Colorimetry & spectrophotometry

COLORIMETRY And SPECTROPHOTOMETRY

1. COLORIMETRY
And
SPECTROPHOTOMETRY
Amit Kumar Singh
Asst. Professor (Clinical Biochemistry)
USMAS, Rayat Bahra University, Mohali Punjab 23/05/2021

2. Introduction
•Photometryisthemostcommonanalytical
techniqueusedinthebiochemicallaboratory.
Itisdesignedtomeasuretheintensityofa
beamoflight.
•Photometricprinciplesareappliedtothe
severalkindsofanalyticaltechniques:
(a)whereabsorbedortransmittedlightismeasured:
•Colorimetry
•Spectrophotometry
•Atomicabsorption,and
•Turbidometry
(b)whereemittedlightismeasured:
•Flamephotometry

3. Introduction
•Thecomponentsofmostphotoelectric
colorimetersarebasicallythesameand the
basicmethodofoperationisalsosimilarfor
alltheinstruments.
•Inanalyticalchemistry,Colorimetryisa
technique“usedtodeterminethe
concentrationofcoloredcompounds
(analytes)insamplesolution”atvisible
spectrumoflight(400–700nm).

4. PropertiesofLight
Lightis thevisiblespectrumof
electromagneticradiation,emittedinthe
formofwavesofdifferentwavelengths
rangingfrom380nmto750nm.

5. ElectromagneticSpectrum

6. Colors&Wavelengths
COLOR WAVELENGTH (λinnm)
Ultraviolet <380
Violet 380–435
Blue 436–480
Greenish-blue 481–490
Bluish-green 491–500
Green 501–560
Yellowish-green 561–580
Yellow 581–595
Orange 596–650
Red 651–780
NearInfrared >780

7. PrincipleofPhotometry
Substancetobemeasuredbyphotometrymustbe
coloredtobeginwithorcanbemadetoproduce
colorderivativesbyusingcertainreagentsand
reactions.
Intensityofcolourproducedispropotionaltothe
concentrationofthecolourproducingsubs.present
insolution.
Coloredsubs.absorbslightof aparticularwave
lengthandtheextentoflightabsorptiondependson
theconc.ofcolorproducingsubs.insolution.

8. Acharacteristicwavelengthofabsorptionspectrum
isisolatedfromlightpassingitthroughfilter
monochromator
Solutionwithcoloredsubs.iskeptinacuvet&
allowedthesubs.toabsorblight.
Degreeoflightabsorptionbyasoluteofunknown
conc.Ispropotionaltodegreeoflightabsorption
bysamesoluteinasolutionof knownconc.
Subs.ofunknownconc.ismeasuredbycomparing
withsamesubs.inanothersolu.Ofknownconc.

9. Colorimetry
Principle
.Coloredsolutionshavethepropertyof
absorbingcertainwavelengthoflight
whenamonochromaticlightispassed
throughthem.
.Theamountoflightabsorbedor
transmittedbyacoloredsolutionisin
accordancewithtwolaws:
Beer’slaw
Lambert’slaw

10. Beer’slaw
Thislawstatesthat,theintensityof
transmittedlightdecreasesexponentially
withtheincreaseinconcentrationof
coloredsubstanceinthesolution.
i.e.theamountoflightabsorbedbya
coloredsolutionisdirectlyproportionalto
theconc.Ofsubstanceinthesolution.

11. Lambert’slaw
Thislawstatesthat,theintensityof
transmittedlightdecreasesexponentially
withincreaseinlengthoflightpathway.
(diameterofthecuvette)
i.e.theamountoflightabsorbedbyacolored
solutionisdirectlypropotionaltothelength
oflightpath.

12. Transmittance
⚫Transmittance:Itistheratioofintensityof
transmittedlight(It)totheintensityofincident
light(lo)acrossasolution.Itisexpressedas%
⚫Transmittance(T)=It/1o
⚫Transmittanceisinverselyandlogarithmically
proportionaltotheconcentration.i.eTαlog1/C.

13. Absorbance
Opticaldensity:itistheamountoflight
absorbedbythecoloredsubstance. OD
maybedefinedasthelogarithmicratio
ofincidentlighttothatoftransmitted
light.
•SoA= log(I/T);=log
10(100/T);=2-
log
10T
Absorbaceisdirectlyandlinearly
propotionaltocon.

