Biological Spectroscopy lain D. Campbell and Raymond A. Dwek University of Oxtord ‘The Benjamin/Cummings Publishing Company, Ine.oar GiOtes see es Re ate tea ‘ae Baja Cumin Pa Preface ‘The molecule hat make up the ving cell continue fo excite ret interes. ‘The methods availabe for iesigain these molecu hae improved lama cally he lst decade Some ofthe os powerful new techniques tnvoWe the ‘ppt ofeleomagnetie adation. These techukguss nce mice mag etic resonance and vine spevrs, which detect trations between cersy level, and others such as misroscopy, sealtetng, and fraction. In this book the tem spectroscopy i wed fo cove all thee methods, The elas laonehipe "tong thee tpi are rarely erphasae in extbooke; te reading level often tithe oe serial or too deta for most readers. Ths book est bring out the recurring concep inthis eld ado sere course etme he specialist et andthe intoauctony ‘Our overall si toad undergraduates, graduate tudens nd other rad esto an appreciation ofthe cure rature, Many people fod the concep Involved in speeoscopy athe diffi 50 ou approach bee if use worked ‘xamples and problems fo emphasize important points and ustat Bloc ‘spplaton rater than complex mathematical Jervations, Most chapters in his text deal with one partcular cchique and can thus be eead independently of tthe chapters, bu ne hive abo inloded a central chapter om comers and ‘efinions and varioos appendices designed give more information on diiul "We owe thanks to many people who have gen advice and ead chapters of te manuscript, Among thee ne should mention D, Ashford, P. Atkins, J Boyd, S. Easterbrook Smith, Z. Luz M, Moody, R. Parekh . Pion, S. Pe. ‘in, R Perit, TW. Rademacher, W.G. Richard. Sai, RI. Spon, B. Sutton, and A: Wats, We aso thank D. Kozlom, 8 Hoel, and F.Caddek forContents Prlace Lescr ' Teiemaehaietnscocay 2 ‘Spectroscopy: The Study ofthe Interaction of Bectomaguatc Radiation wih Mate 1 atest Polarization 7 Frequeney, Wavelength, nergy and Wavenumber. 9 ‘Type of Elctompnaic Radiation 10 tne 0 Tnterpartcle Forces nd aegis 13 ney Levels 14 "The Bec ofan Applied Magnetic Field (Csication of Enersies 18 Probing Different Pere with Different Ranges ‘of Blcromagnetie Radiation 15 Population of Enerey Levels 15‘acted t ‘Seutering and Inference 19 ‘Seaton and Refraction 21 ‘Blastc and Inelastic Satering 23 Absorption 24 Transition Probebiteand Selection Rules 28 Absorption Depends onthe Populations of Energy Levels 27 “Absorplon Spectra Depend on Concentration 27 ‘The Linewith ofon Absorption Line Depend on Lifetine 28 Absorption Spectra Depend onthe Direction ofthe Transition Dipole Moment 23. Opicaly Active Moteales Differently Absorb Lef- and "Right Creal Polarized Ration 29 Absorption Spectra Can Ars from the Reorenation of Magnetic Moments ina Magnetic ed 30 Basin 32 ‘Fuoresconce and Phorphoresence Are Two Particular Kinds ‘of Spontoncous Euston 34 ree 35 See omnes a vidos 9 pra oanterlnndspsie uuene etsy ‘Conventional Double Beam Specirometer 39 Pialicliitspers 1 Molecular Vibrations Tre Morse Curve 2 Vibrational Energy Levels. 43 Zero-Point Energy 44 Vibrational Energy Level Transtions 44 Poyiewions ‘Number of Vibrations 45 Vibrational Sess of Polyatomie Molecules 48 Solvent Etects on Spel Transitions 47 Sigestangte lanolin 2 omic isn Taner ace yp Cxb Cont eee evo cei ts rine 7 4 vn veers 8 in arin Fesmikad Sates Besoiebnins nT Serbs ‘Symmetry Considerations 66 Forbidden Transitions 66 Spin Considerations 6 ‘einen aging onan 8 Peptide Bonde snd Amino Ads 6 Prine and Pyrimidine Basesin Nace Acs 70 Highly Conjugated Systems 70 tet spe 12 id-edTranstions 72 ‘Charge-Transtr Spectra 7 Seba 1 Aleister 1hPeps aii sorte Db Mae ‘nites BeueTn Liacar Dichroism of Oriented Samples «2 ‘Dewrminaiono the Orenatin ofthe ronson Dipole 82 Determining the Orientation ofa Grow 83 ‘Resling Utrvilet Bands 83 Evclion Spliting 85 Hypoctromism and Hyperchromism 7 Pree ah 5 rns 8 omnes Piseieus 6 etsnmtEnsnge Terps 36 cuxunind Foeciaty 57 Fuseswcastin 8 espn ntact lfm foams Environmental ifetson 2 Environmental ffetson Quantum Yield 105 ‘Specie Quenching Proeses 105 Environmental Efectson Lifetimes 106 sore pene 7 ‘Dynamie Quenching: The Ster-Wolmer Reltonship 108 Florence Depolarization 110 SteadySiare Fuorescnee Depolarization: Photoreetion 110 Motional Depolarization 110 Time Resolved Depotesation of Puorescence nr tosmes berms yPasncngyT 1) (Caeaation of Ry 116 Focus 1 Pose ote 12 6 sees ® nner ttn a DaPernne ab Magia 19 econ 0 syctaPne aM 1S testy 133 Chemica Shite 134 Spinspin Coupingsnd Mule Stuctre 137 ‘The Reauation Tine and Linewidh 139 ‘The Reastion Time 14) acne? 1 7 Retaxation Proce 142 ‘Make Ondanu et eli Peace 1 Shit and Relation Prober 1 ApesineoMintoey 5) “The Assignment Problem i NMI Seles ‘ofMacromoleciles IS! Anaya! Usesof NMR 186 and Binding o Macromolecules 156 Ionization States andpHt 159‘Chemica Exchange Anas 162 ‘Concentration Versa Time 168 Suet Studies by NMR 164 | Molecular Motion 167 The Obsersarion of Powder Spectra from Membranes 169 Spatial Dseibation 172 ros 1s 7 ect . Onin 8 een Taemerdin 8) ase 9 SyesaPeamies ie alte He Fie te itis 18 Mungersmewsin rca pt Ss 8 pti a6 “Anisotropy ofthe pValue 186 Anisotropy ofthe Nuclear Hypesfine Interaction A189 | Tetons Diferat EPR Parameter May Have Ditferent Time Seles 191 Soba Effect of Rate of Motion on Spin-LabelSpecira 193 AnitoropeSpa-Labe Motion 195, ‘Quanttation of Amplitude of Maion: The Order Partmter 197 Lateral Ditasonin Membranes 19 ‘1 Contents cl Spinel Ligands Can Probe the Dimensions an Riiity ‘Binding Ses 200 Spn-Laba HypeineSpitings AreSensivetoPlarisy 202 smaton ofthe Separation Heeween Two Paramapnts Centrs 202 tein Spin-Orbit interaction: gValuesand Low Temperatures 205 ypertine Sucre 206 ‘eo-ldSpliting 207 eros 9 ‘Spin Trapping 210 peti 22 B sea ae min #7 indo 21 ‘thai onntstacith acip ‘eho | edie agn aig 8 Deveney ‘eset eae ow rm eran 2) ‘Scattering oma Rigid Array 220 Solton Scattering an Information on Molelar Weight 221 Santon Legis 2 Nid oacpisceng 2 Piston, 2 ‘The Guinier Plot 224 The Zinm Plot 225 ateurmbaeateetige ES Senge 20 Cont nnkawoesetog 2eBlog 2 “Tubicity 231 Refracive Index 232 ‘The Frequency Dependence ofthe Refractive Index Dispersion 233 ‘The Directional Properties of the Refractive nde: Biefingonce "244 Dyan isaneng Specrum Analyzer or Otel Mining Spectroscopy 235 Intensity Flastation or CoreationSpeoscons 257 eee at Y searstg ® nner 28 oat Steg 20 Peete ‘con cansetng 20 noe ernteng 20 puineoantee 2 Peston 243 Imensity 244 painless) ‘Conventional Raman Satering 246 Resonance Raman Scattering 247 Pre Revonasce Raman Scuterng 250 ede 2 10 oressy = oo mo Pr conte pe set Pg 28 nab 57 annaetslO Vensenet1 8 Fara rane ety st Cys oops 28 crating Chaney Tian cDDei ey Sve amotio ie 8 lesan 0 te erse0py noe 28 ottin 28 econ Tahletniin Fasligtone a enmenaion Tenses 282 Magnitition 283 “The Ligh Microscope 284 ‘TheBlesron Microscope 204 DePsrendserpeeMy 2 Cries “The Light Microscope 288 ‘TheElectron Microscope 290 stings "The Light Microscope 290 ‘TheEleccon Microscope 291Contents hastTrigas oe wp) 82 Underocusag 0 Produse Phse Contrast 293 ‘Optical Fikering Teebriques 293 Image Reconstruction 294 Combination of Fleciron Ditton with Miconony 294 TeFurnceconge 8 bene 6 12 cin * nee toe cides Ditraction 200 Laces 300 ‘The Reciprocal Latise 302 Fourier and Opa Transtorms 302 Convolution 303 The Braga Eavaion 205 unnnliace Sources of Rasiaton 305 “The Sampeln the Diteacton Experiment 306 Detection ofthe Dittaction Pattern 306 agian Direc Inerpretation of itiation Data 307 Dewrminaion of Molecular Weight 207 The Symmetry of the Molecule 308 Digfracton Pairs fram Fibers 308 Determination of Molecule Stratire 311 Fowler Synths, Fourier Transformation and “Paterson Functions 311 he Phase Problem and Ie Solution 314 Determination of Molecular Structure from px 2). 317 Refinement 218 Contents sl esr m0 ‘Methods Ued to Depist -D Potsin Streets 322 CCasficaton of Proteins 324 ‘Temperature Factors and Protcin Mobility 325, The Validity of Protein Crystallography 326, psyco Tenis 2 Ceytalosraphy of Nucleotides nd Polymuclecdes 526 tens 1 3 Other Spectroscopic Methods 3 awe Spt) Peet Speman Pact Snap) rebecca a hy eon ase AS) CrrhtanltnLmkecowe ER) Mt A Stony Appendixes | Maamatia Functions 348 Complex Variables 43 Exponential and Trigonometi Functions 348 Vestors 44 4 Oseiators 245 Simple Harmonic Osllaion 8 Dampsd and Fors! Onions 346 IM Waves and Tor Sepopostion 98 NV Wave Mectanies 51 Vom and Molecular Oitals 358 VL Some Concepts of Cyst Fel Theory 58Vil Fourier Transm and ComotonFuctins 359 Vil Magnetic Properties of Mater 61 1K Dipols andthe Inrctn Between Them 62 Tannen Bomemoes “Two Parallel Doles 363, ‘Two Dipols ins Oeneral Conformation 365 X Spec of nterating Diners 96 General References 969 Solutions to Problems 873 Index 391 Biological SpectroscopycHaTer | Introduction Mos objects we se in every ie ae vse only because they remit part of {height that falls them ro some source, sch asthe sun erpeation of Is eflected or transmit light cap yield a wealth of information not only ‘Bout the color std shape ofan objet but ao about the atomic and molecular imechaissiavelved when lit intra wt the objet. Tigh infact a form of electromagaete radiation and this Book is con- cerned with ie study ofthe interaction of tromagnetic waves with mate and ow thiscan be wed to extract information abou ological molecules and els ‘letomagnsis radation ‘covers an enormous Tange of wavelengthy (eversiey, Irequenci). The two exaemes iti ange ae way taken © be aslo waves, with wavelngts around [0"* mand gamma rays, wih wave Tenehs around 10", Vinble ight overs aves sal range, 47x 10~ ‘When Tight ieracs with an object, we can normally se only refeted or «anited radiation. Tice phenomena tht occur when electromagnetic ads tion imtracte with ater can be defined more precely as eatterng (he Sys blue Because uctuatingpartelesin the atmosphere scatter lu light more ‘han eight absorton, eg eg absorb by apiece f gas cases he {tanuited ight fo apes us), andemison (ea. fluorescent dye may emi {dwn light afer aborbng Blue Up). Another eat ofthe iteration of ee ‘Homagnei radiation wth malteris photochemistry. Tis obwousy eteely Important in logy (chain vison and pbetonyabes) but thi aspect snot ‘Sat vith bere, "We thus define spectroscopy at she study of the interaction of electromagnet le cation with rater, excluding chemical effects. For the purposs of this books neutrons and elrone are considered to gv set eestromapnet adi ton, although this ena cy coeect ee Chapter 2) “Al he techngusedesrbed inthis 800k vole (radiation of «sample ian some fort of lsttomaenetic radiation; 2) measurement ofthe catering, stuopton, or emison in term of some measured parameters (6. eating Inteny at Some ange fy extinction coeficet st pica wavelength, oF2 Conaptert inoaveton ww faa Spcxcny ie iain nga wane mot \iteeshown esate process ot fuorercerenmitom asecton an NAR (sheomnon sncum aspetan ard tracton arom aca ‘ont ot sanotonessn ar scater ast ee rma ‘uorscent etme); and) he interpretation ofthese measured parameters to tive uel biologie information, This lat sage requtessome understanding of the physical bass of the iteration, whether ite seterin by eeetrons oul, bsorpton by excitation toa higher vbatonal evel, or emision fom ripe Ste. Aspects ofthese thee aes of spectroscopy are usteated i Pgwe I. “The book is aranged as a seis of chapters a differen tecaigu, which ‘measre diferent