1
EXPERIMENTB6:PKOFANINDICATOR
LearningOutcomes
Uponcompletionofthislab,thestudentwillbeableto:
1) Collectdatausingaspectrophotometer.
2) Evaluateabsorptionspectraandchoosethewavelengthsatwhich
absorbancemaximamaybefound.
3) MeasurethepKofanindicatorusingaspectrophotometer.
Introduction
IndicatorsareweakacidsorbasesthatundergocolorchangesincertainpHranges.
Thesecolorchangesareduetotheindicatoracceptingorreleasingaproton(H+),
whichinturncausesasignificantchangeinthestructureoftheindicator.Inthe
titrationexperiment(CHEM1A:ExperimentA7),phenolphthaleinwasusedasan
indicator.Phenolphthaleiniscolorlessinanacidicmediumandpinkinabasic
medium.Likewise,mostcommonindicatorsusedinlaboratoryexperimentsexhibit
colorchangesinthevisiblespectrum,asthepurposeofanindicatoristobeableto
monitorthepHofsolutionbysimplevisualobservation.Sincethesecolorchanges
occurinthevisiblespectrum,theycanbestudiedusingaUV-Visspectrophotometer
Supposethatanindicatorisaweakacid.Thestructureoftheindicatorcouldthusbe
representedbytheformulaHIn.Inanacidicmedium,theindicatorwouldexistinits
protonatedform(HIn),whileinabasicmedium,theindicatorwouldbeneutralized
andwouldexistinitsconjugate,deprotonatedform(In–).Theequilibriumbetween
theprotonatedanddeprotonatedformscanberepresentedasshownbelow:
HIn ⇔ H++In−
Atanygiventimeinthisequilibrium,boththeacid(HIn)anditsconjugatebase(In−)
willbepresent.Iftheaciddissociationconstantfortheindicatorisassumedtobe
€
Ka,then:
[H + ][In − ]
Ka=
[HIn]
Theaboveequationcanberearrangedtosolveforthe[H+]andmanipulatedas:
€
K [HIn]
[H+]= a − [In ]
€
2
⎛ K [HIn] ⎞
-log[H+]=-log ⎜ a − ⎟ ⎝ [In ] ⎠
⎛ [HIn] ⎞
pH=-log(Ka)-log ⎜ − ⎟ ⎝ [In ] ⎠
€
⎛ [HIn] ⎞
pH=pKa-log ⎜ − ⎟ ⎝ [In ] ⎠
€
⎛ [HIn] ⎞
log ⎜ − ⎟ =-pH+pKa
⎝ [In ] ⎠
€
Sincetheacidinquestionintheaboveexpressionistheindicator,thepKamayalso
bewrittenaspKIN.Thereforetheequationisrewrittenasshownbelow:
€
⎛ [HIn] ⎞
log ⎜ − ⎟ =-pH+pKIN
Equation1
⎝ [In ] ⎠
Iftheprotonatedanddeprotonatedformsoftheindicatorarepresentinequal
concentrations,thentheratiooftheirconcentrationsisone,soEquation1simplifies
€
asfollows:
log(1)=-pH+pKIN
0=-pH+pKIN
pKIN=pH
Equation1b
ByexaminingEquations1and1b,itcanbeshownthatifthesolutionpHislessthan
pKIN,thentheprotonatedformoftheindicator(HIn)willpredominate,whileifthe
solutionpHisgreaterthanpKIN,thenthedeprotonatedformoftheindicator(In–)
willpredominate.
