THEMOLECULA R PHOTOPHYCICS OF CHLOROPHYLL
A STUDY OF ITSTRIPLE T STATE
CENTRALE LANDBOUWCATALOQUS
OOOO 0086 9574 Ditproefschrif tme tstellinge nva n
JOHAN FREDERIKKLEIBEUKE R
doctorandus ind echemie ,gebore nt eGroninge no p1 9februar i 1948,i sgoed - gekeurd doord epromoto rdr .T.J .Schaafsma ,hoogleraa r ind emoleculair e fysica.
De Rector Magnif-icus van de Landbouahogeschool,
J.P.H.va nde rWan t
Wageningen, 15 april 1977 A/Al$Zo/ 68?
J.F.KLEIBEUKE R
The molecular photophysics of chlorophyll
A STUDY OF ITS TRIPLET STATE
proefschrift
terverkrijgin gva nd egraa d vandocto ri nd elandbouwwetenschappen , opgeza gva nd eRecto rMagnificus , dr.ir.J.P.H .va nde rWant , hoogleraari nd evirologie , inhe topenbaa rt everdedige n opvrijda g3 jun i197 7 desnamiddag st evie ruu ri nd eaul a vand eLandbouwhogeschoo lt eWageninge n
/r/?- /p¥ 3/f ~* 1.-„ J BIBLIOTHEEK DEI? LANDBOUV/HOOKSCHOOL WAGENINGSN A/A/ 8&os 68^ Stellingen
1 Eenverder e integratie vand emoleculair efysic ai nhe tonderwij se nonder - zoekaa nd eLandbouwhogeschoo lza lee npositiev einvloe dhebbe no pd etoe - passingva nfysisch etechnieke nbi jhe tlandbouwkundigonderzoek .
2 Detoepassin gva nhe texcitonformalism eo pd etriplettoestan dva nchlorofyl - aggregateni sallee ntoegestaan ,indie nd eeffecte nva nd edonor-acceptor - interactiesi nd ebeschouwinge nworde nbetrokken ,zoal sdi too ki sgedaa n doorShipma ne tal .bi jd ebeschrijvin gva nd eabsorptie-spectr ava ndez e molecuulaggregaten. R.H.Clarke ,R.E .Connors ,H.A .Frank ,J.C .Hoch ,Chem .Phys .Lett. , 45,52 3 (1977). L.L.Shipman ,T.M .Cotton ,J.R .Norris ,J.J .Katz ,J .Am .Chem . Soc, 98,822 2 (1976).
3 Inhe tdoo rHuan ge tal .opgesteld emode lvoo rd enulveldsplitsin gva nd e baan-ontaardetriplettoestan dva nmetalloporfyrine se nftalocyanine sword t tenonrecht everondersteld ,da td espin-spininteracti e inall egevalle n kleini st.o.v .d espin-baankoppeling . T.H.Huang ,K.E .Rieckhoff ,E.M .Voigt ,E.R .Menzel ,Chem .Phys. , 29,2 5 (1977).
4 Hetva nelkaa raftrekke nva nspinlabel-ESR-spectra ,gemete nvoo rloodrecht e enparallel eorientati eva nee ngedeeltelij kgeorienteer dmonste rt.o.v . hetmagneetveld ,zoal si stoegepas tdoo rCha oe tal. ,leid tnie tto tee n meerdoorzichtig eanalys eva nd emeetresultaten . Y.Y.H.Chao ,A .Holtzer ,Biochemistry ,J_4 ,216 4(1975) .
S Deaanmam eva nShid ae tal. ,da td egrondtoestan dva nhe tmononegatiev e anionva ncoronee ne nva ntrifenylee nnie tontaar dis ,i snie ti novereen - stemmingme texperimente dresultaten ,verkrege nui tMC Dmetingen . T.Shida ,S .Iwatw ,J .Am .Chem .Soc ,95 ,347 3(1973) . P.J.Zandstra ,D.J .Scholtens ,R.E .Koning ,J .Chem .Phys. , _57 3821 (1972). 6 Hetverwaarloze nva nhe t effectva nd ekristalveldsplitsin g opd e liggingva n deenergieniveau s ind e laagstekwartettoestan dva nkoper-octaethylporfin e enee nnie tbeweze naannam eomtren the tkarakte rva nd ekwartet-sub-niveau s leidenShatwel le t al.o pee ndwaalspoo rbi j de interpretatie vand © magnetischgeinduceerd e circulairepolarisati e vand eemissi eva nCtCEP . R.A. Shatwell,R . Gale,A.J .McCaffery ,K .Sichel ,J.Am . Chem. Soc, 97,701 5 (1975).
7 i Deverklaringen ,gegeve ndoo rGafn i etal .e ndoo rBrocho ne tal. ,voo rhe t niet-exponentieleverva lva nd epyridine-nucleotid e fluorescentie,toal sdi e wordt gevondenvoo rhe tvrij ecoenzy mi nvisceuz e oplossinge nvoor!he t eiwit-gebonden coenzym,houde nnie tvoldoend e rekeningme toplosmid^el - reorientatie effecten. A. Gafni,L . Brand,Biochemistry ,^_5 ,316 5 (1976). J.C.Brochon, Ph .Wahl ,J.M . Jallon,M . Iwatsubo,Biochemistry , K5,325 9 (1976).
8 vI nee ndrog eherfs tka nee nkort e strengevorstperiod e dewarmtestr
9 ; Detectiegrenzenva n ion-selectieveelektrode n dienenbetrekkin g telliebbe n opd etotal econcentrati e vanhe tbetreffend e ione nnie t opd evrij e ionen- concentratie. Documentatieva nPhilips :Io n selective solid-state electrode for sulfide and silver,typ e IS55 0 S/Ag. Documentatie vanOrion :Guid e to specific ionelectrodes!an d instrumentation.
10 Hetprincipiel e verschil ind ebetekeni sva nhe twoor d trapezium inhe t "Amerikaans-Engels"e n inhe t "Oxford-Engels"ka ngemakkelij k leidento t begripsverwarring. The Concise Oxford Dictionary. Webster's New International Dictionary.
Proefschrift vanJ.F .XLeibeuker . Wageningen,me i1977 .
Voorwoord
Vijfjaa rgeleden ,toe ni knaa rWageninge nkwam ,wa s fotosynthese voormi jee nvolledi g onbekendproce se nwa s chlorophyll eenwa t grootuit - gevallenmolecuul . Zonderd emedewerkin gva nd evakgroe p Plantenfysiologisch Onderzoekbi jmij neerst ekennismakin gme td efotosynthes e end ekenni sva n mijnpromoto rm.b.t .d ebasi sstructuu rva nd e fotosynthetischepigmente n washe tonderzoek ,zoal sbeschreve n indi tproefschrift ,voo rmi jnie tmoge - lijkgeweest . Hetonderzoek ,da t isuitgevoer d ophe tLaboratoriu mvoo rMole - culaireFysic a teWageningen ,wa s alleenmogelij kdankzi jd emedewerkin gva n velenbinne ne nbuite ndez e afdeling.D evel eexperimente nwerde nmogelij k gemaaktdoo rhe tvoorbereiden dwer kva nHenn yva nBeek ,Wille mva nBerkel , dehee rJansen ,Adri ed eJager ,Simo nMaasland ,Jiller tSantem ae nHenn y Zoutendijk,waarvoo r ikaa nalie nmij noprecht edan kwi luitspreken . Bijd eexperimente ne nd edaaro pvolgend ediscussie she b ik altijdvee lsteu ngeha d aand ebelangstellin gva nall emedewerker sva nd e vakgroepMoleculair e Fysica.D emedewerkin gva nRoelo fPlatenkamp ,Pie tGeers e enAri eSonnevel d isonontbeerlij kgewees tvoo rd e afrondingva nhe tonder zoek. Inhe tbijzonde rwi l ikTjeer d Schaafsmabedanken .Hi jinteres - seerdemi jvoo rd e fotosynthese,motiveerd emi jvoo rhe t tripletonderzoe k enwa s tealie ntijd eberei dmi jme traa d endaa d ter zijdet estaan .D evel e discussiesme t jou,Tjeerd ,zij nd e grondslagva nhe tonderzoek ,da tme the t schrijvenva ndi tproefschrif ti safgerond . Tenslott ewi l ikmij nwaarderin guitspreke nvoo rd ewijz e waaropJann y Siebring,d ehee rHoogevee ne nd emedewerkster sva nd e afdeling Tekstverwerkinghebbe nmeegewerk taa nhe t totstan dkome nva ndi tmanuscript . Ikbe nd eNorth-Hollan d Publishing Companyerkentelij kvoo rd etoestemmin g tweei nChemica lPhysic sLetter s gepubliceerde artikelen indi tproefschrif t temoge nopnemen . Contents
List of abbreviations 11
Chapter 1 GENERAL INTRODUCTION 13
Chapter 2 STATIC TRIPLET STATE PARAMETERS 18 2.1 INTRODUCTION 18 2.2 THEORY 18 2.2.1 Electronspin ,it sfunction san doperator s 18 2.2.2Th ephysica l concepto fth etriple t state 23 2.2.3 Definitiono fstati c triplet stateparameter s 24 2.2.4Th etriple t stateparameter s inrelatio nt oth e electronic structure 25 2.3 METHODSO FMEASUREMEN T 26 2.3.1 Optical transitions 26 2.3.2Microwav e transitions 27 2.3.2.1 Zerofiel dmagneti c resonance 27 2.3.2.2Hig h fieldmagneti c resonance 27 2.4 REFERENCES 31
Chapter 3 KINETIC TRIPLET STATE PARAMETERS 32 3.1 INTRODUCTION 32 3.2 THEORY 32 3.2.1 Intersystem crossingprocesse s 32 3.2.2 Relaxation 35 3.2.3 Definitionso fzer ofiel dkineti cparameter s 36 3.2.4 Relationbetwee nhig h fieldan dzer o field kineticparameter s 36 3.3 OPTICALELECTRO N SPIN POLARIZATION 38 3.3.1 Signsan dmagnitude so ftriple tES Rpeak s 38 3.3.2 Electronspi n polarizationpattern s 39 3.3.3 Electronspi n polarizationrati o 40 3.4 TIME DEPENDENT HIGH FIELDELECTRO N SPINRESONANC E 42 3.4.1 Principles 42 3.4.2 Mathematical analysis 43 3.4.3 Analogue electronic simulation 46 3.5 REFERENCES 48 Chapter4 THE ELECTRONIC STRUCTURE OF CHLOROPHYLLS AND MODEL SYSTEMS 49 4.1 INTRODUCTION 49 4.2SPECTROSCOPI CCHARACTERIZATIO N 49 4.3THEORETICA LCALCULATION S 50 4.3.1 Freeelectro nmode l 50 4.3.2Th e4-orbita lmode l 51 4.3.3Mor eextensiv ecalculation s 51 4.4REFERENCE S 52
Chapter5 A SPECTROSCOPIC STUDY OF Mg-TETRABENZOPORPHIN I. EXCITED SINGLET STATE PROPERTIES 54 5.1 INTRODUCTION 54 5.2EXPERIMENTA L 55 5.3LIGATIO NO FTH EMAGNESIU MIO N 56 5.4EXCITE DSTAT EPROPERTIE SO FMgTB P 59 5.5DISCUSSIO N 61 5.5.1Effect so fligatio no nelectroni cdistributio n 61 5.5.2Effect so fmolecula rstructur eo nth eequilibriu m constantfo rbiligatio n 65 5.5.3Jahn-Telle reffect s 67
5.5.4S 2-fluorescenc eo fMgTB P 71 5.6CONCLUSION S 72 5.7ACKNOWLEDGEMEN T 72 5.8REFERENCE S 73
Chapter 6 THE TRIPLET STATE OF MgTBP 75 6.1 INTRODUCTION 75 6.2RESULT S 75 6.3DISCUSSIO N 77 6.4REFERENCE S 79
Chapter7 SPIN POLARIZATION IN THE LOWEST TRIPLET STATE OF CHLOROPHYLL 80 7.1 INTRODUCTION 80 7.2EXPERIMENTA L 80 7.3SPI NPOLARIZATIO NWIT HCONTINUOU SILLUMINATIO N 81 7.4TRANSIEN TSPI NPOLARIZATIO N 83 Acknowledgementan dReference s 86 Chapter8 OPTICALLY INDUCED ELECTRON SPIN POLARIZATION IN THE TRIPLET STATE OF CHLOROPHYLL AND ITS MODEL COMPOUNDS 87 8.1 INTRODUCTION 87 8.2EXPERIMENTA L 88 8.3RESULT S 88 8.4 DISCUSSION 90 Acknowledgement and References 91
Chapter 9 THE TRIPLET STATE OF PHOTOSYNTHETIC PIGMENTS I. PHEOPHYTINS 92 9.1 INTRODUCTION 92 9.2MATERIAL SAN DMETHOD S 94 9.3RESULT S 94 9.4DISCUSSIO N 98 9.4.1 ZFSparameter s 98 9.4.2Electro nspi npolarizatio n 104 9.4.3 Kineticparameter s 105 9.5CONCLUSION S 113 9.6REFERENCE S 113
Chapter1 0 PROPERTIES OF THE LOWEST EXCITED TRIPLET STATE OF CHLOROPHYLL 115 10.1INTRODUCTIO N 115 10.2PHOSPHORESCENC EO FCHLOROPHYLL S 115 10.3ZF SPARAMETER SO FCHLOROPHYLL S 116 10.4TRIPLE TSTAT EKINETIC SO FCHLOROPHYLL S 117 10.5CONCENTRATIO NDEPENDENC EO FTRIPLE TSTAT EPARAMETER S 118 10.6SOLVATIO NO FCHLOROPHYLL S 118 10.7GENERA LDISCUSSIO N 120 10.8REFERENCE S 127
Appendices 129 APPENDIXA 129 APPENDIXB 130
Summary 132
Samenvatting 133 10 List of abbreviations
AO atomic orbital Bchl bacteriochlorophylla Bph bacteriopheophytina Chia chlorophylla Chib chlorophyllb CI configuration interaction CNDO completeneglec to fdifferentia l overlap ESP electron spinpolarizatio n ESR electronspi nresonanc e FC Franck-Condon FEMR fluorescencedetecte dmagneti c resonance FE free electron HOMO highest occupiedmolecula r orbital IEH iterative extendedHiicke l ISC intersystem crossing LCAO linear combinationo fatomi c orbitals LuTO lowestunoccupie dmolecula r orbital (Mg)TBP (magnesium) tetrabenzoporphin MO molecular orbital MTHF 2-methyltetrahydrofuran Pha pheophytina Phb pheophytinb PMMA. polymethylmethacrylate PPP Pople,Parise ran dPar r Py pyridine ligand SCF selfconsisten t field SLR spinlattic e relaxation SOC spinorbi t coupling tol/pyr toluene-pyridinemixtur e(9:1 ) TPP tetraphenylporphin TPC tetraphenylchlorin ZFS zerofiel d splitting
11 1 General introduction
Theproces s inwhic h lightenerg y isconverte d intochemica l energy byplants ,alga ean dbacteri ai scalle d photosynthesis. Thephotosynthesi s canb edivide d inthre ekind so fprocesses ,whic har eessentiall y different: (i)absorptio no flight ,transpor to fexcitatio nenerg yan dinductio no fa charge separationusin gth eexcitatio n energy,togethe r formingth eso-calle d primary events; (ii)transpor to fcharg e alonga chai no fdifferen t intermediate compounds; (iii)conversio no fth eelectro-chemica l energy,store d inth eseparate d charges,int ochemica l energy. Inthi s thesisw ewil l limitourselve s toth eprocesse s described under(i) ; recentreview s coveringth eothe rprocesse sca nb efoun d elsewhere |1|.
Thephotosyntheti c pigments,especiall y thechlorophylls ,bein g thesubjec to fthi s investigation,ar eessentia l forth eprimar yphoto - synthetic events.I nvie wo fthei r role,th evariou spigmen t systemsar e divided inthre eclasses : (i)ligh tharvestin g (bulk,antenna )pigments ,responsibl e forth eabsorptio n oflight ; (ii)pigment swhic har eabl et ochanne l excitation energy fromth eligh t harvestingpigment st oth ereactio ncenter ; (iii)reactio ncente rpigments ,participatin g inth efirs tste po fcharg e separation. Inplant san dalga echlorophyl la (Chia )i sth emos t important pigmenti nal lthre eclasses ,wherea s chlorophyllb (Chib )participate s mainly inth eproces so fligh tharvesting . Inphotosyntheti cbacteria , probablybacteriochlorophyl l a (Bchl)i sth emos t importantpigment ;i n greenbacteri achlorobiu mchlorophyl lprovide s theligh tharvestin g facility. Sinceth edifferentiatio no fpigment s-i nvie wo fthei r rolei sno tonl ya resulto fdifference s inmolecula r structure,i tha st ob eattribute dt o different surroundings.Th eenvironmenta l effectsca nb eunderstoo db y considering themolecula r structurea sgive ni nfig .1 .
13 C0CH3
C2H5
(a) (b)
Fig. 1 (a)Molecula r structure of chlorophylls;X =CH ,fo r Chi a, X =CH O for Chib . (b)Molecula r structure of bacteriochlorophyll a.
Themai nskeleto no fth echlorophyll s isa porphyrin-lik erin g system. InCh ia an dCh ib on epyrrol erin gi shydrogenate d (ringIV )an di n Bchlrin g IVan drin gI Iar ehydrogenated .Al lchlorophyll s containa M g - ioni nth ecente ro fth ering ,whic hca nb ereplace db ytw oprotons ,result ingi nth ecorrespondin gpheophytins .Th eir-electron sar edelocalize d over thetetrapyrrol emacrocycle ,excep tth ehydrogenate dbonds .Als o carbonyl groupsca nparticipat ei nthi sderealization ,e.g .th erin gV ket ogrou pi n allchlorophylls ,th ealdehyd egrou pi nrin g IIi nCh ib an dth eacety lgrou p inrin gI i nBchl .Th eir-electro ndistributio ni sresponsibl efo rman y relevantmolecula rproperties ,suc ha sth elowes texcitatio nenergie san dth e ionisationpotentials .Thi sdistributio nca nb eaffecte db yinteraction swit h thesurrounding s throughhydroge nbondin gwit hcarbony lgroup san dligatio n ofM g .Furthermore ,th epresenc eo felectro ndonatin ggroup ssuc ha sth e carbonyl groups,an da nelectro nacceptin ggroup ,th eM g -ion,i non e moleculeprovide sth epossibilit yt ofor mdimer san doligomers . Therol ean dth esurrounding so fchlorophyll s in-vivo |2|,a swel l asth epossibl estructur eo faggregate s in-vitro |3|hav ebee nstudie d extensively. Inthi s investigation,w ehav eaime da ta deepe r insightint o theelectroni cstructur eo fmonomeri cchlorophyll s insolution ,especiall y w.r.t.th elowes texcite dstates .Absorptio nan dfluorescenc espectr ao fth e differentchlorophylls ,bein grelate dt oth elowes texcite dsingle tstate so f
14 thesecompounds ,hav ebee ncollecte db y Goedheer |4|.I na recen t review, Weissha s correlated these spectrawit hquantu m chemical calculations|5| . Another excited state of interest forstudie s ofth e electronic structure isth e lowestexcite d triplet stateT^ .Th emai ndifferenc e between T„ and the lowestexcite d singlet state S..i s thepresenc e ofa total spin angularmomentu m S= 1i nT. ,wherea s forS. ,S = 0 .Tw o consequences of thesedifferen t S-valuesshoul db enoted : (i)th e triplet state ismetastabl e and (ii)magneti c resonancemeasurement s provide thepossibilit y tostud y the electrondistributio n inT „ |6|.Whe nw e started this investigation only littlewa sknow nabou t the lowest excited triplet state ofth echlorophylls . Triplet state lifetimes hadbee ndeduce d from triplet-triplet absorption spectroscopy |7|an d some tripletES R studiesha dbee nreported , considering only theso-calle d Am= 2transition s |8|. The firstA m = 1triple t ESR spectrum ofa chlorophyl lwa s reportedb y Lhoste |9|. This author measured the tripletES Rspectru m forCh ib inethanol ,usin gmodulate d optical excitation andphase-sensitiv e detection. Interest inchlorophyl l triplet states strongly increased when Duttonan dLeig h |10|reporte d aA m= 1triple t ESR spectrum ofBch l in bacterial reaction centers,whic h couldno tb e accounted forb y theory existing attha t time.Paralle l tomor e extensive studies of the in-vivo triplet states inbacteri a |11|,chloroplast s |12|an d algae |13|,result s havebee nreporte d on the tripletstat e ofvariou s chlorophylls in-vitro. Clarkee t al. |14|use d fluorescence detected zero-fieldmagneti c resonance for themeasuremen t ofth e zero-field splitting (ZFS)an d the triplet state kinetics ofCh ia ,Ch ib and therelate d Zn-compounds |15|,a swel l as of Bchl |11|.Thurnaue r et al. |16|deduce d the ZFSparameter s ofa serie s of chlorophylls by ESR,usin gmodulate d optical excitation andphas e sensitive detection. Similarmeasurement s havebee nreporte db y Levanone t al.|17| . Recently theenerg y levels ofth e lowestexcite d triplet state ofCh ia and b aswel l as therelate dpheophytin s invariou s solvents havebee n determined by Krasnovskii |18|usin g phosphorescence. Thebasi c skeletono fth ephotosyntheti c pigments,th eporphi n ring, hasbee nstudie d extensively |19|.A sha sbee nshow nb yWeis s |5|,th eknow ledgeo f theelectroni c structure ofporphyrin s isver y important forsuccess - fullstudie s ofchlorophyll ,sinc e the introduction ofsid e groups,a swel l as thehydrogenatio n ofpyrrol e rings canb e accounted fora s relatively small perturbations onth eelectroni c structure of theporphyri n ring. Invie w of theseconsideration s wehav e studied inadditio nt ophotosyntheti c pigments,
15 thespectroscopi cpropertie s ofMg-tetrabenzoporphi n (MgTBP),whic hha s the advantage ofbein g astabl e compoundwit hD. , symmetry. InChapte r 5i twil l be discussed that insom erespect sMgTB P isa goo dmode l compound forCh i a.
Apart froma scientifi c curiosity forth epropertie s of triplet states ofsuc h complex compounds as thechlorophylls ,th e triplet state study ofchlorophyll swa s initiatedhavin g threeaim s inmind : (i)t o find ananswe r toth equestio nwhethe r the triplet state isa n essential intermediate inphotosynthesis ; (ii)t ocharacteriz e the triplet stateo fvariou s chlorophylls and the effects ofsurrounding s onth e triplet stateproperties ,t ob e able tous e the triplet statea sa natura l internal label,probin g the in-vivo surround ingso f thephotosyntheti cpigments ; (iii)t ochec k currentquantu m chemical calculations onth e electronic structure ofchlorophyll sb y comparisono ftheoretica l resultswit h the experimental triplet stateparameters . Although the firstquestio n isth emos t interesting oilefo r biologists,thi s investigationdoe sno tyiel d anoutcome .Recentl y Parsone t al. |20|hav epropose d amechanis m forth eprimar y steps inbacteria lphoto synthesis inwhic h the triplet statedoe sno tparticipat e inth e charge separationwhe n thepathwa y forelectro n transport isunblocked . Several studieshav eprovide d indications that thetriple tstat e canac t asa "sink", draining excess excitationenerg y from thephotosyntheti c unitb y coupling tocarotenoi d triplets |21|. Theparameters ,characterizin g atriple t statean d the techniques for theirmeasuremen t arediscusse d inChapter s 2an d 3.Th emai nfeature s of theelectroni c structure ofporphyri n likemolecule s isbriefl y reviewed in Chapter 4,emphasizin g Gouterman's4-orbita lmode l |22|,whic h isuse d throughout this investigation for theexplanatio no f theeffec t ofmolecula r structure onth e lowestexcite d singlet and triplet states.Th e results of the investigations onMgTB P arepresente d inChapter s 5an d 6,wherea s the dataobtaine d forphotosyntheti c pigments arereporte d anddiscusse d in Chapters 7-10. Sinceon eo fth epossibl e interactions betweenth e chlorophylls 2+ 2+ and its surroundings isth e ligationo fM g ,pheophytin s lackingM g have been included inth e investigation (Chapter9) . Special attention isgive n toth e interactiono f thepigmen t moleculeswit h thesurroundings .Th e effects of ligationo nth e spectroscopic properties ofchlorophyll s isrelate d tothos e found forMgTB P (Chapter5) . 16 Hydrogenbondin gi sdiscusse di nChapter s9 an d1 0b ycomparin git seffec t withtha to fintroductio no fa naldehyd egrou pa si nCh ib .
REFERENCES
1.Bioenergetic so fphotosynthesis ,ed .Govindjee ,Ne wYork ,1975 . 2.W.W .Parson ,R.J .Cogdell ,Biochem.Biophys.Acta ,416 ,10 5(1975] . 3.A.D .Trifunac ,J.J .Katz ,J.Am.Chem.Soc. ,96 ,523 3(1974) . 4.J.C .Goedheer ,i n"Th echlorophylls" ,eds .L.P .Vernon ,G.R .Seely , NewYork ,1966 . 5.C .Weiss ,J.Mol.Spectrosc ,44 ,3 7(1972) . 6.S.P .McGlynn ,T .Azumi ,M .Kinoshita ,"Th emolecula rspectroscop yo f thetriple tstate" ,Englewoo dCliffs ,N.J. ,1969 . 7-G.R .Seely ,i n"Th echlorophylls" ,eds .L.P .Vernon ,G.R .Seely , NewYork ,1966 . 8-G.T .Rikhireva ,L.A .Sibel'dina ,Z.P .Gribova ,B.S .Marinov , L.P.Kayushin ,A.A .Krasnovskii ,Dokl.Akad .Nauk .SSSR ,Jjn ,148 5(1968) . 9-J.M .Lhoste ,Studi aBiophysica , VZ, 135(1968) . 10-P.L .Dutton ,J.S .Leigh ,M .Seibert ,Biochem.Biophys.Res.Commun. ,46 , 406 (1972). 11.R.H .Clarke ,R.E .Connors ,H.A .Frank ,Biochem.Biophys.Res.Commun. , 21,67 1 (1976). 12-A.J .Hoff ,J.H .va nde rWaals ,Biochem.Biophys.Acta ,4JT2 ,61 5(1976) . 13-S.J .va nde rBent ,T.J .Schaafsma ,J.C .Goedheer ,Biochem.Biophys.Res . Commun.,71_ ,114 7(1976) . 14-R.H .Clarke ,R.H .Hofeldt ,J.Chem.Phys. ,6 K 4582 (1974). 15-R.H .Clarke ,R.E .Connors ,T.J .Schaafsma ,J.F -KLeibeuker , R.J.Platenkamp ,J.Am.Chem.Soc. ,98 ,367 4 (1976). 16-M.C .Thurnauer ,J.J .Katz ,J.R .Norris ,Proc.Nat.Acad.Sci .USA ,72 , 3270(1975) . 17-H .Levanon ,A .Scherz ,Chem.Phys.Lett. ,31_ ,11 9(1975) . 18-A.A .Krasnovski iJr. ,N.N .Lebedev ,F.F .Litvin ,Dokl.Akad.Nau kSSSR , 216,140 6(1974) . 19-M .Gouterman ,i n"Excite dstate so fmatter" ,ed .C.W .Shoppee ,1973 . 20.W.W .Parson ,R.K .Clayton ,R.J .Cogdell ,Biochem.Biophys.Acta ,387 , 265 (1975). 21. R.J. Cogdell, W.W. Parson, M.A. Kerr, Biochem.Biophys.Acta, 430, 83 (1976) 22. M. Gouterman, J.Mol.Spectrosc, 6, 138 (1961). 17 2 Static triplet state parameters
2.1 INTRODUCTION
Thewavefunctio no fa nelectro ni na nato mca nb echaracterize d by fourquantu mnumbers ;thre eo fthe m (n,1 , m)defin eth espatia ldistri butiono fth eelectrons ,wherea sm definesth espi nstate .T ointerpre t splittingsi natomi c line spectra,Uhlenbec kan dGoudsmi t[ 1] introducedth e concepto fspin :fo relectrons ,havin g spinangula rmomentu m quantum number S= I, thespi nfunction s |a> an d| &> describ etw ostate swit hm =+1 , -\, respectively.I na nexterna lmagneti c field thesetw ostate shav e different energies,sinc eth espinnin g electronha sa magneti c moment v= gB S (where g^ 2.0 , isa dimensionles sconstant ,calle dth eelectro n g-value,8 = 9.27x10 J/Gaussi sth eBoh rmagneto n and3 i sth espi nangula r momentum), whichca nb eoriente di ntw oway sw.r.t .th eexterna lmagneti c field[2] . The existenceo fa nelectro n spini scrucia lfo runderstandin g theelectro ndistributio ni natom san dmolecule s becauseo fPauli' s rule[3] . Inaddition ,th eelectro n spinprovide s anothermean st ostud y this electron distributioni nfre eradical san dtriple t statesb yelectro n spin resonance (ESR).Th eenerg y differencebetwee nth etw ospi nstate s dependso nth e effectivemagneti c fielda tth esit eo fth eelectro nan di srelate dt oth e localsurrounding so fth eelectron s [2] . Aftera brie f introductiont ospi nfunction san dspi noperators , wewil ldiscus sth ephysica l concepto fth etriple t statean dth eparameter s characterizing this state.Th esecon dpar to fthi s chapteri sdevote dt o experimental techniquest omeasur e theseparameters .
2.2 THEORY
2.2.1 Electron spin, its functions and operators
The one electron spin functions |a > and |&> are eigenfunctions of the one electron spin operators S and S [2] .
18 2 h2 S^|ms> = S(S+1)h |ms> = I |ms >,
Sz |ms> = ms h |ms> =+ I h|ms >, where |m >denote s thespi n function,S an dm are the aforementioned quan tumnumbers .Th e operatorsS _an d S arerelate d to the spinangula r momentum operator S,S ^= S.S,S =S x +S +jv . The energy Eo fa nelectro nwit h spin S= | i na nexterna lmagneti c field H//z isgive nb y the Schrodinger equation for the spin function:
gBH z Sz |ms> =E |ms> withE =+ jgB H .Definin g theste poperator s S =S + iS , Ct, P 2 X Y S_"= S x - iS y itca nb e shown that
Because of thesenon-zer omatri x elements aradiofrequen t electromagnetic radiation field (rffield) ,wit h itsmagneti c fieldcomponen t H.. perpendicular to theexterna l fieldfi ca n induce transitionsbetwee n thespi nstates ,i f the frequency fits theenerg y difference.Thi s canb e seenwhe n considering the timedependen tperturbatio nhamiltonian ,resultin g from ther f field,
1 JC =g(5H rS (1)
Ife.g .H..//X ,the n
1 x + <
19 XS =72- (|a(D 6(2)) - |a(2)S(1 )> ) (2)
|a(1)aC2 ) > XT ={ 72- ( |o.(1)6(2 )> + |a(2)6(1 )> ) (3) |6(1)6(2 ) >
S Forth eantisymmetri c spinfunctio nx onefind s
SQ 2 xs -= n0 .
This corresponds toa spi nquantu mnumbe r S= 0 .Sinc eth emultiplicit y of thisstate ,give nb y (2S+1),equalsone ,thi s state iscalle d a singlet state. Foral lthre e symmetricspi nfunction s onefind s
S2 xT= 2h , corresponding toa spi nquantu mnumbe r S= 1an da multiplicity;(2S+1 )= 3 : thesethre estate s together form the triplet state.Usin g theeigenvalues , nu, ofth etriple tstat espi nfunction sw.r.t .th eoperato r S ,thes efunc tionsar enormall y denoteda s
|+1> = |a(1)a(2 )>, |- 1> = |B(1)6(2 )> and
|0> = TJ- (|a(1)6(2 )> - |a(2) 0(1) >).
A timedependen tperturbatio nhamiltonian ,relate d toth emag neticfiel dcomponen to fa nr ffield ,a sgive ni neqn . (1),ca ninduc eth e transitions |+1>+-*|0> and |-1>-*-» •| 0> ,becaus e thematri x elements + - + <+1|S |0>, <0|S"|+1 > ,<-1|S |0> ;an d<0|S |-1> arenon -zero ;thes e transitions followth eselectio nrul eA m =1 . s The aforementioned tripletspi nfunction s areonl ygoo d functions ina sufficientl y strong externalmagneti c field,whe nspin-spi n interactions canb eneglected .Th eful lspi nhamiltonia ni sgive nb y 2 SrS- 3(S .r)(S?.r) X= gB H(S -+ S ) + g V {-V ^ " —T^ > (4) r r
H isth eexterna lmagneti c field;S. .an dS ? define thetw oon eelectro nspi n
20 angularmomentu moperator san dr i sth evecto rconnectin gth eposition so f thetw oelectrons .Whe nH = 0 ,th ehamiltonia ngive ni neqn . (4),reduce st o thespin-spi n interactionhamiltonia n K .Thi shamiltonia nca nb ecas t into thefor m
2 2 2 =-XS -Y S -Z S Xss —x —y —z whereX ,Y an dZ ar eth eexpectatio nvalue so fth er dependen toperators ,
22 , r 2- 3x 2^ 22 r 2- 3y 2, 22 ,r 2- 3z 2^ ,,., X= g B (. 5— J, Y = g e ( •v ^-r J ,Z= g g I 7 j ) (5) r r r
(x,y an dz ar eth ecomponent so fr alon gth ethre emutua l perpendicular principal axeso fth ezerofiel d splitting tensor),usin gth eorbita lpar to f thetriple telectroni cwavefunctions .S , S andS areth e total (two electron)spi noperators .Th espi n functions
|TX> = 72- (ISO) (3(2)> - |a(1)a(2 )> ) (6a)
|Ty> = 72~ (|e(1)g(2 )> + |a(1)a(2 )> ) (6b)
|xz> = 72- (|a(1)3(2 )> + |a(2)B(1 )> ) (6c) areeigenfunction so f5 C withth eeigenvalue sX ,Y an dZ respectively . BecauseX + Y + Z = 0 ,th erelativ e energy levelso fth ethre e spinstate s canb edefine db yth ezerofiel d splitting (ZFS)parameter s
D= 1(X+ Y )- Z , E= -1( X-Y ) (7)
The spinfunction s |T> , | T >,an d| T> al lhav eth eeigen value2 h fo rtota lspi noperato rS 2.Furthermore ,i tfollow s fromth etrans formationpropertie so fangula rmomentum ,
S IT >= i IT > S2 IT >= h 2 IT> —z ' x ' y —z' x ' x 2 2 —Sz„ 'I Ty > = - i IT >' x S IT—z >' = yh IT> ' y
S|T>=0 S2|T>=0% —z ' z —z ' z
21 From this,i tca nb econclude d that IT >an d |T >ar en oeige n functions ofS ;so ,fo r'thes e functions m isno ta goo dquantu ' x m number.Fro mth e z s relationsS T > S T >= S T > 0,i tca nb econclude d that 1 X 1 Z -y 'y -z the spinmove s ina plan e perpendicular toth ex ,y an dz axe sfo r |T > [T >an d T >, respectively .Th emagneti c field componento fa r ffiel d canagai n induce transitions betweenth etriple t spinstates ,followin gth e perturbation hamiltonian giveni neqn . (1).Th enon-zer omatri x elementsar e now,however , ( T |S. .|T, _ „ ),.,
+1 > (|Vlx x > + 1 X >) (8a)
0> X > (8b) 1 Z
ix >) (8c) 1-1 > 1 X Iy whenth eexterna lmagneti c fieldi salon gth ez-axi so fth eZF Stensor .Th e aforementioned selection ruleA m =1 onl yhold swhe n |+1> , | 0>, an d|- 1 > areth eexac t functions,i.e .i na ninfinitel y strong field;i nth einter mediate case,whe n X mayno tb eneglecte d w.r.t.gBfl. S (eqn.4),|+ 1> , |0>, an d|- 1> ar eapproximat e functionsan dA m =2 transition sar eals o allowed. Infig .1 th eeffec to fa nexterna lmagneti c fieldo nth eenerg y levelso fth ethre e spinstate sfo rH//x ,iV/ yan dH// zi spresented .
Fig. 1.Magneti c field dependence of the energy levels of the three triplet states for the three canonical orientations H//x,y,z,wher e x,y,z are the principal axes of the ZFS tensor. |TX> , |T> , and|rz>are the zero-field states; |+1>, |0> and |-1> are thehigh-fiel d spin states.Arrow s indicate resonance field positions for constant rf-frequency.
22 2.2.2 The physical oonaept-of the triplet state.
