IntroductiontoSpectrophotometry

FocusedReading:PropertiesofLightpg.138140.Stop@Lightabsorptionandbiological
Bringacalculatortolab
Introduction
Thepurposesofthislaboratoryaretointroduceyouto:
1.Conventionsusedinmakingsolutions:molarity,andpercent.
2.Spectrophotometryandtheuseofthemicroplatereader.
3.Proceduresforobtaining,recording,andanalyzingdata.
4.Conventionsusedinpresentingdataingraphs.
5.Proceduresforplanningandworkingthroughaseriesofrelatedexperiments.
Note:Remembertorecordyourdata,answerstoquestionsandproblems,andnotesontheblankpagesof
thislabmanual.Keepitbesideyouandwriteinitasyouwork.
Concentrationsofsolutions
Itisimportantthatyouunderstandtheunitsofthemetricsystem(i.e.milliandmicro).Ifyoudonot,
pleasereviewtheseunitsofmeasurementaswellastheCelsius(centigrade)temperaturescaleintheback
ofyourtextbook(frontinsidecoverofPurvestext).
Intheinstructionsbelow,thesoluteisthesubstancedissolved,thesolventistheliquidinwhichthesolute
isdissolved,andtheresultingmixtureisthesolution.
MolarConcentrations
Intechnicalterms,amoleofacompoundis6.02x1023moleculesofthatcompound.Practicallyspeaking,
amoleisthecompound'smolecularweightingrams.Aonemolar(1.0M)solutionhasonemoleofa
compound(thesolute)dissolvedinsolventsothatthefinalvolumeis1000ml(oneliter).Themolecular
weightofNaClis58.54.Therefore:
1.0MsolutionofNaClhas58.54gNaCldissolvedindH20withafinalvolumeof1000ml(or5.85gin
100ml).
0.1MsolutionofNaClhas5.85gin1literor0.585gin100mlor0.058gin10ml.
0.2MsolutionofNaClhas11.71gin1literor1.17gin100mlor0.117gin10ml.
ConcentrationinPercent(Weight/Volume=w/v)
Bydefinition,percentmeans"inahundred"andbyconvention,a10%w/vsolutioncontains10gramsof
asoluteinatotalvolumeof100mlofthesolution.Aw/vsolutionisnotmadebyadding10gramsofa
soluteto100mlofsolvent,butinsteadbydissolving10gramssoluteinenoughsolventtodissolvethe
solidandthenmoresolventisaddedtoreachatotalvolumeof100mlofthesolution.Notethe
differencesinresultsasyouthinkthroughthefollowingmentalexercise.
Experiment1:
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Hereisademonstrationexperimentforyoutoobserve.ThedirectionsinStep1werefollowedexactly.
Examinethegraduatedcylinderonthefrontbenchandrecordtheresultsinyourlabmanual.
Stepnumberonehasbeendoneforyou:
1.5grams(g)ofsodiumchloride(NaCl)wasobtainedandaddedtoexactly50mlofdistilledwater
(dH20)inagraduatedcylinder.Thesaltwasstirredvigorouslytodissolveit.
2.Determinetheexcessvolumebyreadingthevolumeofthegraduatedcylinder.Thisprocedurewasa
failedattempttomakea10%w/vsolution.Thenumberofmlinexcessof50representsthevolume
displacedbythe5gofdissolvedNaCl.
3.WhatistheactualpercentageofNaCl(w/v)?
ConcentrationsinPercent(Volume/Volume=v/v)
Aqueoussolutesmaybespecifiedaspercentsolutionsvolume/volume(v/v).Onehundredmlofa5%
v/vsolutionwillhave5mlsolutedilutedto100mlwithsolvent.Thus,a5%v/vaqueoussolutionof
ethanolismadebydiluting5ml100%ethanolwithenoughdH20(95ml)tomakeatotalof100ml(orby
diluting5liters100%ethanolwith95litersdH20).
Experiment2:
Onthefrontbenchisastocksolution(1.0M)ofadye,neutralred.Make2mlofa4%v/vsolutionfrom
thisneutralredstockusingdH20.Labelyourtubeandsavethissolution;youwilluseitlatertoday.

