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Varian(Agilent)500MHzNMROperationInstructions(HaverfordCollege)

PreparingyourNMRSampleTube

1. Dissolve(i.e.noparticulates)yoursamplein1mLofdeuteratedNMRsolvent(CDCl3,D2Oord6-DMSOarerecommended).

2. Placesolutionintoa9”longNMRtubeandcapit.• 7”tubesarehardtoremovefromthecarousel,

andareolderandoflowerquality.

3. Findagrayspinnerforyoursample.• Seefigure1.• Theseareusuallykeptinthetopdrawerright

behindtheNMRcomputer.• TheyshouldnotberemovedfromE107.

4. Placethegrayspinnerintothedepthgauge(Figure2)andthencarefullypushtheNMRtubeintotheopeningatthetopofthespinner.• Itisatightfit;holdthefragileglassNMRtube

closetothebottomwhilepushingthetubestraightdown.

5. Pushthetubedownuntilittouchesthebottomofthedepthgaugewhilethespinnerisstillseatedfirmlyatthetop.• Besurethattheliquidsamplecoverstheentire

dottedrectanglemarkedontheinsideofthedepthgauge(ifyouused1mLofsolvent,itwill).

• TherectangleindicateswheretheRFcoilwillfitaroundyoursample

6. Holdingeitherthetopofthetubeorthetop(black)ringofthespinner,removethetubeandspinnerfromthedepthgauge.UseaKimwipetoremovefingerprints,etc.fromtheoutsideofthetube(belowthespinner)andthespinneritself.• TheNMRtubemustextendbelowthespinner

bytheproperlength,sodon’tletthetubemoveupordown(withrespecttothespinner)afterremovingitfromthedepthgauge.

• Insertingatubeorspinnerthatisdirty(ontheoutside)willdegradetheNMRspectraoffuturesamples(aswellasyourown).

Figure1:spinners

Figure2:NMRtubeinspinner,properlypositionedindepthgauge

ßspinner

ßtopofNMRtube(cap)

depthàgauge

ßbottomofNMRtube

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PlacingyourSampleintheCarouselandsigningin

CAUTION:Donotapproachthemagnetifyouhaveapacemakerorothermedicaldevicethatmaybeincompatiblewithstrongmagneticfields.Youmaywishtoremovecellphonesandwallets(creditcards)fromyourpockets.

7. Withthespinnerinhand,climbtheladderandlookatthetopofthecarousel.Selectanyfreeslotexceptforslot#1ortheactivesampleslot(theoneclosesttoyou)(seeFigure3).• Explanation:thereisalreadyasampleinthe

magnet,anditneedstomoveupintothisactivesampleslotbeforeyourtubecandropin.

8. HoldthetopoftheNMRtubeandcarefullylowerthetube(+spinner)straightdownintotheslot• Donotleanagainstthemagnet–ifyoudo,it

willwobblebackandforthsinceitissupportedonacushionofair,toshieldagainstvibrations.

• TheNMRtubewillstickupabout1.5”abovethetopofthecarousel.

• Notethenumberoftheslotyouused.Unfortunately,thesamplenumbersareatthebottomofthecarousel.

9. Signintothelogbookwiththedate,yourname,solvent,andtypeofexperimentyouplantodo.Alsoincludethecarouselslotthatyouareusing.

• Thatissowecancomefindyouifyouforgettoremoveyourtubefromthecarousellateron.

Figure3:NeveraddaNMRtube+spinnertoslot#1orthecarouselslotthatisovertheyellowcautiontriangle(theactivesampleslot).Whenthispicturewastaken,slot#1WAStheactivesampleslot,butthisisnotalwaysthecase.

StartingtheVnmrJsoftwarewiththecorrectoperatorname

10. OntheNMRcomputer(youmayneedtomovethemousetowakeit),ifauserisalreadyloggedinandtheVnmrJsoftwareisrunning,clicktheStarttab(Figure4,#6)toseetheoperatorname(Figure4,#7).Ifitiscorrect,youmayskiptothenextsection(“SettingupanewStudy”).• ItIsimportanttobeloggedinasthecorrectuserandtobeusingthecorrectoperatorname

sothatyoucanfindyourdatafilelater.

11. Tochangeoperatorname,exitfromtheVnmrJsoftware(usingtheFilemenu)andlogoutofthesystem(usetheSystem:LogOutoptionfromthelinuxdesktop).

12. Loginwiththecorrectloginname(“research”or“superlab”or“classes”)andpassword(passwordscanbefoundinthepurple“runlog”notebook).