The totalwavefunctio nt|i ,describin gth espatia ldistributio n ofth eelectron sa swel la sthei rspi n state,ca nb ewritte na sa produc t ofa functio n(j> ,onl ydependen to nth espatia l electronic coordinatesan da spinfunctio nx , ty= 4>-x-Du e toPauli' s rule[3 ], 4 o rx ha st ob eanti symmetricw.r.t .electro npermutation ,th eothe rbein g symmetric. Sincei n thefollowin gw econside r onlyth egroun d statean dth elowes texcite d states obtainedb yon eelectro npromotions ,th ediscussio nwil lb econfine dt oa twoelectro n- tw oorbita lproblem ,usin gth eM Oapproximatio n [3] .I nth e groundstate ,bot h electronsoccup yth elowes tM O (molecular orbital) tob ecombine dwit h xS resultingi na single tstate ,an da nantisymmetri con e 1A * (1,2)=7 J Ua(D*bC2)- *a(2)*b(1)), T tob ecombine dwit hon eo fth ex 's (eqns.3 o r8a-c )resultin gi na triple t state.Therefore ,w eca nconclud etha tth esingle tan dtriple texcite dstates , occupyingth esam eMO' sno tonl ydiffe ri nthei rspi n functionbu tals oi n spatialwavefunctio n 23 2 -3 state with a lifetime TT ^ 10 - 10 sec, as contrasted with the lifetime 0 -7 -10 of the lowest excited singlet state TQ ^10 - 10 sec. 2.2.3 Definition of static triplet state parameters In fig. 2 the energy levels relevant for triplet state spectro scopy are depicted. The static parameters characterizing a triplet state are: a) EL , energy level of the mean of the three triplet spin states w.r.t. io the ground state S and AEfS - T ), the energy level of T w.r.t. the & o oo o lowest excited singlet state S1. b) Dan d E, the ZFS parameters defining the energy separation between the three triplet spin states, resulting from spin-spin interaction. As can be derived from (6) and (7) 3z i EV<^- (10a) E = I 2K2,/ (10b) where< > denote s thatth eexpectatio nvalue ,usin g theorbita lpar to fth e wavefunction,ha st ob ecalculate db yintegratio nove rth espace ;r i sth e distancebetwee nth eunpaire d spins;x ,y an dz it scomponent s alongth e principal axeso fth eZF Stensor . e ES1H -sr >x>- -x 2E _Y E T0"| -Td -o u' Z>-L -Z OJ-Sr Fig. 2.Energ y level diagram representing the energy level of the lowest excited triplet state T w.r.t. the lowest excited singlet state Sj;an d the ground state SQ (lefthan d side of the diagram) and the zero-field splittings between the three triplet spin states |T> , |T> ,an d |T>(righ t hand side of the diagram). Note that the twovertica l scales (energy e)ar e different. For definition of X, Y, Z,D , and E see text. 24 2.2.4 The triplet state parameters in relation to the electronic structure Asshow ni nth elas tsections ,th estati c triplet statepara metersI L ,D an dE ar efull ydetermine db yth eorbita lpar to fth etota l wavefunction.I nthi s sectionth eeffect so fmolecula rstructur eo nthes e parameterswil lbriefl yb ediscussed . Inth eM Oapproximatio ntw ofactor sdetermin eth etriple tstat e energy levelE .Firstl yth eenerg y difference,A E ,,betwee nth eMO's , 1 3.—D occupiedb yth etw ounpaire d electrons;secondly ,th eexchang e integralK , giveni neqn . (9),s otha t ET =A E ,- K andE c =A E ,+ K ,wher e Ec irt a—D o-i a—D o* isth eenerg yo fth elowes texcite d singletstate .Th esimples tmode l des cribingth eelectroni cwavefunction s istha to fa particl ei na bo x[ 3] , especiallywhe nth eelectron sar edelocalize d overth emolecul eo ra tleas t * overa par to fth emolecul ea si nth ecas eo fth ei m states consideredi n thisstudy .Thi smode lpredict sa decreas eo fA E ,, whe nth emolecule ,an d consequentlyth esiz eo fth ebo xi sincreased .Th efacto r 1/r1? inth eex change intergral (eqn. 9)predict s alsoa decreas eo fK whe nth esiz eo fth e molecule,o rmor especificall yth eexten to fth eMO' soccupie db yth eun pairedelectrons ,i sincreased .Th epredicte ddecreas eo fE „ -E™ , =2 K i s confirmedb yexperimenta l resultsfo ra serie so faromati chydrocarbon s[ 4 ] Whenth esymmetr yo fth emolecul ei schange do rhetero-atom s (N,0 )ar eintro - duced}morerefine d calculations arenecessar yt ocorrelat e theoreticalan d experimental results.I nchapte r4 w ewil l givea noutlin eo fsom eapproxi mationsuse d inth ecalculation so nporphyrins . TheZF Sparameter sD an dE als odepen do nth einterelectroni c distancer..- ,a si sshow ni neqns . (10a)an d (10b). Inaddition ,D an dE dependo nth e shape ofth eelectro ndistribution ,a sca nb econclude d from thex ,y an dz dependence .Fo ra spherica l electrondistributio nD an dE bot h equal zero;molecule swit ha tleas ta 3-fol daxi so fsymmetr yhav eE = 0 [ 4] . TheZF Stenso ro fplana raromati chydrocarbon san dporphyrin sha s itsz-axi sparalle lt oth enorma lt oth emolecula rplan e[5 ];the neqn .(10a ) reducest o 2 ? 1 D= I gV<-3->. r 2 2 since< r > » 25 typeo fmolecule sa decreas eo fD whe nth eexten to fderealizatio no fth e unpaired electronsi sincreased .Experimenta l results,however ,indicat e thateffect so fa chang eo fshap eo fth eelectro ndistributio no nD ,du et o changedelectro nwavefunctions ,ca ndominat eth eeffec to freducin gth e electronderealizatio n (seee.g .chapte r9) . TheZF Sparamete rE ha sbee nofte nreferre dt oa sa measur eo f thedeviatio nfro m3-fol do rhighe r symmetry.Eve na qualitativ e consideration ofth eeffec to fmolecula r geometryo nthi sparamete ri sver ydifficult , sinceth evalu eo fE strongl ydepend so nth eorientatio no fth eZF Stenso r axesw.r.t .th ein-plan emolecula r axeso fa plana rmolecul e withtw onon - equivalent in-plane axes,suc ha si softe noccurrin g inporphyrins .Fo rthi s reason,wherea sE-value shav ebee nmeasure d forsevera lcompounds ,w ewil l limitou rdiscussion s toth eeffect so fsiz ean dshap eo fth eTr-electro n distributiono nth eZF Sparamete rD . 2.3 METHODS OF MEASUREMENT Inth efollowin g sectionsw edescrib eth edifferen t techniques formeasuremen to ftriple tstat eparameter srelevan tfo rthi sstudy .I n 2.3.1w egiv ea brie fdiscussio no fphosphorescenc e spectroscopy,wherea si n 2.3.2 and2.3. 3 zero-fieldan dhigh-fiel dmagneti c resonancear econsidered . 2.3.1 Optical transit-ions Becauseo fth eoptica lselectio nrul eA S= 0 [ 3] , absorptio n spectroscopy isno ta nappropiat e techniquet omeasur eth eenerg y levelo f thetriple tstate ,a si snormall ydon efo rsingle t states.Althoug h thedeca y T„ •*• Sfii sals ostrictl y forbidden,th erelativ e long lifetimeo fth etriple t statemake sdetectio no frelate d emission (phosphorescence) possible. Thedifficulties ,relate dt oth emeasuremen to fphosphorescence , whichi swea kw.r.t .fluorescenc e (S1 ->S n emission)ca nb eovercom eusin g thedifference sbetwee nth etw okind so fluminescence : a)E ~ 26 Both principles have been used in the home-built phosphorimeter and have been described before [6] . 2.3.2 Miorowave transitions 2.3.2.1 Zero-field magnetic resonance Withouta nexterna l fieldth espi nfunction so fth ethre e triplet statesar egive nb y |T >, |T >,an d |T > (eqns. 6a-c).Microwav e radiation withth eappropiat e frequency (v= 10 0MH z- 2 GH zfo rporphyrins )ca n in duce transitionsbetwee n these states,a sdiscusse d insectio n 2.2.1. The absenceo fsensitiv e detectors forth edirec t measurement ofsmal lmicrowav e absorptions favours theus eo findirec tdetection .I tha s beenpreviousl y shown that transitions betweentw oo fth ethre e triplet spin statesca ninduc ea chang ei nth ephosphorescenc e [7] and/or fluorescence [8] intensity.Especiall y thelatte rmetho dha screate dth epossibilit yt o measureth eZF Sparameter s forth ephotosyntheti c pigments,withou t anex ternal field,i nspit eo fth ever ywea kphosphorescenc e ofthes ecompounds . Tous ethes e techniques,however ,i ti snecessar y thatth erat eo fspin - latticerelaxatio n (SLR), inducing Boltzmanequilibriu m betweenth espi n states,i ssmall .Thi s conditionca nb efulfille d onlywhe nth eexperiment s arecarrie dou ta tver y lowtemperatur e (T= 1-1 0K) . Athighe r temperatures iti snecessar y tous ea nalternativ e technique,hig h field electron spin resonance (ESR). 2.3.2.2 High field magnetic resonance Ina nexterna lmagneti c fieldth espi n functions |x >, |T >, x y and |T >ar en olonge rth eeigenfunction s ofth espin-hamiltonian ,give n ineqn . (4).Whe nth ezero-fiel d splitting canb eneglecte dw.r.t .th eexterna l field,i.e .whe n g@H> >X ,Y ,Z ,an dH// za nappropriat e descriptiono fth e spini sgive nb yth ehigh-fiel dspi n functions |+1>, | 0> and|- 1> defined ineqns . (3)an d (8a-c).A sdiscusse d insectio n 2.2.1,transition s between spinstate s |+1> an d| 0> an dbetwee n |0> and|- 1> (Am =1 )ca nb ein ducedb ya nr ffield ,circularl y polarized inth eplan eperpendicula r toth e external magnetic field[ 2 ]. I nth eintermediat e case,whe nX ,Y ,an dZ canno t beneglected ,i nadditio nt oth eA m= 1 transitions ,th eso-calle dA m= 2 transition |+1> -*-» •|- 1> isals oallowed ,resultin g ina half-fiel dsignal . 27 Resonance conditions,a twhic h transitions canb e induced,ca n berealize d intw oways : a)variatio no f thefrequenc y of the rf field atconstan tmagneti c field; b)variatio no f theexterna lmagneti c fieldwit h aconstan t rf frequency. Because ofexperimenta l difficultieswit hmetho d (a) (seee.g . section 2.3.2.1), inpractic e onlymetho d (b)i suse d [9]. Thus,a nelectro n spinresonanc e (ESR)spectru m isobtaine d by sweeping themagneti c field,whil e keeping the rf frequency constant.Fo ra single molecule init s triplet state,thi s results inthre e transitions (oneA m = 2transitio n and twoA m = 1 transitions] atdifferen tmagneti c fields.A s long as allmolecule s have thesam eorien tation,a s canb e realized ina singl e crystal,onl y these three transitions willb e found.When ,however ,th emolecule s arerandoml y oriented, transitions will takeplac e over a range ofmagneti c field strengthvalues .Sinc eal l samples considered inthi s study containrandoml y orientedmolecules ,thi s problemwil lb e discussed inmor edetail . To find theresonanc e conditions asa functio no fth e orientation of themagneti c fieldw.r.t .th emolecula r frame,firstl y theeigenvalue s of thehamiltonian ,give nb y eqn. (4)hav e tob e calculated.Wit h spin functions diagonalizing thespin-spi n interactionhamiltonian ,th ehamiltonia nmatri x becomes -igBHcos e igBHsin e sin* -igBHsin e sin<(> -igBHsin e cos<|> Z where themagneti c field isdecompose d init scomponent s along theZF Stenso r sine cos? H H sine sinth eangl ebetwee n thecomponen t of H inth ex- y plane and ththex-axis .Th eprincipa lvalue s E1 ? ,o f thishamil - tonianmatri xar egive nb y 28 E3 -E {g2e2H2 - (XY+ Y Z +XZ) } +g 2g2H2 (Xsin 29 cos2* +Y sin2e sin2* +Z cos 2e) - XY Z =0 C11) Theresonanc econdition s are fulfilledwhe n thedifferenc ebetwee n tworoot s ofeqn . (11)equal s 6= hv ,wher eh isPlanck' s constant andv th er ffre quency.Thi s conditionlead s toth eresonanc eequation : g262H2 {Xsin 29 cos2(f> +Y sin29 sin2* +Z cos 29}= -3/2 7 7 7 7 7 =XY Z- 3 ' {(6^+ X Y +X Z+ YZ )- g 0H }x {-&*• - 4(XY+ Y Z +XZ )+ + 4g2B2H2P (12) Fig. 3arepresent s thedependenc e ofresonanc e fieldvalue s onth eorientatio n ofth emagneti c field. Itshoul db enote d that foreac h field orientation threeresonanc econdition s occur.T oge t theabsorptio nspectru ma sa functio n ofmagneti c field fora nensembl eo frandoml y orientedmolecules ,th espectr a ofmolecule swit h allpossibl e orientationshav e tob esuperimposed .Whe n the magneticfiel ddependenc e ofth e transitionprobabilit y isneglected ,thi s results ina spectru m asshow ni nfig .3b ;fig .3 cgive s thefirs tderivativ e ofthi sabsorptio nspectrum ,th efor m inwhic hES Rspectr aar enormall ypre sented. Itca nb eshow n[ 4] that themathematica ldiscontinuitie s inth e Am =1 par to fth eabsorptio nspectrum ,an dconsequentl y theobservabl epeak s inth efirs tderivativ e spectrum,occu ra t fieldpositio nwher e theresonanc e conditions are fulfilled formolecule swit h oneo fthei rZF Stenso raxe s oriented along themagneti c field: for HT,H// iwit h i= x,y,z ;H//x,y, zar e theso-calle d canonical orientations.Thes e fieldposition s are givenb y the equations, (gSFr;)2= (hv+ D JE ) (hv+ 2E), (13a) CgSH^)2 = (hv +D + E ) (hv+ 2E), (13b) (gBH^)2 = (hv+ D) 2 * E2, (13c) whereh v is ther fenerg y andD an dE ar e the ZFSparameters . 29 Fig. 3.Am =1 triple t ESR spec trumo fa nensembl eo frandoml y orientedmolecules , (a)Relatio n between resonance field valueH and orientationo fth eexterna l magnetic fieldw.r.t .ZF Stenso r axesx ,y ,an dz ;8 denote sth e angle between Han dth ez-axes , <(>th eangl e between thecompo nento fH i nth ex- yplan e with thex-axis .Full y drawn lines represent thedependenc eo fH o n 6 ata fixe d valueo f$ . (b) ESR absorption spectrum derived from (a)b ysummatio no fabsorptio n profiles foral lpossibl e orien tations, (c)Firs t derivative spectrumo f (b),correspondin g to experimental presentation. For (b)an d(c )th etransitio n probability istake n tob e independent ofH ;i naddition , Boltzman equilibrium betweenth e spin levels isassumed . Hz Hx K When |gSH|> > |D|,|E| ,eqns . (12a-c)ca nb ereduce dt o gB (Hz -H~ )= 2|D| , (14a) gS (Hx -H x)= |D|+ 3|E| , (14b) gB (H H) = Dl- 3| E (14c) y Eqns. (14a-c)ar euse dt oextrac tth evalue so fth eZF Sparameter sfro mth e tripletES Rspectr ameasure di nthi sinvestigation . 30 2.4 REFERENCES 1. G.E.Uhlenbeck ,S .Goudsmit ,Naturwiss. ,]_3 ,95 3(1925 ) 2. A.Carrington ,A.D .McLachlan ,Introductio nt omagneti cresonance , NewYork ,1967 . 3. H.Eyring ,J -Walter ,G.E .Kimball ,Quantu mChemistry ,Ne wYork ,1944 . 4. S.P.McGlynn ,T .Azumi ,M .Kinoshita ,Molecula rspectroscop yo fth e tripletstate ,Englewood sCliff sN.J. ,1969 . 5. W.G.va nDorp ,M .Soma , J.A.Kooter ,J.H .va nde rWaals ,Mol .Phys. , 28,155 1 (1974). 6. J.D.Winefordner ,P.A .St .John ,Anal- .Chem. ,42 ,63 9(1970) . 7. J.Schmidt ,J.H .va nde rWaals ,Chem .Phys .Lett. ,2 ,64 0(1968) . 8. C.B.Harris ,R.J .Hoover ,J .Chem .Phys. ,56 ,219 9 (1972). 9. C.P.Poole ,Electro nspi nresonance ,Ne wYork ,1967 . 31 3 Kinetic triplet state parameters 3.1 INTRODUCTION Oneo fth emos tstrikin gpropertie so fth elowes texcite dtriple t -3 2 stateT „i sit srelativel ylon glifetime :x ^ 1 0 -1 0 sec.Fro moptica l studies (phosphorescencean dtriplet-triple tabsorption )i tha sbee nfoun d thatT depend so nth emolecula rstructur ean dth emediu mi nwhic hth emole culesar eincorporate d[1 ,2 ] . Suc hoptica lstudies ,however ,onl yyiel d informationo nth emea ndeca yconstan tK „5 1/ 3I k. ,wher ei = x ,y ,z an d k.ar eth edeca yconstant sfo rth ethre etriple tspi nstate s |T >, |T >, and |T >. Usin gmagneti cresonanc etechnique si ti spossibl et omeasur eth e decayconstant sk .an dth erelativ epopulatin grate sP .fo reac ho fth ethre e spinstate sseparately ;th erelevan tparameter sar epresente di nfig .1 an d fig.2 .Apar tfro mth epopulatin gan ddepopulatin gprocess ,transition sbe tweenth ethre espi nlevel sinduce db yspi nlattic erelaxatio n (SLR)ar eo f importancefo rth ekinetic so fth etriple tstate .I nthi schapte rw ewil l givea theoretica lintroductio nt oth emechanisms ,whic hpla ya rol ei nth e populatingan ddepopulatin gprocesses .I nth esecon dpar texperimenta ltech niquesan dmethod sfo ranalysin gth edat awil lb ediscussed . 3.2 THEORY 3.2.1 Inter aystem crossing processes Transitionsbetwee nth esingle tan dtriple tmanifold s(inter - systemcrossing ,ISC )ca nb einduce db yspin-orbi tcouplin g (SOC).Th espin - spininteraction ,discusse di nsectio n2.2.1 , onlymixe sspi nstate swit h thesam equantu mnumbe r S, butwit hdifferen tquantu mnumbe r m; spin-orbi t interactionsca nmi xstate swit hdifferen tspi nmultiplicity ,a sfo rexampl e singletan dtriple tstate san dtherefor ei soperativ ei nradiativ ea swel la s non-radiativetransition sbetwee nsingle tan dtriple tstates .I nth efollowin g wewil ldiscus sth emai nfeature so fspin-orbi tinteractions ;furthermor eth e 32 SOCmatri xelement sfo rth ethre espi nlevel so fth elowes texcite d triplet statewil lb econsidere dan dthei rrelatio nt oIS Crate sfo rth evariou s spinlevel swil lb ementioned . Thespi nquantu mnumber sS an dm areonl y goodquantu mnumbers , whenth eelectron ,bearin g thespin ,i sabsolutel y freean dpossesse sn o orbitalangula rmomentum .Fo relectron shavin gbot hspin -an dorbita langula r momentum,onl yth etota langula rmomentu mJ = L + S i squantifie d[ 3] . If , however,th ecouplin gbetwee nspi nan dorbita langula rmomentu mi sweak ,th e spindescriptio nma yb eretained ;th eeffec to fSO Cca nb einclude db ya smalladmixtur eo fstate swit hothe rspi nmultiplicities .Th eeffec to fSO C onth ehamiltonia nca nb eunderstood ,considerin gth eenerg yresultin gfro m themotio no fa magneti cmomen t ina nelectri c field.Usually ,onl yth e interactiono fa nelectro nspi nS(i )wit hth eaverag e field,dependin go nth e positionan dvelocit yo felectro ni ,i sretained .Thi sresult si na SO C hamiltonian whereS(i )i sth espi nangula rmomentu moperato ran d£_ K(i)th eorbita langu larmomentu m operatoro felectro ni w.r.t .nucleu s K.Th esummatio nove rth e nucleiK ,instea do f integration overful lspac ei spermitte dbecaus eo f thestron gdependenc eo f y„ onth edistanc er. „betwee nelectro ni an dnucleu s r K: Yif(O -K [4]; Y K isdefine da sth eatomi cSO Cconstan tfo rnucleu sK . Thesingl enucleu s (atomic)SO Chamiltonia nca nb ewritte na s[ 5] = Y ^SO i!Ci)-S(i )= Y I (!x(i)-Sx(i)+ «. y(i).Sy(i)+ «_ z(i).Sz(i)) . Itha sbee nshow n[ 5]tha tth ex-componen to fthi sSO Chamiltonian , ^ = Yflz(i).Sx(i) cancoupl ea triple tstat ewit hspi nfunctio n |T >wit ha single tstate , whereasth eorbita lpar to fthi soperato r (8 (i)) simultaneously cancoupl e theatomi corbital s 2p and2 p. Analogousl y x£Q and 3cL~coupl e singlet stateswit hth etriple tspi nstate s |T >an d |x >,togethe rwit ha couplin g of2 p and2 p, an d2 p and2 p, respectively . Consideringth ematri xelement so fW ™ betweenth elowes texcite d 33 triplet statean d singlet states,< S FCQITQ >, i tshoul db enote d that only single electronpromotion s have tob e taken into account. Since only effectso fJfc no nth e twounpaire d electrons are important [6 ], th ewav e function ofthes e electrons has tob e considered for thecalculatio n of thematri x elements.Al lmolecule s tob e discussed inthi s studyhav e a inr lowest excited triplet state,s otha t themolecula r orbitals occupied byth etw ounpaire d electrons inT „onl y contain 2p atomicorbitals . Retaining onlyon e electron-one center integrals,a spropose d byMet z[ 7] , thespi nstate s |x > and |x > canb e coupledwit h singlet states;th e onecente r SOCoperato rJfL .couple s only 2p and 2p atomicorbitals . Singlet states,couple d toth e triplet spinstate s |T > and |x > by3cJ Lan d xL,, respectively,mus t contain 2p and 2p orbitals. ybusu * * * y x Thisrequiremen t isme tb y air, •ua andm r excited singlet states. Itha s beenshow nb yAntheuni s [8 ] andMet z[ 9 ] that themixin g of these states with T^ ismainl y responsible for the triplet populating anddepopulatin g processes foraromatic san daza-aromatics .Non-zer opopulatin g anddepopulatin g rates for the spinstat e |T > canb e attributed to SOCwit h singlet inr states, retaining the smallmulticente r integrals [8 ] or to second order effects: Taking intoaccoun t SOCbetwee n singlet and triplet air, ir a and/orn-r r states,als oJcL ,result s innon-zer o oneelectron-on e center integrals [8]. Which singlet states cancoupl ewit h the lowest excited triplet stateca nb e deduced fromgrou p theoretical arguments.When ,fo r example, the lowest excited triplet stateo fa molecul ewit hD- , symmetry,ha s orbital symmetry B~ ,th e symmetries ofth e totalwav e functions T„ ,T n and Tn (T0x=VlTxU>' etc -)ar e §iven^ B1ufo r T0x>A u£o rV ** B3u£o r 4- The symmetry ofeac ho fth etota lwav e functions isth eproduc t ofth esym metries ofth e orbital-an d corresponding spinfunctions .Sinc eJC „ istotal lysymmetric ,th ematri x elements< S |JC„n|Tn> arenon-zer o only for coupling ofT „ with a B1 singlet statean d similarly,fo rcouplin g ofT „ and Tn withA andB , singlet states.A and are antisymmetric w.r.t. reflection u 3u 6 u B, J. 1u^ ^ ' atth eplan e z=0an d soaris e from ua ,ai r orni r excitations;B , states, which aresymmetri cw.r.t .tha tplane ,aris e from TTW excitations,i nagree mentwit h thediscussio n givenbefore . Invie w ofth estud y ofth edeca y rates ofsom e photosynthetic pigments,w e discuss inChapte r 9 the effects ofelectroni c structure on ISCprocesses .Fo r themomen t themai nconclusio n from the foregoing 34 discussioni stha tth epopulatin gan ddepopulatin g ratesfo rth ethre espi n levelsar epredicte dt ob egenerall ydifferent ,becaus eo fdifferen t coupling of these levelst oth esingle tmanifold . Itshoul db enote d thatth esam e SOCmatri xelement s occuri nth erelation sfo rth epopulatin gan ddepopula tingprocesse so fth etriple t state [8],s otha ton eexpect s roughlyth esam e ratiosbetwee nth ethre edepopulatin g rate constants (k: k:k ) andbetwee n the threepopulatin g rates (P: P: P) o fth etriple tstate .Thi s is,becaus e theP.' san dk-' sar ebot hproportiona l toth esquar eo fth eSO Cmatri x elements[ 8 ] , P k S 2 i' i" l< nl*S0lToi^ - 3.2.2 Re taxation A thirdproces so fimportanc efo rth ekinetic so fa triple t statei sth espin-lattic e relaxation (SLR), inducing transitionsbetwee nth e threespi nlevels .A tleas t threemechanism s havebee npropose dfo rth ecoup ling,betwee nphonon so fth ematrix ,i nwhic hth emolecul e isembedded ,an dth e spinstates .Fo rparamagneti c ions,i tha sbee nshow ntha tth erelaxatio n occurspredominantl yvi ath eva nVlec kSO Cmechanis m[ 10] ,i nwhic hphono n modulationo fth ecrysta l field affectsth eorbita lmotio nan dconsequentl y theSO Co fth eunpaire d electrons.Th eSL Ri nth etriple t stateo faromatic s hasbee nattribute d toa modulatio no fth espin-spi n interactiono rth e hyperfine interactionbetwee nth eunpaire d spinsan dnucle ii nth emolecul e [10, 11] .T odecid ewhic ho fthes emechanism s isoperative ,th eSL Rha st o bemeasure d atdifferen t temperaturesan deffect so fsid egroup so risotop e substitutionhav et ob edetermine d[ 1 0] . I nthi s studyth emeasurement so f thekinetic shav ebee ncarrie dou ta ton etemperatur ean da naccurat edeter minationo fth erelaxatio nrate swa sno tpossible .Som e features,however , deserve attentionan dcomment : (1)Th erelaxatio nbetwee nth ezero-fiel d spinlevel s |T >, | x >,an d |T >differ sfo reac ho fth epossibl etransi tionst1 2] . (2 )Wit ha nexterna l fieldth erelaxatio nrate sdepen do nth e orientationo fth emagneti c fieldw.r.t .th eaxe so fth eZF Stenso r[11 ,1 3] . (3)I nzero-fiel da swel la si nhigh-fiel d therelaxatio ni sstrongl y temperature-dependent [11, 12]. 35 3.2.S Definitions of zero-field kinetic parameters The population ofa stat ei sdefine da sth enumbe ro fmolecule s intha tstate ;N „i sth epopulatio no f SQ,N. .o fS, ,N xo fTg x,N ofT -, andN ofT „ .Th e populating rate P-i sdefine da sth enumbe ro fmolecule s enteringth estat eT n-pe rsecon d [i= x ,y ,z ). Th e populating rate constant p.i sth erati oo fP .an dth epopulatio no fth eleve lfro mwher eth emole cules,enterin gT n.,originate .Th etransition sS -»-S ,an dT •+T nar emuc h fastertha nth eIS Crate s O 10 vs< 1 0 sec. ).Therefor eal lthre e tripletspi nstate sar eassume dt ob epopulate dfro mS 1.Fo rth emolecule s tob ediscusse di nthi sinvestigation ,thi sassumptio nha sbee nprove nt o becorrect :optica lexcitatio nint oS 2result si nth esam erati oo fpopula tingrate sa sexcitatio nint oS 1.Thus ,th epopulatin grat econstant sar e givenb yp .= P-/N- ,( i= x ,y , z).Wit hth emethod suse di nthi sstud yN. . cannotb edetermined ;onl yth erati oP :P: P =p :p: p canb emeasured . j\. y 2/ A. y Zi The depopulating rates K.ar edefine da sth enumbe ro fmolecule s leavinga stat epe rsecond ,an dth edepopulatin grat econstant sk .ar ede finedb yk .= K-/N- .A swil lb eshow nbelow ,ou rexperimenta lmethod syiel d directlyth eabsolut evalue sfo rk. . Inth ecas etha t |T >ha sa highe renerg ytha n |T >, w is defineda sth eSL Rrat econstan tfo rth etransitio n |T >- >| T >.Relaxatio n isa therma lproces sresultin gi na Boltzma nequilibriu mbetwee npopulation s ofth espi nstates ,whe nth eP-' s andk.' sca nb eneglecte dw.r.t .th eSL R rateconstants .Consequently ,th eSL Rrat econstan tfo rth etransitio n |TX> + -|r y> , w isgive nb y -AE/kT w =e .w saw, yx xy xy' where AE is the energy separation between the two spin states |T > and |T >. The other relaxation rate constants are defined in a similar way. In fig. 1 the zero-fieId kinetic parameters for a triplet state are depicted. 3.2.4 Relation between high-field and zero-field kinetic para meters In a strong external magnetic field ft (i.e. ggH >> X, Y, Z in eqn. (4) in Chapter 2) |T >, |T >, and |T >ar e no good functions for the spin ->• / z states. With HHz the functions are given by |+1 > = 1//2 (|T > + i|T >) , 36 10> = IT >,an d1- 1> = -yi - (IT.> - il x >)(se esectio n 2.2.1).Th eSO C matrixelement sdiscusse di nsectio n3.2. 1 are theonl yspi nstat edependen t factorso fth epopulatin g anddepopulatin g rateconstant s[8 ]s othat , P+l,k+1 (0 iKsn|*S0|T0x> •i = 5( k2K +k ) , P^ = | ( P 2+VP ) J K,+1 x y' 1+1 x y (2) Furthermore,fo rth eothe rrat econstant son e finds, k P p (3) -1 +r -1" +r 0 k and ?n z 0 WhenH// xo rH// y similarrelation shold .Th ezero-fiel d spinstat erelate d toth eaxis ,whic hi sparalle lwit hH ,remain spure ,wherea s theothe rtw o zero-field spinstate sar emixed ,resultin gi nth e |+1> an d|- 1> high-fiel d spinstates . Whereas therelation sbetwee n the zero-fieldk' san dP' san d theirhigh-fiel d counterpartsar ei nfirs torde r independento fth estrengt h ofth emagneti c field[ 1 4] - a slon ga sge H» X,Y ,Z - ,th eSL Ri sexpec tedt ob estrongl ydependen to nth emagnitud eo fH ,becaus eo fit sdependenc e onth eenerg yseparatio nbetwee nth espi nstates .Becaus e therei sn otheor y forth edependenc eo fth eSL Ro nmolecula r structure,relate d spinstates , and theenerg y gapbetwee nth espi nstates ,i ti sno tpossibl et odeduc ea relationbetwee n zero-field SLRrat econstant san dthos ea thigh-field . Thehigh-fiel dkineti cparameter s fora triple tstat ear ede - -Px Fig. 1 Zero-field triplet statekinetics . k. and P. denote the depopulating rate l*> constant and the populating rate of the spin level IT.)( i= x,y,z). W.. is the SLR l*/^ Wzx rate constant for the transition r IT.) ~*I T.>• y> i J W;x z w-zy l*z> 37 Fig. 2 High-field triplet statekinet ics,k and P are the depopulating rate ••> constant and populating rate for the spin levels I+ 1> an d I-1> k andP k,/W, •> pictedi nfig .2 .Th erati obetwee nth erelaxatio n rateconstant sfo ru pan d downtransition s isagai ngive nb yth eBoltzma n factor a= ex p (-AE/kT), assuming thatth erelaxatio nbetwee n |+1> an d| 0> an dbetwee n |-1> an d |0> i sequal ,independen to fth edifferenc ei nenerg y separationbetwee n |+1> an d| 0> an dbetwee n |-1> an d| 0> . 3.3 OPTICAL ELECTRON SPIN POLARIZATION 3.3.1 Signs and magnitudes of triplet ESR peaks InChapte r2 onl yth eposition so fth epeak s ina Am= l triplet ESR spectrumhav ebee nconsidered .A slon gas ,unde r continuous optical excitations,Boltzma n equilibriumbetwee nth ethre espi n levelsexists , thepopulatio ndifferenc ebetwee nth elevel s |+1> an d| 0> an dbetwee n |0> and |-1> i sequa lan da r ffiel dwil l inducemor e transitions |+1> •* -| 0> and |0>-s -|- 1> tha ni nrevers edirection ,becaus eo fth elarge r population of |0> w.r.t . |+1> an do f|- 1> w.r.t . |0>, respectively ,resultin gi na nearly equalne tabsorptio no fenerg yfo rbot h transitions.A spectru m obeying thesecondition s iscalle da normal tripletES Rspectru man di sshow ni nfig . 3c,Chapte r2 . Inth eabsenc eo fBoltzma n equilibrium,th esig nan damplitud e ofth epeak s dependo nth erelativ epopulatio no fth espi n levelsan dthu s onth ekineti cparameters .I nth efollowin gw ewil l firstlydiscus sth ecas e thatth eSL Rma yb eneglecte d w.r.t.th epopulatin gan ddepopulatin grates ; next,w ewil l considerth eintermediat e case,whe nSL Rma yn olonge rb e neglected,bu ti sno tfas t enought oinduc ea Boltzma nequilibriu mbetwee n 38 thespi nstates . 3.3.2 Electron spin polarization patterns WhenSL Rca nb efull yneglected ,th epopulatio no fth espi n statesfo rft//z ar egive nb y P+ P + P p p N = N = 1 xN y = and HN N = = o z +1 -1F T= TT-n F+T1= T TT-n^ x 0= FT y F F0 F 0 z (seesectio n3.2.3) .If ,fo rexample ,N „> N +1 ,transition sinduce dbetwee n |0> an d|+1 >wil lresul ti nabsorptio no fenerg yan dth etransition s |-1 >-*-* |0> wil l result in emission.I nth efollowing ,absorptiv ean demissiv e charactero ftransition swil lb edenote db yth esymbol sA an dE,respectively . Becauseth eZ —peak si na A m= 1 triple tES Rspectru mo fa nensembl eo f randomlyoriente dmolecule sar eassociate dwit hth etransition so fmolecule s withH// z(se eChapte r2) ,fro mth esig no fthes epeak sth erelativ epopu lationo f |0> an d |+^1> ca nb ededuced : ifZ ~i sA an d 1 isE ,the nN L> N (ft//z); + ifZ ~i sE an dZ i sA ,the nN + 1 >N Q (Ify/z). Notingtha tfo rH// zH zi srelate dt oth etransition s |+1> - w |0> an dfo r H//x,y H andH correspondt oth etransition s |0> -*-* •|- 1> (seefig .2 , x y _>. Chapter 2),i tca nb eshow ntha tth efollowin grelation shol dfo rH// xo r ify/y: + + ifX" ,Y "i sA an dX ,Y isE ,the nN Q + + ifX" ,Y ~i sE an dX ,Y isA ,the nN +1 Itshoul db enote dtha tth epeak sH 7an dH. ,relate dt omolecule swit hH// i (i= x ,y ,z )alway shav ea nopposit echaracte r (i.e.A vs .E )an dequa l magnitude,a slon ga sSL Rma yb eneglected . Therelativ epopulation so f |0> and |+1> fo rth ethre e canonicalorientation sar eno tindependen to feac hother .A sa result ,no tal l electronspi npolarizatio n (ESP)pattern s (apatter ni nwhic hth echaracte r 39 Table1 PossibleES Ppatterns ,whe nmono-molecula r kinetic processes,a s defined insection s 3.2.3,4, areactiv e only,an dth ecorrespondin g relative spin statepopulations .H- rdenote sth epea k positionan d Ean dA correspon d toemissiv ean dabsorptiv e charactero fth e peaks;N .i sth epopulatio no fth ezero-fiel d spin state |T. >. _ _ _ + + + H H H H H H relativespi nstat e populations X y y X z E E E A A A N ,N >N x y z A A A E E E N ,N 3.3.3 Electron spin polarisation ratio Inth eintermediat e case,whe nSL Ri sno tfas tenoug ht oin duceBoltzma nequilibrium ,bu tth eSL Rrate sma yno tb eneglecte dw.r.t .th e populatingan ddepopulatin g rates,th epopulatio no fth ethre e tripletspi n levelsi nth ehigh-fiel d limiti sgoverne db yth efollowin gequations , dN. +1 P +N (-k - W -W )+ N aW +N_ 2aW , (4a) ~at~ 1 +1 1 1 2 Q 1 1 2 dN„ " =P +M W +N f-k -a W -W 1+ N aW (4b) 1RT 0 +11 0l 0 1 1J -1 T dN -1 -^- =P 1 +N +1W2 +N 0W.,+ N_ 1 (-k1- aW 1 -aW 2). (4c) Thekineti c constants,P. ,k .an dW ar edefine d insectio n 3.2.2,fig .2 .Th e 40 steady statecondition ,obtaine dunde r continuous illumination, isgive nb y dN ., dNn dN, -air -ai- 'itir =° (5) The amplitudes ofth epeak s foron eo fth ethre e canonical orientations arerelate d to thespi nleve lpopulation s forthi s orientation by + S (0 NQ -N +1, S" (:) N_1 -N Q (6) Introductiono feqns . (5)an d (6)i neqns . (4a-c)yiel d asimpl e relation for theES Prati o R: D -S + -S ~ _P 0k1 "P 1k0 ,_. where 6= 1- a = 1- exp(-AE/kT )^ AE/kT .Furthermore ,i ti s assumed that 2 W =W 1 =V\L ;i nth ederivatio n ofeqn . (7)6 hasbee nneglecte dw.r.t .6 andk .. ha sbee nneglecte dw.r.t .W . Itshoul db enote d thatth esam e result isobtaine dwhe n therelation s forA N =N -N .an d AN =N 1 -N_ ,deduce d byWinsco m[ 1 5] areuse d tocalculat e R. Itfollow s fromthi s derivation that iti sno tnecessar y to setW - =W_ ,sinc eW ~ iseliminate d from there lationwhe nk +.