A low-E magic angle spinning probe for biological solid state NMR at 750 MHz

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A low-E magic angle spinning probe for biological solid state NMR at 750 MHz
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  A low-E magic angle spinning probe for biological solid state NMR at 750 MHz Seth A. McNeill b , Peter L. Gor’kov c , Kiran Shetty c , William W. Brey c , Joanna R. Long a, * a Department of Biochemistry and Molecular Biology and McKnight Brain Institute, University of Florida, Box 100245, Gainesville, FL 32610-0245, USA b Department of Electrical and Computer Engineering, University of Florida, FL 32611, USA c National High Magnetic Field Laboratory, Tallahassee, FL 32310, USA a r t i c l e i n f o  Article history: Received 9 August 2008Revised 2 December 2008Available online 14 December 2008 Keywords: Solid state NMR Magic angle spinningMembrane proteinsProbe designLoop gap resonatorLow-ERf heating a b s t r a c t Crossed-coil NMRprobesare auseful tool for reducing sampleheatingfor biological solidstateNMR. Inacrossed-coil probe, the higher frequency  1 H field, which is the primary source of sample heating in con-ventional probes, is produced by a separate low-inductance resonator. Because a smaller driving voltageis required, the electric field across the sample and the resultant heating is reduced. In this work wedescribe the development of a magic angle spinning (MAS) solid state NMR probe utilizing a dual reso-nator. Thisdual resonator approach, referredtoas ‘‘low-E,” was srcinallydeveloped toreduceheatinginsamples of mechanically aligned membranes. The study of inherently dilute systems, such as proteins inlipidbilayers, viaMAStechniques requires largesample volumes at highfieldtoobtain spectrawithade-quate signal-to-noise ratio under physiologically relevant conditions. With the low-E approach, we areableto obtainhomogeneous andsufficiently strongradiofrequency fields for both 1 Hand 13 Cfrequenciesin a 4mmprobe with a  1 H frequency of 750MHz. The performance of the probe using windowless dipo-lar recoupling sequences is demonstrated on model compounds as well as membrane-embeddedpeptides.   2008 Elsevier Inc. All rights reserved. 1. Introduction Performing NMR experiments at high magnetic fields increasestheir applicability to systems that are resolution or sensitivity lim-ited. Polarization enhancement estimates suggest the increase insignal-to-noise (S/N) should be on the order of   B 07/4 , decreasingacquisitiontimesbyafactorof  B 07/2 .Withtheadventofstablehighfield instruments with proton frequencies of 700 to over 900MHz,the application of solid state NMR (ssNMR) spectroscopy to a widevariety of biomolecular systems has become increasingly feasible.However, realizing these sensitivity gains for a variety of samples,nuclei, and pulsed experiments is not straightforward since the in-crease in signal is accompanied by an increase in the isotropic andanisotropic chemical shifts. This results in increased spectralwidths requiring the generation of more powerful  B 1  fields to ex-cite all frequencies of interest. Additionally, as at lower magneticfields, many ssNMR experiments require proton decoupling withrf field strengths above ( x 1 /2 p )=100kHz on the proton channelfor optimal resolution.With traditional ssNMR probe circuits utilizing multiply-reso-nant solenoidal coils, achieving efficient and homogeneous  B 1 fields is complicated by the electrical length of the sample coil athigh proton frequency approaching a quarter of the rf wavelength[1,2]. The study of biomolecules under physiologically relevantconditions substantially alters the probe performance by loadingthe coil withsamples containing highsalt concentrations. Creatingstronger  B 1  fields also creates stronger electric ( E  ) fields within thesample leading to more heating and, ultimately, sample degrada-tion. The generation of   E   fields and their contributions to sampleheating in ssNMR spectroscopy have been extensively studied inrecent years [3–14]. This