Spectrophotometry
Asolution,suchasneutralred,appearscoloredbecauseitabsorbscertainwavelengthsoflightinthe
visiblespectrumandtransmitsorreflectsothers.Eachsolutionwithadifferentsolutehasitsown
characteristicabsorptionpropertiesor"spectrum."Aspectrophotometerisanopticalmachinethat
measuresandletsyousee(sense)howmuchlightenergyistransmittedbyasubstanceinsolutionat
differentwavelengthsofradiantenergy.Biologistsusethespectrophotometerfortwodifferentpurposes
(wewilldobothinthelaboratorytoday):
1.todeterminetheabsorptionspectrumofapuresubstanceinsolution
2.todeterminetheconcentrationofasolution.
Aspectrophotometerconsistsofawhitelightsource(lightofallvisiblewavelengths),aprismor
diffractiongratingthatseparatesthelightintodifferentwavelengths,aslitthroughwhichanarrowbeam
ofthedesiredwavelengthpasses(theincidentlight,I0),asamplesolutionholder,aphotosensitivetube
whichmeasurestheenergyoflighttransmittedthroughthesolution(I),andarecordingdevicethat
displaystheamountoftransmittedlightenergydigitallyoronadial.SeeFig.1below.

I0.012

digital
0
refracting
wave
photoelectric
sample
Flight length
C
A
T
source
display
prism
selector
tube

Figure1.Aschematicdiagramofthecomponentsofaspectrophotometer20.Thearrowsindicate
thepathwayoflight.
Transmittanceistheratioofthetransmittedlightenergy(I)totheincidentlightenergy(Io);percent
transmittanceis100Xthatratio.Transmittance,however,isnotproportionaltosoluteconcentration,soit
isusuallyconvertedintoabsorbancewhichisproportionaltosoluteconcentration.Digital
spectrophotometershavereadoutsforbothpercenttransmittanceandabsorbance,butwewillalways
measuretheabsorbance.

%T=(IIo).100

Abs.=log10(100/%T)

MicroplateReader
Aboveisasimplifieddiagramofaspectrophotometerthatcanmeasureonesampleatatime.Inour
experiments,wewillbeusingamicroplatereaderthatiscapableofmeasuringtheabsorbanceof96
samplesinabout8seconds.Thebasicdesignisexactlythesame;aselectedwavelengthoflightpasses
throughthesamplesandaphototubemeasurestheamountoflighttransmittedthroughthesample,which
theplatereaderconvertstoabsorbance.However,thesamplesarelocatedinmicrowellsthatarearranged
inan8x12matrixinoneplasticplate(seefigure2).Youcanputyoursamplesinanyorallofthe
microwells.Theplateismovedoveranarrayof8fiberopticslightsourcesand8phototubes.Eachrowof
8isscannedandthentheplateadvancesbyonerowandtheprocesscontinuesuntilall12rowsare
scanned.Theabsorbancedatathenaredisplayedonascreeninan8x12array.Thesedatacanbesaved
inthememorytobeprintedlater.Thistechnologyisbasedonthesameprinciplesasolder
spectrophotometers,butnowwecanmeasuremoresamplesinlesstime.Youalsocanprogramtheplate
readertomeasuretheabsorbanceofall96samplesattimeintervalsofyourchoice(e.g.every30
seconds).Youshouldtakeadvantageofthesecapabilitieswhenyoudesignyourexperimentsfornext
week.
0
9
8
7
6
5
4
3
2
1
H
G
F
E
D
C
B
A
photoelectric
photoelectric
fiber
optics cables
tube H
A
B
C
D
E
F
G
A-H
8x12display