= k_ 1 =k -an dP +1 =P_ = P..[ 1 5] . InChapter s 7an d 8th eus e ofeqn . (7)t oth e Am= 1triple t ESRspectr awil l bediscussed .Her ew ewil l give somecomments .ES Pwil l occuronl ywhe npopulatin g and/or depopulatingprocesse s are anisotropic (L i'k.,P ./ P.) . As discussed insectio n 3.2.1 therati obetwee n the three populating rates andbetwee nth ethre edepopulatin g rate constants isexpecte d tob enearl y equal,s otha t (PQk1 -P-kJ/f P + 2PJ « IC.Thu snon-zer o ESP ratioswil l occur only if 6W< K„ . 6an dW are temperature dependent;wherea s S (:) 1/T,th etemperatur e dependence ofW isassume d tob egive nb y 5 9 W(:)T -T [11],s o thatwit h increasing temperature 5Wwil l increase. ESPca nb e obtained atrelativel y high temperatures (T> 100K )whe n IC,i s relatively fast (asfo rBchl ,IC ,^ 200 0 sec" )o rwhe nW isrelativel y slow asfo rpolyme rmatrice s (seeChapte r8) . Finallyw enot e that,whe nn o full ESPoccurs ,th eES P pattern canb ededuce d from therelativ e amplitudes, S(HT), ofcorrespondin gpeaks . Undercondition s that allpeak s areabsorptiv ebu tR f 0,th epeak swhic h are 41 emissivewhe nSL Rca nb eneglected ,wil lb esmalle r thanth e corresponding intrinsically absorptivepeak s (i.e.peak swhic h remainabsorptiv eunde ral l conditions) :i f S(H.)>S(H.),th e transitions atH . andH . are intrinsically A andE ,respectively . Therevers e istru ewhe n S(H.)>S(H.)- 3.4 TIME DEPENDENT HIGH-FIELD ELECTRON SPIN RESONANCE 3.4.1. Principles Many studies onth ekinetic s ofth etriple tstat ehav ebee n performedb y opticallydetecte d zero-fieldmagneti c resonance at 1.2 -4. 2 K [1 6] .Althoug h this techniqueha s greatadvantag e above thehigh-fiel d technique tob ediscusse d inthi ssection ,i nsom ecase s timedependen thigh - fieldES Ri sa mor eappropriat etechnique .Fo rexample ,whe n therelaxatio n rates cannotb eneglecte d evena tth elowes tpossibl e temperature,suc ha s hasbee nfoun d inglass y sampleso rbiologica lmaterial ,o rwhe nfo rtechni calreason smeasuremen t at 1.2 -4. 2 K isno tpossible .Furthermore ,wit h high-field ESR iti spossibl e tomeasur e thetemperatur edependenc e ofth e variouskineti c constants overa large temperaturerange . Thefollowin gpar tprovide s abrie f introduction totim ede pendent ESR. Inth enex tsection s themethod s foranalysin g thesignal swil l bediscussed . Forsample swit h randomly orientedmolecules ,th esi xpeak s inth eA m= 1triple tES Rspectru m correspond to transitions ofmolecule s with oneo fth ecanonica lorientation s (seeChapte r 2). The timedependenc e ofth epopulatio no fth etriple t spinlevel s formolecule swit hH//x ,H// y orH// z canb edetermine d bymeasurin g thetime-dependen t amplitude ofth e ESRpeak ,S(t) ,a t H =H ~ ,H - or H~ ,usin g square-wave amplitudemodula tiono fth eexcitin g lightcreatin g tripletpopulation .W econside r in particular thebehaviou r ofS(t )immediatel y after theexcitin g light is turned ono roff .I nthi smanne rw e obtainth etime-dependenc e ofAN + =N n -N +1 andA N =N -- N „ for eacho fth ethre ecanonica l orientations after starting andstoppin g thepopulatin gproces s of thetriple tstate .W edenot e thetime-dependen tbehaviou r ofth eES Rpeak sa s transients. Fig.3 present s sometransient s forCh ia inMTH Fa t 100K . 42 ESR signal amplitude Fig. 3 Transients atH =H z, Hx and H~ in theA m= 1triple t ESR (arbitrary units) spectrum of Chi a inMTH F at 100K . The lower part of the figure repre sents the time dependence of the H= H z intensity (I)o f the exciting light. Experimental conditions:microwav e frequency 9.1 GHz,microwav e power 2mW , fieldmodulatio n frequency 100KHz ,fiel d modulation amplitude 16Gauss ,spectromete r RC-time 0.3 msec. 3.4.2 Mathematical analysis Thefirs tmathematica lanalysi so fth etransien tbehaviou ro f Am= 1 triple tES Rtransition so fmolecule swit hcanonica lorientation swa s givenb ySchwoere ran dSix l[ 1 4] .Thes eauthor sstarte dwit hthre ekineti c equations,comparabl et oeqns . (4a-c).Whil ethei ranalysi scoul daccoun tfo r theexperimenta lresult si na qualitativ eway ,a goo ddescriptio no fth e kineticso fth etriple tstat eca nb egive nonly ,i fth edifferentia lequatio n forth epopulatio no fth esingle tstate si stake nint oaccount ,a swa sdon e byLevano nan dVeg a[ 1 7] . Sinc eth eequilibriu mdistributio nwithi nth e singletmanifol di sestablishe do na muc hshorte rtim escal etha nbetwee nth e singletan dtriple tmanifolds ,al lsingle tlevel sca nb etake na sa singl e statewit htota lpopulatio nN- . Usingth eapproximatio nP - =P . =P. ,k +1 -1 W„ andk „. . < S (t)= sj , (1- X 1 +S* (1- e" X+'* ) (8a) u 11 e~ -- ) 9 12 43 X 1 1 S2i(t)= S ^ (1- e~ -' ) + s\2 (1- e"^- ) (8b) fortransient s afterswitchin g lighton ;fo rligh tof f (8a)an d (8b)hav et o bemodifie db yreplacin g (1- exp(- A .t))an d (1- exp(- A .t))b yexp(-X_.t ) andexp(-A +.t). Ineqns . (8a,b )S. .. an dS~ .denot eth etim edependen tA m= 1 tripletES Rsignals ,correspondin g toth etransition s |0> •*- *|- 1> an d |+1> +-» •| 0> , respectively ,fo rmolecule swit hH// i (i= x ,y , z).Th etime - independentconstant sar egive nb y C. hvc k..- k „ C. hvp k1 -k „ c1 c1 1 r 1 0.1 0i ,-„„-, whereas thedeca y constants aredefine db y A_ = Kp, A*= Kj,+ 3W1. (10) In eqns. (9a-c) C.i sa constant dependent onth eligh t intensity, themea n ISC rate andth eorientatio n ofth emolecule s w.r.t.H ;v £ isth er f frequency, h isPlanck' s constant andk g isBoltzman' s constant;k. .„ ,P. .„ an dW are the high-field kinetic triplet state parameters forH//i . Eqns. (9a-c)ca nb e rearranged,yieldin g s1 -s 1S* „ hv,hv,p 1 k1 - k1 = s21 b11 . 9W , (11a) K K S1 +S 1 1 o 511 b21 1 1 1 1 pi _ pi bS Kp+ 3W S -S hv *1 0 12 2hvE b11 E b21 3 (11b) 2P1 + P0 s1 +S 1 •3k7 • Kp S1 +s 1 b11 b21 b11 " V b21 Inprincipl e thevalue s forW andK p canb ederive d from thedeca y constants A andA +, sotha t combination ofeqns . (11a,b )fo reac h ofth ecanonica l 44 orientationswit h eqns. (2)an d (3), defining therelation sbetwee nhigh - fieldan d zero-fieldkineti c tripletstat eparameters ,yield s the zero-field kineticparameter s k ,k andk and P„(0 Pv(0 ?z- Inpractice ,however , thetim econstan t ofth eES Rspectromete r limits therang eo fmeasurabl e relaxationrates .Thi s canb e shown,considerin g theeffec to fa nRC-filte r onES Rtransients . Whena time-dependen tvoltag e S(t)i ssupplie d toa RC-filter, thefollowin grelatio nbetwee nS(t )an d thecharg eQ o nth ecapacito r (capacitanceC )applies : s(t)=RdjLW +CLCti; whereR isth eresistance .Th evoltag eove r thecapacitor ,give nb y S (t)= Q(t)/C,i sth emeasure dvoltage .Whe nS(t )i sgive nb y eqn. (8a)th esigna l aftera RC-filter canb eobtaine dusin g aLaplac e transformation,yieldin g s1(t)= 1 - 1 T 1 - 1. T 0- e *-' )+1 _ l\ x (1-e"V )- { + } (1 e ) " 1- A_. T 1- A +.T " W wheret isth etim econstan to f theRC-filter .Fo r theVaria nE- 6 ESRspectro meteruse d inthi sstud yT ha sbee n lowered toa valu eo f3.1 0 sec. With thistim econstan tan dth evalue s for X_ and A+ deduced fromanalogu esimula tionso fth etransient so f Chia i nMTHF ,on e finds A .x^ 0.2 ,A .T^ 2 0 and eqn. (12)ca nb e approximateda s 1 1 tA S (t)= S n d-e"*-- ) +S 12 (1- e" ), sotha tth esecon dexponentia l isdetermine db y the instrumental timecon stantan dno tb y A .Therefore ,relaxatio nrate s cannotb edetermined .Sinc e themathematica l analysisresult s inseve nrelation s for thethre ecanonica l orientations together,wherea s therear eeigh tunknow nparameter s (IC,,k / k , x z k/k , Pv/P, P/ P ,W ,W ^an dW ) iti simpossibl e toobtai nth eparameters , as longa sSL R istake nt ob e fully anisotropic (Wx f W f W ).A s canb e seenfro meqn . (12),th eRC-filte r affectsno tonl yth edeca yconstants ,bu t alsoth epre-exponentia l factors.Thi swil lb eparticularl y importantwhe n A_.T << 1n o longerapplies . 45 Themos t completemathematica l analysiso fth etriple tES R transientsha sbee ngive nb yWinsco m[15] . This authordi dno tus eth e approximationsW 1 =W- ,k +1= k_ 1 andP +1= P_ 1.Whe nth eapproximatio n k * =k 1 andP . . =P 1 ar eintroduce d inhi sresults ,i tfollow stha tW .i s eliminated fromth ekineti c equationsan dth eresul ti sth esam ea stha t obtainedb yLevano n[ 1 7]. These approximations arepermitte da slon ga sth e spinfunction s |+1> , | 0>an d |-1> ar eth epur ehigh-fiel dfunctions .Whe n the ZFSparameter sD an dE ar enon-zero ,th eexac trelatio nbetwee nhigh -an d zero-fieldspi nfunction s are,fo rexampl efo rH// x [1 8] 1 -J l iU - l r^ "-2S i y' "2S •> ' z I"1> = 72 -1 °+ -25T -> 2I V> " ^ - -l^-y K > 2 2 - i wherea = [ J( D+ E ) +(ggH )] 2. Using theseequation son efind s thefollo wingrelation s fork +1, v -v =~( D +E ^ fk -k 1 K+1 -1 2ggH lKy z> anda comparabl erelatio nfo rP +1.I nthi s investigation |D+ E |< 0.0 5c m sotha t( D+ E)/2gg H< 0.08 ; thus,i nfirs torde r thepropose d approximations are allowed. 3.4.3 Analogue electronic simulation Thekinetic so fa triple tstate ,consistin go fthre espi n levels,whic h arepopulate db yP. ,depopulate db yk .an d coupledt oeac h otherb yW ,ca nb esimulate db ya nelectroni cnetwork ,a sgive ni nfig .4 . Thethre ecapacitor s (C)correspon dt oth ethre espi nstates .Eac h capacitor canb e loaded bya nindependent ,adjustabl ecurren t source (P.)an ddeca y througha nadjustabl e resistor (k.)i nth efor mo fa nRC-circuit .Furthermore , theSL Ri ssimulate db yadjustabl eresistors ,connectin g thecapacitors . The differential equations,describin g thetim edependenc eo fth echarge sc mth e capacitorsar eidentica lt oeqns . (4a-c)fo rth ekinetic so fth etriple t state.Th echarg edifference sbetwee nth ecapacitor s correlateswit h thetime - dependentpopulatio ndifference sbetwee nth etriple tspi nstates .Thes e differences canb edetermine db ymeasurin g (V -V ) an d( V -V ), whereV . 46 Fig. 4 Electronic network used for analogue simulation of transients. The three indepen dently adjustable current sources (Pj 23) are drivenb y a square wave'modulated voltage V^.Th e charges on thecapaci tors Cj 2 3> corresponding to the spinstat epopulations ,ar e deduced from the voltages V| 2 3afte r correcting for the Boltzman factor (see text),k ^ andW are adjustable resistors corresponding to decay and SLR. isth evoltag eacros sth ecapacito rC. . Untilnow ,th ecapacitanc e ofth ecapacitor s isno tconsidered . Whenth ethre espi nlevel sshoul dhav eequa lenergy ,th ecapacitor s should havet ob eequal .Becaus eo fth esmal lenerg y separationsbetwee nth espi n levels,th ecapacitor shav et ob eslightl ydifferen tt ointroduc eth eBoltz manfactors .Sinc ew ear eintereste di nth echarg eo nth ecapacitors ,measure d voltageshav et ob ecorrecte dfo rth edifferenc e incapacitanc eo fth eC's . Inpractic eth eBoltzma nfactor sar ever y small (y 0.005)an di ti sno t feasiblet oselec tcapacitor swithi nsuc hnarro w limitso ftoleranc e that thissmal ldifferenc eca nb eaccounte d for.Th evoltag e correction,however , canb eadjuste ds othat ,unde r circumstances thata nES Rspectru mwithou t spin-polarizationwil lb efoun d (6W> > K~),th evoltag edifferenc eV. .- V .= V2 -V andequal s «> 0.005 {1/30^ +V 2 +V 3)h Althoughth eful leffec to f theBoltzma nfacto ro nth etransien tca nonl yb esimulated ,whe n sufficiently accuratecapacitor sar eused ,th eapproximatio ndescribe d aboveha sn olarg e effectso nth etransients ,sinc e therei sonl ya nappreciabl e effecto fth e Boltzmanfacto rwhe nth epopulatio ndifference sbetwee nth espi nlevel si s inth eorde ro fthi sfactor . Toderiv eth ekineti c tripletstat eparameter s fromth ehigh - field transients,th etransient sar esimulate db yth eabov edescribe delec tronicnetwork ,th espi nleve l |+1) correspondingt oleve l (1),[ 0> t o(2 ) and |-1> t o(3) .Sinc eth eexperimenta l transientsar emeasure d througha RC-filter,th esimulate d transients alsopas sa RC-filterbefor e theyar e visualizedo nascope .Varyin gP's ,k' s andW i nth eelectroni cnetwork , optimumagreemen ti ssough tbetwee nexperimenta lan dsimulate dtransients . Forthi spurpose ,bot htransient sar evisualize do nth escree no fa H P5480 B signalaverager . 47 Foreac ho fth etransient s there areman y sets ofvalue sfo r k's,P' san dW simulating theobserve d transient.Th enumbe r ofpossibilitie s arerestricted ,however ,b y thefollowin gconditions : a)Fo reac ho fth ecanonica l orientationsk (k+1)= k,(k_.. )an dP. .(P. )= P,(P i),wherea s thetransient s for the |+1> -*- *| 0> aswel l asfo rth e |0> •*-* |-1> transitionshav e tob e simulatedwit h a single set ofparameters . b)Th epopulatin g -an ddepopulatin gparameter s for thethre e canonical orientations arerelate d toeac hothe r asdiscusse d insectio n3.2.3 . Whenthes etw ocondition s are taken intoaccount ,fo ral l compoundsdiscusse d inthi sstud yonl y alimite d rangeo fvalue s foral l kineticparameter s isfound ;i nal lcase s thevariatio n ink' s andP' s was considerable smaller thanfo r therelaxatio nrates . 3.5 REFERENCES 1.M.P .Tsvirko ,K.N .Solovev ,A.T .Gradyushko ,S.S .Dvornikov ,Opt . Spectrosc,38 ,70 S (1975). 2. G.R. Seely,i n"Th echlorophylls" ,eds .L.P .Vernon ,G.R . Seely,Ne wYork , 1966. 3.H .Eyring ,J .Walter ,G.E .Kimball ,Quantu m Chemistry,Ne wYork ,1944 . 4.J.C . Slater,Quantu m theory ofatomi c structure,Vol .II ,London ,1960 . 5.S.P .McGlynn ,T .Azumi ,M .Kinoshita ,Molecula r spectroscopy ofth e tripletstate ,Englewood s CliffsN.J. ,1969 . 6.D.S .McClure ,J.Chem.Phys. ,V7_ »66 5 (1949). 7. F.Metz ,S .Friedrich ,G .Hohlneicher ,Chem.Phys.Lett. , }6, 353 (1972). 8.D.A .Antheunis ,J . Schmidt,J.H .va nde rWaals ,Mol.Phys. ,27 ,152 1 (1974);D.A .Antheunis ,thesis ,Universit y ofLeiden ,1974 . 9. F.Metz ,Chem.Phys.Lett. ,22 ,18 6 (1973). 10.J.P .Wolfe ,Chem.Phys.Lett. ,J_0 ,21 2 (1971). 11.M .Schwoerer ,U .Konzelman ,D .Klipper ,Chem.Phys.Lett. , V5_, 111 (1972). 12. D.A.Antheunis ,thesis ,Universit y ofLeiden ,1974 . 13.U .Eliav ,H . Levanon,Chem.Phys.Lett. ,36 ,37 7 (1975). 14.M .Schwoerer ,H . Sixl,Z.Naturforsch. ,24a ,95 2 (1969). 15.C.J .Winscom ,Z.Naturforsch. , 30a,57 1 (1975). 16.J . Schmidt,D.A . Antheunis,J.H .va nde rWaals ,Mol.Phys. ,22 ,1 (1971). 17.H .Levanon ,S .Vega ,J.Chem.Phys. , 61_,226 5 (1974). 18.J.H .va nde rWaals ,M.S .d eGroot ,i n"Th e tripletstate" ,ed . A.B. Zahlan,Cambridge ,1967 . 48 4 The electronic structure of chlorophylls and model systems 4.1 INTRODUCTION Whereas inChapter s 2an d3 th emethod san dtechniques , usedi nthi s investigation,hav ebee n introduced,i nthi sChapte rw ewil l considerth esubjec to fstudy :th eelectroni c structureo fphotosyntheti c pigments.Recen tX-ra y studies[ 1] hav eproduce d accurate information about theposition so fth enucle i inth epigment .Abou tth eelectro ndistribution , however,whic hi sver y importantfo rth efunctio no fth epigment s inphoto synthesis,muc h lessi sknown .Th eelectroni c structureo fchlorophyll san d somemode lcompound sha sbee n studiedb yspectroscopi cmethod sa swel la sb y quantum chemical calculations.Extensiv e information aboutth eresult so f suchstudie s isavailabl ei nth efor mo freview s [2]. Thi s Chapter gives only an introductiont oth emai n featureso fth eelectroni c structureo fporphyrin - likecompounds ,treate di nmor edetai li nChapter s 5,9 ,an d 10. 4.2 SPECTROSCOPIC CHARACTERIZATION Thevisible-nea r U.V. absorption spectrumo fa porphyrin - likecompoun d containstw omai n absorption regions:th eQ-band s (500- 90 0nm ) andth eB -o rSore tband s (350- 45 0nm) ,correspondin g toth etransition s S1 +•S n andS _-< -S n,respectively . Inmeta lporphyrin sth eexcite d singlet states S1 andS ~ar ebot h orbitallydegenerat e andsingl e Q(0-0)- andB(0-0) - bands occur.Fo rfre ebas eporphyrins ,a swel la sfo rdihydro -an dtetrahydro - porphyrins,th edegenerac y islifte dan dth eabsorptio nband sar esplit . Whereasth esplittin gbetwee nQ andQ (thetw ocomponent so fth eQ-band )i s relatively large,a ver y smallo rn osplittin go fth eSore tban d isfoun dfo r most systems [3] . For freebase -an dmetallo-porphyrin sth eextinctio nco efficiento fth eQ(0-0 )band si smuc h smaller thanfo rth eB(0-0 )band s(fo r -z 3 _i _1 Cu-porphin9.3x1 0 vs330x1 0 l.mol .cm [4]);th evibroni c componentso f theQ-band s haveth esam eo rlarge r intensity than Q(0-0).Whe non eo rtw o 49 pyrrole ringsar ehydrogenated ,th eextinctio n coefficients ofth eQ(0-0 ) bandsan dth eSore tban dar eo fth esam eorde ro fmagnitud e[ 3] A striking feature,foun d from optical studiesi sth efac t that absorption spectra exhibit only small changeswhe n sidegroup sar eintro ducedo nth etetrapyrrol e macrocycle.Eve nhydrogenatio n ofon eo rtw opyrrol e rings leaves thespectr abasicall y unchanged [5]. Fro m these observations, itca nb econclude d thatth echromophori cgroup ,whic hi sresponsibl efo r the spectroscopic properties ofporphyrin-lik ecompounds ,i sonl y slightly perturbedb yintroductio no fsid egroup so rreductio no fpyrrol e rings. Bearing thisi nmind ,w ewil l limitou rdiscussio n aboutth equantu m chemical calculations ofporphyrin-lik esystem st oa nintroductio nt otw osimpl emodels . 4.3 THEORETICAL CALCULATIONS 4.3.1 Free electron model Applyingth efre eelectro n (F.E.)mode lt oth eir-electron s offre ebas eporphin ,Simpso n[6 ] assumed that 18electron smov e alonga ring.Fro mth e2 6ir-electrons ,eigh tar esuppose d tob emor eo rles s localized: fouro nN-atom s bearing thefre ebas ehydrogen s andfou r forming localized C -C doublebond si ntw oopposit epyrrol e rings (forbasi c structurean d nomenclature C-atoms,se efig .1 ,Chapte r 5). Formetallo-porphyrins ,th e 18electron s areassume dt omov e alongth e16-membere d inner ring,wherea s theothe r eight ir-electronsfor m four localized C.- C .bond s[ 7] . P p The four lowestexcite d states inth efre e electron des criptionar ei nfirs t order degenerate.Thes e four statesca nb edivide di n two sets: S-wit h L =+ 9 (L isth eorbita l angularmomentum )an dS -wit h L =_ +1 .Whe nelectron-electro n interactions aretake nint o account,S - will have lowerenerg y thanS ~becaus eo fHund' s rule [6].Th eoptica l selection ruleA L =+ 1 predict s thatth etransitio n S-+ •S. .i sforbidde nan dS -+ •S ~ is allowed.Thre epoint s shouldb enoted : (i)th eF.E .theor y qualitatively explains theobserve d spectra; (ii)th erestrictio nt o1 8electron so nth e 16membere drin gi si nagreemen twit hth eobserve d insensitivity w.r.t. changesi nmolecula r structurea tth eoute rring ; (iii)wherea su pt ono wth e value L =9 ha sno tbee nreporte dfo ran yporphin ,relativel y highvalue so f Lz arefoun dfo rS.. :L =5.0fo rZn-porphi n[8' ], L =7.9fo rcytochrom ec [9]. SO 4.3.2 The 4-orbital model A general approach toobtai nth eelectroni cwavefunction sfo r molecules,i sformin gmolecula r orbitalsa sa linea r combinationo fatomi c orbitals[ 1 0]. Thi s approach (LCAO)i suse d inth esimpl e cyclic polyenemode l[1 1] ,a swel la si nHiicke l[ 1 2] an dPP Pcalculation s[ 1 3] . As shewnb yGouterma n[ 1 4] , th espectroscopi c properties andth eeffec to f sidegrou psubstitutio no nthes eproperties ,ca nb etreate dusin gth e two highestoccupie dMO' s (HOMO's)an dth etw olowes tunoccupie dMO' s (LUMO's) only (inD. , porphina 1 ,a „ ande ,e ,respectively) .I nth e4-orbita l model,th eexcite d states S.an dS ?,calculate d using configuration inter action (CI),ar esuppose d toconsis to fth epur e excited states,obtaine db y one-electronpromotion s betweenth etw oHOMO' san dtw oLUMO's .A further restrictiono fC Iresult s from symmetry considerations.Onl y stateswit hth e samepolarizatio nca nb emixed : la. e >wit h la,, e >an d[a , e > r '1 u gyx '2 u gxx '1 u gyy with |a_ e > (|a- e >denote sth eexcite d state obtainedb y aone - tM gxy IU gx electronpromotio n froma 1 toe ;th esubscrip t indicatesth epolarizatio n ofth erelate d transition).I nAppendi xA an dChapter s 5,9 an d10 ,th e effectso fchange si nmolecula r structureo nth eenerg y levelso fth etw o HOMO'San dtw oLUMO' sar ediscussed . Theenergie san delectro ndistributio no fth efou rMO' s arededuce d from calculations using all2 p AO'so fth eC -an dN-atom so fth e ring,wherea sal l2 6ir-electron sar etake n intoaccoun t[1 4] . A swil lb e discussed inth enex tsection ,extensiv e calculations show thatth erestrictio n toth e4-orbita l modeli sallowed ,becaus eo fth erelativ e isolationo fth e twoHOMO' san dtw oLUMO' sw.r.t .th eothe rMO' san dtha tth eTr-electro nde - localizationi smainl y restricted toth e16-membere drin g[ 1 5] . 4.3.3 Move extensive calculations Many calculationso nth eelectroni c structureo fporphyrin - likesystem s havebee nreported .Usin gCI-SCF-PP Pmethods ,Weis se tal .[ 1 3] havecalculate dth eenergies ,charg edistributio nan dbon dorder so fa serie s ofmolecule s related toporphin .Tw ofeature s shouldb enoted : (i)Restric tiono fC It oth e4-orbita l pure excited states results inlowes t excited stateenergies ,onl y slightly different from those obtainedwit h fullCI ; (ii)th eir-electro nbon d ordero fth eC -C .bon di srelativel y small,indi cating isolationo fth eC „- C .bon d ir-electronsw.r.t .th e1 6membere d inner fc> p 51 ring ir-electrons.Th eCI-SCF-PP Pmetho dha s alsobee napplie d tochlorophyl l [1 6] . Fro mthes ecalculation s itca nb e concluded that the4-orbita lmode l provides agoo ddescriptio nfo rth eQ-ban d inth echlorophylls ;fo rth eSore t bandmor erefine d calculations appeart ob enecessary . Whereas inth eaforementione d PPPcalculation s onlyth e ir-electronsar econsidered ,i nth eiterativ eextende dHiicke l (IEH)metho d [1 7] , th epee lelectro ntheor y[ 1 8] an dth eal lvalenc e electronCND O calculations n,a an dmetal-io nelectron sar eals o takenint oaccount . Whereas the IEHmetho dpredict s thepresenc e ofa nitroge n lonepai rM Obe tweenth eTr-electro nJC' s a. ande infre ebas eporphin ,th epee l electron theory andCNDO/ Scalculation s result ina norderin g ofenerg y levelsfo rth e two HOMO'S and twoLUMO' s comparable tothos epropose d inth e4-orbita lmodel ; thisi stru efo rporphin ,a swel l asfo rdihydro -an d tetrahydroporphin[ 1 9] . Thesecalculation s show againtha t thetw oHOMO' San d twoLUMO' s areclearl y separated fromal lothe rMO's .A s shownb yMaggiori ae t al.[1 9] ,especiall y theS 1 stateca nb e fairlywel ldescribe d inth e4-orbita lmodel :mor e than 95I o fthi sstat eoriginate s fromth e4-orbita lpur eexcite dstates . Recentab-initi o calculations [2 0] als oconfir m the assumptionsmad e inth e4-orbita lmodel .W e are therefore on safegroun d to use the4-orbita lmode l asa basi s for theinterpretatio n ofth eresult s of thisstudy . 4.4 REFERENCES 1.H.C .Chow ,R . Serlin,C.E .Strouse ,J.Am.Chem.Soc. ,97 ,723 0 (1975). 2.Bioenergetic so fPhotosynthesis ,ed .Govindjee ,Ne wYork ,1975 . 3.J.E . Falk,Porphyrin s andMetalloporphyrins ,Amsterdam ,1964 . 4.U .Eisner ,R.P .Linstead ,J.Chem.Soc , 1955,3749 . 5.M .Gouterman ,J.Chem.Phys. ,30 ,113 9 (1959). 6.W.T .Simpson ,J.Chem.Phys. , V7> 1218 (1949). 7.J.R . Piatt,i n"Radiatio nBiology" ,ed .A .Hollaender ,Ne wYork ,1956 . 8.G.W . Canters,G .Jansen ,M .Noort ,J.H .va nde rWaals ,J.Phys.Chem. ,80 , 2253, (1976). 9.J.C . Sutherland,M.P .Klein ,J.Chem.Phys. , 57,7 6 (1972). 10.H .Eyring ,J .Walter ,G.E .Kimball ,Quantu m Chemistry,Ne wYork ,1944 . 11.W .Moffit ,J.Chem.Phys. ,22 ,32 0 (1954). 12.H.C .Longuet-Higgins ,C.W .Rector ,J.R . Piatt,J.Chem.Phys. ,^8 , 1174 (1950). 52 13.C .Weiss ,H .Kobayashi ,M .Gouterman ,J.Mol.Spectrosc. , 16, 415 (1965). 14.M .Gouterman ,J.Mol.Spectrosc ,6 ,13 8 (1961). 15.K .Tomono ,K .Nishimato ,Buil.Chem.Soc.Japan ,49 ,117 9 (1976). 16.C .Weiss ,J.Mol.Spectrosc ,44 ,3 7 (1972). 17.M .Gouterman ,i n"Excite dstate so fmatter" ,ed .C.W .Shoppee ,1973 . 18.M .Sundbom ,Act aChem.Scand. ,27 ,131 7 (1968). 19.G.M .Maggioria ,L.J .Weimann ,Int.J.Quantu mChem. ,Quantu mBiolog ySymp . I, 179 (1974). 20.D .Spangler ,R .McKinney ,R.E .Christoffersen ,G.M .Maggioria , L.L.Shipman ,Chem.Phys.Lett. ,36 ,42 7 (1975). 53 5 Aspectroscopi c study of Mg-tetrabenzoporphin I. Excited singlet state properties Joop F. Kleibeuker, Tjeerd J. Schaafsma 5.1 INTRODUCTION Theparticipatio no fpigment s containingth eporphi n ring systemi na numbe ro fimportan tbiologica lprocesse sha sprovoke dth estud y ofthes ecompound sfo rquit esom e time[1,2] ;fig .1 arepresent sth ebasi c porphinstructure . (Throughout thispape r "porphin"indicate sth eporphi n macrocyclewit ha forma lcharg eo f-2 ; ifi ti sno texplicitl y stateother wise,n ointeractio no fa centra lmetal-io no rtw oproton swit hth erin gi s included).I nphotosyntheti cpigments ,suc ha schlorophyl la an db an d bacteriochlorophylls,on ean dtw opyrrol ering sar esaturated ,respectivel y [1] , whereasa Mg-io ni scoordinativel yboun dt oth efou rnitroge natoms ; thechlori nstructur egive ni nfig .1 bform sth ebasi so fchlorophyl la an d b. Thebenzoporphin sar enon-naturall y occurringporphyrin swit h oneo rmor ebenzoi d groups condensedwit hth epyrrol ering sa sshow nfo r tetrabenzoporphin (TBP)i nfig .1c .W ewil l showtha tth eperturbatio no fa porphinrin gb yhydrogenatio no fon eo rmor epyrrol ering sessentiall yha s thesam e effectsa sth eintroductio no fbenzoi d groups.A sWeis s[ 3 ] indicated,on ema yexpec tth esam econfiguratio n interaction (CI)patter n fordihydroporphi nan dTBP .Thi si son eo fth ereason swh yMgTBP ,whic hi sa relatively stablecompound ,ca nb econsidere da sa nattractiv emode lfo r chlorophyll[ 4 ] . The absorptionspectr ao fporphyrin s containtw otransition s inth evisibl eregion :on ea t^ 40 0n m (Soreto rB-band )an don ea t^ 60 0ru n (Q-band)[ 1 ].Fo rTB Pan ddihydroporphin sth eintensit y ratio Info-m/^Rfn-oi isconsiderabl y larger thanfo rporphyrin swit hunreduce dpyrrol ering san d withoutbenzoi dgroup s[ 1] • As shownb yStor me tal. [5] , fora serie so fporphyrins ,th e positiono fth eSore tban ddepend so nth ecoordinatio nnumbe ro fMg .Evan s etal.[6 ]reporte da chang eo fth epositio no fth eQ bandi nbacteriochloro - phyllan dchlorophyl la upo naddin gpyridin et oa toluen esolution ,whic h 54 Pig.l Molecular structures of porphin (a),dihydroporphi n (b)an d tetrabenzoporphin(c) . theseauthor s attributed to thecoordinatio n ofa nextr a ligand to thecen tralMg-ion .Th e coordination properties ofMgTB P are found tob e similar tothos e ofchlorophyll s rather thant othos e ofothe rMg-porphyrins . Qualitative considerations,base d onth e4-orbita lmode l ofGouterma n[7 ] canexplai nth e spectroscopic resemblance betweenhydroporphin s andbenzo - porphins,a swel l as thedifferenc ebetwee nMg-porphyrin s andMgTB Pwit h respect toth estabilit y constants of their bi-ligatedcomplexes . This study is aimed toanswe r threequestions : 1.Ho w good amode l compound isMgTB P for chlorphyll? 2.Ar e splittings,observe d inth e excited singlet states and the lowest tripletstate s ofmetallo-porphyrin s insoli d solution,cause db y the presence ofdifferentl y coordinated species orb y the lifting ofth e degeneracies ofenerg y levels ofon e and the samemolecule ? 3. Is itpossibl e tocorrelat e theexcite d statemolecula r structurewit h theobserved ,bu tunusual ,S fluorescence and theU.V . absorptions of MgTBP? The following abbreviations willb euse d inthi spaper : (Mg)TBP is (Magnesium)tetrabenzoporphin,MTH F is 2-methyltetrahydrofuran , tol/pyri sa mixtur e of toluene andpyridin e (9:1),Ch i a/b is chlorophyll a/b,Bch l isbacteriochlorophyl l a. 5.2 EXPERIMENTAL MgTBPuse d inthi sstud ywa s agif t from Dr.J . C. Goedheer. A purity-check wasmad eb y its absorption spectrum and thin layer chromato graphy.Th e compoundwa s found tob e sufficiently free from impurities for usewithou t furtherpurification .A small amount of anunknow n impurity, also reportedb y Bayemae t al.[8], absorbing at 455n m andemittin g at65 0nm , 55 didno tinterfer ewit hou rmeasurements .Al l solvents (ethanol,2-methyl - tetrahydrofuran (MTHF),pyridine ,toluen ean dmethanol )wer eo fhig hpurit y (proanalysis ) grade.A sjudge d from spectroscopic analysis,MgTB P solutions werestabl efo rman yweek supo nstorag ea t-1 8C ;n odegradatio nwa sfoun d duringmeasurements . Absorption spectrawer emeasure d usinga Cary-1 4 spectrometer, whereas aPerkin-Elme rMP F2a ,a swel l astw ohome-buil t fluorimeterswer e usedfo rfluorescenc emeasurements ;phosphorescenc e spectrawer e determined usinghome-buil t instruments. 5.3 LIGATION OF THE MAGNESIUM ION Empirically,i tha sbee n found thatth ecoordinatio n number ofM g inporphin-lik ecomplexe s is5 o r6 [5] , i.e.a tleas ton eextr a ligand iscoordinativel y boundt oth ecentra lmeta l ion,apar t fromit sfou r coordinationbond s toth eporphyri nnitrogens .I na complexin gmediu m like pyridine,a biligate d species,wit h six-coordinated Mg iseasil y formed. Fora serie s ofporphyrins ,wit hpyridin ea sligands ,Stor me tal.[5 ]foun d theequilibriu m constantK t ovar ybetwee n0.0 7an d0. Sl.mo l forth e equilibrium PL+ L -* • PL,,,wher eP = magnesiu m porphyrin,L = ligand . Such Fig.2Absorptio n spectra ofMgTB P inethano l at 293K (a),22 3K (b) and 123K (c).Th e spectra areno t corrected for cone, changes due to changes of specific volume of the solutionwit h temperature. 440 610 x(nm)— > 56 dataca nb eobtaine dbecaus eth eshap eand/o rpositio no fth eSoret-ban d dependo nth enumbe ro fligand san d- t oa les sexten t- o nth estrengt ho f themetal-ligan dinteraction . Inaceton e[ 4 ]an dethano l (fig.2a)th eSoret-ban d forMgTB P at29 3K exhibit sa splittin g intotw oclearl y separatedbands ,wherea si n tol/pyra singl enarro wban di sobserved ;i nMTH Fan di nmethano la broadening occurso nth eshor twavelengt h sideo fth emai nband . Wehav ecarrie dou ttitratio n experimentso nth eequilibriu m MgTBP.Py + Py % MgTBP.(Py), (1) (Py= pyridine )i nth efollowin gway :a smal lamoun to fMgTBP-pyridin e complex,obtaine db yevaporatin ga solutio no fMgTB Pi ntol/pyr ,wa sdis solved intoluene .B ythi sprocedure ,a minimu m ratio[ P y]/ [MgTB P]i nth e Fig. 3Absorptio n spectra ofMgTB P (•^2x 10~5M) in toluene pyridine mixtures: (a)MgTBP-pyridin e complex inpur e toluene,T = 293K (see text) (b)a s (a),bu twit h 8x 10_3M pyridi ne; (c)a s (a)bu twit h 0.23 M pyridine; insert: (d)MgTB P in tol/pyr (9:1)a t 293K (e)MgTB P in tol/pyr (9:1)a t 123K . Note differentwavelengt h scaleo f insert. 