High Resolution Magic Angle Spinning Spectroscopy
Contents
1. 2 Insert the sample into the rotor Screw the cylinder head screw M2 x25 into the upper spacer but do not tighten it figure B 3 Use the screw as a handle Spray the upper spacer with coolant spray or dip it into liquid nitrogen and press it completely into the bore Excessive sample will spill out through the center hole and the thread Version 001 BRUKER 47 55 Figure B 3 Spray upper spacer with coolant spray Upper Spacer Cyl Head Screw HZ3909 M2 x 25 Sample 7 H Ee 3 Put the grub screw on a screwdriver and screw it completely into the upper spacer figure B 4 Figure B 4 Screw grub screw completely Grub screw Screwdriver 3mm ie When the assembly is done correctly the distance between the end of the upper spacer and the end of the rotor will be 3mm The short end of the rotor packer K8111001 is 2 9 mm long which will allow you to easily check for the correct fit 4 4 Push the rotor cap KELF all the way into the rotor figure B 5 Sample Figure B 5 Push the rotor cap into the rotor Upper Spacer Sample Grub Rotor Ca Lower Spacer SCREW H6304 i 48 55 BRUKER Version 001 References 10 11 12 13 14 15 Version 001 H D H St ver and J M J Fr3 4chet Macromolecules 1991 24 883 J D Gross P R Costa J B Dubacg D E Warschawski P N Lirsca P F Devaux and R G Griffin J Magn Reson 1995 106 1872. T T T T T T T T T T T T T T T T T T T T T ppm 8 6 4 2 0 500 MHz gradient H H COSY spectrum of an N FMOC N Boc L Lysine deriva tized Wang resin swollen with CDCIs MAS 3 5 kHz 1 ms gradients 10 G cm Gradients are frequently used in inverse detected heteronuclear correlation exper iments to select the magnetization from protons coupled to a 136 spin and sup press the uncoupled protons magnetization The spectra below show H C hr MAS HMQC figure 7 13 and HMBC figure 7 14 spectra of a Wang resin used in combinatorial chemistry Note the excellent suppression of t1 noise in the gradi ent HMQC spectrum figure 7 13 left versus the phase cycled spectrum figure 7 13 right and the clean noise floor in the HMBC spectrum figure 7 14 Note BRUKER 39 55 Application Examples that no attempts were made to improve the spectral quality e g by t4 ridge sub traction Figure 7 15 shows another example of a gradient HR MAS HMBC spectrum The spectrum is obtained from a human lipoma tissue and demonstrates the useful ness of this type of high resolution MAS spectroscopy to the study of biological tis sues Figure 7 13 400 MHz H C HMQC spectra of an N FMOC N Boc L Lysine Poa 100 pes 40 55 Ln Tun T 7 T 4 2 0 derivatized Wang resin swollen with CDCls The left spectrum is acquired using gradients to suppress the magnetization from uncoupled prot
3. ll l uo MI BE APC iN IN 4 N am OK L U Nuar T l EM Jl an An i kans T BEE EN E W TT m En een e ACA Raa 80 78 76 74 l 72 70 i 68 66 i 64 i 62 60 ppm obtained with a single pulse top and with a CPMG sequence bottom The reso nances of the resin are suppressed in the lower spectrum by employing a CPMG 32 55 BRUKER Version 001 Hr MAS of small organic molecules sequence T n t n with t 21 5 ms and n 20 Avance 400 MHz 3200 Hz spin ning 2 mg sample on Tentagel substrate swollen in deuterated chloroform Figure 7 8 Figure 23 400 MHz proton spectra of a single 100 um bead M M 8 4 8 0 7 6 7 2 6 8 6 4 ppm top separated under the microscope volume 0 5 nl 1056 scans weight 600 picograms and 2mg of the same sample swollen in CDCI 8scans Avance 400 3200 Hz spinning Figure 7 4 2D J resolved proton spectrum of 2 mg sample on a Tentagel resin e d ln d E ae ate teintes d M swollen in CDCI Avance 400 MHz 4 scans MAS 3200 Hz Version 001 BRUKER 33 55 Application Examples 34 55 Figure 7 5 Double Quantum filtered Cosy of a small organic molecule on a Tentagel resin Avance 400 MHz 16 scans 2 mg sample MAS 3200 Hz In a different application of hr MAS to the study of small organic molecules spots were scratched off a TLC plate and made into a slurry with DoO Figure 7 6 sh
4. High Resolution Magic Angle Spinning Spectroscopy User Manual Version 001 BRUKER The information in this manual may be altered without notice BRUKER accepts no responsibility for actions taken as a result of use of this manual BRUKER accepts no liability for any mis takes contained in the manual leading to coincidental damage whether during installation or operation of the instrument Un authorised reproduction of manual contents without written permission from the publishers or translation into another lan guage either in full or in part is forbidden This manual was written by Frank Engelke March 4 1998 Bruker Elektronik GmbH Rheinstetten Germany P N Z31437 DWG Nr 1147001 Contents GONIEMIS saunas hennen nnen eenen bend she iii 1 IHTOGUCHON user 5 1 1 Introduction u armata ee nern 5 2 System Description iris cria aon aaa Ra ora ai aas ra Rmus 7 2 1 Probe Description Gava dead oag aa n AA E 7 2 2 Site and Parts Requirements ee ee ee 8 3 Installation of the Probe and Peripherals 9 3 1 Probe Installation unne TK Vev eneen ennen 9 3 2 Pneumatic connections sss eee eee ee eee eee eee 9 3 3 Electronic connections ee eee ee e eee ee 11 3 4 MAS Sample Changer see eee eee sss 11 4 Operation of the HR MAS System 13 4 1 MAS Rotors Caps and Inserts sese eee 13 4 2 Sample loading sese eee eee 14 4 3 Cap r movalzi dende Mente M i
5. L L Cheng C L Lean A Bogdanova S C Wright Jr J L Ackerman T J Brady and L Garrido Magn Reson Med 1996 in press K Millis W E Maas S Singer and D G Cory Magn Reson Med 1997 in press R C Anderson M A Jarema M J Shapiro J P Stokes and M Ziliox J Org Chem 1995 60 2650 P Keifer L Baltusis D M Rice A Tymiak and J N Shoolery J Magn Reson 1996 A119 65 S K Sarkar R S Garigipati J L Adams and P A Keifer J Am Chem Soc 1996 118 2305 A N Garroway J Magn Reson 1982 49 168 A Sodickson and D G Cory submitted to J Magn Reson W E Maas F H Laukien and D G Cory J Am Chem Soc 1996 118 13085 R E Hurd and B K John J Magn Reson 1991 91 648 W E Maas F H Laukien and D G Cory J Am Chem Soc 1996 118 13085 M Piotto V Saudek and V Sklenar J Biomol NMR 1992 2 661 P C M van Zijl and C T W Moonen J Magn Reson 1990 87 18 High Resolution MAS Spectroscopy 27 BRUKER 49 55 50 55 BRUKER Version 001 Figures Version 001 1 Introduction 5 Figure 1 1 500 MHz proton spectra of a human Lipoma tissue 6 2 System Description 7 Figure 2 1 Upper probe chamber with MAS stator in magic angle position Q 7 3 Installation of the Probe and Peripherals 9 Figure 3 1 Pneumatic connections of the HR MAS probe 9 Figure 3 2 Diagram for the pneu
6. Sensing of bearing Drive aut Eject out Figure 3 3 Set up for low temperature operation B VT 2000 or Heater Thermecauple back panel 3000 Spinrate front panel Heater Thermocouple front panel KS magicangle vertical MAS pneumatic eject unit drive a bearing gas heat exchanger ta ambient autput o enas KOPEN EU ERE NR EEEE pneumatic unit The bearing gas preferably dry No gas is cooled by an heat exchanger im mersed in liquid No and is directly fed into the glass dewar inlet at the bottom of the probe 10 55 BRUKER Version 001 Electronic connections Electronic connections 3 3 Connect the X channel of the probe to the X BB HPPR preamplifier using a proton reject or a low pass filter e Connect the H channel of the probe to the proton using a proton band pass filter Connect the H lock channel of the probe with the lock preamplifier Connect the spin rate cable from the MAS unit For variable temperature operation connect the heater cable and the thermo couple from the rear panel of BVT 2000 unit or the front panel of the BVT 3000 unit Connect the gradient cable to the gradient connector on the side of the probe Connect the Y channel to the probe via a low pass filter MAS Sample Changer 3 4 Version 001 The Bruker MAS sample changer is designed to allow the acquisition and pro cessing of MAS data from up to 20 samples without ope