phenomena, except for Chapter 2, which deals wih enezp tnd deiniins. Each chapter canbe rea akmos independent, hot wl sm times beneesary abd helpful to wie Chaper? andthe Append “To normation valle rom Spectroscopy 3 ‘THE INFORMATION AVAILABLE. FROM SPECTROSCOPY etalled study of satel, absorption, ad emission yids biological itor ‘on of varow kinds. Ts information ean be broadly clase sutra, {nami energie ad anal Te inforsaion avalable depends om th in ‘Xrumen uted tomas the menseremens. While the eelsexceptonal powerful {ind venatle,inrumente sich at the microscope or the spectrometer on ‘ihanee and quaniy the information discernible, Each echaigue ba ferent. Sthantges and advantages, bot experimentally and i the interpretation of the measurement. Some sesroncopie tecnigues ae raed (sing ars) in ‘Table - om thelr bility to give the diferent kinds of information "Tn ba techniques for determining srbture othe coordinates of log cal system are rosony and fraction. Light microscopy ia technique that {in ge structural information dieey and noninvasively abou ving 92m, butte resolution that cam be achleved (~ xm fen suet to std ini {al molecules. Electron mlrorcoy can aie higher resolution (~2 am), but {he stmple mst be stoi faa vacuum and Is normaly covered witha malic Sti, whlch cases a problem with eqard tothe integrity ofthe uct dtr nino Diffaction studies of cytle of pre macomolerles ange stoctral Fnformaton tothe stoic level (~0.15 nm), bat hi achaigue require crystal, fnd the structure leno longer obtained dey Buc must be inerpreed from a observed faction patter. ‘Some ecnigues that cu be used to study macromolecular structure in so tion ncade epic aivy measurements, which pv information on secondary Structure of proteins, forscens and lear magne resonance (NMR), ‘thick can give information sbou interactions betwee pis of enters: elton ‘aramagneticreonance (EPR), and resonance Raman, which can ingerprint”™ ‘iain types of structure around etal on or chromophore; ad solution set {erngs which an give iaformation abou the overall shape ofa molecule nso tion The srsctsal formation avaiable from hse methods nearly always auivora although te ofen very uel nd important becuse ean be ob {sind ia olution and can be combined wih other information om energcis and ‘yma ‘Dynamic information about bolgial stems can be obaind in vate cof ways: The best mathods are hose that gv informatio a slut, Examples Se dynamic light scattering ster of chemotaxis by baci, fuorscence de- Polaraation sade of the rotational difuson of macromolecules, EPR sin Tat studies of ip Midi, and studies of the movement of flurescent label with the faorexence mirocope Information about energetics can be obaine by studying the in uence of environment, such as temperature, ligand concentration, pH, ad fone tenth ‘or the syste The techniques of UV ible spectroscopy, Naorescene, opal Stviy, and NMR are al good fr disngshing beeen bound and unbound forms ofa lgand or 8 macromolecule, diferent lonization sas, and diferent sHrctural form of a macromecle.T° The information Available from Spectroscopy 5 “The bes methods to obtain anata! formation, by which we mean the ‘deniication of a parcula compound and the determination of concentra tin, are UVisisile spectroscopy, NM, and omic absorption spectroscopy (AAS), should also be noted that different methods operate bes init ‘Soveeiraton ranges. NMR s best for studying change nthe milimolat fang, le orescence Is used to study much lover concentration (~1 pM}. Irom he different metods. A deal study of Holga system wil nvlve the application of Several diferet techniques Serre Hal, li Flas a2 § inCHAPTER, 2 Spectroscopy: The Study of the Interaction of Electromagnetic Radiation with Matter WHAT IS ELECTROMAGNETIC RADIATION? -lestomagntic radiation can be considered o behave a wo wave motions (= ‘Apron Xa ight angler to each oter (Figure 2.1), One ofthese waves ‘Mogntc Gh) andthe other elec ().Eletomapnsi waves ae generated {yrosllaing electric or magnetic poles and are propagated through avacaum ihe vloty of gh (c,h energies associated sth # and Mare equal, but now opti effects are concerned with the electric Wave.” Polarization. Since the E- andthe Micomponeats ar alvays perpendicular fo voter ulin, n man eases, to consider ony the component inde Fee 21esvomsanete rations tog eat by 1 Wh ansptor2 Spectroscopy: f@ C) @ owe 22 Orecton ote crc vetripotran td np hn ‘olreegh or arty clare gmt onshore paca a Secrest dstorettor pas pay ae scribing the wave. Although the amplitude ofthe F-wave shown in Figure 2.1 os lates inthe plane, it could osllate equal wel in any dietion perpendic lac tothe dirstion of propagation). Unpoarized light contin ossiations of the components in al dections perpendicular fo the drt of propagation. Plane, o:Hneart, polarize radiation has E-xilations in only oe plane, tis convenient to inroduce a parameter ald the degree of plaiation (P) to describe situations where the radiation ie patalypolared (Pgure 2.2 wat) Gan where Fad, ate the fetes of the eatin resolved in dzetion parte 2nd perpendicular wo the dreton of paral polaration Plane-goarzed radiation ofthe sr pictured in Figure 2.20) can be cond ‘ted to aie fom a soure that osiltes parallel othe xn I the sure als ‘slates paral othe aus inthe same se, then the wo wives superimpose (Gee Appendin ID to produce another plane plained wave, Forename ithe ‘wo waves have he same amplitude the new wave willbe oie at 45" he = fs. Whe the oiations are nota the sme pate, then the superposition of "he two oscllalons does nt fal to afte dren for For xan, he phase diferene Is x/2 a8 shown in Figure 2.3, then the path ofthe econ Fle, When the amplitudes ofthe two waves are egal ad ifthe phase fer. ences +/2, then theresa wave ld tobe srealary polarized, I the two somone of esilation along x an yee unequal in ampltude, heaters ‘ant wae is elpteally polarized eee o (a) E, & y 2 Fg, Maton now ans lesley rene Frequency, Wavelength, Energy, and Wavenumber ‘The frequency (and wavelength (R) ofa wave ae elated by the equation where cs the veoety of propagution af he wave. For tromagnetic radiation Ina vacuum, o= 3 10" mes". Frequency can ako be converted dire t0 ‘nits of energy, sing the relationship #'~ fy, where hw Planck’s contant (= 663 10 Jos). Units of energy (Jomo, fequeney (Hz), and wave lenght (a), ae all wen dcssone of sletomagniti adn “Expression of the radiation asa frequency Ha) vs results with very large sumbes; therefore it is eommon to find, partulry for eetromaetie ad tin inthe mirowave to Xa) rane, te frequency exposed ara wavenumber (co), The wavenumber") deflned as the inverse a he wavelength in cen ave ‘The wavenumber i thus the number of waves per centimeter.ier sean 10 Chapter? Specwoscopy Worked Example 1 Wavenumbero Energy S\ghe t aaa a ese pee ae eee tae ane i 4 tion 5 i eee re Wand wd “ Hy i cary i i yi es HEE Hal Ae also lide Aen B= 609105 669 10 Lx PF eal 212 5-mo! s i i rats = RE tt aa fp og @ ak ferrpanompmepronene eunueneney (eH ei ea uHtey Table 22 some of the asocated curs) scales. The names of the diferent ‘ypes—Xrays, visible aco Trequency, abd So forth—have largely asen trom the origin ofthe dation andar somewhat arbitrary. There is sharp tant tion between these nes oe betneen properties ofthe radiation “The inclusion of neutrons and electrons these ables ot scl coret, since these particles havea ite res mass), whe elaceomagaetic radiation has er rest mas, Pais of mater can, however, also be deseibed by waves (Se next scton); hence the term wave-parte daly. The wavelength aad the Did by a, aie momentum (p) of a parce are related by the equations h= h/P, where Do=(E*~ meh for a parce of mass m and A= Be for slocromagnie. ‘ation, Inthe context ofthe Bookie convenient to conser nelson wes nd elaron wes fopether with sletomagnetic adation, WHAT IS MATTER? ‘The mater we are concerned with sthevng cel, which consists oa wide are: {9 of molecules in iferentenvionmens, To develop some of the consi Doran fr ths took, i convenient fo conser Brifly some rest of wave ‘mechan, which, nial, s the moa powerfel model known fr describing ‘ater apd issummatzed by the Seroaiager wave cquation (ce Appendix). ‘This equation can besoved o gle te dsytbution ofa parle in space and ime ands energy However, porous application of this equation to even an lated molecule of reatonable se a formidable ask and the dist sppliation fo i2 Conapter2_ Spectroscopy — Common Us Name and (Aporox) per Proton Frequency Warne)‘ Lng for Tinea kadasoe “otk” “Na “Coop wr ey ie pe w = 1 tern) ee a 7 o molecules ofthe oll i not yt faible, Tere ae, howeh, a Feast two concepts ting from wave macs that are very important tous hee 1. The sribution of partie in spac ven bythe square ofits wae faction. Thi leads (oan understanding of orbit Gee Append). 2, neoyy sates are quanta hus any jem has cen characteristic energy vaso eels ‘We hal ce latr tat the dssibution of a pate in spaces mportant in inter prstng and understanding senttering, nd he conep energy lel she bass of absrption and emission spectro. ‘An sindersanding of the properties of mater, whether they ae describable ‘by wave meena or not, depends oa knowledge abou Is energy edits coOr- nates apace and ane (ha, shape and dynamic properties). is impr ‘ato reaie tha the thee properties of eneay shape, abd ynamis are closely rer ttt eet atte atte? 13 interdependent. Thus, slution of the Schrosinger equation to ve posible diy aes canbe obaled only if tain constraint ze paced onthe wave {fon Fo example asa model for an electron ine chemlea bo, the ea {oncom be soled for a pate of mas Is constaned to be a one {Tepslonal bor of width Ie tues ou ha he only energies avalable tothe Sal are Ey = 7m (here = 1, 2, 3) the energies ate ead ‘ape he width ofthe bon, ). Two spl exampls of the itrelatinship fecneen shape apd energy are (1) the angle of 105" betwen the H atoms andthe ‘Oston ofthe H,O moles, which arises because this gies an ney nium {othe molecu and (2 the fldg of «polypeptide chai insolation to give & ‘felldfined globular protein, which also depends onthe energy of the system Giewe 29. Moles insolation have Kinetic energy Because they undergo Brownian oti, tat they rota and diffe laterally (rant) In addon, the moe {es orate becaae of thie hema energy, Thus, nami nd ene peop hls are alo vlad. in the remainder of the sction we sal brief describe some ofthe charae- tei ofthe lteractons of molecls with each eer and wih stati magnetic {els We sal also discuss the clifton of iferea ypes of ences and some ofthe conseences of diferent ener and enty levels Interparticle Forces and Energies ‘Te foes between various paride, such as a nuleus and an electron oF ttncen molester, often resale na characteristic behavior forthe energy ofthe ‘tem. Thiet represented bythe dag shown in Figure 2S. 1-ewo particles {cfr apr, there to interaction bswoen them, Ae the pasteles approach, there may be die atacon of pose and netve charges a thee maybe a ‘hang in charge drtibation on the partie, eesulng inane attraction. The neta of the system decreases ntl an equlbrium vale of the distance between {he arises i etched, Ar he paris et close han this they ben C0 repel ‘ach other andthe energy Increases. This Sr of behaviors iypial of many = Stone, Examples neuds a electron orbiting mls, he neraton between tome inthe formation of chemeal bonds and crystals, the interaction between ‘molecules in gases and liquide, and the specie tindlag of a substrate to an“ nape? Spectronsopy Sania wine sean nergy Levels “Tae ey le repre th share tl of hole. The poner ‘softs arcane ae doe sy ft moka oe ‘cr sce nd tthe gc any hal pce te ‘otc ay une, Tae pnd aise as cone poems scape ‘oka he marek ond se aS thigh! ene cal xe ie rm of he moe me he an uma ae of cow, rob eerie deny nyt rene Gro Srl by in on ose oxen, mck ea ci or gree fi tea ‘The Erfect ofan Applied Magnetic Field Aelrons an sone tcl poss oer Kowa pT rope fei toscana cm ean te ocrvalanin the prc of mage New es noe ‘edhe muy sens a enya Se tral pin of te slom or sre The pan ae vals In fa the i vents ean wed Quant) atone Sagat fa thr ning ee) lec Te xa of Ma Me a Powe oy sn pe or Fque28 theeoagy ots otpe te” on ina magia. ison hs. ‘Sten sin fansraciarsn te matty aang roman secon oh ‘Sand nat tom ne acre gh 1 ergy ees shorn _ eee nats matter? 