Page 1
ThisMSWordfileisstoredintheChemDeptfacultyGoogleDrivefolder.Printed 8/28/16 6:06:00 PM
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
Page 2
-NMRinstructions,p. 2 -
Printed 8/28/16 6:06:00 PM
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
Page 3
-NMRinstructions,p. 3 -
Printed 8/28/16 6:06:00 PM
• Ifyouareinsuperlaboranotherclass,chooseyoursemester.Filesassociatedwiththeseoperatornameswillgenerallydisappearaftertheendofthesemester(researchgroupfilesmaystayslightlylonger,butdon’tcountonlongtermstorage)
Figure4.VnmrJscreenshot.Numbersareusedintheinstructions.
Page 4
-NMRinstructions,p. 4 -
Printed 8/28/16 6:06:00 PM
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).
Page 5
-NMRinstructions,p. 5 -
Printed 8/28/16 6:06:00 PM
• 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.
Page 6
-NMRinstructions,p. 6 -
Printed 8/28/16 6:06:00 PM
• 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.
Page 7
-NMRinstructions,p. 7 -
Printed 8/28/16 6:06:00 PM
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.