7. A Shipping List 45 B Spherical Inserts 47 Figure B 1 Spray lower spacer with coolant spray eee ee 47 Figure B 2 Inserting the rotor is now easier eee ee ee e e 47 Figure B 3 Spray upper spacer with coolant spray eee eneen 48 Figure B 4 Screw grub screw completely see eee annen eeen eenn 48 Figure B 5 Push the rotor cap into the rotor nnee 48 C References 49 BRUKER Version 001 Tables Version 001 1 Introduction 2 System Description 3 Installation of the Probe and Peripherals 4 Operation of the HR MAS System 5 Preparing the Probe for Experiments 6 Gradient HR MAS Spectroscopy 7 Application Examples A Shipping List Table A 1 Shipping List nanne eeens B Spherical Inserts C References BRUKER 13 19 25 53 55 Tables 54 55 BRUKER Version 001 Version 001 BRUKER 55 55
8. For both samples to adjust the angle the sample has to be spun at about 5 to 6 kHz Note that either sample can be used to set the angle irrespective of what nucleus will be observed in the HR MAS experiments For the proton sample a spectrum is acquired and Fourier transformed The mag ic angle setting is then slightly changed by turning the micrometer screw at the bottom of the probe Take another spectrum and repeat the procedure until the magic angle setting is optimal see figure 5 1 BRUKER 19 55 Preparing the Probe for Experiments 20 55 Figure 5 1 Proton NMR spectra of BaCIO3H2O BaCl0 HO off magic angle H MAS NMR EBENEN UU Us PEE N E in magic angle at magic angle f l di hod ER A SSN M E et nee Nam m id U LJ MA L LJ T Lt Obtained off the magic angle top and obtained with the correct angle setting bottom Experimental parameters ns 1 swh 125kHz pulse program zg p1 3us pl1 0 dB d 5s spinning rate 6000 Hz The Br resonance frequency is very close to the 13C resonance frequency thus no or just a slight tuning of the RF circuit is necessary Furthermore KBr provides a very sensitive signal with a very short T4 Already at moderate spin rates the satellite transition will be split into sidebands which have to be as narrow as pos sible for the optimum angle setting With a rotor completely fi
9. are anisotropic and impose a dependence on the NMR frequency based on the orientation of a spin or molecule with respect to the main magnetic field direction Furthermore the magnetic sus ceptibility of the sample and susceptibility differences within the sample lead to a broadening of the resonances In liquid state spectroscopy the rapid isotropic motion of the molecules averages the anisotropic interactions resulting in an isotropic chemical shift frequency and a disappearance of the line broadening due to dipolar couplings Furthermore the sample geometry a cylinder parallel to the main magnetic field is chosen such that the susceptibility broadening is minimized In solid samples on the other hand the lack of molecular mobility results in broad lines This line broadening can be reduced by spinning the sample rapidly around an axis which is oriented at an angle 6 54 7 with the direction of the magnetic field By spinning at this so called Magic Angle the angle between the z axis and the 1 1 1 vector at a rate larger than the anisotropic interactions these in teractions are averaged to their isotropic value resulting in substantial line nar rowing In addition magic angle spinning removes the magnetic susceptibility broadening In addition to pure solids or pure liquids there is a wide range of materials which exhibit either reduced or anisotropic mobility Examples include polymer gels lipids tissue samples swoll
10. requires an excessive shim cur rent reduce the current and continue shimming by adding current to another shim from the same group as shown in the table For instance if the current in ZX is too high reduce the value and optimize the line shape by adding current to the Xo Y2 Z shim If the probe is not exactly aligned with the xz plane see figure 5 4 then a small amount of Y and YZ may be needed for optimal shimming A second shim set should be obtained for rotors without spacers and the optimal shim settings for these will be somewhat different Under normal conditions the shim values obtained as described above will be close to optimal for all other samples When changing samples one usually only needs to adjust Z and ZX Both shim value sets should be stored on disk Make sure when the shim values are stored that the HR MAS probe is defined as the current probe in the EDHEAD command then shims can be recalled with the correct lock conditions like phase and power Choosing the appropriate spinning speed 5 4 The correct spinning speed depends strongly on the sample under study A mini mum spinning speed is recommended which depends on the field strength of the magnet 300 MHz 2400 Hz spinning 400 MHz 3200 Hz spinning 500 MHz 4000 Hz spinning 600 MHz 4800 Hz spinning Version 001 BRUKER 23 55 Preparing the Probe for Experiments 24 55 These spinning speeds are chosen to prevent spinning sidebands to fall into re
11. since the reactions can be monitored without cleaving off the substrate High resolution NMR spectra are obtained by swelling the beads in a deuterated solvent in combination with Magic Angle Spinning An example from this field is illustrated in figure 7 1 The proton spectrum is ob tained from 2 mg of a meta and para disubstituted aromatic ring on tentagel res in swollen in d chloroform and acquired with a CPMG sequence The broad resonances of the polymer resin are distinguished from the substrate resonances by exploiting the differences in proton To s using the CPMG sequence as is dem onstrated in figure 7 2 Further information on these compounds is obtained from two dimensional NMR techniques Figures 7 4 and 7 5 show a J resolved and a double quantum filtered Cosy spectrum of the same compound respectively BRUKER 31 55 Application Examples Figure 7 1 1H hr MAS NMR spectrum of small organic molecules en O bead n O N O Y n 10 15 Polystyrene resin Linker Substrate a para disubstituted aromatic b meta disubtituted aromatic c ig 72 71 70 69 68 67 66 65 ppm including a meta and para disubstituted aromatic ring on tentagel resin obtained under the following NMR conditions DRX400 2mg in 4mm rotor with spherical spacer swollen in d chloroform cpmg 40msec 3200Hz spinning smallest line width at half height 2 7 Hz Figure 7 2 Comparison of proton spectra L 1