Theindicatorthatwillbestudiedinthisexperimentisbromocresolgreen.The
ionizationequilibriumofbromocresolgreenisshownbelow:
Br
Br
Br
O
OH
Br
Br
– H+
+ H+
Br
O
Br
SO3–
Br
O
O
Br
Br
SO3–
O
Br
SO3–
In– (blue)
HIn (yellow)
Br
3
Bromocresolgreenundergoesacolorchangewhenitisdeprotonatedbecauseofthe
extensivedelocaliztionfoundintheresultinganion.Asdiscussedabove,the
protonatedformofbromocresolgreen(HIn),whichisyellow,willbethe
predominantformatlowpH(muchbelowthepKIN)andthedeprotonatedformof
bromocresolgreen(In−),whichisblue,willbethepredominantformatpHvalues
muchhigherthanthepKIN.AtallintermediatepHvalues,boththeacidform(HIn)as
wellasitsconjugatebase(In−)willbefound.AccordingtoEquation1,the
[HIn]
concentrationsofHInandIn−andthereforetheratio
dependonthepHofthe
[In − ]
solution.
Theconcentrationsofchemicalspeciesthatarecoloredcanbedetermined
€
spectrophotometricallyusingBeer’slaw(alsoreferredtoastheBeer-Lambertlaw).
AccordingtoBeer’slaw,forsubstancesthatabsorblightinthevisibleregionofthe
electromagneticspectrum(~400to800nm),theamountoflightabsorbedorits
absorbance(A)isdirectlyproportionaltothemolarconcentration(C).The
mathematicalformofthislawisshownbelow:
A=ε×C×l Equation2
Asidefromabsorbanceandconcentration,thetwootherquantitiesinEquation2
are:ε-themolarextinctioncoefficientandl-thepathlengththroughwhichlight
travelsinthesample.Thepathlength,“l”isusuallyafixedquantityforaparticular
spectrophotometerandismostfrequently1.00cm.Themolarextinctioncoefficient,
ε,whichrepresentsthesensitivityofthecompoundtolightdependsinsteadonboth
thesampleandthewavelength,λ,atwhichtheabsorbanceismeasured.
So,eventhoughtheconcentrationsofapuresampleofHInandIn−maybe
measuredrelativelyeasilyusingaspectrophotometer,assumingεHInandεIn-are
known,theconcentrationofamixtureofHInandIn−ismorechallengingtoobtain.
ThereforeinordertouseEquation1todeterminethepKINsomeamountof
mathematicalmanipulationisrequired.
ManipulationofEquation1toDeterminepKIN
⎛ [HIn] ⎞
log ⎜ − ⎟ =-pH+pKIN
Equation1
⎝ [In ] ⎠
Atanygiventimeintheequilibrium(otherthanatextremelylowandhighpH)both
HInandIn−willbepresent.Thereforethetotalconcentrationoftheindicator,Inis
€
givenas:
[In]=[HIn]+[In−] Equation2
4
Assumingthatthemolarextinctioncoefficientoftheindicatorisε,theabsorbance
ofthemixtureisthengivenas:
A=ε×[In]×l
Equation3
Thetotalabsorbanceisacombinationofthetwoformsoftheindicator.Therefore
thetotalabsorbanceAcanalsobewrittenas:
A={εHIn×[HIn]×l}+{εIn-×[In−]×l}
Equation4
CombiningEquations3and4:
ε×[In]×l={εHIn×[HIn]×l}+{εIn-×[In−]×l}
ε×[In]={εHIn×[HIn]}+{εIn-×[In−]}
Equation5
Keepinmindthatonlythetotalconcentrationoftheindicator,[In],isameasurable
quantityandtheindividualconcentrationsofthetwoformsoftheindicator,[HIn]
and[In−],arenoteasilymeasured.
