 19 1 Principle of 2D Data Sampling in STMAS Experiments The blue filled rectangle on the left symbolizes the first pulse which starts the evolution period t4 After each revolution of the rotor rotational echo s show up User Manual Version 002 BRUKER BIOSPIN 245 327 STMAS which are indicated by the red filled circles The open rectangles symbolize the second pulse one pulse at the end of each individual t4 increment They must al ways 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 experiments but can be completely suppressed by a double quantum fil ter The contribution of the ST rides on top of the CT signal like spikele
274. oldburg offset cnst31 Spinning speed in Hz 11 for 2 40 msec Number of rotor cycles for mixing time F2 direct C Left column td 4k Number of complex points sw 200 ppm Sweep width direct dimension F1 indirect C Right column td 256 Number of real points sw Rotor synchronized sweep width or 2 sw 140 327 BRUKER BIOSPIN User Manual Version 002 RFDR Table 10 1 Acquisition Parameters in f1 dw or rotor period Synchronised sampling avoids sidebands FnMode STATES or STATES TPPI Spectral Processing 10 4 Table 10 2 Processing Parameters Parameter Value Comment F1 acquisition 13c 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 STATES or STATES TPPI ph_mod pk Phase correction if needed bc_mod no Automatic baseline correction Figure 10 3 1 C Histidine Signal Decay as a Function of the RFDR Mixing Time User Manual Version 002 BRUKER BIOSPIN 141 327 RFDR 180 160 140 120 100 80 60 40 20 ppm Figure 10 4 2D RFDR Spectrum of 13c fully Labelled Histidine RFDR mixing time 1 85 ms 142 327 BRUKER BIOSPIN User Manual Version 002 Proton Driven Spin Diffusion PDSD PDSD is a 2D experiment that correlates a spin 1 2 nucleus to another spin of the
275. olution preamplifiers use actively switched pin diodes for this purpose and are therefore broadbanded so there is no exchangeable box Pulsing with high power into an RF circuit which is not properly set up to pass this frequency may result in damage to the RF circuit in this case the matching box or to the transmitter This applies to filters preamplifiers and matching boxes Es pecially if liquids preamps are used for solids work as well power limitations as frequency limitations must be strictly observed RF Connections Between Preamplifier and Probe 3 2 20 327 These connections must be done with high quality cable with suitable length It should be short but not too short so that the cable must not be severely bent Higher quality cable is fairly stiff the flexible ones are of less quality N B It is extremely important that RF cables are not bent to a radius of less than 30 cm and that no force is exerted on the RF connectors Adapters should be avoided since every connector may change the impedance to deviate from the required 50 Q Cables with loose connectors should be discarded unless they BRUKER BIOSPIN User Manual Version 002 RF Filters in the RF Pathway can be repaired by a skilled RF engineer BNC connectors should be avoided they are usually off 50 O As pulses in solids NMR can be rather long and rather high power it is also nec essary to consider the preamplifier s power limitations
276. on pro cess Therefore it is useful to perform a calibration run like the glycine measure ment before analyzing an unknown sample and to set up the complete experiment very carefully in order to calibrate the experiments The above shown simulated REDOR curves can now be used to demonstrate the second moment approach in analyzing REDOR experiments Here a parabolic fit is used to describe the first few points of each curve In the case of the isolated two spin system of the glycine this parabola is defined by AS _4 2 Vs Pan OT a Eq 12 3 Using this equation you end up with the values given in Table 12 2 for the dis tances and the second moments The Mo values given in brackets for the simulat User Manual Version 002 BRUKER BIOSPIN 165 327 REDOR ed curves are calculated using the second moment approach in order to demonstrate the error margins you have to expect by the M approach 1 0 I m E 0 8 m 0 6 o 2 o4 0 2 m m Experiment 0 04 M2 Parabel 0 000 0 002 evolution time s Figure 12 9 Experimental data with the corresponding M parabolic analysis Figure 12 9 shows the calculated parabola for the experimental data set As you can see in the corresponding table the experimental setup reflects the theoretical Mp within an error margin of 40 while this transforms to an overall distance error of about 10 Afterwards the calculated M can be transformed into the dipolar coupling con stant by usin
277. on 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 296 327 BRUKER BIOSPIN User Manual Version 002 CRAMPS 2D Pulse Sequence Diagram 24 6 Phases 11 622 doo ha us 134 el Pulses and delays taul 3 4 pll pmig pli taul 3 4 Pulse power p17 pli2 sp2 pl2 pl7 p112 spl mm Biel pe Ee te DQ excitation evolution DQ reconversion detection PC7 pmlg DUMBO PC Gef aq E Figure 24 5 Pulse Sequence Diagram When applied to X nuclei like 1C the RF field during this sequence must be care fully 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 forgiv ing 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 required 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 re quired RF field is then 7 14000 98000 Hz The known proton 90 degree pulse is 2 5 usec 1 4 2 5e 6 2100000 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
278. onger than SB probes Probe lengths are the same up to 400 MHz the same for 500 and 600 MHz and longer for higher fields SB probes VTN and DVT type probes also major differences between older and more modern probes Furthermore probes with sample insert eject and probes without insert eject exist The major difference between DVT and VTN WVT probes is that for VTN WVT probes the bearing gas is used for temperature control whereas for DVT probes bearing drive and VT gas are separate User Manual Version 002 BRUKER BIOSPIN 27 327 General Hardware Setup Wide Bore WB Magnet Probes 1 VT gas only input into dewar 3 Drive gas in 2 Two thermocouple connectors 4 Bearing gas in Figure 3 15 WB DVT Probe MAS Tubing Connections 1 VT plus bearing gas 3 Drive gas 2 One thermocouple connector at stator inlet Figure 3 16 VTN Probe MAS Tubing Connections Note WVT Probes are VTN Type Probes 28 327 BRUKER BIOSPIN User Manual Version 002 MAS Tubing Connections 1 Sample insert eject 5 T piece to insert tube allows the flush gas to be fed in 2 Eject gas in at low temperature to avoid ice formation on spinner cap 3 Insert gas in 6 Shim stack flush connection 4 Flush gas for transfer tube Figure 3 17 WB Probes Eject Insert Connections 1 Thermocouple s 4 PICS cable 7 VT gas in 2 Bearing gas in 5 Heater 8 Spin rate cable 3 Drive gas in 6 Heater cable in 9 Flush gas in Figure 3 18
279. oo ccoo ncccnnccnnncnnncconncononcnnncnnoncnnnncnnnon 214 Figure 17 3 Comparison of 87Rb MAS spectra of RONO3 excited with selective and non selective PISOS mE 217 Figure 17 4 Nutation profiles of selective and non selective pulses ooooccocccnccccccnccncccnccnccnnninn 218 Figure 17 5 Example for popt Set up for Optimization of p1 and p2 ssssssssesss 219 Figure 17 6 Signal Intensities of 87Rb Resonances in RbNO3 as Function of p1 and p2 220 Figure 17 7 2D 87Rb 3QMAS Spectrum of RbNO3 00 0 0 cece eect eect eee esate ee eene nne 223 Figure 17 8 Comparison of Differently Processed 2D 23Na 3Q MAS Spectra of Na4P207 224 Figure 17 9 Calculated Shift Positions dMQ sssssssssssse mee nennen 225 Figure 17 10 170 MQMAS of NaPO3 at 11 7 T 67 8 MHz on the left and 18 8 T 108 4 MHz on the dg ee 228 Figure 17 11 Slices and Simulations of the 18 8 T 170 MQMAS of NaPO3 ccecce 229 Figure 17 12 Graphical Interpretation of the Spectrum from Figure 17 10 230 18 MQ MAS Sensitivity Enhancement 231 Figure 18 1 Hahn Echo Pulse Sequence and Coherence Transfer Pathway sssssss 232 Figure 18 2 Processing of Hahn Echo Left is the Shifted Echo ssee 233 Figure 18 3 Four Pulse Sequence and Coherence Transfer Pathway for the 3Q MAS Experiment 234 Figure 18 4 Three Pulse Sequence and Cohere
280. oooconnccccccnccncccnccnso 249 Table 19 3 Initial Parameters for the Set up of stmasdfgz av ssssssssssssss 250 Table 19 4 Initial Parameters for the Set up of stmasdfqe av cooccoccoocccccncccncccnccnccnncnnnnnnnnnnnnnnnnns 251 Table 19 5 F1 Parameters for the 2D Data Acquisition 252 Table 19 6 Processing Parameters for the 2DFT eene 253 Table 19 7 Values of R and cR pc for the Various Spin Quantum Numbers Obtained in the STMAS Experiment diene e ned ade indie es lest ME ERA RR ERAN AS 254 20 Double CP 255 Table 20 1 Recommended Parameters for the DCP Getunp 260 Table 20 2 Recommended Parameters for the DCP 2D Gerup 265 Table 20 3 Recommended Processing Parameters for the DCP 2 266 21 CRAMPS General 271 22 CRAMPS 1D 275 Table 22 1 Phases RF Levels Timings ccccecece essen eeeen EEN ENER NEEN EEN 275 Table 22 2 PMLG Analog Mode 279 Table 22 3 DUMBO Analog Mode ccccccecceeeeeeeeee tence teen esee t ua EENAA NANAREN EENET ENAREN tanen ia 280 Table 22 4 Parameters for Digital Mode ssssssssse meme nme 282 23 Modified W PMLG 285 Table 23 1 Phrases RF Levels Timings cccccceeeccceeceeeceeeeeee Ires 285 Table 23 2 PMLG Analog Mode aser izsenizeee cocina ite a a AU SE ES Rie aa 287 Table 23 3 DUMBO Analog Mode eee cece eceeeceeeeeeseeeeeeeeeseeseeeseeeeeseeeaeeeaes 288 Table 23 4 Parameters for Digital Mode 0 ccceececeeeeeee eee eee emere 2