15 Ctasitication of Energies For most purpose, its convenient to tet a molecule a It posses several ‘tic eserves of energy. The ttal energy i then sven by Fao ~ Enutemn + Ena Erte * Eee Bacio + En tech Bin the equation represents the appropriate nergy as india by is ain libou a molest has a large numberof othe: types of ene, those ua ae, for our poses, the mos. importa. Th ssluton, a motcuc can obviously anslate, rotate, and vibrate, The ace svociied with each of thew ate guanied, Probably more familie ‘er thar the sletrons have certain allowed energies, whsh are defined by bia Gee Appendix ¥. Probing Different Energes with Different Ranges fr Flecromapnetie Radlation ‘Figure 27 shows some examples ofthe arangement of energy levels typical ‘stem. Although every type of energy is quantized, the sparaton between ‘itoring transaional energy levels so sal that fr practi purposes we an divegard the quaniraon of the tansaional energy. The separation ‘twecr energy levels asocated wth the oventalos that a nuclear o electron TBorcan keto s magnet il reso very small 10"? ml, Bu they ae {ry precise beste thir fetes ae long (ascussed below), Thee oF four rds of magnitude pester are the separations betwen the eneray Teves aio- ‘Sted withthe eifeene rates of rotation of a molecule (10 Jom"). Two to ‘Gre orders of magnitude greater ell are wibeonal energies of ¢ molecule, sich are determined bythe se of the toms and the Netty ofthe chemical ond Joining thom (I-40 KS-nol". The sopattion between electronic eneey Teves even greater (010° KJ mo) ‘ing Worked Example 2-1, we ca ace tha rotational energies ocur inthe inicronave region ofthe sesomagneticspcirum. Simlariy calelton wil how Sat nulear-spin oentaion energies our he rao equency repo, ‘inational eneaier occur i he infrared repion Electronic eneaies (hen ele {toms are moved from one rion oft oleae to another) ocr in the wile fd ltevilt parts of the spect The ang of ectsomagnete radiation ca ha be broken down int eons ‘that are stoi with a partelar type of ener = Table 21. Population of Energy Levels We have disused the rane of diferent typet of energies valle 10 one pat ‘our mola, In any peaceal sade, me deal wih very large numbers ofeee PRT eae mee Reet eet eee eae qmber of molecules in a Upp ate (a) Fltiv 10 the number a lower tar tes) Sven by Boman dstlbutn law ee Figure 28), Maes on =) Reve AT eres the Boltzmann constant (1.38 10°? JK"). When 4 apis o rersne term onthe right besomes exp AE/RT), where Rl ke ee constant he eal lemot 9, eis lear hat when AB << KT, exp(—AEVAT) appcoaches 2, whic i rhe number of molecu in he uppe and lower level then equal. Convers, nen AE >> AT, Me epg WH eS Mo “Worked Example 23 Popudtlons of Energy Levels Jor Different Ferg Separations ‘Secepraton teow ten folow, Ave te temperatures 30K: The 41492107 Joma dn teareton) 1,9 Jeet rain) 6 SRSomot "(bration UV RSot"(lcoie) Solution ‘We 7 Ran /Re = AC AE/ATD nex se. Sinc pen fF al a Steal LPAI (electronic (Vibration (eRetational (4) Translational goose ‘lan lous vtec pera on hai theo sae areas equ populated ico re soon continue 2 fase os oes, At any inte temperate, the molecule wil be ditibued among the ne ‘nergy levels aval them becaseof thermal agtaton. The exact dsb a Bae a 10 tion wil depend on the temperate (ermal energy) and on the separation po betncen energy levels (AE) im the energy Inder Ata pen temperature, the Incomes with () wt tht fr ita a electronic enter be‘THE INTERACTION OF ELECTROMAGNETIC RADIATION WITH MATTER "The sonsequenes ofthe iteration of eleewomagnetic waves with mater are {hat the waves are seattred,ebsrbed, or emitted, With atows mdifiatons a INMIR, for example the sample is placed im a static magnetic fll, and in nerowsopy th sotered waves are refocused wih aes system), Figure 2.9 rp ‘esate basis of al the experiments dscused inthis book, Seatering Scattering is uvaly detected by measuring the intensity of radiation at some fle # to te incieat wave, but k may alo be detected bythe reduction i the transite light wih 6 O this is then often called vari). Satring eves ‘eto reaction abd diffraction phenomena. The frequency of the satred tease is aay but not always the sume as that of the incident wave Elecromagneis wanes intent ona lated atom with an leon loud cause rasatontg "| [fon grote Sactemnarnten nde en anager MRR reer rere ere pe elesroms to oxi about ther equibrium poston, in ytpay wih he Sean om Figure 2.10), The ek an roi slat eis arom inal ation ina pate perpendicular to the oscillation. Some ra oul are ore suceptble to the applied wave than obes, abd their sey to ovulate defied a parameter a, the poaribily. (tbe pa. ‘emit, cluding eos sects and ati scattering factors, ae someties ant cal of a) Electrons are the al seater n molecules, while mice Seiterng and Interference, Blesromagntic waves can superimpose (et See Satered waves, which well all have the same frequency