1

1d TOCSY InaTOCSYspectrum.magnetizationisallowedtopassfromoneprotontoanotherthatis3bondsorlessaway,andtotakesuchstepsrepeatedly.Thus,magnetizationmovesfromanyoneprotontoallothersthroughoutthespinsystemofwhichitisapart.Thisisagreattoolforlearningwhichprotonsareconnectedclosely.TheselectivityoftheTOCSY1Dsequenceisbasedonapairofgradientechoesemployingselectiveinversionpulsesthatwillinverttheresonanceofinterest,sothatitisrephrasedbythesecondofeachpairofgradients.Allotherresonanceswillexperienceaccumulatingdephasingbythesumofallfourgradients.

Beginningwithanexperimentcontainingyourbeautifulcalibrated1d...Copytheparametersintoasecondexperiment.Youwillmodifythesecondexperimenttomakeita1DTOCSYbutyouwillworkinthefirstonetomakeyourshapedpulse.Consideringthatyouhaveyourbeautiful1dinexperimentA(Aisanumber)movetheparameterstoexperimentb(Bisanumber)bytypingmp(A,B).IfyouarecurrentlyinAatthetimeyouissuethiscommand,youneedonlytypemp(B).Setuptheparametersfora1DTOCSYInexperimentB,Convert current parameters to do ....> Selective Excitation Experiments> 1D TOCSY (dpfgse).(Figure2)Youseetwospinechoesinwhichthe180°pulseisselective,andflankedbyapairofequalgradientpulses[1].Youwillneedtomaketheselective180°pulseshape,toinverttheresonancewhichyouwanttointerrogatewithrespecttoconnectivity.Currentlythatpulsehasthenameq3[2].Wewillneedtomakeourownshapetotaketheplaceofq3.Formaximumflexibility,thissequenceallowsustomaketwodifferentshapes,butwe'lljustmakeone.Makeashapedinversionpulse(a180°pulse)GotoexperimentA(exp9forme)bytypingjexpA(jexp9)(Figure3)Putcursorsaroundalineorspectralregionyouwilluseasthesourceofmagnetization[1].Inplainwords,youwillbeaskingwhatotherprotonsareconnectedtothisonebythrough‐bondconnections(anynumberofindividualconnectionsof3bondsorless).Ichoseaclusteroflinesnear7ppmbecausetheyareisolatedinthespectrum.GotoEdit>New Pulse Shapes (Pbox)Againchosethe'MakeWaveform'tab[2]andclickon[NewWaveform][3].For'Shapetype'choose'inversion'[4].For'Shapename'Ilike'iburp'butthecurrentexampleshows'g3'[5].

2

Checkthatthe'Referencepw90'and'Referencepower'arecorrect(andchangethemifneedbe)[6].Clickon[Selectfromspectrum][7]touseyourcursorstocalculatetheBandwidth[8],pulselengthandoffset.Click[AddWaveform][9]..Giveyourpulseausefulnamesuchas'inv7ppm_21Sep10'[10].Click[MakeIt!][11]Thenclick[√]'ShowZ'andsimulate[12].Figure4showstheresult.NotethattheZ‐magnetizationchangessignfrom+1to‐1andthenback,butretainsfullamplitude:cleaninversion,withverysharpspectralboundaries.ClosePbox.Integratingyournewpulseintothe1DTOCSYexperimentNowjointheexperiment(B)inwhichyouwillusetheshapedpulse.BESUREthatthisexperimentusesidenticaltofandswastheoneinwhichyoucreatedthepulse.(ChecktheseinAcquire>ChannelsandAcquire>Acquisition.)GototheAcquire>PulseSequencepage(Figure5).DrawdownEdit>New Pulse Shapes (Pbox)againandchoosethe'UpdateParameters'tabinthePboxwindow[1].DONOTusethe'Setparametervaluesintoexperiment'button.Ifyourpulse'namedoesnotappearinthefilenamebox,typeitin(retain'RF'amongtheoptionsontheright[2]).Afterenteringyourpulse'sname<return>andtherestofthewindowshoulddisplaythedurationofyourpulse[3],thepowerrequired[4]andothercharacteristicsinblueas'values'.Justusethewindowasadisplaypanelsoyoucancopytherelevantvaluesintoyourexperiment's.Thelocationsforthefirstpulse'sname,duration(width)andpowerare[5],[6],[7].(Checkthatyournumbersareconsistentwiththeunitsgivenforthepulsewidths.)Here,Iamusingthesameshapeforbothpulses.Notethatthenamesoftheshapedpulsesarecase‐sensitive.Fornow,don'ttouchthegradientsorthespinlock.Theformeraregenerallyverygoodasprovided.Thespinlockmaybevariedwithcare.Thepi/2pulse[8]usedwascalculatedfromourpw90andpower.Irecommendthatyoutrytohave1/(4*(pi/2))≈sw.ourswis≈4000sowe'dlike250us=4*pi/2ora60uspi/2pulse.Varian'sdefaultisveryclose,sowe'llleaveit.InAcquire>Acquistion,usethe'Arrays'buttonandturnoffthearrayinnt(selectntandclick'Unarray')(Figure6).Leavetwovalues(atleast)ofmix[1]butdonotexceedamixof.1s[2].mixisthetimeallowedformagnetizationtomoveamongprotonsinthespinsystem.Besurethatyourlocklevelis60orabove[3],becausetheuseofgradientsinthisexperimentwillcauserepeatedlossoflock,fromwhichthespectrometerwillhavetroublerecoveringifthelockisnotstrong.Formorefun,arraythemixingtime,from0‐80ms(Figure7)Afterselectiveexcitationofmagnetizationnear7ppm,increasinglylongmixtimesareallowedforthemagnetizationtomigratetoothersites.Thetime‐dependenceof

3

themagnitudeofmagnetizationatanothersite,suchasat8ppm,iscalledthebuild‐up.Weseethataromaticprotonscanpassmagnetizationtootheraromaticprotonsviastepsof3bondsorless(Figure8).Toseewhatthealiphaticprotonsnear1.4ppmareconnectedto,makeanewshapedpulsethatinvertsonlythem.UsingthatForthisoneIusedashapecenteredat1.4ppm(Figure9).TakeyourT2intoaccountinchoosingthemaximumdurationofspinlock(whichisoftencalled‘mix’),TakeyourT1intoaccountinchoosingd1.Forcleanresultswiththissequence,Isuggestnt=8ormultiplesthereof.

TOCSY 1D Figure

© A-F Miller 2010

1

Selective TOCSY:TOCSY1D

TOCSY 1D Figure

© A-F Miller 2010

2

[1]

[2]

TOCSY 1D Figure

© A-F Miller 2010

3

[1]

[2][3][4]

[5]

[6]

[7][8]

[9]

[10] [11] [12]

TOCSY 1D Figure

© A-F Miller 2010

4

TOCSY 1D Figure

© A-F Miller 2010

5

[1][2]

[3]

[4]

[5]

[6]

[7]

[8]

TOCSY 1D Figure

© A-F Miller 2010

6

[1]

[2]

[3]

TOCSY 1D Figure

© A-F Miller 2010

7

TOCSY 1D Figure

© A-F Miller 2010

8

Bottom-to-top, longer mix times allow magnetization from near 7 ppm to migrate to other sites in the molecule.

80706050403020100

mix (ms)

TOCSY 1D Figure

© A-F Miller 2010

9

80706050403020100

mix (ms)

1D TOCSY selective for cluster of resonances near 1.4 ppm.