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TRUCTURALBI
OLOGY

Modul
e-4
SPECTROSCOPI
CTECHNI
QUES

X-
RAYDI FFRACTI ON: SINGLE- CRYSTALX- RAYDI FFRACTI ON
X-
RayCr yst allogr aphy
 X-raycr yst al logr aphyi s at oolused f ori dent i
f ying t heat omi c and mol ecular
structureofacr yst al,inwhi cht hecr yst allineat omscauseabeam ofi ncidentX-
raystodi f f
r acti nt omanyspeci ficdi rect ions.
 By measur ing t he angl es and i ntensi ties of t hese di ff r
act ed beams, a
crystallogr aphercan pr oduce a t hr ee- dimensi onalpi ct ur e oft he densi ty of
electronswi t hint hecr yst al.Fr om t hi sel ectr ondensi ty, themeanposi tionsoft he
atomsi nt hecr yst alcanbedet ermi ned,aswel last hei rchemi calbonds,t heir
disorderandvar iousot heri nf ormat ion.
 Themet hodal sor eveal edt hest ruct ur eandf unct ionofmanybi ol ogicalmol ecules,
including vi tami ns,dr ugs,pr ot ei
ns and nucl ei c aci ds such as DNA.X- ray
crystallogr aphyi sst illt hechi efmet hodf orchar act er
izi ngt heat omi cst ruct ur
eof
new mat erial s and i n di scer ni ng mat erials t hat appear si milar by ot her
exper i
ment s.
 X-ray cr yst alst ructur es can al so account f or unusualel ect ronic or el astic
proper t
iesofamat eri al,shedl ightonchemi cali nter act ionsandpr ocesses,or
serveast hebasi sf ordesi gni ngphar maceut icalsagai nstdi seases.
 Crystalst r
uct uresofpr oteins( whi char ei r
r egularandhundr edsoft imesl arger
thanchol est erol)begant obesol vedi nt hel at e1950s, begi nni ngwi tht hest ruct ure
ofsper m whal emyogl obi nbySi rJohnCowder yKendr ew,f orwhi chheshar edt he
NobelPr izei nChemi st rywi thMaxPer ut zi n1 962.Si ncet hatsuccess, over8681 7X-
raycr yst alst ruct uresofpr otei ns,nucl eicaci dsandot herbi ologicalmol ecul es
havebeendet er mi ned.

Si
ngle-cryst alX- rayDi ffr action
 Single-cr yst alX- r ayDi f
f r
act i
oni sanon- dest r uctiveanalyticalt echniquewhi ch
providesdet ai l
edi nf or mat i
onaboutt heinternall atticeofcr ystalli
nesubst ances,
including uni tcel ldi mensi ons,bond- l
engt hs,bond- angles,and det ai
ls ofsi te-
ordering.
 Directlyr el atedi ssi ngl e-crystalrefinement ,wher ethedat agener atedf rom theX-
rayanal ysi si si nter pr etedandr efi
nedt oobt aint hecrystalst ructure.
 In a si ngl e-cryst alX- ray di f
fraction measur ement ,a cryst ali s mount ed on a
goniomet er .Thegoni omet erisusedt opositiont hecr ystalatsel ectedorientations.
Thecr yst ali si llumi nat edwi thaf inel
yf ocusedmonochr omat icbeam ofX- rays,
produci ngadi ff r
act i
onpat t
ernofr egularl
yspacedspot sknownasr efl
ect i
ons.
 Thet wo- di mensi onali magest akenatdi fferentor ientati
onsar econver tedi ntoa
three-di mensi onalmodeloft hedensi tyofel ect ronswi thint hecr ystalusi ngthe
mat hemat icalmet hodofFour iertransforms,combi nedwi thchemi caldat aknown
forthesampl e.
 Poorr esol ut i
on( f
uzzi ness)orevener ror
smayr esultifthecr ystalsar etoosmal l,or
notuni formenoughi nt heirinternalmakeup.

Fundament
alPrinci
plesofSi
ngl e-
crystalX-r
ayDiff
racti
on
 Max von Laue,in1912,discovered thatcryst
all
ine subst
ances actas t
hree-

1
15BT45 S
TRUCTURALBI
OLOGY
dimensionaldi f
fractiongr ati
ngsf orX- raywavel engt hssimilartot hespaci ngof
planesi nacr ystallat t
ice.X- raydi ffract i
oni snow acommont echni quefort he
studyofcr ystalst r
uct uresandat omi cspaci ng.
 X-raydiffractioni sbasedonconst r uctivei nterferenceofmonochr omat i
cX- rays
andacr ystallinesampl e.TheseX- raysar egener at edbyacat hoder aytube,fi
ltered
to produce monochr omat icr adiation,col l
imat ed t o concentrate,and di rected
towardt hesampl e.
 The interaction oft he incidentr ays wi tht he sampl e produces const ructive
inter
ference( andadi ffractedr ay)whencondi tionssat i
sfyBr agg' sLaw (nλ=2d
sinθ).Thi sl aw r elates t he wavel engt h of el ectromagnet icr adiation tot he
diffr
actionangl eandt helat t
icespaci ngi nacr yst all
inesampl e.
 Thesedi ffractedX- raysar et hendet ected,pr ocessedandcount ed.Bychangi ng
thegeomet ryoft hei ncidentr ays,t heor i
ent at
ionoft hecent er
edcr ystalandt he
detector,al
lpossi bledi ffractiondirect ionsoft hel atticeshouldbeat tained.

X-
rayr eflect ioni naccor dancewi thBr agg'sLaw
 Whenacr yst ali sbombar dedwi thX- raysofaf ixedwavel engt h( si milartospaci ng
oft heat omi c- scalecr yst allatticepl anes)andatcer t
aini ncidentangl es,intense
reflected X- rays ar epr oduced when t hewavel engt hs oft hescat tered X- r
ays
interfereconst r
uct i
vel y.
 Inor derf ort hewavest oi nterf
er econst ructi
vely,thedi fferencesi nt het ravelpat h
mustbeequalt oi ntegermul tiplesoft hewavel engt h.Whent hi sconst ructive
interferenceoccur s,adi f
fractedbeam ofX- rayswi l
lleavet hecr ystalatanangl e
equalt ot hatoft heinci dentbeam.
 Toi ll
ust rat et hisf eatur e,consideracr ystalwithcr ystallatticepl anardi stancesd
(ri
ght ).Wher et het ravelpat hl engt hdi ffer
encebet weent her aypat hsABCand
A'B'C'isani nt egermul t ipleofthewavel ength,const ructivei nterf erencewi l
loccur
foracombi nat ionoft hatspeci ficwavel ength,cryst allatticepl anarspaci ngand
angl eofi ncidence( Θ).Each r ationalpl aneofat oms i n acr yst alwi llunder go
refract i
onatasi ngle,uni queangl e(forX-raysofaf ixedwavel engt h) .
 Thegener alr elati
onshi pbet weent hewavel engt hoft hei nci dentX- rays,angl eof
incidenceandspaci ngbet weent hecr ystallatti
cepl anesofat omsi sknownas
Bragg' sLaw, expr essedas:

nλ=2dsi
nΘ

Wheren( aninteger
)ist he"order
"ofrefl
ecti
on,λisthewavelengt
h ofthe
i
nci
dentX-rays,d i
st heint
erplanarspaci
ng ofthecryst
aland Θi
st heangleof
i
nci
dence.

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15BT45 S
TRUCTURALBI
OLOGY

Fi
gur
e1.
Bragg'
sLawr
efl
ect
ion

Overviewofsi ngl e-crystalX- raydi f

f r
act ion
 Theol destandmostpr ecisemet hodofX- raycryst al
lographyi ssi ngle-crystalX-
raydi ffraction,i nwhi ch abeam ofX- rays stri
kes asi nglecr ystal,pr oducing
scat t
eredbeams.Whent heyl andonapi eceoff i
lm orot herdet ector,thesebeams
makeadi ff
r actionpat ternofspot s;t hest rengthsandangl esoft hesebeamsar e
recordedast hecr ystalisgr adual l
yr ot ated.Eachspoti scal l
edar efl
ection,since
itcorrespondst other efl
ect ionoft heX- raysfrom onesetofevenl yspacedpl anes
wi t
hint hecr ystal .
 Forsi nglecr yst alsofsuf f i
cientpur ityandr egularit
y,X-r aydiffractiondat acan
det er
mi nethemeanchemi calbondl engt hsandangl estowi thinaf ewt housandt hs
ofanangst rom andt owi thinaf ewt enthsofadegr ee,respect i
vely.Theat omsi na
crystalar enotst ati
c,butosci l
lateaboutt heirmeanposi tions,usual l
ybyl esst han
af ew tenthsofanangst rom.X- raycr yst all
ographyal l
owsmeasur ingt hesi zeof
theseosci llations.

Pr
ocedur e
 Thet echni queofsi ngle-cryst alX-raycrystall
ogr aphyhast hr eebasi cst eps.
 Thef ir
st—andof tenmostdi f f
icult—stepi st oobt ainanadequat ecr yst aloft he
mat erialunderst udy. Thecr yst alshouldbesuf fici entlyl arge( t
ypi callylar gerthan
0.1mm i nalldi mensi ons),pur ei ncomposi tionandr egul ari nst ructur e,wi t
hno
signif i
cantinternalimper f ectionssuchascr acksort wi nning.
 Int hesecondst ep,thecr ystali splacedi nani nt ensebeam ofX- rays,usual l
yofa
single wavel engt h( monochr omat ic X-r
ays) ,pr oduci ng t he r egularpat t
ern of
reflections.Ast hecr ystali sgr aduall
yrotated, pr eviousr eflecti
onsdi sappearand
newonesappear ;t
hei ntensi tyofever yspoti sr ecor dedatever yor ientat i
onoft he
cryst al.Mul t
ipledat a set s may havet obecol l
ected,wi th each setcover i
ng
slight l
ymor et hanhal faful lrotationofthecr yst alandt ypi call
ycont ainingt ensof
thousandsofr eflections.

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15BT45 S
TRUCTURALBI
OLOGY
 Inthethir
dst ep,thesedataarecombinedcomput ati
onall
ywithcomplementar
y
chemicali
nformationtoproduceandrefi
neamodeloft hearr
angementofatoms
withi
nthecrystal
.Thefinal,
ref
inedmodeloftheatomicarr
angement
—nowcalleda
cryst
alstr
ucture—isusuall
ystoredi
napublicdatabase.

Fi
gur
e:Wor
kfl
owf
orsol
vingt
hest
ruct
ureofamol
ecul
ebyX-
raycr
yst
all
ogr
aphy.

Instrument at ion
X-raydi ffract omet ers
 X- raydi ffract omet er sconsi stoft hr eebasi cel ement s,anX- rayt ube,asampl e
holder ,andanX- raydet ector.
 X- rays ar egener ated i n a cat hoder ay t ubeby heat ing a filamentt opr oduce
elect r
ons,accel er atingt heel ect r
onst owar dat argetbyappl yingavol tage,and
impactoft heel ect ronswi tht het ar getmat er i
al .Whenel ectronshavesuf ficient
ener gyt odi slodgei nnershel lelectronsoft het argetmat eri
al,char acteristicX- ray
spect raar epr oduced.
 Thesespect r
aconsi stofsever alcomponent s,themostcommonbei ngKαandKβ.
Kαconsi sts,i npar t,ofKα1andKα2.Kα1hasasl ightlyshor t erwavel engt hand
twicet hei ntensi tyasKα2.Thespeci ficwavel engt hsar echar act eri
st i
coft he
targetmat er i
al.
 Fi lt
er ing, by f oils or cr ystal monochr omet ers, is r equi red t o pr oduce
monochr omat icX- raysneededf ordi ffr act i
on.Kα1 andKα2ar esuf fi
cientlycl osei n
wavel engt hsucht hatawei ght edaver ageoft het woi sused.Mol ybdenum i st he
mostcommont ar getmat eri
alf orsi ngl e- cryst aldi f
fraction,wit hMoKαr adi ation=
0.7107Å.
 TheseX- raysar ecol limat edanddi rect edont othesampl e.Whent hegeomet ryof
the i nci dent X- rays i mpi nging t he sampl e sat isfi
es t he Br agg Equat ion,
const ruct ivei nt erfer enceoccur s.A det ect orr ecor dsand pr ocessest hisX- ray
signalandconver tst hesi gnaltoacountr atewhi chi sthenout putt oadevi cesuch
asapr i
nt erorcomput ermoni t
or .X-raysmayal sobepr oducedusi ngasynchot ron,
whi chemi tsamuchst rongerbeam.