12. tee 14 4 4 Spinning the rotor under manual control 15 4 5 Spinning the rotor under computer control 16 5 Preparing the Probe for Experiments 19 5 1 RE TUNING oer eR Het eek bar b ret ve 19 5 2 Adjusting the magic angle nennen eeen 19 5 3 Shimming a HR MAS probe eee nuur 21 5 4 Choosing the appropriate spinning speed 23 6 Gradient HR MAS Spectroscopy 25 6 1 Magic Angle Gradient Design nnen 25 6 2 Setting up the magic angle gradient ussssss 26 7 Application Examples eere ene 31 7 1 Hr MAS of small organic molecules sssss 31 7 2 HR MAS of swollen polymers sese eee e eee 35 7 3 MAS of biological samples narren eneen nennen ven 36 7 4 Gradient hr MAS spectroscopy eee e 39 7 5 Water suppression and Diffusion Studies with MAS gradients 42 A Shipping LISE u nun 45 Version 001 BRUKER iii Contents Spherical Inserts asssssssnervnsnrs ren eru tan deb dessendrandehe 47 FIGTCT e 49 FIQUFES c 51 DIEDIIT o 53 BRUKER Version 001 Introduction Introduction Version 001 1 1 The line width of an NMR resonance depends strongly on the microscopic envi ronment of the nucleus under study Interactions such as the chemical shift and di pole dipole coupling between neighboring spins
13. 001 BRUKER 37 55 Application Examples Figure 7 10 Hr MAS of benign versus malignant human prostate tissue EN en ee Be T o F o er E 2 24 2 i 1 2 T 3 2 8 Bom 6 0 8 Hr MAS of benign top two spectra versus malignant bottom human prostate tissue Avance 400 MHz 4000 Hz spinning The cancerous tissue shows a strongly enhanced lipid content Figure 7 11 TOCSY presat of a living white worm enchytraed in 95 H20 5 D20 Avance 400 8scans 300incr MAS 2800 Hz 38 55 BRUKER Version 001 Gradient hr MAS spectroscopy Gradient hr MAS spectroscopy 7 4 Version 001 The benefits of using magnetic field gradients in high resolution spectroscopy have been well documented In high resolution MAS similar benefits are seen from the use of gradients in particular a reduction in a variety of artifacts such as t ridges and repetition artifacts while also saving time by eliminating the need to complete a full phase cycle A number of gradient high resolution MAS experi ments are shown below on a variety of samples Figure 7 12 shows the gradient COSY of a swollen Wang resin used in combina torial chemistry The use of gradients eliminates the repetition artifacts and t ridg es Note that the spinning sidebands in the f direction are aliased since no bandwidth filters are used in the indirect domain and the spectrum is not symme trized Figure 7 12 Gradient COSY of a swollen Wang resin
14. L H75 H100 8 amp 8 amp e L 25 H150 ppm To To To To me ppm 5 4 3 2 1 of a human Lipoma tissue sample courtesy of Dr Singer Brigham and Women s Hospital MAS 5 kHz 1 ms sine shaped gradient pulses Version 001 BRUKER 41 55 Application Examples Water suppression and Diffusion Studies with MAS gradients 7 5 42 55 Gradients allow for fast and efficient water suppression Figure 7 16 shows spec tra of a hydrogel in which the water is suppressed with the Watergate sequence An alternative gradient method is the so called Dryclean sequence in which a stimulated echo sequence is used to attenuate resonances based on differences in the diffusion rates of the molecules Figure 7 17 shows spectra of a biological tissue in which the water signal is suppressed using Watergate and Dryclean re spectively Figure 7 16 500 MHz single pulse proton spectrum of an acrylamide hydrogel GERM od eee etl B M M 9 0 8 0 7 0 6 0 5 0 4 0 3 0 2 0 1 0 ppm top The spectrum below is acquired with the Watergate method 1 ms sine shaped gradient pulses are use with a strength of 30 G cm sample courtesy of Prof Tanaka Massachusetts Institute of Technology BRUKER Version 001 Water suppression and Diffusion Studies with MAS gradients Figure 7 17 500 MHZ proton spectra of human high grade Pleiomorphic Wh THI A N A e D T AL il KIL ACORN A anco ge
15. ads The magic angle z shims are listed below for orders Z through Z The simplest approach is to use the laboratory shims with the largest coefficient so for instance the X shim is used as the normal high resolution Z shim Complete shimming can then in principle be accomplished using only X ZX ZPX Z and Z as the surro gate nt order on axis shims Note however that the coefficients given for the magic angle shims do not take into account the efficiencies of the shim coils and the use of the other shims listed below may be needed BRUKER Version 001 Choosing the appropriate spinning speed MAS shims Laboratory shims Z 1 2 Q RUSET B WB ze x2 Y y 242ZX 3 2 1 5 5 7 EL L K YZ xX m 2h e ABUS CO E 4 7 Z g4 m 18 5 1 Z ge 643 To shim the HR MAS probe a sample of 3 CHCI in Acetone d6 is suggested using a rotor with a spherical insert Make sure the magic angle is adjusted prior to shimming the probe Spin the sample at a rate suggested in the following sec tion It is always best to shim up the probe already under the conditions used later for the real samples Make sure that the correct gas is used for temperature control and spinning the coils can be optimized for air or nitrogen gas To obtain best results the gas indi cated should be used Lock the sample tune the probe and start shimming using the X ZX Z X Z and Z shims as described above If one of the shims
16. at a frequency of 500 MHz Figure 1 1 500 MHz proton spectra of a human Lipoma tissue 6 0 5 0 4 0 3 0 2 0 1 0 ppm The top spectrum is acquired in a conventional high resolution probe spinning at 20 Hz Bo while the lower spectrum is acquired in a HR MAS probe spinning at 5 kHz 6 55 BRUKER Version 001 System Description Probe Description 2 1 The HR MAS probes have been designed to perform solution type experiments while spinning the sample at the magic angle The probes are either doubly tuned e g H and 18C or triply tuned e g H C and 15N in addition to a H lock channel All three or four channels are operating via a single NMR transmit re ceive solenoid coil located inside the MAS turbine see figure 2 1 The probes are capable of performing either direct or indirect inverse detection experiments Properties of the HR MAS probes include 1 a fF Oo N MAS at spinning rates of up to 16 kHz for 4 mm o d Zirconia rotors for liquid or liquid like samples and up to 6 kHz with spherical volume PTFE or Kel F in serts Sample insertion ejection without removal of the probe up to 600 MHz Magic angle adjustment with micrometer screw at probe bottom Built in heater dewar and thermocouple for VT operation from 20 C to 70 C Optical spin rate counter with trigger signal output for rotor synchronized ex periments Automatic insert and eject of the sample using the MAS sample cha
17. carbon bottom spectra ppm T l 7 i A T A T 180 160 140 120 100 80 60 40 20 ppm of a derivative of poly acethylene swollen in deuterated chloroform MAS of biological samples 7 3 Plant tissue and food samples can be investigated by HR MAS NMR The useful ness of magic angle spinning within these fields of study is demonstrated in the examples shown below Figure 7 8 shows a H 2D TOCSY spectrum of a sample of orange peel under MAS In figure 7 9 the 800 MHz proton spectrum of red perch filet is shown Fig ure 30 shows spectra of benign and malignant prostate tissues obtained via biop sies The cancerous tissue shows a strongly enhanced lipid content Figure 7 11 displays a TOSCY spectrum obtained from a living white worm which allows one to study drug metabolism in vivo 36 55 BRUKER Version 001 MAS of biological samples Figure 7 8 2D TOCSY hr MAS spectrum of orange peel ppm ippm cut to small pieces flushed and measured in DO obtained under the following NMR conditions Avance 400 2800Hz spinning 4mm rotor with spherical insert 2 7mg of sample phase sensitive 2D TOCSY with presaturation of the residual water signal 8 scans 400 increments linear prediction to 800 points in f1 window function shifted sine bell squared in both directions ssb 2 Figure 7 9 8 mg red perch filet cut flushed and measured in D20 Avance 800 128 scans 15KHz spinning Version