14. Relationshipbetween
absorbance andtransmittance
OD %T
OD=2-log
10T

15. CombinedBeer’s-Lambert’s
law
Combiningthetwolaws:
AαCxL
A=KxCxL
LetA
T=absorbanceofthetestsolution
C
T=concentrationofthetestsolution
A
S=absorbanceofthestandardsolution
C
S=concentrationofthestandardsolution

16. C
T=
A
T
A
S
xC
S
Concentration
ofTESTsol.
AbsorbanceofTEST
AbsorbanceofSTANDARD
xCon.ofSTANDARD=
Concentration
ofTEST/100ml
AbsorbanceofSTANDARD
=
AbsorbanceofTEST
x
ConcnofStdX100
Xml
OD
S
OD
T
= xC
S

17. Standard(calibrationcurve)
•Thestandardcurveispreparedtocheck
whetherthemethodofassayingaparticular
substancefollowsBeer’sLaw,i.e.whetherthe
absorbanceofthesubstanceincreasesina
linearwaywithitsconcentration.
•Thestandardcurveisconstructedbyplotting a
vertical axis (y–axis, ordinate) for optical
densities(absorbance)andahorizontalaxis (x
–axis,abscissa)theconcentrationof standard
solution.
•Theconcentrationofthetest/unknowncanbe
measuredfromthegraph(standardcurve).

18. StandardCurve/Calibrationcurve

19. Preparationofsolutionfor
investigation
•Incolorimetricestimationitisnecessaryto
prepare3solutions:
BLANK(B)
STANDARD(S)
TEST(T)
10/3/20164:36PM

20. BLANK•.Tocompensateanynonspecific
color
•.Tosettheinstrument100%T
andzero%OD
WaterBLANK
Reagent BLANK

21. STANDARD
Solutionofknownconcentrationof
thesubstance
Both O.D and
concentrationare
known
Soconcentrationof
unknowncanbe
calculated

22. TEST•Testsolutionismadebytreating
aspecificvolumeofthetest
samplewithreagents
Asperprocedure

23. Complimentarycolor
Wavelengthbetween400nmto700nm
formthevisiblespectrumoflight
Lightpassedthroughasolutionwhich
selectivityabsorbsradiationatfixedwave
lengths,thenthecolorofthe
transmittedlightiscomplementarytothat
of theabsoredlight.

24. Colorsandcomplimentarycolors
ofvisible spectrum
Color ofthe
solution/solution
colortransmitted
Filterused/color
absorbed
Wavelength(nm)
Yellow Blue 450–479
Red Green 505–534
Blue yellow 640–689
Green Red 620-689

25. Colorimeter

26. Componentsof Colorimetry
1.Lightsource:
Thelightsourceisusuallyatungtenlamp,for
wavelengthinthevisiblerange(320–700nm)anda
deutariumorhydrogenlampsforultravioletlight
(below350nm).
a)Tungsten lamp Visiblerange
b)Deutarium/hydrogenlamp(preferred)UVRays
c)Blackbodyradiators(Nerstglower) Infraredradiations

27. Monochromators/Filters
•Thisdeviceusedforspectralisolation(lightof
singlewavelength)
•thismeansofselectingasufficientlynarrow
waveband.
FilterSBW about50nm(widebandpass
monochromator)
Prism SBWis5-10or<5nm(narrow
bandpassmonochromator)
DiffractiongratingSBWis15-20nm.

28. Monochromators
•EarlycolorimetersusedAbsorptionfilters(i.e.glass
filter,Gelatinfilter)thattransmittedawidesegmentof
spectrum(50nmormore).
•NewerinstrumentuseInterfacefiltersthatconsistof
thinlayerof magnesiumfluoridecrystalswitha
semitransparentcoatingof silveroneachside.
•Monochromatorconsistsof:
–Entranceslit
–Absorption/interfacefilterand
–Prismsordiffractiongratingforwavelengthselection
–Exitslit

29. SampleHolder/Cuvette
•Cuvettesarerectangularcell,squarecellor
circularone.
•Madeupofopticalglassforvisiblewavelength
(quartzorfusedsilicaforUV).
•Commononeissquare,rectangulartoavoid
refractionartifacts.
•Opticalpath(length)ofcuvetteisalways1cm.
•Capacitymaybe3ml/2ml/1mldependingupon
thethicknessofthewallofthecuvette.
•Foraccurateandprecisereadingcuvettemustbe
transparent, clean, devoid ofany scratches and
thereshouldbenobubbleadheringtotheinner
surfaceofthefilledcuvette.

30. Photosensitivedetectors
Detectorsarethe transducers,whichconvert
lightenergytoelectricalenagery.Adetector
shouldbepossessfollwingcharacteristics:
Shouldbesensitive,stable
Shouldhavelinearresponse,shortresponse.
•Differentdetectorsusedare:
Barrierlayercells(photocells)
–simpliest
Photoconductive cells
(photodiodes)–newest

31. Readoutdevices
•Thedetectorresponsecanbemeasuredbyany
ofthefollowingdevices:
a)Galvanometer
b)Ammeter
c)Recorder
d)Digitalreadout.
Thesignalmaybetransmittedtocomputerorprint
outdevices.

32. Criteriaforsatisfactorycolorimetric
estimations
Stabilityofcolor
IntensityofcolorThe colorofthe solution
should beintense.
ClarityofthesolutionSubstanceunder
•investigationshouldbecompletelysoluble.
SpecificityColor producedshouldbespecificfor
the desiredconstituent.
ValidityofBeer’slawTheintensityofcolor
•shouldbeproportionaltoconcentration.