ofdata

Figure2.Schematicdiagramofamicroplatereader.Samplesareplacedinthe96microwells,analyzed
bythe8channelspectrophotometer,andtheabsorbancedataaredisplayedinthelargeLEDwindow.
TheAbsorptionSpectrum
Becausesolutionsofpuresubstancesdonotabsorbtheenergyofallwavelengthsoflightequally,a
substancemaybeidentifiedbytheuniquepatternofwavelengthsabsorbed.Thechlorophyllsinplants
absorbstronglyinthebluewavelengths(about450nm)andredwavelengths(about650nm),butreflect
thegreenwavelengths(about525nm).Aplotofabsorbanceversusvisiblewavelengths(400to700nm)
forasolutionofchlorophyllashowstwomajorpeaks,oneat450andoneat650nm,andavalleyfrom
500to625nm(SeeFigure3).Thisspectrumischaracteristicforchlorophyllaandmaybeusedasanaid
initsidentification.
Bymeasuringtheabsorbanceofanuncharacterizedsolutionoverarangeofwavelengthsandplottingthe
absorbancevalueontheYaxisandthewavelengthontheXaxis,onecandeterminetheabsorption
spectrumofasample.Theabsorptionmaximumofanypuresubstanceinsolutionisthewavelength
whereabsorptionisthegreatest.

wavelengthinnanometers(nm)

Figure3.Theabsorptionspectrumofchlorophylla.Theabsorbanceofvisiblelightby
chlorophyllaismeasuredspectrophotometricallyasafunctionofwavelength.Theabsorption
maximumisabout460nm.Comparewithfigure8.7onpage140ofPurves.
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HowtouseaspectrophotometertoAnswerBiologicalQuestions
StandardConcentrationCurveand
DeterminingtheConcentrationofaCharacterizedSubstance
Onecanconstructastandardconcentrationcurveofasolutionbymeasuringtheabsorbanceofseveral
differentknownconcentrationsofthesolutionandgraphingtheresultsbyplottingabsorbanceontheY
axisandconcentrationontheXaxis.Spectrophotometrycanbeusedtomeasuretheabsoluteorrelative
concentrationofacharacterizedsubstanceinsolution.Todeterminetheabsoluteconcentrationofapure
substance,onefirstconstructsastandardconcentrationcurvefromknownconcentrationsandthentakes
theabsorbancereadingoftheunknownconcentration.Theunknownconcentrationcanbedetermined
fromthestandardcurvebydrawingahorizontallineonthegraphparalleltotheXaxisandthroughthe
pointontheYaxiswhichcorrespondstotheabsorbance.Thislinewillintersectthestandardcurve;at
thisintersection,averticallineisdroppedtotheXaxisandtheconcentrationreadfromtheXaxis.
Twofactorsareimportantindeterminingunknownorrelativeconcentrations.Theabsorptionmaximum
shouldbeused,andabsorbanceratherthanpercenttransmittanceshouldbeplottedbecauseabsorbanceis
directlyproportionaltoconcentrationandtransmittanceisnot.

StandardCurvewithImaginaryData
0.6

0.5
0.4

Abs.

0.3

0.2
0.1
0
0

Concentrationinpercent(v/v)
y=0.136x+0.001r 2 =0.994
Figure4.Thisisanexampleofastandardcurve.Abestfitlinehasbeengeneratedand
theresultingequationandr2valueareshownbelowtheXaxislabel.
Experiment3:
Usingthe4%(v/v)neutralredsolutionyoupreparedinexperiment#2,setupthefollowingsolutionsin
microfugetubesusingdistilledwateravailableonthelabbenches.
TABLE1Volumesofneutralredanddistilledwaterusedtopreparesolutionsfortubes16.You
must
calculatethevolumesandfillinthetableforthefirsttwocolumnsbeforeyoubegintheexperiment.
Concentration
of Neutral Red
(M) **

**Thesepartsofthetableshouldbecompletedwhenansweringquestion#6attheendofthe
lab.
Experiment4:
Determinationofabsorptionmaximum.
Usetubes16.
Put200lofthesixdifferentdilutionsofneutralredintosixdifferentwells(wellsH7H12)ofyour
96wellplate.
Youwillcollectthedataforexperiments4and5atthesametime(seebelow).
Experiment5:
Determinationofastandardconcentrationcurveforneutralred.
YouwillgeneratethesedataatthesametimeasthosefromExperiment4.Useonlythedatafromthe
wavelengththatistheabsorptionmaximumforneutralred.Withtheappropriatedata,youwillgeneratea
curvetomeasuretheunknownconcentrationofasolution.Followthedirectionsbelowtocollectthedata.

