Single beam spectrophotometer

Single beam specrophotometer,complete detail_introduction,history,construction or instrumentation,working and applications with references.

1. SingleBeam
Spectrophotometer

2. TownshipCampus,Lahore
Submittedto: Dr.Mehr-un-Nisa
Submittedby: MuhammadKashif
(mcf1700986)
M.KamranNazir
(mcf1701055)
NabeelAhmed
(mcf1701195)
SohaibArshad
(mcf1701178)
Abdul-Qadeer
(1701258)
Programme: M.ScChemistry(E)
SemesterI
Subject: Advanceanalytical
Chemistry
Coursecode: CHEM3114

3. Contents:
.HistoryandIntroduction
.ConstuctionandInstrumentation
.Working
.Applications&Advantages
.References
TheHistoryofSpectrophotometer
Aspectrophotometermeasureseithertheamountoflightreflectedfromasample
objectortheamountoflightthatisabsorbedbythesampleobject.
Itisadevicetomeasurethebrightnessofvariousportionsofaspectra.
Spectrophotometryisthestudyofthespectra,thisstudyisbuiltuponthebeliefthateach
chemicalelementhasitsownspectrum.
Thespectrophotometerwasinventedin1940,byArnoldJ.Beckmanandhiscolleaguesat
NationalTechnologiesLaboratories,thecompanyBeckmanhadstartedin1935.Theywere
ledbyprojectleaderHowardH.Cary.Thespectrophotometerwasthecompany'sgreatest
discovery.
Before1940,thechemicalanalysisprocesswasalongventuretakingweekstocomplete
withonly25percentaccuracyaccordingtotheMIT's"InventoroftheWeek"archive.In
1940,whentheBeckmanDUSpectrophotometerwasintroduced,itsimplifiedtheprocess
greatly,requiringonlyafewminutesforanalysis.Accordingtothesamesource,thistest
offered
99.99percentaccuracyontheanalysis.Thisinstrumentsetthestandardinchemical
analysis
[1]
.

4. Inthebeginningtherewereperformanceissueswiththespectrophotometer.These
problemsledtochangesindesign.ThemodelBspectrophotometerusedaquartzprism
insteadofaglassprismthisimprovedtheUVcapabilitiesofthedevice.ModelCsoon
followedwithchangesthatraisedwavelengthresolutionintheUVandthreesubsequent
ModelCspectrophotometersweremade.In1941theModelD,alsoknownastheModel
DU,wasproducedwithahydrogenlampandotherimprovements.Thisdesignremained
essentiallyunchangedfrom1941to1976whenitwasdiscontinued
[2]
.
In1981CecilInstrumentsproducedaspectrophotometerthatwasmicroprocessor
controlled.Thisautomatedthedeviceandimprovedthespeed.Thisspectrophotometerwas
morereliablethanothersmadeinthisera.From1984to1985,developmentwasmadein
doublebeamversionsoftheinstrumentwhichdevelopedintotheSeries4000model.With
the1990scametheadditionofexternalsoftwarethatprovidedPCcontrolandonscreen
displaysofthespectra.Today,thedevelopmentofthespectrophotometercontinuesandits
applicationsrangefromscienceandmedicinetocrimesceneinvestigationandlaw
enforcement.
TheDR/4000Spectrophotometerisapowerfulscanningspectrophotometerthatgivesyou
advancedcapabilitiesandperformance.Ithasultravioletandvisiblespectrumanalysis
capabilitieswitharangeof190to1100nm,customercalibration,kinetics,timecourseand
multiwavelengthreadingscapabilities
Introdution
Thisiscalledassinglebeamspectrophotometerbecauseallthemeasuresmentsare
basedonthevaryingintensityofasinglebeamoflightinsingleopticalpath.Theprimary
advantageisthatithasfewercomponentandislesscomplicatedthanalternativedesign.
Hence,itiseasiertoconstructandlessexpensive.Withasinglepathandaminimum