18. d line of XWINNMR Version 001 masr masg mash mase masi masrmon set the spinning rate Hz start spinning stop spinning eject sample insert sample MAS spinning rate monitor continuous BRUKER 17 55 Operation of the HR MAS System 18 55 BRUKER Version 001 Preparing the Probe for Experiments RF Tuning 5 1 The RF tuning is performed by centering the dip of the wobble curve on the screen through turning the tuning and matching rods at the bottom of the probe The rods for the H channel are labeled yellow the rods for the 13C channel are labeled blue The tuning bandwidth of the 13C channel is equal to a few MHz The H lock channel is fixed i e no external matching and tuning adjustment is nec essary Always tune the probe while the sample is spinning When tuning the probe pay attention to the wobble curve If modulations are superimposed on the wobble curve this is usually a sign that the sample spinning is not stable Adjusting the drive and bearing pressures or restarting the spinner can stabilize the spinning If problems persist repack the sample Adjusting the magic angle 5 2 Version 001 The precise setting of the magic angle is mandatory to obtain the maximum spec tral resolution obtained by MAS In order to adjust the magic angle a sample is needed with an NMR line that is very sensitive to angle misalignment Suitable samples are BaCIO H O H observe and KBr Br observe
19. e gradient 6 2 26 55 Before the magic angle gradient can be used in a routine fashion a few adjust ments have to be made In order to ensure short rise and fall times of the pulsed field gradients it is necessary to compensate for Eddy currents generated by the gradient pulses and for the switching times of the gradient amplifier This is done through so called pre emphasis adjustment Furthermore the gradient strength BRUKER Version 001 Version 001 Setting up the magic angle gradient needs to be calibrated Before initiating these adjustment procedures make sure that the gradient amplifier has been installed correctly Adjusting the pre emphasis Figure 6 3 Pre emphasis controller window For this procedure use a rotor with H5O D5O preferably doped with CuSO 2 mMol Spin the sample at approximately 3 KHz lock and shim to a proton line width of better than 5 Hz at half height Set the offset a few kHz off resonance from the water peak Use a sweep width of 20 ppm D1 1 2s and choose TD such that the FID has decayed about 50 in amplitude after approximately 1 8 of the acquisition time Next set the pulse program to preempgs for RX22 based systems set the second channel to proton as well Use a gradient program which defines a single square gradient pulse e g gradprog 1 squa and define a variable delay list vdlist pre emp which contains the following values 100ul 300ul 1m 3m 10m 30m 100m 300m Thi
20. e known length of the sample l 0 27 cm and gradient amplifier output 20 of 10A the gradient strength is determined as 5 2 Gem A Figure 6 5 Gradient profile for a spherical sample of water sphericalsample E fillhole T T T T T T 15000 10000 5000 0 5000 10000 15000 Hz The gradient strength is obtained from the width of the profile BRUKER 29 55 Gradient HR MAS Spectroscopy 30 55 BRUKER Version 001 Application Examples High resolution MAS provides an easy means of obtaining high resolution spectra of a variety of samples that would otherwise result in poorly resolved spectra due to residual anisotropic interactions The addition of a HR MAS probe and a MAS pneumatic unit to a standard high resolution spectrometer is all that is needed to open a gate to the world of HR MAS spectroscopy and access to a vast amount of highly interesting samples In this section a number of application examples are shown from a variety of com pounds All spectra are acquired with standard high resolution techniques Hr MAS of small organic molecules 7 1 Version 001 There is a wide variety of conceivable applications for HR MAS probes Among others one important field for pharmaceutical research concerns combinatorial chemistry In combinatorial chemistry small organic molecules are synthesized on resin beads consisting of a polymer matrix and a linker material NMR is advantageous in analyzing these samples
21. en resins used as supports in combinatorial chemistry gt 7 plant and food samples While these samples generally have suf ficient mobility to greatly average anisotropic interactions the spectral resolution for the static samples are still much lower than that which is achieved for liquid samples The excess broadening under static conditions is due to a combination of residual dipolar interactions and variations in the bulk magnetic susceptibility For a variety of samples including the aforementioned examples magic angle spinning is efficient at averaging these left over components of the solid state line width and leads to NMR spectra that display resolution approaching that of liquid samples Such methods have been termed High Resolution MAS HR MAS NMR Bruker has developed a series of dedicated probes for standard bore magnets to accommodate the rapidly expanding field of HR MAS These probes are available in double and triple resonance modes and come equipped with a deuterium lock channel The probes have automatic sample ejection and insertion capability with the availability of an optional sample changer enabling fully automated sample runs A Bo gradient directed along the magic angle is optional BRUKER 5 55 Introduction Figure 1 demonstrates the gain in resolution that can be achieved with a HR MAS probe compared to a standard high resolution probe The spectra are proton spectra of a human lipoma tissue obtained
22. f magic angle vem Balls LLL Lu Shimming a HR MAS probe 5 3 When shimming a high resolution probe one distinguishes between on axis z and off axis x y etc shims Spinning the sample parallel to the magnetic field di Version 001 BRUKER 21 55 Preparing the Probe for Experiments 22 55 rection averages the off axis in homogeneity which may result in spinning side bands In MAS spectroscopy the spinner axis is at an angle 9 4 with the magnetic field di rection and the distinction between the traditional on axis and off axis shims no longer holds The spinning rates in MAS spectroscopy however are typically at least a few kilohertz which is much larger than the magnetic field in homogeneity As a result the amplitudes of the sidebands are small and shimming may be done with a set of shims that is cylindrically symmetric about the MAS spinner axis Such a set of shims can be constructed from combinations of the standard labora tory frame shims via a transformation to the tilted magic angle frame In the sim plest implementation the MAS probe is aligned such that the spinner axis is in the laboratory xz plane figure 5 4 Figure 5 4 The magic angle spinner axis N ee pue _ shim plate y an de x leads The magic angle spinner axis is aligned with the xz plane of the shims by position ing the front plate of the probe parallel to the direction of the shim le
