安捷伦液体核磁共振培训手册

1、0； Agilent Technologies安捷伦液体核磁共振培训手册胡伟 安捷伦核磁共援应用工程算由B箱： wei.hu 手机：185161736031,2,3,4,5,bi培训内容trz核磁共振简介 核磁硬件介绍 VJ软件介绍 样品制备一维谱图采集及处理氢谱 碳谱PRES AT DEPTJune 6, 2014June 6, 20146,二维谱图采集及处理同核相关谱June 6, 2014异核相关谱7,核磁日常维护探头校正 1H PW90简单测试June 6, 20141,核磁共振简介K1,核磁共振技术发展迄今为止共获得5次诺贝尔奖> 1938年Rabi在分子束中测量到核磁共振现象

2、，1944年获得诺贝尔物理学奖。1945年Bloch和Purcell小组将核磁共振技术应用拓展到液体及体领域.1952年获得June 6, 20145诺贝尔物理学奖。1967年Ernst将傅立叶变换运用到NMR谱图中,1991年获得诺贝物理学奖。June 6, 2014#> 1973年Paul和Peter运用核磁共振技术来检视人体组织与器官的状况(MRI), 2003年 获得诺贝尔医学奖。> 1985年Wuthrich利用二维核磁解析生物大分子(BPTI)溶液结构，2002年获得诺贝尔 化学奖。June 6, 20147June 6, 2014#June 6, 2014#12核磁共

3、振的观测核,ILIIN£10Rb 的Cs87222Ti45143Rh PdAlInPbPeriodic Table of the ElementsElements applicableUno>几乎每一个元素都有可采集到NMR谱的同位素> 常用 NMR可观测核:13C, 15N, 31P, 19FK3,核磁共振原理无外部磁场:有外部磁场:June 6, 20149K4,磁化矢量在外磁场中的空间分布June 6, 2014June 6, 2014June 6, 2014K5,宏观磁化矢量June 6, 20148K5,宏观磁化矢量June 7, 2014K6, 90度脉冲iJ

4、BozzJune 6, 201410t7, FID信号产生切割感应线困June 6, 20141.01.0产生电流信号，与时间相关!11仁8, FID信号傅立叶变换（时域信号）（频域信号）傅立叶变换HIcIH-HIc H-H特征指纹峰-不同的官能团在一维氢谱中出峰位置不一样;积分大小与氢原子数目成正比；峰组中裂分距离（耦合常数）与构型紧密相关；June 6, 201412仁2磁共振的分类固体核磁共振无机材料（纳米复合材料、催化剂等）有机固体（高分子、膜蛋白等）液体核磁共振有机化合物天然产物生物大分子核磁共振成像临床诊断的成像仪动植物实验研究用的成像仪June 6, 2014131"核

5、磁共振的优势不破坏样品的结构性质可无偏向性的检测样品内所有物质样品制备简单定量方便A特别适用于MS不易检测的物质（挥发性酮，胺类，和异构糖等）可对未知化合物进行结构测定1.11核磁共振的局限性方法最小检测量(mol)荧光1O-18J0-23质谱10-13_10-21电化学10-15-10-19放射化学1014-1019紫外-可见光吸收1013-1016核磁共振10-9-10n无法检测低浓度物质解决及矜超高场:700 MHz超低温探头:信噪比提高4倍以上使用小的样品体积June 6, 2014152,核磁共振系统介绍2沁核磁系统部件超导磁体（磁场强度:MHz）探头（探测核特异性）机柜射频系统（生

6、成/检测射频信号）工作站+软件（实验设置和数据处理）June 6, 201419安全性最重要1. Outer shell of magnet2. Foil radiation shield helps reduce liquid losses due to radiation. This area is also under vacuum to reduce heat conduction.3. Outer wall of liquid nitrogen dewar container4. Bore tube stand-off keeps the bore tube from physica

7、l contact with the cold5. Liquid nitrogen container6. Inner wall of liquid nitrogen dewar container7. More heat shield foil - also under high vacuum8. Outer wall of liquid He dewar9. Coil and electrical stuff container10. Superconducting coils11. Don't know!12. Outer coils13. Room temperature bo