281. orter 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 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 p1 because the signal intensity is much more sensi tive to this pulse length A suitable set up for the parameter optimization proce dure popt is shown in following figure T store as 2D data ser file l The AU program specified in AUNM will be executed Perform automatic baseline correction ABSF T Overwrite existing files disable confirmation Message l Run optimisation in background OPTIMIZE i pe lid pi PARAMETER OPTIMUM POSMAX POSMAX 05 15 15 45 STARTWAL ENDVAL NEXP VARMOD INC LIN LIN oo N Sa Figure 17 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 parame ters please refer to the manual Figure 17 6 shows the signal amplitudes as func tions o
282. ot shielded From these data filter out the linear magnet drift Determine the number of digits the field value in the bsmsdisp menu must be changed to set the field back to the exact same value as in the first spectrum Recalculate this number to a drift time of exactly 1h 24h Note these values as your magnet drift rate This drift rate will only reach a stable and constant value some time after charg ing so the drift rate measurement should be repeated until the drift value is con stant This may take some weeks to months for high field magnets The magnet drift value will allow to calculate the time dependant component of the field The probe dependant component can be established by shimming every probe and setting the field on the same sample following the same procedure for every probe after shimming When then the shim file is written the current cali brated field position is written to disk with the shim file Executing the AU program or command TopSpin version 3 0 and later probe field will now set the field to the appropriate value according to drift and time tak en from the date of the shim file so the computer clock should be correct and probe shims from the current probe setting so the appropriate probe must be se lected in edhead However this will only work if all shim files contain the precisely determined field value for the same reference compound User Manual Version 002 BRUKER BIOSPIN 87 327 Basic
283. oton resonance frequency in MHz This results from spin rate requirements for 13C observation to avoid rotary resonance conditions as well as excitation bandwidth considerations Spectrometer Setup for 13C 14 2 1 Load a CPMAS parameter set for 13C Load a uniformly labeled glycine sample and rotate at the desired rotation rate see table 1 depending on the recoupling experiment planned and the sample under investigation Consider possible rotational resonance conditions in the sample of interest Tune and match the probe optimize the 13C and 1H pulse parameters for exci tation and decoupling Use the cp90 pulse program with pI11 pl1 to measure the nutation frequency for 13C in order to calculate the recoupling conditions see chapter Basic Setup Procedures Calculate the power levels required by the spin speed see table 1 using calcpowlev 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 14 2 2 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 Load the power level calculated for the necessary 1c recoupling B field into pl11 set p1 as determined in step 4 Set 0215 should be but need not be a multiple of 5 for SPC5 or of 7 for PCT SC14 This determin
284. otor periods it is possible to miss an echo completely For example if the duration of the rotational echo is 1 us it will be missed when the deviation is larger which is the case for a 1 Hz devi ation at 10 kHz but requires a fluctuation of 10 Hz at 30 kHz spinning Table 19 1 Time deviation of the rotor period for spinning frequency variations of 1 and 10 Hz for various spinning frequencies Fluctuation of Hz Ne i ii e See 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 246 327 Typically the spinning frequency must be stable within lt 1 Hz throughout the entire 2D data acquisition Secondly the accuracy of the magic angle setting is extreme ly important The sidebands 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 3cosdsin6d9 which close to the magic angle is v2d0 The magnitude of the interaction that must be narrowed in the pres BRUKER BIOSPIN User Manual Version 002 STMAS ent case is in the order of MHz so even a small deviation causes a severe broad ening in th
285. parameters for 19C CP MAS The gyro magnetic ration of 15N is lower by a factor of 2 5 compared to car bon proton frequency 400 MHz 13C frequency 100 MHz IDN frequency 40 MHz The probe efficiency is about the same for 13C and 15N but not Tun SO one needs about 2 5 times higher RF voltage for the 15N contact pulse than for the C contact pulse if the spin rate and the proton RF field are the same This is equivalent to 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 Precalculating power levels like this will get the pa rameters close enough to see a cp signal on a good test sample so further opti mization is possible See Test Samples for suitable test samples The most efficient way of precalculating power levels for multi nuclear spectrosco py is the following 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 precise 360 pulse make sure it is 360 not 180 or 540 and the associated power level Make a table in your lab notebook as fo
286. particu lar site and the Qis is then given by Eq 17 2 User Manual Version 002 BRUKER BIOSPIN 229 327 Basic MQ MAS 230 327 axis CS fe wm mm mm Figure 17 12 Graphical Interpretation of the Spectrum from Figure 17 10 In the 11 7 T spectrum this gives quadrupole induced shifts gj of 75 ppm and 20 ppm for the two sites respectively At 18 8 T the gis of the lower peak in the 2D spectrum decreases to 30 ppm whereas it cannot be determined graphical ly anymore for the upper peak since the chemical distribution broadens the peak km wm wm al zm zm mm wm e e e bk wm wm d wm i i i J e e wl e m in the F1 dimension more than the theoretical 3 gj of 5 ppm BRUKER BIOSPIN User Manual Version 002 MQ MAS Sensitivity 1 8 Enhancement The MQMAS experiment on half integer quadrupole nuclei is an extremely insen sitive experiment This is due to the low efficiencies of both the excitation of 3Q coherence and their conversion to observable magnetization Several approaches have been taken to enhance the efficiency of the excitation and conversion main ly 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 al ternative phase cycling schemes have also yielded improvements Improving the efficiency of the MQ excitation pulse has been tried but no generally applicabl
287. pe of shape it is recommended to select shapes which are all centred around 50 ampli tude which allows arbitrary amplitude modulation without changing the HH condition For a start generate a ramp shape from 45 to 55 with 100 slices using shape tool stdisp Store the ramp as ramp4555 100 Select this ramp as spnam o 20 e mm pearen Figure 20 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 5 Since the shape is centred around 50 the RF voltage here is down by a fac tor 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 13C but it can also be used on 15N Set DIS to 46 or 24 kHz RF field on IDN Set spnam1 to ramp4555 100 sp1 to 35 kHz RF field on 1C 6 dB Set pI13 pI12 for a start set p16 to 5 msec Optimise the power level pl5 A variation over 1 to 1 dB in steps of 0 2 dB should be ample In order to be sure one can optimise sp1 and pl5 as an array sp7 in steps of 0 5 dB p 5 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 Optimise p16 between 5 and 15 msec See fig 6 for an optimization of pl5 SN square pulse power 7 With a ramp shape for the N C transfer
288. periments 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 homonuclear proton dipolar decoupling are pmighet and wp mighet f DUMBO decoupling is desired the pulse programs are dumbohet or edumbohet These pulse programs use either windowless pulse trains or win dowed pulse trains which can be timed in exact analogy to the CRAMPS type se quences wpmlg 2 and dumbo 2 These sequences also suppress the center ridge efficiently so that the carrier frequency need not be shifted out of the proton range during evolution cnst24 0 In contrary it is possible to shift the carrier to the pro ton 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 setting contact times short Of course this reduces cross peaks from remote couplings more than it reduces cross peaks from directly bonded pro tons However the remote couplings are always present through the homonuclear coupling between all protons These couplings can however be suppressed by ex ecuting the contact with a Lee Goldburg proton offset Then the protons are homonuclear decoupled and the transfer from protons to X only follows the heter onuclear dipolar coupling between those The pulse program Ighetfqlgcp works completely in analogy to Ighetfq