23. gions of interest for typical proton samples For some samples such as for instance in biological tissues higher spin rates may be beneficial Keep in mind however that this may result in irreversible changes in the sample due to the breakdown of cellular structures and that the ro tation may lead to significant sample heating as much as 10 C In the case of very fast spinning it is a good practice to acquire spectra at lower spinning speeds both before and after the fast spinning and compare those spec tra to ensure that no changes have occurred in the sample Figure 5 5 displays proton spectra of a plant leaf acquired at spinning speeds of 4 kHz 10 kHz 19 kHz and 6 kHz The spectrum at 6 kHz is obtained after spinning at 19 kHz and shows that no irreversible changes have taken place Figure 5 5 Proton spectra of a plant leaf Effect of spinning speed on 400 MHz HR MAS proton spectra of a plant leaf cut and flushed in water The spectra are displayed with absolute intensity scaling The spectrum obtained at 6 kHz is acquired after spinning at 19 kHz and shows that no irreversible changes due to spinning have taken place BRUKER Version 001 Gradient HR MAS Spectroscopy The addition of a magnetic field gradient to a high resolution MAS probe leads to further advances in this type of spectroscopy Based on the broad acceptance of gradient methods in high resolution solution state NMR studies it is not sur p
24. hr MAS NMR spectrum of small organic molecules 32 Figure 7 2 Comparison of proton spectra esse e eee ee 32 Figure 7 3 Figure 23 400 MHz proton spectra of a single 100 m bead 33 Figure 7 4 2D J resolved proton spectrum of 2 mg sample on a Tentagel BRUKER 51 55 Figures 52 55 MEI 33 Figure 7 5 Double Quantum filtered Cosy of a small organic molecule 34 Figure 7 6 Hr MAS spectra of TLC plate spots se 35 Figure 7 7 500 MHz proton top and carbon bottom spectra 36 Figure 7 8 2D TOCSY hr MAS spectrum of orange peel 37 Figure 7 9 8 mg red perch filet cut flushed and measured in D O 37 Figure 7 10 Hr MAS of benign versus malignant human prostate tissue 38 Figure 7 11 TOCSY presat of a living white worm enchytraed in 95 H O DDO EE 38 Figure 7 12 Gradient COSY of a swollen Wang resin 39 Figure 7 13 400 MHz H C HMQC spectra of an N FMOC N Boc L Lysine EEn 40 Figure 7 14 400 MHz H C HMBC spectra of an N FMOC N Boc L Lysine L Bs de eene L ar Ba 41 Figure 7 15 500 MHz H C Gradient hr MAS HMBC spectrum 41 Figure 7 16 500 MHz single pulse proton spectrum of an acrylamide hydro EN 42 Figure 7 17 500 MHz proton spectra of human high grade Pleiomorphic 43 Figure 7 18 500 MHz STE diffusion spectra esse eee nennen eenen 44 Figure 7 19 500 MHz STE proton spectrum top and single pulse spectrum tonnen Add MER 44
25. ing List Item Quantity Description HR MAS probe Zirconia rotors each rotor including 3 KEL F rotor caps 1 pair of rotor spacers upper and lower part 1 sealing screw Cylinder screw M2 25 HR spacer tool Small screw driver Rotor packer hand tool Screw driver for tuning rods Marker pen black Ol GW dl oOo a C Cap removal assembly eo k Repair declaration form Installation and Operation Manual Version 001 BRUKER 45 55 46 55 BRUKER Version 001 Spherical Inserts gt Important Note The rotor bore and the spacer must be absolutely clean The lower spacer will be destroyed if it is removed after being inserted into the bore 1 Spray the lower spacer with coolant spray or dip it into liquid nitrogen insert it slightly into the rotor bore and align the rotor axis figure B 1 Figure B 1 Spray lower spacer with coolant spray Rotor Lower Spacer HR Spacer Tool zin HZ3913 P icis Fi sad a Spray the spacer a second time with coolant spray and press it com pletely into the bore Apply more coolant spray if needed figure B 2 or b In the case where the spacer fits very tightly first dip the rotor and spacer in liquid nitrogen and let them cool The spacer can now be inserted much easier into the rotor figure B 2 Figure B 2 Inserting the rotor is now easier
26. ion 001 Installation of the Probe and Peripherals Probe Installation 3 1 3 1 Probe Installation Insert the probe into the lower shim stack For easy shimming it is recommend ed to align the probe with the y gradient direction as described in section 5 3 Shimming the HR MAS probe Lower the sample transfer tube into the top of the shim stack Place the MAS sample changer optional on top of the sample transfer tube see section 3 4 MAS Sample Changer Figure 3 1 Pneumatic connections of the HR MAS probe Pneumatic connections 3 2 Connect the MAS pneumatic unit to the main gas supply see section 2 2 Connect the RS232 port of the pneumatic unit to tty08 on the console Version 001 BRUKER 9 55 Installation of the Probe and Peripherals For the connection of air hoses between the MAS control unit and the probe refer to figure 3 1 and figure 3 2 For low temperature experiments the bearing gas connection has to be re moved and the outlet of a heat exchanger has to be connected instead See figure 3 3 for low temperature operation Please note Kel F caps for Zirconia rotors can be used in the tempera ture range from 10 C to 50 C For a more extended VT range 30 to 70 C the usage of caps made of macor or boron nitride are mandatory Figure 3 2 Diagram for the pneumatic connections for operation at ambient tem perature Vertical Magic angle Ambient bearing pressure
27. lled with KBr the an gle setting procedure can be performed in gs mode on the free induction decay FID In gs mode go to the acqu window separate the real and imaginary part of the FID and adjust for an on resonance decay This can be done by changing o1 or adjusting the field Make sure that the field sweep is off and remains off If the angle is close to the magic angle you will see rotational echoes on top of an exponential decay figure 5 2 Far off angle only the exponential decay is visible Turn the micrometer screw until the rotational echoes last up to 4 ms Write down the setting of the micrometer screw at the probe bottom for the correct angle posi tion Check for the optimal setting by acquiring 32 scans and maximizing the num ber of sidebands in the spectrum figure 5 3 BRUKER Version 001 Shimming a HR MAS probe Figure 5 2 7 Br FID s KBr KBr off magic angle 1 l Br MAS NMR FID nn at magic angle la Khalkhin ve ine ee rper Lu mill I L TTT oot 0005 0 0010 0 0015 0 0020 0 0025 0 0030 0 0035 0 0040 0 0045 0 0050 0 0055 0 0060 0 0065 0 0070 0 0075 0 s amp Experimental parameters for acquisition pulse program zg swh 100kHz td 2k d1 30ms p1 2us DIT OdB spinning rate 6000 Hz 79Br NMR spectra corresponding to the FID s shown in figure 5 2 Figure 5 3 KBr 79 Br MAS NMR spectrum of