OperationoftheMicroplateReader
1.Turnonthespectrophotometerandletitwarmupfor15minutes.
Therearetwomainvariableswhichyoumustpayattentiontowhileyouusetheplatereader:the
ANALYSISNUMBERandtheFORMATNUMBER.Formatiseasybecausewewillalwaysuse
format#1,whichsimplymeansthatallofthewellsaretobeanalyzedforabsorbance.Analysisisthelist
ofoptionsyouwanttousewhenperformingyourspectrophometricanalysisofyoursamples.
Specifically,youtellthemachinewhichwavelengthoflighttouse,tocalculatetheabsorbance,tousea
singlewavelengthoflight,andtomakeonlyonereadingforeachtimeyoupresstheSTARTkey.Today,
wewilluseANALYSISNUMBERS16.
2.Beforeyoucollectanydata,clearthememoryoftheplatereader.Todothis:
pressFUNCTION,CLEARALL;whenitasksyouifthisisOK?,
pressFUNCTION,CLEAR.
3.Startthereadingofyoursamplesbyusingallsixwavelengthsoflightthattheplatereadercanuse.At
theendofeachreading,youwillgetaprintoutofyourabsorbancedata.Whenyouhavefinishedallsix,
tearoffyourpaper,cleanupanymessyoucreatedandremoveyour96wellplate.Toexecutetheanalysis
withall6wavelengths(1=340;2=405;3=450;4=490;5=595;and6=655nm):
a.pressANALYSIS,1,ENTER,START.Whenthereadingiscomplete,
b.pressANALYSIS,2,ENTER,START.Whenthereadingiscomplete,
c.pressANALYSIS,3,ENTER,START.Whenthereadingiscomplete,
d.pressANALYSIS,4,ENTER,START.Whenthereadingiscomplete,
e.pressANALYSIS,5,ENTER,START.Whenthereadingiscomplete,
f.pressANALYSIS,6,ENTER,START.Whenthereadingiscomplete,
g.removeyourpaperprintouts,removeyourplateandcleanupanymess.
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AnalysisoftheDataforExperiments4and5

Experiment4:
Nowthatyouhaveseveralpagesofdata,whatdoyoudonext?Followtheorderoftheexperiments,
beginningwithnumber4;determinetheabsorptionmaximumforneutralred.Findthewavelengththat
producedthehighestabsorbancevalues.Thiswavelengthistheabsorptionmaximumforneutralredand
allsubsequentexperimentsshoulduseonlythedatageneratedwiththiswavelengthoflight.Togenerate
anabbreviatedabsorptionspectrum,plottheabsorbancefortubenumber3inagraphwiththeYaxisas
theabsorbancevalueandtheXaxisasthewavelengthoflight.Thisgraphwillbegraphnumber1.
Experiment5:
Usingtheabsorbancedatathatweregeneratedwiththeabsorptionmaximum,subtracttheabsorbanceof
theblankfromthevaluesforthesamples.Thissubtractioncorrectsfortheamountoflightabsorbedby
theplasticandthewater.Now,constructagraphwiththeXaxisaspercentneutralred(increasingfrom
lefttoright),andtheYaxisasabsorbanceatyourselectedabsorbancemaximum(e.g.Abs666nm)using
youradjustedabsorbancevalues.Plottheresultsfromtubes1through6anddrawastraightline(bestfit)
togeneratethestandardconcentrationcurve.Thisgraphwillbegraphnumber2.
Aftercompletingtheseanalysesandgeneratingtwographs,continuewithexperimentnumber6below.
Experiment6:
Determinationoftheconcentrationoftheunknownneutralredsolutions.
Obtainfourneutralredsolutionsofunknownconcentrationfromyourinstructorandrecordthe
identifyingletterinyourlabmanual.Determinetheconcentrationofyourunknownsbyputting200lof
itinawell,andrecordthewellsidentifyingletterandnumber(e.g.H3H6).Usetheplatereaderatthe
wavelengthoflightthatisabsorbedthebestbyneutralred.Subtracttheabsorbanceofwater(theblank)
fromthesevaluesandusethesecorrectedvaluestodeterminetheconcentrationsofthefourunknowns.