8、re tube14. Bottom of coil container15. Inner wall of liquid He dewar16. Don't know!17. Charging rod for charging the magnet when it is installed18. Liquid helium level sensor19. Discharge resistors20. Liquid helium container主要由超导线圈构成，产生巨大的磁场 磁场强度(B0)单位通常用1H的共振频率MHz表示June 6, 2014212.2,超高场的意义提高灵敏丿

9、增强NOE效应的信号强度 NOE效应应用于提高碳谱的信噪比提高信噪比提高空间分散度(Dispersion)S/NOCB03/2场强400M500M600M700M800M信噪比11.3981.8372.3152.828June 6, 2014#2.4t液体探头-室温探头激发核自旋和探测来自样品的核磁共振信号根据检测的样品的状态，大致分为液体探头和固体探头固体探头咼频核FH/19F)：咼频线圈pttj 低频核(13P-15N):低频线圈mi低频线圈置于内层，高频线圈在外层，提高低频核的 灵敏度反相探头：低频线圈薛外层，高频线圈在内层，提高高频核的 灵敏度一合 探头-低頭内外屛线圈紧贴绕制的新工艺

10、从而同时提 高了两个线圈的填充因子高，低频核都有很高的灵敏度June 6, 2014#2.5,液体探头-超低温探头June 6, 2014252.5,液体探头-超低温探头June 6, 2014#2.5,液体探头-超低温探头>>前放电子线圈(70K)> S/N比常温探头提高约4倍June 6, 2014#2.6,射频系统-机柜频率合成器 (DDS/PTS)功放脉冲场梯度 放大匀场电源June 6, 2014232.7,射频系统信号检测June 6, 2014#2.7,射频系统信号检测o°June 6, 2014#2.7,射频系统信号检测June 6, 2014#2.

11、7,射频系统信号检测前置放大器有两种用途：放大楡测矗NMR詹号(从微伏到毫伏)分离高能RF脉冲与低能NMR信号 前置放大器含有一接收发射开关(T/R),其作用就是阻止高压RF脉冲进 入敏感的低压的信号接收器核磁共振信号频率虽然咼，但是覆盖范围小:：在400MHz系统，lOppm质子信号的频率范围只有4000Hz（399,998400Q02KHz） 第一步混频（中频信号）：19998-20002kHz第二步混频（声频信号）：中频信号参考频率二声频信号399,998-400,002 KHz 19998-20002 KHz-2.0 +2.0 KHzJune 6, 2014252. &射频系统

12、-直接检测Direct Drive Receiver2.9,射频系统锁场实验对磁场稳定性的要求可以通过锁场实现。通过不间断的测量一参照信号（宛信号）并与标准频率进行比较：出 现偏差后，差值被反馈到磁体并通过增加或减少辅助线圈（Z。）的电流 乘进行矫正OJune 6, 20142723射频系统匀场在样品中，磁场强度应该是均匀且单一，以使相同的核无论处于样品 的何种位置都应给出相同的共振峰。为达此目的，一系列所谓匀场线 圈按绕制所提供的函数方式给出补偿以消除磁场的不均匀性，从而得 到窄的线形。低温匀场(cryo-shims)线:补偿环境对磁场均一性的影响June 6, 2014#室温匀场(RTsh

13、ims)线圈：补偿样品对磁场均一性的影响June 6, 2014#June 6, 2014#较好的匀场June 6, 2014293, VJ软件简介3.1, VJ软件作用Prepare sam卩le and select experimentAcquire dataProcess dataDisplay and plot dataJune 6, 2014313.2, VJ软件界面VnmrJVERSION 4.1：姦： Agilent TechnologiesExperiment SelectorChoose one o< more expernnents to tun wa in the

