独家公布~斯洛文尼亚-塞尔维亚【3 9 7 7 . T W 官方指定】

独家公布~斯洛文尼亚-塞尔维亚【3 9 7 7 . T W 官方指定】

1. SPECTROPHOTOMETRY
Dr. S.A. Ojedokun
Department of Chemical Pathology
LTH Ogbomoso
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2. Outline
•Introduction
•Light spectroscopy
•Principles of light Absorption
•Principles of Spectrophotometry
•Classifications/Types
•Interferences
•Quality assurance
•Applications
•Conclusion
•References
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3. Introduction
•Spectrophotometryisamethodthathingesonthequantitative
analysisofmoleculesdependingonhowmuchlightisabsorbedby
colouredcompounds.
•Importantfeaturesofspectrophotometersarespectralbandwidth
(therangeofcoloursitcantransmitthroughthetestsample),the
percentageofsampletransmission,thelogarithmicrangeofsample
absorption,andsometimesapercentageofreflectance
measurement.
•Photometryisthemeasurementoftheamountofluminouslight
(luminousintensity)fallingonasurfacefromasource.
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4. Intro cont’d
•Spectrophotometryisascientificanalyticaltechniquebasedonthe
absorptionoflightbyasolutionataparticularwavelengthwith
relevantpropertiesofthesolution.,e.g.,concentration.
•Spectrophotometrydependsonthelight-absorbingpropertiesof
substancesoraderivativeofthesubstancebeinganalyzed.
•Aninstrumentformeasuringtheintensityoflightinapartofthe
spectrum,astransmittedoremittedbyparticularsubstancesisa
spectrophotometer
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5. Light spectroscopy
•Light is a form of energy propagating into space at a very high speed.
•As an electromagnetic wave travelling into space –it is radiant energy.
•The energy of light oscillates periodically between a minimum and a
maximum as a function of time –like a wave.
•The distance between two maxima or two minima, respectively of the
electromagnetic wave is defined as the wavelength, given in
nanometers (nm).
•Light behaves like discrete energy packets called photons whose
energy is inversely proportional to the wavelength
•The shorter the wavelength, the higher the energy
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6. Light spectroscopy cont’d
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7. Light spectroscopy cont’d
•Thus,thedifferentcomponentsoflightarecharacterisedbyaspecific
wavelength.
•Thesumofallcomponentsi.e.ofallwavelengths,iscalleda
spectrum.
•Morespecifically,aspectrumrepresentsadistributionofradiant
energy.
•Forinstance,theelectromagneticspectrumofvisiblelightranges
fromapproximately390nmuptoapproximately780nmwith
differentcolours.
•Eachcolourhasaspecificwavelength,e.g.redlighthasawavelength
of660nm,whilegreenlighthasawavelengthof520nm.
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8. 8

9. Light spectroscopy cont’d
•Optical spectroscopy is based on the interaction of light with matter.
•The light which is not absorbed by the object is reflected and can be
seen by the eye
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10. Principles of Light Absorption
10
Properties of Light