289. pinning speeds up to about 12 5 kHz sample rotation depending of course on the width of the employed r pulses Figure 7 5 shows for a com parison the results obtained with the 4 pulse sequence with 256 scans BRUKER BIOSPIN User Manual Version 002 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 dA4osr S500 Figure 7 4 CPTOSS243 Experiment on Tyrosine HCI at 6 5 kHz Fiqure 7 4 is a CPTOSS243 experiment on tyrosine HCI at 6 5 kHz sample rota tion using a 4 mm CPMAS triple resonance probe at 500 MHz with 243 accumu lated transients No spinning sideband residuals can be observed with a noise level below 296 peak to peak compared to the highest peak intensity User Manual Version 002 BRUKER BIOSPIN 109 327 Basic CP MAS Experiments Sein Manuali chapter 10 4 jon 1708 Spectrum ProcPars AcquPars Title PulseProg Peaks integrais Sample Structure Fig Phase Callorate Basetine FO Setup CPAS Mast 6505 SELTICS 110 327 fyrosme HC Figure 7 5 CPTOSS Experiment on Tyrosine HCI at 6 5 kHz Figure 7 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 sideband residuals can be observed outside a noise level of approxima
290. precise 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 19 5 Similar considerations for the maximum t4 period determined by the number of FID s to be acquired and the t4 increment can be made as for MOMAS 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 suf ficient initially Processing parameters are described in the next section BRUKER BIOSPIN 251 327 STMAS Table 19 5 F1 Parameters for the 2D Data Acquisition Parameter Value Comments F1 parameters In eda FnMode States TPPI or 2D acquisition mode for stmasdafz av States QF 2D acquisition mode for stmasdafe av TD see text Number of FID s to be acquired 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 fre quency offset is correctly set important for referencing Pulse program parameters In ased D6 0 Used in stmasdafe av only IN6 IN0 8 9 Used in stmasdafe av for 3 2 D7 0 Used in stmasdafe av only IN7 IN0 7 24 Used in stmasdqfe av for 5 2 IN0 28 45 Used
291. ptimized 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 19 ppm Figure 22 8 Optimizing p14 for Minimum Carrier Spike Optimized at 0 6 usec User Manual Version 002 BRUKER BIOSPIN 283 327 CRAMPS 1D womlg CRAMPS ren 4 c CIN itus Is what should be achieved with Oftfe omon 10 LI 1 f N U A A j j Fi fy m N i Fa gt j IX T 4 tf DU J V f j Qi ei 4 T T T T T T T T T T T T T T 10 n 6 4 2 o ppm Figure 22 9 WPMLG CRAMPS After Optimization Digital Acquisition 284 327 BRUKER BIOSPIN User Manual Version 002 Modified W PMLG 2 3 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 ofp arbitrarily This is achieved by a 180 degree phase alternation between consecutive WPMLG pulses The magic angle tilt pulse is then not re quired anymore This reduces setup time and enhances experimental possibilities significantly Reference 1 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 23 1 Phases 1 010010 O10 10
292. r C and N so observation can be changed between C and N without rewiring There should be an X low pass filter or 6 bandpass filter on 13C al N low pass or bandpass on IDN MAS rate 5 10 kHz The MAS spinning speed should be stable within 1 to 2 Hz in order to get a well refocused echo Overall Experimental time including setup procedure 3 5 hours User Manual Version 002 BRUKER BIOSPIN 157 327 REDOR Packing the sample is critical for the success of the experiment check your sam ple is within the central region of the spinner or use a 12ml spinner The quality of the refocusing p pulses is essential this can only be achieved with a center packed sample The coil of a4mm MAS probe has a length of 10 mm the sample should be no longer than 5mm preferably 3mm CRAMPS spinner or 12 ml HR MAS spinner Setup the CP conditions for the 1H magnetization transfer on both coupled nuclei X and Y Use cp90 to determine precise p pulses on both the X and Y channel of your probe Accurately setup the p 2 and p pulses for both C and IDN according to the stan dard setup procedures with an accuracy of at least 0 1ms After the setup the pulse lengths for the different channels should be within the same duration and short enough to not exceed an overall duty cycle of about 5 One can reoptimise the refocusing pulse on the coupled nucleus using a 1D ver sion of redor setting the number of experiments 1 td to 1 Here the number of
293. r Proton Observation 67 Proton Spectrum of Adamantane at Moderate Spin Speed ssssssssssss 68 Setting the Carrier on Resonance emere nens 69 Expanding the Region of Interes 70 Save Display Region to Menu 70 The popt Window ccccc cece eee n cee eee eee eee Ie nemen hene nl hne he nne ninth rn nnn rrr 71 The popt Display after Proton p1 Optimization cccccceecc cece eee eeeceeeeeeeeeeeeeaes 72 Adamantane 13C FID with 50 msec aq setsh Display oooocoocccoccccccnccnccnnccnccnncnnnnnnn 74 Adamantane 13C FID with 50 msec aq setsh with Optimized Z Shim Value 74 A cp Pulse Sequence nhe he rre rn ne rennen 76 Hartmann Hahn Optimization Profile 77 Hartmann Hahn Optimization Profile Using a Square Proton Contact Pulse 78 Display Showing a Glycine Taken Under Adamantane Conditions 4 scans 79 Optimization of the Decoupler Offset 02 at Moderate Power Using cw Decoupling 80 Glycine with cw Decoupling at 90 kHz RF Field sssssse HH 81 Glycine Spectrum with Spinal64 Decoupling at 93 kHz RF field 83 5 Decoupling Techniques 89 Optimization of TPPM Decoupling on Glycine at Natural Abundance 90 Geometry for the FSLG Conditon meme 93 FSLG Decoupling Pulse Sequence Diagramm 94 Adamantane FSLG decoupled showing the downscaled C H J couplings 94 Shape with Phase Gradients
294. r and the number of exper iments nexp is set automatically when clicking on the save button Then save ta ble by clicking on the save button and click on start optimize to start the optimization procedure The parameter value obtained by the program is written into the parameter set of the actual experiment at the end of the optimization In order to stop the execution of popt use the skip or stop optimization buttons Skip optimization will evaluate the obtained data as if popt had finished regularly and writes the parameter into the parameter set Stop optimization will stop with out 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 optimiza tion Popt will generate a data set where the selected expansion part of the spectrum is concatenated for all different parameter values in this case for p7 It will have a procno around 999 To achieve this processing parameters are changed ap User Manual Version 002 BRUKER BIOSPIN 71 327 Basic Setup Procedures propriately Fourier transforming a normal FID in such a window will generate an incorrect spectrum window 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 acans sation nunmno opte on soedborta mono ai ew test town Dist ORE Z pd 0099 Sowclrum ProcPors
295. r coupling that different relax ation is not seen for the different sites If the experiments are set up with short contact times the individual carbon signals will be derived only from directly bond ed protons and thus any differences in proton relaxation within a molecule could be isolated Such indirect observation can be implemented conveniently for both T4 and T relaxation For T4 a proton saturation recovery step is inserted prior to the cross polarization step in a standard CP sequence The proton magnetization immedi ately prior to CP and thus the observed carbon signal depends on the extent of BRUKER BIOSPIN User Manual Version 002 Relaxation Measurements recovery after the saturation so the carbon signal as a function of recovery delay gives the proton T4 value For T4 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 T4 re laxation Indirect Proton T1 Measurements 16 3 1 Data processing 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 T4 e
296. r decoupling pulses p 22 during delays should be high 5 The experiment requires a minimum of 64 transients to complete the phase cy cle Between 32 and 64 experiments are needed for a 2D data set Depending on the choice for the gamma integral more transients per slice may be re quired The recommended value is 4 which increases the number of required transients per experiment to 256 6 Run 1D experiment and make sure everything is set properly 7 Create a new experiment with either iexpno or edc 8 Change to 2D data set BRUKER BIOSPIN User Manual Version 002 SUPER Setup 2D Experiment 13 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 appropri ate FnMode parameter in eda Pulse program parameters are detailed as follows Figure 13 1 shows the pulse sequence ocPars AcquPars Title Pu EVA Figure 13 2 The 123 Icon in the Menu Bar of the Data Windows Acquisition Pa rameter Page The 123 icon in the menu bar of the data windows acquisition parameter page is used to toggle to the different data acquisition modes 1D 2D and 3D if so de sired 9 Make sure the correct nucleus is selected in F1 dimension make sure an ap propriate quadrature detection mode is selected in FnMode TPPI STATES TPPI or STATES 10 Choose the appropriate sampling time tdT so that the required resolut