400 450 x(nm) 600 650 toluenesolutio ni sobtained ,befor estartin gth etitratio nexperiment .Th e roomtemperatur eabsorptio nspectru mo fthi ssolutio ncontain sa nunspli t Soretband a t42 9n m (fig.3a);additio no fpyridin ecause sa secon dban d centereda t44 0n mt oappea r (fig.3b) .Progressiv e titrationo fth e solutionreduce dth eintensit yo fth e42 9n mban dwit ha simultaneou sin creaseo fth eintensit yo fth e44 0n mband .Attributin g the42 9n man d44 0 nmband st oMgTBP.P yan dMgTBP.(Py) 7,respectively ,th etitratio ndat aca n -1 beanalyze di nterm so fth eequilibriu m (1),yieldin gK = 4 5+ 1 01 .mo l . Thevalu eo fK fo rMgTB Pi smuc h largertha nthos efo rothe rMg-porphyrin s 57 Table 1. Relationbetwee n refractive indexn an dth epositio no fth eQ (0-0 ) band (A) i nth eabsorptio n spectrumo fMgTB P Solvent n X(nm ) Methanol 1.33 625+1 Ethanol 1.36 627+1 MTHF 1.41 627+1 Toluene 1.50 630+1 Pyridine 1.51 632+1 [5] , bu to fth esam eorde ra sfo rchlorophyll s [6] . Additiono fpyridin eha s no effecto nth epositio no fth eQ-band ;it sshape ,however ,change s from roughly Gaussiant oflat-toppe d (fig. 3d). Asi sshow ni ntabl e2 ,th eSore tban d shiftswit h temperaturefo r allsolvents ,whic hhav ebee nstudied .Th ebroadenin go nth eshor twavelengt h sideo fth eSore tban di nMTH F solutiona swel la sth e42 2n mban d observed atroo m temperaturefo rth eethano l solution,disappear sa tlowe rtempera ture (seefig .2c) .Th elatte r effecti sa resul to fth etemperatur e For dependenceo fth eequilibriu mbetwee nMgTBP.P yan dMgTBP.(Py) 2- ethanol solutions,th e42 2n man d43 1n mband sar eassigne dt omono -an dbiligate d species,respectively .Th etemperatur e dependent shifto fth eSore tban do f biligated speciesresult s froma nincreas ei nth emetal-ligan d interaction strength (seesectio n 5.5.1).Th epositio no fth eQ-ban d istemperatur e independent.Fro mth erati oo foverlappin g absorptionband so fmono -an dbi ligatedMgTB Pi nethano lth eequilibriu m constantK i sfoun dt ob e^ 0. 1l.mo l andthu si smuc hsmalle r thanfo rpyridin e ligation.W eattribut eth e differencebetwee nth eequilibriu m constantsfo rbiligatio nwit h pyridine andethano lt odifferen tbasicitie so fN an d0 containin g ligands.Besides , theinequalit yo fth ebiligatio nequilibriu m constantsfo rethano lan d methanol (fromth esingl eSore tban di nth eabsorptio nspectru mo fMgTB Pi n methanoli tca nb econclude d thatMgTB Pi smostl ybiligate d inthi s solvent at29 3K )ca nb eascribe dt oth edifferenc ei nsteri chindranc ebetwee nbot h ligandsan dth eporphyri nmolecul e[ 9 , 10]: methanolha sbette racce st oth e Mg -iontha nth ebulkie rethanol . AsEvan se tal .[6 ]hav eshown ,th epositio no fth eQ-ban d ofBch l isstrongl ydependen to nth ecoordinatio nnumbe ro fth eMg-ion .Furthermore , anincreasin g ratiobetwee nbi -an dmonoligate d speciesca nb epredicte dt o 58 result ina neffectiv ere dshif to f theintensit ymaximu mo fthi s composite absorptionQ band.Fro mBch lspectr a (seeWeis s[3] )i ndifferen t solvents itca nb econclude d that X„increase s for theserie sdiethylether-acetone - -ethanol-pyridine,implyin g acorrespondin g increase inligand-bindin g strength.Employin g the intensityrati oo f thetw oSore tband s ofMgTB P in thesesolvent s[ 4] w e derived theorde rdiethylethe r 5.4 EXCITED STATE PROPERTIES OF MgTBP Thepositio no fth eQ-ban d inth eabsorptio nspectr ao fMgTB P ina serieso fsolvent s canb e related toth erefractiv e indexo fth e solvents [11] (Table 1). Theabsenc eo fan yshif ti nth eS 1 +-S n transitionwhe nth e monoligatedMgTB P complex isconverte d into thebiligate d speciesa sfoun d intitratio nexperiment swit hpyridin e (fig.3) ,indicate s thatth eQ-ban d forbot h species coincides.Thisi sconfirme db y theabsenc eo fan y temperature dependence ofth epositio no fthes eband s (fig.2 ,Tabl e2) . As isshow n infig .3d ,th eQ-ban d ofMgTB P intol/py rha sa flat - topped absorptionprofile ,whic h indicates thepresenc e ofa nunresolve d splitting,whic h isindee dpartiall y resolved at 123K . Inth e temperature rangestudie d (293K - 123K )th esplitting ,A = 120+20c m ,an d intensity ratioR o fbot hcomponent s areconstan t (fig.3e) ;a smal lvibroni cban d at 610n mha s thesam esplitting .Fo rsolution s inethano l andMTHF ,onl ya t lowertemperature s there isa n indicationo fa splitting .Fo r thesesolvents , thesplittin g issmalle r and isno tdetectabl e at 293K ,becaus eo fsever e linebroadening .Fro mspectr atake na t lowesttemperature s (123K forethano l _i and9 3K forMTHF )w eestimate d thesplittin g A= 60+2 0 cm ,wherea s the intensityrati oR ^ 1 . At 4.2 Kth efluorescenc e spectraar echaracterize db y sharpsingl e 0-0 bands.Fo rMgTB P intol/py r at29 3K w e observed awea k shoulder separated from themai nQ transitionb y^ 130c m ,o nth eshor twavelengt h sideo fth e0- 0 band.Th e fluorescence excitationspectru m ofMgTB P intol / pyr at7 7K show s acomparabl e splitting (A= 110+20cm ") fo r thedifferen t components ofth eQ-band , inagreemen twit h thesplittin gpreviousl y found inth eabsorptio nspectra . Whereasn oresolve d splitting isobserve d inth eSore tban d ofan y ofth esolutions ,whic hw e studied,th eSore tbandwidt h is largerfo rth e 59 Table2 . Temperaturedependenc eo fth epositio no fQ-band san dB-band si n absorptionspectr ao fMgTB Pi nvariou ssolvents . wavelength(nm ) at T(K): solvent band Shift3 293 223 173 123 ethanol BI 422 BII 43) 432 433 434 + Q 627 626 626 626 0 tol/pyr BII 441 442 443 445 + 630 630 631 0 Ql ) 633 QII > 636 636 636 0 MTHF BII 431 433 435 437 + Q 627 627 627 627 0 Shiftdenote sth esig no fshif tfo rdecreasin gtemperatur e Fordesignation sB , B ,Q ,an dQ TT,se etext . tol/pyrsolution stha nfo rth eMTH Fan dethano lsolution sa t12 3K ,290+2 0cm ' -1 and230+2 0c m ,respectively .A t29 3K th ebandwidt hi sth esam efo ral l solutions,370+3 0c m .W eexplai nthes eresult sb ya nunresolve dsplittin g ofth eSore tband ,tha ti slarge rfo rth etol/py rtha nfo rth eMTH Fan d ethanolsolutions ,i nagreemen twit hth eresul tfo rth eQ-band . Infig .4 w epresen tth eS.-fluorescenc eo fa 1 0 Msolutio no f MgTBPi ntol/py ra t29 3K .Thi sfluorescenc espectru mshow sth esam evibra tionalprogressio na sfoun dfo rth eSore tan dQ-band si nabsorption .Th e excitationspectru mo fthi sfluorescenc efull yagree swit hth eSore tregio n ofth eabsorptio nspectru man dth e S*-fluorescenceexcitatio nspectrum . Fig.4 S -*-S .absorptio nspectru m ( )an aS ." S emissionspectru m ( )o fa 2 x 10 Msolutio no f MgTBP intol/py ra t29 3K . Ifl. 400 , ,440 480 %X(nm) j, 60 Therelativ equantu m efficiency ofth eS„-fluorescenc ewit h respectt oth e SH-fluorescence,measure dwit h excitationi nth eB(0-1 )ban d is^ 0.01 . ' -1 Whereas theQ-ban dha sonl ya smal lStokes-shif t of< 5 0c m ,th eS- - fluorescence isred-shifte db y^ 20 0a n with respectt oth eabsorptio n maximum.S~-fluorescenc eha sbee nreporte d forZnTB Pi nn-octan e at29 3K b y Bayemae tal .[8 ]an dfo rZnTB Pan dCdTB Pi nvariou s solventsb yZalesski i [12],wherea s therecen tresult so fFieldin g[13 ] dono tconfir mthi scon clusion. 5.5 DISCUSSION 5.5.1 Effects of ligation on electronic distribution Duringth elas ttw odecade sman y theoretical studies aboutth e electronic structureo fporphyrin shav ebee npublishe d [1, 7, 14-17] .I nth e following discussionw ewil l interpretou rresult susin ga simpl e qualitative modelan dcompar e theseresult swit hpublishe d SCF-MOcalculations . As foundb yKobayash i[ 1 8], th einne r 16-memberedrin go fmetallo - porphyrinsca nb esuppose d tofor mth emai n7r-electro nconjugatio npath ; Kuhn[ 1 9] argue d that thisi sespeciall y truefo rmetallo-TBP's ,becaus eo f thepresenc eo fth ebenzoidgroups .Thi sviewpoin tha sbee nconfirme db y recentcalculation s carriedou tb yTomon oe tal .[ 20] . F6rth estud yo fth etw olowes texcite d states thefou rorbita l model,a spropose db yGouterma n[7 ],i sa goo d approximation.Onl yth etw o 02u(t>l) a|u(b2l Fig. 5 Electron distribution of the two HOMO's (a„ and a, ) and two LUMO's 2u lu (e and e )o fD, , porphin followingGouterman s 4-orbitalmodel . Thesiz e gy gx _ 4h v v 5 of thecircle s isproportiona l to theatomi c orbital coefficients;dotte d or closed circles indicate their sign. 61 e gy egx e e x e e x i gy g gy 9 -*- c2 !l ? cV a2u a1u ain b, 1 a a 2u 1u a2u bi I (a) (b) (c) (d) | Fig. 6 Energy diagram of the twoHOMO' s (a. and a, )an d theLUMO' s (e 2u lu gx and e ) ina 16-membered polyene ring (a),porphi n (b),TB P (c), an ddi - hydroporphin (d) (bjan d b resp. c and c )[ 1 ,7 ]. Th e diagrams only indicate relevant energy differences and shifts and dono t represent absolute energies. highestoccupie d andth etw o lowestunoccupie dmolecula r orbitals (inth e followingabbreviate da sHOM Oan d LUMO),a ., a , ande ,e ina syste m withD 4h symmetry,ar e consideredi nthi smodel .Followin g Gouterman[7 ], orbitaldesignation s forD 4han dlowe rsymmetr y arecorrelate db ya ? -«-»-b.. , alu*- *b 2, e ++ c^, e •*-+ c^. Thea lu orbitalha shighes t electron-density onC a atoms (fig. 5),wherea s thea 2 orbitalha s thehighes tdensit yo n N-atomsan dmethin ecarbon s (C.). Fora 16-membere dpolyen emacrocycl ewit hD , symmetry ,consistin g of1 6C— H fragments,th eorbital sa . and a_ aswel la se ande are 1u 2u gx gy pairwisedegenerat e (fig.6a )[ 1] . Th eintroductio no ffou rN-atom s inpor phinwil l stabilizeth ea 2u orbital,becaus eo fth ehig h electron-densityo f thismolecula r orbitala tth eN-positions .Th ebranchin ga tth eC -positions , presenti nporphin ,however ,stabilize s thea . orbital,whic hha shighes t electron-densityo nC . Neithe r substitutiono fN-atoms ,no rC -branchin g willremov e thedegenerac yo fth ee ande orbitalsi nth eporphi nskeleto n gx gy f f with D4h sy™ietry- In D4hporphi nth etw oorbital s a., anda , are foundt o be almostdegenerat e (fig.6b )[7 ],thu sth ezer oorde rexcite d states e a e a ande + a e a ar e de enerate g"* " 1u *-' g 1u^ g ' 2u ^ g 2u^ S resultingi ncon figurationinteractio n (CI),whic hmixe sstate swit h thesam epolarizatio ni n equalamounts .I tshoul db enote d thatth eoptica ltransition se -t-a „ and e a ar eDOt n gy 2u r,gxv* " il„u y-polarizedr ,wherea sth e transitionse +•a . ganyd e„1v u gx e *•a 2u arex-polarized .C Icause s thedegenerac yo fth eexcite dstate sS. . andS 2t ob eremove dan d resultsi na lo wS. .-* - SQ radiative transitionproba bility,becaus eo fcancellin go ftransitio nmoments ;fo rS _* S .th etransitio n probabilityi senhance d duet oadditio no fmoment s[ 7] . This agreeswit hth e 62 experimental spectra fore.g .Mg-porphi n[ 2 1] wit h aver ywea k Q(0-0)an d a strong B(0-0)transition . Reductiono fbranchin g ofth e7r-electro nsyste m atC willdesta bilize thea 1 orbital (fig.6c )wit hrespec t toporphin .Then ,equall y polarized transitions fore -«-a 1 ande +•a . arenon-degenerate ,suc h thatth eamoun t ofC I isdecreased ,resultin g innon-zer o S1•* - Sn transition probability.A C-branchin g reduction canb erealize d intw oways : a)reductio n ofth epyrrol e ringsb yhydrogenatio n ofth eC 0 -C „bonds ; P R b)participatio n ofth e Ca- C .bond s inconjugatio npath s other thanth e p P 16-memberedporphi n innerring . Thedihydroporphi n ring system and the opposite-tetrahydroporphin system,obtaine d afterhydrogenatio n ofon eo r twopyrrol e rings,ar e the basicstructure s forCh ia ,b andBchl ,respectively .Th e reductiono f1- 3 pyrrolering sresul t ina los s ofD. , symmetry and a lifting of thee -e degeneracy (fig.6d) .This ,i nturn ,lead s toa differen t amount ofC Ifo r they-polarize dstate s (c.« -b.. ,c -« -b_ )an d thex-polarize dstate s (c?•* -b. , c^b2)[7]. Inth ebenzoporphin s oneo rmor e CR -C bondsparticipat e inth e ir-electronconjugatio npath s ofa benzoidgroup . Introduction of lesstha n fourbenzoidgroup s lifts thedegenerac y ofth eorbital s e and e ,simila r toth esituatio n inhydroporphins .Comparin g thepur e excited states of the benzoporphins,a s calculated byMcHug h[ 1 6] , wit h those ofth ehydroporphins , whichw e deduced fromth eorbita l energies foundb y Gouterman[ 2 2] ,i ti s cleartha tth eperturbation s inth etw oserie sar eo fth esam enatur e (fig.7) ; Fig. 7 Energies of thepur e excited p PH PH4 PH6 states la1,_ e > ofporphi n (P), di- 2 PH8 1,2u gx,y f f i> hydroporphin (PH ),opposite-tetrahydro 40- -40 porphin (PH.)hexahydroporphi n (PHfi)an d 30- -30 octahydroporphin (PHfi)a s calculated by VO- -?() Gouterman[ 2 2] and the energies of these 10- -10 states forporphin ,monobenzoporphi n (MBP), opposite-dibenzoporphin (DBP), tribenzo- -0 porphin (TrBP)an d tetrabenzoporphin (TBP) as calculated byMcHug h[ 1 6] . Fo r porphin 30- -30 four energy levels coincide,fo rPH „an d o c 20- lli TBP theenerg y levels coincide pairwise. 10- -10 0- -0 p MBP DBP TrBP TBP 63 for thehydroporphin s theeffect s are larger thanfo r thebenzoporphins . Study ofth e electronic structure ofporphins ,dihydroporphin s and benzoporphins isnecessar y todecid ewhethe r MgTBP isa suitabl emode l com pound forprotochlorophyll ,a spropose d by Goedheer [4], or forchlorophyll . (Protochlorophyll is identical to thecorrespondin g chlorophyll except for the lacking ofa hydrogenate d pyrrole ring.)- Asw e justhav e shown,th e introduction ofbenzoidgroup sha s aver y similar effect on the electronic structure ashydrogenatio n ofC .- C bonds.Althoug h dihydroporphin has no 4-foldsymmetr y asTB Phas ,fig . 7demonstrate s that thedistanc e between they-polarize dpur e states |c-b1 > and |c_b~> isroughl y the samefo r these twocompounds ,which results ina comparabl e amount ofCI ,a snotice d already byWeis s[ 3] . A s aresult ,th e intensity ratio I[ Q (0,0)]/I[ B (0,0)] is expected tob eroughl y thesam e fordihydro - and tetrabenzoporphins,which is found for e.g. Zn-dihydroporphin and ZnTBP[ 2 2] . Fo r thex-polarize d transitions ofdihydroporphin ,however ,th epur e states |c.b-> and |c.b2> arenearl y degenerate as inporphin ,resultin g ina stron g B and aver y weak 0 transition,du e to 50/50 CI.Fo rsymmetr y reasons TBPha s equivalent x- andy-polarize d excited states.Th e small splitting of theQ-ban d inbi - ligatedMgTB Presult s from asplittin g between thee ande orbitals.Thi s splitting,however ,i ss osmal l thatw ema y safely assume thatx andy transitions stillhav e the sameamoun t ofCI ,resultin g inth esam e intensity forQ andQ bands. xx ^y A change inth eenerg y levels ofon eo f thetw oHOMO' S indihydro porphinha sdifferen t effects onQ andQ bandposition s and intensities, since CIha s differentmagnitud e forbot h transitions;fo rTBP ,th e situation isessentiall y different:here ,Q andQ behave identically. Theeffec t of ligationo fth ecentra lmeta l iono nspectra l changes for chlorophyll and MgTBPprovide s ademonstratio n ofth epreviou s statement.Coordinativ e binding ofa ligan dwit hM g inthes e compounds reduces theelectro n transfer from therin gN-atom s toM g ,causin g theN-atom s tob e less electro-negative. Thus theenerg y ofth ea „ orbital— and toa nappreciabl y less extent the energy of thee orbitals— is increased. ForMgTBP ,th erati ob/ a inth e wavefunctions |B > =ala . e > +b|a e > 1 x,y '2 u g 1'1 u g IQ > =ala , e > +bla - e > lxx,y '1 u g - '2 u g isrelativel y small,a scompare dwit hporphin ,becaus eo frestricte dCI . 64 Therefore,increasin g theenerg yo fth ea ~ orbitalwil lmainl y affectth e Soretban dan dt oa les s extentth eQ-band , inagreemen twit hou rexperimenta l results.Sinc eporphyrin swithou tbenzoidgroup so rsaturate dpyrrol e rings havenearl y 50/50mixin go fth epur e states |a, e >an d |a. e >,resul tingi na rati ob/ a^ 1,on eexpect s small changeso fth ea 2 orbital energy tohav e largeeffect so nth epositio nan dintensit yo fth eQ-ban d duet oa changei nCI .I nagreemen twit h thisprediction ,Stor me tal .[5 ]hav ere portedno tonl ya re dshif to fth eSore tband ,bu tals o appreciable intensity changeso fth eQ(0-0 )ban dfo rsevera lMg-porphyrins . Illustrating theconcep tdevelope dbefore ,y-transition s forchloro phylls (havingrelativ ewea kCI )ar efoun dt obehav e likeMgTBP ,wherea s changesi nthei rx-polarize dtransition s follow thosefo rporphin :whe n Chia an dBch lar etitrate dwit hpyridin eusin g toluenea sa solven t[ 6] , onlymino r effectso nth estron gQ bandwer e found,wherea s appreciable effectso nth eQ transitions areobserved ,i nagreemen twit hou rmodel . 5.5.2 Effects of molecular structure on the equilibrium constant for biligation Thevalu eo fth eequilibriu m constantfo requilibriu m between mono-an dbiligate d MgTBP species isexpecte d todepen do nelectroni ca s wella ssteri cpropertie so fth eporphyri n ringan dth eligand .A sa reduc tiono fC -branchin gwil lhav e onlymino r influenceso nth e total ir-electron- densityo nth eN-atoms ,i ti sno tlikel y thatth elarg edifference s between theequilibriu m constantso fMgTB Pan dchlorophyl l (10-45 1/mol)vs .thos e ofunperturbe dMg-porphyrin s (0,07-0,5 1/mol)result s froma differenc ei n theeffectiv e chargeo nM g .Wit h respect toth esteri chindranc e between theporphyri nrin gan da ligand ,MgTB Pan dchlorophyl l appeart ob emor e favourablefo rbiligatio no nM g thanunperturbe dMg-porphyrins . * Asa nexampl eo fth einfluenc eo fligatio no nspectra l characteristics,du e tochange si nC Iw erefe rt oa recen t reportb yKielma ne tal .[ 2 3] o n temperature dependentMCD .I nth eligh to fou rdiscussio nth eresult s reportedb ythes e authorbecom eclear :wherea sth eequilibriu m constants forth eformatio no fbiligate d Mg-porphyrin complexesi smuc h smaller than forMgTB P[ 5] , lowerin gth etemperatur efo rMg-porphi ni nEP Acause sa shif t ofth eequilibriu mbetwee nmono -an dbiligate d complexes.Sinc eMg-porphi n isi nth elimi to fstron g CI,thi s shiftwil l inducea considerabl e change inth eQ-ban d absorptionan dMC Dspectra . 65 Fig. 8 Diagrams of two conformationso f theMgTBP.(Py) . complex. The upper part represents the topview , the lower part a side view looking along the arrow shown below the top-view diagram. The corners of the square,representin g theporphi n skeleton,correspon d to thepositio n of themethin e carbons; the N-atoms are positioned half-way the sides of the square. a)Bot h pyridine ligands perpendicular to the porphin plane,wit h their planes parallel to the twoperpendicula r "valleys" 2+ of theruffle d porphyrin molecule.M g lies in theplan e containing the four N- atoms. ~ b)A s (a)bu twit hM g below theplan e containing the fourN-atoms . When considering theeffect so fsteri chindranc ebetwee nth epor phin corean dtw oaxia l ligands,Hoar d[2 4] demonstrate d thatth eruffle d conformation,wit hD_ ,symmetry ,o fth eporphi n coreprovide sa sterica l configuration favouringbiligation . Inth eruffle d bi-pyridine complex,bot h ligandsar elikel yt ohav e their longestN— C axisperpendicula r toth e porphinplane ,wherea s thenormal st oth epyridin eplane sar eexpecte d tob e approximately parallel toth ediagonal s connecting oppositemethin e carbons inth eporphi nplan e (seefig .8a) .Reductio no fth eir-electro ndensit yo n theC -C .bond sa sa resul to fth eintroductio no fbenzoidgroup s lowers CX p theenerg ybarrie rfo rdeformatio no fth eindividua lpyrrol e rings, resulting ina mor e flexible structure,tha ni nporphyrin s withnorma lbond-orde rfo r thepyrrol eC -C „bonds . (ForTB PWeis s[ 1 5] ha scalculate d aC -C „ bond ordero f0.38 6a scompare d to0.46 8fo rporphin) .Th eC— C bondi n benzene (1,39A )i ssomewha t larger thantha to fth eC -C bondi nunper turbedporphi n (1,35A) . Therefore,introductio no fa benzoldgrou pwil l lengthenth eC -C bondwhic h easily results ina D- ,rufflin g ofth epor phyrinskeleto nb yrotatin g thebenzoidgroup s outo fth emolecula rplane . Theseprediction s areconfirme db ymolecula rmodel so fth ecomplex .B ycon structing suchmodel sfo rchlorophyll ,on eals o learns that introductiono f apentano nrin gan dhydrogenatio n ofon eo rtw opyrrol e rings inducesa de formationo fth eplana rporphi n ring structure,simila r toth eD~ ,deformatio n proposedfo rMgTBP .Thi s observation isi nagreemen twit h results from X-ray studies[ 2 5] . These findingsal lpoin t intoth esam e direction:th erelativ e 66 largeequilibriu m constantfo rligatio no fMgTB Pca nb eexplaine db yinvokin g a loweringo fth eresistanc e againstdeformations ,necessar yt ogiv e steric accessabilityo fth ecentra lmeta lfo ra secon d ligand.Thi smode lca nals o beapplie dt ospectra lpropertie so fth ecomplex :a sa consequenc eo fth e ruffledstructure ,th ebenzoidgroup sar erotate dou to fth emolecula rplane , thereby increasingth eisolatio no fthes egroup sfro mth eporphi nir-electro n path.Thi si si nagreemen twit hth eobservatio no fbenzoi d absorptionsi n the48.00 0- 54.00 0c m region.Also ,th eisolatio no fth eporphi nir-electro n systemw.r.t .th ebenzoidgroup sma yoffe ra nexplanatio nfo rth erelativel y long triplet lifetime (T= 230^2 0msec )— ascompare dt oT ^ 7 0mse cfo r Mg-porphin[ 2 6] — :introductio no fisolate dbenzoidgroup s reducesth e numbero fC— H bonds effectivei nth eradiationles s transitionT ->S [2 7J . Inth efollowin gpape r (II)w ewil ldiscus si nmor e detailth epropertie so f the lowestexcite d tripletstat e[ 2 8] . 5.S.3 Jahn-Teller effects The temperature independenceo fth eintensity-rati oo fthe ?tw o componentso fth eQ-ban d inth eabsorptio nspectru mo fMgTB Pi ntol/pyr ,a s wella sth eabsenc eo fan ysplittin g inlo wtemperatur e fluorescencean d phosphorescencear eevidenc eo fa splittin go fth efirs t excited singlet state,th egroun d statebein gunaffected .Th etw oQ-ban d components cannot be accountedfo rb yth emono -an dbiligate d speciesi nthermodynami c equili brium,sinc ea chang eo ftemperatur ewoul d affectth erelativ e intensityo f both componentso fth eQ-band , contraryt oou rresults . Anticipatingth ediscussio ni nsectio n5.5.1 ,w enot e thatth e firstan dsecon dexcite dstate s S1 andS ~ar ebot h orbitallydegenerate .A Jahn-Teller couplingwil l separateth epotentia lwell so fth etw ocomponent s ofbot h states alongth enorma l coordinateQ o fth eJahn-Telle r activevi bration,bu tdoe sno tremov eth edegenerac ya sshow ni nfig .9 [ 2 9] . L ithi s diagram,th epotentia lwell so fth etw odegenerat e excited states $ and ij; havethei rminim aa tth enuclea r conformationsQ = Q „an dQ = -Q_,respective - iy- Inth eD. ,porphi nskeleto nther ear etw oJahn-Telle r active vibrationswit h b1 andb ? symmetry,respectively ,inducin g in-planed e- formations.A nexterna l field,e.g .a crystal-field ,wit hnon-equa l components alongth etw oprincipal ,mutuall yperpendicula r axesi nth emolecular 'plan e canremov eth edegenerac ybetwee nth etw odistorte d conformations.Fo ra b 67 (b) Fig. 9 Effect ofJahn-Telle r instability and an anisotropic field on the potential E (Q);Q is theJahn-Telle r activenorma l coordinate. In (a) ( )represent s E (Q)i n the absence ofvibroni c coupling, ( )i s pot E (Q) in thepresenc e ofvibroni c coupling; at Q_ and -Q.E (Q)ha s minima corresponding todistorte d conformationswit h electronic wavefunctions ip and IJJ; W is the stabilization energy of the distorted conformations ri E, J1 w.r.t. a square shapedmolecul e [2 9] . Th e distorted conformations for ab Jahn-Teller active vibration are shown.Dashe d lines through therectangle - shaped molecular conformations represent the locus ofmode s for <(i and I(J. b)Th e effect of an anisotropic field on the energies of the distorted con formations; A represents the energy difference between the two distorted conformations[2 1 ]. distorted conformation theprincipa l axespas s throughth eN-atoms ,wherea s fora b _ distorted conformationth emethin eC-atom s areo nth eprincipa l axes.Canter se tal .[2 1] foun dth eQ-ban do fZn-porphi n ina n-octan e single crystalt ob espli tb y10 9c m ,whic h theseauthor s ascribet oa Jahn-Teller instability inth epresenc eo fa crystal-field ; fromMC Dmeasure mentso nZn-porphi ni nEP AKielma ne tal .[ 2 3] obtaine da zero-fiel dsplit tingo fth elowes t excited stateA = 58+1 0c m . The observed splittingA o fth evisibl e absorptionban do fMgTB P intol/py ri sremarkabl e insevera l respects.Rapidl y fluctuating molecular fieldsi na liqui d solutioncanno t cause sucha splittin gno rca na Jahn - Tellerdistortio n involvingnon-degenerat e vibrations [21 ,3 0] . Eve ni nth e presenceo fdegenerat evibrations ,th evibrationles s lowest excited singlet level (E) canno tb espli t[ 3 0] (highervibrationa l levelsd osplit ,however , whendegenerat e vibrations interactwit hE electronic states).Thus,a resolved splitting,suc ha sobserve d inth epresenc eo fligands ,require sa well-defined crystal-fieId,arisin g froma fixe dmolecula r environment duringa tim ex ,suc h that x> >-r -, wher eA i sth eobserve d splitting; from 68 -12 theobserve dvalu eo fA i tca nb einferre d thatT > >1 0 sec. The temperature independenceo fth esplittin go fth eQ-ban do f MgTBPi ntol/py ri na temperatur erang e coveringbot hth eliqui dan dth e glassy state (transition temperature^ 15 0K )indicate s that this splitting iscause db ya nanisotropi c field arising froma well-define d molecular environment.Th eshor trang e surroundings ofth e16-membere dring ,whic hi s thechromophori cgrou p inMgTB P (seesectio n 5.5.1),i smad eu po ffou r benzoidrings andtw oaxia l ligands.Wherea sth ebenzoidring sar efixed ,boun d andunboun d ligandsca nexchang e insolution .I nth ebiligate d complex,th e chromophoric group iswel l shielded frommediu man dlon g range interactions with solventmolecules .Ascribin g thesplittin gt oa porphin-ligan d inter actioni nth epresenc eo fa Jahn-Telle rdistortion ,leave su swit hth e question:ho wca nsuc h interactionsb edifferen t forth eexcite d statesI| I and ty? An yphysica lmode l explainingou rresult s should containa ninequi - valencyfo rth eligand-porphi n interactionfo rth ein-plan eprincipa l axes ofth eMgTB Pmolecul e inorde rt oge ta sit e symmetry excluding degenerate states. The complexconsistin g ofa ruffle dporphi n corean dtw opyridin e ligands,wit h their longestC— N axisparalle lt oth enorma lt oth eporphi n planean dth eplane so fth epyridin emolecule s alongth etw operpendicula r 'valleys'i nth eruffle dporphi n core (fig.8a) ,ha sD- ,symmetr ys otha t thedegenerac y betweenth estate s \j>an di p isretained . Small ligandre orientations canremov eth edegeneracy .However ,whe nn owell-define d external distorting forcesar epresent ,ther ei sn oreaso nwh ya singl e distorted conformation,necessar yt oobtai na singl evalue d splittingA ,i smor e stable Fig. 10 Proposed cross section of the potential energy surfaces along the normal coordinate Q(a]) containing the displacement 2+ ofM g out of the porphin plane (along the normal to theplane ) for ground state S.an d excited states S and S of abiligate d MgTBP 2+ complex (see text); Q= 0 ifM g is in the molecular plane.Th eminim a of potential energy occur for Q= +_a , +_b ,+ ^c for the states S ,S ,an d S„, respectively. 0 a-'bc 69 thanal lothe rconformations .Thi smake s itunlikel y that the splitting in solutionresult s fromsuc h reorientations ofth epyridin emolecules .I n addition ,th e comparable splitting,foun d for ligationwit hpyridin e and ethanol,indicate s that it isno t the interactionbetwee n the ligands and the porphincore ,whic h isresponsibl e for theobserve d splitting,sinc efo r ethanolth emos t favourable conformation ofth ebiligate d complexha s already a symmetry inwhic h thedegenerac y isremoved . An alternative distortion is thedisplacemen t ofth eMg-io nou t ofth eplan e containing the fourN-atom s of theruffle dporphi n core.Eve n without axial ligands,thi sdistortio n results ina symmetr y ofth e complex inwhic h thedegenerac y canb e removed.Th e absence ofd-orbital san dth e ionicradiu s ofM g support the ideatha t the in-planepositio n ofth eMg - ion isno t themos t favourable one[ 2 4] . I nth enon-ligate d and thebiligate d complex,suc h adisplacemen t ofM g will result ina doubl ewel lpotentia l (fig. 10),wher e thepositio no f theminima ,relate d toth e equilibrium positions of theMg-ion ,depend s onth epresenc e ofan d thestrengt h of interaction ofM g with axial ligands.Fo rth emono-ligate d complex,th e two minimawil lb e inequivalent. Thestronge rbasicit y ofpyridin ew.r.t .MTH F and ethanolwil l result ina large rout-of-plan edisplacemen t ofM g inMgTBP.(Fy ) than in MgTBP.(MTHF)2 orMgTBP.(ethanol) 2.Thi s largerdisplacemen t for MgTBP.(Py)2, being related toa large rdistortio no fD- ,symmetry ,correlate swit h a largerQ-ban dsplittin g forMgTBP.(Py) _ thanfo rMgTBP.(MTHF) 2 orMgTBP . (ethanol)„ as isexperimentall y observed. (MgTBP.(Py). :A = 120±10 cm ; -1 MgTBP.(MTHF)7 andMgTBP.(ethanol) , :A = 60±2 0 cm ). Concluding,a displace - 2+ mento fM g outo fth emolecula rplan e canexplai nth esplittin go f the originally degenerate excited state,a swel l as theamoun t of the splitting. Wenot e that apart fromth eruffle d conformation,show n infig . 8a,ther ema yb e other out-of-planedistorte d conformations,whic h dono t lift thedegenerac ybetwee n ty andi| i states,a s long asM g is inth e porphinplane .Th e relatively broad absorptionbands ,exhibitin g thesplit ting,discusse dbefore ,d ono t allow adistinctio nbetwee ndifferen tout-of - plane conformations. Inthi spaper ,w ehav e limited ourdiscussio n toth e stericallymos t favourableone . Forth eSore tband ,correspondin g to theS ~-« -S n transition,on e doesno tnecessaril y expect thesam esplitting s as forth eQ-ban d because of thedifferen t electronicdistributio n inth e twoexcite d states.I nH-TBP , in 2+ which thereplacemen t ofM g by twoproton s canb e considered asa stron g 70 in-plane anisotropic field,th esplittin go fth eQ-ban d islarge rtha ntha t -1 -1 ofth eSoret-band : A„= 135 0c m andA R =80 0c m [7] . Inagreemen twit h these free-base results,th erelativel y broad Soret-bando fMgTB P seemst o consisto ftw ounresolve d componentswit ha splitting ,whic hi ssmalle r than thato fth eQ-band . 5.5.4 S -fluorescence of MgTBP The Stokes shiftca nb erelate d toth edifferenc e ingeometr yo f theexcite dan dgroun d state.A sdiscusse d previously,th echromophori cgrou p withinth ecomple xi swel l shielded fromsolven t interactions other than ligation,implyin g thatth edominan tmechanis m resulting ina nobservabl e Stokes shift should involve geometrical changeswithi nth ecomple xupo n excitation S.. +• S„o r S? •«-S . Turningno wt oou rresults ,w eobserv e that So exhibitsa large r Stokes shift thanS...S. .