are ‘iat susceptible tothe phenomenon of interference, in which waves can a comtvtely or destucively. or example, co waves difrig ony in ws by IBD efecvely cancel each ode ot A simple example ithe effect. Distr when light waves mereing fom vo sits ntrere, a shown in igure aa etn mie, hota Vee A 7 Wey Spe cramer att‘Chapter2 Spectroscopy (etn ray (oeestconaanosree Several stustons where interference is important canbe identified and four cases wil be Hlarated bere: 1. Large aray of exsentity ris particle, rytals where the wave length of the incident radiation fs uch preter than the dmesions of fhe ary. In ths case there Is very ite observed scattering at angles ‘other than = 0, nce pais of scares can alvays be ound thu cause estrucive imterfereace Gee Figre 2.120). Only forward satering remains, and this eas (0 retraction, 2. Large atays, ab in (1, where the wavelength is much es than che dlmenson of the array. In this ease, comple three densioalintt= ference pavers, usualy calle fraction patterns, ay be peerate. “These can give iformation both about the array (ote) ad the con- observed patters is tha piven By W. L- Bragg” tn replay spaced ‘ray, sch a ery there are planes of atoms or molecules that wll Seat im phase. Consrutveintrereace will be observed when the Base ference betwee he scattering rom these planes sa multiple of 540". As shown in Figute 2.120), the path eiference between the to laos f saterere for waves incident at an angle 8 fo the plane i ‘AO + OB = 2dsin6 1 thi path ference a nepal mulple of he ‘wavelength then constructive nerferenc wll be observed. The rl: tionship 2a sin #= mi own a Bagel tre! ‘The Interaction of Electromagnetic Radiation with Matter =} 44. Fluctuating array, sch at gases and gus, where the dimensions of the scatterers arth ethan. In thi ait sto longer penis sivas find a pale that cane eachother, thus Sextring i baci at ‘neler other than 6 = 0 Gee Figre 2126), This eaten ected the concentration and size ofthe particle and can be analyzed te se rmoeclar weight 4. Apart olution whote dnensions ae greser han the waclength, By analog withthe stuation in (2, there willbe cealn ale wich ‘eset ltrferene takes lace (ce Figure 2.20), This ocr even ifthe particle tumbles in solution causing an averaging oral anges The et ress tat the sealteing dares ap With increstng 9. “This sneuar dependence can give iformation about the cand shape (ofthe sentarng partie, (Note that 6 in Figure 2.148) ~ 8) seatterng and Refection. The phenomena of eefetion and refraction canbe holaned lg seterng theory. Rfletion ests when ight is seated in he {Etestion ppocie ofthat ofthe inedet ihe. Refraction ithe el of radi tion sctered nthe some dection sr that of the snlden wave Ge Figure xa) The phase of the seatred wave i diferent from that ofthe wave that cs sce through the sample. Test two types of wave then recombine tO [rea wave hat as apparendypasedthroogh the spe with iffereat velo {iy The pramcer unt to dexeribe thi phenomenon the refractive Indes 0), = set wave vacuum velocy of wave in sample ™ & Saacercane ipeevergieece Eranunecoames iaretzceeoeechapter? Spectroscory {canbe shown ee Chapter 8, problem 3) hat eration resus inthe rel tionship ym sin sin By where gm the reactive index in passing from Imei {to medium 2 and ands are the ales made between the directions tt theproptgied waver andthe surface separating the two media (ee Figure Sp) The consequence ofthis i famllr: Lenses ean be made that focus some Clectromagnetic waves. The ait to manufactre lenses allows microscopy tobe ‘performed. Not al form of radiation can be focused by lense, X-rays ca ‘ot (Note tat electrons ea be focused by eectric and magnetic fll) "The reltactive inden can be diferent for detent diction of polaris: ‘dona phenomenon known a biringence. "The polrzbiy, and therefore the satring and the etre index, ae frequency dependent ce Blow). Frequency dependent retraction i known 38 ec ear et 4c aera ne fr urs 0 am lg, 0998 crating ee [ em a Se ee x Eben itratn » 4 fe iat soe oes seers foc, rr ‘Shap areton why os pos oncenrains ae aly mere 20, maven th abso pee ia Figure 2.27ES ‘Spectroscopy oT HAPTER 3 na ae Atom pee 227 atte sorion fetes aber of matt moments fo {espn reonanee fern, y= 1700 ™ 1ONT os ana) forthe ‘nine spn resoeane fH atom, = 2693 107 Vr" lanai aka teat of he probe of ptcaneousemison or an genie ‘ranaon a3 (19H apd oon stan 3c 1 Ha) Cakate eee broadening ander "female tha ara Ute or 10" sand 103. Inmagic remance, yotanotemision of radiations elie Thermal ey colon rian ln epee al son st en Many moll tat forex notion refund tophospbrece wes trpp ins a pe, chaps. Why is? ‘res of agate tae sping han ete tana ee eel Tra Sion betes rational ree canna rele ina saan ie Worked Example 26, ys welded NMR wenn orecommoly chee Why th Infrared Spectroscopy OVERVIEW ofa (UR) ssa ee lforaton on motel itn 2. Tae sin experimental petamete Fg lem theposionof he ain oe abspion Han “The cons ntred eon of th spt is 140-409 om” “The piyninl bas or desing oll ion te Movs (goteta crepe Quast mca ec al ee 15. Thegroseestion rae fora atone nated acetate aeaton ‘ist oan a change the cpte moet 6 The miner of es of iration (2 ~ fora malclecossing of ‘Sonn $i te molrleietinen. Fora macromole thr ste that ey ‘Suny shoo asitons May ofthe vibraons ee, howere, teocled ‘Syarar bods or eowpings, nc arte “CO rou. Tis forse ‘of eerie rap fester. 