13. StarttheVnmrJprogram(itsiconwillbeonthedesktop).Youwillbepromptedtoentertheoperatorname:• Ifyouarearesearchstudent,enteryourresearchgroupnameforoperatorname

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• Ifyouareinsuperlaboranotherclass,chooseyoursemester.Filesassociatedwiththeseoperatornameswillgenerallydisappearaftertheendofthesemester(researchgroupfilesmaystayslightlylonger,butdon’tcountonlongtermstorage)

Figure4.VnmrJscreenshot.Numbersareusedintheinstructions.

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Settingupanew“Study”torununderAutomation

Theautomationroutineallowsyoutosetupa“study”foreachsampletubepositioninthecarousel.Each“study”willconsistofoneormoreNMR“experiments”;forinstance,obtaininga1-DprotonNMRspectrumisanexperiment,andobtainingaseriesof13CDEPTNMRspectraisanotherexperiment.

14. Clickonthesmallcircleatposition#4(Figure4)todisplaythesampleslotnumbers(aringof12numbereddots–Figure5).

15. Ifyouarerunningthefirstsampleoftheday,orifyoursampleslotnumberisshowingupasacirclewithacolorotherthangray(indicatingtheslotnumberhasalreadybeenusedbysomeoneelse),choose“AutomationRun[autodir]>NewAutomationRun”fromtheAutomationmenu(Figure4,#1).

16. Iftheyaren’talreadyselected,clickontheProtocolsandCommontabs(Figure4,#2and#3).• DoNOTusetheStd1Dtab

17. Clickon‘NewStudy’in(Figure4,#10)tocreateanewstudy.• A“study”isoneormoreexperiments

donebacktobackonthesamesample

18. Clickonthecirclewiththeslotnumberforyoursample(seeFigure5)

19. Typeinasamplenameandselecttheappropriatesolvent(Figure5,orFigure4,#8and#9).• Itisusefultoincludeyournameorinitialsinthesamplename,butyoudonotneedto

includethesolvent,dateortypeofexperiment.

20. Clickonyourexperiment(i.e.‘PROTON’;Figure5orFigure4#3).• Ifyouwish,youmayclickonmultipleexperiments.Alongerlistofexperimentsisavailable

underthe“Liquids”tab.

Optional:ChangingAcquisitionParametersforanExperiment

ThefirstfewtimesyoucollectNMRspectra,skipoverthissection.Butbychangingacquisitionparameters,youmaybeabletogetbetterqualityspectrawithmoreaccurateintegrations.

21. Doubleclicktheexperimentwherelistedinthe“tobesubmitted”studyqueue(itwillhaveanamelike“PROTON_001_day[0:24]”–upperleftredcircleinFigure6).Waitabout10-15secondsasthedisplayontherightsidechangestoshowtheacquisitionparameters.

22. Ifitisn’talreadyselected,clickontheAcquiretabtotherightoftheStarttab(Figure4,#6).Changeparametersasdesiredforyourspectrum.Someparametersyoumaywishtochangeforaprotonspectrumareasfollows:

Figure5.Thesamplechangerslotdisplayduringsetupofanewautomationstudy.RedcirclesshowbuttonsthatmustbeclickedorfieldstobefilledinwhensettingupanewstudythatincludesaprotonNMRexperiment(seesteps18–20,and24).

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• SpectralWidth(inthe“DefaultH1”list):Bydefaultthisis-2to14ppm,butifyouknowthatallyoursample’ssignalsarebetween0and5ppm,thenarangeof-1to6ppmwillsuffice.Warning:donotattempttoexcludeimpuritypeaksbycuttingdownthespectralrange;ifyoudo,thosepeaksmayshowupas“Fourierfold-over”peaksatthewrongppm.

• Acquisitiontime(ontheleftsideofthe“Acquisition”list):Thedefaultis2-3seconds(itwillchangeifyouchangethespectralrange),buttogetthesharpestpeaks,increasethistoatleast5seconds.

• RelaxationDelay(ontherightsideofthe“Acquisition”list):Theacquisitiontime+therelaxationdelayshouldbeatleast15secondstogetthemostaccuraterelativeintegrations.

• Numberofscans(ontheleftsideofthe“Acquisition”list):Bydefaultthisis8,whichIsusuallysufficientforprotonspectra.Butifyouhaveaveryweaksample,youcanincreaseyoursignaltonoiseratiobyafactorof4byincreasingthenumberofscansbyafactorof42=16(i.e.numberofscans=128).Thisofcoursewilldramaticallyincreasethetimeittakestorunyourexperiment(toatleast32minutes,ifyoualsoincreasedtherelaxationdelayasdescribedabove).

• Youmayalso,ifyouwish,changesomeoftheprocessingparameters.Butthesecanbechangedafteryoucollectyourdata,soitisnotcriticaltochangethemnow.