BasedonEquation2,[In−]=[In]–[HIn].SubstitutingthisinEquation5:
{ε×[In]}={εHIn×[HIn]}+{εIn-×([In]–[HIn])}
{ε×[In]}={εHIn×[HIn]}+{εIn-×[In]}–{εIn×[HIn]}
{ε×[In]}–{εIn-×[In]}={εHIn×[HIn]}–{εIn-×[HIn]}
[In](ε–εIn-)=[HIn](εHIn–εIn-)
⎛ ε −ε − ⎞
In
⎟⎟ [HIn]=[In] ⎜⎜
Equation6
ε
−
ε
− ⎠
HIn
⎝
In
Similarly,basedonEquation2,[HIn]=[In]–[In−].SubstitutingthisinEquation5
gives:
€
⎛ ε −ε ⎞
⎟⎟ [In−]=[In] ⎜⎜ HIn
Equation7
ε
−
ε
− ⎠
HIn
⎝
In
DividingEquation6byEquation7gives:
€
5
[HIn] ⎛ ε − ε In − ⎞
=⎜
⎟
[In − ] ⎝ ε HIn − ε ⎠
Equation8
SubstitutingtherighthandsidefromEquation8intoEquation1:
€
⎛ε −ε − ⎞
In
log ⎜
Equation9
⎟ =-pH+pKIN ε
−
⎝ HIn ε ⎠
ThreeassumptionsarerequiredtofurthermanipulateEquation9.
€ 1) TheabsorbanceatpHfarbelowthepK ,letssayA ,ismostlyfromHInthe
IN
L
predominantspeciesatthispH.
2) TheabsorbanceatpHmuchhigherthatthepKIN,letssayAH,ismostlyfrom
In−thepredominantspeciesatthispH.
3) ThetotalconcentrationoftheindicatoratlowpHissimplytheconcentration
ofHInandthetotalconcentrationoftheindicatorathighpHisthe
concentrationofIn−.
Therefore:
AL
AL=εHIn×[In]×landtherefore: ε HIn =
Equation10
[In] × l
AH
AH=εIn-×[In]×landtherefore: ε In − =
Equation11
[In] × l
€
AtallotherpHvalues,theabsorbanceis:
€
A
A=ε×[In]×landtherefore: ε =
Equation12
[In] × l
SubstitutingEquations10,11,and12intoEquation9givesthefollowing:
€
⎛ A − AH ⎞
log ⎜
Equation13
⎟ =-pH+pKIN ⎝ AL − A ⎠
€
ExperimentalDesign
6
Inthefirstpartoftheexperimentthewavelengthatwhichallabsorbancevaluesare
tobemeasuredwillneedtobedetermined.Inordertodothis,theindicator-
bromocresolgreenshouldbeplacedinasolutionofthehighestpHprovidedandthe
visiblespectrumofthissolutionshouldbeobtainedbymeasuringtheabsorbanceat
10nmintervals.Theplotofabsorbancevs.wavelengthwillshowtwowavelengths
atwhichabsorbancemaximaareseen.Thehigherofthesewavelengthsshouldbe
usedforallsubsequentabsorbancemeasurements,asthiswavelengthcorresponds
towherethecompoundismostsensitivetolightandthereforemostreliably
detected.
Inthesecondpartofthisexperiment,theindicatorbromocresolgreenwillbe
placedinamediumoflowpHvaluetodetermineALinEquation13.Thesame
concentrationoftheindicatorwillthenbeplacedinamediumofhighpHto
determineAHinEquation13.Thenthetotalabsorbanceoftheindicator,Awillbe
measuredatdifferentpHvalues;keepingtheconcentrationoftheindicatorthe
samethroughouttheexperiment.Thedataoftotalabsorbance,Avs.pHwillbeused
inEquation13tographicallydeterminethepKIN.
ReagentsandSupplies
Bromocresolgreen,solutionsA(boricacid/citricacid)andB(sodiumphosphate)
(combinationsofwhichwillbeusedtogeneratesolutionsofpHvaluesfrom1to
14),spectrophotometer
(SeepostedMaterialSafetyDataSheets)
Procedure
7
PART1:DETERMINATIONOFWAVELENGTHATWHICHABSORBANCEVALUESARETOBEMEASURED
1. Obtainaspectrophotometerandturnthepoweronandlettheinstrumentwarm
upforabout10minutes.