7, The rina equ roa bon can Be aprosinay desi by he BV eres ont and theredaed mas. The erm pcan be Shang by oti sistaon Ine abnpon ty ste aia le settee ad Intaed pcr rent ample einormaion on the deston oe {tnsion pols momen beans the pet ae bo Thre specacan b ow8 ‘Chapter Infrared Spectroscopy ‘orate wih molest conformation bathe pc interptetaion canbe ‘Somplsed sna of inracons betwen segboring aston das, 10, Temi cope apts involve seating Hand cating Ring {maemo probing kop bos ope Seti change ‘hd om reed sams, tec cotoraton INTRODUCTION ‘The energy of most molecular ibeatons corresponds to that of the infrared revion ofthe estuomagnei spesrum. These wbatons may be detected and Imenered ther diet in an infrared spectrum or indice (a 2 Raman ‘catering Specrum (ie Chigter 9). The main Boshemica aplcaons have ‘been to monitor vibrations of sleced rouns, either on ligands or macromole- ‘ules, the liga binds othe pH changes. The technique has lo een used to follow HD exchange. With oiented spl, soch as fibrous proeins and poly shards, dshrosm mensaement sie information oa the eeaton of the trancton dipole moment, wach can then be corzlated wich molec confor ‘maton. OF the ota iafraredspectum, only the ange 1400-400 emi wef {a eadly dvving any Information. In this epion the selected vibrations Tom ‘many charactenste groups, e£., C=O, can be iene. However, the {600-1400 cm region contains very many bands, netuding tov fom shea ‘iran, ao threo is sometimes fl “ngerpiatng” molecles, ‘Whi’ the absorbance ofa saple s determined bythe Bee-Lambet Law, ‘te eninesioncoeTcent of a Ban is rarely used as a pacameer of infrar ‘spectra. The ewtincioncoeiciemt rele the transition probabil) which i Fated 0 the magnitude of the transition dipole moment (ace Chapter 2 for ‘efntion). Tis it sons to eironmeat thal vibeasons rom characterise ‘groups in diferent molecules show enormous varlation in thelr extinction uefficlents. Forti eason the intenses of infared peaks are often indeated ‘nly by he (upeserip) leering, trong), meu, (wea), Note ha, ‘ontrat extinction cece in trail spectra ar the as for wing inten ‘ie of lr band to messre concentration EXPERIMENTAL PARAMETERS OF AN INFRARED SPECTRUM An ntared spectra aul onsets of plot ofthe absorption of ation sa function af wavenumber ands characterized oly interns ofthe postion of the maxima of cach of the absorption bands Fy, expressed i mt ‘Bo amen ih tot ila tise a of ae SS nea a pret Fr essuroment of Intrared Spectra FD i i gon 31 The ole baum sonoma, MEASUREMENT OF INFRARED SPECTRA ‘Conventional Double Beam Spectrometer ese ee Lo a Soci perenne a ta Fe arora teeter ree ec ro ee tetera Haein tad hei essence aarels ier ay tes Seaenen CL Ono aan Sra le i he one a coer ee erie a Si es Dees Seen si lh ec rer he eee ar! Sra oa caeer hae cr pare secre igen a enema vee iaee Sioa erirciee Pati ah ee Se es bee ee Berri mah eter ats eras see pi pi ohne ee scene0 Chapter’ Infrared Spectroscopy “The Fourier Transform Method Is oer much more eet co cols infrared spectea ina different way trom that described above. Figure 3.2 shows device invented in 1880 by Michelson. A hleecting mitor splits che beam nto two eaual pars The to Beas then traverse ferent paths, are reiected at diferent iors, a are recombined a A detetor. The pat length of oe ofthe enn varied By moving the approp the two path lengihs are he same, then there wil Beno phase diterence beencen the beams an they wl combine constructively fr al requencies pes tin the oil beam. For eferet path length, the amplitde ofthe revo bined signal il depend on the frequency and the distance the mirror moves. For trap tow frequencies wl interfere destructively (have a phase shift of 18°) for relaively lage movements of the miror, whereas high fequenses il requierelatay sll movement for hs contin to ecu. The esl of ts jasis of infrared Spectra Sapcenauniniisansttny. 00 pve agen poatrctn grated mw seed fe oe connote tid ra gan, Fae ae a eerie ove! io aasform Infea Re (FTIR is aalgous tothe method wed ia sien a etna ser Fe tl te a apn Ce et rane tiie el ioe aaa ee cote hea ies coe ie a i tae tps ete a i Fae a ee es eee He seth, mn ae poner ae ree onan need ese = aa PHYSICAL BASIS OF INFRARED SPECTRA Molecular Vibrations ‘Asingle way of relating energetics and structure is provided by the ample of the potential ney curve of 4 diatomke molecule (wth atoms A and B). The {arc in Fgure 3 shows how the energy’ ofa moleul changes ste stance Seweun its all changes, "Atte mimi ofthe poteaal-nergy curve the ataction due to various sper forces is balanced by the frees ftom charge repulsion. The srething ‘r contacting of he Bond then sila othe Behavior of spin, ine theres {Tesoring fore. The wbrations ofthe bond cn in the ist instance be analyzed in tern of the molecules wadergoing simple harmonic oxcilations abouts lis bond Teng The wbrational frequency sven By (ee APE eth2 ‘Chapter. Infrared Spectroscopy a Fae Aap ancy ‘iru Cond lengin he mele ‘Repose woah ‘Shp homo cacaton south ge he fest te he at f he ‘molecule AB, which is given by. ie where My and fy are the stomie masses of A and 8. Worked Example 1 Meaning of Reduced Mast 4 wana dace Ahem A ait, Wish Sottion 1 inet, heme single atom Dah by rings ek wl 2th omen rind te as ae, sea © ItAaed Bae thesane mas we soul xpos lr: Tread mt athena yf xing ‘ antl wa oxigen ne srenty pet v= Vite Torte hg tities Wen, Hated ttn = hye aot aie th ial recs a en ye eae a Ve of Infrared Spectra “3 orked Bxample 3.2 Changing the Reduced Masrby opts caval theredved mass of 8 0-H otc and anO—D “mole ‘alt anon waon! freuen) Between thee 07 soltion “Te redesed mat oe OH “make een by Fou 61 sing ht he fore cna remain the sae each ime, teen ees ae eee tne firs ee re oeh eee trues 9 not of. ‘The Morse Curve Rel molecules do not obey the lave of simple harmon motion exactly. The po Tearenergy curve shown ia Fire 3.5 clearly more complex than tha for & “Sinpieharmonic oat (which hae the parable she sown in Fie 3.0. More derived an empirical expression to fic the experimentally observed curve For ditomie molecule Vibrational