297. ramatically 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 Compressors 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 as sure probe performance 56 327 BRUKER BIOSPIN User Manual Version 002 Basic Setup Procedures Setting the Magic Angle on KBr 4 2 RF Routing For all following steps generate new data sets with appropriate names using the edc command to record all individual setup steps 4 2 1 The spectrometer usually has 2 or more RF generation units SGU s transmitters and preamplifiers In order to connect the appropriate SGU to the appropriate transmitter and the transmitter to the associated preamp where the probe chan nels are connected there are several routing possibilities In order to minimise er rors in hardware connections the routing is under software control where possible Where cable connections need to be done manually the software 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
298. re 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 perfor mance 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 extended as time permits and as it is required by new development in NMR The manual is written primarily for Bruker AVANCE III instruments but the experimental part will be identical or similar for AVANCE and AVANCE II instru ments For example pulse programs will have slightly different names 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 looking 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 ex periments within a given type since the same information can often be obtained with pulse sequences differing by subunits only or in using a totally different prin ciple The
299. re the line width of the KBr central peak and compare it with the line width of the 5 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 typ ing peakw and then repeating this with the 5 sideband to either side Most cp mas probes are tunable over a large range of X frequencies It can some times be fairly 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 tun ing position of the probe into the lab notebook and start retuning to the new nucle us frequency from this frequency on following the probe response over the whole frequency range using a large wbsw of 50 MHz Alternately check the microme ter 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 mea sured nuclei User Manual Version 002 BRUKER BIOSPIN 85 327 Basic Setup Procedures 86 327 Remember which way to turn the tuning knob to tune to higher and lower frequen cies On most probes turning the adjustment counter clockwise tunes to higher frequency Do not change the matching adjustment until you have found the cur rent t
300. relaxation de lay along with the fit and calculated relaxation parameters Note that any peaks with integrals or intensities too close to zero will be omitted from the analysis by 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 gener al parameters should be determined automatically but ensure that the limits for baseline correction are set to cover the whole spectrum The fitting function de pends 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 experiment 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 calcu late multi exponential fits but data with very good signal to noise is required for this to be accurate Unless there is obvious overlap of peaks the assumption is usually that each peak corresponds to a single nuclear site and thus a single T value BRUKER BIOSPIN User Manual Version 002 Relaxation Measurements Fatascatuvs gy T Tg 1 9 e Ztcket Zugang wien 9008 cu Bore sno i Fiting type V Ermonentist de y Tay n d m E intensity 5 1
301. requency For the high frequency range 9F and 1H two different types of solids preamps are available the older HPHPPr 19F H and the recent replacement HPLNA User Manual Version 002 BRUKER BIOSPIN 15 327 General Hardware Setup High Power Low Noise Amplifier which is strictly frequency selective either 19 or H The connections into the back of the preamp stack should normally not be changed For broadband high power preamplifiers it is important to insert the ap propriate matching box into the side of the preamp RF cables from transmitter RS 485 control DC voltages in tune and lock RF in RF signal out to receiver gate pulses for preamplifier control multi receive setup only The orange colored cable is the high voltage supply for the HPLNA pream plifier Figure 3 1 All Connections to the Back of the Preamplifier 16 327 BRUKER BIOSPIN User Manual Version 002 Connections to the Preamplifier The lock preamplifier is located at the bottom of the stack the transmitter cable carries the lock pulses For sol ids this preamp is normally not re quired When the transmitter cables are re wired to different preamp modules the changes must be entered into the edasp routing type edasp setpre amp NMRSU password required Figure 3 2 Transmitter Cables only Wired to Back of the Preamplifier User Manual Version 002 BRUKER BIOSPIN 17 327 General Hardware Setup frequency logical c
302. ression 256 tran sients were recorded Hasc experiments 5 1 Cda Ou boda jas OS Ble R al L E E Ei 7 os Bara 4 jas c experiaents P Civera siMrbrxsb 30530805 CA X os Bsasc_experzuents 6 l CitidartaS0Orbrxbb 3050005 or Ju en 8 AR UN BE t Ss r r T r r r r a T r 1 1 Figure 7 8 Cholesterylacetate Spectrum Using Sideband Suppression Figure 7 8 is a cholesterylacetate spectrum using sideband suppression with the SELTICS sequence at 5 Hz sample rotation upper spectrum The lower spec trum is the CPMAS spectrum at 5 kHz sample rotation 112 327 BRUKER BIOSPIN User Manual Version 002 Basic CP MAS Experiments Non Quaternary Suppression NQS 7 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 heteronuclear dipolar in teraction For the dephasing delay d3 one uses between 30 and 80 us gt 93 heteronuclear decoupling Figure 7 9 Block Diagram of the Non quaternary Suppression Experiment The non quatemary suppression experiment is also called the dipolar dephas ing experiment Use glycine or tyrosine spinning at 11 kHz as before Table 7 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 002 BRUKER BIOSPIN 113 327
303. rn cose equency KOCH channe WF S066057 MHZ HEH x10 Drot 6C 67997 Mec np son RP x ors 1650 d Hr ft MPH KEEP E BE SON Win Mier wen ee SFU SU 197547 1H 103 wr Ore fra 7 NEID STP OF 126 764920 Mite MACH S gt X 500 W PHP Ek SFOS 125 166853 mz ra sous P oro 20240 tz oc 1 200 ore 300 190 Miz NICA mm was sro 500 150 Mer F4 SGUs ops foo Mz utr bs cable wg settings possible RF routing F snow recener rong Gre Steh FVFZ Zeg FF fic a ngical channel Deno a logical channel Refuse infa Para m Clow Figure 3 41 Short Display Pulse Routing Only for C N H DCP or REDOR Experi ment observing 130 above and 15N below 52 327 BRUKER BIOSPIN User Manual Version 002 EDASP Display Software Controlled Routing In the figures above is a short display pulse routing only for a C N H DCP or REDOR experiment observing C and observing N without any hardware change Green dots indicate CORTAB linearization 13 routed via 500W trans mitter since 1 C requires less power than 15N a gx M crne rote treamone ege cera ere am Own FU Den E LECH pia Den rra Era 1 09c a crane Qat re Gees som Figure 3 42 Long Display Pulse and Receiver Routing The figure above is a long display with pulse and receiver routing Green dots CORTAB done for this path transmitter linearized Dotted green lines Possible hardwired routing are shown
304. roscopy Nature 420 98 102 2002 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 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 W Luo X Yao and M Hong Large Structure Rearrangement of Colicin la channel Domain after Mem brane Binding from 2D 13c Spin Diffusion NMR J Am Chem Soc 127 6402 6408 2005 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 002 Proton Driven Spin Diffusion PDSD Pulse Sequence Diagram 11 1 oy O10 1H SPINAL 64 SPINAL64 decoupling decoupling cw DARR pl14 62 Qs os prec X t mixing t2 63 00000000 Qrec 02201331 65 00221133 du 0 20023113 Figure 11 1 CPSPINDIFF Pulse Sequence Basic Setup 11 2 1 On a standard sample i e glycine determine HH match and decoupling pa rameters 2 Check the X 90 hard pulse with cp90 on the standard sample 3 If the 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 h
305. rt with slightly reduced power set tings 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 display 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 decou pling p 12 pl12W and associated pulse lengths p3 pcpd2 Value of proton contact power level in dB and watt sp0 spOW Value of carbon contact power level pl pI1W and associated pulse length pl S N value obtained on glycine SR value for shift calibration line width on a carbon in Hz BRUKER BIOSPIN User Manual Version 002 Basic Setup Procedures Field Setting and Shift Calibration 4 9 Note It is essential that if spectra taken at different times and or taken with differ ent probes need to be compared the shift calibration is executed correctly If the magnetic field was not the same the spectra will have diff
306. s within this manual will have to be modified to comply with this The setting will also be possible on a dB scale however with an absolute reference Power level changes will therefore be calculated properly using calcpowlev Where pulse power recommendations are given in this manual they will still apply if given in watts they will however not ap ply in the future version of TopSpin if given in dB We will try to release a new version of this manual when the new TopSpin version is available whereas possible inconsistencies will be removed There is no incon sistency with TopSpin vs 2 1 1 1 Any hardware units mentioned in this manual should only be used for their intend ed 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 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 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 10 327 For further technical assistance please do not hesitate to