4
15BT45 S
TRUCTURALBI
OLOGY
 Single-cr yst aldi ff
r act omet ersuseei ther3-or4- circl egoniomet er s.Theseci rcles
refert ot hef ourangl es( 2θ,χ,φ,andΩ)t hatdef inet her elat
ionshi pbet weent he
cryst allat ti
ce,t hei nci dentr ayanddet ect or .Sampl esar emount edont hingl ass
fibers whi ch ar eat tached t obr ass pi ns and mount ed ontogoni ometerheads.
Adj ustmentoft heX,YandZor t hogonaldi rect i
onsal lowscent eringoft hecr ystal
wi t
hi ntheX- raybeam.
 X-rays l eave t he col limat orand ar e di rect ed att he cr ystal.Rays ar e either
transmi tt edt hrought hecr yst al,reflect edof fthesur face,ordi ffractedbyt he
cryst allat t
ice.
 Abeam st opi slocat eddi rectlyopposi tet hecol li
mat ort oblockt r ansmi ttedr ays
and pr eventbur n-
outoft hedet ector.Ref l ected r aysar enotpi cked up byt he
detect orduet ot heangl esinvol ved.Di ff ract edr aysatt hecor rector i
ent ati
onf or
theconf igurat ionar et hencol lect edbyt hedet ector.
 Moder n si ngl e-cryst al di ffract omet ers use CCD ( charge- coupl ed devi ce)
technol ogyt ot ransf ormt heX- rayphot onsi nt oanel ect r
icalsignalwhi char ethen
sentt oacomput erf orpr ocessing.

Fi
gur
e:Schemat
icdi
agr
am ofX-
rayDi
fract
omet
er

Pr
ot eincr yst allization
 Pr oteincr yst al l
izationi st hepr ocessoff ormat ionofapr ot
eincr yst al.Whi l
esome
pr otein cr yst als have been obser ved i n nat ure,pr ot
ein cr yst allizat
ion i s
pr edomi nant lyusedf orscient ificori ndust rialpur poses, mostnot abl yforst udyby
X- ray cryst allogr aphy.Li ke many ot hert ypes ofmol ecules,pr ot ei
ns can be
pr ompt edt of orm cr yst alswhent hesol utioni nwhi cht heyaredi ssol vedbecomes
super sat ur ated.
 Undert hesecondi tions, i
ndi vidualpr oteinmol ecul escanpacki nar epeat ingar ray,
hel dt oget herbynoncoval enti nt eract i
ons.Thesecr ystal
scant henbeused i n
st ructuralbi ologyt ost udyt hemol ecularst ruct ur eoft hepr otein,orf orvar ious
indust rialorbi otechnol ogicalpur poses.
 Pr oteinsar ebi ologicalmacr omol ecul esandf unct ioni nanaqueousenvi ronment ,so
pr oteincr yst allizationi spr edomi nant lycar riedouti nwat er
.
 Pr otein cr yst alli
zat ion i s t radit ionally consi der ed chal l
engi ng due t o t he
rest ri
ct i
ons oft heaqueous envi ronment ,di f f
icul ties i
n obtaini ng hi gh-qual it
y
pr oteinsampl es, aswel lassensi tivi t
yofpr ot einsampl estotemper ature, pH,ionic
st rength, andot herf actors.

5
15BT45 S
TRUCTURALBI
OLOGY
 Proteins var y greatl
y in t heir physicochemi cal charact
erist
ics, and so
crystallizationofapar ti
cularprotei
nisrarel
ypr edictable.
 Determi nationofappr opri
atecr yst
all
izat
ioncondi tionsforagivenpr ot
einoften
requiresempi ri
caltesti
ngofmanycondi t
ionsbef oreasuccessfulcrystall
izat
ion
conditioni sf ound.

Pr
inciplesofpr otei ncr ystallizat ion
 Thesol ubi lityofpr otein mol ecul es i s subj ectt omanyf act or s,especi allyt he
int
er act ion wi t h ot hercompounds i n sol ut i
on.Mostpr otei ns ar e sol uble at
physi ologi calcondi t i
ons,butast heconcent rationofsol ut esr ises,t hepr otein
becomesl esssol ubl e,drivingi tt ocr ystallizeorpr ecipitat e.Thi sphenomenoni s
knownas" sal tingout ".
 Count eri nt ui ti
vel y,atver yl ow sol ut econcent rations,pr ot einsal sobecomel ess
soluble,becausesomesol utesar enecessar yf ort hepr oteint or emai ni nsol ution.
Thisconver sephenomenoni sknownas" sal tingi n".
 Mostpr ot eincr yst al l
izationt echni quesf or m cr ystal sbysal tingoutt hepr otein
int
ocr yst al s,al thoughsomeexper i ment alset upscanpr oducecr yst alsusi ngt he
salti
ngi nef fect .
 Thegoalofcr yst allizationi st opr oduceawel l
-order edcr yst alt hati sl ackingi n
contami nant s whi le st il
ll arge enough t o pr ovide a di ffract ion pat t ern when
exposedt oX- rays.Thi sdi ffract ionpat t
er ncant henbeanal yzedt odi scernt he
protein’st er ti
ar yst r
uct ure.
 Protein cr yst al li
zat ion isi nher ent ly di fficul tbecauseoft hef ragi l
enat ur eof
proteincr yst als.Pr oteinshavei r regul arlyshapedsur f
aces,whi chr esul tsint he
format ionofl ar gechannel swi t hinanypr ot eincr yst al.Ther ef or e,t henoncoval ent
bondst hathol dt oget hert hel at ticemustof t enbef ormedt hr oughsever allayer sof
solventmol ecul es.
 Inaddi tiont oover comi ngt hei nher entf r
agi li
tyofpr oteincr yst als,anumberof
environment alf act orsmustal sobeover come.
 Duet ot hemol ecul arvar iationsbet weeni ndi vidualpr oteins,condi ti
onsuni quet o
each pr ot ei n must be obt ained f or a successf ulcr yst allizat ion.Ther efor e,
attempt ing t o cr yst all
ize a pr ot ein wi thout a pr oven pr ot ocolcan be ver y
challengi ngandt imeconsumi ng.

Cr
ystalli
zat ioncondi tions
 Manyf act orsinfluencet hel i
kelihoodofcr ystalli
zationofapr oteinsampl e.Some
ofthesef actor
si ncl udepr ot
einpur i
ty,pH,concent rationofpr otein,t
emper atur e,
precipitantsandaddi t
ives.
 The mor e homogeneous t he protei
n solut i
on is,t he mor el i
kelyt hati twi ll
crystall
ize.Typi cally,pr ot
einsampl esabove97% pur ityareconsi deredsui table
forcryst alli
zation,al t
hough hi gh puri
tyi snei thernecessar ynorsuf f
icientf or
crystall
izat i
on.
 Soluti
onpH canbever yi mportantandi next r
emecasescanr esultindif ferent
packing or ientations.Buf fers,such as Tr i
s-HCl,ar e often necessaryf ort he
maintenance ofa par ti
cularpH.Pr ecipi
t ants,such as ammoni um sulfat e or
polyethylenegl ycol,areusuallyusedt opromot ethef ormationofpr otei
ncrystal s.

Met
hodsofpr
otei
ncr
yst
all
izat
ion

6
15BT45 S
TRUCTURALBI
OLOGY
Vapordiffusi on
 Vapordi ff
usi onist hemostcommonl yempl oyedmet hodofpr oteincryst allization.
Inthismet hod,adr opletcont ainingpur if
iedpr otein,buf fer,andpr eci pi tantar e
all
owed t o equi l
ibrat e with al ar gerr eservoi rcont ai
ni ng similarbuf f
er s and
precipi t
ant sinhi gherconcent rat ions.
 Init
ially,thedr opletofpr oteinsol utioncont ainscompar ativelylowpr eci pitantand
proteinconcent rations,butast hedr opandr eservoi requi l
ibrate,thepr eci pitant
andpr oteinconcent rationsincreasei nt hedr op.
 Iftheappr opriatecr ystall
izationsol ut i
onsar eusedf oragi venpr ot ein,cr yst al
growt hwi lloccuri nt hedrop. Thismet hodi susedbecausei tall
owsf orgent leand
gradualchangesi nconcent rationofpr ot einandpr ecipitantconcent r ation,whi ch
aidint hegr owt hofl argeandwel l-order edcr ystals.
 Vapordi ffusioncanbeper f
ormedi nei t
herhangi ng- dr
oporsi tting-dr opf ormat .
Hangi ng-dr opappar atusinvolveadr opofpr ot einsol utionpl acedonani nver ted
cover sl i
p, whi ch i s t hen suspended above t he r eservoir. Si tting-dr op
crystallizationappar atusplacet hedr oponapedest althati ssepar at edf rom t he
reservoir.Bot h oft hese met hods r equi re seal ing oft he envi r
onmentso t hat
equilbrationbet weent hedropandr eser voircanoccur .

Microbatch
 Itisat ypeofbat chcr yst all
izati
onmet hodinwhi challthecomponent saredi rectly
combi ned i nto a singl e,super satur at
ed pr otein solut
ion,whi ch i st hen left
undist urbed.Thet echni quecanbemi ni
aturi
zedbyi mmer singpr ot
eindr opletsas
smal las1µli ntoani nertoi l
.Theoilpr eventsevapor ati
onoft hesampl e.
 Thisi st hesocal led‘ mi crobat
ch’met hod.Besi dest hever ylimitedamount sof
sampl eneeded,t hel attermet hodhasasf urtheradvant aget hatthesampl esar e
protect edfrom airbor necont amination,astheyar eneverexposedt ot heairdur i
ng
theexper i
ment .

Microdialysis
 Mi crodialysi
stakesadvant ageofasemi -per
meablemembr ane,
acrosswhichsmall
mol eculesandi onscanpass,whi leprot
einsandlargepolymerscannotcross.By
establishingagr adientofsoluteconcentrat
ionacr
osst hemembraneandallowing
thesyst em t
opr ogresstowar dequi l
ibr
ium,thesystem canslowlymovet oward
super saturat
ion,atwhichpoi ntprotei
ncrystal
smayf or
m.

 Microdialysi
scanproducecr ystal
sbysaltingout
,empl
oyinghighconcentrat
ions
ofsaltorothersmal
lmembr ane-permeabl
ecompoundsthatdecreasethesol
ubili
ty
ofthepr otei
n.Veryoccasionall
y,somepr otei
nscanbecr yst
alli
zedbydi al
ysis
salt
ing in,by dial
yzing against pure water,r
emovi
ng solutes,dri
ving self-
associati
onandcrystal
lizat
ion.