2. Inalargetesttube,combine1.80mLofsolutionA,6.00mLofsolutionB,and
0.30mLoftheindicatorandthoroughlymixthecontents.Thiswillbethe
“sample”.
3. Inanotherlargetesttube,combine1.80mLofsolutionA,6.00mlofsolutionB,
and0.30mLofdeionizedwaterandthoroughlymixthecontents.Thiswillbethe
“blank”.
4. Obtaintwocuvettes.Cleananddrythecuvettesandbesuretowipethesidesof
thecuvetteswithakimwipes.
5. Theinstructorwilldemonstratetheproperuseofthespectrophotometer.
6. Setthewavelengthofthespectrophotometerto370nm.
7. Withthesamplechambercompletelyempty,closethelidandadjustthe
absorbancereadingtozerobyusingtheappropriateknob.
8. Placethecuvettecontainingthe“blank”insidethesamplecompartment,align
theguidemarkonthecuvettewiththeguidemarkatthefrontofthesample
compartment,closethelidandadjusttheabsorbancereadingto100usingthe
appropriateknob..
9. Nowplacethecuvettecontainingthe“sample”insidethesamplecompartment,
aligntheguidemarkonthecuvettewiththeguidemarkatthefrontofthe
samplecompartment,closethelidandrecordtheabsorbance.
10. Changethewavelengthto380nmandthenrepeatsteps7through9.Continue
thisprocessin10nmincrementsofthewavelength,untilabsorbancevalues
havebeenrecordedat700nm.
11. Emptythecontentsofthecuvettesintoalargebeakerlabeledas“Waste”.Rinse
andcleanthecuvettes.
12. PlotagraphofAbsorbance(y-axis)vs.wavelength(x-axis).
13. Fromtheplotdeterminethewavelengthatwhichthesecondabsorbance
maximumisfound.Thiswavelengthwillbeusedforallsubsequent
measurements.
8
9
PART2:DETERMINATIONOFABSORBANCEOFINDICATORATDIFFERENTPHVALUES
1. Obtainaspectrophotometerandturnthepoweronandallowtheinstrumentto
warmupforatleast10minutes.
2. Obtain14largetesttubesandnumberthemfrom1to14.
3. CombinesolutionA,solutionB,andtheindicatorineachofthetesttubes
accordingtothefollowingtable.Mixthecontentsofthetesttubesthoroughly.
Thesewillbethe“sample”testtubes.
SolutionNumber pH SolutionA(mL) SolutionB(mL) Indicator(mL)
1
2.0
7.80
0.00
0.30
2
3.0
7.00
0.80
0.30
3
3.5
6.60
1.20
0.30
4
4.0
6.20
1.60
0.30
5
4.5
5.80
2.00
0.30
6
5.0
5.40
2.40
0.30
7
5.5
5.10
2.80
0.30
8
6.0
4.80
3.20
0.30
9
6.5
4.40
3.50
0.30
10
7.0
4.00
3.80
0.30
11
8.0
3.40
4.40
0.30
12
9.0
2.80
5.00
0.30
13
10.0
2.20
5.60
0.30
14
11.0
1.80
6.00
0.30
4. Obtain14largetesttubesandnumberthemfrom1to14andprepare“blank”
solutionscorrespondingtoeachsamplepreparedinstep3.
5. SetthewavelengthofthespectrophotometertothevaluedeterminedfromPart
1oftheexperiment.
6. Recordtheabsorbancevaluesofeachofthe14samples(usethemethod
describedinPart1(steps7through9).