28. matic connections for operation at ambient temperature 1 etaed teet epar uta ee eite ere e rad ae nd anne 10 Figure 3 3 Set up for low temperature operation nnen eenen eenn 10 Figure 3 4 Installation of the MAS sample changer 12 4 Operation of the HR MAS System 13 Figure 4 1 Zirconia rotor with Kel F cap upper spacer cylinder head lel X 13 Figure 4 2 Schematic drawing of the rotor with spherical insert 14 Figure 4 3 Schematic drawing of the cap removal tool 15 Figure 4 4 MAS pneumatic unit control window see 17 5 Preparing the Probe for Experiments 19 Figure 5 1 Proton NMR spectra of BaClO3H2O seen 20 Figure 5 2 OBIAEID S KBr eet iten ti Be nieten 21 Figure 5 3 Br NMR spectra corresponding to the FID s shown in figure bar uu cc ML E e LM LT E 21 Figure 5 4 The magic angle spinner axis sse ee eee ee e e 22 Figure 5 5 Proton spectra of a plant leaf annen enen 24 6 Gradient HR MAS Spectroscopy 25 Figure 6 1 Use of a z gradient in MAS spectroscopy sssss 25 Figure 6 2 Gradient field is directed along the magic angle spinner axis 26 Figure 6 3 Pre emphasis controller window eee 27 Figure 6 4 8 consecutive FID s esse sees serere eenn 28 Figure 6 5 Gradient profile for a spherical sample of water 29 7 Application Examples 31 Figure 7 1 1H
29. n in the probe press stop Then the packed rotor can be dropped into the probe via the transfer tube with the cap up Close the insert tube and press the button insert on the pneumat ic unit This will automatically set the stator into the magic angle position after 10 seconds if the pneumatic unit is operated in automatic mode In manual mode you have to press the stop button again Spinning the sample If the pneumatic unit is set to operate in automatic mode you may start spinning the sample by setting the desired spin rate Vd and pressing the go button on the control panel In case the rotor does not reach the desired spin rate in automatic mode the rotor has to be spun up in manual mode For spinning up 4 mm Zirconia rotors in automatic mode set the start bearing pressure to about 800 mbar the default setting of 2000 mbar is too much for most samples This can be done by pressing the bearing button in automatic mode and reducing the displayed Bp start to the desired value For manual spinning of the 4 mm Zirconia rotors use 800 mbar bearing Bp and 150 mbar drive pressure Dp to obtain a spin rate above 2 5 kHz If the spinning rate achieved at these pressures is lower slowly decrease BP to 100 mbar and immediately increase BP as soon as the rotor accelerates Standard samples will BRUKER 15 55 Operation of the HR MAS System easily spin in a 4 mm system problems can be encountered for conductive mate rials or very heavy
30. na uA NAM EAP Su s AS Version 001 25 515 pom 3 0 4 5 4 0 3 9 3 0 ZA 250 1 275 1 20 Liposarcoma tissue sample courtesy of Dr Singer Brigham and Women s Hospi tal MAS 5 kHz Top single pulse excitation center Watergate bottom Dry clean The combination of magnetic field gradients and hr MAS spectroscopy allows one to measure the diffusion constants of molecules in tissue samples under high res olution conditions Figure 7 18 displays a series of proton spectra of a Liposarcoma tissue obtained with a Stimulated Echo STE sequence The spectra are acquired with increasing gradient strengths Apart from measuring diffusion the stimulated echo sequence is used for spectral editing by attenuating the resonances from mobile compo nents in the sample see figure 7 19 BRUKER 43 55 Application Examples Figure 7 18 500 MHz STE diffusion spectra of a high grade human Pleomorphic Liposarcoma tissue sample courtesy of Dr Singer Brigham and Women s Hospital MAS 10 kHz 10 ms gradients 0 40 G cm TE 100 ms Figure 7 19 500 MHz STE proton spectrum top and single pulse spectrum 9 8 7 6 5 4 3 E 1 0 bottom of a high grade human Pleomorphic Liposarcoma tissue sample courte sy of Dr Singer Brigham and Women s Hospital MAS 10 kHz 10 ms gradi ents 30 G cm TE 100 ms 44 55 BRUKER Version 001 Shipping List Table A 1 Shipp
31. nger Figure 2 1 Upper probe chamber with MAS stator in magic angle position Version 001 BRUKER 7 55 System Description Site and Parts Requirements 2 2 8 55 Air requirements 1 2 3 Free of oil and dust 0 01 micron filter Dew point of at least 30 C 4m h flow of air at more than 6 bar for stable fast spinning Dry nitrogen gas for low temperature operation either from a pressurized liquid nitrogen tank with a nitrogen boil off device a 60 liters gas cylinder 200 bar lasts for approximately 1 hour B VT 2000 or 3000 variable temperature unit for all variable temperature VT operation and a 25 liters liquid nitrogen dewar for low temperature operation MAS equipment 1 MAS pneumatic control unit for sample spinning display of the spinning rate as well as sample insert and eject Set of air hoses and spinning rate cable 3 Pneumatic sample transfer system for standard bore magnets 4 MAS sample changer for automatic multiple sample insert and eject optional RF amplifiers HR MAS probes are designed to operate with the standard RF transmitters No high power transmitters are needed BO gradient option For HR MAS probes equipped with a magic angle gradient in addition to the above mentioned items a gradient amplifier is needed Gradient amplifiers capa ble of preemphasis and 10 A output current are recommended such as the GRE AT TM or ACCUSTAR BRUKER Vers
32. ntrol window shown in fig ure 4 4 Start by selecting the correct probe parameters or reading them in from a previously saved probe setup file Press Eject Sample to flip the stator in the upright position and eject a rotor which might be in the probe After the eject air is off insert the rotor and click Insert Sample to flip the stator to the magic angle position Enter the desired spinning rate and select spinning on to start spinning By press ing Continuous Update the actual values of the bearing and drive pressure and the spinning rate will be updated every 10 s After the rotor reaches the desired spinning rate and the spinning is stable the Spin Locked parameter will be set to Yes Exit the menu by pressing cancel The spin rate can still be monitored on the com puter screen by selecting MAS rate monitor from the windows menu of XWIN NMR Spinning can be halted by re entering the MAS menu and selecting spinning off note that when you enter the MAS window spinning is always set to off however you will have to activate this to stop the spinner Press Continuous Update to monitor the spinning rate Once the spinner is stopped press Eject Sample to re move the sample BRUKER Version 001 Spinning the rotor under computer control Figure 4 4 MAS pneumatic unit control window Command line control In addition to using the MAS window figure 4 4 the following commands can be used to control the spinner from the comman
33. on spins while the spectrum on the right is obtained with phase cycling No attempt was made the improve the appearance of the spectra MAS 5 kHz 1 ms gradients 10 10 and 5 G cm BRUKER Version 001 Gradient hr MAS spectroscopy Figure 7 14 400 MHz H C HMBC spectra of an N FMOC N Boc L Lysine H25 900 H50 0 06 Ge OE R A 0 DL KR L vw fs UW vua bulo L v h EE he L 100 Ve oe dd n F Tan toy nts R tse M a estat y x T wert gt L 0 i To sesta egli a al 108 F yo C FD M gede o p CRI RAE N L u E E B E ro 0 I F de D POE LET EN DON i FU i NT M m Ns D m Es N L m WA pe ns e L L L150 D S Ms T OTR N DON D E Ma 4 TTE amp QUAgP A L wk do PP doas 6 0 L 9 css DE nh n A Dis L i T Pas wi ay its IRA d MAL ae ue nial 1 Lh RMN qao a d b ai agi t L EH KEM aant Py tot ju t dem OE T M Mr M mi T T T T T ppm 8 6 4 2 0 derivatized Wang resin swollen with CDCI A J evolution time of 50 ms is used to reveal long range proton carbon correlations No C decoupling is applied dur ing acquisition The right spectrum shows the noise floor and demonstrates excel lent artifact suppression MAS 5 kHz 1 ms gradients 10 10 and 5 G cm Figure 7 15 500 MHz H SC Gradient hr MAS HMBC spectrum Lo e o o 6 6 F 6 F id o o N 00 es R H25 o o0 o 8 b H50 080 o