7
15BT45 S
TRUCTURALBI
OLOGY

Fi
gure:Threemet
hodsofpr
epar
ingcr
yst
als,A:Hangi
ngdr
op.B:Si
tti
ngdr
op.C:
Micr
odial
ysis

Li
mi t
ations
 Ast hecr ystal'
sr epeat inguni t,itsuni tcel l
,becomesl argerandmor ecompl ex, t
he
atomic- levelpi ct urepr ovidedbyX- raycr ystallographybecomesl esswel l-
resolved
(more" fuzzy" )foragi vennumberofobser vedr eflections.
 Two l imi ting cases of X- ray cr yst allography—" smal l-molecule" and
"macr omol ecular " cr yst allography—ar e of t
en di scerned. Smal l
-molecul e
crystal logr aphyt ypical l
yi nvolvescr ystalswi thf ewert han1 00at omsi nt heir
asymmet ri
cuni t;such cr yst alst r
uct uresar eusual lysowel lresolved thatt he
atomscanbedi scer nedasi solated" blobs"ofel ect r
ondensi ty.
 Bycont r ast,macr omolecul arcr ystal l
ogr aphyof teni nvolvest ensoft housandsof
atomsi nt heuni tcel l
.Such cr yst alst r
uctur esar egener allylesswel l-
resolved
(more" smear edout ");theat omsandchemi calbondsappearast ubesofel ect ron
densit y, rathert hanasi solatedat oms. Ingener al,smal lmolecul esar ealsoeasiert o
crystal lize t han macr omol ecules;however ,X- ray cr ystallography has pr oven
possibl eevenf orviruseswi thhundr edsoft housandsofat oms. Thoughnor mallyx-
raycr yst allographycanonl ybeper f
or medi ft hesampl ei sincr ystalform,new
resear chhasbeendonei ntosampl i
ngnon- cryst alli
nef ormsofsampl es

FI
BERDI FFRACTI ON
 Fiberdiffr
actioni
sasubar eaofscatteri
ng,anareainwhi chmol ecul
arst ructur
ei s
determinedf r
om scat teri
ngdat a(usuall
yofX- r
ays,electronsorneutrons).Infiber
diffr
actionthescatteringpat terndoesnotchange,ast hesampl eisrotatedabouta
uniqueaxis( t
hefiberaxi s).Suchuni axi
alsymmet ryisf r
equentwi t
hf i
lamentsor
fi
bersconsi sti
ngofbi ologicalorman-mademacr omolecules.
 Incrystall
ographyf ibersymmet ryisanaggr avat
ionr egardingthedet ermination

8
15BT45 S
TRUCTURALBI
OLOGY
ofcr yst alst ruct ur e,becauser ef l
exi onsar esmear edandmayover l
api nt hef iber
di ff
r act ionpat tern.Mat er ial ssci enceconsi der sf ibersymmet ryasi mpl ifi
cat ion,
becauseal mostt hecompl eteobt ai nabl est ruct ur ei nf ormat ioni si nasi nglet wo-
di mensi onal( 2D)di f fr act ionpat t er nexposed onphot ographi cf i
lm orona2D
det ect or .2inst eadof3co- or dinat edi rect ionssuf fi
cet odescr ibef iberdi ffr
act ion.
 Thei dealf iberpat ter nexhi bits4- quadr antsymmet ry.Int hei dealpat ter nthef iber
axi si scal l
edt hemer idi an, t heper pendi cul ardi rect ioni scal ledequat or .
Incaseof
fibersymmet r y,manymor er ef lexionst hani nsi ngl e-cr ystaldi ffract i
onshowupi n
the2D pat ter n.I nf iberpat ternst heser ef lexionscl ear lyappearar rangedal ong
lines( l
ayerl ines)r unni ngal mostpar al lelt ot heequat or .
 Thus,i nf iberdi f fract ion t he l ayerl i
ne conceptofcr yst allogr aphy becomes
pal pabl e.Bent l ayer l ines i ndi cat et hat t he pat tern must be st raight ened.
Ref lexi onsar el abel ledbyt heMi lleri ndexhkl ,i.e.3di gits.Ref lexionsont hei -th
layer l ine shar e l =i. Ref lexions on t he mer idi an ar e 00l -
ref lexions. I n
cr yst allogr aphyar tifici alf iberdi ffr act ionpat ter nsar egener at edbyr otatinga
singl ecr yst alaboutanaxi s( rot atingcr yst almet hod) .
 Non- idealf iberpat ter ns ar e obt ai ned i n exper i
ment s.They onl y show mi rror
symmet ryaboutt hemer idi an.Ther easoni st hatt hef iberaxi sandt hei nci dent
beam ( X- rays,el ect r
ons,neut rons)cannotbeper fect lyor ient edper pendi culart o
eachot her .Thecor r
espondi nggeomet ricdi stor tionhasbeenext ensi velyst udi ed
by Mi chaelPol anyii nt r oduci ng t he conceptofPol anyi '
s spher ei ntersect ing
Ewal d'sspher e.
 Rosal indFr ankl inandRaymondGosl i
nghavecar riedoutt heirowngeomet rical
reasoni ng and pr esent ed an appr oxi mat iveequat ion f ort hef ibert iltangl eβ.
Anal ysi sst art sbymappi ngt hedi st or ted2Dpat ternont her epr esent at ivepl aneof
thef iber .Thi si st hepl anet hatcont ai nst hecyl inderaxi sinr ecipr ocalspace.
 Incr yst allogr aphyf ir stanappr oxi mat ionoft hemappi ngi nt oreci pr ocalspacei s
comput edt hati sr efi nedi ter at i
vel y.Thedi gitalmet hodf r equent lycal l
edFr aser
cor rect ionst ar tsf rom t heFr ankl i
nappr oxi mat ionf ort het iltangl eβ.I tel iminat es
fibert ilt,unwr apst hedet ect ori mage,andcor rect st hescat teringi ntensi ty.The
cor rectequat i onf ort hedet er mi nat ionofβhasbeenpr esent edbyNor ber tSt ri
beck.

NEUTRONDI FFRACTI ON
 Neut rondi ffractionorel ast ic neut r
onscat ter ing i
st heappl icationofneut ron
scat teri
ng tot hedet ermi nation oft heat omi c and/ ormagnet ic structureofa
mat erial.Asampl et obeexami nedi spl acedi nabeam oft hermalorcol dneutronsto
obtain a di f
fract ion patter nt hatpr ovides i nformat i
on oft hest ructureoft he
mat erial.
 Thet echniquei ssi milart oX- raydi ffractionbutduet ot heirdi fferentscat ter
ing
proper ti
es,neut r
onsandX- rayspr ovidecompl ement aryi nf ormat ion:X-Raysar e
suitedf orsuper ficialanal ysis,strongx- raysf r om synchr otronr adiationaresuited
forshal low dept hsort hi nspeci mens,whi leneut r
onshavi ng high penet rati
on
dept har esuitedf orbul ksampl es.
 Neut rondi ff
ract i
onexper i ment sdet erminet heat omicand/ ormagnet i
cst r
ucture
ofamat eri
al.Thi st echni quecanbeappl iedt ost udycr yst alli
nesol i
ds,gasses,
li
qui dsoramor phousmat er i
als.
 Neut rondi ff
ract i
oni saf or m ofelast icscat ter i
ngwher et heneut ronsexi tingthe
exper imenthavemor eorl ess thesameener gyas t hei nci dentneut r
ons.The
techni queissi mi lartoX- r aydi ff
ract ionbutt hedi fferentt ypeofr adiationgives

9
15BT45 S
TRUCTURALBI
OLOGY
compl ement
aryinf
ormat i
on.A sampl et obeexamined is pl
aced in a beam of
thermalorcold neutrons and the i
ntensit
y pat
ter
n around the sample gives
infor
mat i
onoft
hestructureofthematerial
.

I
nstrument alandsampl er equi rement s
 Thet echni quer equi resasour ceofneut rons.Neut r onsar eusual lyproducedi na
nuclearr eact ororspal lat ionsour ce.Atar esearchr eactor,othercomponent sar e
needed, includi ngacr yst almonochr omat orsaswel lasf i
lterst oselectthedesi red
neutronwavel engt h. Somepar tsoft heset upmayal sobemovabl e.
 Ataspal lati
onsour ce,thet imeoff lightt echni quei susedt osor ttheenergiesof
theinci dentneut rons( higherener gyneut ronsar ef ast er
),sonomonochr omat oris
needed,butr atheraser iesofaper tureel ement ssynchr onizedt ofilt
erneut ron
pulseswi ththedesi redwavel engt h.
 Thet echni quei smostcommonl yper formed aspowderdi ffracti
on,which onl y
requiresapol ycryst alli
nepowder .Forsi ngl ecr yst alwor k,t hecr ystalsmustbe
muchl argert hant hoseusedi nX- raycr yst allography. Itiscommont ousecr ystals
3
thatareabout1mm .
 Themai ndi sadvant aget oneut rondi ff r
act i
oni st her equi rementf oranucl ear
reactor.Forsi nglecr ystalwor k,t het echni quer equi r
esr elativelylargecryst als,
whichar eusual lychal lengi ngt ogrow.Themai nadvant agest ot hetechniquear e
many -sensi tivityt ol ightat oms,abi li
t yt o di stingui sh isot opes,absence of
radiationdamage, aswel lasapenet rationdept hofsever alcm

Nuclearscat ter ing

 Neut ronsi nt er actwi th mat terdi ffer ent l
yt hanx- rays.X- raysi nter actpr imarily
wi ththeel ect roncl oudsur roundi ngeachat om.
 Thecont r i
but iont ot hedi ffract edx- rayi ntensi tyi st her efor el argerf orat omswi th
al argeat omi cnumber( Z)t hani ti sf orat omswi t
hasmal lZ.Ont heot herhand,
neut ronsi nt er actdi rectlywi tht henucl eusoft heat om, andt hecont ribut iontothe
diffractedi ntensi tyi sdi fferentf oreachi sot ope;f orexampl e,r egul arhydr ogen
anddeut er iumcont ributedi ffer ent ly.
 Iti s also of ten t he case t hatl ight( low Z)at oms cont ribut e st rongl ytot he
diffractedi nt ensi tyeveni nt hepr esenceofl ar geZat oms.Thescat teri
ngl engt h
var i
esf r om i sot opet oi sot oper at hert hanl inear lywi tht heat omi cnumber .An
elementl ikeVanadi um isast rongscat tererofX- rays, buti tsnucl eihar dlyscat ter
neut rons, whi chi swhyi tof tenusedasacont ainermat erial. Non- magnet icneut ron
diffractioni sdi rectlysensi tivet ot heposi tionsoft henucl eioft heat oms.
 Amaj ordi ffer encewi t
hX- raysi st hatt hescat ter ingi smost l
yduet ot het i
nynucl ei
oft heat oms. Thatmeanst hatt her ei snoneedf oranat omi cf ormf act ort odescr ibe
theshapeoft heel ectroncl oudoft heat om andt hescat t er i
ngpowerofanat om
doesnotf allof fwi tht hescat teringangl easi tdoesf orX- r
ays.
 Diffractogr amst her eforecanshow st rongwel ldef ineddi ffract i
onpeaksevenat
high angl es,par ticularlyi ft heexper imenti sdoneatl ow t emper at ures.Many
neut ron sour ces ar e equi pped wi thl i
qui d hel ium cool ing syst ems t hatal low
collectingdat aatt emper atur esdownt o4.2K.
 Thesuper bhi ghangl e( i.e.hi ghr esol ution)i nf ormat ionmeanst hatt hedat acan
givever ypr eci seval uesf ort heat omi cposi tionsi nt hest ruct ur e.Ont heother
hand,Four iermaps( andt oal esserext entdi ffer enceFour iermaps)der ivedf rom
neut rondat asuf ferf rom ser iest er mi nationer r ors,somet imessomucht hatthe

1
0
15BT45 S
TRUCTURALBI
OLOGY
r
esul
tsar
emeani
ngl
ess.

X-
rayabsor ptionf inest ruct ure( XAFS)
 X-rayabsor ptionf inest r ucture( XAFS)i saspeci ficst r
uct ureobser vedi nX-r ay
absor ption spect roscopy ( XAS) .By anal yzing t he XAFS,i nformat i
on can be
acqui redont hel ocalst r uct ureandont heunoccupi edl ocalel ectronicstat es.
 Theat omi cX- rayabsor pt ionspect rum (XAS)ofacor e-leveli nanabsor bingat om is
separ atedi ntost atesi nt hedi scretepar tofthespect r um called" boundsf inal
states"or" Rydber gst at es"bel ow thei onizat
ionpot ent i
al( IP)and" stat esi nthe
continuum"par toft hespect rumabovet heioni
zat i
onpot ent ialduetoexci tationsof
thephot oelectroni nthevacuum.
 Abovet heI Pt heabsor pt i
oncr osssect ionat t
enuat esgr aduallywi tht heX- r ay
energy.Fol lowi ngear lyexper i
ment alandt heoreticalwor ksi nthet hirt
ies,i nthe
sixtiesusi ngsynchr ot ronr adiationatt heNat ionalBur eauofSt andar dsi twas
establishedt hatt hebr oadasymmet ri
cabsor ptionpeaksar edueFanor esonances
abovet heat omi ci onizat ionpot ent i
alwher ethef inalst at esar emanybodyquasi -
boundst ates( i
.e.,
adoubl yexci tedat om)degener atewi t hthecont i
nuum.