307. sed first with a sub sequent 2D interactive phase correction BRUKER BIOSPIN User Manual Version 002 STMAS The STMAS experiment for half integer quadrupole nuclei is a 2D experiment to separate anisotropic interactions from isotropic interactions In the NMR of half in teger quadrupole nuclei the dominant anisotropic broadening of the central 1 2 lt gt 1 2 transition CT and symmetric multiple quantum MQ transitions is the 2 d order quadrupole interaction which can only partially be averaged by MAS The satellite transitions ST e g the 3 2 gt 1 2 transitions however are broad ened 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 averaged 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 20d order broadening of the CT can only be narrowed by a factor of 3 to 4 so a signal is ob served that still reflects this 2 order broadening The 2D STMAS experiment exploits the fact that the 2nd order broadening of the ST transitions e g 3 2 lt gt 1 2 in a spin 3 2 is related to the 214 order broad ening 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 tran sitions 3 2 lt 1 2 and the 1 2 gt 1 2 single quantum c
308. sen too small Processing is done in complete analogy to the FSLG experiment as for all follow ing sequences User Manual Version 002 BRUKER BIOSPIN 129 327 Modifications of FSLG HETCOR Table 9 1 Acquisition Parameters for pmlg HETCOR on tyrosine HCl f T E l H 2 amp Poe Lu hm IM ro GA L pa y Lo c 1 e i i E L Le c 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 d 200 150 F2 ppm Figure 9 2 HETCOR Using Windowless Phase Ramps The figure above shows the HETCOR using windowless phase ramped shapes for proton homonuclear 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 Chem Phys Lett This allows reduced measure ment times Pmlghet wpmlghet dumbohet and edumbohet should give rather similar spectra 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 130 327 BRUKER BIOSPIN User Manual Version 002 HETCOR with DUMBO PMLG or w PMLG Using Shapes
309. set the X tuning range and the proton tuning frequency In such a probe changing the coil would throw off the proton tuning totally so a coil change is not possible Extending the tuning range of a CP MAS probe can be done in the following ways Figure 3 34 1 User Manual Version 002 Switch the proton transmission line between A 4 low range and A 2 mode high range The proton transmission line is also part of the X circuit and is higher lower in capacitance in MA A 2 mode only 400 MHz and up Add a parallel capacitance to the X tuning capacitance which makes the ca pacitance bigger tunes to lower frequency This is normally done to shift the tuning range to or below 15N Add a capacitance in series to another capacitance This reduces the total ca pacitance and shifts to higher frequency A capacitance in series to the AA line will reduce its total capacitance and shift the X tuning to higher frequency Add a parallel coil to the NMR coil This reduces the total inductance and shifts to higher frequency however at the cost of filling factor The bigger the parallel inductance the smaller the high frequency shift and the loss BRUKER BIOSPIN 43 327 General Hardware Setup 1 X frequency in 4 lambda 4 switch 7 Serial capacitance 2 X tuning 5 Parallel capacitance 8 H tuning 3 NMR Coil 6 Parallel inductance Figure 3 34 Possible Modifiers for Probe Tuning Ranges 400 MHz and up only
310. signal is finally detected on 13C under suitable proton decoupling The purpose of this experiment is to gain information about the C N dipolar 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 2 5 kHz This has some ex perimental consequences 1 There is no need to decouple 15N while observing 13C since the coupling is spun out already 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 behaves 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 con ditions during the cross polarization S Hediger et al or provide only selective polarization transfer Specific CP Baldus et al References 1 J Schaefer T A Skokut E O Stejskal R A McKay and J E Varner Proc Nat Acad Sci USA 78 5978 1981 2 J Schaefer E O Stejskal J R Garbow and R A McKay Quantitative Determination of th
311. sition 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 SI 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 222 327 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 t4 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 program 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 performed 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
312. ss x iniecit eoi eod tod EP epa a pude ped n site 35 Figure 3 26 WB Wideline or PE Probe Connections ernennen nenn 35 Figure 3 27 Low Temperature Heat Exchanger for VTN Probes old style sssesssss 36 Figure 3 28 Low Temperature Heat Exchanger for DVT Probes ssss nennen 37 Figure 3 29 Low Temperature Liquid N2 Dewar with DVT Probe Heat Exchanger 38 Figure 3 30 Bottom view of Low Temperature DVT Probe Heat Exvchanger 39 Figure 3 31 Low Temperature Setup with B CU X or B CU OD 40 Figure 3 32 Low temperature setup with BCUN eh enr 41 Figure 3 33 RF Setup of a Wideline Single Frequency Probe ssssssee nnne nenn 42 Figure 3 34 Possible Modifiers for Probe Tuning Ranges 400 MHz and up only 44 Figure 3 35 A 4 low range and A 2 Mode high range 400 MHz Probe 45 Figure 3 36 A M4 only probe left and a A 4 A 2 probe right sse HA 46 Figure 3 37 Without with Parallel Capacitance to Shift the Tuning Range to Lower Frequency 47 Figure 3 38 Parallel Coil to Shift the Tuning Range to Higher Frequency sessese 48 User Manual Version 002 BRUKER BIOSPIN 309 327 Figures Figure 3 39 Figure 3 40 Figure 3 41 Figure 3 42 Figure 3 43 Figure 3 44 Figure 4 1 Figure 4 2 Figure 4 3 Figure 4 4 Figure 4 5 Figure 4 6 Figure 4 7 Figure 4 8 Figure 4 9 Figure 4 10 Figure 4 11 Figure 4 1
313. t the shims x y z 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 Save the shims using the command wsh followed by a suit able name Before the shims are saved it is recommended to reset the field value in the bsmsdisp menu to be exactly on resonance with your shift reference sample of choice protons of adamantane or water in D20 or silicon rubber This allows the command probefield TopSpin version 3 and up to set the field according to probe shims and magnet drift see below and Field Setting and Shift Calibration on page 87 Note For long acquisition times aq gt 0 05 s the decoupling power level pl12 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 pro gram 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 4 5 In TOPSPIN as well as in the XWIN NMR 3 5 release the frequency list for NMR nuclei follows the IUPAC recommendations 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 1 C low field signal of adamantane to 38 48 ppm This will set the param eter S
314. t this name for spnam1 The effi ciency should be noticeably better Reoptimise the first HH contact decoupling and p16 More than 50 should definitely be obtained EE UI 14 1 nl 3 du nung TangAmplitudeMod 4 8 molifude Parameters 100 Bize of Shape 5500 0 deta MAS 4000 dipolar coupling RF field at match Ampl Scaling Factor Use general notation Figure 20 5 Shape Tool display with a tangential shape for adiabatic cross polar ization The amplitude factor is 50 corresponding to 50 RF field or a power level change of 6 dB 4 fold power in TopSpin 3 0 and later since the amplitude cor responds 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 BRUKER BIOSPIN User Manual Version 002 Double CP BB sy prose 22 393 Casatoria jns0507 Spectrum ProcPars ArquPars Tite PuseProg Peaks rtegak Sample Structure Fid Acqu 4 6 8 Lad aaa aac LA 2 T T PT FS TT T T T T T T T am bm T ES T T U T T T T T T T T T 3 2 1 0 E 2 ppm Figure 20 6 Double CP optimization of PL5 in increments of 0 1 dB Note how narrow the optimum DCP conditions are However with diligent prepa ration 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 CP
315. ted using edlist and the name of the list set as the parameter vdlist Table 16 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 d1 3s Needs only to be 3x proton T1 pl1 X HH contact power standard cp setting pl11 power for 90 degree pulses usually pl11 lt pl1 for short pulses p1 Measured 90 X pulse length adequate for required excitation bandwidth at pl11 Ns 2 Should be enough to see a reasonable spectrum 204 327 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 z magnetization and in the second scan it creates z The phase cycling of the receiver means that the difference between the two scans is record ed 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 magnetiza tion 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 positive peaks give