AnalysisoftheDataforExperiment6
Usingthestandardcurve(graphnumber2),determinetheconcentrationofyourunknowndilutionsof
neutralred.Foreachunknown,theunknownconcentrationcanbedeterminedfromthestandardcurveby
drawingahorizontallineonthegraphparalleltotheXaxisandthroughthepointontheYaxis
correspondingtotheabsorbance(aftersubtraction)fortheunknown.Thislinewillintersectthestandard
curve;atthisintersection,averticallineisdroppedtotheXaxisandtheconcentrationreadfromtheX
axis.
Experiment7:
+
DeterminetheabsorptionmaximumforNADPHandNADP.
Overthenexttwoweeks,wewilluseNADPHandNADP+,andweneedtoknowwhichwavelengthof
lighttouse.Youwillwanttoperformaseriesofexperimentssimilartoexperiments4and5.Record
whichwavelengthisabsorbedthebestandverifyyourresultswiththeinstructor.
Experiment8:
Cleanup.Leaveyourworkstationasyoufoundit.Althoughthisstepmayseemlikeastupidthingtolist
inaprotocol,itisquiteimportant.Itisbasiclaboratorycourtesytoleavetheworkspaceascleanasyou
founditwithequipmentbackinitsproperplace(s).
10

Beforeyouleavelab:
Makesureyouhaverecordedalldataandobservationsandplottedbothofyourgraphsforthisweek:
1)Maximumabsorptionwavelengthofneutralred,
2)Standardcurve,anddeterminationofconcentrationofunknownsolutions.
3)DeterminetheabsorptionmaximumforNADPH.
Questionstoansweronyourowntime
1.HowdoyouaccountfortheexcessvolumeyouobservedinExperiment1?
2.Thesolutionmadeinexperiment1doesnotconstitutea10%w/vsolutionofsodiumchloridebut
somethinglessthanthat.Calculatetheactualpercentagefromyourdata.
3.Whydoyouhavetodissolvethesoluteinavolumeofsolventlessthanthefinalvolumeyou
eventually
want?Doesitmatterifthesaltisaddedfirstorsecondtothegraduatedcylinder?
4.Describepreciselyhowyoumadeupthe4%v/vsolutionofneutralred.
5.Doesitmatterwhetherthepropervolumeofsoluteisaddedfirstorsecond(relativetothewater)
tothegraduatedcylinder?
6.Theaqueousstocksolutionofneutralredisa1.0Msolution.Determinethemolarconcentrationof
thesixsolutionsyoumade.Ifneutralredhasamolecularweightof87,howmanyg/larecontained
in
your4%(v/v)solution?Calculatetheconcentrationofneutralredineachtube(g/l)andaddthese
data
tothetablefromexperiment3.
7.Describehowtoprepare50mlof70%ethanolwhenyouronlysourceisastockcontainerof95%
ethanol.
8.Whatisthemolarityofyour10%w/vNaClsolution?
9.Whatisthepercentconcentrationofa2MNaClsolution?
10.ThemolecularweightofNa2C03is106.Describehowyouwouldmakeup100mlofa0.15M
solution.
11.WhatistheabsorptionmaximumforNADPH?

11