Spectraofmol ecul esandcondensedmat ter

Edgeregi on
 Theedger egionusual l
yext endsi nar angeoff eweVar oundt heabsor pti
onedge.
 Thespect ralf eaturesi nt heedger egioni )ingoodmet alsar eexci tat
ionst of i
nal
delocal i
zedst atesabovet heFer mil evel ;i
i)ininsulatorsar ecor eexci tonsbelow
thei oni
zat ionpot ent i
al;i ii)inmol ecul esar eelectronictr ansitionst ot hef i
rst
unoccupi ed mol ecularl evel sabovet hechemi calpot ent
iali nt hei nitialstates
whi char eshi f t
edi nt ot hedi scr etepar toft hecoreabsor ptionspect r
um byt he
Coul ombi nter act i
onwi tht hecor ehol e.
 Mul ti-el
ect ronexci t
at ionsandconf igurat i
oni nt
eractionbet weenmanybodyf i
nal
statesdomi nat etheedger egioni nst rongl ycorrel
atedmet alsandi nsulators.For
manyyear st heedger egi onwasr eferredt oast he“Kosselst ructure”butnow i tis
known as " absor ption edge r egi on"si nce t he Kosselst ruct urer efers onlyt o
unoccupi ed mol ecularf i
nalst ateswhi ch isacor rectdescr i
ptiononl yf orfew
parti cul
arcases: mol eculesandst rongl ydisorderedsyst ems.

X-
rayAbsor pti
onNearEdgeSt ruct ure
 TheXANESener gyregionext endsbet weent heedger egionandt heEXAFSr egion
overa50- 100eVener gyr angear oundt hecor elevelx-rayabsor pt iont hreshold.
 Bef ore1 980t heXANESr egionwaswr ongl yassignedt odi fferentf inalst ates:a)
unoccupi ed t ot
aldensi tyofst ates,orb)unoccupi ed mol ecul aror bitals( kossel
struct ure)orc)unoccupi edat omi cor bit
al sord)l owener gyEXAFSosci l
lations.In
thesevent i
es,usingsynchr otronr adi ati
oni nFr ascat iandSt anf ordsynchr otron
sour ces,i twasexper imentallyshownt hatt hef eaturesi nt hisener gyr egionar e
duet omul tiplescattering resonancesoft hephot oel ectroni nananocl usterof
variabl esize.
 Ant onioBi anconii n1 980i nvent ed theacr onym XANESt oindi cat et hespect ral
regi ondomi natedbymul ti
plescat t
eringr esonancesoft hephoel ectronint hesof t
x-rayr ange andi nt hehar dX- rayr ange. Int heXANESener gyr anget heki netic
ener gyoft hephot oelectronint hef i
nalst ateisbet weenf eweVand50- 100eV.
 Inthi sr egimet hephot oelectronhasast rongscat teringampl it
udebynei ghbor i
ng

1
1
15BT45 S
TRUCTURALBI
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atoms i n mol ecules and condensed mat ter,its wavelength isl arger t han
interatomi cdi stances, it
smeanf r
eepat hcoul dbesmal l
erthanonenanomet erand
finallythelifet i
meoft heexci tedstateisint heorderoffemtoseconds.
 TheXANES spect ralf eatures aredescr ibed byf ullmultiplescatteri
ng t heory
pr oposedi ntheear lysevent ies.Thereforethekeyst epforXANESi nterpretationis
thedet ermi nat i
onoft hesi zeoft heat omi cclusterofneighboratoms,wher et he
finalst atesar econf i
ned,whi ch couldr angef rom 0.
2nm t o2nm i ndi f f
erent
syst ems.
 Thi sener gyr egionhasbeencal l
edl at
er( i
n1 982)alsonear-edgeX-rayabsor pt i
on
finest ructure( NEXAFS) ,whi chi ssynonymouswi t
hXANES.Dur i
ngmor et han20
year stheXANESi nt erpretationhasbeenobj ectofdiscussionbutrecentlyther eis
agr eementt hatt hef inalstatesar e"multi
pl escatter
ingresonances"andmanybody
finalstatespl ayani mpor tantrole.

I
ntermedi
ateregion
 Therei
sani nt
ermediat
eregionbet
weentheXANESandEXAFSr
egi
onswher
elown-
bodydist
ributi
onfunct
ionsplayakeyr
ole.

Ext
endedX- rayabsor ptionf inest ructure
 Theosci l
latorystruct ureext endingf orhundr edsofel ect
ronvoltspastt heedges
wascal l
edt he“Kronigst ructure”afterthesci enti
st ,RalphKr onig,whoassi gned
thi
sst r
uct ureinthehi ghener gyr ange(i.e.
,foraki neticenergyr ange-largerthan
100eV-oft hephoel ect r
oni nt heweakscat t eringregime)t othesi ngl
escat ter
ing
oftheexci tedphot oelect ronbynei ghbouri
ngat omsi nmol eculesandcondensed
mat t
er .
 Thisr egimewascal ledEXAFSi n1 971bySayer s,Ster nandLittleandi tdeveloped
onlyaf t
ert heuseofi nt ensesynct rotronradiationsour ces.

Applicationsofx- rayabsor ptionspectroscopy

 X-rayabsor ptionedgespect r
oscopycorrespondst othet ransi
tionf r
omacor e-
level
toanunoccupi edor bitalorbandandmai nlyr ef
lectst heelect ronicunoccupied
states.EXAFS,r esulti
ngf rom theinterf
erencei nthesingl escat teringprocessof
thephot oel
ect ronscat ter
edbysur roundingat oms,pr ovidesi nf ormat
ionont he
localst ructure.
 Informat ionont hegeomet ryofthelocalstructureisprovidedbyt heanalysisofthe
mul ti
pl escat teringpeaksi nt heXANESspect ra.TheXAFSacr onym hasbeenl ater
introducedt oindi cat
et hesumoft heXANESandEXAFSspect r
a.

CI
RCULARDI CHROI SM (
CD)SPECTROSCOPY
 Intheear lynineteenthcent urytheFr enchphysi cistJeanBapt i
steBi otobser ved
that solutions of some or ganic mol ecules appear ed t or otatet he pl ane of
polari
zationofpl anepol ari
zedlight ,aphenomenonr ef er
redt oasopt icalrot atory
dispersion
 Electr
omagnet i
cr adi
at i
onconsist sofanel ectri
candmagnet icfieldthatosci l
late
perpendicul artooneanot herand t othepr opagat ing di r
ection.Whi l
el inearly
polari
zedl ightoccur swhent heel ectr
icfi el
dvect orosci ll
atesonl yinonepl ane
andchangesi nmagni tude,cir
cularlypolarizedl i
ghtoccur swhent heel ectr
icf ield
vectorrotatesaboutitspr opagationdirectionandr etainsconst antmagni tude.
 Hence,itf ormsahel i
xi nspacewhi l
epr opagat ing.Forl eftcircular
lypol ar i
zed

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2
15BT45 S
TRUCTURALBI
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li
ght( LCP)wit
h propagati
ontowardstheobser
ver,theelect
ricvect
orrotat
es
countercl
ockwi
se.Forrightci
rcul
arlypol
ari
zed l
ight(RCP),t
heelect
ricvector
rotat
esclockwi
se.

Fi
gur
e:Typesofpol
ari
zedl
ight

 Weknowt hatthisi saconsequenceoft hefactthateachenant i

omerofanopt i
call
y
activemoleculeinter
actsdiffer
entlywi t
hleftandr i
ghtcircularl
ypolar
izedlight.
Byconvention,l
aevorotat
ionisdesignatedas( −)whil
edextrorotati
oni
sdenot edby
(+).A50:50mi xtureofequalamount sofdandL- enant
iomer sdoesnotr ot
atethe
planeofpolari
zationnordoesasolutionofanachi r
almoleculesuchasglycine.

Figure:OpticalRotat
oryDispersi
on
(a)L-
ent i
omer srot
atetotheplanetot
heleft
(b)D-entiomersrotat
etotheplanetother
ight

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3
15BT45 S
TRUCTURALBI
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Fi
gure:Ci
rcul
arDichr
oism of(
a)pr
oteinconf
ormat
ions(
Alpha,Bet
aandCol
i)(
b)DS
DNAvaryingwit
hG+CContent(
26%,72%,and1
00%)

 Lightpassingt hr
oughachr omophor esolut
ionmayi nter
actwi t
ht hesampl eintwo
mai nways.Thel i
ghtmayber efractedordel ayedonpassaget hrought hesolut
ion
ori tmaybeabsor bed.Refracti
oni squantifiedbyt heref racti
veindex,n,oft he
solutionwhil
eabsor ptioni
squant if
iedbyt hemol arexti
nct i
oncoef fi
cient,ε.
Ifthe
lightisplanepol ari
zedandt hesampl eisopt ical
lyactive,eachenant iomermay
interactdi
fferentlywiththeleftandr ightcircular
lypolarizedcomponent softhe
lightbeam.

 ORDarisesf
rom t
hefactthatt
her
ei saspeci
ficr
efr
act
ivei
ndexf
orl
eft(
nL)and
ri
ght(
nR)cir
cular
lypol
ari
zedli
ght:

nL≠nR-Equat
ion1

 Thedi
ffer
enceinr
efracti
veindexatanywavelengthmaybeexpr
essedas∆n.An
ORDspectrumi
saplotof∆nagainstwavel
engt
h( λ)
.

 Simil
arly,opt
ical
lyacti
vesampleshavedisti
nctmolarext
incti
oncoeff
ici
entsfor
lef
t(εL)andr i
ght(εR)cir
cul
arl
ypolar
izedli
ght.Thi
siscalledcir
cul
ardichr
oism
(CD):

εL≠εR-Equat
ion2

 The difference bet ween εL and εR may be expr

essed as ∆ε.Combini
ng the
Equations(1 )and(2)(the
 Beer–Lamber tlaw)meanst hatt her
eisadiff
erenceintheabsorbanceofl
eftand
ri
ghtci rcularlypolari
zedlight(∆A):

∆A=∆ε·
c·l

 If∆εor∆A orelliptici
tyis plot
ted againstwavel
ength(λ),aCD spectr
um is
obtai
ned.
 TheCDspectrum ofoneenantiomerisami rr
orimageofthatoftheotherandis
rel
atedtothecor
r espondi
ngORD spect rum (andvicever
sa)byamat hemati
cal

1
4
15BT45 S
TRUCTURALBI
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t
ransf
ormat
ioncal
ledt
hegener
alKr
oni
g–Kr
amer
str
ansf
ormat
ion

Pr
inci ple
 Ci r
cul ardi chroism ( CD)spect roscopyi s an opt i
calt echni quet hatal lows t he
det ect i
onandquant i zat i
onoft hechi ralit
yofmol ecul arst ructur es. I
talsopr ovides
infor mat i
onaboutt hesecondar yandt ertiar ystructur esofpr otei ns.
 Ci r
cul ardi chr oism ist hedi fferencei ntheabsor pti onofl eftandr ightci r cular l
y
pol arizedl ight .
Aper iodi cvariationi nt hepol ari
zationoft hel ightbeam i si nduced
byt hepol arizationmodul atort hroughal lelliptict iesf rom l eftci rculart hr ough
elliptical,unchangedl inearandel li
pticalt or ightcir cular .
 Thi spol ar izedl ightpassest hrought hesampl et oaphot omul t i
plierdet ect or.If
thesampl ei snotopt icallyact i
ve, thel i
ghtbeamdoesnotvar yt hr ought hiscycl e.
 Wi tht hei nt roduct i
onofanopt icallyact i
vesampl e,apr eferent ialabsor pt ioni s
seendur i
ngoneoft hepol ari
zat ionper i
odsandt hei nt ensityoft hetr ansmi t
t ed
lightnowvar i
esdur ingt hemodul at i
oncycl e.
 Thevar i
at ioni sdi rect lyrelatedt ot heci rculardi chr oism oft hesampl eatt hat
wavel engt h.Successi vedet ectioni sper f ormedatvar i
ouswavel engthsl eadst o
thegener at i
onoft hef ullCDspect rum.ForCDt obeexhi bi
tedf orasampl e,t he
sampl emustbeopt ical lyactiveandnotsuper posabl eoni t
smi rrori mage.