•< -S -an dS •*• S.excitation s correspondt oelectro n promotionse •*• a., ande -<-a - ,respectively . Referring tofig .5 ,w enot e thatth efirs t electronpromotio n results ina n -increase ofir-electron - densityo nth eporphin-nitrogens ,wherea sth esecon d involvesa decrease . Thispredict s thatth emagnitud e ofinteractio n betweenM g andth eporphi n nitrogens decreases inth eorde r S1 >S n >S 9.I tshoul db estresse d that 2+ thisorderin gpertain st oth einteractio nbetwee nM g andth eporphin-nitro gens,whic hdoe sno tnecessaril y implyth esam e relative orderingo finter actionsbetwee nth eligan dan dth eporphi n ir-electronsystem .Thi s results indifferen t displacemento fM g outo fth eporphi nplane ,suc htha tit s distance fromtha tplan edecrease s forth eserie s S~,S nan dS 1.Sinc eth e change inelectroni c distributiono nth eN-atom s islarge rfo re -*- a_ than fore +•a - oneexpect s also thatth egeometr yo fth ecomple x inth eS. . state resemblesmor e closely thato fS .tha ntha to f S?. Ourobservation s agreewit h thesepredictions ,i fw erelat eth eobserve d Stokes shiftfo r S- •*•S „an dS _-* - SQ transitions toa Mg-displacement .Suc ha situatio ni s depicted infig . 10representin ga vibrationa l cross-sectiono fth epotentia l energyprofil eo fth eC, v complex alonga na- inorma l coordinate,whic hin volvesM gdisplacemen tperpendicula r toth eporphi nplane .Th eseparatio no f potentialminim afo rS n,S. .an dS _ states reflects thequalitativ e conclusions whichw ereache dbefore .Thi sno wcoul dexplai nth eunusua l S~-» •S n fluor escence:th eradiativ e S?- *S nproces sca ncompet ewit hth eradiationles s transition S2 ^> S.becaus eo fth esmal l Franck-Condon factoro fth elatter . 71 A similar situationma y apply toth etriple tmanifold . Thesolven tdependenc e ofth erati obetwee nth eS~ -fluorescenc e andth eS..-fluorescence ,reporte db y Zalesskiie t al.[1 2] ,ca nb e easily understood inthi smodel .Th edifferenc e inbasicit yo fth evariou s ligands, bound toth ecentra lmetal-io ngenerall ywil l affect theequilibriu m dis placement ofth ecentra lmetal-io nw.r.t .th eporphi nplan e forth ethre e excited states toa differen t degree,resultin g inunequa l Franck-Condon factorsfo rth eS _ -»•S n and S? -*S 1 transitions forth evariou s ligated species. 5.6 CONCLUSIONS 1.Gouterman' s fourorbita lmode l satisfactorily explains thechange s inir - electronstructur e ofporphin supo ncondensatio nwit hbenzoidgroups . A comparisonwit hhydroporphin s canb emad e inth esam e framework. 2.MgTB P isa goo dmode l compound forchlorophyl lw.r.t .ligatio nan dsom e ofit sspectra leffects :optica l transitionspolarize d along one ofth e in-plane axiso fchlorophyl lbehav e as inMgTBP ,th eothe r in-plane polarized transitions follow aporphi npattern . 3.Th eelectroni cstructur e ofMgTB P allows amolecula r conformationfavour ingbiligatio n ofM g and isolationbetwee n theTT-electro nsystem so f benzoidgroups and the inner 16-memberedporphi n ring,resultin g inbezoi d absorptionbands . 4.Th epropose d structuralmode l forMgTB Pwit h out-of-planedisplace dM g explains theoccurrenc e ofS - fluorescence and theobservatio n ofJahn - Tellerdistorte d S1 andS- ,states ,spli tb y ananisotropi c 'internal' field. 5.7 ACKNOWLEDGEMENT TheMgTB Pwa s agenerou s gifto fDr .J.C .Goedheer ;th eauthor s areindebte d toth eBiochemistr y Departmentfo rusin gthei r lowtemperatur e absorptionapparatus .Discussion swit h Dr.M . Goutermanhav e stimulated our interest inth epeculiaritie s ofMgTBP ,wherea s R.J. Platenkampha sbee n a welcomepartne r indiscussion s during theearl y stageso fthi s investigation. Toou rrelie fmis sJ . Siebringwa swillin g totyp e themanuscript . 72 5.8 REFERENCES 1. G.P.Gurinovich ,A.N .Sevchenko ,K.N .Solov'ev ,Spectroscop yo fchloro phyllan drelate dcompounds ,NTIS ,Springfield ,U.S.A. ,1968 . 2. K.M.Smith ,Porphyrin san dMetalloporphyrins ,Elsevier ,Amsterdam ,1975 . 3. C.Weiss ,J.Mol.Spectros. ,44 ,3 7 (1972). 4. J.C.Goedheer ,J.P.J .Siero ,Photochem.Photobiol. ,6 ,50 9 (1967). 5. C.B.Storm ,A.H .Corwin ,R.R .Azellano ,M .Martz ,R .Weintraub ,J.Am . Chem.Soc,88 ,252 5 (1966). 6. T.A.Evans ,J.J . Katz,Biochem.Biophys.Acta ,396 ,41 4 (1975). 7. M.Gouterman ,J.Mol.Spectros. ,6 ,13 8 (1961). 8. L.Bayema ,M .Gouterman ,C.B .Rose ,J.Mol.Spectros. ,39 ,42 1 (1971). 9. B.C.McLees ,W.S .Caughey ,Biochemistry ,7 ,64 3 (1968). 10.S.S .Eaton ,G.R .Eaton ,R.H .Holm ,J.Organometal.Chem. ,39 ,17 9 (1972). 11.G.R .Seely ,R.G .Jensen ,Spectrochim.Acta ,2J_ ,183 5 (1965). 12.J.E .Zalesskii ,V.N .Kotlo ,A.N .Sevchenko ,K.N .Solov'ev ,S.F .Shkirman , Dokl.Akad.Nauk SSSR,210 ,31 2 (1973)(Sov.Phys.Dokl .18 ,32 0 (1973)). 13.P.E .Fielding ,A.W.H .Mau ,Aust.J.Chem. ,29 ,93 3 (1976). 14.J.R .Piatt , 'Electronicstructur ean dexcitatio no fpolyene san dpor phyrins'i n 'RadiationBiology' ,ed .A .Hollaender ,1956 . 15.C .Weiss ,H .Kobayashi ,M .Gouterman ,J.Mol.Spectros. ,J_6 ,41 5 (1965). 16.A.J .McHugh ,M .Gouterman ,C .Weiss ,Theor.Chim.Acta ,24 ,34 6 (1972). 17.D .Spangler ,R .McKinney ,R.E .Christoffersen ,G.M .Maggiora ,L.L . Shipman,Chem.Phys.Lett. ,36 ,42 7 (1975). 18.H .Kobayashi ,Nippo nKagak uZasshi ,83 ,116 7 (1962). 19.H .Kuhn ,Forsch.Chem.Org.Naturstoffe , ]!_, 404 (1959). 20.K .Tomono ,K .Nishimato ,Bull.Chem.Soc .Japan ,49 ,117 9 (1976). 21.G.W .Canters ,G .Jansen ,M .Noort ,J.H .va nde rWaals ,J.Phys.Chem. , 80,225 3 (1976). 22.M .Gouterman ,G.H .Wagniere ,L.C .Snijder ,J.Mol.Spectros. , V\_, 108 (1963). 23.E.C.M .Kielman-va nLuyt ,H.P.J.M .Dekkers ,G.W .Canters ,Mol.Physics , 32,89 9 (1976). 24.J.L .Hoard , 'Stereochemistry ofporphyrin san dmetallo-porphyrins 'i n 'Porphyrinsan dmetallo-porphyrins' ,ed .K.M .Smith ,Amsterdam ,1975 . 25.H.C .Chow ,R .Serlin ,C.E .Strouse ,J.Am.Chem.Soc. ,97 ,723 0 (1975). 26.G .Jansen ,J.H .va nde rWaals ,Chem.Phys.Lett. ,43 ,41 3 (1976). 27.R .Englman ,J .Jortner ,Mol.Physics ,^8,14 5 (1970). 73 28.J.F . Kleibeuker,R.J . Platenkamp,T.J . Schaafsma,t ob e published. 29.J.T . Hougen,J.Mol.Spectrosc . Vi, 149 (1964). 30. G.Herzberg ,Molecula r spectra andmolecula r structure,Vol . Ill, VanNostrant, Ne wYork , 1966. 74 6 The triplet state of MgTBP 6.1 INTRODUCTION SinceMgTB Pturn sou tt ob ea goo dmode l systemfo rchlorophyll s w.r.t.som epropertie so fth efirs texcite d singlet state,on ema ywonde ri f itstriple tstat epropertie s alsoresembl e thoseo fth echlorophylls .Thi s questionha sprompte du st omeasur ephosphorescenc ean dA m= 1 triple tES R spectrao fMgTBP ,an d- t oa limite d extent- fluorescenc edetecte dmagneti c resonance (FDMR)spectr aa tzer omagneti c field |1|. Forexperimenta l conditionsw erefe rt oChapter s5 ,7 ,8 an d9 . 6.2 RESULTS The (0-0)band i nphosphorescenc e spectrao fMgTB Pi nvariou s solvents (MTHF,methanol ,polymethylmethacrylat e (PMMA.)an dtol/pyr )i s invariably founda t80 0^ 5nm .Tw ofeature so fth espectr ashoul db enoted : (i)th ephosphorescenc e spectrahav ea norma l Franck-Condonprofile ,i.e . the (0-0)ban dha sappreciabl e larger intensity thanhighe rvibroni cbands , ascontraste dwit h spectrareporte dfo rothe rporphyrin s |2|; (ii)althoug h noaccurat equantu mefficienc ymeasurement shav ebee ncarrie dout ,th e phosphorescence intensity indicatesa relativel y largephosphorescenc eyield . TheA m= 1 triple tES Rspectru mo fMgTB P inMTH Fa t10 0K consist s oftw oshar pZ-peak san dbroa dband si nth eX- Yregions ,exhibitin gsom e structure indicatingth epresenc eo fX an dY peak s (seefig .1c) .T ofin dth e origino fthi sunusua lX/ Ybands ,th etemperatur edependence ,a swel la sth e solventdependenc eo fth eA m= 1 triple tES Rspectru mo fMgTB Pha sbee n studiedi ndetail .Fo rstudie si nth ehig htemperatur eregim e (100- 30 0K ) thecompoun dwa sincorporate d insoli dPMMA ,wherea sfo rT = 5 - 10 0K ,th e measurementswer ecarrie dou tusin gMTHF ,tol/pyr ,methano lan dethano l solutions;fig .1a- frepresen ta numbe ro ftypica l spectra.Fo ral lfou r solventsth eexperimenta l spectraar ever ysimilar .Th emai n featureso fth e spectraar e(i )th eshar p Z-peakswit ha nalmos t temperature independent width, (ii)th esingl eX/ Ypeak sa t30 0K ,wherea sa t5 K doubl eX an d 75 Fig. 1 Am = 1triple t ESR spectra ofMgTB P invariou s solvents and at different temperatures. (a)MgTB P inPMM A at 315K ; (b)MgTB P inPMM A at 100K ; (c)MgTB P inMTH F at 80K ; (d)MgTB P inMTH F at 40K ; (e)MgTB P inMTH F at 15K ; (f)MgTB P in tol/pyra t 5_K; H ,H etc.mar k the field positions for the various peaks. Note the two sets of X and Y peaks in (e) and (f). The samples are illuminated with red light (A >57 0 nm). Experimental conditions:microwav e fre quency 9.1 GHz,microwav e power 5m W (a)— 0.2 mW (f), fieldmodulatio n frequency 100KHz ,modulatio n ampli tude 10-16Gauss . A- u-'324 0G u* u* " *£ ^ll ^yi 1 ^yii I 7*1? z 76 doubleY peak sar efoun di nth eX- Yregion san dfinally , (iii)th eabsenc e of appreciable electronspi npolarizatio n (ESP),eve na t5 K ,th elowes t temperaturewhic h couldb eexperimentall y obtained.Th elo wtemperatur e spectraca nb ethough tt ob ea superpositio no ftw oA m= 1 triple tES R spectra,characterize db yth esam eD-valu ebu tdifferen t E-values.Th eZF S parameters obtainedfo rth evariou s solutionsar ecollecte d intabl e 1.Fro m phosphorescence,a swel la sfro mth etim edependenc eo fth eES Rsignal s followinga puls eo fexcitin g light,th elifetim eo fth elowes texcite d triplet stateha sbee ndetermine dt ob eT = 23 0+ 2 0msec . Upt ono ww edi dno tsuccee d inmeasurin g opticallydetecte d zero- fieldmagneti cresonanc eo fMgTB Pa t4. 2K i nMTHF ,ethano lo rtol/py r glasses,monitorin g either fluorescenceo rphosphorescenc e detection.Fo r solutionso fMgTB Pi nn-octane ,containin ga smal lamoun to fMTHF ,FDM R spectraar eeasil yobtained ,whe ndetectin ga tth eMgTB P (0-0)fluorescenc e banda t62 7nm .Th eobserve d resonancesca nb edivide d intotw osets : (i)measurin gwit h 1m Wmicrowav epower ,resonance sar efoun da t1220,74 0an d 480MHz ,correspondin gt oD = 32 7x 1 0 cm andE x 10"4cm1 : (ii)measurin gwit h 10m Wmicrowav epower ,i nadditio nt oth eaforementione d resonances,transition sar eobserve da t144 0an d60 2MHz ,mos tprobabl y -4 -1 -4 -1 correspondingt oD = 34 4x 1 0 cm andE = 16 0x 1 0 cm . Detecting,however ,a t64 7n m (fluorescenceo fa nunidentifie d impurity|3|) , microwavstatewit heD resonance= 30 8x 10'4scoccu m 1an rda E t 110 8an d74 0MHz 6,1correspondin x 10~4cnf1. g toa triple t 6.3 DISCUSSION Theunusua lshap eo fth eMgTB PA m= 1 triple tES Rspectru man dit s temperature dependence isno tfoun dfo rphotosyntheti cpigment s (Chapter 7-10). Furthermore,i nvie wo fth erelativel y long lifetimean dth eabsenc eo f Table 1 Zero-field splittingparameter so fMgTB Pi ndifferen tmatrices . E andE „refe rt oth etw oset so fX an dY peaks . 4 -1 4 -1 4 -1 Solvent Dx 10c m E x 10c m E2x 10c m tol/pyr 334+ 2 65+ 2 38+ 2 MTHF 341+ 2 69+ 2 37+ 2 ethanol 341+ 2 71+2 37+ 2 methanol 339+ 2 72+ 2 38+ 2 PMMA 334+ 3 - - 77 appreciable ESP,eve na ttemperature swher e thisphenomeno n isusuall y observed,w ema y conclude thatMgTB Pdoe sno t represent agoo dmode l system forchlorophyl lw.r.t .triple t state studies.Th e origino f theunusua l and interesting Am= 1triple tES Rspectra ,a swel l as their temperature dependencewil lb e discussed elsewhere,sinc e it isoutsid e the scope of this thesis. Inth efollowin gw ewil l only consider the effects ofligands , bound toM g ando fth ebenzoidgroup s onth e ZFSparameters . Only onese to f ZFSparameter s obtained from FDMR is insatisfac tory agreementwit h theES R results:FDM Ryield s D = 327x 10~4cm~1, E =8 0x 10"4cm-1,wherea s from ESRa t SK w e obtain D= 334- 34 0x 10~4cm"1 and E1 =6 5 - 72x 10 cm .I nn-octane ,containin g asmal l amount ofMTHF , mono- andbiligate dMgTB P (MgTBP.Lan dMgTBP.L~ )ar eexpecte d tob epresent . As reported inChapte r 5,w e found fromoptica l studies that the S. energy level isequa l forbot h complexes,s ow e attribute the twoset s of ZFSpara meters detected at the62 7n m (0-0)fluorescenc e band toth emono -an d biligated compound. SinceMgTB P inpola r solvents atT <_ 100 Ki s expected tob e completelybiligated , the correspondence between ESR,measure d using polar solvents,an d FEMRresult s indicates that forMgTBP.L ~ D= 32 7 x 10~4cm"1 andE = 80x 10~4cm"1,wherea s forMgTBP. L D =34 4x 10_4cm and E = 160x 10 cm .U p tono ww ehav en oexplanatio n forth e strong increase ofE forMgTBP. L w.r.t. MgTBP.L_.Th e largerD-valu e inMgTBP. Lca nb e 2+ attributed toa largereffectiv e electronegativity ofM g inMgTBP.L , because of thepresenc e ofonl y one instead of twoelectro ndonatin g ligands. Then,th e largerelectro nattractio n ofM g inMgTBP. L vs. MgTBP.L. results ina decreas e ofth e extento fth e tr-electronderealizatio n and consequently ina largerD-valu eo fth emonoligate dcompoun d (seeChapte r2) . Recently comparable effectshav ebee n found forCh ia |4|. TheD-valu e forMgTBP. L isonl y slightly smaller than that reported forMg-porphi n - lackingbenzoidgroup s - inn-octane ,containin g asmal l -4-1 amounto fethano l (D= 35 0x 10 cm ) |5|. Ifth eunpaire d electrons in MgTBPwoul db e delocalized over theporphi n ring and the fourbenzoidgroups , onewoul dpredic t theD-valu efo rMgTBP. L tob e smaller thanfo r monoligated Mg-porphin.Th e similarity of theD-value sfo rbot h compounds indicates - inagreemen twit h theresult s obtained fromoptica l studies -tha t the dominant u-electronconjugatio npat h inTB P is located on the 16membere d ring,whic h iselectronicall y isolated from thebenzoidgroups . 78 6.4 REFERENCES 1. S.J. vande rBent ,A .d eJager ,T.J . Schaafsma,Rev.Sci.Instrum. ,47 , 117 (1976). 2. M.P.Tsvirko ,K.N .Solovev ,A.T .Gradyushko ,S.S .Dvornikov ,Opt . Spectrosc,38 ,40 0 (1975). 3. L.Bayeraa ,M . Gouterman,C.B .Rose ,J.Mol.Spectrosc. ,39 ,42 1 (1971}. 4. R.P.H.Kooyman ,T.J . Schaafsma,J.F . KLeibeuker,Photochem .Photobiol. , tob epublished . 5. G.Jansen ,J.H .va nde rWaals ,Chem.Phys.Lett. ,43 ,41 3 (1976). 79 Volume 29,numbe r 1 CHEMICAL PHYSICS LETTERS 1 November 1974 7 Spin polarization in the lowest triplet state of chlorophyll Joop F. KLE1BEUKER and Tjeerd J. SCHAAFSMA Laboratoryof MolecularPhysics, Agricultural University, Wageningen, The Netherlands Received 18Apri l 1974 Revised manuscript received 21 July 1974 Chlorophyll-bi n glassy solution hasa spin-polarized lowest triplet state at and above 77 K. The magnitude of the effect isdifferen t for MTHF and ethanol as solvents, in contrast to what isfoun d for the porphin free base. Oiloro- phyll-a does not exhibit spin-polarization under identical conditions asfo r chlorophyll-b. Zero-field parameters are found to be: chlorophyll-a (MTHF)£>= (281± 6) X10" 4 cm"1;*' =(3 9± 3 ) X lO^cm"-'; chlorophyll-b (MTHF)D =(28 9* 4) X 10^ cm"';£ = (49± 3 ) XlO^ 1 cm"1. From ESR signal kinetics it follows that for chlorophyll-b,populatio n and depopulation mainly involve-th e spin level \y), describing a spin moving in aplan e perpendicular to the molecular plane: kr = 240 ± 40 s" 120 s" kz < 75 s-', wherePj an dfy denot e populating and decay rates. Thus, the kinetic scheme for the chlorophyll triplet is different from that of porphyrins with heavier metal ions,bu t very similar to that of the porphin free base. The spin-lattice relaxation time isfoun d to be anisotropic and shorter than the decay rates of individual spin levels. Nevertheless, spin polarization can be observed, essentially because the ESR signal ampUtude depends on population differences. 1. Introduction amount of spectral data [1,8,9] are available. Eveni f the triplet state isno t an essential intermediate inbio Upon illumination of photosynthetic pigments logical energy conversion, it provides an attractive both in vitro and invivo ,significan t steady-statelevel s probe to study primary processes in photosynthetic of triplets can be formed [1-3]. Furthermore, it has model compounds and in the in vivo unit. For exam been found that triplet state kinetics affect the popu ple,Dutto n and Leigh [3]hav e recently demonstrated lations of the ground and excited singlet stateso f that the presence of triplets isclosel y connected to chlorophyll [4] and of the porphin free base [5]. On the primary photochemistry in the photosynthetic re the other hand, the triplet lifetime of chlorophyll at action centre of rhodopseudomonas spheroides. room temperature isi n the millisecond region [1], In this letter we present kinetic data on the lowest thus exceeding the period which issignifican t for the triplet state of chlorophyll-a and b, together with that photosynthetic energy conversion. Therefore, it is of the porphin free base, obtained from the steady- still a matter of debate as to whether the lowest trip state and transient behaviour of their low temperature let state of chlorophyll ispar t of the energy transfer ESR spectra. pathway in photosynthesis [3,6], and more kinetic studies are needed to settle this question. Also from the molecular point of view, the triplet 2. Experimental state of chlorophyll iso f interest, since it belongs to the class of porphyrins, for which well-developed the ESR spectra of the porphin free base, chlorophyll- ories on electronic structure [7] aswel l asa larg e a and b, dissolved in dry and oxygen-free 2-methyl- 80 tetrahydrofuran (MTHF) and ethanol were takenwit h tudesS for each of the six Am - ±1transition s at a slightly modified Varian E-6 spectrometer, equipped H; !•» where fractional population ratesp 0 and P]ar ede fined by PQ± j = PQ± \I(PQ +2/^) . Note that polariza w3 W, (1+25) tion is observed if k^p0 - k^p^ is of the same order of *,ll*5| 1 w, magnitude asw'S, eve n if w > k0,k^. Aswil l be shown below, spin polarization in the f-4 |0> porphin free base and chlorophyll-b arises because both the populating and decay rates are different for the three zero-field spin states \i),resultin g in p0 =£p j W, (1+5) = ( P_l)and k0=t kl (=k_{) for //II x, y, orz. Since one may neglect the radiative contribution to the to tal decay rates, the kinetics of the high-field spin com \->. ponents are entirely governed by radiationless pro cesses.Then , the populating and decay mechanism are rather similar in nature, asha sbee n shown by the Fig.1 . Kineticschem efo r tripletmagneti csublevel s|0 > and ory and experiments [15] on aromatic and aza-aromat- l±l>a thig hfield . Fordefinitio n ofsymbols ,se etext . icmolecules . Quantitatively, this means that the dif- 81 H2P in Me-THF Rigid solution T«77"K 0=9.123 GHz Fig. 2. Triplet ESR spectrum of the porphin free base in MTHF (4X 10" 5 M)a t 77 Kindicatin g the presence of spin polarization. Microwavepowe r 0.05 mW,modulatio n amplitude 16G , modulation frequency 100 kHz. Spectrum recorded with continuousil lumination. CHLOROPHYLL-B in MTHF Fig. 3.Triple t ESR spectra of chlorophyll-b in MTHF (2X 1 0 M)a t two different temperatures recorded with continuous illumi nation; the central radical signali s omitted. Microwave power is:0. 5 mW(a ) and 5 mW(b) , microwave frequency 9099 MHz,mo dulation amplitude 16 G, modulation frequency 100 kHz. In this region the shape of the observed spectrum isindependen t of mi crowave power. 82 ference betweenPg/pj and AgAl 's rather small for Table 1 each of the canonical field directions. Therefore, the Zero-field parameterso f theporphi n free basean d chloro difference frjPg - ^rjPi can only have appreciable phyll-aan db .D valueswer ecalculate d from theseparatio n of theoute rpeaks ;£ valuesfro m theaverag eo f the separa magnitude with respect to w'8 for short-lived triplets, tionbetwee nx andy positionsi nth elo wan dhig hfiel dpor i.e., when k0 and Ajar elarge . tionso f thespectrum . All measurementswer ecarrie d outa t In most solvents at « 77 Kth e spin system is only 77K , except for chlorophyll-a,wher eT- 95K . The varia partially polarized due to fast relaxation and oneob tionsi nD andE i n thistemperatur e rangear ewithi nth e servesn o emissive transitions, but an asymmetric in measuring error tensity distribution asi nfigs. 2 an d 3.Th e compo Compound Solvent OUO^cm-' E(.lO-*cm-1) nent out of apai r of transitions (e.g.,hT x and Hx) with lowest intensity will appear asemissive if relaxa porphin ethanol 437 ± 7 66 + 5 tion issufficientl y slowed down. MTHF 436 ± 6 65 + 3 The spectra offigs. 2 and 3giv e the following po chlorophyll-b ethanol 287 ± 5 36 + 4 larization ratios: MTHF 289 ±4 49 ± 3 chlorophyll-a MTHF 281 ± 6 39 ± 3 porphin free base (77 K,MTHF ) Rx =+0.3O(±0.05) , R = -0.50(±0.06). Rz « 0 ; agreement with previous conclusions from Am = 2 chlorophyll-b (94 K,MTHF ) spectra [17]. The amount of spin polarization Rj remains con /? =+0.26(±0.03) , R =-0.59(±0.06), R « 0 . x z stant upon achang e of solvent in the case of the por Chlorophyll-a does not show spin polarization under phin free base, in contrast with chlorophyll-b, where the same conditions asch'lorophyll-b . Rj islowe r in ethanol than in MTHF.Althoug h more Applying eq. (3), these results imply that both for data are needed to substantiate such a conclusion, it the porphin free base and chlorophyll-b, transitions at is conceivable that the lowering of both E andRj upon H~an dHt, ar e emissive and those atH* x andH~ y are a change in solvent have a common origin, if it is as absorptive in the absence of relaxation. Anticipating sumed that the amount of inequivalence of themolec further results from transient ESR responses, follow ular* andy axes, reflected by the magnitude ofE, ingbelow ,w efind p >px> pz. Combining thisre also affects spin relaxation, and thusRj. Afas t spin sult with the steady state behaviour of transitions at relaxation, accompanied by alo wE value, could also Hy. and /C, we conclude that the spin-component \y) explain the absence of spin polarization in chloro decays faster than \x) and/or |z>.Thi s is very different phyll-a, whichi s surprising in view of its triplet life from the kinetic scheme observed for Zn-porphin time being shorter than that of the -b compound. [16],wher e |z> is the dominant level for populating Achang e in solvent viscosity has a different effect and decay. It follows from (3) that for isotropic re onRj and E, asi sshow n byfig. 3 : asmal l tempera x z laxation (w = wy = w ) ture increase does not affect D orE, but spin polari zation isnearl y wiped out. Even if no spin polariza R +R R = 0 x y- z - W tion is observed, i.e., the Am =1 ES R spectrum hasa Spin relaxation must be anisotropic, since eq. (4) symmetric intensity distribution around the center does not hold experimentally. field,anisotropi c spin relaxation can affect the rela Table 1 summarizes the zero-field parameters for tive intensities of the three pairs of transitions ina the three compounds of interest dissolved inMTH F different way asi sapparen t in triplet ESR spectra of and ethanol. Results agree with existing data obtained some metalloporphyrins [19]. from high-field Am = 2 ESR spectra [11,17,18] and with zero-field transitions observed for the porphin free base [5]. Note that D andE of the porphin free 4. Transient spin polarization base are insensitive to a change in solvent, whereas chlorophyll-b changes itsE value by «* 30%. Chloro With standard ESR detection, quantitative infor phyll-a has alowe rE value than itspartne r -b,i n mation on the kinetic scheme of fig. 1canno t beob - 83 tained in asimpl e manner. Therefore, following lution which isprobabl y limited by the 0.3 ms re Lhoste [20] and Levanon andWeissmann [21,22], we time of the spectrometer. After switching off the ex have employed a chopped light source and phase- citing light, the S signal changes sign from absorp sensitive detection with reference to the chopping tion (A) to emission (E) and then decays to zero. The frequency. It appears to be useful to study both the same phenomenon, but lesspronounced , is found for time dependence of each of the Am =± 1 transitions, S.. This means that (orHfy k0 > kj. When switching aswel l as the shape of the spectrum after a certain on the exciting light, signalSj initially grows propor time has elapsed after the exciting light has been tional toPj — i(Pj+P k) (i, j, k = x, y or z). Thus switched on or off, usinga boxca r integrator [23]; such a spectrum iscalle d agate d spectrum. Treat S(H)= lim ment of our results goes along the same lines asi n pre / S(t)dt Af-0 vious studies on slower systems by Sixlan dSchwoere r [13,14] and Clarke [24].Solvin gth e differential equa represents a spectrum in statu nascendi with normal tions leads to a sum of exponentials for the timede ized signal amplitudes reflecting the difference in the pendence of the various signal amplitudes. If the relax populating ratesp 0 andp±1. ation rates are of comparable magnitude or larger than Aconvenien t experimental method to obtain a the decay rates, signalsS, - and Sj~ have unequal ampli spectrum in statu nascendi is to measure a gated spec tude [13].For//ll.x , y and z this iswha t we observe. trum with the gate-open-period of the boxcar integra Fig.4 represents the observed time dependence for tor directly after the"switchin g on of the exciting the signal amplitudesS x, S* and 5*;wit h a time rose light. For chlorophyll-b such a gated spectrum is shown in fig. 5.Not e the large amplitude of theS~ signal resulting from py >px,pz. By evaluating the initial normalized slopes of the transients Sx(t), Sy(t) andS z (f), a first estimate of the populating rates can be obtained: Px'-Pv •3:>6:<2. The opposite behaviour of atransitio n with continu ous or pulsed excitation can now be understood. In the former caseth e Sy transition, for instance,woul d be emissive in the absence of relaxation, even when theonset ofth e same transition isabsorptive. Thisi s s' due to the fact that, although pg> pj, p0/A:Q 5)JiS 84 Gated ESR Spectrum of CHLOROPHYLL-B in MTHF 32500 160H z light mod Fig. 5. Gated ESR spectrum of chlorophyll-b in MTHF. Response time ESR spectrometer 0.3 ms;gatewidth : 1 ms;dela y with re spect to leading edge of light pulse: 1 MS.Respons e time gated amplifier: 10 s.Choppin g frequency: 160Hz . sent from the spectrum, asi s observed for the chloro phyll-bS J and SJ transitions at 160H z chopping fre quency. Finally, kinetic constants p,-,k i and w' can beob tained by electronic simulation of the ESR transients. Asw' > k/,Pj, all transients decay to equilibrium with exponentials containing the averages l^yi^i an(* 3S/ fy n^]> and in this respect do not provide data on the individual spin components. However, the signsan d relative amplitudes of5/"(f ) and S,r(/) both for continuous illumination and at ashor t time after the leading and trailing edges of the applied light pulse aredetermine d by kit Pj,an d w'. Fig. 6 repre sents a superposition of the experimental and simu Fig.6 . Superposition of simulated and observed transient Sy lated transient S~(t). Usingth e same values offc,- an d of chlorophyll-b in MTHF at achoppin g frequency of 15Hz , Pifo r thefive remainin g transients leadst o close ESR response time 0.3 ms. Accumulation of « 1000 tran agreement with experimental results, if wi s taken to sients. Timebase : 5 ms/div.; A= absorption ; E= emission. be different for each of the three pairs of transitions S*,S * and S$. We find that ESR value of 300 s_1.W ecombine d the last number with the ratio kx :k:k2 from the simulation experi- ment, yielding = 0.30(±0.02) :0.60(±0.04 ) :0.09(+0.04 ) ; K = 240 ± 40 s~ *y = 600: 120 s' k, <75s" in agreement with previous conclusions from the :fc :* = 3.1(+0.5):7.8(±1.5): <1. z steady state spin polarization. In actual simulation, The value of j^y; £,-> determined from the simulation the error limits are smaller than quoted above, since experiment, is 320 ±5 0 s~' in agreement with the the choice of kj and p, must be consistent with the 85 magnitude of the steady state polarization. Spin- References lattice relaxation rates are found to be of the order of 104 s-'..Furthermore wx « w? < w2, in qualitative G.P. Gurinovich, A.N. Sevchenko and K.N. Solovyov, agreement with the results obtained from (3) using ex Spectroscopy of chlorophyll and related compounds perimental values for k and p,-. Probably, the absence (Science and Engineering Publishing House, Minsk, ( 1968) [English transl. Natl. Techn. Inform. Service, of spin polarization for//II zis due to the very small Springfield] ch. 7. value of kyPQ- k0p\ in (3) and not to fast relaxation. A.T. Gradyushko, A.N. Sevchenko, K.N. Solovyov and Simulation was carried out with a fictitious Boltz- M.P.Tsvirko , Pliotochem. Photobiol. 11(1968 ) 387. mann difference 5 =0.1. Since the shape of the tran P.L. Dutton, J.S. Leigh and D.W. Reed, Biochem. Bio- sient is largely determined by w'5, if w' >fc,-,p,-, thi s phys. Acta 292 (1973) 654. R.H. Clarke, Abstracts Intern. Conf. Excited States of does not affect the results, as long as w'5 is kept un Biological Molecules, Lisbon (1974), Contribution nr. changed with respect to the actual ESR experiment, C6. and o < 1. W.G. van Dorp, T.J. Schaafsma, M.Som a and J.H. van Our results show good agreement with data recent der Waals,Chem . Phys. Letters 21 (1973) 221. ly obtained by Clarke [4], using optically detected A.K. Chibisov, Dold. Akad. Nauk USSR 205 (1972) 142. magnetic resonance in zero magnetic field, and are M.Gouterman , in: Excited states of matter, ed. C.W. qualitatively similar to experimental results on the Shoppee, Grad. Studies Texas Tech. Univ. 2:1-174, porphin free base under conditions of slow relaxation 1973, and references therein. [25]. L.P. Vernon and G.R. Seely, eds., The chlorophylls Summarizing, we conclude that the kinetic behav (Academic Press, New York, 1966). J.E. Falk, Porphyrins and metalloporphyrins (Elsevier, iour of the lowest triplet state of chlorophyll-b, as de Amsterdam, 1964). rived from steady state and transient spin-polarization, [10 U. Eisner and R.P. Linstead, J. Chem. Soc. (1955) 3749. is characterized by the presence of a fast decaying in- |H J.M. Lhoste, Compt. Rend. Acad. Sci. (Paris) D(1968 ) plane spin component which also has the largest pop 1059. ulating rate. In this respect, chlorophyll-b resembles [12 J.H. van der Waalsan d M.S.d e Groot, in: The triplet state, ed. A.B. Zahlan (Cambridge Univ. Press, London, the porphin free base rather than porphyrin with 1967) p. 125. heavier metal ions. [13 H. Sixl and M.Schwoerer , Z. Naturforsch. 24a (1969) Both the presence of a different side group as in 952; 25a (1970) 1383. chlorophyll-a and a change of solvent affect the zero- [14 M. Schwoerer, in: Proc. XVII Congress Ampere, ed. V. field parameter E, as well as the observed amount of Hovi (North-Holland, Amsterdam, 1973) p. 143. [15 D. Antheunis, Thesis, Leyden (1974). spin polarization, which also depends on the direction [16 I.Y. Chan, W.G.va n Dorp, T.J. Schaafsma and J.H. van of the magnetic field with respect to the molecular der Waals, Mol. Phys. 22 (1971) 741,753 . frame, due to anisotropic spin—lattice relaxation rates. [17 G.T. Rikhireva, L.A. Sibel'dina, Z.P. Gribova, B.S. Marinov, L.P. Kayushin and A.A. Krasnovskii, Dokl. Akad. Nauk USSR (Biophysical Section) 181 (1968) 103. G.T. Rikhireva, Z.P. Gribova, L.P. Kayushin, A.V. Umrikhina and A.A . Krasnovskii, Dokl. Akad. Nauk USSR 159(1964) 196. J.M. Lhoste, C. Helene and M.Ptak , in: The triplet state, ed. A.B. Zahlan (Cambridge Univ. Press, London, Acknowledgement 1967) p. 487; J.F. Kleibeuker and T.J. Schaafsma, unpublished results. We wish to thank Dr. J.H. van der Waals for per [20 J.M. Lhoste, Stud. Biophys. Berlin 12 (1968) 135. mission to refer to results obtained in his group prior [21 H. Levanon and S.I. Weissman, J. Am. Chem. Soc. 93 to publication. One of us (T.J.S.) is greatly indebted (1971)4309. [22 H. Levanon and S.I. Weissman, Israel J. Chem. 10 to Drs. J.H. van der Waals and M. Gouterman for in (1972) 1. spiring discussions on the electronic structure of por [23 M.Plat o and K. Mobius, Messtechnik 8(1972 ) 224. phyrins. Mr. P.A. de Jager has provided us with valu [24 R.H. Clarke, Chem. Phys. Letters 6 (1970) 413. able technical assistance. [25 M.Soma , private communication. 