23. Onceyouaredoneeditingtheacquisitionparameters,click‘Save.’onthelineofbuttonsabovetheparameters.Youmaynowaddorchangeparametersforasecondexperimentifyouwish.

SubmittingyourStudyforAutomation

24. Clickthegreen‘Submit’button(lowerleftofFigure5).Yourstudyhasnowbeenaddedtotheautomationqueue.Thesampleslotnumberonthedisplayshouldturnyellow.

25. Ifyoursisnotthefirstsampleoftheday,dataacquisitionmaystillbeinprocessonsomeoneelse’ssample.Butotherwise,withinafewseconds,expecttoseeandhearsomeactivity:• Afterafewseconds,thesamplechangershouldejectthesampleinthemagnetbefore

rotatingtoyoursampleslotanddroppingyourtubeintothemagnet.Thesampleslotnumberonthedisplaywillturnpurpletoindicateithasbecometheactivesample

Figure6.Modifyingparametersforanexperimenttobeincludedinanewstudy.

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• Ifnothinghappensafter30seconds(orerrormessagesappearbelow),tryrightclickingonthesampleslotnumberandselect“DeleteStudy”.Thenchoose“DeleteDay&NightQueue”.Then,makesurethatthecorrectsampleslotisstillselectedandsubmityourjobagain.

• Ifstillnothinghappens(andyouaresurethatnobodyelseiscurrentlycollectingdataontheirsample),youmaytry“AutomationControls>AbortAutomation”atthebottomoftheAutomationmenu.Thengobacktostep15andstarta“NewAutomationRun”.

• Ifstillnothingishappening,seekhelp.

AftersubmittingyourStudy

TheadvantageofusingtheAutomationQueueisthatyoucansetupadditionalstudieswhiledatacollectionisinprocess.

26. ToensureefficientuseoftheNMRtime,don’twaitfordatacollectiontobedonebeforesettingupthestudyforthenextsample.

27. Ifanotheruseriswaitingtosetuptheirsample,letthemdoso.Iftheyareusingadifferentoperatorname,theymaycloseVnmrJandrestartittochangetheoperatorname.Datacollectionwillcontinueonyoursample.• Unfortunately,ifyouchangeloginnames(research,classes,superlab),youcannotaddnew

studiestotheautomationqueue.

28. Afteraddingstudiesforallsamplestoberun,addastudyconsistingofjustaprotonexperimentforthelocksample,keptinsampleslot#1.Usesamplename“Lock”andsolvent=D2O.• ThelocksampleisjustatubecontainingD2O.Itisbesttoleaveasampleinthemagnetat

alltimes(exceptduringsamplechanges).• Puttingthelocksampleattheendofthequeueensuresthatyoursampletubeswillbe

ejectedsoyoucangetthembackattheendofdatacollection.

DataProcessing

Whenyourstudyhascompleted,thepositionofyoursamplewillturngreen.Atthispointyoucanprocessyourdatabydouble-clickingonthedatafile(listedundercompletedstudiesatthelowerleft)andgoingtotheProcesstab.Thisgivesyoulotsofoptionsforintegrals,plotting,etc.

29. VnmrJwillautomaticallyintegrateyoursample.GotothedataprocessingtoolbarandgotoIntegralsDeleteIntegrals.

30. IfyoucannotseetheintegralsgotoIntegrals ShowPartialIntegrals

31. Definetheregionsyouwouldliketointegratewiththedefineintegralsscissors.

32. GotoProcess Integrationandselecttointegrateby‘SinglePeak.’

33. Movethecursortoapeakthatrepresentsaknownnumberofprotons.

34. Enterinthenumberofprotonsthatcorrespondstothechosenpeak.

35. Click‘SetIntegralValue’andthen‘ShowIntegralValues’todisplaytheintegration.

36. ToprintyourspectragotoPlot AutomaticPlotPage.

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Figure7.DataprocessingusingVnmrJsoftware.Redcirclesindicatesomeoftheiconsthatareusedinsettingupintegrationcurves.YoumayfinditeasiertouseMestreNovasoftwaretoprocessyourdata(seeseparateinstructions).InordertofindyourdatatousewithMestreNova,youwillneedtoknowtheloginname,operatorname,andthesamplename.

Whenyouaredone…

37. Carefullyremoveyoursample(s)fromthecarousel.Besuretoliftthetubestraightupsoyoudon’tsnapoffthebottom.

38. Removethetubefromthespinnerandreturnthespinnertoitsbox.

39. Ensureyou’vefilledoutthelogbookentrycompletely–addcommentsifanythingunusualhappened.

40. TaketheNMRtubecontainingyoursamplewithyou–youareresponsibleforcleaningit.