11. Beer’sLaw
•Theabsorbanceoflightisdirectlyproportionaltoboththe
concentrationoftheabsorbingmediumandthethicknessofthe
medium.
•InSpectrophotometrythethicknessofthemediumiscalledthepath
length.
•Beer’slawallowsthemeasurementofsamplesofdifferingpathlength
andcomparestheresultsdirectlywitheachother.
•Inbasicterms:Absorbance=Concentration×Pathlength
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13. Lambert’sLaw
•Theproportionoflightabsorbedbyamediumisindependentofthe
intensityofincidentlight.
•Asamplewhichabsorbs75%(25%transmittance)ofthelightwillalways
absorb75%ofthelight,nomatterthestrengthofthelightsource.
•Lambert’slawisexpressedasI/Io=T
WhereI=Intensityoftransmittedlight
Io=Intensityoftheincidentlight
T=Transmittance
•Thisallowsdifferentspectrophotometerswithdifferentlightsourcesto
producecomparableabsorptionreadingsindependentofthepowerofthe
lightsource.
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14. Beer-Lambert law
•When light, passes through a transparent cuvette filled with sample
solution, the light intensity is attenuated proportionally to the sample
concentration. In other words, a highly concentrated sample solution
will absorb more light.
•In addition, the attenuation is also proportional to the length of the
cuvette; a longer cuvette will lead to a higher absorption of light.
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15. •Mathematically,
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16. This relationship is called the Lambert-Beer law where:
1. The sample concentration is c.
2. The path length, d of the cuvette.
3. The extinction coefficient ε(epsilon) is a sample-specific constant
describing how much the sample is absorbing at a given wavelength
•When the path length is 1 cm and the concentration is 1% w/v, the
extinction coefficient is called specific absorbance (E )
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17. •TheLambert-Beerlawallowsforthedeterminationofthesample
concentrationfromthemeasuredabsorbancevalue.Iftheextinction
coefficientεandthepathlengthdareknown,thenconcentrationc
canbecalculatedfromabsorbanceAasgivenbelow:
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18. Beer’s law holds if the following conditions are met
•Incident radiation on the substance of interest is monochromatic.
•The solvent absorption is insignificant compared with the solute
absorbance.
•The solute concentration is within given limits.
•An optical interference is not present.
•A chemical reaction does not occur between the molecules of interest
and another solute or solvent molecule.
•The sides of the cell are parallel
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19. Calibration curve/ standard curve
•A solution of known concentration is prepared.
•Serial dilutions up to about 5 solutions of different concentrations.
•Spectrophotometric absorbance is set at zero using a blank.
•Measurement of absorbances of the solutions.
•Plotting of absorbances(y axis) against concentrations (x axis).
•Determination of the concentration of unknown solution using the
graph.
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20. •Calibration curve for glucose assay
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21. PrinciplesofSpectrophotometry
The spectrophotometry technique is used to measure light intensity as
a function of wavelength using a spectrophotometer.
And this is achieved through:
1.Diffraction of the light beam into a spectrum of wavelengths
2.Directing the diffracted light onto an object
3.Reception of the light reflected or returned from the object
4.Detecting the intensities with a charge-coupled device
5.Displaying the results as a graph on the detector and then the
display device
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22. Basic components of spectrophotometer
•Light source
•Monochromator
•Cuvette or sample cell
•Photo detector
•Readout device
•Recorder
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23. 23
Basic components of a spectrophotometer