5. numberofopticalcomponentssingle-beamsystemtypicallyprovideveryhighlight
throughput.
Theprimarylimitationisthatitprovidesnomeanstocompensateforinstrumental
variationsduringananalysissuchaschangeinsourceintensity.Theresultingsignal
variabilitycanlimittheproformancecapabilitiesofasinglebeamsystem.
UV-VIS230UV-VisibleSpectrophotometeristheoneofthebestsinglebeam
spectrophotometerstillavailbleinthemarketandithahighefficiency.TheUV230isan
intelligentultra-violet/visiblespectrophotometerthatiswellsuitedtothequalitativeand
quantitativeanalysesrequiredbytoday'slaboratories.Withitswidewavelengthrangeand
narrowbandwidthdesign,theinstrumentprovidesextraordinaryflexibilityandhigh
resolutionatanexceptionalprice.Itfeaturesastandardprinterportforhardcopy.TheUV-
VisSpectrophotometeralsofeaturespowerinterruptionprotection,easyoperation
[3]
.
TheUV/VisSpectrophotometeroperatesoverawavelengthrangeof190-1100nm
witha5nmbandwidth.Thespectrophotometerfeaturesastandardprinterportanda
standardRS-232serialport.TheUV/VIS230hasthecapacitytoanalyzeuptofour
cells.
.InstrumentationandConstruction:
Spectronic20isdesignedforuseinthevisibleregionofthespectrum.Itconsisitsof
followingparts:

6. 1.Tungstenlamp
Tungstenlampisamajorsourceofprovidinglight.Itislinkedwiththephototubewhichis
usedtomeasureintensityoflight.Thelightisemittedfromthislampandisenteredinthe
entranceslit.
2.DiffractionGratting
Lightrayspassthroughobjectivelensandstrikewiththediffractiongrating.Diffraction
gratingisusedasabarrieranditislinkedwiththewavelengthcamwhichisusedtoadjustits
position.
3.Lightcontrol:
Lightcontrolisbasicallyametalrodwhichisusedtoadjustthewavelengthofrays.Itis
linkedwiththeanoccluder.Anoccluderissituatedontheuppersideoflightcontrol.
4.Sample
Itconsistsofsoluteandsolventbutitsmajorpartconsistsofsolute.Zeroreadingshows
solventandfinalreadingisthereadingofthesolute.Zeroreadingontherightsideisusedfor
thesolventandreadingofsoluteisobtainedonthedigitalreader.
5.Photodetector
Photodetectorisusedtoconvertelectromagneticwavesintoelectricalwaves.Theyarealso
presentinoureyes.Theypasselectricalraystowardstheamplifier..Ampliferenhancesthe
powerofthephotorays.
6.Digitalreader:
Itisusedtoshowthefinalreadingofelectricalrays(lightrays).Andfinalreadingisobtained
fromrighttoleftsideofthereader
[4]
.
Rangeofspectronic20spectrophotometer:
Therangeoftheabovementionedspectrophotometerisfrom340nmto950nmand
accuracyofwavelengthis+2.5nm(Pictureofinstrumentisgivenonnextpage).

7. Wavelength
CamLight
Control
Sample
Measuring
Phototube
Filter
Occluder
Diffraction
Grating
Lamp
Reference
Phototube
Objective
Lens
EntranceSlit
Field
Lens
LightGate
Diagram:Internalinstumentationofspectronic20
Diagram:Generalconstructionofsinglebeamspectrphotometer

9. SPECTRONIC20Spectrophotometer
Thesequenceforsamplemeasurementis:
a.SelectwavelengthusingWavelengthControl.
b.Withsamplecompartmentemptyandcoverclosed,adjustZeroControl
sothatthemeterreadszero(ontheleftsideofthedial).
c.InsertreferenceblankintothesamplecompartmentandsetAbsorbanceControltozero
(ontherightsideofthedial).
d.Insertunknownsampleintothesamplecompartmentandread
measurementfrommeterinpercenttransmittanceorabsorbance
[5]
.

10. GeneralWorking:
Alampprovidesthesourceoflight.Thebeamoflightstrikesthe
collimatororlens.Thelensthenmakestheseradiationsparallelandfallthem
ontheprismorsometimesdiffractiongrating(Inoptics,adiffractiongrating
isanopticalcomponentwhichsplitsanddiffractslightintoseveralbeams
travellingindifferentdirections.).Theprismorgratingthenseparatesthe
lightintoitscomponentwavelengths.Thegratingisrotatedsothatonlya
specificwavelengthoflightreachestheexitslit.(Theentranceslit,prismand
exitslitcollectivelyformthemonochromator).Thenthelightinteractswith
thesampleplacedinthetransparentcuvette.Heresomeoftheamountoflight
isabsorbedbythesamplesolutionplacedinthecuvette.Thenthelightthatis
notabsorbedbythesamplestrikeswithdetectorwhereitstransmittanceis
measured.Transmittancereferstotheamountoflightthatpassesthroughthe
sample.Absorbanceisameasurementoflightthatisabsorbedbythesample.
Thedetectorsensesthelightbeingtransmittedthroughthesampleand
convertsthisinformationintoadigitalvalueasshowninfigure
[6]
.
Prismsplittinglight Wavelengthofdifferentcoloursofvisiblelight