I
nstrumentat
ionandwor ki
ngofCDspectr oscopy
 CD spectr
aar emeasur ed i
n aspecialt ypeofspect
rophotometercal
led aCD
spectr
opolar
imeterofwhichanoutl
inedesi gni
sshowninFigurebel
ow.

Fi
gur
e:CDSpect
roscopy

 SinceCD dependsondi ffer ent ialabsor bance,ameansofsel ect i

velyexposi ng
sampl et oleftandr ightci r
cul arlypol arizedl i
ghti snecessary.Thisi sachi evedby
passi ngabeam ofpl anepol ar izedl i
ghtt hroughaphot oelasticmodul atorwhi chi s
normal l
yaquar t
zcr ystalsubj ect edt oanosci ll
atingelectr
icf ield.Theef fectof
thisi stovar ytheci rcularpol arizationoft hebeam passi ngt hrought hemodul ator
alternat elyfrom leftt orightwi t
haf requencyofsome50kHzwhi l
stmai nt aininga
const antl ightintensi ty.
 Different ialabsor ptionofl ef tandr ightci r cularlypolari
zedl ighti sdetect edata
phot omul t
iplierandconver tedi ntoellipt i
ci ty,θ,whichhasuni tsofmi l
lidegr ees.
Thi st erm ar isesf r
om t hef actt hatsel ect iveabsor pti
onofoneoft heci rcularly

1
5
15BT45 S
TRUCTURALBI
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polar i
zed component s ofpl ane pol arized l ighthas t he consequence thatthe
resultantelect ri
cvectort r
acesanel lipt icalpat har oundtheaxisofthebeamandi s
saidt obeel l
ipt i
call
ypolar i
zed.
 InaCDspect r opol
arimeter,thet wol i
ghtbeamsar enotinfactrecombinedbuta
phot omul t
iplierdetectorconver tsinci dentl ighti ntensi
tyintoanelectri
ccur r
ent
composed par tl
y ofalternating cur rent( AC)and par t
ly ofdirectcurrent(DC)
component s.TheDCcomponenti sr elatedt ot otalli
ghtabsorptionbythesampl e
whi letheACcomponenti sadi rectmeasur eofCD.
 Thi sar r
angementf acil
itatessepar at eabsor pt i
onmeasur ementsoft herightand
leftcircularl
ypol ari
zedcomponent sofpl anepol arizedli
ght.

Applicati onofCi rcul arDi chr oi smSpect r

oscopy
 Circul ar di chr oi sm i s exhi bited by bi ologi cal mol ecules,because of t hei r
dext ror otaryandl evor ot ar ycomponent s.Evenmor eimpor tanti st hatasecondar y
struct ur ewi llalsoi mpar tadi st inctCDt oi tsr espect i
vemol ecul es.Ther efore, the
alphahel ixofpr otei nsandt hedoubl ehel ixofnucl eicaci dshaveCD spect ral
signat ur es r epr esent at ive oft hei rst ruct ures.The capaci t y ofCD t o gi ve a
represent at i
ve st ruct ur al si gnat ure makes i t a power ful t ool i n moder n
biochemi strywi t happl icat ionst hatcanbef oundi nvi rt
ual l
yever yf i
eldofst udy.
 CDspect racanbeusedt oest imat ethef ract ionofamol ecul et hati si ntheal pha-
helixconf ormat ion,t hebet a-sheetconf or mat i on,t hebet a- tur nconf ormat ion,or
someot her( e.g.randomcoi l
)conf ormat ion
 CDcanbeusedt ost udyhow t hesecondar yst ruct ureofamol ecul echangesasa
funct ion of t emper atur e or of t he concent r ation of denat ur ing agent s,e. g.
Guani di nium hydr ochl ori de or ur ea. I n t hi s way i t can r eveal i mpor tant
thermodynami ci nf or mat ionaboutt hemol ecul e( suchast heent hal pyandGi bbs
freeener gyofdenat urat ion)t hatcannotot her wi sebeeasi l
yobt ained.
 UV CD spect rapr ovi dest ruct ur alinfor mat ion on t henat ur eoft hepr osthet ic
groupsi npr oteins, e.g.,t hehemegr oupsi nhemogl obinandcyt ochr omec.
 Itisal sousedt ost udyt ert iaryst ructureofpr ot ei n
 VisibleCD spect roscopyi s aver ypower f
ult echni quet ost udymet al–pr otein
interact i
onsand canr esol vei ndi viduald–d el ect ronict r ansi tionsassepar ate
bands.
 CDcanbeusedt ost udyal ar genumberofsol ventcondi ti
ons, var yingt emper atur e,
pH, sal inity,andt hepr esenceofvar i
ouscof act or s.
 CD spect roscopy i s usual l
y used t o st udy pr oteins i n sol ut i
on,and t hus i t
compl ement smet hodst hatst udyt hesol idst at e.Thi si sal soal imi tati
on,int hat
manypr oteinsar eembedded i nmembr anesi nt heirnat ivest at e,and sol ut i
ons
cont aini ngmembr anest r uct ur esar eof tenst r ongl yscat ter ing.CD i ssomet imes
measur edi nthi nf ilms.
 CD al so used t o st udy t he nucl eic aci d conf ormat ion:Enhanced CD t hati s
represent at i
veoft her elat ivebase- baseor ient at ion

NMRSPECTROSCOPY( STRUCTUREDETERMINATION)
Nucl
earMagnet
icResonance(
NMR)Spectr
oscopy

1
6
15BT45 S
TRUCTURALBI
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 Nucl earMagnet ic Resonance ( NMR)spect r
oscopy ist he mostpower fult ool
availabl efordet ermi ningt hestructureofor ganiccompounds.
 Thist echni quer eli
esont heabi l
ityofat omi cnucleitobehaveli
keasmal lmagnet
andal ignt hemwi t
hanext ernalmagnet icfield.
 Wheni rradiatedwi thar adiofrequencysi gnalt henucleiinamolecul ecanchange
from bei ngal ignedwi tht hemagnet i
cf iel
dt obei ngopposedtoi t
.Ther efore,i
ti s
called“ nuclear ”forthei nst r
umentwor ksonst imulati
ngt he“
nuclei”oftheatomst o
absor br adiowaves.
 Theener gyf requencyatwhi chthisoccur scanbemeasur edandisdi splayedasan
NMRspect rum. Themostcommonnucl eiobser vedusingt hi
stechni queare1Hand
1
3
so31P,19 29
C,
butal F, Siand77SeNMRar eavai lable.
 NMRSt andsf orNucl earMagnet i
cResonance

Pr
oper
tiesoft
heNucl
eus
 Nucl
earspi
n
 Nucl
earmagnet
icmoment
s

TheNucl
eusi
naMagnet
icFi
eld
 Precessionandt
heLarmorf
requency
 NuclearZeemaneff
ect&Bol
tzmanndist
ribut
ion

WhentheNucleusMeett
herightMagnetandr
adi
owaveManyat
omi
cnucl
eibehave
asi
ftheyspi
nonanaxisofr
otati
on
 Nucl eiarepositivel ychar ged
 Thesespi nningnucl eigenerateti
nymagnet i
cfiel
ds
 Tinymagnet sinter actwi thanexter nalmagnet i
cf i
eld,denot edB0
1 13
 Pr ot
on(H)and car bon(C)ar ethemosti mportantnucl earspi nstoorgani
c
chemi sts
 Nuclearspinsar eor ient edrandomlyint heabsence(a)ofanext er
nalmagnet
icfiel
d
buthaveaspeci fi
cor i
ent ati
oninthepr esence(b)ofanext ernalfiel
d,B0
 Somenucl earspi nsar eal
ignedpar al l
eltotheexternalfield
 Lowerener gyorient at i
on
 Somenucl earspi nsar eal
ignedant i
par al
lelt
otheext er
nalf iel
d
 Higherener gyorient ation

1
7
15BT45 S
TRUCTURALBI
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 When nucl eit hatar e al i
gned par all
elwi t
h an ext ernalmagnet icfield are
irr
adiat edwi t
ht hepr operf requencyofel ectromagnet i
cr adiationtheenergyi s
absorbedandt henucl ei“ spin-flips”tot hehigher-energyant iparall
elali
gnment
 Nucleit hatunder go“spi n-flips”inr esponset oappliedr adiationaresaidtobei n
resonancewi t
ht heappl i
edr adiation-nuclearmagnet icr esonance
 Frequencynecessar yf orr esonancedependsonst r
engt hofext er
nalfieldand
theident it
yoft henucl ei
 Theener gydi fference∆Ebet weennucl earspinstatesdependsont hest r
engt h
oftheappl iedmagnet icf ield.
Manynucleiexhi bitNMRphenomenon
 Allnucleiwi t
hoddnumberofpr ot ons
 Allnucleiwi t
hoddnumberofneut rons
 Nucleiwi t
hevennumber sofbot hpr otonsandneut ronsdonotexhi bitNMR
phenomenon
 Absor ptionofener gywi t
hf requencyconver t
sanucl eusf r
om al owertoahi gher
spinst ate.

 Theabsor ptionf requencyisnotthesamef orall1Hand13Cnuclei

 Nucleiinmol eculesar esurroundedbyel ectrons
 Electr
onssetupt inylocalmagnet i
cf ieldst hatactinopposi t
iontot
heappl ied
fiel
d,shieldingt henucl eusfromthef ulleffectoftheexternalmagneticfi
eld
 Theef fecti
vef ieldact uall
yfeltbyt henucl eusistheappl i
edf i
eldreducedby
thelocalshieldingef fects

J-
Coupli
ng
 Some of the most usef ulinformation for structure det
erminati
on in a one-
dimensionalNMRspect rum comesf rom J-couplingorscalarcoupling(aspecial
caseofspin-spincoupl
ing)betweenNMRact ivenuclei.
 Thiscoupl i
ng ari
sesfrom thei nteracti
onofdi fferentspinstatesthrough the
chemicalbondsofamol ecul
eandr esult
si nt hesplitti
ngofNMRsi gnals.These

1
8
15BT45 S
TRUCTURALBI
OLOGY
split
tingpat ter
nscanbecomplexorsi
mpleand,li
kewise,
canbest r
aight
forwar
dly
int
erpr etabl
e ordecepti
ve.This coupl
ing pr
ovides detai
led i
nsightintothe
connect i
vityofatomsi
namolecule.

Instr
umentat ionofNMRspect r
oscopy
Theessentialcomponent sofNMRi nst
rumentincl
udet
hef
oll
owi
ng.
 Ahighl yst abl
emagnet
 Sampl epr obe
 Radiofr equencygenerat
or
 Phasesensi ti
vedet
ectorandampli
f i
er
 Datapr ocessingunit&displ
aydevise

Magnet
 Itisthehear tofalltypesofNMRspect r
omet er
s. Thesensi ti
vityandresolut
ionofa
spectr ometercriti
cal l
ydependsont hest rengt handqual i
tyoft hemagnet .Since
sensitivityandresol uti
onbothincreasewi thf i
el dstrength,itisadvantageoust o
operat etheinstr
umentathi ghestpossiblef i
eldst rength.
 Inaddi ti
on,thefieldmustbehomogeneous, uniform andr eproducibl
e.Threet ypes
ofmagnet shavebeenusedi nNMRspect romet er s;t
hesear epermanentmagnet ,
elect
r omagnetandsuperconduct i
ngsol enoid.

TheSampl ePr obe

 Iti sakeycomponenti nNMRspect romet erwhi chnotonl yhol dst hesampl eina
fixedposi tionint hemagnet icf iel
dbutal socont ainsanai rt urbinet orotat e(spin)
thesampl e.Thesampl eisspi nnedsoast oensur et hatitexper i
encesauni form
field.
 Inaddi tiont othese,i thousesacoi lforgener atingr adiofrequencyf orexci tation
andal sof ordet ect ionoft heNMRsi gnal .EarlierNMRspect romet ersusedt ohave
separ at et ransmi tterandr eceivercoilsper pendi cul artoeachot herf orexci tation
oft hepul seanddet ect i
onofthesi gnal .
Moder nspect r
omet erscont ainasi nglecoil
pr obewhi ch i ssi mpl eand mor eefficient.Si ncemostoft hecont inuouswave
spect romet ersusef iel
d sweep mode,af i
xed f requencyi sgener ated f rom t he
radi ofrequencygener atorandf requencysynt hesi zer .
 Thespect romet erisknownbyi tsoper atingf requencye. g.,
a60MHz, a1 00MHzora
500MHzi nst rumentandsoon. However ,inFour iertr ansformspect ra,thesampl eis
irradiat edwi thapul seconsist ingofar angeoff requenci essuf ficient l
ygr eatt o
exci tenucl eus havi ng differentr esonancef requenci es.Thepul seofr adiation
pr ovides a r el
ativel y broad band of f requenci es cent er ed on t he osci l
lator
frequency.