86 Volume 41, number 3 CHEMICAL PHYSICS LETTERS 1 August 1976 8 Optically induced electron spin polarization in the triplet state of chlorophyll and its model compounds Joop F. KLEIBEUKER, Roelof J. PLATENKAMPan d Tjeerd J. SCHAAFSMA Laboratory of MolecularPhysics, AgriculturalUniversity, Wageningen, TheNetherlands Received 7Apri l 1976 Optically induced electron spin polarization (OEP) isreporte d for the triplet state of chlorophyll-aand -b in an organic glassa t 15 Kan d for tetraphenyl porphyrin (TPP) and -chlorin (TPC) free base in a solid polymer up to = 200 K. Such polarization results in emissive transitions in the Am= ± 1 ES R spectrum during continuous illumination of these com pounds. From the experimental results it is concluded that both chlorophylls have dominant steady-state population in the top zero-field level,associate d with an arbitrary in-plane molecular axis.Th e amount of spin-polarization isstrongl y affected by the rigidity of the medium asi s demonstrated by the observation of emissive transitions in the Am = 1triple t spectra of TPPan d TPC up to >* 200 K. 1. Introduction ation between spin states Tfj can be made sufficiently slow, such that emissive Am = 1transition s also can At low temperature, and in the presence of ahig h be observed during continuous optical excitation. magneticfield, opticall y induced electron spin polar The observation of "anomalous" emissive Am = 1 ization (OEP) hasbee n observed in the photo-excited transitions of the triplet state of bacterial photosyn molecular lowest triplet state [1].B y noting that thetic reaction centers during continuous illumination, high-field spin states T{j(p =± 1,0) contain zero- by Leighan d Dutton [10-13], has considerably stim fieldspi n states TQ (X= x, y, z) (referred to the prin ulated interest in OEPa sa means for obtaining infor cipalaxi ssyste m of the zero-field splitting tensor mation on the mechanism of in vivo energy tiansfer. {x,y, z}) having different spin-orbit coupling with This paper reports emissive transitions in the spin- (excited) singlet states (S„) [2],populatin g and de polarized triplet ESRspectru m of chlorophyll aan d populating rates S„ -> Tgan d T{j - S0 are expected b in an organic glassa t 15K an d of two model com to be different for /J =± 1 and 0 [3].Previously , it pounds, tetraphenylporphyrin free base (TPP)an d has been reported that OEPresult s in an asymmetric tetraphenylchlorin free base (TPC) *dissolve. ! in a absorption pattern in the Am = 1 triplet ESR spectrum solid polymer, at relatively high temperature. Our ex of photosynthetic pigments at 95 K;immediatel y periments demonstrate that spin relaxation in the low after applying an exciting light pulse,som e of the est triplet state of these compounds is strongly affected Am = 1 transitions were observed to have emissive by the rigidity of the medium. From the interpreta character [4].Transition s with apparent emissive tion of the polarization pattern in the low tempera character are obtained when the exciting light is ture Am =1 spectra of both chlorophylls under con modulated and the resulting ESR spectrum is recorded tinuous illumination we obtain an ordering of steady- using phase-sensitive detection with the light-modula state populations of the zero-field spin levels, differ tion asa reference signal [5-8]. ent from that published recently by Katze t al. [7,8], Under favourable conditions, such aslo w tempera based on OEP patterns of light-modulated Am - 1 ture (< 4.2 K) or usinga lattice with weak electron- spectra. phonon coupling — e.g. a Shpolskii-matrix [9] - relax * Systematic name: tetraphenyldihydroporphyrin free base. 2. Experimental at 15-33 Kwer e carried out on a Varian E-9 spectrom eter equipped with an Air-Productsvariabl e tempera Chlorophyll a and bwer e prepared and purified ture helium flow system. Other experimental condi following methods of Strain et al. [14];2-methyl - tionswer e the same asi n ref. [4]. tetrahydrofuran (MTHF) waspurifie d by vacuum Populating and depopulating rate constants were distillation from asodiu m mirror. ESR measurements evaluated from ESR transients following a light pulse, usinga n electronic analog simulation [15]. H„ H I I ITI 1I 7 3. Results 100G Figs, la and lc represent Am= ± 1 (first derivative) triplet ESR spectra of chlorophyll-a and- bi n MTHF glassa t 15K . Emissivetransition s are labelled by E, V 6 L \ t, 0 -2 - 4 a Hu Hy Hv rU Iy 3240G I y G 1 0 30 SO 70 90 . I I JT I 1 1 V 100G \ 6 - \ \ 4 —0— 0 - 2 / / - k * i • 6 1 1 b - 8 i 1 Fig. 1.X-ban d Am =± 1 triple t ESR spectra of chlorophyll-a -10 in MTHFa t (a) 15 K;(b ) 95 Kan d of chlorophyU-bi n MTHF -12 i at (c) 15 Kan d (d) 95 K. Field positions are labelled assuming the energieso f the zero-field spin states to be ordered as E(7$) >E(T$) >£(J$) asi s usualfo r irir* states;x and y Fig. 2. Temperature dependence of OEPa s measured by are two arbitrary directions in the molecular plane,z is the Rj(X) for (a) chlorophyll-aan d (b) chlorophyll-bbot h in out-of-piane axis. MTHF. o:Rx;X:Ry;a:Rz. those withenhance d absorption by A;thei r absolute MTHFa t » 120K ,ca nb e suppressed by using solid signfollow s from the phase of a free radical signali n polymethylmethacrylate (PMMA)a sa solvent for the spectral center. Both spectra are rather similar, TPPan d TPC.Fig .3 represent s TPCA m = 1triple t except for the absence of a transition atH~ for spectra at 95 and 173K ; fig. 4 shows similar spectra chlorophyll b (probably sincei t iso n theverg eo f for TPPa t 103 and 213K . For both compounds, turning from absorptive to emissive) and a different transitions at different canonical orientations exhibit intensity-ratio S*/S~. different changes with temperature: e.g.fo r TPC + At 15K bot h compounds have atriple t ESRspec (fig. 3) the signalamplitud e of the emissive Y tran trum with an EEA/EAA pattern;fro m figs, lb and Id sition only slightly decreases with increasing tempera + it canb e concluded that at « 100K ther e isstil l no ture, whereas the Z transition turns from emissive thermal equilibrium among the triplet spin levels, al into (weakly) absorptive. though no emissive transitions areobserve d (in con TPPi n the same matrix exhibits considerable spin- trast,bacteriochlorophyll- a in MTHFa t 100K ha s polarization,eve n at 213K ,wher e Y*an d Y~ inten emissive X- and Y*transition s [15]). sitiesar estrongl y asymmetric and the X~ transition The magnitude of spin-polarization canb e ex isstil l weakly emissive.A t 103 Kth e triplet spectrum pressed as the ratio [4] of TPPi nPMM Acontain s strong emissive transitions atit andH^. , in contrast with what hasbee n re /?,= (St-S,r)/(5t+S,-) , ported for this compound inethano l glassi n the same whereSf measures signalintensit y at a pair of canon temperature range [17]. icalfield position sHj for i= x,y andlf z for i= z ; Sji stake n to be positive when the transition isab TPC IN PMMA sorptive and negative when it isemissive .I f 1/?,|> 1, A A emissive transitions are observed. Fig. 2show sth e J\ change of Ry versus temperature for chlorophyll-a T= 173 K ^J and -bi n MTHFt o be markedly different. Table 1 collectsth e effects of achang e of solvent on the Y" magnitude of Rx and Ry for both chlorophylls. T= 95K The effect of solvent viscosity on triplet spin- j-—N f 1-100G- i lattice relaxation hasbee n studied for TPPan d TPC . J Z" *~ f X* Z* free base,whic h are structurally related to chlorophyll, 1 H 1 i 1 1 A 1 E E and for which triplet state kinetics have been deter A E . mined [16]. Fig.3 .X-ban dA m = ± 1 triplet ESRspectr ao ftetraphenyl - Intramolecular motion and molecular reorientation, chlorinfre ebas ei nsoli dPMM A at95andl73K. such asrotatio n and libration, caninduc e transitions between triplet spin states by modulation of the non- secular part of the dipolar interaction. The contribution of molecular reorientation to the spin-lattice relaxation, evident from the rapid disap pearance of OEParoun d the softening point of Table 1 Solventdependenc echlorophyl la/ bOE P at9 5 K Compound Solvent chlorophyll-a MTHF 0.45 -0.25 «0 chlorophyll-a ethanol 0.09 -0.29 "0 chloiophyll-b MTHF 0.30 -0.47 •»0 Fig. 4.X-ban d Am ± 1 triplet ESRspectr ao ftetraphenyl - chlorophyll-b ethanol 0.30 -0.07 ~0 porphyrinfre ebas ei nsoli dPMM A at 103an d21 3 K. 89 4. Discussion taken into account or when CJ 90 PMMA following excitation by a light pulse [4,17] |9] E.V.Shpolskii ,Sovie t Phys.Uspekh i 3(1960 ) 372;5 indicates that relaxation rates in this medium are con (1962) 522;6 (1963)411. siderably lower than in low-melting glasses at the [10] P.L. Dutton, J.S. Leigh and M.Seibert , Biochem.Bio - phys. Res.Commun . 46 (1972) 406. same temperature. This can be helpful in the study of [11] P.L. Dutton, J.S. Leigh and D.W.Reed , Biochim. Bio spin polarization in aggregated or strongly ligated pig phys.Act a 292 (1973) 654. ments where relaxation is fast [26]. [12] C.A.Wraight ,J.S . Leigh,P.L .Dutto n and R.K. Clayton, Biochim. Biophys.Act a 333 (1974)401 . [13] J.S. Leigh and P.L.Dutton , Biochim.Biophys .Act a 357 (1974) 67. Acknowledgement [14] H.H.Strai n and W.A.Svec ,in : The chlorophylls,eds . L.P.Verno n and G. Seeiy (Academic Press,Ne w York, We want to thank P. Geerse for providing us with 1966) p. 22. the model compounds and the Staff of the Physical [15] J.F. Kleibeuker, P.A.d eJager , R.J. Platenkamp and Chemistry Machine Shop for technical assistance. T.J. Schaafsma, to be published. [16] S.J. van der Bent and T.J. Schaafsma, Chem.Phys . We are indebted to Dr. R. Wever for providing us with Letters 35 (1975)45 . facilities on an ESR spectrometer equipped with a [17] H.Levano n and S. Vega,J . Chem. Phys.6 1 (1974) 2265. helium-flow cryostat. [18] T.J. Schaafsma, J.F. Kleibeuker, R.J. Platenkampan d P.Geerse ,in : Proceedings of the 12th European Con gresso n Molecular Spectroscopy, eds. M.Grossmann , S.G. Elkomossan d J. Ringeissen (Elsevier, Amsterdam, References 1976) p. 491. [19] R.H.Clark e and R.H. Hofeldt, J.Chem . Phys.6 1 (1974) [1] H.Six lan d M.Schwoerer ,Z .Naturforsch . 25a (1970) 4582. 1383. [20] R.H.Clarke , R.E.Connor , T.J. Schaafsma, [2) J.H.va n der Waalsan d M.S.d e Groot, in: The triplet J.F. Kleibeuker and R.J. Platenkamp, J. Am.Chem . state,ed . A.B.Zahla n (Cambridge Univ.Press , London, Soc (1976),t ob e published. 1967) p. 125. [21] C.J. Winscom, Z. Naturforsch. 30a (1975)571 . [3] H.Six lan d M.Schwoerer , Chem.Phys . Letters 6 (1970) [22] R.H.Clarke , R.E. Connors, J.R. Norrisan d 21. M.C.Thurnauer , J. Am.Chem . Soc.9 7 (1975) 7178. [4] J.F. Kleibeuker and T.J. Schaafsma, Chem.Phys . Letters [23] R.A. Uphaus,J.R . Norrisan d J.J. Katz,Biochem .Bio 29 (1974) 116. phys.Res .Commun . 61 (1974) 1057. [5] J.S.Brinen , J. Chem.Phys .4 9 (1968) 586. [24] U.Elia van d H. Levanon,Chem . Phys.Letter s 36 (1975) [6) H. Levanonan d S.Weissman ,Israe lJ . Chem. 10 (1972) 377. 1. [25] J.R. Norris,R.A . Uphaus, H.L. Crespian d J.J. Katz, (7) J.R. Norris, R.A. Uphausan d J.J. Katz, Chem.Phys . Proc.Natl . Acad. Sci.U S6 8 (1971) 625. Letters 31 (1975) 157. [26] S.J. van der Bent and T.J. Schaafsma, to be published. [8] M.C.Thurnauer , JJ. Katzan d J.R. Norris,Proc .Natl . Acad.Sd . US7 2 (1975) 3270. 91 9 The triplet state of photosynthetic pigments I. Pheophytins J.F.Kleibeuker ,R.J .Platenkamp ,T.J .Schaafsm a 9.1 INTRODUCTION Thetriple tstat eo fchlorophyll san drelate dcompound sha sbee n studiedextensivel y |1-6|.Th ediscover yo ftriple tstat ebacteriochlorophyl l inphoto-excite dphotosyntheti cbacteri ab yLeig he tal . |7| providedne w interesti nchlorophyl ltriple tstate so fbot h in-vitro andothe r in-vivo systems.Althoug hth erol eo fth echlorophyl ltriple tstat ei nth eprimar y stepso fphotosynthesi si sprobabl yo fsecondar yimportanc eunde rnatura l conditions,i tca nb euse da sa natura lan dnon-pertubin gprobe ,sensin g short-rangeinteraction so fpigment swit hthei r in-vivo surroundings.A n importantinteractio ni sligatio no fth ecentra lMg-ion ,an di ti stherefor e interestingt ostud yth eeffec to fth epresenc eo rabsenc eo fM g onth e tripletstat eparameters . Inthi spape rw erepor tth eresult so fa stud yo fth etriple tstat e ofpheophytins ,whic hca nb ederive dfro mth ecorrespondin gchlorophyll sb y replacingM g bytw oprotons .Fig .1 represent sth emolecula rstructur eo f thesepigments .Bacteriopheophyti nha sbee nshow nt ob epar to fth ephoto - CH.CH3 COCH3 C2 H5 C2H5 Fig. 1 a)Molecula r structure of pheophytins;X =CH „ for Ph a, X =CH O for Phb . b)Molecula r structure of bacteriopheophytin. 92 Fig. 2 Triplet state parameters inzer omagneti c field. Zero-field splittings between spin levels |T> , |x> , and |T> ar e defined by D and E;P ,P and P are thepopulatin g rates and k ,k and k thedepopulatin g rate constants of thespi n levels |T> , |x> ,an a |xy>, respectively; thespi n levels can be connected by spin-lattice relaxationw ,whic h has been omitted from this figure,fo r reasons of clarity. syntheticuni t inbacteria lreactio ncenter s |8|; forpheophytins ,derive d fromchlorophyl la an db ,i ti sno tclea ri fthei rpresenc e inpreparation s ofphotosyntheti c pigments fromplant s isnatura lo rartificial . Absorption spectrao fth epheophytin san drelate d chlorophylls exhibit slightdifferences .Krasnovski iha spublishe d phosphorescence spectra ofpheophytin sa an db wit h emission-maxima nearly coincidingwit h thoseo f thecorrespondin g chlorophylls 191.Apar t fromth eenerg y separation AET „ 10 betweenth elowes t triplet stateT. .an dth egroun d state SQ,ther ear e otherparameters ,whic har eo finteres tfo ra qualitativ e understandingo f theelectroni c structureo ftha ttriple t state:th ekineti c constants defining populating-an ddepopulatin g processesan dth ezero-fiel d splitting(ZFS ) parameters,define d infig .2 . ZFS-parametersca nb eobtaine d fromES Rusin g continuous ormodulate d optical excitationo fth elowes t triplet state.Kineti cparameter so ftriple t statesca nb eobtaine db yanalysin gth etransien tbehaviou ro fth etriple t ESRtransition susin gpulse d optical excitation|10| . Our results clearly demonstratea paralle l betweenstati can d kinetic triplet stateparameter s forchlorophyll san drelate dpheophytins . Thisparallel ,a swel la sth eeffect so fpyrrol e ring reductionan dsub stitutiono fcarbony l groupso nth eoute r ringo nth eZF Sparamete rD ar e explainedi nth eframewor ko fth e4-orbita lmodel ,s osuccesfull y appliedt o porphyrinsb yGouterma n |11, 12|. Longuett-Higgins'concep to flong -an droun d fieldmolecule s |13|whic h is,i nprinciple ,equivalen tt oth e4-orbita l 93 model,wil lb e frequentlyuse d inou rdiscussion s forreason so fclarity . Whereas ZFS-parametersca nb e related toth esiz ean dshap eo fth e ir-electroncloud ,a treatmen to fkineti cparameter s requiresa nadditiona l feature:th emixin go fni r orc m states intoth esinglet -an dtriple tstate s of interest,whic har eassume d tob emainl y of TTTT character. Throughout thispape rw eus e the followingabbreviation s forcom poundsan d solvents:Ch ia/ b =chlorophyl l a/b,P ha/ b =pheophyti n a/b, BChl= bacteriochlorophyl l a,BP h =bacteriopheophyti na ,tol/py r= toluene / pyridinemixtur e 9:1,an dMTH F= 2-methyltetrahydrofuran . 9.2 MATERIALS AND METHODS Pheophytinswer eobtaine dfro mth ecorrespondin g chlorophylls following Smithe t al. |14|. Chia an db wer eprepare d andpurifie d following Straine tal . |15|;Bch lwa s agif t fromDr .J.R . Norris. Solvents (MTHF, ethanol,toluene ,pyridine )wer eanalytica l grade.Al lsample swer e degassed -4 to 10 Torrbefor eillumination . ESRexperiment swer ecarrie d outo na Varia nE- 6 spectrometer; tripletswer eproduce db y illuminating thesampl ethroug h aslotte dwindo w inth eES Rcavity .W euse da Varia nVI X 150U VXeno n lamp,whic hcoul db e square-wavemodulate dwit hth eai do fa home-buil t electronic circuit.Th e timedependenc eo fth eES Rsignal swa s recordedo na H P 5480B signa laverager . Allmeasurement swer eperforme d at 100K ,usin ga ^-variabl e temperature flowcryostat .Hea t input,a sa resul to f illuminationwa smini mizedb yusin g appropriateoptica l filters;th e temperaturedifferenc e in lighto nan d lightof fperiod samounte d toles stha n 1K . 9.3 RESULTS Thetriple tES Rspectru mo fP ha intol/py ra tT = 100K usin g continuous illumination (fig.3 )differ s fromtha to fCh ia |5|i ntw o respects:firstly ,a s isapparen tfro mlin epositions ,P ha ha sa largerD - valuean da smalle rE-valu etha nCh ia (Table 1).Th esam e ZFS-parameters havebee n foundwhen ,instea do fcontinuou s illumination,th eexcitin g light ismodulate dan dth eES Rsigna l isphase-sensitiv edetecte d|5| . Secondly,Ch ia an dP ha ar e found tohav edifferen telectro nspi n polarisation (ESP)patterns .I nsuc hpattern sw edenot eb yA orE th eabsorp tiveo remissiv echaracte ro fan yo fth esi xpeak so fa A m= 1triple tES R 94 Hz- Hx- Hy. _L_ Jt _1_ _L_ H(Gauss ) Fig. 3 First derivative Am = 1triple t ESR spectrum ofP ha _i n tol/pyr at 100K , using continuous optical excitation. H -,H - etc.mar k field positions for transitions of molecules with canonical orientations H//z,H// x or H//y. Experimental conditions:microwav e frequency V = 9.1 GHz;microwav e power 2mW ;modulatio n frequency 100kHz ;modulatio n amplitude 16G . spectrumo fa nensembl eo frando moriente dmolecules ,measure dunde r conditionsa twhic h spinlattic erelaxatio n (SLR) canb eneglecte dw.r.t .th e populatingan ddepopulatin grate s |5,10| . If,however ,SL Rcanno tb eneglecte d theES Ppatter nca nb ededuce d fromth erelativ e intensitieso fpeak sa t thecanonica l fieldposition s |2,5| .I na nincompletel y relaxed spectrum, thepeaks ,whic har eemissiv ewhe nSL Rca nb eneglected ,hav ea smalle rin tensitytha ni na completel yrelaxe d spectrum,wherea sth eamplitud eo f intrinsicallyabsorptiv epeak s islarge rtha ni na relaxe dspectrum .Al l chlorophyll tripletES Rspectra ,obtaine dwit hcontinuou s illumination exhibitth epatter nEEAEA A 15, 161.Th epolarisatio npatter no fpheo - phytina i ntol/py rca nb ededuce d fromth etriple tES Rspectru ma t10 0K (fig.3 )t ob eA A E A E E .Not etha ti nth espectrum ,show ni nFig .3 ,Y is Table 1. ZFSparameter s of pheophytins and corresponding chlorophylls 4 -1 4 -1 4 -1 Solvent Compound Dx10 cm ExlO cm Compound Dx10 cm Ex1 0 cm MTHF Ph a 341+3 33+3 Chia 280+2 40+1 tol/pyr M 340+3 30+2 II 279+2 40+1 ethanol !l 334+3 26+2 It 272+4 32+2 MTHF Phb 358+8 46+5 Chib 294+3 48+1 j-54+5 MTHF BPh 256+4 BChl 232+4 58+3 ^37+5 95 emissivean dX haszer o intensity sincei ti so nth everg eo fturnin g from absorptive intoemissive . ForMTH Fan dtol/py rsolution s ZFS-parameterso fP ha ar ever y similar;th eamoun to fspin-polarisatio n inMTH F issomewha t less thani n tol/pyr:th eY transitioni sabou tt odisappear ,wherea sX isweakl y absorptive.W eobserv ea considerabl e changei nth etriple t state parameters ofpheophytin ,whe nusin g ethanola sa solvent . ZFS-parameters Dan dE ar e reduced (seeTabl e 1)an dth etriple tES Rspectru ma tT = 10 0K exhibit sa smallamoun to fspin-polarizatio n inth eY-line sonl y (Y 1QOG 3230G a e a Vwfa a e &f e<- — a e T !! A V w tV t >r V H2- HX- H Hy+ Hx* Hz* , r 1—H(Gauss i 1 ) > Fig.4 First derivativeA m= 1 triplet_ES R spectrumo fBp hi nMTH F at10 0K using continuous optical excitation.H ,H etc.hav e thesam emeanin ga s inFig .3 .Th espectru mca nb edecompose d intotw ospectra ,characterize db y the sameH + peak sbu tdifferen tH + an dH + peaks ;th eH andH peakso f 2— Y v—- X V both separate spectraar emarke d with (-)an d( );a an ae denot e absorptive and emissive character ofES Rtransition . Experimental conditions asi nFig .3 . 96 observed forP ha .Usin g 1kH zoptica lmodulatio nan dphas e sensitive detection, we findth eES Rspectru mo fBP hi nMTH Freveal sth epresenc eo fonl yon e species.It sspectru mi salmos t identicalt otha to fBP hi ntol/py ra sreporte d byThurnaue re tal . 131. Whereas fieldposition so fth esi xA m= 1 transition sar edetermine d byparameter sD an dE ,thei rrelativ e intensities (includingth esign )ar e determinedb ytriple t statekinetics .Fro m spinpolarizatio npatterns , observeddurin g continuous optical excitation,th erelativ e orderingo fstead y state spinleve lpopulation sca nb efound .O nth eothe rhand ,populating - anddepopulatin g ratesca nb eobtaine dfo reac hspi nleve lfro mth etransien t behaviouro fth etriple tES Rtransition s afterswitchin g lighto no rof f |2, 10|.A sshow nb yLevano n |10|th etim edependen t behaviouro fa triple t ESRtransitio n containstw oexponentials : -K-t -(3W + L) t S(t)= A e l +B e l (D whereA an dB ar econstant s determinedb yth erelativ epopulating -an d depopulating rates foreac ho fth ethre e spin levels;IC ,i sth emea ndeca y constant (1/3Z k i)o fth ethre e triplet spinlevel san dW th eSL Rrat e constant. Since"W turn sou tt ob efast ,th esecon d exponential in(1 )canno t bedetermine dwit h sufficient accuracy invie wo fou rinstrumenta l time- resolution,thu spreventin g completemathematica l analysiso fth etransien t S(T).A sa nadditiona l complication,th eSL Rrat edepend so nth eorientatio n ofth emagneti c fieldw.r.t .th emolecula r axes |2,17| .Usin ga nelectroni c circuitfo rsimulatio no ftransient s |2,16| ,w eobtaine dth erelevan t triplet statekineti cparameters .O fcourse ,a singl e transientca nb esimulate di n manyways .However ,a uniqu e solutionyieldin g one seto fkineti c parameters canusuall yb efound ,i fi ti srequire d thati tshoul db epossibl et osimulat e all6 transient sb ya singl ese tkineti cparameter san dassumin ga tth esam e timetha tW (|+ 1 > «- +| o > )= W (j- 1 > -s- >| o > )irrespectiv eo fth e orientationo fth emagneti c fieldw.r.t .th emolecula r frame.B yelectroni c Table2 Relative populating ratesP .an ddepopulatin g rate constantsk .o f the triplet stateso fP h aan dP hb _i nMTHF ,measure d with time dependent ESRa t10 0K . -i Compound sec % k k k P P P z z X y X y Ph a 1040+50 1300+100 820+100 31+2 43+5 26+6 Phb 590+50 870+150 420+150 30+3 48+10 23+9 97 simulationi ti sfoun dtha ta tT = 100 K W> 500 0se c ,i naccordanc ewit h previousmeasurement so nsimila rsystems .Tabl e2 collect sth eresult sfo r Pha an dP hb i nMTHF .Du et oth efas tdeca yo fBPh ,ou rinstrumenta l resolutionlimit sth edat ao nthi scompoun dt oit smea ndeca yconstan tK p (seeTabl e 3). 9.4 DISCUSSION 9.4.1 ZFS-parameters Forporphyrin slackin ga heav ymeta lion ,ZFS-parameter sar efull y determinedb yth espin-spi ninteractio n |18|;spin-orbi tinteractio nca nb e neglected. Wenot etha tth eZFS-parameter sD an dE reflec tth edistributio no f electrondensit yi nth etriple tstate ,sinc ethes eparameter sar erelate dt o themutua ldistanc ebetwee ntriple tspins : 3z D i gV < ^ 12 C2) L12 y x E= Ig V< 12" 12 (3) l12 Fig. 5 Molecular structure of porphin (a),dihydroporphi n (b)an dopposite - tetrahydroporphin (c);centra lmeta l iono r protons are omitted. ( ) denotes their-electro nconjugatio n path. 98 en a 01 V c en P5 ^O vO >Cl) fi cj •r-t .—t •H (3 (44 44 •H CD en CM CO A «^o + V4 eel Pi -C*M CM CO CO CM•• CN ^D — w>* X CO 44 CM P. C ^dl C (C)J CM w 4^= * 1 S3 CO CO CN no P 0—0 00 00 O—^ X S en + ^ 0^0 ~ o 1 o o o T) to VJ S C 6-—0 o p 3 sn T. I- M-t O <44 0 v« P — -' I 8 c en 4-t 6 o 1 99 Herer 17,x 17,y 17an dz 1? defineth emutua l distancebetwee n spinsan dit s components along threemutuall yperpendicula r axes.Fo rchlorophyll san d related compounds,x an dy ar etw oarbitrar y oriented axesi nth emolecula r planean dz i sperpendicula r totha tplane ;g i stake nt ob eth efre e electron value, gi sth eelectroni c Bohrmagneto nan d< >denote sth eexpectatio n valuecalculate d usingth eorbita lpar to fth etriple t statewav e functions. < D(: ) •gyW) «gx(£2) I b2 b2 b2 b2 °2u(t>1)°1u (a) (b) (C) (d) (e) Fig.6 Energy levelso fth etw oHOMO' s (b.an db„ )an dtw oLUMO' s (c.an dc„ ) of porphin (a),dihydroporphi n (b),P h a (c),P h b_(d )an dopposite-tetra - hydroporphin (Bph)(e) ,a sfollow s from thebasi c featureso fGouterman' s 4-orbitalmodel .Arrow s denotey-polarize d transitions important forC Io f the lowest excited state.Interactio n with twocentra l protonso ra meta l ionar eignored . 100 Fig. 7 Electron distribution of the twoHOMO' s (a, and a„ )an d two lu 2u LUMO's (e and e )o f D,, porphin gx gy 4h as calculated byGouterraa n |I2|.Th e size of the circles is proportional to the atomic orbital coefficients; dotted or closed circles indicate their sign. 02u(bl) Q1u(b2) sucha sporphin ,t oa typica l "long-field"molecule ,suc ha sBph .Th econcep t "long-field"/"round-field"ca nals ob eapplie dt oth eeffec to fsubstituents , whenthes esubstituent s appreciablyparticipat e inth e-rr-electro ndistribution . Piatt's theorypredict sa re dshif to fth eQ-ban d anda simultaneou s increase ofit soscillato r strengthfo rth eserie sporphi n-* •Bph ,i nagreemen twit h theexperimenta l data (seetabl e3) . Theeffect so fa chang ei nth elong-field/round-fiel d charactero n theZF Sparamete rD i nth eabov ementione d seriesi ssimila rt otha tobserve d forplana raromatic s |20|. Infirs torde rth eparamete rD depend sonl yo n thecoordinate so fth etw ounpaire d electrons inth etriple t state.Bearin g thisi nmind ,w ewil l limitou rdiscussio nt oth etw ohighes t occupied (HOMO)an dth etw olowes tunoccupie d (LUMO)molecula r orbitalsa sproposed , by Goutermani nth e4-orbita lmode l |12|.Th eparalle lbetwee nth e4-orbita l modelan dth eround-field/long-fiel dconcep ti sdemonstrate db yth efac t thatbot happroache spredic t strongconfiguratio n interaction (CI)fo rth e lowestexcite dstate so fporphi nan dwea kC Ifo rth ecorrespondin g stateso f Bph. Fig.6 arepresent sth eenerg y levelsfo rth etw oHMD' s (a1 and a2 )an dtw oLUMO' s (e ande )fo rD. ,porphin .A sdiscusse d inappendi xA , reductiono fon eo rtw opyrrol e ringsresult si na modificatio no fenerg y levelsa sshow ni nfig .6 b ,e . (Becauseo freductio no fsymmetry ,th elevel s aredenote db yb 1 (a^), b2 (a1u),^ (e ), andc 2 (e ) |12|.)Th eeffec t 101 ofsid egroup s -i nparticula r carbonyl groupspartakin g inth eir-electro n delocalisation -ca nb eunderstoo d following the samereasonin g asgive n in appendixA . Infirs torder ,a carbony l groupwil l stabilize thoseMO' swhic h have electrondensit y onth epositio nwher e this group is introduced; the amount ofstabilizatio ndepend s onth eelectro ndensit y onthi sposition . Taking intoaccoun t theelectro ndistribution s ascalculate d by Gouterman |12| (fig. 7), theeffec t ofth erin gV keto-group inP ha w.r.t . unsubstituted dihydroporphins and theeffec t ofth ealdehyd e group inP hb w.r.t.P ha o nth eenerg y levels canb e qualitatively understood. Fig.6 c,d shows therelativ epositio no fth eenerg y levels forth e twoHMD' s andtw o LUMO'sfo rP ha an d Phb . As shownb yGouterma n 1121,a goo ddescriptio n ofth e lowest excited state ofporphyri n likemolecule s requires the inclusiono fCI ,whic h mayb erestricte d toth e four toporbitals .A s discussed inappendi xA , for the lowest excited states ofdihydroporphin s and tetrahydroporphins it suffices toconside r theC Ibetwee n c.b . > (pureexcite d stateobtaine db y electron promotionfro mb. .t oc ^ and |c 2 b2 >•Sinc e theamoun to fC Idepend s on the energydifferenc ebetwee nth e interacting states,fig .6 a- e learns that CI between thesepur e statesdecrease s for the seriesporphi n -dihydropoxphi n- Phb -P ha - Bph . Itshoul db enote d that,sinc ew e didno t consider the size ofth e effects,th erelativ epositio no fP hb w.r.t . dihydroporphin cannotb e settled onth ebasi so fthi s argument alone;th e experimental results,a swil lb ediscusse d hereafter,poin t to adecreas e of CIfo rP hb w.r.t.dihydroporphin . Assuming thatth e lowest excited singlet andtriple t state approximately have thesam e zero-order electronicwav e functions,w ewil l showtha ta systemati c decrease ofth e ZFStriple tparamete r Dfollow s from the4-orbita lmodel ,whe nC I is included. Suchprediction s havebee nverifie d by Boorsteine t al. |20|fo rth e ZFSparameter s ofplana r aromatics. As demonstrated byVa nde rWaal s etal . |21|an d illustrated by VanDor p etal . |22|fo rporphi n freebase ,i ti snecessar y to incorporate many singly excited states forsufficientl y reliable calculations ofZFS - parameters.Wherea s infirs torde rmixin g ofth e states |a, e >an< ^ |a1 e >i nD. , porphin isprohibite d forsymmetr y reasons |18|,secon d order effects canmi x thestate s appreciably |23|.Fo r Cu-porphin |23|an d free-baseporphi n |18| itha s beenshow ntha t there isappreciabl e CI inth e lowest triplet state,wit h considerable contributions frombot h 4-orbital 102 excitedstate s |a~ e >an d |a. e >,formin gtogethe r^ 9( H fromth emixe d state.A sw e areprimaril y interested ina qualitativ e considerationo fth e effecto fmolecula r geometryo nD ,w ewil l restrict theC It oth e4-orbita l model. Withinth e frameworko fth e4-orbita l model (i.e.neglectin g CI with states involvinghighe rMO's )th eeffec to fC Io n ZFS-parametersca nb e qualitativelypredicte d using 2 2 - ^T =a |c.b1>T + (1-a) |c 2b2>T (5) asth eorbita lpar t ofth etriple twav e function ineq n (2);i t isreadil y seentha t 2 2 2 D =a D1 + (1-a )D2 +2a/(1-a )D12 (6) whereD. ,= D1 andD 2 tob enearl y equal,implyin g that achang e inD a s aresul to fa change inC Imus tb emainl y due toa variatio no fth ethir d term ineq n (6) only.Fro mthi sequation ,i ti sreadil y found thatth econtributio no fD. 2 hasa maximu mwhe na = 1/2,i.e .wit h 50/50 CImixing , inth ecas e that D1=V Under theseconditions ,eq n (6)predict s thata decrease inC Idu e toreductio no fon eo rmor epyrrol e rings inth eporphi n skeletonresult s in l 2 2 a decrease ofth eobserve d Dvalue ,whe nth eter m 2a(1 -a )D 12ha sth e same signa sD.. ,D 2- Forporphyri n systems this lastconditio n in fulfilled |18|.Thi spredictio n is inagreemen twit h theresult s collected intabl e 3 bearing inmin d thedecreas e ofC I forth eserie sporphi n• >bacteriopheophyti n mentionedbefore .Furthermore ,a sca nb esee n fromeq n (6),th eeffect s of changes inC Io nD ar e largestwhe nC I issmal l (a^ 0,a ^ 1)an dar e negligible forsmal ldeviation s from 50/50mixing . ZFSparameter s forporphi nfre ebas e are found tob e virtually independent fromth esolvent .D an dE value so fchlorophyll s |3,5 |an d 103 pheophytins,o nth econtrary ,ar efoun dt ob esmalle r forethano la scompare d toMTH Fa sa solvent .Becaus e thesam eeffect sar e found forchlorophyl lan d pheophytin,w eca nexclud e thepossibilit y thata chang eo fth e interaction between solvent andM g inchlorophyl li sresponsibl e forthi s effect.I t isplausibl e thathydrogen-bonding sar e formedbetwee n therin gV ketogrou p and thesolvent ,onl yoccurrin gi nethano l solution.Becaus e sucha ninter action stabilizes extra electrondensit yo nth erin gV ketogrou p oxygen, the effecto fthi sketogrou po nC Ii senhanced ,resultin gi na furthe r decrease ofD ,i nagreemen twit h theresult s collectedi ntabl e 1. Since the ZFSparamete rE i smor e sensitivet o local electron distribution and orientationo fth e in-plane axeso fth e ZFStenso r thanD , no reliable analysiso fth e effecto fC Io nE seem st ob epossibl ei nth e frameworko fth e4-orbita lmodel .Fo ral l systems,excep t bacteriopheophytin, we finda singl eE-value ,independen to fth emetho do fdetection .Unti lno w we haven osatisfactor y explanation forth e2 E-value sfoun d forbacterio pheophytin. Becausebot h spectracharacterize db ydifferen t E-values,hav e fullspi npolarization ,th ecorrespondin g transitions cannotoriginat e from a dihydroporphino rporphin-lik eimpurity ,sinc e for thisclas so fcompound s wed ono t expect full spinpolarization ,i nvie wo fth erelativel y slow triplet decay. 