7. Recordthecolorofeachsamplesolution(labeled1to14)
8. TheabsorbancevalueofSolution1willbeusedasAL.
9. TheabsorbancevalueofSolution14willbeusedasAH.
DataTable
10
PART1:DETERMINATIONOFWAVELENGTHATWHICHABSORBANCEVALUESARETOBEMEASURED
Wavelength(nm) Absorbance
370
380
390
400
410
420
430
440
450
460
470
480
490
500
510
520
530
540
550
560
570
580
590
600
610
620
630
640
650
660
670
680
690
700
11
PART2:DETERMINATIONOFABSORBANCEOFINDICATORATDIFFERENTPHVALUES
Solution
Color
pH Absorbance
1
2.0 2
3.0 3
3.5 4
4.0 5
4.5 6
5.0 7
5.5 8
6.0 9
6.5 10
7.0 11
8.0 12
9.0 13
10.0 14
11.0 DataAnalysis
€
12
PART1:DETERMINATIONOFWAVELENGTHATWHICHABSORBANCEVALUESARETOBEMEASURED
1. DrawagraphofAbsorbance(y-axis)vs.wavelength(nm).
2. Fromtheplot,identifythewavelengthatwhichthemaximumabsorbance
occurs.Therewillbetwoabsorbancemaximainthisspectrum.Usethelargerof
thetwowavelengthsforallsubsequentmeasurements.
PART2:DETERMINATIONOFABSORBANCEOFINDICATORATDIFFERENTPHVALUES
Muchoftheanalysisforthispartcanbedoneusingaspreadsheet.Thegoalisto
determinethepKINandthiswillbedoneusingEquation13:
⎛ A − AH ⎞
log ⎜
⎟ =-pH+pKIN
⎝ AL − A ⎠
Acomparisonoftheaboveequationtotheequationofastraightline:y=mx+b
indicatesthatthevariablesyandxintheequationofthelinecorrespondto
€
⎛ A − AH ⎞
⎛ A − AH ⎞
log⎜
⎟ andpH,respectively.Therefore,aplotof log⎜
⎟ (onthey-axis)
⎝ AL − A ⎠
⎝ AL − A ⎠
andpH(x-axis)whenfittotheequationofastraightlinewillyieldaslopeof-1
(negativeone)anday-interceptcorrespondingtopKIN.
€
ThevalueofAHistheabsorbancevalueofsolution14andthevalueofA
Listhe
absorbancevalueofsolution1.
Thefollowingtableshowstheentryofinformationinthespreadsheet.
13
A
B
C
D
E
F
G
H
AH
Enter value
AL
Enter value
Solution
Absorbance
(A)
A - AH
AL - A
(A-AH)/(AL-A)
log((A-AH)/(AL-A))
log((A-AH)/(AL-A))
2
Enter value
Enter
=(B6-B1)
Enter
=(B2-B6)
Enter
=(C6/D6)
Enter
=log(E6)
pH
Enter
pH
value
here
3
Enter value
4
Enter value
5
Enter value
6
Enter value
7
Enter value
8
Enter value
9
Enter value
10
Enter value
11
Enter value
12
Enter value
13
Enter value
Enter =F6
Toplotagraphandobtaintheequationofthecurvefitline:
1. Selectthehighlightedcellsfromthetable(thevaluescontainingthexandydata
thatwillbeplotted).
2. Select“InsertChart”
3. Choose“XYScatter”
4. Clickonanydatapointonthescatterplotthatappears.
5. Fromthe“Chart”menu,select“AddTrendline”
6. Inthe“Options”menu,check:1)displayequationand2)displayR-squaredvalue
7. They-interceptoftheequationisthepKIN
14
Results
1.
2.
3.
4.
5.
IncludethegraphdrawnusingdatafromPart1oftheexperiment.
Thewavelengthchosenformeasurementofabsorbancevalues=______________nm
Includethespreadsheetandtheplotofthedatashowingthecurvefitline.
TheexperimentalvalueofthepKINforbromocresolgreen=_______________________
ThetheoreticalvalueofthepKINforbromocresolgreen=__________________________
(Indicatesourcematerialusedtoobtainthisvalue)
Source:
6. Thepercenterrorinthemeasurement=________________________________________
(Showcalculationforpercenterror)