11. .Whysomeoflightisabsorbedbythesamplesolution?
Someamountoflightthatiscomingfromtheexitslitisabsorbedbythe
samplesolution.Thereasonisthattheelectronsofthemolecuespresentinthe
samplesolutionabsorbsomespecificamountofenergyfromthephotonsof
thelightwhichhavespecificwavelengthandgointoanexcitedstatei.e
intohigherenergylevel.Inthisway,lightenergyisconvertedintoheatenergy
[7].
Inthiswayelectronsarepromotedtoahigherenergymolecularorbitalafter
absorbingthatmonochromaticlight.
ExplanationaccordingtoBeer-Lambert’sLaw:
SpectrophotometersworkontheprincipleofBeer-Lambert’slaw.Withthe
helpofthislaw,onecalculatetheTransmittanceoflight.Andso,absorbance
canalsobecalcultulated.
Transmittance:
Theratioofintensitiesofthetransmittedandincidentlightgives
transmittance.Inotherwords,
Transmittanceistheamountoflightenergythatistransmittedthroughthesolution
withoutabsorbing.ItismeasuredinpercantageT(i.e%transmittance)
[8]
.
Transmittance=IT/I0×100

12. WhereIT= Intensityoftransmittedlight
throughthesample
AndI0=Intensityoftheincidentlight(i.eblankcuvettehaving
purewater)
A100%valueofTrepresentsatransparentsubstancepresentinthe
cuvettewhilezerovalueofTrepresentsthatobjectistotallyopaquemeaning
thatithasabsorbedallwavelengthoflightandhasnottransmittedany
wavelengthoflight.
Absorbance:
Absorbanceistheamountofenergyofspecificwavelengthin
termsofphotonsthatisabsorbedbytheelectronsofmoleculespresentinthe
sample.Itisgivenas:
Absorbance: -log10T
Wecanalsowriteitas: -log10IT/I0
HereTresentsthetransmittanceofasubstance[4].
Beer-Lambert’sLawisgivenas:
Absorbance∝b.c
A=ε.b.c
Where‘ε’ispropotionalityconstantknownasmolarabsoptivity
coefficient.Wecanalsousehere‘a’whichmeanssamething.“b”isthepath
lengthand“c”istheconcentrationofthesample.
Thisequationstatesthatthequantityoflightabsorbedbyasubstancedissolvedin
afullytransmittingsolventisdirectlyproportionaltotheconcentrationofthe
substance(i.e‘c’)andthepathlength(i.eb)ofthelightthroughthesolution.
Itmeansthatifthereismorepathlength(i.e‘b’)ofthecuvetteinwhichsample
isplaced,moreabsorbanceoflightwilltakeplaceandasaresultlesstransmission

13. willbeobtained.Becausemoredistancehastobecoveredbythephotonsoflight
whilepassingthroughthesolutionandtherewillbemoreinteractionbetweenthe
photonsandabsorbingsubstances.Hencetheywillbeabsorbedmore(i.etheir
energywillbetakenupandabsorbedbytheelectronsoftheabsorbing
molecules).Andlesstransmittancewillbeobtained.
SimilarlyIfthereismoreconcentration(i.ec)ofabsorbingmaterialsinthe
sampleofcuvette,theresultwillbethesame(meaningmoreabsorbanceandless
transmittance)
[9]
.
Units:
Inuvspectroscopy,theconcentrationcofthesamplesolutionismeasuredinmolL
-1
andthePathlengthbofthelightpathincm.Thus,giventhatabsorbanceisunitless,
theunitsofmolarabsorptivityεareLmol
-1
cm
-1.
[10]
c=molL
-1
b=cm
ε=Lmol
-1
cm
-1
EXAMPLE:
Guanosinehasamaximumabsorbanceof275nm.ϵ275=8400M
−1
cm
−1
.ϵ
275=8400M
−1
cm
−1
andthepathlengthis1cm.Usingaspectrophotometer,we
findthatA275=0.70.Whatwillbetheconcentrationofguanosine
[11]
?
SOLUTION:
Since,
Absorbance =A=0.70
Molarabsoptivity=ϵ=8400
M
-1
cm
-1
)
Pathlength =b=l=1cm
Concentration=c=?
Tosolvethisproblem,weuseBeer'sLaw.