Energy Levels ‘Quan mecha tls w that only ceian vats ofthe eneray ar allowed ‘Tht values ae represented by the rizonal ine onthe More curve in Figure 415 Values of energy inbetween these eneray levels ae 6 permied and the ‘acing betwen enerey levels becomes smal at higher enety vali. As the ‘herayinrees, the alome move farce! from tet equim postions snd ence ener a nonparabolic region ofthe Morse curve Therefor, the iations “anions tate esponsble or the eolr of transition metal complexes Spin Considerations In dealing wit lstons, we Have 1 remeber that they also have he ntsc property of spin. A molar ota may contin up to two eestrons, bu they ume for an Electronic Transition See eae ve opposed spin oretations- One selection rule sates that during an els sett sca cne fronn s n i Se a pein onion a ren thd ‘Seimorientaton transitions (or flips) can oocur only a8 result of interaction of ms i atnionin Seca er tesa rae ee ee lon Donan ty och tla tans seer Ue as oy eh ano ee wea Worked Example 14 Spin-Alowed Transons ‘Whchof he fetwingtanonbtuenshe ener vein itr 42 ein Bowe? das re oa Ma ak ae Fawn aa Sotion Nie oven of he pin das nt change an thers allows 1 tas is in fokien ansion. The renin ofthe sin canes. sia Thetwo elton ina molec ein ms have apo pn {is knw ain aa pr). Th nl sate cannot ex 1 Thisis siti aon. The eeu sinh nial ste cou ate tn when tin ated id. nica bead ei woud Eeipt norte set Te tranion woul beset "THE TIME, FOR AN ELECTRONIC TRANSITION lsronicransions between wo sates osc whea.a transition dipole oats ‘rear th wo states involved The resonance condtion = hr mans that he frequency of oelation i. The tne taken to cla between the states forthe emion to oocr—isthur Is, A typical electronic transitions 420 am {elt equncy of 7.14% 1H witht take (7.14 105, tau ies eer Chapter 4 Utraviolet and Visible Absorption Spectroscopy [Worked Example 4.5 Fletonie Transit are Faster than ‘Nucla Vlbvtions ‘Compare tine for an eto triton a wavelet 420 with that ora ‘opel nuclear ation a 150 Solution 1.4 tor Forte vation weet cnn 110m" 80 regan. ice PZateywcotaing = Hops 2% 10s so a5 IDM Thetie ora Snir tore (45 10) = 22510 22> 10". Tenge ha he cit hve bine to sone pace ising the sro enon ‘The prvous worked example prowdes the bass of the Franek-Condon rincne hich at tat an electron ranaiton curso rap tat during it Ihe mice arta Since vibrational tensions by defini equtes change Itternucear tance, and ince this cant occur ovr the te Sale ofan ee ‘tonetranstion, follows tht elaconi ansdons axe not accompanied Dy V+ ‘rational transis. Fire 43 shows the Morse curve forthe ground an frst excited electronic state. Te briana poson of the upper curve depends onthe bond natn (ay toe tower nate gents Monesunasarne oad cdc ig {eimai anon tern te apart tant bow at r pooping of Chvomophores ry seitcd ste compared withthe band length i the round state, The two to fhe bon sta necked a cr he esa yng nies tate We cn soe ay hat tent pound wear of he ou on sss An the mice ration in which the nce donot move can be rpresentd bya ver= 10 eal Stypot a mtod for followin he Kins of th maton AHS NADY EES as Hee NADH Sontion ‘xn befolone ty monorng hs wae eee ete tememntrees. 7 iorkedEcagle 8 Cand Binding fans do NAY, ed bd ae ee tSayes hasan sonveaetas soup Boa i foo oo nem a owe 410 . Calas evnton fen a 340 for NADH whe ov 10th ‘Stone: Path eat tem) Fie 4.101 aston oft tence in abortane st MO am Bewese NADH nde sue conceataon of NADH nthe resco ed concen: traonotenyme 24.298), Thee nce ont uta corer ‘ye rao ota Inept sigan ote Te af NADH bound othe eee whe te sorta 08s O10 Sonuion 1 From igre 10), weasetht be sbnrtasce a 40am for heb fr i 14 = 429.3 «10 1K) CoA emo" “Tis ees tthe bound extinction cout, which me sl call state preene of he ene, the arbace ms ross NADH 0 pos Anal nel ap Aa obd+ 467 aWorked Example £8 (conthuad) Inte cwete without enye, the arn yn Thue difecee spec Aas “ ‘Tethumon caren Fgue 10) thas esi of ding of NADIE {heenzyne (6) The ctrve eget tt moe of NADIE nd ey {er thecnre wh hen ontes ah preay ‘Snceya)= 15 fal then when t= 04,6 = 27 and when At =o. 6 Mise. and alo Bch Biophys, rv, 39175) 208-8, Worked Example 49 Probing Catbte Subunits of Aspartate Trascarbamytse Unrate dir spectoony hasbeen eto wd theca setup ‘artanyphowatefoeentme, tay dee ‘hetmgtde thespeara of he tern) compl, ks ee Forte Recmaretettenaes 00 tee stantially greater than the sum of the oi mee complexes formed by each substrate. oe 300 tam ‘natn actnchanee gy 434 ‘caine isd chan, ar sre inh eeary compe futher ‘eye KD. Caine AG. Shy. Be Chm 34199 1865187). “Frorked Example 4.10 Solvent Perturbation to Determine Moncey of Tptopan Reside nso Soe pttalo sented io poo eminent yin or Soveylan uno et at eae irda pas in {ofa reid ote ra: be pad aps ‘simian otto ees neon bonita ‘be peedatcaonnen, Tor ie dapy lw eco the Sos eou chore totaal Tseeinoes meiod eee et este of enter and 20 of sear save chee ret [ier dane Tarsumbe ofp teu bed omopirrcr 5d rm ra often ne atowespnr ees nie ed ran oc aoe tbe am ero en eet Am ote ea Sit genres Surana amen st etecietreienssone Stents eae tecagpromatin wate a cuvarr ste gq potsar) i. Sotuion “shee arte asumpson imps Intheolven prtrbaon metho ove (Onl exon eeomeptores tected (No petri ese eg suouncing tbe ec ayaa i ponents 70 290-30 tera ire 2 nm) The peat 292m nthe sont set oo ais = oninay fom the ep ctomoptves ee gate 48). Thea of th as ec (.01/0.0% gst that he ee export Teypontan edo ‘i bsnyme iO. ht yao ne ypeoghn resi). ore important poet sta hs method measure ol te erage ‘eal chose reeds, such stb ented pat, fore 10 epee ‘nda, Theslven petaratin method shou avs bene witha ‘ion(or futher det se for example £3. Waban ad NL. Low, 4 Bod. Che, 240 (96S 35003580),