24. Lamp source
1. Incandescent lamps
UV spectrum
•Deuterium-discharge lamp
•Mercury arc lamp
•Xenon arc lamp
•Hydrogen lamp
Visible and near infrared region
•Tungsten halogen lamp containing
•iodine or bromine
2.Laser(Lightamplificationbystimulatedemissionofradiation)
•This is a device used in spectrophotometry, which transform light of various frequencies into an extremely
intense, focused, and nearly non divergent beam of monochromatic light.
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25. Important factors for a light source
•Range
•Spectral distribution within the range
•Stability of radiant energy
•Source of radiant production
•Temperature
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26. Monochromator
•Necessary to isolate a desired wavelength of light and exclude other
wavelengths
•Wavelength isolation is a function of the type of device used and the
width of the entrance and exit slits.
•Devices used to obtain monochromatic light include
•Filters (colored glass and interference)
•Prisms
•Diffraction gratings
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27. Colored glass filters
•Least expensive
•Simple, although not always precise
•Usually pass a relatively wide band of radiant energy
Interference filters
•Produces a monochromatic light based on the principle of constructive
interference of waves
Prism
•A narrow beam of light focused on a prism is refracted as it enters the denser
glass.
•The prism can be rotated, allowing only the desired wavelength to pass through
an exit slit.
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28. Diffraction gratings
•Most commonly used as monochromators.
•Diffraction (the separation of light into component wavelengths), is
based on the principle that wavelengths bend as they pass a sharp
corner. The degree of bending depends on the wavelength
•Diffraction grating consists of many parallel grooves (15,000-30,000
per inch) etched onto a polished surface.
•Because the multiple spectra have a tendency to cause stray light
problems, accessory filters are used.
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29. Cuvette/sample cell
•Are small vessels used to hold liquid samples to be analysedin the
light path of the spectrophotometer.
•Can be round or square
•Square cuvettes have advantages over round cuvettes in that there is
less error from lens effects, orientation and refraction.
•It can be made of Glass (Visible range), or quartz(UV & Visible range)
•Light path must be kept constant
•Cuvettes with scratched optical surfaces should be discarded as they
scatter light.
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30. Photodetector
•Detects transmitted radiant energy and converts it into an equivalent
amount of electricity
Types include:
•Photocell/Barrier layer cell
•Phototube
•Photomultiplier tube
•Photodiode
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31. Photocells/Barrierlayercell:
•Leastexpensiveanddurable
•Composedofafilmoflightsensitivematerials:Seleniumonaplateofiron
coveredbyathintransparentlayerofsilver
•Whenexposedtolight,electronsinthelightsensitivematerialsareexcited
andreleasedtoflowthroughhighlyconductivesilver
•Resistancepreventsionsfromflowingintheoppositedirectiontowards
theironthusformingabarrier.
•EMFisgeneratedfromtheresistancewhichcanbemeasured.
•Itistemperaturesensitiveandbecomesnonlinearatveryhighorverylow
levelsofillumination.
•Outputisnoteasilyamplifiedsoitisusedininstrumentswithillumination
levelssuchthatthereisnoneedtoamplifythesignals.
•Light-sensitive
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32. Phototube
•Similartophotocellbutneedsanoutsidevoltagetooperateit.
•Containscathodeandanodeenclosedinaglasscase
•Cathode:madeupoflithiumorrubidiumthatactsasaresistorinthe
darkbutemitselectronswhenexposedtolight.
•Theemittedelectronsjumpovertothepositivelychargedanode
wheretheyarecollectedandreturnthroughanexternalmeasurable
circuit
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33. Photomultiplier(pm)tube
•Detectsandamplifiesradiantenergy,hencemoresensitivethanthe
phototube.
•Incidentlightstrikesthecoatedcathodeemittingelectrons
•Theelectronsareattractedtoaseriesofanodesknownasdynodeseach
havingasuccessivelyhigherpositivevoltage
•Thesedynodesareofamaterialthatgivesoffmanysecondaryelectrons
whenhitbyasingleelectron
•Initialelectronemissionatthecathodetriggersamultiplecascadeof
electronswithinthePMtube
•Theaccumulationoflightstrikingtheanodeproducesacurrentsignal
measuredinamperes
•Theyareusedininstrumentsdesignedtobeextremelysensitivetovery
lowlightlevelsandlightflashesofveryshortduration
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34. Photodiode
•Inaphotodiode,absorptionofradiantenergybyareverse-biasedpn-
junctiondiode(pn,positive-negative)producesaphotocurrentthatis
proportionaltotheincidentradiantpower.
•Photodiodearray(PDA)detectorsareavailableinintegratedcircuits
containing256to2,048photodiodesinalineararrangement
•Eachphotodioderespondstoaspecificwavelength,
•Itsexcellentlinearity(6–7decadesofradiantpower),speed,and
smallsizemakethemusefulinapplicationswherelightlevelsare
adequate
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35. Readout devices
Analog
•Uses deflector pin on a meter
•Zero error is common
•Parallax error
•Easily affected by current/light voltage
•No longer popular
Digital
•Now common with newer spec
•Limit zero error
•No parallax error
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36. Classifications
A.Electromagnetic forms
UV, ViS, & IR
B.Geometry designs
Scanning & Array
C.Optical pathways
Single & double beam
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37. A. Based on electromagnetic form
1. UV spectrophotometry
•Uses light over the UV range (180-
400nm)
•A prism of suitable material and
geometry will provide a continuous
spectrum in which the component
wavelengths are separated in space
•In addition to prisms, diffraction
gratings are also employed for
producing monochromatic light
•Quartz cuvettes used to hold
samples
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38. 2. ViSSpectrophotometry
•Uses the visible range (~400-700nm) of the electromagnetic radiation
spectrum
•Plastic or glass cuvettes can be used for visible spectrophotometry
3. Infrared spectroscopy(IR)
Infrared spectrum refers to a spectrum greater than 760nm, which
is the most commonly used spectral region of organic compounds,
and can analyze a variety of conditions (gas, liquid, solid) of the sample.
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B.BasedonGeometrydesigns
1.Scanningspectrophotometer
•Theworkingprincipleofaconventionalscanningspectrophotometer
isbasedonthemeasurementofthetransmittancevalueateach
singlewavelength.
•Thetransmittanceatthisspecificwavelengthisrecorded.Thewhole
spectrumisobtainedbycontinuouslychangingthewavelengthofthe
light(i.e.scanning)incomingontothesamplesolutionbyrotatingthe
grating