9.4.2. Electron spin polarization From theelectro n spinpolarizatio n (ESP)patter n found forP ha inMTH Fan d tol/pyrth eorderin go fth epopulatio no fth espi n levels canb e deduced: Ny ,' N z>N x whereN , N andN areth esteady-stat e populationso fspi n levels IT> , I t> and IT> inth eabsenc eo fSLR .Whe n relaxation canb e 1y 'x ' z neglected thesteady-stat e populationi sgive nb y \ =Pi/k j (7) where P.i sth epopulatin g ratean d k. thedepopulatin g rateconstan tfo r spin level |T-> .Fro m thekineti c constants giveni ntabl e2 on e derives relative spin level populationsi nagreemen t with themeasure d ESPpattern . 104 ForBp h inMTHF ,on e findsAA AEE E (1)an dAE AEA E (2)resultin g in (1)N z >N x,N y and (2)N z,N x >N y. Itha sbee nshow ntha t theamoun to fES Pdepend so nth e ratioo f the SLRrat econstan t andth emea ndeca y rateconstan t K™ |2|. Sinceth e decayconstant s forP ha inMTH Fan dtol/py rar ealmos tequal ,th edifferenc e inES Pha st ob eattribute d tounequa l SLRrate s inth etw osolvents .B y comparisono fth eamount so fES Pobserve d forbot hsolvents ,w e findtha t the SLRrat e inMTH F is^ 2time s larger than intol/pyr .Th e fullES P found forBp hca nb eunderstoo dbecaus eo f itsrelativel y fastmea ndeca y constant V 9.4.3 The kinetic -parameters Table 3compile s theresult sobtaine d inth epresen t investigation andpreviou s reports |2,6 , 24,25| , illustrating theeffect so foute rrin g substitutionan dreplacemen t ofM g by twoprotons ,o nth ekineti cconstants . 2+ Therear e severalstrikin g features inthes edata :substitutio no fM g by twoproton s significantly increases themea ndeca y constant K™an dsimulta neously increases therelativ econtributio no fth espin-leve l |x> i nth e intersystemcrossin gpathway .Furthermore ,th erati obetwee nkineti c constants forP ha an db iso fcomparabl emagnitud e totha tbetwee nth e corresponding chlorophylls. Ingeneral ,ther e isa remarkabl eparallelis mbetwee nth e magnitudeo fth ekineti c constants for theserie sporphi n free-base •*•Bp h andthei rM g containinganalogs .Finally ,i ti st ob enote d thatth e effecto freplacemen to fM g byproton s onth ekineti c constants decreases inth eserie sporphi n free-base- *Bph . Fora nunderstandin g ofth eeffect so fM g substitutionb y two protons andperturbatio nb y sidegroup so nkineti c constants ofth etriple t state,w emak eus eo fth econcept s outlinedb yAntheuni s |26|an dMet z|27| . Denoting the non radiative intersystemcrossin g (ISC)rat econstant s (i.e. populating anddepopulatin g rateconstants )t oan d fromtriple t spinleve l |x> b yK ,w ema yus ea sa startin gpoin t forou rdiscussio n the statement ofAntheuni s |26|,tha tther ear etw ofactor sdeterminin g themagnitud eo f K: a)Franck-Condo n (FC)overla p integralsbetwee nth e iso-energetic states T„ (v)an d S,(0) forth epopulatin gproces s andth estate sT„(0 )an d S„(v)fo rth edeca yo fth e lowest triplet state.Thes e integrals are independent ofth espi nstat e |x> . (Thesymbo l inparenthese s (o,v) denotesth evibrationa l state.) 105 b)A spin-orbi tcouplin g (SOC)facto rdependin go nth einteractio no fa particularspi nstat e |x> wit hth erelevan tsingle tstate . Inth efollowin gw ewil lb emainl yintereste di nth edeca ymechanism , sincew ehav eonl ydetermine dabsolut erat econstant sfo rdecay .Th eF Cfacto r dependenceo fth edeca yrat econstant sk isrepresente di neq n(8) . k 2i r C E S S 2 S E u -£ u l (V°^ o^)! $T (o) "v S (v)) K W -\TV u11 * > 'J 0C\ K } formulatedb yEnglma nan dJortne r |28|.C isth eSO Cmatri xelemen tbetwee n theelectroni cstate san dS| T (o);S (v)|i sth efamilia rF Coverla pintegra l betweenth evibration-les striple tstat eT (T isproduc tfunctio no fspi n andorbita lparts :T =T.| T >)an dth evibrationall yexcite dleve lo fth e groundstat eS withenerg yE< , , -.;summatio nextend sove rth evibroni cstate s coupledt oT .Eq n(8 )ca nb eworke dou tt ogiv eth es ocalle d energy gap law k C 2 u- u MhVAE ) exp(-YAE/hWm) (9) whereA Ei sth eenerg yseparatio nbetwee nth eelectroni cstate sT andS ; w isth efrequenc yo fth evibrationa lmode(s) ,whic har erelevan tfo rth e radiationlessproces san dy i sa parameter ,dependen to nth erelativ edis placemento fth epotentia lenerg ysurface si nth etw oelectroni cstates ,an d whichi sestimate dfo rplana raromatic st ob ei nth erang e0.50-1.3 1|28| . 2 i i Itca nb eshow ntha tonl yth eter mC isspin-stat edependen t |26|;thi s meanstha tth e relative participationo fth espi nlevel s |T> , |T> an d |x> isdetermine db yth ematri xelemen tC exclusively.Fo rth emea ndeca y constantKp ,C aswel la sth eA Edependen tpar to feq n (9)hav et ob etake n intoaccount . Eqn (9)ca nb erearrange di nth efollowin gway , In{k u.(AE^}= C ,- C 2 .A E (10) C 2 whereC ,= I n{ ^ (^)h m and C2= Y(ft») m)~• Fig.8 represent sa plo to fth edat afo rth eserie sMg-porphin/Ch la ,a swel l asfo rth eserie sporphin/P ha ,accordin gt oeq n (10).Th evalue sfo rA E areobtaine dfro mphosphorescenc edat a |9,29j .A sshow ni nthi sfigure ,th e 106 meandeca yconstant ,L = 1/3 Jk ,fo r theradiationles s decay ofphoto - syntheticpigment s and somemode l compounds follow the energy-gap lawwit h 3 y/ wm *10~ . Assuming thatt o corresponds toa CHstretchin gvibration ,suc h as hasbee npropose d byEnglma ne t al. |28| forplana r aromatics,on e finds Y^ 3wit h1 0 ^ 3000c m .Thi svalu e for y issignificantl y larger than found for aromatic compounds and also about oneorde r ofmagnitud e larger than thevalu ecalculate d forporphyrin s following Englman's theory |28|. This discrepancy indicates that it isprobabl y incorrect toassum e that radiationless T..- +S „transition s inporphyri n like compounds are determined byC— H stretchingvibration s only.A spointe d outb y Henry and Siebrand |30| it isno t only theC Hstretc hvibratio nwit hhighes t frequencywhic hha s to be considered; formolecule swit h a small energy gap,a s forporphyrins , C—C skeletonvibration sbecom e also important for the FC factor ineq n(9) , 106! 105. 104. 'KTj(AE>'2 (see"1, cm-'*) •AEx10-3 Fig. 8 Energy gap-law as given ineq n (10),definin g thedependenc e of tripletdeca y constant L,o nA E iE( T E(S ),applie d to photosynthetic pigments.Dat a points for the freebas e compounds:P h a inMTH F (A . = 935 ph + 10n m |9|,K ^= 1050+ 100se c ),P hb inMTH -1,F (A 900+ 10n m 9|, IC =63 0+ _12 0se c ),porphi n free-base inn-octan e (A 795+ 15n m 29 K„ 105+5 sec |24|)ar e indicated by crosses;dot smar k the data for theMg-containin g compounds:Ch i a_i nethano l (A, = 987 +_7 nm ) |9,16| , K^,= 1250+ 150sec" ' [16 1) , Chi a inMTH F (Afa = 96 0+ 5n m |16 1, 1 K = 630+ 4 0 sec" |16|), Chib inethano l (Ah = 929 + 10n m |16|, IC =41 0+ 30 sec"' |16|), Chib inMTH F (Ah=913+5nm |16|, KT=290+ 30 sec |2, 16|)an dMg-porphi n inn-octane , containing ethanol (A, = 730 + 15n m |29|,K = 14+_3 sec j251) . Error limits are indicated by rectangles. 107 ifthe yar erelate dt oa larg edisplacemen to fth epotentia lenerg ysurfac e ofth etriple tstat ew.r.t .th egroun dstate .Th eparticipatio no fC— C skeletonvibration si nth eradiationles sdeca ywil linfluenc eth econstan t Cji neq n(10) .When ,fo rexample ,instea do fu =300 0cm " oneassume s in tob e %140 0c m (frequencyo fC— Cskeleto nmodes )on efind sfro mfig .8 Y ""1.4 ,wherea sthi svalu efo rw , introduce di nth eexpressio nfo r ya s givenb yEnglma ne tal .|25| ,give s y ^ 0.5- 1 dependen to nth enumbe ro f modesan dth edisplacemen to fth epotentia lenerg ysurface . Inth eforegoin gi twa ssuppose dtha tth epre-exponentia lfacto r 2 C (eqn(9) )wa sconstan ti nth eserie sMg-porphin/Ch la .On eshoul drealiz e however,tha tth ereductio no fa pyrrol erin gan dth eintroductio no fcarbony l 2 groupsca naffec tth efacto rC becauseo fth eintroductio no fne wvibrations , inducingvibroni ccoupling swhich ,a swil lb ediscusse dhereafter ,determin e theradiationles sdecay .Th epresenc eo fthes eeffect si sclearl ydemonstrate d byth efac ttha tth erelativ eparticipatio no fth espi nlevel s| T>an d |T> i nth eIS Cprocesse si sno tth esam efo ral lcompound si nbot hseries , y 22 whichca nonl yb eexplaine db ya chang eo fth erati o| C|/| C| becauseth e FCfacto ri neq n(9 )i sspin-stat eindependent .I nvie wo fthi seffect , the linearrelationship ,show ni nfig .8 i srathe rsurprising ,sinc eth eserie s Mg-porphin/Chla canno tb econsidere da sa nidea lmode lfo ra chec ko fth e energyga plaw . Onepoint ,however ,i sclear :th echang ei nIC ,upo nsubstitutio n 2+ ofM g bytw oproton scanno tb eexplaine db ya chang ei nenerg yga ponly . Sinceth eM g /2H substitutionma ychang eth eenerg ylevel so felectroni c states,a swel la sth enumbe ran dth enatur eo fvibration soperativ ei nISC , 2 inth efollowinf c gw ewil lshortl ydiscus sth eorigi no fth efacto rC in eqn (9). Expressingth ewavefunctio nfo rth egroun dstat eS. ,an dth e lowestexcite dtriple tstat eT (u=x ,y ,z )i na Herzberg-Telle rexpansio n andretainin gonl yth eone-cente rone-electro nintegrals ,Antheuni se tal . |26|hav eshow ntha teq n(8 )ca nb eexpande das : T S C0) 2 0] 2 2 V u * Q} =? |CU | -F +£ |CU | .F + *L |Cu^| . F (11) whereF i sth eFranck-Condo n(FC )facto rcontainin gth eA Edependenc e resultingi nth eenerg yga pla wan dC ^ Jthroug hC ^ ^represen tmatri x elementsfo rvibroni cspin-orbi tcouplin gu pt osecon dorder . 108 As all compounds,discusse d inthi spaper ,hav e a TTTT lowest excited singlet and triplet state,th eter mC ^J canb eshow n tovanish ,a s longa s only onecente r integrals areconsidere d and anynon-planarit y isneglecte d |27|.Th eter mC ,however ,wil lb enon-zer o especially for thespi n levels f1 1 |T >an d |T> . Itca nb e shown |26|tha t C ^ x contains,besid e terms related to thevibroni c dependence ofth espin-orbi t interaction,product s of SOC matrixelement s <3fq,>,vibroni c couplingmatri x elements 1 <3C > . Retaining onlyone-cente r one-electron SOC integrals,th evibroni c coupling of S with singletai r, TO andrnr state swil l resultonl y innon-zer o contributions toC *• i andC *• ,C ^^ vanishe s inthi s approximation. For x thespi nleve l |T> ,i C *• J is(2 th)e firs' tnon-zer o term; itscontributio n to thedeca y constant k isexpecte d tob e smaller thanth econtributio n ofth e m termC ,sinc eher evibroni c coupling has to involve Sn aswel l asTV, . Thus,apar t from thedependenc e ofK ~o n theenerg y gapAE ,a s expressed by theenergy-ga p law,ther e are threepossibl emechanism s which canaffec t IC,,whe n themolecula r structure ischanged : (a)a chang e iny and/o ru will affectth e FCfacto r (seeeq n (9)); x x x 3t (b)a chang eo f theenergy-ga pbetwee nrnr , ai r or TO states and TTTT states affects the factorsC ^ and C ^ (seeeq n (12)); (c)a chang e inth enatur eo rnumbe ro fmodes ,responsibl e for thevibroni c couplingmatri x element 110 Untilno ww ehav eonl yconsidere d themea ndeca yconstan t K™. Wewil lno wprocee dwit hdiscussin g thedeca y ofth e individual spinstates . Sincew ed ono tkno wth eorientatio no fth e in-planeaxe so fth e ZFStenso r w.r.t.th emolecula r frame,w e cannot correlate thechange s inth erati o k /k withmolecula r structure.A sdiscusse dbefore ,theor ypredict stha t loweringo frnr states ,a swel la schange s invibroni ccouplin gmatri x elements,wil lmainl y influenceth e ISCprocesse s of |T> and/o r |T> becaus e ofa non-zer o firstorde r termC *• ' forthes e states (cf.eq n (11]].There fore,on ewoul d expecta decreas eo fth erelativ eparticipatio n ofth espi n level |T> ,whos e contribution toK -depend so nth e second orderter mC ineq n (11], inth etriple tdecay .Th e experimental results,however ,sho w an increaseo fk /K ™o nM g /2H substitution. Inth efollowing ,w ewil l give a tentativeexplanatio nfo rthi seffect . Inth e free-basecompounds ,tw oN-atom s dono tparticipat e inth e mostprobabl econjugatio npat h |12|.Thi sresult s inlocalize dTr-electro n densities onthes eN-atoms ,s otha tmulti-cente r TTTT SOC integralsn o longer canb eneglecte d as inMetz ' |27|an dAntheunis ' |26|work . Sincethes e integralsmainl y attribute tok 1261,th e increaseo fk /Kj,ca nb e attributed toth e introductiono fsuc hrelativel y localizedir-orbital san dno t toth e loweringo fth ernr state so rth echang e inth ematri xelement s Finally,w ewil ldiscus sshortl y theeffect so fpyrrol erin g reductionan d sidegrou psubstitutio no nth etriple tdeca ymechanisms .A s showni ntabl e 3,th erati oo fdeca yconstant s forMg-containin g andfree - basecompound s increases forth eserie sMg-porphin/porphi n• *BChl/BPh .Thi s effectca nb e fullyexplaine da sa resul to fchange s inth eenerg y gap AE (E(TQ] -E(S„]].A Smentione dbefore ,substitutio no fM g by 2H inporphi n results ina decreas eo fth etriple tstat eenergy ,wherea s asimila rsubstitu tioni nChl/P hcause sa sligh t increaseo fA E |9|,s otha tth eeffect so fth e substitutiono nth eA Edependen tpar to feq n (9]an dth eC dependentpar t onK ™reinforc eeac hothe rfo rporphin ,wherea s inChl/P hthes eeffect swor k inth eopposit edirection .Becaus esufficientl y reliabledat afo rth etriple t energy levelso fCh ia/P h aan dCh ib/P hb are lacking,w e cannot correlate the increaseo fK^/K ^ forCh ia/P h aw.r.t .Ch ib/P hb wit h shifts in tripletstat eenerg y levels.I tshoul db enoted ,however ,tha t thechang e in singletstat eenerg yo nM g /2H substitution (AO ,se etabe l3 ]i ssmalle r forCh ia/P h atha nfo rCh ib/P hb . Iti sinterestin g tonot e that fromth e increaseo fK p^/IC ,2 on ederive sa simila rchang e inth etriple t stateenerg y levels, 111 Whenconsiderin g theeffect s ofpyrrol e ringreductio n and side groupsubstitution ,w ewan t topoin tou t that thesechange s inmolecula r structurewil l inducea n inequivalence between twoset so fopposit e N-atoms inth echlorophyll s anddihydroporphin ,comparabl e toth eeffec t of the introduction oftw oproton s inth ecente r ofth ering . Forexample ,i nBCh l themos tprobabl e conjugationpat hfo rir-electron swil lno tcontai nth etw o N-atoms ofunsaturate d pyrrole rings.Th eir-electro norbital s localized on theseN-atom s can induce arelativel y largecontributio n ofth e |T> stat e to thetriple t decay,a s for the free-base compounds.A s is shown intabl e 3, experimentally iti s found indeed thatk /K ~ increases inth eserie sMg - porphin/BChl. Theredistributio n ofth eir-electro ndensit y forth eserie sMg - porphin/BChl,responsibl e for theinequivalenc y ofth etw oset s ofopposit e N-atoms,i sals oreflecte d inth echang eo fth epositio n ofth eQ-ban d (40 ) and thechang eo fth e ZFS-parametersD (Er^/D^),whe nM g is substituted by twoprotons .Bot hA O and YT&/VT2 decrease,a s shown intabl e 3.Thi s canb eunderstoo dwhe nw e realize thatth eeffec to f theproton so n thein equivalency between thetw oset so fopposit e N-atoms decreases inth e series Mg-porphin/BChl,becaus e ofth e increasing inequivalency already present in theMg-containin g compounds.A O evenchange s sign,sinc ebeside s the effect of induced inequivalence ofth e twoset so fN-atoms ,th e electronegativity ofth etw oproton s together islarge r thanth eelectronegativit y ofM g .As pointed outalread yb y Gouterman |11|thi sresult s ina blue-shif to fth e Q-bandan d thus ina negativ e contribution toA O . Comparing thevalue s of AO and IC,&/Y*Jh forCh ia/P h aan d Chib/P hb ,a swel l ask /K-fo rCh ia an d Chib ,w e conclude thatth e increase ofround-fiel d character,induce d by the introduction ofth ealdehyd e group in Chib/P hb ,i sprobabl y notrestricte d toth e fourorbitals ,considere d in Gouterman'smodel .Fro mth eresult s intabl e 3an d thebefor e mentioned theory,th eir-electro ndistributio nw.r.t .th e fourN-atom s ismor e symmetrical inCh ib thani nCh ia ,resultin g ina large r effect ofth e Mg /2H substitution inCh ib . 112 9.5 CONCLUSIONS 1. Theeffect so nth eZFS-pararaete rD o fsubstitutio nb ysidegroup so r reductiono fpyrrol erings ,occurrin gi nphotosyntheti cpigments ,ca n bequalitativel yunderstoo db yconsiderin gth econfiguratio ninteractio n betweenexcite dstate sconstructe dfro mth efou rorbital si nGouterman' s model|8| . 2. Steadystat epopulation so fth ethre espin-level so fpheophytins ,durin g continuousoptica lexcitation ,hav ebee nfoun dt ob edifferen tfro mthos e forth erelate dchlorophylls :th eout-of-plan e |T> spi nleve li n pheophytinscarrie sa relatively largerpopulatio ntha ni nth ecorre spondingchlorophylls . 3. Foral lthre espinlevel so fpheophytin sth eradiationles sdeca y constantsar efoun dt ob elarge rtha nfo rth ecorrespondin gchlorophylls . 4. Thedifferenc ebetwee nradiationles sdeca yconstant so fpheophytin san d correspondingchlorophyll scanno tb eexplaine db yconsiderin gth espin - independentpar to fth eenergy-ga plaw ,bu tca nb eunderstoo db y 2+ + includingth eeffect so fM g /2H substitutiono nvibroni ccouplin gan d theenerg yleve lo fni r states,relevan tfo rthi scoupling . 5. Arelativ eincreas eo fk inpheophytin sw.r.t .chlorophyll sca nb e explainedb ya tentativ emode linvokin glocalize d ITorbitals ,givin g riset onon-negligibl emulticente rTTT T SOCintegrals . 9.6 REFERENCES 1. R.H.Clarke ,R.H .Hofeldt ,J .Am .Chem .Soc ,61_ ,458 2(1974) . 2. J.F.Kleibeuker ,T.J .Schaafsma ,Chem .Phys .Lett. ,29 ,11 6(1974) . 3. M.C.Thurnauer ,J.J .Katz ,J.R .Norris ,Proc .Nat .Acad .Sci .USA , 72,327 0 (1975). 4. R.H.Clarke ,R.E .Connors ,T.J .Schaafsma ,J.F .Kleibeuker ,R.J .Platen - kamp,J .Am .Chem .Soc ,98 ,367 4(1976) . 5. J.F.Kleibeuker ,R.J .Platenkamp ,T.J .Schaafsma ,Chem .Phys .Lett. , 41_,55 7(1976) . 6. R.H.Clarke ,R.E .Connors ,H.A .Frank ,Biochem .Biophys .Res .Commun. , n, 671 (1976). 7. P.L.Dutton ,J.S .Leigh ,M .Seibert ,Biochem .Biophys .Res .Commun. , 46,40 6 (1972). 8. D.M.Tiede ,R.C .Prince ,G.H .Reed ,P.L .Dutton ,FEB SLett. ,65 ,30 1 (1976). 113 9. A.A.Krasnovski i jr.,N.N .Lebedev ,F.F .Litvin ,Dokl .Akad .Nau kSSSR , 216,140 6 (1974). 10. H.Levanon ,S .Vega ,J .Chem .Phys. ,61_ ,226 5 (1974). 11. M.Gouterman ,J .Chem .Phys. ,30 ,113 9 (1959). 12. M.Gouterman ,J .Mol . Spectrosc,6 ,13 8 (1961). 13. H.C.Longuet-Higgins ,C.W .Rector ,J.R .Piatt ,J .Chem .Phys. , _18» 1174(1950) . 14. J.H.C.Smith ,A .Benitz ,i n"Modern eMethode nde rPflanzenanalyse" , vol.IV ,eds .K .Paeck ,M.V .Tracey ,Berlin ,1955 . 15. H.H.Strain ,W.A .Svec ,i n"Th echlorophylls" ,eds .L.P .Vernon , G.Seely ,Ne wYork ,1966 . 16. J.F.Kleibeuker ,Thesis ,Agricultura lUniversity ,Wageningen ,1977 . 17. U.Eliav ,H .Levanon ,Chem .Phys .Lett. ,36 ,37 7:(1975) . 18. S.R.Langhoff ,E.R .Davidson ,M .Gouterman ,W.R .Leenstra ,A.L .Kwiram , J.Chem .Phys. ,62 ,16 9 (1975). 19. J.R.Piatt ,J .Chem .Phys. ,J_8 ,116 8 (1950). 20. S.A. Boorstein, M.Gouterman ,J .Chem .Phys. ,39 ,244 3 (1963). 21. J.H.van derWaals ,G .te rMaten ,Mol .Phys. ,8 ,30 1 (1964). 22. W.G.va nDorp ,M .Soma ,J.A .Kooter ,J.H .va nde rWaals ,Mol .Phys. , 28.155 1 (1974). 23. B.Roos ,M . Sundbom ,J .Mol .Spectrosc ,36 ,8 (1970) . 24. W.G.va nDorp ,W.H .Schoemaker ,M .Soma ,J.H .va nde rWaals ,Mol .Phys . 30,170 1 (1975). 25. G.Jansen ,J.H .va nde rWaals ,Chem .Phys .Lett. ,43 ,41 3 (1976). 26. D.A.Antheunis ,J .Schmidt ,J.H .va nde rWaals ,Mol .Phys. ,Tl_, 1521 (1974);D.A .Antheunis ,Thesis ,Universit y ofLeiden ,1974 . 27. F.Metz ,S .Friedrich ,G .Hohlneicher ,Chem .Phys .Lett. ,J[6 ,35 3 (1972);F .Metz ,Chem .Phys .Lett. ,22 ,18 6 (1973). 28. R.Englman ,J .Jortner ,Mol .Phys. ,J_8 ,14 5 (1970). 29. M.P.Tsvirko ,K.N .Solovev,'A.T .Gradyushko ,S.S .Dvornikov , Opt.Spectrosc ,38 ,40 0 (1975). 30. B.R.Henry ,W .Siebrand ,i n"Organi cMolecula rPhotophysics" ,ed . J.B.Birks ,London ,1973 . 31. S.Volker ,J.H .va nde rWaals ,Mol .Phys. ,32 ,170 3 (1976). 32. S.J.va nde rBent ,T.J .Schaafsma ,Chem .Phys .Lett. ,35 ,4 5 (1975). 33. U.Eisner ,R.P .Linstead ,J .Chem .Soc , 1955,3749 . 114 10 Properties of the lowest excited triplet stateo f chlorophyll 10.1 INTRODUCTION Inth eprecedin g Chaptersw ehav epresente dan ddiscusse d mostexperimenta ldata .Thi s Chapter containsa genera l outlineo fth e propertieso fth etriple tstat eo fchlorophylls ,base do nthes ean dsom e additionalresults . 10.2 PHOSPHORESCENCE OF CHLOROPHYLLS Phosphorescence spectrao fCh ia an db ,reporte db yKrasnovski i [1], leadt oth econclusio n thatth epositio no fth elowes t triplet isstrong lysolvent-dependent ,similarl yt oth ebehaviou ro fothe r triplet statepara meters (Chapter7 ,9) .W ehav e carriedou tsimila rmeasurements ,especiall y forcompariso no fth etriple tstat eenergie so fphotosyntheti c pigmentsi n MTHFan dethanol .I ntabl e1 th eresult sfo rCh ia an db ar ecollected .I n ethanolth eS„(0 )-< -T„(0 )transitio noccur sa ta longe rwavelengt htha ni n MTHFfo rbot hcompounds .Th epresenc eo fa naldehyd egrou pi nCh ib raise s thetriple tstat eenergy ,wherea si treduce sth esolven tshift .U pt onow , wedi dno tfin dan yphosphorescenc efo rBch lbelo w 1100run ,th elimi to fou r apparatus.Usin gth eenerg yga plaw ,a splotte di nfig .8 ,Chapte r9 ,an dth e meandeca yconstan t K~o fBchl ,measure db yClark e[2 ] (K„= 210 0se c ),th e phosphorescencewavelengt ho fBch lca nb eestimate dt ob e^ 110 0nm . Table I Phosphorescencewavelengt h (nm)o f Chi aan d b solvent compound MTHF ethanol Chi a 959+3 987+5 (985*) Chib 914+3 929+10 (912*) Krasnovskii [I] 115 Table2 ZFSparameter so fchlorophyll s (T= 10 0K ) 4 -1 4" -1 compound/solvent D x 10 cm E x 10 cm Chib inMTH F 294+3 48+1 tol/pyr 293+3 51+2 ethanol 286+3 37+1 Chi a inMTH F 280+2 40+1 tol/pyr 279+2 40+2 ethanol 272+4 32+2 Bchl inMTH F 232+4 58+3 tol/pyr 227+3 55+3 10.3 ZFS PARAMETERS OF CHLOROPHYLLS Preliminaryvalue so fZF Sparameter so fCh ia an dCh ib hav e beenpresente di nChapte r7 .Mor eaccurat emeasurement shav eresulte di nth e valuesgive ni ntabl e2 .Fo rbot hCh ia an dCh ib i nMTH Fth eZF Sparameter s aretemperature-independen ti nth erang e1 5- 10 3K ;fo rth eothe rsolvent s notemperature-dependen tstudie shav ebee nperformed .Furthermore ,optica l excitationo fCh ia i nth eQ-band ,havin ga relativel ylarg eextinctio nco efficient,result si nth esam eD an dE value sa sexcitatio ni nth eSore t band.Th eD -an dE-value sfo rCh ia an db i nethano lan dtol/py ragre ewit h thosegive nb yNorri s[ 3] ; forBch li ntol/py rw efin da slightl yhighe r valuetha nreporte db ythi sautho r[3 ]. Thedecreas eo fth eD-valu ei nth eserie sCh ib i nMTH F- Chib i nethano l- Ch ia i nMTH F- Ch ia i nethano l- Bch li nMTH Fparallel s thedecreas eo fD fo rth ecorrespondin gserie so fpheophytins .Simila rt oth e situationi npheophytin s (seeChapte r 9), thisreductio no fD ca nb erelate d toa decreasin gtriple tstat eC Ii nthi sseries .Again ,th eeffect so fC Io n E-valuesar emor edifficul tt ointerpret .Fro mth edefinitio no fE ,give ni n Chapter2 ,i tca nb esee ntha tcalculate dE-value sar estrongl ydependen to n theorientatio no fth ein-plan eZF Stenso raxe sw.r.t .th emolecula rframe . Asnote di nChapte r8 ,i nCh ia an db thes eaxe sma yb eoriente dquit e differently.Als ooptica lmagnetoselectio nexperiment sprovid eevidenc efo r arotatio no fth eZF Stenso raxe si nCh ib w.r.t .Ch ia [ 4] . 116 Table3 Kinetic triplet stateparameter so fchlorophylls ;k .represent sth e depopulating rate constantan dP .th erelativ e populating ratefo rspi n leve 1 |Ti> (i= x ,y , z); KT =1/3 Zk._ ; (e th. ethanol), -1 sec % compound solvent k k k P P P KT z X y z X y Chib MTHF 290+30 270+30 570+30 40+20 35+3 58+3 4+2 eth. 410+30 310+30 850+30 65+20 27+3 66+3 7+3 Chia MTHF 630+60 620+80 1120+20 140+40 31+3 60+3 11+3 eth. 1250+100 710+ 2710+ 370+50 25+4 65+4 10+2 100 100 10.4 TRIPLET STATE KINETICS OF CHLOROPHYLLS Usingth eelectroni cnetwork ,describe d insectio n 3.3.3,th e relativepopulatin g ratesP .an dth edepopulatin g rateconstant sk .hav ebee n deduced fromth eES Rtransient sb yanalogu e simulation (seeChapte r3) . Table3 present sth edat afo rCh ia an db i nMTH Fan dethano la t10 0K . From transient analysisusin g analogue simulationw ecoul d notderiv eaccurat evalue sfo rth eSL Rrat econstants .Generally ,thes erat e constantsar elikel yt ob edifferen tfo reac ho fth ethre ecanonica lorien tations,bu tno tmuc hi sknow nabou t thisSL Ranisotropy .Fo rCh ia i nMTH F we foundth efollowin g rangesfo rth eSL Rrat econstants ,whic hwer econsi stentwit ha satisfactor y simulationo fth etransient swit hP .an dk .value s 1 i i _i giveni ntabl e3 :220 0se c < W -<1340 0se c ,340 0se c -1 ."'< W *< 1000 0se c , 5000sec " 117 support suchconclusions ,sinc e itappear s tob ever y sensitive tosmal l changes inkineti cparameters .A s anexample ,th ecalculate dES Prati oR forOi la i nMTHF ,usin g theerro r limitso fk . andP . given intabl e 3, -1 -1 1 1 ranges from 72.3x (6W). to-91. 2x (6W) .Therefore ,n oreliabl einfor mationo nchange s ink's ,P' s orW' sca nb eobtaine d fromES Pratio salone . 10.5 CONCENTRATION DEPENDENCE OF TRIPLET STATE PARAMETERS Thedat acollecte d intable s 2an d 3refe r to 10-3 - 10~4 M chlorophyll solutions,wherea s thephosphorescenc edat apresente d intabl e 1 areobtaine dwit h^ 5x 10 M solutions.I norde rt ochec k concentration effects ontriple tstat eparameters ,w ehav emeasure d ZFSparameter s aswel l asY andY transientso fCh ia inMTH F and ethanol inth e concentration range 5x 10 -2 x 10 M. Inthi srang e theZF Sparameter s arefoun dt o be concentration-independent.Also ,th e transients arequalitativel y the samefo ral lconcentrations ;becaus eo f lowS/ N ratio,especiall y forth e mostdilute d solutions,w e couldmak ea qualitativ e comparisononly . Invie w ofth epolarit y ofth esolvent s andou rexperimenta l results,w e conclude thatou rES Ran dphosphorescenc edat arefe r tomonomeri cchlorophylls . 10.6 SOLVATION OF CHLOROPHYLLS Titrationexperiment sb y Evans et al.[5 ] haveshow ntha tth e Mg-ioni nchlorophyll s issix-coordinate dfo r^ 90° &o fth edissolve dmole cules intol/py rsolution s at 293K .Takin g intoaccoun t the temperature dependence ofth eequilibriu mbetwee nth emono -an dbiligate d complex,a s discussed forMgTB P inChapte r 5,i tseem s reasonable toassum e thata t T <10 0K chlorophyll s intol/py r arealmos tfull ybiligated . Theoccurrenc e ofbiligatio n isclearl y seen inth eQ -band shift inth eabsorptio nspectru m ofBch l[ 5] . Fo r themonoligate dspecie s thisban d isfoun d at ^ 580nm ,shiftin g to^ 61 0n m onbiligation .Fo rBch l inMTH F at29 3K w e found theQ -band at^ 59 0nm , indicating thatmos t molecules arestil lmono-ligated .Upo ncoolin g thissolutio n to 100K thi s band shifts to60 8nm ,whic hw e takea s evidence for thepresenc eo falmos t exclusivelybiligate dBchl .Sinc eal lES Rexperiment s arecarrie dou ta t thiso rstil l lower temperatures,th etriple tparameter s determined from these experiments arethos e ofbiligate d Bchl inMTH F aswel l as intol/py r solutions. 11* ForCh ia an db theeffec t ofbiligatio n isles s clearlyde fined.Comparin g theabsorptio nspectr ao fbot h chlorophylls inMTH Fan d tol/pyr,w eexpec tappreciabl ymor ebiligatio n forth elatte rsolvent ,a s was found forMgTB Pan dBchl .Ther ear e threemai ndifference s inspectra l behaviour oftol/py rw.r.t .MTH Fsolutions : a)a re dshif to fth eSore tband ; b)a smal lre d shifto f theQ -band; c)a smal lbu tdistinct,ban d appearing at63 3n m inth eabsorptio n spectrum ofCh ia i ntol/pyr ,whic hwa s attributed toQ byEvan s et al.[5] . All threeeffect s arei nagreemen twit h thetheoretica l discussion given in Chapter 5,indicatin g anappreciabl e amounto fbiligatio n ofCh ia an db in tol/pyra t 293K .Again ,a sfo rBchl ,w e expect thata tth etemperatur e of ourES Rexperiment s (T< 100K )Ch ia an db arebiligate d intol/pyr .T o determine theirstat eo f ligation inMTH Fan dethano l at 100K ,w ehav e studied thetemperature-dependenc e ofth eabsorptio n spectrao fCh ia i n thesesolvents .A s shown intabl e 4,lowerin g the temperatureha s roughly thesam eeffect sa sdissolvin g intol/pyr .Som ecautiou s commentshav et ob e madehowever .Apar t froma re d shift ofth eSore tban d forCh ia inMTHF , thisban d issplit ,whic h canb eattribute d toth epresenc e ofmono -an dbi ligated Chia inMTH Fa t 100K o rt oa splittin g of theSoret-ban d inB and B components.I ti sgenerall y accepted thatth eSoret-ban d atroo mtempera turecontain sbot htransition s[ 6] . A s discussed inChapte r 5,th eeffec to f ligation isexpecte d tob edifferen t forth ex an dy polarize d transitions, whichma y result inth eobserve d splitting.Furthermore ,th etriple t state parameters forCh ia intol/py r andMTH F areexactl y thesam ean dw ema y concludetha t inMTH FCh ia isalmos t fullybiligate d like intol/pyr .Recen t results ofKooyma ne tal .[7 ] usingODM Rtechniques ,indicat e thatth e ZFS parameters formonoligate d Chia diffe r appreciably from thoseo fth ebili gated species.Th epossibilitie s ofth epresenc e ofmonoligate d Chia i n MTHFa t 100K ,however ,canno tb ecompletel y eliminated.Unti lnow ,n o Am= 1triple tES R spectrum of themonoligate d Chia a t 100K wa smeasured , perhapsbecaus e ofa ver y lowstead y state triplet statepopulation . Inconclusion ,experimenta l tripletstat eparameter s are attributed tobiligate d Chia ;th eorigi no fth esplittin g of the Soret-band inth eabsorptio n spectrum cannotb e solvedwithou t furtherexperiments . Forethanoli c Chia solution s no splitting of the Soret-band isfoun d inth etemperatur e range 100-300K .Whe n thetemperatur e is lowered, there dshif to f theSoret-ban d aswel l aso f theQ-band , islarge r in 119 Table 4 Wavelength (nm)o f absorption bands (B I,B II,Q and Q )o f Chia -5 x y (^5 x 10 M) inMTH F and ethanol atdifferen t temperature (T). The two components of the Soret-band inMTH F at low temperature are denoted as B Ian d B II (see text); when only oneban d ispresen t it isdenote d as BI . compound/solvent TOO BI B II % Qv , Chi a inMTH F 293 432 - - 663 173 436 442 (626) 666 93 439 443 633 670 Chi a in ethanol 293 431 - - 664 173 444 - 643 670 93 449 - 649 675 ethanol thani nMTHF .Furthermore ,a strikin g difference withth eabsorptio n spectrao fOi la i nMTH Fa t10 0K i sth epositio no fth eQ -band .Wherea s inMTH F (100K )an dtol/py r (293K )thi sban di slocate da t-v -63 3nm ,i n ethanol (100K )th ecorrespondin g bandi sfoun da t64 9nm .Delayin gth edis cussion aboutth edifferenc ebetwee nMTH Fan dethano l solutionst oth enex t section,w eno wconside rth eagreemen tw.r.t .th etemperatur e dependence. There dshif to fth eSoret-ban d (430- *45 0nm )a swel la sth eappearanc eo f aQ -ban da t10 0K indicate sth epresenc eo fmostl ybiligate d Oila i n ethanola tthi s temperature,jus ta sw efoun dfo rth eMTH Fan dtol/py r solu tions. 