316. tely 2 peak to peak compared to the highest intensity The residual sidebands have up to 5 6 intensity compared to the highest resonance 7 3 Like the TOSS experiment SELTICS Sideband ELimination by Temporary Inter ruption of the Chemical Shift is an experiment for spinning sideband suppression Pulses on the 19C channel are driven with pl11 and pulse times are rotor synchro nized For optimum suppression the shortest pulse 1 24 of the sequences where 1 is the rotor period should be a 7 2 pulse or stronger Choose pl11 ac cordingly Unlike TOSS SELTICS is only 0 5 rotor periods long BRUKER BIOSPIN User Manual Version 002 Basic CP MAS Experiments heteronuclear decouplin 1 93 H 2 bs e 798 Prec t 12 1 12 1 12 G 1 24 1 124 Figure 7 6 Pulse Program for SELTICS In Figure 7 6 one can see that the SELTICS experiment takes only 75 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 7 7 SELTICS at 6 5 kHz Sample Rotation on Tyrosine HCI User Manual Version 002 BRUKER BIOSPIN 111 327 Basic CP MAS Experiments In Figure 7 7 the amplitude of the spinning sidebands are reduced to more than 1096 compared to the original spectrum without sideband supp
317. ter the 180 pulse is incremented proportionally to the t4 period a split t1 experiment as described in chapters 16 and 17 for some MQMAS experiments will be performed h3 h2 h4 dO p4 p2 Op p4 d7 pl21 pl11 pl21 Figure 19 3 Four pulse sequence and coherence transfer pathway Four pulse sequence and coherence transfer pathway for the double quantum fil tered STMAS experiment with shifted echo acquisition stmasdqfe av Pulses p1 and P2 are non selective pulses 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 us respectively 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 ph1 0 180 90 270 ph2 0 4 90 4 180 4 270 4 ph3 0 16 90 16 180 16 270 16 BRUKER BIOSPIN User Manual Version 002 Experiment Setup STMAS ph4 0 receiver ph3 ph1 ph2 19 3 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 e A known MAS spectrum With sufficiently good sensitivity to facilitate the set
318. th ex periments If w pmlg is used and optimized for the direct detect experiment the same parameters will work with w pmlghet provided that both experiments are done with the same probe Table 9 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 pr Power level channel 1 for contact pulse pl12 Power level channel 2 TPPM SPINAL decoupling pl13 Power level channel 2 w PMLG decoupling spo Power level channel 2 for contact pulse 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 132 327 BRUKER BIOSPIN User Manual Version 002 HETCOR with DUMBO PMLG or w PMLG
319. tion 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 tgge can be determined whereas for a non selective pulse this is not the case User Manual Version 002 BRUKER BIOSPIN 217 327 Basic MQ MAS 05 10 15 20 25 30 35 ppm 20 40 60 80 ppm 218 327 Figure 17 4 Nutation profiles of selective and non selective pulses Left diagram shows signal intensity of Rb resonances in RbNO3 as a function of a non selective 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 dis played 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 iexpno and the MQMAS pulse program can be loaded Available pulse programs are mp3qzqf and mp3qzfil The first is a 3 pulse sequence the second a 4 pulse sequence 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 methods like DFS or FAM see MQ MAS Sensitivity Enhancement on page 231 describing sensitivit
320. tion 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 windowed 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 Leskes Michal Madhu P K Vega Shimon Supercycled homonuclear decoupling in solid state NMR towards cleaner 1H spectrum and higher spinning rates J Chem Phys 2007 in press The Sequence pmighet 9 2 1 This sequence uses windowless phase ramped shapes One can write these shapes as multiples 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 carrier 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 pres ence of proton spinning sidebands along F1 which may inappropriately fold in if the spectrum window along F1 is cho
321. to the eject tube which would lead to formation of ice on low temperature runs This requires to maintain some overpressure above the spinner usually by applying some flow to the insert gas line Heat exchangers must be dried with a flow of dry gas before use so there is no water left in which will ice up the exchanger loop After use they must be warmed up and dried with a dry gas flow so that there is no water in leading to corrosion The following figures show the various connections to different probes 2 VT gas in 4 Frame flush gas Figure 3 21 WB Probe MAS VTN and WVT and DVT Probe Connections BRUKER BIOSPIN User Manual Version 002 Additional Connections for VT Operation 1 Probe heater connector 3 TC connector s 2 VT gas in 4 Frame flush gas Figure 3 22 WB Probe MAS DVT Connections In the figure above the upper thermocouple connector read located at stator out lower thermocoupler connector regul located at stator inlet are connected The VT unit must have the auxiliary sensor module to read more than one temper ature Only the TC labelled regul is used for regulation User Manual Version 002 BRUKER BIOSPIN 33 327 General Hardware Setup 1 Probe heater connector 3 TC connector s 2 VT gas in 4 Frame flush gas Figure 3 23 SB Probe MAS VTN J heater and TC sek conn Figure 3 24 SB Probe MAS DVT Connections 34 327 BRUKER BIOSPIN User Manual Version 002 A
322. 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 per form the 2D FT Figure 17 8 compares the same 2D 3Q MAS spectrum pro cessed with no shift and an additional 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 reenters into the spec tral 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 d E d M o MX E S e 10 a 15 ames ia 10 ES H d 0 e s PE DEA CH Fam ee TAE EU oe IS 20 D 20 40 60 ppm 40 20 0 20 40 60 ppm Figure 17 8 Comparison of Differently Processed 2D 3Na 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 Manu
323. ts Since MAS efficiently averages the 1st order quadrupole interaction of the ST the corre sponding MHz broad signal is now narrowed into a huge number of spinning side bands These coherency originating from the ST dephase rapidly and refocus into rotational echoes with each rotor cycle A pulse precisely on top of such a rota tional echo can transfer the SQ coherency from the ST to SQ coherency of the CT the signal from which can then be acquired under standard MAS conditions The evolution in the indirect 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 realiza tion of the STMAS experiment Firstly the spinning frequency must be kept abso lutely constant The duration 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 rotor period varies from that specified in the parameters the calculated delay in the pulse program is incorrect and the pulse misses the echo top so less or no signal intensity is obtained Table 19 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 t4 increment accumulates to as much as 100 r
324. tter should be wired to the probe directly via the proton bandpass filter without going through the pre amp if a high power proton preamplifier is not available For HP HPPR modules 1H 9F this is not absolutely necessary but recommended For HPLNA 1H mod ules it is not required to bypass Note that when bypassing the preamp which at tenuates by about 1 dB the proton power levels 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 4 11 Click on SwitchF1 F2 to set for 13C observation with proton decoupling Load the pulse program hpdec Set cpdprg2 cw Set pl12 to the power level that yields a 4 5 usec proton pulse Set p 7 such that in ased the power displayed is 200W for 13C 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 pro ton 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 protons as found above accumulate 4 scans Set the carrier fre quency between both adamantane 6 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 4 14 for the larger of the two peaks Define the plot limits and determ
325. u can also use the au program dumbo See instruc tions 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 User Manual Version 002 BRUKER BIOSPIN 97 327 Decoupling Techniques References 1 D Sakellariou A Lesage P Hodgkinson and L Emsley Homonuclear dipolar decoupling in solid state NMR using continuous phase modulation Chem Phys Lett 319 253 260 2000 2 Lyndon Emsley s home page http www ens lyon fr STIM NMR NMR html Transverse Dephasing Optimized Spectroscopy 5 3 Decoupling optimized under refocused conditions 3 heteronuclear decoupling 2 4 Prec 13 C t Figure 5 6 Pulse Program for Hahn Echo Sequence Transverse Dephasing Optimized spectroscopy G De Paepe et al 2003 uses a spin echo sequence for optimizing heteronuclear decoupling The idea behind it is simply the removal of the normally dominant Jg term describing coherent residual line broadening effects in the transverse relaxation rate R2 A Abragam chapter 8 With the normal CP experiment the observed line broadening coherence de cay time T3 might be caused by other heterogeneous effects such as distribu tion of chemical shifts or susceptibility effects an