heating can be overcome by coolingsam-ples down to where the heating does not disrupt the system [3],but often this means cooling the samples well below biologicallyrelevant temperatures which can alter protein conformation or re-move the molecular dynamics of interest. Lowering the conductiv-ity of the samples is another method of reducingheating [14]. Thisispossibleforsomesamples,butagainmayleadawayfrombiolog-ically relevant conditions. Common spectroscopic approaches tominimizingsampleheatinginclude the useof very lowdutycyclesandutilizingverysmallcoils, withasubsequentreductioninscansper unit time and sample volume, respectively, leading to poorerS/N and/or longer acquisition times, particularly for concentra-tion-limited samples.More recently, probe design efforts have focused on the morefundamental issue of modifying the coil design by reducing induc-tance and/or adding shielding to reduce sample heating. Tradi-tional multinuclear ssNMR probe designs employ a single,multiply-resonant solenoid as this maximizes the filling factorfor the various frequencies and (when wavelength effects can be 1090-7807/$ - see front matter    2008 Elsevier Inc. All rights reserved.doi:10.1016/j.jmr.2008.12.008 *  Corresponding author. Fax: +1 352 392 3422. E-mail address:  jrlong@mbi.ufl.edu (J.R. Long). Journal of Magnetic Resonance 197 (2009) 135–144 Contents lists available at ScienceDirect  Journal of Magnetic Resonance journal homepage: www.elsevier.com/locate/jmr  neglected) helps ensure rf overlap. Several clever modifications tothe solenoid have been proposed to reduce heating while preserv-ing as much as possible the efficiency of both high and low-fre-quency channels. These designs also allow commercial probesuppliers to continue to use their well-developed multichannelmatching networks. Scroll coils [15] offer a robust solution to theproblem of sample heating as they have a lower inductance thansolenoids and their geometry creates a built-in Faraday shield forthe  E   field since the inner turns shield the sample from the  E   fieldgenerated by the outer turns [4]. Both these factors reduce sampleheating and improve stability and performance on the protonchannel. The  1 H efficiency of scroll coils with lossy samples cansurpass that of solenoids. However, scroll coils are less efficientthan solenoidal coils at lower frequencies due to their low-induc-tanceandlow Q   [4].Scrollcoilsalsopresentchallengesduetotem-perature dependant tuning changes inherent to the largecapacitance between turns [7,11]. This capacitance also limits theavailablesamplevolume[9]. TheZ-coil,consistingofacentralloopwith two spiral coils on the ends of the loop [10], lowers sampleheating by more than an order of magnitude relative to a solenoi-dal coil and has an rf efficiency that is independent of sample con-ductivity. However, unlike the scroll, the rf efficiency of the Z-coilwith a lossy sample is just comparable to that of the solenoid, andthere is also a penalty in sensitivity and efficiency at the lower fre-quencies. Most recently, Krahn and co-workers have shown thatheating can be reduced by inserting a conductive shield betweenthe sample and the solenoid [13]. Precise manufacturing of theshield led to an effective decrease in heating at a modest cost insensitivity due to the decrease in sample filling factor comparedto an unshielded solenoid. However, the close proximity of theshield to the sample coil can be expected to the limit the voltages,and hence the achievable  B 1 , for larger sample volumes. Thesethree single-coil alternatives to the solenoid have been shown toreduce heating at some cost to rf efficiency. While the gains in rf and temperaturestabilitycertainly outweighthe loss in sensitivityforlossysamples, analternativeapproachwhichdoesnot compro-mise the sensitivity and efficiency of the lower frequencies wouldbeattractive,particularlysincethebulkofbiologicalssNMRexper-iments rely on direct detectionof lowgamma nuclei due to protonresolution limitations at slow to intermediate magic angle spin-ning