14、 foe卽GsW er ccnfi$i(e experremtt to run Utdf m tho Study Quouc.Use the other Vlenical Paneb co dtsotay and n wiipv late mukipte Spectra 3»de by skte vr in overlay.Coilinland Line u話 tl>o ccmmand line to: dxect occ63$ to VnmrJ commjMa. mros anti paramcm.Grap hies CanvasS|)ec!ra arp <fl($pl

15、ay«d iri this area IrnerMi with viur 幺彳心 mkig ifx» tools h the Gcaphks Toolbar Vo perfenm loaning and pannmQ. adjust EicH scale, find and dispUy fUeg idls and pick peaks.Study QueueU id the Sb知 QiiAue ta build, zg”朋 aid siittmt collociions ot o<p«rimcn($ and io rzw spectra that hav

16、e afraciY tieen measured”qa7&lOlittirjAAIDPhargMM*Experiment Parameter PanelsUse the Experirrent Parameter Panels to configure ocojisiLion paromrtefs and to conval dotaik ol th«>such 歸 wh杯 to porfotm 3utwhiin»ni«ig. Pametcnfeom one orpennxmt can be carne-l ovor to ratod experim

17、ents 1cr easy setup Tho p$nvk Also indnd« tcilffiws io control lhf»incidi ng Mtning spin and CpmgieThe PameKr Panels die organized irto "tn. and e»:h tab contaiii oiw cr inore pages For example ihg Process Tab »ov<ks options for wecwaJ prgessing and plotting The Action 3

18、provides quiet access to ccmnncnlr used lincticrs tor the task at bond1 )l AcnPA Mr,i>26x4tfr Enc 匕Graphics Too-barJune 6, 201433核磁样品制备June 6, 2014#41,待检物、溶剂及浓度要求待检物要求：待检物可以是单一化合物或者混合物待检物性质稳定（如果不稳定可以采用特殊核磁管）待检物不能含有顺磁性物质溶解性好，溶液澄清根据相似相容原理选择合适克代溶剂，注意：克代溶剂不能与待检物反应克代溶剂最好采用小瓶装，一次一瓶特殊样品必须用特殊的克代溶剂浓度要求 05

19、mg50mg/ml9总体积600ul左右，如果体积过少，可以采用特 殊核磁管在高浓度条件下，如果谱图匀场效果差或者谱图质量不理想，可以 考虑降低浓度June 6, 2014354 2,样品高度调节及手动进样June 6, 2014#4 2,样品高度调节及手动进样June 6, 2014#5.1, 一维氢谱采样參数设置4, 一维实验设置和谱图处理点击"Experiments” ,在下拉菜单中选择"Proton”June 6, 2014#5.1, 一维氢谱采样參数设置June 6, 2014#5.1, 一维氢谱采样參数设置June 6, 2014395.1, 一维氢谱采样參数设

20、置2,在参数面板"Start-Sample Info"中填写样品名称，选择溶剂，浓 度，对应的实验记录本信息以及待检物描述信息VnmrJEik Edit Yiew Experiments Acquisition Automation Process Tools HelpIH回 H! | alfa sx to 20 - from th电 probe filQ5Da<5eq PRO TOMIndec 1V£10dl42SzrtlAiquirJfRog訂 旦Ul ect “宀附仙険匕)(巫Sample Info LockShim Spin/TernpOperato

21、r: vnmrlSample information1 ClearSample name "5【Sample direciory (2st.20：1404M0-01)Solventcoc 13DMSO|CDCI3 020 OtherEmail |gL6m comCommenlsThis sample is fonest oniv|ConuntmionNotebookPage0mMjio5 4Wlemp spin Lock sunpie VnmrlProbeATB3InactiveHEifa set to 20 - from the probe file3,调谐匹配探头后在参数面板“S

22、tart-Lock”中点击FindZO,系统将进行自动锁场VnmrJE«le Edit 乂Experiments Acquisition Automation process Tools hklp曰團画E3ma aal fa set to 20 - horn the probe fileSWAafv -oldssaLJo£llodM±A国 Exp: 3Seq PROTONindex： 1C13Q XStart Acquire Process Jnsen jf Auto lockGradient shim Logout JMluQnsMu二5Sample InfoL