33. ApplicationsOfColorimeter
•Estimationofbiochemicalcompoundsinblood,
plasma,serum,CSF,urine,etc.:
–Glucose
–Urea
–Creatinine
–UricAcid
–Bilirubin
–Lipids
–TotalProteins
–Enzymes[e.g.ALT,AST,ALP]
–Minerals[Calcium,Phosphorusetc.]etc….

34. Spectrophotometry:
Instruments&Applications

35. Principleof Spectrophotometer
–Solutesinasolutionshowcharacteristicabsorption
spectruminUVorvisibleorinfraredregionof
electromagneticradiation.
–Characteristic
isolatedby
absorptionspectrumcanbe
passingtheelectromagnetic
radiationthroughaprismmonochromator.
–Degreeofabsorptionofelectromagnetic
radiationdependsonthecondensationofsolute
insolution.

36. TheSpectrophotometer

37. TheSpectrophotometer

38. Introduction
•Spectrophotometer:
a)Single-beam
b)Double-beam
4[41]

39. Instruments
•Light source:provideasufficientoflightwhichis
suitableformarkingameasurement.
•Thelightsourcetypicallyyieldsahighoutputof
polychromaticlightoverawiderangeofthe
spectrum.[4]
42

40. Commonmonochromators:
⚫Filter
⚫Prism
⚫Diffractiongrating
⚫Interferencefilter
•Monochromator:Acceptspolychromaticinputlightfrom
alampandoutputsmonochromaticlight.
•Monochromatorconsistsoftheseparts:
I.Entrance slit
II.Collimatinglensormirror
III.Dispersion element
IV.Focusinglensormirror
V.Exit slit[6]
43

41. Instruments
•Dispersiondevices:Aspecialplatewithhundreds
ofparallelgroovedlines.
•Thegroovedlinesacttoseparatethewhitelightinto
the visiblelightspectrum.
44
Themorelines
thesmaller
thewavelength
resolution.[5]

42. Instruments
•Focusingdevices:Combinationsof lenses,slits,
andmirrors.
•relayandfocuslightthroughtheinstrument.[2]
45

43. Instruments
•Cuvettes:designedtoholdsamplesforspectroscopic
experiments.madeofPlastic,glassoropticalgrade
quartz
•shouldbeasclearaspossible,withoutimpuritiesthat
mightaffectaspectroscopicreading.[2]
46

44. Instruments
•Detectors:Convertradiantenergy(photons)intoan
electricalsignal.
Thephotocellandphototubearethesimplest
photodetectors,producingcurrentproportionaltothe
intensityof thelightstrikingThem.[1],[2]
47

45. Instruments
•Displaydevices:Thedatafromadetector are
displayedbyareadoutdevice,suchasananalogmeter,
alightbeamreflectedonascale,oradigitaldisplay,or
LCD.
•Theoutputcanalsobetransmittedtoacomputeror
printer.[3]
48

46. Applications
1.Concentrationmeasurement
–Preparesamples
–Makeseriesof standardsolutionsof known concentrations
[4]
49

47. Applications
–Setspectrophotometertotheλof maximumlight
absorption
–Measurethe absorptionoftheunknown,andfromthe
standardplot,readtherelatedconcentration[4]
50

48. Applications
2.DetectionofImpurities
•UVabsorptionspectroscopyisoneofthe
bestmethodsfordeterminationof impuritiesinorganic
molecules.[7]
51
Additionalpeakscanbe
observedduetoimpurities
inthesampleanditcanbe
comparedwiththatof
standardrawmaterial.

49. Applications
3.Structureelucidationoforganiccompounds.
•From thelocationof peaksandcombinationofpeaks
UVspectroscopyelucidatestructureoforganic
molecules:
othe presenceorabsenceofunsaturation,
othe presenceofheteroatoms.[7]
52

50. Advantages:
Ensurehigherdegreeofspectralpurity.
Minimumstraylightintotheexitbeam
(wavelengthoutsidethedesiredlightis
calledstraylight).
Greateraccuracy.
Moresensitivity,specificityandprecision.

51. Sourceoferrorsin spectrophotometer
Straylight
Lowresolutionoflightsource
Lackinglinearity
Variationintemp.
Lowsamplevolume

52. Difference between colorimeter and
spectrophotometer
Traits Colorimeter Spectrophotometer
Monochromator Filter Prism
Spectralbandwidth Broadband Narrowband
Spectralpurity Less More
Spectralisolation Filterhastobe
changed
Desiredwavelength
canbeadjusted
Straylight More Minimum
Accuracy Less More
Sample LargervolumeneededSmallvolumeneeded
Cost Cheaper Morecostly
Lightsource VisiblerangeoflightBeyondvisiblerange