speeds.One such solution to achieve the desired rf performance at theproton frequency and simultaneously reduce sample heating is touse separate coils for the low and high frequencies. There are sev-eral benefits to this design: using two coils allows the individualcircuits and coils to be optimized for each frequency range; havingonecoilinsidetheotherallowstheinnercoiltoactasapartialFar-aday shield for the outer coil; the rf fields generated by the twocoils can be designed to be orthogonal, which increases channelisolation and therefore efficiency; and, when the coil assembly isrotatedformagicangleapplications, theuseof orthogonalcoilsre-sults in a compromised rf field on only one coil.An advantageous approach for using crossed coils in MASprobes is to place a low-inductance, segmented  1 H saddle coil in-side a solenoid tuned for the lower frequency channel [7]. Withthis configuration, the  1 H coil shields the sample from some of the  E   fields created by the inductance of the solenoid. The inverseconfiguration, in which the low-frequency sensitivity is improvedby placing a solenoid within a loop gap resonator (LGR), has beenused effectively to reduce heating in large volume static probes[9]andisthefocusofthisproject.TheLGRisacoilgeometrywhichworks well at proton frequencies due to its low-inductance, lower E   fields, and short electrical length; LGRs have been used exten-sively in EPR for high frequency applications [16] as well as inMRI [17]. Previously, we have shown that they also work well forhigh field ssNMR applications when combined with an orthogonalinner solenoid for the lower gamma nuclei [9], a design we havechristened‘‘low-E”duetoitsfavorablemitigationof  E   fieldswithinthe sample space. In such designs, the sample is placed within asolenoidal coil to maximize sensitivity and homogeneity for thelow-frequency channel; an LGR optimized for the proton channelis orthogonal to and surrounds the solenoid. In this configuration,thesolenoidfurtherlowersthe E   fieldbyactingasapartialFaradayshield between the sample and the LGR. The outer  1 H resonator isslit strategically to cancel low-frequency eddy currents whichwould otherwise reduce the efficiency of the inner coil. The lossof filling factor for the proton channel in a crossed coil setup likethis is largely made up for by the improved efficiency of the sin-gle-frequency  1 H matching network. And since the solenoid isnot called upon to produce a  1 H field, its length and number of turns can be increased to improve sensitivity. For a MAS probe,the fact that the rf field of the LGR can be made orthogonal tothe polarizing magnetic field  B 0  further improves  1 H efficiency rel-ative to a multiply-tuned solenoid.An additional benefit of the LGR   1 H coil is that its homogeneityis excellent. The high  B 1  homogeneity on both channels of the  1 HLGR/ 13 C solenoid configuration is of critical importance for theapplication of cross-polarization (CP) and multipulse experimentsto samples which are concentration-limited. In multipulse recou-pling experiments, especially long windowless experiments, theaccumulation of phase errors from different parts of the samplenutating at different rates leads to reduced excitation efficienciesandphaseerrors intheresultingsignals. Theincreasedhomogene-ity of the LGR at the proton frequency and the solenoidal coil atlower frequencies can increase the efficiency and final signalstrength of multipulse experiments, particularly experimentswhich utilize double quantum filtering.In considering concentration-limited samples, the  1 H LGR/ 13 Csolenoid configuration allows reasonably straightforward scalingof the sample volume even at high frequencies. For samples whichare inherently dilute (i.e. membrane proteins in lipid vesicles orproteins adsorbed on to solid substrates), it is often preferable touse larger sample volumes. This is because sensitivity per unit vol-ume scales as  (1/ d ) with respect to the rotor diameter while fullrotor sensitivity scales as  d  2 , so a larger sample diameter presentssignificant