23、ock Shim SpinTemp spinLockLock status ( ftSelect lock signal.tczslua »lempspinVnmrJ Find zOAutolock_ AutomaticSamplezo onryj Unlocked© Not usedRun autolockLock samplePiobeATB3AlInactive4,在参数面板“Start-Lock”中点击“Find ZO” ,系统将进行自动锁场，完成后，硬件面板Lock按钮将出现蓝色数值，表明场被锁住。VnmrJE«le Edit 乂Experiments

24、Acquisition Automation process Tools hklp曰團画E3ma aal fa set to 20 - horn the probe fileSWAafv -oldssaLJo£llodM±A国 Exp: 3Seq PROTONindex： 1C13Q XStart Acquire Process Jnsen jf Auto lockGradient shim Logout JMluQnsMu二5Sample InfoLock Shim SpinTemp spinLockLock status ( ftSelect lock signal.

25、- lempspinVnmrJ Fine! zOAutolock_ AutomaticSamplezo onryj Unlocked© Not usedRun autolockLock samplePiobeATB3AlInactive5,在参数面板"Start-Shim”中点击“Start shim” ,默认为Z方向上 自动匀场。VnmrJFile Edu ViqwExp eriments Acqui sitionAutcmaaonProcess Toolsip !Q from ttw probe filQPlPWVInseitAcquire ProcessGiadien

26、t shim J ( Logout iAuto lock jIock5<'an"Autoshi inSpin /TempStart shimStop shimLockIfkPh逊胡Lk Gain .& Sample InfoE Lock上 ShimEjea.pcksoyiStartFID sonid cknum snm SavelRead shims from 卩巧Spin on ,sPin 曲 fSpin off 5ave2 'wTam 卩 | 一 vnmnSpinLackSampleProbeAFB3Inactive6,在参数面板“Start-Spi

27、n/Temp"在'Spinner:liuidsr下输入所需转 逋；在"Temperature”下输入所需温度。vnmrjFile Edit View Experiments Acquisition Automation Process Tools Help甩 al fa set to 20 - fi om theprobe fileExp 3Seq. PROTONIndex: 11X>JijO.42Pw/AStart Acquire Processa xln«rt ” 耳getSample Info LockShimSpinner liquids1C

28、onirol spinner from this panel only O Abort after spinner error Warn after spinner error Ignore spinner errorRegulate «pee(i|20Spin/TernpiCurrentOffspin orrTempeiatureControl temperature from this panel only(Rcqulate tampAbort after lempcrature errorIf25=n1Aam after temperature errorCurrentOffT

29、emp off Io no re temperature erroraule£J±UJqns>p 三CmalSMii?.i 小."2Auto lock)(cad 注 nt丿 (Logout JlempspinLocksimpleProbeVj l|ATB3Inactheaifa set to 20 - from the probe file6,在参数面板 uAcquire-Default HV在"Number of scans” 下输入所 需采样次数。默认“Autogain”前面打勾，点击"Go"即可。VnmrJFile Edi

30、t ViewExperiments Acquisition Automation Process Tools HelpEB0®(tai alfa set to 20 -J二 a 牙IIfrom the probe fileseq: protonIndex: 1Stait AcquireProcessDefault Hl Acquisition Pulse Sequence ChannelsFlagsFuture Actions OverviewShow lime-1Experiment: PROTON Solvent cdd3Acquisition optionsppmppm卜20

31、|3atzSpectral widih (select) (.or enter) Number of scans Relaxation delay Pulse angledegreesTempSpinLockSampleProbewATB3Esl pw90 Aujls Sequence diagram < Sequence help jObserve HlDecoupler: C13Receiver gain (dB)v Auto gainInactivealfa set 10 20 - from the probe fileJune 6, 2014475.2, 一维氢谱谱图处理7,在参