34. ows the proton spectrum of approximately 15 ug of salicylic acid from a TLC plate BRUKER Version 001 Figure 7 6 HR MAS of swollen polymers Hr MAS spectra of TLC plate spots N ml N M p TRE v MA val DP Ny n y nu d M MIN i j NI NN EE Te N Ny i M 4 5 4 0 ppm rn as The compound is 15 micrograms of salicylic acid scratched of the TLC plate and slurried with DO Avance 600 MHz 64 scans 4200 Hz spinning HR MAS of swollen polymers Version 001 7 2 Some polymers are nearly insoluble and as a result obtaining high resolution NMR spectra may become impossible An example is a range of poly acethylene derived polymers These samples show an extremely low solubility in most com mon solvents and attempts to obtain solution spectra result in low signal to noise ratios and long measurement times Solid state spectra on the other hand can be obtained but generally display a poor resolution A solution to this dilemma is to swell the polymer with a solvent and to acquire spectra while spinning at the magic angle Figure 7 7 displays proton and carbon spectra of a derivative of poly acethylene swollen in chloroform The carbon spec trum was obtained in about two hours BRUKER 35 55 Application Examples Figure 7 7 500 MHz proton top and
35. r parallel to the direction of the magnetic field gradient figure 6 1 This would result in a partial averaging of the gradient field as experienced by the spins and would necessitate the need to synchronize one s experiments with the spinner rotation which may be difficult or even impossible due to the finite rise and fall times of the pulsed gradient fields A better solution is to implement gradient spectroscopy experiments in a MAS probe in such a way that the gradient should introduce a resonance that is not temporally modulated This may be accomplished with a time independent gradi ent field where the gradient is oriented such that the z component of the magnetic field increases along the axis of the spinner and the z component of the magnetic field gradient is uniform in the planes perpendicular to the spinner axis see figure 6 2 Figure 6 2 Gradient field is directed along the magic angle spinner axis rE sias If in MAS spectroscopy the gradient field is directed along the magic angle spinner axis then a rotating spin will always sample the same magnetic field strengths The HR MAS probe is optionally equipped with a magic angle gradient The gra dient coil design is compatible with commonly used MAS stators for widebore and standard bore probes and does not interfere with the automatic sample ejec tion and insertion capabilities Gradient strengths of up to 45 G cm at 10A can be achieved Setting up the magic angl
36. rator intervention The sample changer is mounted on top of the sample transfer tube and controlled via the insert and eject pneumatic lines of the MAS controller To install the device re move the upper sleeve of the sample transfer tube and place the sample changer on top of the tube Connect the air lines as indicated in figure 3 4 The rotors are stacked in the sample changer with the rotor caps upward and af ter ejection are gathered in the collection tray The rotor collection tray may be re moved and emptied and additional rotors may be inserted into the sample changer while the system is in operation allowing for indefinite operation with only infrequent operator intervention In order to distinguish the samples numbered ro tor barrels are available BRUKER 11 55 Installation of the Probe and Peripherals Figure 3 4 Installation of the MAS sample changer sample changer gt 77 v 5 BROKER one MAS unit LIDS SAMPLE CHANGER s sample transfer INSERT tube To Probe MAGNET 12 55 BRUKER Version 001 Operation of the HR MAS System MAS Rotors Caps and Inserts 4 1 The rotors most commonly used for MAS are made of zirconium oxide Zirconia Rotors can be either filled entirely with sample or they can be used with rotor in serts Inserts are provided to improve shimming and RF in homogeneity and are
37. rising that gradients can be of use in HR MAS measurements particularly since the NMR methods are adopted from the liquid state rather than from the solid state class of methods As in high resolution liquid state studies the gradient is essential for removing t noise and is extremely helpful in coherence pathway se lection Magic Angle Gradient Design 6 1 Version 001 In conventional high resolution spectroscopy with gradients the gradient field most commonly used is aligned with the main magnetic field direction a so called z gradient Although the sample is usually stationary in gradient spectroscopy for reasons discussed later it is in principle possible to rotate the sample since the isoplanes of the gradient field are perpendicular to the rotation axis see figure 6 1 This means that a rotating spin will always experience the same magnetic field strength Figure 6 1 Use of a z gradient in MAS spectroscopy If in conventional high resolution spectroscopy with a z gradient the sample would be spun then the spins will always experience the same magnetic field strength In MAS spectroscopy however the use of a z gradient would cause a rotating spin to sample different values of the magnetic field BRUKER 25 55 Gradient HR MAS Spectroscopy If a z gradient would be applied to a sample spinning at the magic angle the rotat ing spin would pass through different isoplanes since the axis of rotation is no longe
38. s pulse program will acquire 8 FID s and display them sequentially in a single acquisition window see figure 6 4 Each FID is acquired at a time vd after a gra dient pulse Set the gradient pulse length to 50 ms and its strength to 2096 The pre emphasis is controlled by a set of three time constants and amplitudes These values can be addressed through the Pre emphasis Controller window which is invoked by setpre figure 6 3 Select for the time constants 20 ms 2 ms and 2004 and start with 50 for all time settings and 0 for all gain settings Start the acquisition in gs mode and observe the FID s in the acquisition window With the time constants set as described above the first FID s will seem distorted as can be seen in the example of figure 6 4 top spectra Adjust the pre empha sis values by changing the time constants and gain settings Start with the set tings for the slowest time constant and adjust the ST and 6 FID so that they appear similar to the last two FID s Repeat the procedure for the mid and fast BRUKER 27 55 Gradient HR MAS Spectroscopy time constants for FID s 3 and 4 and FID s 1 and 2 respectively If all values are set correctly all FID s should appear equal figure 6 4 bottom The pre emphasis values are dependent on both the probe and the gradient am plifier and once correctly set up will remain the same The settings can be stored on the computer by saving the set up from the file menu in