14. A=ϵlc
0.70=(8400)(1cm)
Next,dividebothsideby[(8400)(1cm)],wegettheanswer
c=8.33x10
-5
mol/L
Graph:
Wecanplotagraphbetweenconcentrationandabsorbance.Drawing
ConcentrationinmolL
-1
onX-axisandabsorbanceonY-axis.Astraightlineis
obtainedwhichindicatesthatasconcentrationofasampleisincreased,absorbance
alsoincreasesinthethesameratio.Similarlygraphbetweenpathlengthband
absorbanceccanbeplotted
[12].
Graph1:Graphbetweenconcentrationc(inmolL
-1
)andabsorbance
A(nounits)indicatingdirectlyproportionalrelation.

15. Graph2:Graphbetweenpathlengthb(orc)incmabsorbanceA.
>ApplicationsofSingleBeam
Spectrophotometer
Beforethespectrophotometerwasdevelopedscientistshadnoreliableandrapidmethodto
determinethechemicalmakeupofasubstance.Spectrophotometerradicallychangedallof
that.Someapplicationsofspectrophotometeraregivenas
1.Itisusedextensivelyinastronomy
2.Itisalsousedinarcheology
3.Itisusedtodeterminethedeviationoflightbyaglassprism
4.Itisusedtodeterminerefractiveindex
5.Itisusedtostudythechemicalbondingandtodetermine
concentrationofsubstances(nucleicacidforexamplewhichdonot
absorbvisiblelight)
6.Spectrophotometersareusedtoidentifytheunknowncompounds
7.Itisusedtostudyatmosphereofdistantplanets
8.Theyarealsousedinchemistrytofindoutwhichchemicalsare
presentinchemicalsamples
9.Spectrophotometerscanalsobeusedforcolourdeterminationwithin
rangeof380-700nm
10.Itisalsousedinforensicsciencesforinvestigationpurposes
11.Itisalsousedinmicrobiologytostudyofmicro-oragnismslikebacteria
12.Itisalsousedinpharmaceutical,foodandpaintindustries
13.Itisusedinthediagnosisofdiseaseswhichisdetectedbythesamplestaken
fromthebodyareanalysizeinthreeareasChemistry,Hematologyand
microbiology

16. 14.Itisusedtoexplainabsorbancevstimegraphinkinetics
15.Itisusedinthesinglepointcalibrationforequilibriumconstant
determination
16.Itisalsousedinquantitativeanalysisoforganiccompounds
17.Itisalsousedforstudytheprogressofachemicalreaction
Advantagesanddisadvantages:-
Therearefollowingadvantagesofsinglebeamspectrophotometer
Ithassimpleconstruction
Ithaslesscomplicatedsystem
Itiseasytouse
Itisnotverymuchexpensive
Ithasfewerpartswhichcanbeeasilyhandled
Ithashighlightthroughput
Itmeasuresaccurately
Therearefollowingdisadvantagesofsinglebeam
spectrophotometer
Itconsumesmuchtimeascomparetootherdesigns
Wecannotmakevariationsduringexperiment
References:
1.TheJournalofBiologicalChemistryAClassicInstrument
2.BeckmanDUSpectrophotometerandItsInventor,Arnold
InventoroftheWeekArchive:ArnoldBeckman
3.CecilInstruments:HistoryofCecilInstruments
4.AnalyticalChemistrybyD.ASkoog,D.MWestF.JHollerandS.RCrouch,,8th
edition
5.https://web.stanford.edu/class/chem184/lectures08/Zare_Spectroscopy
6.https://www.lsteam.org/projects/videos/how-does-spectrophotometer-
work

17. 7.https://www.andor.com/learning-academy/what-is-light-an-overview-
of-the-properties-of-light
8.https://cellbiologyolm.stevegallik.org/node/7
9.https://chem.libretexts.org/Core/Physical_and_Theoretical_Chemistry/
Kinetics/Reaction_Rates/Experimental_Determination_of_Kinetcs/Spect
rophotometry
10.https://life.nthu.edu.tw/~labcjw/BioPhyChem/Spectroscopy/beerslaw.
html
11.https://chem.libretexts.org/Core/Physical_and_Theoretical_Chemistr
y/Spectroscopy/Electronic_Spectroscopy/Electronic_Spectroscopy_Basi
cs/The_Beer-Lambert_Law
12.https://public.iorodeo.com/docs/colorimeter/lab_2.html