32、数面板“Process-Basic"点击“Autoprocess” ,系统自动完成 谱图处理。VnmrjFile Edit Vi«v/ &<prime nts Acquisition Auto mation Process Tools HelpJune 6, 2014495.2, 一维氢谱谱图处理June 6, 2014#5.2, 一维氢谱谱图处理Et5“dspaip<一 £dss=o£toaA#>aweu:.cu2n9p3o %JL7SUWcalc mis s/n ratio = 4324.3Exp：3Seq： PROTONc

33、ursor0.51in彳eg 5<tle1083 5meg EseB9.1Start Acquire ProcessBasicDefaultWeightingDisplayMore IDIntegrationCursors/Line ListsPlotText Outputindex. 1deltappmjJlTransform) Autoprocess ；i Display spectrumSample InfoSample: EthvtindanoneSolvent cdc!3Sample owner: vnmrlComments:Ethvl indan one standard t

34、esi sample Recorded on 400-MR wiih OneNMR probe and PZT tuningi Clear scieen )( CancelProcess OptionsFT data size| LineBroadenPlot Option%aEJune 6, 2014#5.3, 一维氢谱谱图打印June 6, 2014515.3, 一维氢谱谱图打印8,在参数面板“Process-plot"选择打印参数，点击uAuto plot preview”系统生成pdf文件。VnmrJ£ile Edit Ybw Experiments Acquisi

35、tion Automation Process Iools kklpEJBS画3 ascalc rms $/n ratio = 4324.3UlTTaE Exp:3Seq: PROTONIndex* 1I一_厂厂y4 ibl心丨<1|KhI-H a.aIr11187 64321mica8S.1ini«9 scil«cursordaltxLO63.50.51spectrum )lk Clear screen )( CancelIStarTTe Process空”更还回血亟BasicDefaultAuto plot 卩agePlot setupFIDsWeighting

36、Display More ID Integration Cursors；Line Lists PlotText OutputAuio plot previewSend this plot ioHz to mm: 13.7Screen positionFulluniQiLeft I RightTempSpinLockSampleATB3Plot AS aispl43ParametersBasic (left)Hot parametersintegral values；Se9d, horiz.ShowPI oiPeak frequenciesProbeManual plotPlot s卩ectru

37、m scaleMISC(Plot 2*(Plot Hsi or mwqralsPlot moleculesInact hePlot FIDPlot FID scalePlot FID sealPlot pulse sequence、1 7Clear plotJune 6, 2014#5.4, 一维碳谱采样參数设迁1,点击"Experiments”在下拉菜单中点击"Carbon” ,调入碳谱脉 冲。和氢谱设置相似，探头调谐匹配，锁场，匀场，设置转速及温 度。Eile Edit 里iew:3ExpEKperimencs Acquisition Automation Eroces

38、s Iools Help. Setup New Paiameters for.ProtonCarbonFluorinePhosphorusOth«r NucleusHl Relaxationo.o一 o-dsmos:Convert Cuiient Parameters To Dq.Standard ID ExperimentsSolvent Suppression - Select Peakso.o,d2"tfoa 盍>Homonuckar Correlations J-CorrelationsIndirect Hwronuchar Corr-alaxions (Ba

39、sic) rtastSampleLock ShimAcquinInfo sIndirect Heteronuclear Correlations (More)Indircct Hewronuchar CPJ SIS2Selective ExcitationAuto lock( Cradi电nt him丿SpinnempSpinJ X-H Muliipliciiy Deirminaiion H HeteronuckarCorrelations j 13C-13C Correlations 19F-LH ExperimentsJ Relaxation Measurement DOSY Experi

40、mentsControl spinnerfrom this panel on”Abort after spinner errorWarn after spinner error ' Ignore spinner errorConuol lemperaiure from ihls panel only| Hadamard ExperimentsCurreni0疔飞 Temp ofT jLock Sample 內 ob 电AFB3Abort after temperature error Warn after lemperature error ignore wmpQraiurQ erro

41、rInactiveale rms s/n ratio = 4324.329 在参数面板"Acquire-Default C13"在"Number of scans"下输入所 需采样次数。默认“Autogain"为30,点击"Go"即可。VnmrJ£ile Edit yiev/ EiPnments Acquisition Automation Process Tools khlpEJBS画E3 ascalc rms $/n ratio = 4324.3Exp:3Seq: CARBONIndex: 1o.ouottMa