10.7 GENERAL DISCUSSION Chlorophylls contain functional groups,whic har esuitabl e forman y kindso finteractio nwit hth esurrounding so rwit h other chlorophyll molecules, (seee.g .fig .1 ,Chapte r 1). Especially,whe ndimer sar econ sidered,th einteraction s betweenth etw oir-electro ncloud sha st ob etake n intoaccount .Thes e effectsar eo fimportanc ewhe ndime r formation through ligationo fth ecentra lMg-io ni simpossible ,suc ha si npheophytin s [8]. Usingpola r solventsw ehav e avoidedth eformatio no fdimer san doligomers . Thereforeth eeffec to fTT-T Tinteraction swil lno tb ediscusse d inthi s study. Inpola r solventstw okind so fpigment-solven t interactions havet ob econsidered . Firstly,th einteractio no fsolven tmolecule s with 120 thecentra lmetal-io ni nchlorophylls ;secondly ,interaction s with the carbonyl sidegroup s of thechlorophylls .Ther e are twomai n differences betweenthes e interactions:ligatio no fM g results ina ne t electrontrans ferfro m the solventmolecul e tochlorophyll ,s o that the strength of the interaction depends on thebasicit y of thesolven tmolecules .Th e carbonyl groups,o nth eothe rhand ,ar eelectro ndonatin g and interaction can take placeonly ,i faci dgroup s arepresent ,e.g . oxygenboun d hydrogen atoms inethano l or theM g -iono fa secon d chlorophyll molecule. Inaddition , the ligationo fM g isi nprincipl e anaxia lperturbatio n andwil lno tin fluence theshap e of theelectro ndistributio n in themolecula rplane , whereas e.g.hydroge nbondin gwit hon e ormor e carbonyl groups canhav e appreciable effects onth e in-planeelectro ndistribution . Both effects areno t only important inpola r solventsbu t also inin-viv o systems,wher e e.g. carbonyl groups ofprotein s can ligateM g , whereas (acid)hydrogen s are abundantly available for interactionswit hcar bonyl groups.I nth e following,bot h types of interactionswil lb e discussed, togetherwit h intramolecular effectshavin g comparable influences onth eir - electrondistribution . To study theeffect s of theabove-mentione d interactions separately, wehav e looked formethod s toexclud e oneo fth etw okind s of interaction.Th ehydroge nbondin gwit h carbonyl groups ispossibl eonly ,i f thesolven t contains appropriate hydrogen atoms.Consequentl y inMTH F and tol/pyr this interaction isabsent ;i nethanol ,however ,O Hhydrogen s easily formhydroge nbond swit h carbonyl groups of thephotosyntheti c pig ments. Iti sno tpossibl e toexclud eM g ligationb y choosing the appropriate solvent,sinc e carbonyl groups ofothe r chlorophyll molecules interactwit h thisM g -io nformin gdimer san d oligomers [9] , if thesolven t isno t able to ligatewit hM g .T oeliminat e solvent-Mg ligation inphotosyntheti c pigments,w ehav e substitutedM g by 2H ,th eresul tbein g apheophytin . Theconsequence s ofM g /2H substitution areextensivel y discussed in Chapter 9. The effects of ligation ofM g on theoptica lpropertie s of Mg-containing porphyrin systems are considered inChapte r 5.O n thisbasi s andnotin g theful l agreementbetwee n thetriple t stateparameter s for the chlorophylls inMTH F and tol/pyr at 100K ,w e concluded that the experimental tripletES R spectra refer tobiligate d species (seesectio n 10.6). ForMgTB P (seeChapte r 6)a swel l as for chlorophylls,th etriple t stateparameter s are nearly independent onth ekin d of ligandsboun d to thecentra lmeta l ion. 121 Forth emono-ligate dcomplexe s ofMgTB P (seeChapte r 6)an dOi la [ 7] , however,a larger ZFSparamete rD isfoun d thanfo r thebiligate dspecies . 4 1 4 1 (MgTBP.(MfflF) 2:D = 32 6x 10" cm" ,MgTBP .(MTHF) :D =34 4 x 1(f cm" in n-octane at4.2 .K ;Ch ia .L , :D = 280x 10-4 cm"1,Ch ia .L :D =30 9x -4-1 ~ l ~ 10 cm ,i nn-octan e at4. 2 K,wher e L is ligand (probably H?0)). Foral lphotosyntheti cpigments ,whic hw ehav e studied,a n appreciable change ofth espectroscopi cparameter s occurswhe nth epigment s aredissolve d inethano l instead of inMTH Fo r tol/pyr (seee.g . Chapter 7, 9). Since themode lcompound sporphi nfre ebas e andMgTBP ,lackin g carbonyl sidegroups ,d ono tsho wcomparabl e solventeffect s (seeChapte r 5,6 ,7) , we attributethes esolven t effects forbot h chlorophylls andpheophytin s to hydrogenbondin g of thecarbony l groupswith ethanol . InCh ia thre ecarbony l groups arepresen t (seefig .1 , Chapter 1). Only therin gV ket ogrou pparticipate s inth eir-electro ndis tributiono f thetetrapyrrol emacrocycle ,s otha thydroge nbondin gwit h this groupca naffec tth e^-electro npropertie s of themacrocycle .Althoug hhydro genbondin gwith theothe rcarbony l groupsma y occur,w e expect that these bondingswil lhav eonl ymino reffect so nth eTr-electro ndistributio no fth e ring andw ehav eneglecte d theseeffects . Beforeapplyin g themode ldevelope d inChapte r 9t oth eeffect s ofhydroge nbonding ,w e summarizewha t isobserve d experimentally: a)a re d shifto fth e0 absorptionban d[ 1 0] , especiall y at 100K an d a shift inth esam edirectio no fth efluorescenc e andphosphorescenc e bands [1]; b)a decreas e ofth eZF Sparameter ; c)a n increaseo f themea ntriple tdeca y constantK™ ; d)a shif to fth eQ -band inth e absorptionspectru mo fCh ia, measure d at 100K (forP ha n o effecto nQ isobserved) . Iti sinterestin g tocompar e theeffect s ofhydroge nbondin g with therin gV ket ogrou pwit h thoseo fth e introductiono fa naldehyd e groupsuc ha s inCh ib .Hydroge nbondin g toa carbony l group effectively increases thene tdisplacemen t ofelectroni c charge from therin gt othi s groupan d thereforeon eexpect s that suchbondin g or the introductiono f an extracarbony l group,conjugate dwit h theTr-electro nsystem ,wil l infirs t orderhav e similareffect so nth eir-electro nproperties .This ,however ,i s incontras twith theexperimenta l data:w.r.t .point s (a),(b) ,an d (c)th e effecto f introductiono f thealdehyd e group isopposit e toth eeffec to f hydrogen-bonding ofCh ia ;th epositio no fth eQ -band ofCh ib at 100K i s 122 unknown.A fullunderstandin g ofthes edat aca nb eobtaine d onlywhe nth e positiono fth eperturbatio no nth erin g istake nint oaccount . According toth econcep t of long-field/round-fieldcharacter ,a s proposedb yPiat t[11 ] and Longuet -Higgins [12], hydrogenationo fon eo r twoopposit epyrrol ering so fa porphi nmolecul eresult s ina nincreas eo f thelong-fiel dcharacte ro f the7r-electro nclou d (seee.g .fig .5 ,Chapte r9) . The introductiono fa carbony lgrou p indihydroporphi na trin g III (seefig . 1,Chapte r 1)wil l increase thelong-fiel d character ofth eir-electro nsystem , whenthi scarbony lgrou pparticipate s inth eTr-electro ndistributio no fth e ring.Th eviny lgroup ,o npyrrol erin g I,presen t inCh i awil l furtheren hance thelong-fiel d character ofthi smolecule . UsingCh ia a sa startin gpoint ,w ewil l consider theeffect so f hydrogenbondin go fth erin gV ketogrou pan dth eintroductio no fth ealde hydegrou po nth eTr-electro nsystem .Th ehydroge nbondin gresult s ina stabi lizationo felectro ndensit yo nth ecarbonyl-oxyge natom .Consequently ,th e ^-electrondistributio nattain s aslightl ymor e long-field character.O nth e otherhand ,th eintroductio no f thealdehyd egrou p inrin g IIcause s adis placement ofsom eelectro ndensit y towards thispyrrol e ring,resultin g in anincreas eo f theround-fiel d character ofth eTr-electro ncloud .So ,withi n thiscrud emodel ,hydroge nbondin gan d introductiono fa naldehyd egrou par e expected tohav eopposit e effects,i nagreemen twit hth eexperimenta ldata . Furthermore,th edirectio no fth eeffect s onth epositio nan d intensity of theQ-ban di sexactl y as isexpecte d fromPiatt' s theory[1 1 ]. I nChapte r 9w ementione d thata decreas e ofD upo na n increaseo f thelong-fiel d character isno trestricte d toth eporphyrin ,bu t alsoha sbee nfoun d for aromatichydrocarbon s[ 1 3]. Concluding ,i tca nb estate dtha tth elong-field / round-field conceptca nexplai nqualitativel y theeffect so fsid egrou psub stitutiona swel l as interactiono fsid egroup swit h thesurrounding s onth e propertieso fth elowes texcite dstates . Using Gouterman's 4-orbitalmode l[ 1 4] , th edat aca nb e cast into amor equantitativ e framework.A s discussed inChapter s 4,5 ,9 an dappendi x A, inthi smode l only thetw oHOMO' S and twoLUMO' s aretake nint oaccount . ForD. ,porphi nth eHQMD' s aswel l asth eLUMO' s aresuppose d tob edegener ate (seefig .6 , Chapter 9). Asbefore ,w e limitth efollowin gdiscussio nt o they-polarize dtransitions .Th estate s ofinteres t inth e4-orbita l descrip tion,ar e *?= aS|b1C1>S- (1 -as)i|b2C2>S ™ 123 T b c (1b * =a r|b1c1 >T - (1- 4^l 2 2 >T ' ) S T wherei K and ty areth efirs texcite d singlet and triplet state,respectively ; a„an da T areth eC Icoefficient s forth esingle t and triplet statesan d |b1c1 >, |b_c ~> representpur eexcite d states;th esubscript sS an dT denot e singletan d tripletcharacte ro fthes epur estates .W eassum e that1( 1 andij/. . correspond toth esam eone-electro npromotions ;a swil lb eshow nbelow ,th e experimental resultsprov e thisassumptio nt ob ecorrect . Defining EQ = |[E(|b1c1 >) +E(|b 2c2) ) ]an d 265 |E(|b1c1 >)- E(|b_c 2> ) with E(|b..c..> )bein g theenerg yo fth epur e excited state It^c.> w.r.t .th egroun d state,th eenerg yo fS. .i sgive nb y [14] E =E - (62+ z^y =E 2 , (2) bl U U 2 2ag (1- ap~ where ei sth enon-diagona lmatri xelemen t< c-b "|JC|c ?b->. Fo rD. ,porphin , with 6= 0 ,C I ismaximu m and a„= 2~2.A s shownb yGouterma n[ 1 4] , th e shifto fS 1,resultin g fromsmal lperturbation s ofth eporphin-rin gir-electro n distribution,ca nb eaccounte d forb y eqn. (2)wit hE „an d econstant . Although thehydrogenatio n ofon eo rtw opyrrol ering so rth e introductiono f carbonylgroup s ismor e thana smal lperturbation ,i nfirs torde rw ekee p E. and econstant .Then ,th eincreas eo f 6,resultin g ina nincreas eo fa,, , causes E„1 todecrease ,i nagreemen twit h theexperimentall y foundre dshif t ofth eQ-ban d forth eserie sMg-porphi n— Mg-dihydroporphin— Chib — Chia — Bchl (forenerg y levels,se efig .6 ,Chapte r9) . TheC Idependenc eo fth eZF Sparamete rD isgive nb y (seeChapte r9 ) D =D 0 +2a T(1 - a^y.-Du , (3) ifw esuppos e thatth eD-value sfo rth etw opur eexcite d statesbot hequa lD_ . D12 isth enon-diagona lter m< c.b. .|D|c_b ->. Asdiscusse d forth epheophytin s inChapte r9 ,th edecreas eo fD forth eserie s porphin— dihydroporphin— Phb — Pha — Bphca nb eattribute d toa decreas eo fth eC Ii nth etriple tstate . Supposing thatth eC Ii nth elowes texcite d singletstat ean d thelowes t excited tripletstat e isequa l (a,,= a^), fromeqn . (2)an d (3)i tfollows , D = D0+ E^T^- D12 W 124 -1 Infig .1 D i splotte dv s (En - £„,) .ro rt „tn evalu eobtaine dto r por- phini suse d toeliminat ea smuc ha spossibl e effectso fC Io fS _wit hothe r states,whic h isespeciall y importantfo rchlorophyll san dpheophytin s[6] . Thesurprisingl y goodfi to fth edat at oeqn . (4),indicate s thati nfirs t orderC Ii nth elowes texcite d tripletstat ei so fcomparabl emagnitud et o thati nth elowes texcite d singletstate . Asmentione d inChapte r9 ,Gouterma n[ 1 5] an dLanghof fe tal .[1 6] excludedth epossibilit yo fC Ifo rth elowes t excited triplet stateswithi n the4-orbita lmode lfo rsymmetr y reasons.Wherea s second-order effectsca n induceappreciabl eparticipatio no fbot h 4-orbital excited statesi nT [17 ], Goutermane tal .[1 8] suppose d thatth elowes texcite d triplet statei sa relativelypur estate .Thi s ideaha sreceive d support fromrecen t calculations byVa nDor pe tal .[ 1 9] t odetermin eth eD-valu ea swel la shyperfin esplit ting constantso fporphi n freebase .Th efollowin gpoints ,however ,suppor t theide atha tth eC Ii nT „parallel s thati nS. : a)Phosphorescenc e spectrao f"normal "Mg-porphyrins ,wit h strongC Ii n S1,ar echaracterize db ya 0- 0ban dwit h equalo rweake r intensity thanth e 0-1ban d[ 20 ], indicatin g thatth etransitio nT S is orbitally forbidden 0 justa si sfoun dfo rth etransitio nS Sn. This orbitalforbiddenes sca n onlyb eaccounte d forb ystron gC Ii nT ~[ 1 4] . Fo rMgTBP ,wit h relativewea k CIi nS 1 (seeChapte r 5), thestron g0- 0ban di nphosphorescenc e spectra[ 2 0] pointst owea kC Ii nT. . b)Ther ei sa paralle lbetwee nth eshif to fth e0- 0ban do fth efluorescenc e andth e0- 0ban do fth ephosphorescenc e (fig.2 )whe nth emolecula r structure ofa porphyri n systemi schanged ,e.g .i nth eserie sMg-porphi n— Chib — Chia ,o rwhe n interactionswit h solventmolecule s takesplac e (e.g.Ch ia inMIrI Fan dethanol) . Fig- ' Relation between energy level of JDx104em-1 ,.' lowest excited singlet state Eq] and ZFS parameter D following eqn. (4),fo r the 400 •/ series porphin freebase , dihydroporphin 1 / free base,P h b, Ph a, and Bph (.) and for the seriesMg-porphin ,Ch i b,Ch i a_,Bch l 1 / (x). For definition of E,.se e text. f / 300 1/ " (Eo-Es1)x10*cm 200 15 2.5 125 Fig. 2 Relation between energy levelo f (Mg-porphin) / the Lowest excited singlet state E„. and ETX10 " the energy level of the lowest excited triplet state E forMg-porphin , 13- MgTBP,Ch i b inMTH F (1J°Ch i b in ethanol (MgTBP)0 (2), Chi a inMTH F (3), and Chi a in ethanol(4) . 12 // //(2) ' /i3) ESlx10- Jcm-' / ,/(4) * 10 —i— -1— —i— 14 15 16 17 Asmentione d before,th ereductio no ftw oopposit epyrrol e ringsan d the introductiono ftw ocarbony l groups,a si nBch lw.r.t .Mg-porphin ,canno t be treated asa smal lperturbatio no nth eenerg y levels only.Thes e changes inmolecula r structurewil l affectth eM Owavefunction s andconsequentl yth e parameters e,D „an dD- .i neqn . (4).Bearin g thisi nmind ,th eagreemen t betweenth eexperimenta l resultsan deqn . (4)i squit e surprising.Fo rsmal l changes,e.g .th eintroductio no fth ealdehyd e groupo rhydroge n bondings withth erin gV ket ogroup ,th emode l clearly canexplai n theexperimenta l data.I naddition ,usin g thismodel ,w eca nunderstan d therelativ e smallD - valueo ftetraphenyl-porphi n (TPP)fre ebas ean dth eabsenc eo fa decreas e ofD whe non epyrrol e ring ishydrogenate d [2 1] . Thepresenc e ofelectro ndonatin g phenyl groups,boun d toth e methinecarbon s slightly destabilizes theM Ob 1 w.r.t.b- ,resultin g ina n energy levelschem ea sgive ni nfig .3a .C Iwil ln olonge rmi x |b1c1 >an d Fig. 3 Diagrams representing the relative <=1 energy levels of the twoHOMO' s (bj, b2) c and the twoLUMO' s (ci,c 2)o f tetraphenyl- 2 porphin freebas e (a)an d tetraphenyl- dihydroporphin freebas e(b) . b bi bi 2 b2 (a) (b) 126 Fig. 4 Relation between ZFSparamete r D and CI coefficient |a_| (a)an d relation between lowest exited singlet state energy 0L5- level E andC I coefficient |a |, according to eqns.(3) and (2).Th edashe d part of the curve (E„.-En)/£ in (b)represent s a region where thetheoretica l expression (2) is expected tobrea k down.Fo r definitions of E-, D_ and£ seetext . |c,b~ inequa lamoun ta sfo rporphi nfre ebase .Becaus eo fth elowe renerg y of |c?b, ,thi sstat ewil lcontribut emor etha n50 1t oS .an dT.. ,wit ha~ , a™< 2 2 ineqns . (1a,b) .Followin gAppendi xA ,th ereductio no fon epyrrol e ringresult si nth eenerg ylevel so fb, ,b ?,c .an d c? asshow ni nfig .3b . NowC Iwil lresul ti na majo rcontributio no f |c.b.. ,s otha ta„ ,EU , >2~2. Fromth eoutlin egive nabove ,i tca nb econclude dtha tfo rTP Pth ereductio n ofon epyrrol erin gresult si na shif to fD an dE„ 1 overth eto po fth e curvesgive ni nfig .4a ,b .Experimentall yi ti sfoun dtha tbot hD an dE^ , arenearl yequa li nTP Pan dTP Cfre ebas e 21,2 2, indicatin gtha tth e amounto fC Ii sth esam efo rbot hcompounds ,bu ttha tth eparticipatio no f thetw opur estate si sdifferen tfo rTP Pan dTPC .Jus ta sfo rth ecompound s discussedbefore ,th eamoun to fC Ii nT -i scomparabl et otha ti nS.. . 10.8 REFERENCES 1. A.A.Krasnovski ijr. ,N.N .Lebedev ,F.F .Litvin ,Dokl .Akad .Nau kSSSR , 216,140 6 (1974). 2. R.H.Clarke ,R.E .Connors ,H.A .Frank ,Biochem .Biophys .Res .Commun. , 71_,67 1 (1976). 3. J.R.Norris ,R.A .Uphaus ,J.J .Katz ,Chem .Phys .Lett. ,_3J[ ,15 7(1975) . 4. M.C.Thurnauer ,J.J .Katz ,J.R .Norris ,reporte da tth eBrookhave n symposiumabou t"Chlorophyll-proteins ,reactio ncenter san dphoto - syntheticmembranes" ,Jun e7-9 ,1976 . 5. T.A.Evans ,J.J .Katz ,Biochem .Biophys .Acta ,396 ,41 4(1975) . 6. C.Weiss ,J .Mol .Spectrosc ,44 ,3 7(1972) . 7. R.P.H.Kooyman ,T.J .Schaafsma ,J.F .Kleibeuker ,Photochem .Photobiol . tob epublished . 127 8. G.P.Gurinovich ,A.N .Sevchenko ,K.N .Solov'ev ,Spectroscop yo f chlorophyllan drelate dcompounds ,NTIS ,Springfield ,U.S.A. ,1968 . 9. J.J.Katz ,G.L .Closs ,F.C .Pennington ,M.R .Thomas ,H.H .Strain , J.Am .Chem .Soc ,85 ,380 1 (1963). 10. G.R.Seely ,R.G .Jensen ,Spectrochim .Acta ,21_ ,183 5 (1965). 11. J.R.Piatt ,J .Chem .Phys. ,JJS ,116 8(1950) . 12. H.C.Longuet-Higgins ,C.W .Rector ,J.R .Piatt ,J .Chem .Phys. , ^18, 1174(1950) . 13. S.A. Boorstein, M.Gouterman ,J .Chem .Phys. ,39 ,244 3(1963) . 14. M.Gouterman ,J .Mol .Spectrosc ,6 ,13 8(1961) . 15. R.L.Ake ,M .Gouterman ,Theoret .Chim .Acta ,J_S ,2 0(1969) . 16. S.R.Langhoff ,E.R .Davidson ,M .Gouterman ,W.R .Leenstra ,A.L .Kwiram , J.Chem .Phys. ,62 ,16 9 (1975). 17. B.Roos ,M .Sundbom ,J .Mol .Spectrosc ,36 ,8 (1970) . 18. M.Gouterman ,D.B .Howell ,J .Chem .Phys. ,6J_ ,349 1 (1974). 19. W.G.va nDorp ,M .Soma ,J.A .Kooter ,J.H .va nde rWaals ,Mol .Phys. , 28,155 1 (1974). 20. M.P.Tsvirko ,K.N .Solov'ev ,A.T .Gradyushko ,S.S .Dvornikov ,Opt . Spectrosc,38 , 400(1975) . 21. S.J.va nde rBent ,T.J .Schaafsma ,Chem .Phys .Lett. ,35 ,4 5(1975) . 22. G.Peychal-Heiling ,G.S .Wilson ,Anal .Chem. ,43 ,55 0 (1971). 128 Appendices APPENDIX A EFFECTS OF PYRROLE RING HYDROGENATION ON SPECTROSCOPIC PROPERTIES IN VIEW OF GOUTERMANS 4-ORBITAL MODEL Inporphi nwit hD. , symmetry theHOMO' S a* anda _ aresuppose d tob eaccidentall y degenerate75 ,wherea sth e twoLUMO' se ande „ havet ob e gx gy degenerateb ysymmetr y reasons (se efig .6 ,Chapte r9 ). Inlowe r symmetry, a1 ,a ~, e ,an de aren olonge r appropriate symbols for theMO' san dw e use thenotatio n b1(a~ ), b?(a1 ),c ,( e ), andc,( e ).Fig .7 i nChapte r9 givesth e electron distributiono fth e fourorbital sa scalculate db y Gouterman 1. Describin g thepur e excited states (e +-a ? ),( e -<-a )1 , (e -«-a - 1,an d( ev -<-a „) b yl e >, l e a,> , Te „a . >,an d| e a- > , gy 1ir' g x 2u^ ' a„'g y2 uy ' ' gx1 uy ' ' gy1 ux ' ' gx2 ux ' whereth esubscript st oth ebracket s define thepolarizatio no fth e optical transition,w enot e thatth e states le a0 > and'l e a. >,a swel la s 'g y2 uy ' gx1 uy ' 1lg ey 1 a.ux > and'l eg xa.2 ,u> x ar edegenerat 6 e inD„ ,4 porphinhF ^ .Th etransitio n probability for all four transitions betweenpur e states are equal.C Imixe s transitionswit h equal polarization, thus le a-> andl e a >,a swel la s H v ' gy2 uy 1 ' gx1 uy ' |e a^ > and| e a,> . Agai n forD. , porphin,C Iresult si n50/5 0mixin g and,consequently ,th elo wenerg y (Q-ban d )transitio n receives zero- transitionprobability ,wherea s thehigh-energ y transitionbecome s highly allowed. Reducing oneo rtw opyrrol e rings,a si ndihydroporphi n andtetra- . hydroporphin removes thedegenerac y between thee and e aswel la sbetwee n thea - and a? MO's.Th e changei nenerg y canb eeasil ypredicted : because the a., M Oha s electrondensit yo nC atoms (an dt oa les s extento nC „1 lu a 8 (se e fig. 7,Chapte r9 ) ,i twil lb edestabilize d w.r.t.th ecorrespondin g porphinMO ;th e a~ MO,however ,i sno t affectedb ysuc ha pyrrol e reduction. Similarly,on eo fth ee MO's,sa ye ,i sdestabilize db ypyrrol e ring reduction,wherea s itspartne re isno t affected.Thu sx -an dy-polarize d 129 transitionsd ono tbehav ei nth esam ewa yanymore .Fo ry-polarize dtransition s apyrrol erin greductio nresult si na re dshif to fth eQ -ban dan dasimul taneousincreas eo fth etransitio nprobability ,du et oth edecreas eo fC I between|c..b ,> and|c~b_ >. Th e|c-b ?> and|c~b.. > statesremai nalmos t degenerate,however ,an dC Ii sstil llarg eenoug ht oresul ti na ver ysmal l Q-ban dtransitio nprobability . Sinceth ereductio no fa secon dpyrrol erin gopposit et oth eothe r reducedrin greinforce sth eeffec to fth ereductio no fth efirs tring ,fo rth e seriesporphin-dihydroporphin -tetrahydroporphi nth eC Ifo rth elowes texcite d statedecreases ,resultin gfro ma nincreas eo fth eenerg yga pbetwee nth epur e states|c. b> an d|c~b,,> . 1.M .Gouterman ,J .Mol .Spectrosc ,6 ,13 8(196 1 ). APPENDIXB POSSIBLEES PPATTERN SI NA m= 1 TRIPLE TES RSPECTR A Theconcep to fth eelectro nspi npolarizatio n(ESP )pattern sha s beenintroduce di nChapte r3 .Ou to fth e2 =8 ESPpatterns ,whic hca nb e constructedwhe nzer opea kintensit yi sexcluded ,th epattern sA E E A A E andE A A E E A ar eno tallowed ,whe nonl ymonomolecula rprocesse sar eeffec tivei nth etriple tstat ekinetics .I nth efollowin gthi swil lb eprove nfo r thepatter n AE E A A E ;fo rth eothe rpatter nth eproo fgoe salon gth e samelines . Ifth epea kH isemissive ,the nN „ >N_ 1fo rH// xan dthus , Pn P P- P +P 0= x 1= y z m E F E k+ k • U3 Ox 1 y z Furthermore,whe nH ~i semissive ,the nN ~> N_ 1fo rH//y ,o r P P +P T? >F ^ C2) y x z Summationo fth einequalitie s(1 )an d(2 )result si n P +P p k +k > F~ x y z orN +1 >N .fo rH//z .Fro mthi sinequalit yi tfollow stha tH hast ob e 130 emissive andH " has tob e absorptive,bearin g inmin d that theH peak z + z corresponds to thetransition s |+1 «-»•| 0 andH to the transitions |0 *-•|- 1 •Consequently ,whe nbot hH andH areemissive ,onl y theES P x y pattern EE EA AA is allowed. 131 Summary The lowestexcite d triplet state T of chlorophyll a, chlorophyll b,bacteriochlorophyl l and corresponding pheophytins has been studiedb ymagneti c resonance and optical spectroscopy. Zero field splitting (ZFS)parameter s D and E,populatin g rates,an ddepopulatin g rate constants for the three triplet spin states of thesepigment s havebee n obtained. The magnitude of thetriple t stateparameter s for thevariou s pigmentsha s been correlated with thedifference s inmolecula r structure,especiall ywit h the extent and shape of the -rr-electron cloud. Inparticula ri tha sbee n shown thateffect s ofmolecula r structure on the ZFSparamete r D and on the energy ofT „ canb e accounted forb y Gouterman's4-orbita lmode lwhe n configuration interaction (CI)i nT „ and the lowestexcite d singlet state S1 iso fcomparabl e magnitude.Furthermor e itha sbee n found that the effect of substitution of the centralM g -ionb y twoproton s isessentiall y different from the effects of changes in the structure of theoute rporphyri n ring. The considerable increase of themea n triplet decay constant on substitution ofM g by 2H cannotb e accounted forb y the energy-gap law. Thepresenc e ofoccupie d non-bonding orbitals,a swel l as ofN- H groups inducing additional promoting vibrational modes,ar e assumed tob e responsible for the large increase of thenon-radiativ e decay rate.Ther e is experimental evidence thatbot h effects are important. Therelativel y stable and highly symmetric compound Mg- tetrabenzoporphin (MgTBP)ha sbee n found tob e a goodmode l compound for chlorophylls w.r.t. the effects of ligation on the lowest excited singlet states,bu tno t for the triplet state T„. Some specialpropertie s ofMgTB P are reported and discussed, i.e. the S~-fluorescencean d the splitting of the lowestexcite d statesS 1 andT „b yJahn-Teller "coupling . 132 Samenvatting Ditproefschrif t beschrijftd eresultate nva nee nstudi eaa nd e laagste aangeslagen toestandenva nee naanta l fotosynthetischepigmente ne n eenmode lverbinding ,Mg-tetrabenzoporphin e (MgTBP). Daarbij isi nhe tbij - zonder aandachtbestee d aand elaagst e aangeslagen triplet toestand T„va nzowe l chlorophyla ,b (Chia ,b )e nbacteriochlorophy l a (Bchl),al sva nd ecorre - sponderende pheophytines (Pha ,b e nBph) . Zoweld estatisch eparameter s (nulveld splitsingparameter sD e nE end eenergi eva nT „ t.o.v.d egrondtoestan d S„),al sd ekinetisch e parameters (bevolkingsnelhedenP .e nverva l snelheidsconstantenk .va nd edri e spincom - ponenten |T.> ,i = x ,y ,z )va ndez eparamagnetisch etoestan dT „ zijngemete n m.b.v.optisch e spectroscopie (fosforescentie)e nmagnetisch eresonanti e (ESR). Deeffecte nva nveranderinge ni nd emoleculair e structuure nva ninteractie s vanhe tmolecuu lme td eomgevin go pd everschillend e parameters isgecorre - leerdme td eelectrone n structuuraa nd ehan dva nhe tzgn ."lon g field/round field"mode le nGouterman' s4-orbita lmodel .I nbeid ebenaderingswijze n worden alleend eir-electrone nbeschouwd ,waarbi jword t aangenomenda tver anderingeni nd emoleculair e structuur,zoal sreducti eva nee npyrroo l ring en introductieva nee ncarbony l groep,evenal sd einteractie sme td eomgeving , kunnenworde nopgeva tal sstoringe no phe tir-electro nsystee mva nd e16-ledig e binnenrin gva nhe tporphin e skelet,da td ebasi svorm tvoo r allebestudeerd e pigmenten. MgTBPblijk tee ngoed emode lverbindin g voor chlorophyl,voorzove r hetd einvloe dva nligande no pd elaagst e aangeslagen toestanden betreft (Hfdst.5) .Voo rd estudi eva nd etriple t toestand gaatdi tnie t op,daa rd e eigenschappenva nd elaagst e aangeslagen triplet toestandva nMgTB P sterkver - schillenva ndi eva nd everschillend e chlorophylmolecule n (Hfdst.6) .I n hoofdstuk5 worde n tevens enkele speciale eigenschappenva nhe tMgTB Pbe - schreven,n.l .d eS - fluorescentie end eopsplitsin gva nd elaagst eaange slagen toestanden t.g.v. Jahn-Tellerkoppeling . Detriple t toestandenva nd efotosynthetisch e pigmenten zijn alleen inoplossin gbestudeerd . Naastd eMg-bevattend e chlorophylverbindingen ,zij n 133 de corresponderende pheophytinesonderzocht ,waari nhe tM g -ion isvervange n door 2protonen .Me tbetrekkin g totd estatisch eparameter s isdaarbi j het volgende gebleken: (a)d e substitutieva nM g door 2H doetd enulvel d splitsingsparamete r D toenemenvoo r allebestudeerd e systemen,terwij lhe t energienivea uva nT „ voorP ha e nP hb hoge r ligtda nda tvoo r de corresponderende chlorophylver - bindingen; (b)binne nd e reeksbestudeerd e chlorophyl-verbindingene nbinne n de reeks bestudeerde pheophytinesword tee nverban d gevonden tussend e energieva n S., de energieva nT „ end ewaard eva n D; (c)he t onder (b)genoemd everban d kanworde ngeextrapoleer d naard emode l verbindingen porphine endihydroporphin e eni s ookva n toepassing opd e effectenva nwaterstofbrugvorming ; (d)he t effect vand ereducti eva n eeno ftwe epyrroo l ringenva n hetporphin e skelet,he teffec tva nd e introductieva ncarbony l groepen enva nd ewater stofbrugvorming met carbonyl groepen,o p zoweld e energieniveau sva nS 1 en T„,al so pD kunne n inhe t 4-orbitalmode lworde nbeschreven .Hierbi j wordt aangenomenda td econfigurati e interactie inS 1 enT _ ongeveer gelijkis . Deonderdele n a-d zijni nd ehoofdstukke n9 e n 10nade ruitgewerkt . Ookd ekinetie kva nd etriple t toestandword t doord ebovengenoemd e veranderingen vand emoleculair estructuu rbeinvloed .Allereers t wordt dit duidelijk uitd e electron spinpolarisati e (ESP)va n Am= 1triple t ESR spectravoo rd everschillend e pigmenten,verkrege n onder continue bestraling met licht.Ui tES Pka nechte r geendirect e eenduidige informatieove rd ek.' s enP.' s wordenverkregen ;d emat eva nES Phang t sterk afva n spin rooster relaxatie (Hfdst. 7e n 8). Dekinetisch e parameters k. enP . zijnbepaal d uit de tijdsafhankelijkheid vand eES R signalenn ahe t aan-e nuitschakele n van het excitatie lichte nvergeleke nvoo r deverschillend e pigmenten. Hierbij kwamvas tt estaa ndat : (a)d e gemiddeldeverva l snelheids constante K„ster k toeneemtal she tenergie - verschil tussenT _ end egrondtoestan d kleinerwordt .Di t geldt,zowe l binnen deireek sbestudeerd e chlorophylverbindinge n alsbinne nd ereek spheophytines . Deze "energy-gap law"word tnade rbesproke n inhoofdstu k 9. (b)d everhoudin g tussen deverva l constanten voord edri e componenten slechts weinigverander t t.g.v.d ebovengenoemd e veranderingen ind e moleculaire structuur. (c)voo r allebestudeerd e pigmenten deverhoudin g tussen debevolking s snel- hedenva nd edri e spin componenten ongeveer gelijk isaa nd everhoudin g tussen 134 dedri everva l constanten. (d)naa r analogieme twa t gevonden isvoo r destatisch e parameters,d esub - stitutieva nM g door 2H niet tevergelijke n isme t de invloed vanver - anderingen aand ebuitenkan t vand eporphyrin e ring.Di tka nworde ntoege - schreven aand e aanwezigheidva n gevulde,niet-bindend e banen ind epheo - phytines enaa nd e extravibrati emogelijkhei dt.g.v .d e ind e pheophytines aanwezigeN— H groepen (Hfdst.9) . Debeschreve n resultaten illustreren datd e triplet toestand een gevoelige "probe"i svoo rklein everanderinge n ind emoleculair e structuur vand e fotosynthetische pigmenten,evenal svoo r interactiesva ndez epig - mentenme td e omgeving.D ewaargenome neffecte nkunne n kwalitatiefme t een eenvoudigmode lworde nverklaard ;di tmode lka n ooknaa rno gnie t bestudeerde systemenworde n geextrapoleerd. 135 Curriculum vitae In196 5he bi khe tH.B.S.- B diploma gehaald aanhe tRijk s Lyceumt eMeppel .Hetzelfd e jaarbe ni kd estudi e chemieaa nd eRijk sUniversi - teitt eGroninge nbegonnen .N ahe taflegge nva nhe tkandidaat s exameni nd e Fysische Chemie in1968 ,he bi kmi jo pd eafdelin gva nProf .Dr .J .Kommandeu r voorbereid ophe tdoctoraa l examen.Onderwij lhe bi ki nhe tkade rva nhe t bijvakBedrijfseconomi eee nstag egelope nbi jd eShel lRaffinaderi j tePernis . Nahe tdoctoraa l examen,afgeleg d indecembe r 1971,e nee n kortdienstverban dbi jd eRijk sUniversitei tt eGroningen ,be ni k1 augustu s 1972i ndiens tgetrede nbi jd eLandbouwhogeschool ,alwaa ri kt ewer kwer d gesteldbi jd ei noprichtin g zijndevakgroe pMoleculair e Fysica. Gedurended eeerst edri emaande nbe ni kwerkzaa mgewees tbi jd e werkgroepMolecule ni nd eAangeslage n Toestandaa nd eRijk s Universiteitt e Leiden. Naasthe tonderzoek ,beschreve n indi tproefschrift ,ha di kal s onderwijstaakhe tgeve nva nd ecollege s ChemischeBindin ge nFysic avoo r Biologen. 136