advantages for concentration-limited samples if suffi-cient rf strength and homogeneity can be achieved [18].In this paper we present the design and characterization of assNMR magic angle spinning (MAS) probe that utilizes a  1 H LGR placedorthogonallytoa 13 CsolenoidimplementedonanNMRsys-temwitha17.6Tmagnet(750MHz 1 Hfrequency). Thedesignwasoptimizedforintrinsicallydilutesamplesbyutilizinga4mmrotor.The use of two separate coils allowed us to significantly increaseboth the length and number of turns in the solenoidal coil, makinghighly homogeneous  B 1  fields achievable even with the increasedvolume of the coil. 2. Probe design and performance characterization  2.1. Sample coil assembly and integration into a MAS stator  Theprobedesigndescribedandcharacterizedinthispaperisanadaptation of a previously reported static low-E probe [9]. The coil assembly consists of two rf coils which are orthogonal to eachother. The outer coil is a rectangular LGR tuned for the  1 H circuitand the inner coil is a solenoidal coil for the low gamma nucleus.In previous work this assembly was optimized for PISEMA experi-ments on static, oriented membrane-embedded protein samples.For the present MAS application, the coil assembly (Fig. 1a) wasmodified so that it could be integrated into a 4mm MAS stator(model AMP4023-001, Revolution NMR, Inc., Fort Collins, CO) witha top spinning speed of 18 kHz. In MAS implementation of low-E 136  S.A. McNeill et al./Journal of Magnetic Resonance 197 (2009) 135–144  coils, the sensitivity of the detectionchannel benefits froma statordesign where MAS bearings are placed further apart as this pro-vides space for additional turns in the observe solenoid. The coilcavity available in the stator measures 11  12mm in cross-sec-tion and 20mm in length, which is substantially longer than the12.7mmlengthofourcoilassembly.Thisstatoriscompatiblewithstandard Varian 4.0mm Pencil style rotors (Revolution NMR p/nAMP4088-001 or Varian p/n MSPA003006). The exact physicaldimensions for both coils are provided in Fig. 1a. Regulation of sample temperature is accomplished by VT gas delivered throughthe side of the stator. A photograph of the fully assembled probehead is shown in Fig. 1b.The loop gap resonator was fabricated by forming a 0.25mmthick, 9.0mmwide copper striparound a 12.2  8mmrectangularblock. The ends of the strip were terminated with non-magneticchip capacitors (100B series, American Technical Ceramics) tocomplete the LGR. The resonator was attached to the  1 H matchingnetwork using low-inductance leads threaded through a Teflonplatform that centers the coil assembly in the stator housing(Fig. 1). The inner, low gamma coil is an 8-turn 4.6mmID  8.3mm long cylindrical solenoid. The solenoid was madefrom 0.6mm round copper wire (American Wire Gauge #22).Locating the low-frequency coil closest to the sample maximizessensitivity for direct detection. The homogeneity of the  B 1  fieldwas improved by using variable spacing between the coil wind-ings. The solenoid leads were also threaded through the Teflonplatform which centers the coil with respect to the LGR and theMAS stator.  2.2. Rf matching network The double-tuned X– 1 H matching network implemented in ourMASprobe isshownschematicallyinFig. 2. Thedesignandperfor-mance of this rf circuit has been thoroughly described [9]. For thepurposes of the applications described below, the detection chan-nel was tuned to  13 C. It can be re-tuned to any other low gammanuclei by exchanging capacitors  C  7A  and  C  8 . Variable capacitors C  5 ,  C  6 , and  C  7  in the low-frequency circuit are 1–10pF trimmers(NMNT10-6, Voltronics Corp., Denville, NJ). In the proton channel, C  1  is a 1 to 6pF trimmer (Voltronics NMQM6G),  C  2  and  C  3  are 0.3–3pFtrimmers(RP-VC3-6, PolyflonCo. Norwalk, CT). Non-magneticfixedcapacitorsemployedintheprotonmatchingnetworkareVol-tronics11serieschips.Inthelow-frequencychannel,weusednon-magnetic 100C series chips from American Technical Ceramics,Huntington Station, NY.The chip capacitor values for the proton LGR ( L 0 – C  0 ) had to bechosentoresonateit slightly abovethe Larmorfrequency. This self resonance frequency is affected by the dielectric material of thestator and the coil platform which are in close proximity. A smallloop was inserted through a side hole in the stator to pickup theresonant frequency  f  0  of the entire stator assembly. Chip values( C  0 ) were chosen to place  f  0  between 780 and 790MHz.  2.3. Power efficiency and homogeneity of rf fields The low-E resonator MAS probe was fully characterized viaNMR experiments using a 750MHz Bruker AV2 system with an Fig. 1.  (a) Physical dimensions of the coils (in millimeters) and their integration into a MAS stator using a Teflon coil centering platform; (b) photograph of the probe head.The Teflon coil platformand two pairs of leads can be seen at the bottomof the stator. The observe solenoid is made using a variable pitch winding which is not reflected inthe drawing. S.A. McNeill et al./Journal of Magnetic Resonance 197 (2009) 135–144  137  89mm bore magnet and a CPC MRI Plus model 19T300  1 H ampli-fier. The power going into the probe was measured using a direc-tional coupler and an rf power meter (Agilent E4416A meterwith an E9323A power sensor). Adamantane (Acros Organics)was used for direct observation of the  13 C and  1 H resonances for B 1  field and homogeneity measurements since it is a low-lossmaterial and its dipolar couplings are inherently small due tomolecular motion and can be removed to first order by MAS atmoderate rates. It was also used for calibrating chemical shiftsand lineshape measurements. For restricted volume measure-ments, Kel-F spacers were used to reduce the sample length andto center the sample within the coils.  1 H and  13 C nutation experi-ments utilized a variable length single pulse on the observe chan-nel and, for  13 C experiments, CW proton decoupling (83kHz) wasapplied during acquisition.  13 C homogeneity was determined byirradiating and monitoring the carbon resonance at 38.48ppm; 1 H homogeneity was determined by irradiating and monitoringthe unresolved proton resonances at an average position of 2.6ppm.The maximum  1 H power available on the spectrometer is220W, which is belowthe power limit of the probe. With our lim-itedavailablepower,themaximum 1 Hdecouplingfieldisachievedby bypassing the duplexer and connecting the amplifier output di-rectly to the probe, and this is the setup we typically use in ssNMR experiments. To measure maximum  1 H nutation rates achievableby this setup using NMR, we prepared a sample of chloroformsealed in a 1mm capillary with 5min epoxy. The capillary was in-serted inside a thick-walled rotor along with ground KBr to stabi-lize the spinning at   1kHz. The  1 H nutation rate was thenmeasured via indirect detection. B 1  homogeneity measurements as a function of adamantanesample length are shown in Fig. 3a. Homogeneity is reported asthe ratio of the signal intensities after 810   and 90   pulses (810  /90  ). Rf field strengths ( x 1 /2 p ) were measured to be 104 kHz at220 W of input power for  1 H (750.2 MHz) and 72 kHz at 75 W of input power for  13 C (118.6 MHz). As expected, the LGR coil forthe proton channel, the increased number of turns in the solenoi-dal coil, and restricting the sample length to within the coils leadsto enhanced  B 1  homogeneity at both low and high frequencies.Example nutation profiles for both  1 H and  13 C can be seen inFig. 3b. The length of sample in Fig. 3b is 81% of the length of the solenoid. Isolation between probe channels was measured usinga HP8752C Vector Network Analyzer (Hewlett Packard). Withoutexternal filters, the isolation achieved between the  13 C and  1 Hports is 45 dB at the  1 H frequency and 24dB at the  13 C frequency.Placementofthe 1 Hloopgapresonatorastheoutercoilleadsinprincipleto less efficient performance on the high frequency chan-nel, but this compromise is offset by orienting the resonatororthogonal to  B 0  and by the high rf homogeneity of the LGR. Bychoosing this geometry, the  1 H  B 1  field in the  x –  y  plane is notattenuated by rotation of the coil assembly from a static orienta-tion orthogonal to the external magnetic field to an orientationinwhichthesolenoidalcoil axisisat themagicangle.Moreimpor-tantly,theplacementofthesolenoidalcoilinsidetheassemblyim-provesthefillingfactorontheobservechannel. Thishelpstooffsetthe loss of   B 1  field in the solenoid due to its magic angle orienta-tion. Because the length of the solenoid is not limited by  1 H wave-length effects, we are able to utilize an 8-turn solenoid, whichfurther improves the performance of the  13 C channel relative tomultiply-tuned solenoids containing fewer turns. C 3 L 0 L 1 3.3 C 8 1.5 C 7A 1.0 C 7 C 6 C 13 C 0 4.32.7 C 5 1.52.0 L 3 L 3 C 2 L 4 L 4 12 H 1 C 1 L 2 synch C 4 3.8 Fig. 2.  Schematics of the double-tuned rf matching network.  L 0 – C  0  forms the  1 Hloopgapresonatorwiththedetectionsolenoidinside( L 1 ).Inductors L 3  and L 4  (5–10nHeach) represent flexible leads connecting sample coils to the  1 Hand  13 C circuits. C  1 ,  C  2 , and  C  3  are variable capacitors for, respectively, matching, balancing, andtuning the  1 H LGR. In the low-frequency channel,  C  5  is used for matching while  C  6 and C  7  tuningcapacitorsareconnectedtoasingletuningrodviaagear mechanism.Retuningtodifferent observenuclei (e.g.  15 N) isdonebyreplacinga tuningchip,  C  8 ,and a balancing chip,  C  7A . A low-voltage  1 H rejection trap,  L 2 – C  4 , is placed at theentry of the  13 C rf cable. 0 10 20 30 40 50 60 70 80 90 100 0 2 4 6 8 10 12    B    1    H  o  m  o  g  e  n  e   i   t  y   (   8   1   0   /   9   0   ) Sample Length (mm) 1H 13C    1   3    C   C  o   i   l   L  e  n  g   t   h 0 5 10 15 20 25 30 35 1 H 0 5 10 15 20 25 30 35 13 C Pulse Length ( µ s) Fig. 3.  (a)  B 1  homogeneity characteristics for the  1 H (black squares) and  13 C (graydiamonds) channels as a function of sample length. The  13 C solenoidal coil length,8.3 mm, is indicated by the dashed vertical line. As expected, homogeneity is highand improves when the sample is confined within the coil. (b) Nutation profiles forthe  1 H (top) and  13 C (bottom) channels collected using a 6.7mm long adamantanesample. Each peak corresponds to the monitored resonance as a function of thepulse length in 0.5 l s increments.138  S.A. McNeill et al./Journal of Magnetic Resonance 197 (2009) 135–144   2.4. Power handling and stability The probe’s power handling capabilities were bench tested todetermine if long, high-power pulses led to either arcing or detun-ing of the resonant circuits. No arcing was observed during 80mslong pulses in the  1 H channel at powers exceeding 280W, whichcorrespondsto( x 1 /2 p )  117kHz.However,thefirstimplementa-tion of the probe exhibited detuning of the  1 H resonance by asmuch as 0.9 MHz once the decoupling pulse length exceeded 20ms. The chip capacitors in the  1 H LGR are heated up by the highcurrent needed to produce strong decoupling fields, and this canlead to small changes in capacitor values. This problem was nar-roweddowntoalackofcoolingmechanismforthesechips.Tocor-rect it, a channel was cut in the Teflon platform underneath thechip capacitors, allowing the gases circulating inside the samplecompartment to flow around the chips and cool them on all foursides. This measure significantly decreased detuning of the  1 Hchannel to a much smaller, comfortable level, which would not re-quire tuning adjustments during NMR experiments. A subsequenttest has shown that high-power detuning in the  1 H channel can beeliminated if the 100B series chip capacitors in the LGR are re-placed by their temperature-compensated NP0 counterparts, suchasnon-magneticversionof700Bseries. The 13 Cchannelwasstableunder high-power conditions with pulses up to 20 ms in length atpowers exceeding 75 W ( x 1 /2 p  72 kHz) and 5 ms long pulses at117 W ( x 1 /2 p  90 kHz).  