39. solid samples Stopping sample spinning In automatic as well as in manual operation the rotation can be stopped by press ing the stop button If you want to stop rotation manually from the keypad de crease the drive pressure Dp first and then lower the bearing pressure Bp slowly In order to stop spinning after variable temperature experiments make sure that the heater is off and that gas of ambient temperature is used for the bearing Sample Ejection After the rotor has stopped press the eject button on the pneumatic unit The sta tor will switch back to the vertical position and the rotor be ejected For this proce dure the eject air pressure has to be adjusted by a little adjust knob on the rear of the pneumatic unit such that the pressure is high enough for the rotor to lift off but not too high such that the cap is not damaged when the rotor knocks against the closure of the transfer tube Spinning the rotor under computer control 4 5 16 55 The MAS window In order to operate the MAS pneumatic unit from XWINNMR the unit has to be run in remote mode which can be selected on the keypad on the front of the unit The set up of and communication with the pneumatic unit is established by typing cfmas on the XWINNMR command line and answering the questions in accor dance with your set up The control of the pneumatic unit from the host computer is initiated by typing MAS on the command line This brings up the MAS co
40. t the rotor into the barrel clamp and remove the cap by simultaneously rotating and pulling the barrel clamp Unscrew the cap clamp and push the cap out with the push pin Be careful not to damage the caps For more complete in structions please refer to the guidelines delivered with the cap removal tool BRUKER Version 001 push pin Spinning the rotor under manual control Figure 4 3 Schematic drawing of the cap removal tool tool body cap clamp barrel clamp Spinning the rotor under manual control 4 4 Version 001 Prepare the pneumatic unit for operation by setting the proper probe parameters such as rotor size BL4 magnet SB and rotor material e g ZrO Consult the manual for the pneumatic unit and or section 4 4 of this manual For manual operation of the MAS pneumatic unit set the unit in the local mode by pressing the local remote button on the keypad of the pneumatic unit Choose ei ther manual or automatic for spinning control see below Itis also possible to operate the pneumatic unit from your host computer For this please refer to the XWINNMR manual and section 4 5 Inserting the rotor In order to make sure that the rotor will properly drop into the stator press the eject button on the pneumatic unit first This will set the stator into a vertical position and ensure that a rotor which might be inside the probe will be ejected After the eject air is on and after removal of a rotor which has bee
41. the Pre emphasis Con troller window Figure 6 4 8 consecutive FID s Acquired at different times after the application of a gradient pulse Top without pre emphasis bottom with pre emphasis properly adjusted Calibrating the gradient Gradient calibration is performed by measuring the gradient profile of a sample of known size Usually a proton spectrum of a sample of water is acquired The profile is obtained with a gradient spin echo which can be acquired with the pulse program calib Set the gradient amplifiers to a low output e g 10 or 20 and acquire 4 scans Multiply the FID with a sinebell ssb 0 Fourier transform and represent the data in magnitude mode mc The resulting profile represents a projection of the sample s density along the magic angle spinner axis For a sam ple of known length the gradient strength can be determined by measuring the width of the spectrum Aa and dividing it by the length of the sample I in cm by the gyromagnetic constant of protons y 4250 Hz G and by the current of the gradient amplifier I in A 28 55 BRUKER Version 001 Version 001 Setting up the magic angle gradient Ao PEN G in Gem A ylI In figure 6 5 below water in a spherical insert is used to calibrate the gradient strength The profile represents the spherical sample volume plus a small amount of water in the fill hole of the inserts From the width of the profile Aw 12000 Hz and th
42. useful when the amount of sample is limited The maximum spinning rate for 4 mm Zirconia rotors is about 15 kHz but is limited to lower speeds when using the spacers A full rotor has a sample volume of about 80 ml with a spherical insert the volume is reduced to about 25 ul Numbered rotors are available to easily dis tinguish between different samples The function of the rotor cap is twofold firstly to close the rotors and secondly to provide the driving of the rotor There are several types of caps available The standard caps are made of Kel F which can be used in a temperature range from 10 C to 50 C This material will shrink at lower temperatures and soften at more elevated temperatures However for a more extended VT range 30 C to 70 C caps made from macor or boron nitride can be used Those materials have tem perature coefficients similar to Zirconia dk 4 1 Zirconia rotor with Kel F cap upper spacer cylinder head screw adu dun TID UTD HERE TEE and sealing grub screw to ude a spans sample volume Version 001 BRUKER 13 55 Operation of the HR MAS System Sample loading Cap removal 14 55 The most common type of rotor spacers provide a sample volume that is approxi mately spherical in order to improve the shimming of the probe see figure 4 1 and figure 4 2 Appendix B is an example of an instruction sheet showing how to handle this type of insert For other inserts please refer to the accompan
43. ying in structions provided with the spacers Figure 4 2 Schematic drawing of the rotor with spherical insert Upper spacer Lower Spacer SALLI LAAk E Mi ZU KAZ UM ZA BD ZZZ 2 A Av p ap a 4 2 In order for the rotor to spin stable and fast the rotor has to be well balanced Load the sample loosely into the rotor barrel and tap the rotor lightly on a hard surface Do not attempt to pack the material tightly into the spinner If you wish to add more sample into the rotor it is better to spin up the rotor first so that the sample is packed against the walls of the barrel and then add additional material Liquid or viscous samples are easily loaded using a syringe or a small pipette Solvent swelling can be done with the powdered sample in the rotor Push the rotor cap on all the way if the cap is improperly positioned the rotor may not spin Clean the outside of the rotor with a tissue so that no sample ends up in the MAS stator Mark half of the beveled rim on the bottom of the rotor with a black marker for optical spin rate detection For loading samples using spherical inserts see Appendix B or the instruction sheet that came with the inserts 4 3 The rotor cap is easily removed with a dedicated cap removal tool see figure 4 3 Screw the tool body loosely to the cap clamp Insert the cap all the way into the teeth of the clamp Screw the tool body snugly but not too tight onto the clamp Inser
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