326. uency both in the X nuclei range A triple probe X F H only has 2 RF connectors because 1H and 19F are tuned to the same RF connector simultaneously Likewise a quadru ple probe is an X Y F H probe with the X channel tuned to X and Y and the pro BRUKER BIOSPIN User Manual Version 002 Probe Setup Operations Probe Modifiers ton channel tuned to 9F and H so there are only 3 RF connectors Check Figure 3 9 Figure 3 10 Figure 3 11 Figure 3 12 for the connection of such probes So the double tuning of the X channel into an X and a Y channel is an optional op eration available for WB probes SB probes are always fixed multi channel probes with no option to insert remove an RF channel Such probes have 2 complete X tuning circuits almost identical in construction Activating the second channel means Insert a filter which is tuned to reject at the exact X frequency exactly to 19C frequency for a 19C 18N 2H probe The X chan nel frequency is fixed to the specified frequency in this case 130 while the Y fre quency has a broader tuning range in this case 9N H With different filter inserts that same probe can be modified to different frequency combinations with the following restrictions 1 A frequency outside the tuning range of the probe in double mode cannot be reached in triple mode for instance if the probe in double mode does not tune to 31P a triple insert for 91P 18C cannot be provided without a freque
327. ulse program doubcp AVIII Topspin 2 1 only else use doubcp doubcp av nuc1 13C Nucleus on f1 channel o1p 100 ppm 13C offset nuc2 1H Nucleus on f2 channel 02p 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 sp1 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 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 H TPPM p15 3 5 msec Contact pulse first contact p16 5 12 msec Contact pulse second contact f1 f3 channel d1 5 10s histidine Recycle delay 4s a glycine spnamO Ramp for 13 CP step e g ramp 80 100 sp0 Power level for Ramp HN contact pulse 1H spnam1 ramp45 55 tcn5500 or ramp tangential contact pulse ten5500 square 100 on C or square spnam2 square 100 or ramp45 square on N or ramp tangential pulse 55 tcn5500 cpdprg2 SPINAL64 SPINAL64 decoupling ns 2 4 or 16 Number of scans 260 327 BRUKER BIOSPIN User Manual Version 002 Double CP 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 ty
328. ulses P1 and P2 are exactly an in teger 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 spinning frequency This 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 dO is negative Optimization of the pulses P1 and P2 can be done us ing popt in full analogy to the optimization of the pulses in MQMAS Table 19 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 2120 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 19 3 2 User Manual Version 002 Once the pulses are calibrated the 2D data acquisition can be used to find the cor rect and
329. uning 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 x 10 MHz when the probe is tuned close to the de sired 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 com pared to a probe with a spinner in When a probe has not been used over an extended period humidity may collect inside the turbine 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 accumulated 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 also be performed after a probe repair Since a repair may re sult in a more efficient power conversion sta
330. up and A 2d order quadrupole interaction of the order of the one expected for your sample of interest In a first step a low power selective pulse must be calibrated in a single pulse ex periment After this the STMAS experiment can be optimized using the 2D pulse sequence for the first t4 increment Setting Up the Experiment 19 3 1 Sample There are a large number of crystalline compounds that can be used to set up the experiment Please refer to Table 19 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 fre quency 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 set ting is extremely critical Table 19 2 Some Useful Samples for Some Nuclei with Half Integer Spin Nucleus Spin passis d di s 2 Sample Comments 170 5 2 67 78 2 NaPO gt 10 enriched 1B 3 2 160 42 gt 5 H3BO3 23Na 3 2 132 29 10 Na HPO 2 27 Al 5 2 130 32 5 YAG 87Rb 3 2 163 61 0 5 RbNO3 93Nb 9 2 122 25 1 LiNbOs In MHz at 11 7 T i e 500 13 MHz proton frequency 2 Alternatively Na5HPO 2H50 can be used For anhydrous NagHPO the sample should be dried at 70 C for a couple of hours before packing the rotor in order to eliminate crystal water completely 3 Recycle del
331. usec a 2 7 usec pulse requires about 4 5 dB more power corresponding to almost 4 times more power 80 327 BRUKER BIOSPIN User Manual Version 002 Basic Setup Procedures With p3 properly set a spectrum like in Figure 4 25 should be obtained with about 93 kHz decoupling RF field File Edi View Spectrometer Processing Analysis Options Window Help Danann AALS EX MUS E gt Or TFOs im M2 2RERTEMRARQKRHABMew e atta Figure 4 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 inter action to 4N At fields above 9 4 Tesla 500 MHz and higher the residual line width is mostly determined by chemical shift dispersion and insufficient decou pling 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 4 26 is obtained User Manual Version 002 BRUKER BIOSPIN 81 327 Basic Setup Procedures Table 4 1 Summary o
332. used for recoupling pulses which is insensitive to offsets While in cpredor and cpredori the p pulses should be executed close to resonance this is not required nor desired in the XY 8 version because the offset dependence is well compensated Additionally to the cross polarization pulse programs all REDOR sequences also exist as a direct excitation version without a cp Step These are the programs re dor redori and redorxy8 The setup procedure of these sequences is identical to the ones explained earlier but you can skip the CP optimization procedure which is replaced by a p 2 pulse on the observe channel User Manual Version 002 BRUKER BIOSPIN 159 327 REDOR Data Processing 12 2 2 The following refers to the sequence cpredori where the REDOR experiment and the reference experiment are executed in an interlaced mode which is less likely to be subject to systematic errors The acquired NMR data are arranged in a 2D like structure every odd row 1 3 5 contains the REDOR data set with additional p pulses on channel S ex periment every even row 2 4 6 contains the corresponding echo experi ment Sg experiment with the same evolution period t after the RAW Data is processed with the XF2 command see Figure 12 2 For further processing you can then either use the function T1 T2 Relaxation in the analysis part of Top spin in order to do the integration and or find the peak maxima of the Sg and S
333. uted MUST be set in order to force recalculation of the shape for each step of the optimization In this case acquisition is run with the 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 18 6 shows results of the variation of the RF power level of sweeps with different durations Experiments have been run at 20 kHz and optimization procedures for 1 0 5 0 25 and 0 125 rotor peri ods 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 reduced the required RF field ampli tude 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 User Manual Version 002 BRUKER BIOSPIN 237 327 MQ MAS Sensitivity Enhancement 1 1 rotor period one rotor period and the spinning frequency is increased and hence the rotor pe riod decreased On real life samples the differences between signal intensities at one rotor period compared to e g rotor periods will be more pronounced than on a crystalline model compound Since the spinning frequency is usually deter mined by the spectrum itself the only degree of freedom is the amplitude of the sweep In the
334. value obtained for the center peak see Figure 4 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 4 9 This is most easily seen with the carrier exactly on reso nance 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 4 9 FID and Spectrum of the 79Br Signal of KBr used to Adjust the Magic Angle lit TA Z ep Calibrating 1H Pulses on Adamantane 4 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 H decoupling as shown in the follow ing figure User Manual Version 002 BRUKER BIOSPIN 65 327 Basic Setup Procedures frequercy logic al channe amplifier preamp Sr 150 907083 MHz NUCI E I SFO 150 916083 MHz m JL ses S x x 300 W HPH fix orsi 000 0 He HPH BF2 600 147 MH HMC 7L 1H 1000 HH SFO 600 1465 MHz R sev Y 1H 100 W HPH orsz 6000 H in He BF3 600 147 MHz NUCS SFO3 600 147 MHz F3 S6U3 x HL x 300 w orsa fo 0 Hz ort BF4 600 147 MHz NUCA SFO4 600 147 MHz F4 so IH
335. ve 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 IH Far homonuclear decoupling Far homonuclear decoupling Pul amp 4 n37547 300 Frequency of observe channel Proton power level during contact Proton power level during contact Shape for contact pulse ramp 100 Phase alignment of freq offset in SPO Offset frequency for SPO User Manual Version 002 FSLG HETCOR In the figure above the frequency offsets for the FSLG part are shown as cnst22 cnst23 They are different because cnst24 shifts the center frequency by 2000 Hz Table 8 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 ph 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