2.5. Shimming  The probe shims adequately without spending extensive time.The 13 Cfullwidthathalfheightforadamantaneis9Hzatasamplelength of 3.7 mm; the 0.55% linewidth is 83 Hz. For the full rotorlength, 11.7 mm, the half height linewidth is 11 Hz. A small footis observed in the  13 C signal which is similar to inhomogeneousbroadening we have observed in a commercial XC4 probe fromDoty Scientific. Our lineshape would likely be improved by usingzerosusceptibilitywireinthesolenoidwhichisclosesttothesam-ple, but the opportunity to test this hypothesis has not arisen. An-other source of inhomogeneity may be the capacitors for the  1 Hcoil. However, they are more physically distant from the sample,so we expect their contribution to the observed broadening to beless, relative to the  13 C coil wire. 3. Measurements of sample heating   3.1. Rf-induced heating  To characterize rf performance with typical biological samples,test samples containing either D 2 O or 0.15M NaCl in D 2 O wereprepared. Experimentswereperformedusingthefull rotorvolume(57  l L for a thick-walled rotor) as well as more restricted samplelengths. Rotors were sealed with PTFE tape gaskets and samplelengths were varied using Kel-F spacers.To measure rf-induced heating, aqueous samples describedabove were doped with 20mM thulium 1,4,7,10-tetraazacyclod-odecane-1,4,7,10-tetrakis(methylene phosphonate) (TmDOTP 5  )(Macrocyclics) as the temperature dependencies of the exchange-able proton chemical shifts in TmDOTP are sensitive, linear, andwell documented [19]. In particular, the H(6) proton provides a ni-cely resolved resonance for monitoring temperature changes. Be-cause of its high ionic strength, the relatively small concentrationof TmDOTP 5  isexpectedto contributesignificant rf loss. The sam-ple rotation rate was regulated at 2kHz, and bearing and drive airwere supplied at room temperature. The sample temperature wasregulated by means of an air stream cooled by a Bruker BCU-05refrigeration unit and controlled by a BVT-3300. The rf heatingexperiment was run as follows: a presaturation pulse was appliedtotheprobefor40msatthetestpowerlevel;thiswasfollowedby5 ms of signal recovery before a standard pulse and acquire se-quence. The duty cycle was maintained at a constant 3.8% whilethe presaturation power was varied. Before signal averaging, 256dummy scans were run (taking   5min) in order for the sampleto reach a steady state temperature.Thesampletemperaturerisedueto rf irradiationcanbeseen inFig. 4a. Even for the full rotor with 150mM NaCl added to the 0100200300051015  Average RF Power (<kHz 2 >)       ∆    T   (   K   ) Fig. 4.  (a) Average rf sample heating at three different sample lengths and two saltconcentrations. Black filled circles: 20 mM TmDOTP 5  with a 3.7 mm samplelength. Black filled squares: 20 mM TmDOTP 5  and 150mM NaCl with a 3.7 mmsamplelength. Grayfilledsymbols correspond tothe samesolutions witha 6.7mmsample length, and the empty symbols correspond to the same solutions with an11.7 mm sample length. Lines are linear fits to the data as a visual guide. Note thatbecause of its high ionic strength, small concentrations of TmDOTP 5  stillcontributesignificantly torf loss. The rf heatingremains under 15 Kfor all samples,and either decreasing the sample length or the salt concentration further reducesthe heating. Average power was varied by keeping the duty cycle constant at 3.8%and varying the presaturation pulse power. A delay of 5 ms between thepresaturation pulse and the acquire pulse was used and 256 dummy scans (   5min) were run before acquiring to make sure the sample had reached equilibriumtemperature. (b) Frictional heating due to MAS. The sample temperature wasmonitored using a sample containing 10%lead nitrate (Pb(NO 3 ) 2 ) diluted with NaClto reduce its density. The gas lines were kept at room temperature. The line is aquadratic fit through the origin. The initial temperature drop is due to Joule-Thomson cooling. Heating at 13 kHz MAS reaches 20 K, which is enough to be aproblem for biological samples, but it can be mitigated by cooling the rotor withchilled VT gas. S.A. McNeill et al./Journal of Magnetic Resonance 197 (2009) 135–144  139
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