Det
ectorSyst em
 Highf r
equencyr adiosignalisfirstconvertedtoanaudi ofrequencysignalwhich
canbet houghtofasbei ngmadeupoft wocomponent s;acar r
iersignalwhichhas
thefrequencyoft heoscill
atorthatisusedt oproduceitandasuper imposedNMR
signalfromtheanal yte.
 Theanal yt
esi gnaldi f
fersinfrequencyf r
om thestandardbyaf ewppm.Incaseof
protonspect rum,chemi calshiftsaret ypical
lyinther angeof1 -10ppm.Thus
protonmagnet icr esonancedat ageneratedby200MHzspect rometerwouldli
ein
thefrequencyr ange200,000,000Hzt o200,002,
000Hz.

1
9
15BT45 S
TRUCTURALBI
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 Iti
si mpracti
caltodigit
izesuchasmal ldif
ference.Inpractice,adiff
erencesignal
is obtai
ned thatlies inthe audiofrequency in kilohert
zr ange.The moder n
spectrometerscontainaquadrupolephasesensi t
ivedet ect
orwhi chi scapableof
sensingthesignoff r
equencydiff
erence.Thisall
owst hedet erminati
onofpositive
andnegat i
vediff
erencebetweenthefrequencyofst andardandsampl e

Fi
gur
e:Schemat
icdi
agr
am ofNMRSpect
roscopy

RepresentationofNMR
 TheNMRspect rum isrecordedonachar tpaperwithX-axisrepr esent i
ngchemi cal
shift(δ)inppmandY- axisasintensi
ty.Theδvaluesincreasef r
omr ighttoleft
.
 The val ue of zer o on the X axi s corresponds t ot he i nternal standard,
tetramethylsilane( TMS)si gnal.Low δvaluescorrespondt ohi ghf iel
dandvi ce
versa.
 TheNMR spect raar enormallyrecorded i
nt womodes;absor pt ionand integral
modes.Theout putconsi st
soft wot r
acesonei sthespect rum i.e.
,t heabsorption
signalsatdi f
fer entδvaluesandt heothercall
edintegrat
iont r
ace.

Examples:
NMRspect rumofEt hylal cohol
1
 H NMR spect rum ( 1-dimensi
onal)ofet hanolpl ott
ed as si
gnali nt
ensity vs.
chemicalshift
.
 TherearethreedifferenttypesofHat omsi nethanolregardi
ngNMR.
 Thehydr ogen(H)ont he-OH groupi snotcoupl ingwi ththeotherH atomsand
appearsasasi nglet,butt heCH3-andt he-CH2-hydr ogensarecoupli
ngwi t
heach
other,
result
inginat ripletandquart
etr espectivel
y.

20
15BT45 S
TRUCTURALBI
OLOGY

Fi
gur
e:NMRSpect
raofet
hylal
cohol

Appl i
cat i onofNMRi nSt ruct uralBi ology
Studyofpr oteins
 Much oft hei nnovat ionwi t hinNMR spect roscopyhasbeenwi thint hef ield of
prot einNMRspect roscopy, ani mpor tantt echni quei nst ructur albiology. Acommon
goaloft hesei nvest igat ionsi st oobt ainhi ghr esolut i
on3- di mensi onalst ruct ur es
ofthepr ot ein.
 NMR spect roscopyi susual lyl imitedt opr otei nssmal lert han35kDa,al though
largerst ruct ureshavebeensol ved.NMR spect roscopyi sof t
ent heonl ywayt o
obtai nhi ghr esolutioni nf ormat iononpar tiallyorwhol lyint ri
nsi callyunst ruct ur ed
prot eins.
 I ti s now a common t ool f or t he det ermi nation of Conf ormat ion Act ivity
Relat ionshi pswher ethest ruct urebef oreandaf terinteract ionwi t
h, forexampl e,a
drugcandi dat eiscompar edt oitsknownbi ochemi calact ivity.
 Pr ot eins ar e or ders of magni tude l ar
ger t han t he smal lor gani c mol ecul es
discussedear lierint hi sar t i
cle,butt hebasi cNMRt echni quesandsomeoft he
NMRt heor yal soappl i
es.
 Becauseoft hemuch hi ghernumberofat omspr esenti napr oteinmol ecul ei n
compar i
sonwi t
hasmal lor gani ccompound, t
hebasi c1 Dspect rabecomecr owded
with over lappi ng si gnal st oanext entwher edi rectspect raanal ysi s becomes
untenabl e.Ther ef ore,mul tidimensi onal(2, 3or4D)exper i
ment shavebeendevi sed
todealwi t
ht hispr oblem.
 Themosti mpor tantmet hodusedf orst ruct ur edet er minat i
onofpr ot einsut i
lizes
NOEexper iment st omeasur edi stancesbet weenpai r sofat omswi thi nt hemol ecul e.
Subsequent ly,theobt ai neddi stancesar eusedt ogener at ea3Dst ruct ureoft he
mol ecul ebysol vingadi st ancegeomet rypr obl em.

St
udyofcar bohydr atesandNucl ei
cacids
 Carbohydr ate NMR spect r
oscopy addr esses quest i
ons on the structure and
conformat i
onofcar bohydrates.
 NMRspect r
oscopyi susedt oobtai
ni nfor
mat i
onaboutt hestruct
ur eanddynami cs
ofpolynucleicacids,suchasDNAorRNA.
 Asof2003,near l
yhal fofallknownRNAst ructur
eshadbeendet erminedbyNMR
spectroscopy.Par amet er
st akenfrom NMSRspect rum canbeusedt odetermine
localstructuralfeaturessuchasgl ycosidi
cbondangl es,di
hedr alangles(using
theKarpl usequation),
andsugarpuckerconf ormations.

21
15BT45 S
TRUCTURALBI
OLOGY
 NMRi salsousefulforinvesti
gat ingnonstandardgeomet ri
essuchasbenthel ices,
non-Watson–Cr i
ckbasepai ri
ng, andbasest acking.I
thasbeenespeci al
lyusefuli n
probing the structure ofnat uralRNA ol igonucl
eotides,which t end to adopt
compl exconformat i
onssuchasst em-loops.
 NMR i sal sousef ulforprobing t hebinding ofnucleicacid moleculest oot her
molecules,suchaspr ot
einsordr ugs,byseeingwhichr esonancesareshi f
tedupon
bindingoft heothermolecule

Anal
ysisofVar i
ouselement s
 Though NMR i s notr outinely used forthe determinat
ion oftot
alel emental
concentr
ationyet,inprincipleitcouldbeusedf orgivenNMRact i
venucl eusina
sample.
 TheintegratedNMRi ntensi ti
esoft heprot
onpeaksf oralargenumberofor ganic
compounds can be used f orthe accurat
e quantit
ative det
ermi
nation oftotal
hydrogenatomsinanor ganicmi xture.

Or
gani cf unctionalgroupanal ysis
 A usef ulappl i
cation ofNMR i sthe deter
minati
on offunctionalgroups such
hydr oxylgr oups (-OH)i n alcohols(ROH)and phenol s( C6H5OH)
,al dehydes,
carboxyl i
c aci ds,olefinic and acet
yleni
chydrogens,amines and amides wi t
h
rel
at iveerrorof1to5%r ange.

I
NFRAREDSPECTROSCOPY( IRSPECTROSCOPY)
 Infraredr aysar eelectromagnetr adiationhavingwavelengt habovemi crowavebut
below vi siblel ight:bet ween 700 nanomet er
s( 0.7 micr ometer) and 100,000
nanomet er s(1mi l
limeter).
 Infraredr aysar er adi
at edandabsor bedbyal lbodiesatabovet heabsolute-zero
temper atur e.
 Infrared l ight lies bet ween the vi sible and mi crowave por tions of t he
electromagnet i
cspect r
um.I nf
rared li
ghthasar angeofwavel engths,justlike
visibleli
ghthaswavel engt hsthatrangef romredli
ghtt oviolet.
 Ther earet hreemai nregi ons:
1.NearI R
2.FarI R
3.Mi dIR

Fi
gur
e:I
Rregi
onofel
ect
romagnet
icspect
rum

 Neari
nfr
aredl
ighti
scl
osesti
nwavel
engt
htovi
sibl
eli
ghtandf
ari
nfr
aredi
s

22
15BT45 S
TRUCTURALBI
OLOGY
closert ot hemi cr owaver egionoft heel ectromagnet icspect rum.Thel onger ,far
infrar edwavel engt hsar eaboutt hesi zeofapi nheadandt heshor ter,nearinf r
ar ed
onesar et hesi zeofcel ls, orar emi croscopi c.
 Infrar edspect roscopy( IRspect roscopy)i st hespect roscopyt hatdeal swi tht he
infrar ed r egi on oft he el ect r
omagnet i
c spect r
um t hati sl i
ghtwi th al onger
wavel engt handl owerf requencyt hanvi siblelight .
 Itcover sar angeoft echni ques, most lybasedonabsor pt ionspect r
oscopy. I
tcanbe
usedt oident ifyandst udychemi cals.Acommonl abor at oryinst r
umentt hatuses
thist echni quei saFour iert ransformi nfrared( FTI R)spect romet er.
 Four iert ransf or mi nfr aredspect roscopy( FTIR)i sat echni quewhi chi susedt o
obt ainani nf rar edspect r um ofabsor ption,emi ssi on,phot oconduct ivityorRaman
scat ter i
ngofasol id, l
iqui dorgas.
 Four iert ransf or mi nfr ar edspect roscopyor iginat esf rom t hef actt hataFour i
er
transf orm( amat hemat icalal gorithm)i sr equir edt oconver tther aw datai ntot he
act ualspect rum.
 Infrar edvi br at ionalspect roscopyi sat echni quewhi chcanbeusedt oi dent i
fy
mol ecul esbyanal ysisoft heirconst ituentbonds.
 Eachchemi calbondi namol eculevi bratesataf requencywhi chi schar acteristic
oft hatbond.Agr oupofat omsi namol ecul e(e.g.CH2)mayhavemul tiplemodesof
osci l
lat ioncausedbyt hest retchingandbendi ngmot ionsoft hegr oupasawhol e.
 Ifanosci llat ionl eadst oachangei ndi polei nt hemol ecul e,theni twi llabsor ba
phot on whi ch has t he same f requency.The vi brati onalf requenci es ofmost
mol ecul escor respondt ot hef requenci esofi nfr aredl i
ght .Typically,thet echnique
isusedt ost udyor gani ccompoundsusi ngl ightr adi ationf rom 4000–400cm−1 ,the
mi d-inf r
ar ed.
 Aspect rum ofal lt hef requenci esofabsor ptioni nasampl eisrecor ded. Thiscanbe
used t ogai ni nfor mat i
on aboutt hesampl ecomposi t i
on int er ms ofchemi cal
groupspr esentandal soi tspur it
y( f
orexampl eawetsampl ewi l
lshowabr oadO- H
absor pt ionar ound3200cm−1 )
.

Pr
incipleofIRspect roscopy
 Infrar
ed spect roscopy expl oit
s t he f act that mol ecules absor b specif
ic
frequenci
est hatarecharacteristi
csoft heirstr
ucture.
 Theseabsor ptionsar eresonantf requencies,i.
e.thef requencyoft heabsorbed
radiati
onmat chest hefrequencyoft hebondorgr oupt hatvi br
at es.Theener
gies
are deter
mi ned by the shape oft he mol ecul
arpot entialener gy surf
aces,the
massesoft heat oms,andtheassoci atedvibroniccoupli
ng.

