Working of spectrophotometer

Working of single beam spectrophotometer.

1. GeneralWorking:
Alampprovidesthesourceoflight.Thebeamoflightstrikesthecollimator(or
diffractiongrating),whichworkslikeaprismandseparatesthelightintoitscomponent
wavelengths.Thegratingisrotatedsothatonlyaspecificwavelengthoflightreachesthe
exitslit.(Theentrancelit,prismandexitslitcollectivelyformmonochromator).Thenthe
lightinteractswiththesample.Heresomeoftheamountoflightisabsorbedbythe
samplesolutionplacedinthecuvette.Thenthelightthatisnotabsorbedbythesample
strikeswithdetector.whereitstransmittanceismeasured.Transmittancereferstothe
amountoflightthatpassescompletelythroughthesample.Absorbanceisa
measurementoflightthatisabsorbedbythesample.Thedetectorsensesthelightbeing
transmittedthroughthesampleandconvertsthisinformationintoadigitalvalue[1].

2. .Whysomeoflightisabsorbedbythesamplesolution?
Someamountoflightthatiscomingfromtheexitslitisabsorbedbythesample
solution.Theelectronsofthemolecuespresentinthesamplesolutionabsorbsome
specificamountofenergyofspecificwavelengthandgointoanexcitedstatei.e
intohigherenergylevel.Inthisway,lightenergyisconvertedintoheatenergy[2].
MoleculesarePromotedtoaMoreEnergeticState:
AbsorptionofUV-vislightresultsinanelectronpromotedtoahigherenergymolecular
orbital.
.sàs*
transitioninvacuumUV
.nàs*
saturatedcompoundswithnon-bondingelectrons
.nàp*,pàp*
requiresunsaturatedfunctionalgroups
(eq.doublebonds)
mostcommonlyused,energygoodrangeforUV/Vis
ExplanationaccordingtoBeer-Lambert’sLaw:
SpectrophotometersworkontheprincipleofBeer-Lambert’slaw.Withthehelpofthis
law,onecalculatetheTransmittanceoflight.Andso,absorbancecanalsobecalcultulated.

3. Transmittance:
Theratioofintensitiesofthetransmittedandincidentlightgivestransmittance.
Inotherwords,
Transmittanceistheamountoflightenergythatistransmittedthroughthesolution.Itis
measuredinpercantagetransmittance(i.e%transmittance)[3].
Transmittance=IT/I0*100
WhereIT= Intensityoftransmittedlightthroughthe
sample
AndI0= Intensityoftheincidentlight(i.eblankcuvettehavingwater)
A100%valueofTrepresentsatransparentsubstancewhilezerovalueof
Trepresentsthatobjectistotallyopaquemeaningthatithasabsorbedallwsavelengthof
lightandhasnottransmittedanylight.
Absorbance:
Absorbanceistheamountofenergyofspecificwavelengthintermsofphotons
thatisabsorbedbytheelectronsofmoleculespresentinthemoleculesof
sample.Itisgivenas:
Absorbance:-logT
Wecanalsowriteitas:-logIT/I0
HereTresentsthetransmittanceofasubstance[4].

4. Beer’sLawisgivenas:
Absorbance▶b.c
A=ε.b.c
Where‘ε’ispropotionalityconstantknownasmolarabsoptivitycoefficient.Wecanalso
usehere‘a’whichmeanssamething.“b”isthepathlengthand“c”istheconcentration
ofthesample.
Thisequationstatesthatthequantityoflightabsorbedbyasubstancedissolvedinafully
transmittingsolventisdirectlyproportionaltotheconcentrationofthesubstance(i.e‘c’)and
thepathlength(i.eb)ofthelightthroughthesolution.
Itmeansthatifthereismorepathlength(i.e‘b’)ofthecuvetteinwhichsampleis
placed,moreabsorbanceoflightwilltakeplaceandasaresultlesstransmissionwillbe
obtained.Becausemoredistancehastobecoveredbythephotonsoflightwhilepassing
throughthesolutionandtherewillbemoreinteractionbetweenthephotonsandabsorbing
substances.Hencetheywillbeabsorbed(i.etheirenergywillbetakenupandabsorbedbythe
electronsoftheabsorbingmolecules).Asaresultlesstransmittancewillbeobtained.
SimilarlyIfthereismoreconcentration(i.ec)ofabsorbingmaterialsinthesampleof
cuvette,theresultwillbethesame(meaningmoreabsorbanceandlesstransmittancewillbe
obtained).
Units:
Inuvspectroscopy,theconcentrationcofthesamplesolutionismeasuredinmolL
-1
andthePathlengthbofthelightpathincm.Thus,giventhatabsorbanceisunitless,the
unitsofmolarabsorptivityεareLmol
-1
cm
-1
.
C=molL
-1
b=cm
ε=Lmol
-1
cm
-1

5. EXAMPLE:
Guanosinehasamaximumabsorbanceof275nm.ϵ275=8400M−1cm−1.ϵ275=8400M−
1cm−1andthepathlengthis1cm.Usingaspectrophotometer,youfindthatA275=0.70
Whatistheconcentrationofguanosine?
SOLUTION:
Since,
Absorbance =A=0.70
Molarabsoptivity=ϵ=8400M
-1
cm
-1
)
Pathlength =b=c=1cm
Tosolvethisproblem,weuseBeer'sLaw.
A=ϵlc
0.70=(8400M
-1
cm
-1
)(1cm)(cc)
Next,dividebothsideby[(8400M
-1
cm
-1
)(1cm)],
c=8.33x10
-5
mol/L
Graph:
Wecanplotagraphbetweenconcentrationandabsorbance.DrawingConcentration
inmolL
-1
onX-axisandabasorbanceonY-axis.Astraightlineisobtainedwhichindicatesthat
asconcentrationofasampleisincreased,absorbancealsoincreasesinthethesame
ratio.Similarlygraphbetween

6. Graph1:Graphbetweenconcentrationc(inmolL
-1
)andabsorbanceA(nounits)
indicatingdirectlyproportionalrelation.
Graph2:Graphbetweenpathlengthb(orc)incmabsorbanceA.

7. References:
1.https://www.lsteam.org/projects/videos/how-does-spectrophotometer-work
2.http://www.andor.com/learning-academy/what-is-light-an-overview-of-the-
properties-of-light
3.http://cellbiologyolm.stevegallik.org/node/7
4.https://chem.libretexts.org/Core/Physical_and_Theoretical_Chemistry/Kine
tics/Reaction_Rates/Experimental_Determination_of_Kinetcs/Spectrophoto
metry