336. version 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 1 190 327 BRUKER BIOSPIN User Manual Version 002 Symmetry Based Recoupling Figure 14 6 PC7 2d SQ SQ correlation on tyrosine HCl Spectral Processing 14 7 Processing parameters as above Parameter Value Comment F1 acquisition 16 left column SI 2 Ak 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 i u fi A _ f 1 e Li d P E z 7 Lg ex s t RE s E lt s B 8 gt e F 8 Lg S af g o eg ld e es D i 4 D f b ao He Lg 4 s t s H u T3 Tt TT mr TT TT 160 140 120 100 so 0 F2 ppm 162 146 120 190 6o F2lppe 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
337. which are nor mally smeared out by extensive homonuclear dipolar couplings between pro tons Setup a 2D data set with the pulse program Igcphetfq The figures below com pare spectra taken with and without LG offset during cp Ft el en ue on Ft ppm Ir s A hh ul Il IBI dua E 10 re e u to m wo M eo F pp 160 140 120 10 20 F2 ppm Figure 9 3 HETCOR on tyrosine HCI without left and with LG contact 1msec contact BRUKER BIOSPIN User Manual Version 002 RFDR Radio Frequency Driven Recoupling RFDR with longitudinal magnetization ex change is a homonuclear dipolar recoupling experiment This easy setup tech nique is a zero quantum recoupling sequence that achieves chemical shift correlation under MAS conditions The time dependence of the cross peak ampli tudes can be employed to determine inter nuclear distances With short dipolar recoupling times only spins in close spatial proximity lead to cross peak facilitat ing 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 homonuclear dipolar recoupling is implemented via the application of rotor synchronised 180 degree pulses one inversion pulse per rotor period The phas es of the 180 degree pulses are cycled with Gullion s compensated
338. which is a mul tiple of Tg 2 as it is shown in Figure 12 6 The x axis is therefore calculated by 1 MASrate 2i 2 Eq 12 2 where i is the number of the corresponding data point So S So the number of the Sy S set within the 2D data set In this experiment MAS spinning speed was 10 kHz resulting in data points every 200ms Note only the analysis of the low field shifted peak is shown here corresponding to the alpha glycine modification of the measured sample BRUKER BIOSPIN User Manual Version 002 Setup 1 2 1 0 Le Pr NEE an 0 8 e e gy 0 6 D e 7 0 4 e Experimental Data 0 000 0 002 0 004 0 006 0 008 0 010 0 012 0 014 evolution time s Figure 12 6 Plot of the Normalized Signal Intensity Versus the Evolution Time There are different methods for the interpretation of the experimentally measured REDOR curves In the case of isolated two spin systems like in this case of 15N 13C glycine it is generally possible to fit the experimental dephasing curve by us ing a combination of bessel functions This is called the REDOR transformation and gives you direct access to the dipolar coupling information for the measured spin system for details check reference 6 The more common way for the interpretation of the experiment is the second mo ment approach which is also suitable for multiple spin systems Here the begin ning of the REDOR curve can be fitted by a parabolic approximation up to norm
339. xperi ment should suffice Saturation parameters can be set as for carbon saturation previously 20 5 100 and d20 1 50 ms Acquire a spectrum with these parameters and verify that there is again no signal Make a new data set with iexpno and set parameters for 2D acquisition as for the previous experiments D1 can be short with the same proviso about duty cy cle as the X saturation recovery 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 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 polar ization from the same proton 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 alpha 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 User Manual Version 002 BRUKER BIOSPIN 211 327 Relaxation Measurements 212 327 BRUKER BIOSPIN User Manual Version 002 Basic MQ MAS Introduction Pulse sequences 17 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 in teger quadrupole nuclei the dominant
340. xperiment 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 1C is selected as nuc in the F1 dimension set FnMode STATES TPPI BRUKER BIOSPIN 185 327 Symmetry Based Recoupling 12 13 14 Set the spectral window along 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 un interpretable because frequently the folding does not lead to cross peak over lap 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 in termediate sampling rate along F1 can be achieved by incrementing the evolu tion period synchronised to the phase shifted blocks of the sequence one PC7 block being 21 7 T rotor period This will also not g
341. y For SB probes and lt 400 MHz different hardware is used A set of 1H transmitter bandpass preamp and F transmitter bandpass preamp is re quired Note a standard H 9F preamplifier will not allow long 19F pulses through it For short pulses it is ok for decoupling it must be bypassed or a dedicated 19 pre amp must be used Figure 3 11 19F 1H Combiner Filter Set BRUKER BIOSPIN User Manual Version 002 Connections for Probe Identification and Spin Detection X BB HPHPPr 1H HPHPPR Y BB HPHPPr X pass Y stop Figure 3 12 Quadruple Resonance HFXY Experiment WB probes 2 400 MHz 19F HPHPPr only Connections for Probe Identification and Spin Detection 3 4 Most solids probes were delivered without Probe Identification System PICS Probes delivered since 2007 are equipped with PICS Please refer to Figure 3 4 to identify the PICS port at the preamplifier cover module The probe connections for the spin rate cable and the PICS cable are shown in Figure 3 13 for a WB and in Figure 3 14 for a SB probe 1 PICS probe connector 2 Spin rate monitor cable Figure 3 13 PICS Probe Connector and Spin Rate Monitor Cable on a WB Probe User Manual Version 002 BRUKER BIOSPIN 25 327 General Hardware Setup On SB probes the location may differ but the connectors are if present in the case of PICS easily identified by the type of connector e FN Figure 3 14 Spin Rate Monitor Cable Connector
342. y enhancement methods In Table 17 2 the starting parameters for the set up are displayed This table gives typical values for the pulses and powers that should be close to the final val ues confirmed by the optimization 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 Optimi zation will be done on the first increment of the 2D sequence i e d0 1 us Two strategies for the optimization procedure can be followed either the pulse lengths p1 and p2 or the power level pl11 can be optimized for maximum signal ampli tude However the latter can be disadvantageous because 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 002 Basic MQ MAS Table 17 2 Initial Parameters for Setup Parameter Value Comments Pulprog mp3qzdf av or Pulse program mp3qzfil av NS 12 n zaf Full phase cycle is important 96 n zfil DO 1u Or longer t4 period D1 5 T4 Recycle delay use dummy scans if sh
343. y Powder patterns by Effort less 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 experiment but provides better compen sation for experimental imperfections such as B4 in homogeneities and pulse im perfections 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 ver sion As a consequence the SUPER experiment does not require high spinning speeds to fit the F1 lineshape into the rotor synchronized spectral window or very strong 136 pulses SUPER has several advantages First of all it covers a large bandwidth for the isotropic chemical shift Secondly no requirements exist for 1H decoupling during the recoupling pulses because it uses 360 pulses instead of the 180x pulses in Tycko s experiment Exact 360 pulses automatically decouple the heteronuclear dipolar interaction so that no or only weak 1H decoupling is required during the re coupling 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 moderate spinning speeds of up to 6 5 kHz can be chosen so that experiments can be per formed without serious problems on high field instruments The limiting factor in the choice of the spinning speed is the rotor synchronization r
344. y clicking the marked button in the up per icon bar Figure 12 5 Here the value for number of points has to be set to the td2 value and the list file name has to be switched to auto otherwise the data User Manual Version 002 BRUKER BIOSPIN 161 327 REDOR 162 327 preparation will fail compare Figure 12 5 By clicking OK topspin will automati cally pick all the intensities for your measured REDOR experiments These values are then saved in the processed data directory data user nmr experi mentlexp pdata 1 in the file t1t2 dx The intensities are saved in two columns for each peak while the first column represents a arbitrary x scale the measured intensities are within the second column Remember because of the used pulse program cpredori every odd line contains an intensity value for an REDOR spec trum S and every even line the corresponding intensity of the ECHO experiment So Relaxation glycine _4nwe_redor 13C_15N 7 1 C sewe_200012 Fitting Function wmnmrtt 1 0 0010 ncrement auto v to pick data points nitr l parameters juesse Additional Parameters Figure 12 5 Setting the Correct Analysis Parameter After importing this file into Excel or any other program using either Origin or Igor is recommended for calculating the values for 85 S Sg these normalized intensities are plotted versus the evolution time of each spectrum

AVANCE Solids User Manual - UC Davis Nuclear Magnetic

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