Numberofvi brationalmodes
 Amol eculecanvi brateinmanyways, andeachwayi scal l
edavi brat i
onalmode. I
tis
associatedwi thwi thchangesi ntheper manentdi pole.SoitiscalledI Racti
ve.
 Formol eculeswi t
h N at omsi nthem,l i
nearmol eculeshave3N – 5degr eesof
vibr
at i
onalmodes, wher easnonl i
nearmol eculeshave3N–6degr eesofvi brati
onal
modes( alsocal l
edvi brationaldegr eesoff r
eedom) .
Asanexampl eH2O, anon-li
near
molecule,willhave3×3–6=3degr eesofvi brat
ionalfreedom, ormodes.
 Simpledi at
omi cmol ecul eshaveonl yonebondandonl yonevi brationalband.Ifthe
moleculeissymmet rical,e.g.N2,thebandi snotobser vedint heIRspect rum.
 Unsymmet r
icaldi atomi c mol ecules,e.g.CO,absor bi nt he IR spect r
um.Mor e
compl ex mol ecules have many bonds, and t heir vibrational spect ra are

23
15BT45 S
TRUCTURALBI
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corr
espondi
ngl
ymor
ecompl
ex,i
.e.bi
g mol
ecul
eshavemanypeaksi
nthei
rIR
spectr
a.

TypesofVi
brat
ionalmode

 TheatomsinaCH2group,
commonl yfoundi
norgani
ccompounds,canvibr
ateinsix
dif
fer
entways:symmet r
ic and ant
isymmetr
ic str
etchi
ng,sci
ssoring,rocki
ng,
waggingandtwi
sti
ng

Fi
gur
e:Vi
brat
ionmodesi
ndi
ffer
entmol
ecul
es

I
nstrument at ionandwor kingpr inciple
 TheI Rspect romet erwor ksbyemi ttingt helightt hr oughasampl echamberand
through asol ventr ef erencechamber ,thenmeasur ing t heamountofr adiation
absor bedbyt hesampl eascompar edt othatabsor bedbyt hereference.
 Adet ect orpl otstheabsor bance( or%t ransmi ttance)asaf uncti
onofwavenumber .
Thi s pr ocess gi ves a spect rum f ort he sampl e whi ch may be used t ol earn
informat ionaboutt hesampl e.
 InIRSpect roscopy,t her adi ationsour cebeam i sspl i
tbyami rrorinordert opass
throughbot hthesampl eandt herefer encechamber s.Thel ightisreflectedusi ng
mir ror si ntoamonochr omat or( splitteront hediagr am)whi chonl yall
owsl ightofa
singl ewavel engt hatat i
met or eacht hedet ector.
 Thedet ect orr eceivest hesi gnal sfr om bot ht hesampl ebeam andt her efer
ence
beam.Thi si nfor mationgoesi ntot hepr ocessorwhi cht ranslat
est hei nf
or mation
intoapl otwi thwavenumberont hex- axi sandi ntensi tyont hey- axi
s.I ntensityis
measur ed ast heper centt r ansmi t
t anceoft heI Rr adiat i
onwi thr espectt ot he
refer ence.
 A1 00% t ransmi t
tance means t hatt he sampl e absor bed t he same amountof

24
15BT45 S
TRUCTURALBI
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radiati
onast herefer
ence.A0%t ransmitt
ancemeanst hatt
hesampleabsorbedal
l
ofther adi
ati
on.
 Thepl otshows1 00%t r
asmittanceatt hetopand0%att hebott
om.Ther esul
tisa
plotwi thseveralpeaksi nthedownwar ddirection.Thesepeakscorrespondto
frequenciesoflightthatwer eabsor bedbymol eculesbecausetheymatchedthe
frequenciesofthenaturalvi
brationofthemolecularbonds.

Fi
gur
e:Schemat
icsofat
wo-
beam absor
pti
onspect
romet
er

Appl
icat
ionofI
Rspect
roscopy

1
.Identif
icationoff unct ionalgroupandst ructureeluci
dat ion
 EntireIRr egi oni sdi vided int
ogr oup f requencyr egionand f ingerprintr egion.
Rangeofgr oupf requencyi s4000-1 500cm- 1whilethatoff ingerprintregionis1 500
-400 cm- 1.
In gr oup f requency r egi on,t he peaks cor r
espondi ng to di f
f erent
functionalgr oupscanbeobser ved.Accor di
ngt ocorrespondi ngpeaks,f unct ional
groupcanbedet er mined.
 Eachat omoft hemol eculeisconnect edbybondandeachbondr equir
esdi fferentI R
regionsochar act eri
sticpeaksar eobser ved.ThisregionofI Rspect r
um iscal led
asfingerpr intregi onofthemol ecul e.Itcanbedet erminedbychar acteristi
cpeaks.

2.
Identif
icationofsubst ances
 IR spectroscopy is used t o establi
sh whet hera gi ven sample ofan organic
substance isi denti
calwi th anotherornot .Thisi s because l
arge numberof
absorpti
on bands i s observed int heI R spectraofor gani
c molecul
es and the
probabil
itythatanyt wocompoundswi llproduceidenticalspect
raisalmostzero.
 Soiftwocompoundshavei denticalIRspect r
at henbot hofthem mustbesampl es
ofthesamesubst ances.IRspect raoftwoenat iomericcompoundsar eidenti
cal.So
IRspectroscopyf ai
lst odistinguishbet weenenantiomer s.

3.
Studyingtheprogr essoft her eacti
on
 Progressofchemi calreactioncanbedet erminedbyexami ni
ngthesmallporti
onof
thereacti
onmi xt
ur ewithdrawnf r
omt i
met otime.
 Ther ateofdisappear anceofachar acteri
sticabsorpti
onband ofther eactant
groupand/ ort
her ateofappear anceoft hechar acter
ist
icabsorpt
ionbandoft he
productgroupduet oformat ionofpr oductisobserved.

25
15BT45 S
TRUCTURALBI
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4.
Detect
ionofimpur it
ies
 IRspectrum ofthet estsampletobedeter
minediscompar
edwiththestandar
d
compound.Ifanyaddi ti
onalpeaksar
eobser
vedint
heIRspectr
um,
theniti
sduet o
impur
iti
espr esentinthecompound.

5.
Quantit
ativeanalysi
s
 Thequantit
yofthesubst ancecanbedet er
mi nedeit
herinpureform orasami xt
ure
oftwoormor ecompounds.I nthis,char
acterist
icpeakcorrespondingtot hedrug
subst
ance is chosen and log I
0/ I
tofpeaks f orstandar
d and t estsampl eis
compared.Thisi scalled baselinetechniquet odeter
minet hequant i
tyoft he
subst
ance.

UVSPECTROPHOTOMETER

26
15BT45 S
TRUCTURALBI
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Fi
gur
e:Theel
ect
romagnet
icspect
rum

27
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28
15BT45 S
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29
15BT45 S
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Fi
gur
e:Schemat
icr
epr
esent
ati
onofUVSpect
rophot
omet
er

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LASERRAMANSPECTROSCOPY
 Thephenomenonof" RamanScat t
er ing"wasdi scover edi n1 928byI ndi anphysi cist
,
Chandr asekharVenkat aRaman,popul ar
lyknownasCVRaman,whor eceivedt he
NobelPr izeforPhysi csin1 930, andwasl at erawar dedt he" Bhar atRat na"
 Whenl i
ghti st ransmi t
tedt hroughmat ter ,iti sscat teredi nr andom di rections.
Mostoft hescat ter ed lighti s oft hesamewavel engt h( orf r
equency)as t he
incidentl ight .Thi si sknownas" Rayl eighscat t er
ing"andi sconsi der edelast icas
thescat teredphot onhast hesameener gyast hei nci dentphot on.
 Somel ighti si nel asticallyscat ter ed—scat t
er edphot onhavi nghi gherorl ower
ener gyt hani nci dentone—atadi ffer entwavel engt h( orf requency) .Thi siscal led
"RamanScat ter ing". Thisshi ft
ingoff requencyi salsocal led" RamanEf fect",whi ch
arises when t he i ncident l ight exci tes mol ecul es i n t he sampl e,whi ch
subsequent l
yscat terst helight .
 Itisaspect roscopi ct echni queusedt ostudyvi brational ,rotat i
onal,andot herl ow-
frequencymodesi nasyst em.
 Ramanspect roscopyi saspect r
oscopi ct echni queused t oobser vevi br ati
onal,
rotational ,
andot herl ow- fr
equencymodesi nasyst em.
 Itr eli
es on i nelast ic scat tering,orRaman scat tering,ofmonochr omat icl ight,
usual lyfromal aseri nthevi sible,neari nfrar ed,ornearul t
raviol etrange.
 Thel aserl ighti nter actswi thmol ecul arvibr ations, phononsorot herexci tat i
onsi n
thesyst em, resul tingi nt heener gyoft helaserphot onsbei ngshi ftedupordown.

33
15BT45 S
TRUCTURALBI
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 Theshi fti
nener gygivesinf or
mat ionaboutt hevibrat
ionalmodesi nthesystem.
Inf
raredspectroscopyyieldssi milar,butcomplementary,i
nfor
mat i
on.
 Typicall
y,asampl ei sil
luminat edwi thal aserbeam.Lightfrom theill
uminated
spotiscoll
ectedwi thalensandsentt hroughamonochr omator.Wavel
engthsclose
tothelaserli
neduet oelasticRayl eighscatteringar
efilt
eredoutwhi l
etherestof
thecoll
ectedlightisdispersedont oadet ector.

Pr
inci pl e
 Ramanspect roscopyi sausef ult echni quef ort hei dent i
f i
cat ionofawi der angeof
subst ances-sol i
ds, liqui ds, andgases.
 Itisast rai ght for war d,non- dest ruct ivet echni quer equi ringnosampl epr epar ation.
Ramanspect roscopyi nvol vesi llumi nat ingasampl ewi thmonochr omat icl ightand
usi ngaspect romet ert oexami nel ightscat ter edbyt hesampl e.
 Att hemol ecul arl evelphot onscani nt eractwi thmat terbyabsor ptionorscat tering
pr ocesses.
 Scat ter i
ngmayoccurei t herel ast i
cal ly,ori nel ast ically.Theel ast icpr ocessi s
ter med Rayl eigh scat ter i
ng,whi lst t he i nel astic pr ocess i st ermed Raman
scat ter i
ng.
 Theel ect ricf i
el dcomponentoft hescat teringphot onper t
ur bst heel ect roncl oud
oft hemol ecul eandmayber egar dedasexci tingt hesyst emt oa‘ virtual’ stat e.
 Ramanscat ter i
ngoccur swhent hesyst em exchangesener gywi t
ht hephot on, and
thesyst em subsequent lydecayst ovi br ationalener gyl evelsaboveorbel owt hatof
thei nitialst ate.
 Thef requencyshi ftcor respondi ngt ot heener gydi fferencebet weent hei nci dent
andscat ter edphot oni st ermedt heRamanshi f t
.Dependi ngonwhet hert hesyst em
hasl ostorgai nedvi br ationalener gy,t heRamanshi ftoccur sei therasanup-or
down- shi ftoft hescat tered phot on f requencyr elativet ot hatoft hei nci dent
phot on.
 Thedown- shi ftedandup- shi f
tedcomponent sar ecal ledr espect ivel yt heSt okes
andant i
-St okesl i
nes.Apl otofdet ect ednumberofphot onsver susRamanshi ft
from t hei nci dentl aserener gygi vesaRamanspect rum.Di ffer entmat erial shave
di ffer entvi br ationalmodes,and t her ef orechar act erist i
cRamanspect r a. Thi s
makesRamanspect roscopyausef ult echni quef ormat erialident i
ficat ion.
 Ther ei sonei mpor tantdi st inctiont omakebet weent heRamanspect raofgases
andl iquids,andt hoset akenf rom sol ids-i npar ticular ,cryst als.Forgasesand
liqui dsi ti smeani ngf ult ospeakoft hevi brat ionalener gyl evel soft hei ndi vidual
mol ecul eswhi chmakeupt hemat erial.
 Cr yst al sdonotbehaveasi fcomposed ofmol ecul eswi th speci f icvi br at i
onal
ener gyl evel s,inst eadt hecr yst allat ticeunder goesvi bration.Thesemacr oscopi c
vibr at ionalmodesar ecal ledphonons.