40. Scanning spectrophotometer
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2.Arrayspectrophotometer
•Inthisconfiguration,thesampleinthecuvettesimultaneouslyabsorbs
differentwavelengthsoflight.Thetransmittedlightisthendiffractedbya
reflectiongratinglocatedafterthecuvette
•Subsequently,thediffractedlightofvariouswavelengthsisdirectedonto
thedetector.
•Thedetector,withitslongarrayofphotosensitive,semiconductor
material,allowsforsimultaneousmeasurementofallwavelengthsofthe
transmittedlightbeam.
•Thisdesignisalsoknownas"reverseoptics"

42. Array spectrophotometer
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C.Basedonopticalpaths
1.Singlebeamconfiguration
Thelightbeamisdirectlyguidedthroughthesampleontothe
detector.Acuvettecontainingonlysolventhastobemeasured
firsttodeterminetheblankvalue.Aftermeasuringtheblank
value,thesolventcuvetteisreplacedbyacuvettecontainingthe
sampletomeasuretheabsorptionspectrumofthesample.

44. 2.Double-beamconfiguration
Inadouble-beamconfiguration,thelightbeamissplitintoa
referenceandasamplebeam.
a)Simultaneousintime:Thelightbeamofthelampissplitintotwo
beamsofequalintensities.Eachbeampassesthroughadifferent
cuvette;oneisthereferencecuvette,whereasthesecondcuvette
containsthesamplesolution.Theintensitiesofbothbeamsare
measuredsimultaneouslybytwodetectors.
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45. Simultaneous-in-time
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b)Alternating in time: This configuration is achieved by directing
the light path with an optical chopper (OC), which is a rotating
sectional mirror. The light is directed alternately through a
sample and a reference cell. A unique detector measures both
light beams one after the other.

47. Interference
•Interference is phenomenon that leads to changes in intensity of the
signal in spectrophotometry.
Types of interference
•Optical interference is a phenomenon in which two wavelengths
superimpose to form a greater or lower wavelengths.
•Chemical interference arises out of the reaction between different
interferents and the analyte.
•Physical interference are due to physical properties of the sample e.g.
impurities in the solution
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48. Quality assurance
Wavelength accuracy
•Checked using standard absorbing solutions or filters with maximal
absorbance of known wavelength.
Stray light
•Refers to any wavelength outside the band transmitted by the
monochromator. Most common causes are light reflection from scratches
on optical surfaces or dust particles in the light path. Stray light is detected
and eliminated by using cutoff filters.
Linearity
•Refers to the difference between the actually measured value and the
value derived from the equation. It is checked using colouredsolutions of
different concentrations labelled with expected absorbances.
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49. Applications of Spectrophotometry
•Chemical reactions
•End point reaction
•Kinetics reaction
•Fixed time
•Two-point absorbance
•Fixed wavelength
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50. •Bio applications
•Use in clinical laboratory analysis
•Dissolution/ in vitro releases assay of drugs
•Quantification of DNA, RNA and proteins
•Dye, ink and paint industries
•Heavy metal and organic matter from environmental/agricultural
samples
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51. •Other applications
•Quantifying concentrations of compounds.
•Determining the structure of a compound.
•Finding functional groups in chemicals.
•Determining the molecular weight of compounds.
•Determining the composition of materials
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52. Conclusion
•Theuseofspectrophotometersspansvariousscientificfields,
such as physics, materials
science,chemistry,biochemistry,chemicalengineering,
andmolecularbiologysemiconductors,laserandoptical
manufacturing,printingandforensicexamination,aswellasin
laboratoriesforthestudyofchemicalsubstances.
•Spectrophotometrycontinuestoenjoywidepopularityduetothe
commonavailabilityofitsinstrumentationandsimplicityof
procedures,aswellasspeed,precisionandaccuracyofitstechnique.
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53. References
•Tietzfundamentals of clinical chemistry, 2008 fifth edition chapter 4
pg63-80.
•Cosimo A. De Caro, Haller Claudia. UV/VIS Spectrophotometry -
Fundamentals and Applications. 2015
https://www.researchgate.net/publication/321017142
•The principles of use of a spectrophotometer and its application in
the measurement of dental shades Chapter 11 Spectrophotometer
•Mass spectrometry
https://www2.chemistry.msu.edu/faculty/reusch/virttxtjml/spectrpy/
massspec/masspec1.htm
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Thank you