42、.A 2U12-UHdss七 o.£Acquire亠一一 ulqnpwo,Default C13Acquisitio n Pulse Sequence ChannelsFlagsFuture Actions Overview1.0gJLz/uaShowtimediExperiment: CARS ON Sob/em: cdcBrAcquisition options-Pulse angleCheck S/N?Temp | Spin Lock I SampleVnrr»rld2QQAngAnavs Sequence diagram ' Sequence helpObs

43、erw C13Decoupler HLNumber of scansRelaxation delayHl decouplingSpectral vridth (select)PPm(.or enier)ProbeReceiver gain 9B)ATB3Inact heJune 6, 2014555.5, 一维碳谱谱图处理3,在参数面板“Process-Basic"点击“Autoprocess",系统自动对谱 图进行处理。VnmrJFile Edit Vkw Experiments Acquisition Automation Process Tools HlpenD2ds

44、paAe.t<-2dss£odtioaMd>aEEHtiUJar-s>p=5s!ta$wretrieving and processing: Ethylindanone_CARBON_01.fidSeq CARBONIndex: 1V.position220V.KXJc20cursor180delti1GCStart Acquire ProcessBasicDefaultWeightingDisplayMore IDIntegrationCursors/Line Lists PlotText OutputSample Infosamp I e Eihylindano

45、naSolvent: cdc!3Sample owner '/nmrlcomments:Transforma XProcess OptionsPlot OptionsPlot Pt A14 QW120w*>ft aaia size J LincBroadenAutoprocess ) Display spectrum Clear screenEthindanone standard tesi sampleRecorded on 400-MR wlih OneNMR prooe and PZT wmngTempSpinLocksampleProbeATB3Cancel iL28KZ

46、F0.5一-|hzParametersBasicOff1 1 "111ManeIntegrals Peak. ValuesInactiveJune 6, 2014575.6, 维碳谱谱图打印4,在参数面板“Process-plot"选择打印参数，点击uAuto plot preview”系统生成pdf文件。and畧 retrievingprocessing: EthylindanoneC.AKBONOLfid220"0160V_po<ttiondimcure orV.ceali44667.3Exp： 3Seq CARBONIndex: L70b（）120 1

47、00ppm144-Start Acquire ProcessTran stormAutoptocess H Display speafum Clear screen cancel1 H! vnmrjFile Edit Vie/ Experiments Acquisition Automation Process Tools Help曰o画x回Plot setupManual plotFIDS-oldss±p£lloawsBasicDefault WeightingDisplay More IDIntegration Cuisors/Line Lilts PlotText O

48、utputI Spin I Lock I SampleProbeAuio plot pageAuto plot previewSend ihis ploi toH2(o mm: 712Plot As displayed Parameters®asic(ieH)( Plot paiameters 'Iniegral values n：门二|7Peak frequenciesPloi spQCUiim ) 、上iwpgu皿Plot spertnim scaleMiscnot text j :Plot list of integralsPlot moleculesPloi FID、

49、i即皿加邯例Pint HI) sole(Plot PUS£2eqUenClCIqm plotSi_A|inactive厂國閲则眼剑ML乩酣加制畏耐5.7, PRESAT实验设置1,参照51中氢谱设置方法，采集氢谱，点击“Experiments-SolventSuppression-Select Peaks-PRESAT”。5.8, PRESAT实验设2,效果：将CDCI3溶剂峰压制完全。同理，选择 “Multi-Freq PRESAT” 模式，利用 “BOX/Cursor” ,再 "Select"可以压制多组峰进行压制。VnmrJJune 6, 201461Fih Edit ViQ* Experiments Acquisition Automation Process Tools HelpBQ XJUsStart! dscaeShow TimeDefaultTemp .ShangHai File BrowserIy jExpenment PRE5AT Solvent: cdd3Observe HlDecouple