34
15BT45 S
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Fi
gur e:Energyleveldi agr amshowi ngt hest at esinvolvedinRamansi gnal
 Achangei nthemol ecularpolari
zationpot ent i
al—oramountofdef ormationoft he
electron cloud — wi thr espectt ot he vi br ati
onalcoordinateisr equired f ora
mol eculetoexhi bitaRamanef fect.
 Theamountoft hepol ari
zabili
tychangewi lldeter
minet heRaman scat t
er ing
intensity.Thepat ternofshi ftedfrequenci esi sdeterminedbyt her ot
at i
onaland
vibrationalstatesoft hesampl e.
 Ramanspect roscopyi sausef ultechni quef ortheidenti
ficati
onofawi der angeof
subst ances-sol i
ds,l iquids,andgases.I ti sast rai
ghtforward,non-dest r
uct ive
techni que requi r
ing no sampl e pr epar at i
on. Raman spect roscopy i nvol ves
ill
umi nating a sampl ewi th monochr omat icl i
ghtand usi ng a spectromet ert o
exami nelightscat teredbyt hesampl e.

I
nst
rument
ati
on

Fi
gure:Schematicdiagram ofaRamanspectrometer
 InmodernRamanspect rometer
slasersareusedasaphot onsourceduetotheir
highl
ymonochr omati
cnat ur
e,andhighbeamfluxes.
 Thisisnecessaryast heRamanEffectisweak,typi
cal
lyt
heSt okesli
nesar
e~1 05

35
15BT45 S
TRUCTURALBI
OLOGY
timesweakert hantheRayleighscat t
eredcomponent .
 Inthevisi bl
espect ralr
ange, Ramanspect rometersusenot chf il
ter stocutoutthe
signalfrom aver ynarrow rangecent r
edont hef requencycor respondi ngtothe
laserradiation.
 MostRaman spect rometersf ormat eri
alchar acterisation useami croscopeto
focusthel aserbeamt oasmal lspot(<1-
100mmdi ameter).
 Lightf rom t he sample passes back t hr
ough t he mi croscope opt ics intothe
spectromet er.Ramanshi ftedradiati
oni sdetectedwi thachar ge-coupl eddevice
(CCD)det ectorandacomput erisusedfordataacqui sitionandcur vefitt
ing.These
factor
shavehel pedRamanspect r
oscopyt obecomeaver ysensi tiveandaccurate
technique.

Applicati ons
 Raman spect roscopy i s:Non dest ruct ive,Non i nvasi ve,No need of sampl e
prepar ation,I nf or mat i
onr ich,Needonl ysomemi ll
igram ( orpi cogr am)ofmat eri
al
(gas, liqui dorsol id)
 Ramanspect r oscopyi scommonl yusedi nchemi stry,sincevi br ationalinfor mat ion
is speci fict ot he chemi calbonds and symmet ry ofmol ecul es.Ther ef ore,i t
provi desaf inger pr i
ntbywhi chthemol ecul ecanbei dent ified.
 Raman gas anal yzer scan be used i n medi cine f or r eal -time moni tor i
ng of
anaest het icandr espi ratorygasmi xt uresdur ingsur gery.
 Ramanspect r oscopycanbeusedt oobser veot herl owf requencyexci t
ationsoft he
solid,suchaspl asmons, magnons, andsuper conduct inggapexci tations.
 Ramanspect roscopyi sbei ngi nvest igat edasameanst odet ectexpl osi vesf or
airpor tsecur i ty.
 Ramanspect roscopyhasal sobeenusedt oconf irmt hepr edi ctionofexi st enceof
low- f
r equencyphonons.
 Ramanspect roscopyhasdemonst rated t hesensi ti
vityt odi stinguish bet ween
cancer ous, pr e- cancer ousandnor malt issues, andi t ssensi t ivit ytochangesi ncel l
met abol i
tes and pr otein st ructures el evat ei tabove compet ing spect r oscopi c
techni ques.
 It can be al so used i n var ious appl i
cat i
on i n Phar maceut i
cal
indust ry,Pet rochemi stry, indust rial chemi st r
y, pol ymer s, Mat erial sci ence,
Semi conduct ors,phot ovoltai cmat er ials,Li fesci ence( human,ani malandpl ants:
biology, bi ochemi stry, bi ophysi c, vi r us, t umour ), Geol ogy and mi ner alogy,
gemmol ogy,Nucl ear sci ence,i sotopi c ef fect s,For ensi cs sci ences,f or ensic
sciencel abor at ories
 Study and i dent ification of var iou sbi ol
ogcal mol ecul es such as pr otein,
car bohydr tes, lipi dsandnucl eicacids.

ELECTRON SPI N RESONANCE SPECTROSCOPY or ELECTRON PARAMAGNETI C

RESONANCESPECTROSCOPY( ESR/EPR)
 Electr
onspi nr esonance( ESR)isasophi sticat
edspect roscopictechniquethat
detectsfreeradicalsorinorganiccompl exesi nchemi calandbi ol
ogicalsystems.
 Electr
on Spin Resonance Spect roscopy or El ectron Par amagnetic Resonance
Spectroscopyi sat echniqueusedf orst udyingchemi calspeciesthathaveoneor
mor e“
freeradical”orunpairedelect rons.
 Itisamagnet icresonancet echniqueusedf orthest
udyofpar amagnet icmateri
als,
thosethatcont ai
nunpai redelectrons

36
15BT45 S
TRUCTURALBI
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 Thepar amagneticspeciesfoundi nbiol
ogi calmateri
alsincludefreeradicalsand
transi
tionmetals.
 EPRmeasur esthet r
ansiti
onfrequencybet weendifferentel
ectronspinstates.
 Mostst abl
emol eculesonl
yhavepai redelectronswithoutanyf r
eeradicals,soESR
Spectroscopy is used less often than NMR ( Nuclear Magnet i
c Resonance)
Spectroscopy,theusualtechniqueforstudyi ngchemi calbonds.Thisalsomeans
thatESRSpect r
oscopyi sahighlyspecifi
ct echnique.

Pr
incipleandi nstrument ation
 Everyelect ronhasamagnet icmomentand spi nquant um numbers=1 /
2,wi th
+ −
magnet i
ccomponent sms= 1 /2and ms= 1 /
2.I nthepr esenceofanext ernal
magnet i
cf i
eld with str ength B0,t heel ect
ron's magnet i
c momental i
gns itself
− +
eitherpar al
lel(ms = 1 /2)orant iparall
el(ms = 1 /
2)t othef ield,eachal ignment
havingaspeci ficenergy. Thisi scall
edZeemansspl itt
ingorZeemanef f
ect .
 Thepar allelali
gnmentcor respondst othelowerener gyst ate,andt hesepar ation
betweeni tandt heupperst atei s∆E=geμBB0, wher egeistheel ectron'sso-call
edg-
factorandμB ist heBohrmagnet on.Thisequat i
oni mpliesthatt hespl i
tti
ngoft he
energylevelsisdi rectlypr opor ti
onaltot hemagnet icfiel
d'sst r
engt h,asshowni n
thediagrambel ow.

Figure:
ZeemanSpl
itt
ing Fi
gur
e:EPR spect
rum of t
he CH3
radical

 Anunpai r
edelect
roncanmovebetweenthetwoenergylevel
sbyeitherabsorbingor
emitt
ing elect
romagnet
icradiat
ion ofenergy ε =hν such t
hatt her esonance
condit
ion,ε=∆E.
 Substitut
inginε=hνand∆E=geμBB0 l eadstot hefundamentalequati
onofEPR
spectroscopy:
hν=geμBB0
 WhereμB isPlanksconstant,μB Bohrmagnet on,geisgfactor,B0i
smagnet icfi
eld
str
engthandνi sthefrequency.
 Thefactorgistreat
edasaspect roscopicvar i
ablewhichisachar acteri
sti
cofthe
paramagnet.
 Experi
ment al
ly,thi
s equation per mit
sal ar ge combi
nation off r
equency and
magneticfiel
dvalues,butthegr eatmaj or
ityofEPRmeasur ementsar emadewith
microwavesinthe9000–10000MHz( 9–10GHz)r egi
on.

37
15BT45 S
TRUCTURALBI
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Fi
gur
e:Di
agr
amofESR/
EPRSpect
romet
er

 Ther adi ationsour ceusual lyusedi scalledakl ystron.Klyst r

onsar evacuum tubes
known t o be st able hi gh power mi crowave sour ces whi ch have low- noise
charact er i
st icsandt husgi vehi ghsensi tivi
ty.
 A maj ority of EPR spect r omet ers operate at appr oximat el y 9.
5 GHz,whi ch
corresponds t o about32 mm.The r adiation may be i ncidenton t he sampl e
continuousl y( i
.e.,
cont i
nuouswave, abbreviatedcw)orpul sed.
 Thesampl eispl acedi nar esonantcavi tywhi chadmi tsmi crowavest hroughan
iri
s.Thecavi t yisl ocat edi nt hemi ddleofanel ectromagnetandhel pst oampl ify
theweaksi gnal sf romt hesampl e.
 Numer ous t ypes ofsol id-
st atedi odes ar esensi tivet omi cr owaveener gy and
absor ptionl inest henbedet ect edwhent hesepar ationoft heener gylevelsisequal
orver ycl oset ot hef requencyoft heincidentmi crowavephot ons.
 Inpr actice, mostoft heext er nalcomponent s,suchast hesour ceanddet ector,are
containedwi thinami crowavebr idgecont rol.Addi ti
onally,othercomponent s,such
asanat t enuat or,fieldmodul ator,andampl ifier,arealsoi ncl udedt oenhancet he
performanceoft hei nst rument .

Applicat ionofESR/ EPR

 ESRspect roscopyi smostof t
enusedi nmedi ci
neandbi ologyt ostudyf r
eer adical
elect rons’pr oper t
iesthatchemi calandphysi calreact i
onspr oduce.Forexampl e,
whenhi ghl evelsofener gyfrom heat,light,ormi crowaver adiati
onbombar dice,
freer adi calssuchasHandOHar ecreatedwhi lethei ceisconver tedtowater .Free
radi calscanal sobeat tachedt oaspeci fi
clocationwi thinacel landcanbeusedt o
anal yzet hecel l’
senvironmentwhenESRi sappl i
ed.
 Radi at ion-sterilizedfoodshavebeenexami nedwi thEPR spect roscopy,theai m
bei ng t odevel op met hods todet ermi neifa par t i
cularf ood sampl ehas been
irradiat edandt owhatdose.
 Becauseofi tshi ghsensi ti
vity,EPRwasusedr ecent lytomeasur ethequant i
tyof
ener gyusedl ocal l
yduringamechanochemi calmi l
lingpr ocess.

38
15BT45 S
TRUCTURALBI
OLOGY
 EPRspectroscopyhasbeenusedt omeasur epr
oper
tiesofcr
udeoil
,inparti
cular
asphalt
eneandvanadi um cont
ent.EPR measur
ementofasphal
tenecontentisa
funct
ionofspindensi
tyandsolventpol
arit
y

AdvantagesofESR
 ESR spect roscopy i st hepr eferred means ofdet ecti
ng and analyzing free
radicalelectr ons f
ormanyr easons.Forexampl e,ESR spectroscopycan be
performed ver yquickly,of t
eni nf ifteent ot wentymi nut
es,and pr ovidesan
accurateanal ysisoffreer adicalact ivities.
 ESR spect roscopy r esults ar eusual ly very easy tounderst
and and can be
recordedast hef reeradicalel ectronsar estudied.
 Ot herspect roscopyt echni quescani dent i
fyfreeradicalsandpairedel ectr
ons;
ESRspect roscopyi dent if
iesf reer adical sonly,afuncti
onthatisveryi mportant
inthestudyofunpai redel ectrons.

Di
sadvantagesofESR
 While ESR spect roscopy has severaladvantages overot herspect roscopy
techniques,i
talsohast hedisadvantageofrequi
ringlowt emper at
uresinorder
todetectfreeradicalelectr
ons.
 Thisisbecauseel ectronstypicallymovetoofastforanaccur ateanalysistobe
made. ItmeansthatESRspect roscopymustbeper for
medi nal aborat
orythatis
specifical
lydesignedt oprovidetheselowtemperatures.

39