Uv vis spectroscopy- part-2

Uv vis spectroscopy instrumentation

1. UV-Visible spectroscopy
Part-2 Instrumentation
By-Dr. Mrs. P. S. Chaudhari
JSPM’s Charak College of Pharmacy & Research, Wagholi

2. Instrumentation
The essential components of spectrophotometer are:
1.Suitable source of electromagnetic radiation
2.Lenses, mirror, slits which collimate &focus the beam on
the sample
3.Filter or monochromator
4.Sample containers
5.A detector
6.Recorder or display

4. 1. Light Sources
1. Tungsten filament lamp 2.Hydrogen discharge lamp
3. Deuterium Lamp 4. Mercury Arc Lamp
5. Xenon Lamp

5. 1. Light sources:
•Widerangeofsourceswhichprovideradiantenergyare
available.Radiationsourcesneedtobecontinuousoverthe
rangeofwavelengthsofinterest.
•Youneedalightsourcewhichgivestheentirevisible
spectrumplusthenearultra-violetsothatyouarecovering
therangefromabout200nmtoabout800nm.
•Youcan'tgetthisrangeofwavelengthsfromasinglelamp,
andsoacombinationoftwoisused–
fortheUVspectrum-Deuteriumlamp
Forvisiblepart-ATungsten/Halogenlamp

6. Sources of radiant energy should have following
characteristics:
•Itmustbestableanddonotshow
fluctuation
•Itshouldprovideincidentlightof
sufficientintensity
•Itshouldemitacontinuousspectrumof
highanduniformintensityoverthe
entirewavelengthregion.

7. 1. Tungsten filament/halogen lamp(for visible radiation 350-2500nm ):
•Thisismostcommonsourceinvisible&nearIRradiation.
•Thefilamentisheatedbyastabilizedpowersupplyorbyastoragebattery.
•Tungstenorhalogenlampscontainasmallquantityofiodinewithina
quartzenvelopethathousesthetungstenfilament.
•Thelifetimeismorethenordinarylamp.
•Togetbestresults,itisimportantthatthetungstenlampshouldemit
radiantenergywhichremainconstantoverlongperiodsoftime.
•Thiscanachievebyemployingconstantpowersupply.

9. Construction
•Incandescentlightbulbsconsistofanair-tightglassenclosure(theenvelope,or
bulb)withafilamentoftungstenwireinsidethebulb,throughwhichanelectric
currentispassed.Contactwiresandabasewithtwo(ormore)conductors
provideelectricalconnectionstothefilament.
•Smallwiresembeddedinthesteminturnsupportthefilamentanditslead
wires.
1.Outline of Glass bulb
2.Low pressure inert gas (argon, nitrogen, krypton, xenon)
3. Tungstenfilament
4. Contact wire (goes out of stem)
5. Contact wire (goes into stem)
6. Support wires (one end embedded in stem; conduct no current)
7. Stem (glass mount)
8. Contact wire (goes out of stem)
9. Cap (sleeve)
10. Insulation (vitrite)
11.Electrical contact

10. How it works?
•Anincandescentbulbusesheatcausedbyanelectricalcurrent.When
electricalcurrentpassesthroughawire,itcausesthewiretoheat.
•Thewire,orfilament,getssohotthatitglowsandgivesofflight.Everyday
incandescentlightbulbshaveafilamentmadeoftungsten.
•Thehotfilamentisprotectedfromoxidationwithaglassorquartzbulbthat
isfilledwithinertgasorevacuated.
Thebulbisfilledwithaninertgas,toreduceevaporationofthefilamentand
preventitsoxidationatapressureofabout70kPa(0.7atm)
Thepresenceofgasmoleculesknockstheliberatedtungstenatomsbackto
thefilament,reducingitsevaporationandallowingittobeoperatedat
highertemperaturewithoutreducingitslife

11. Tungsten/halogenlampscontainasmallamountofiodineina
quartz"envelope"whichalsocontainsthetungstenfilament.
Quartzisrequiredbecauseofhighoperatingtemperatureofthe
lamp(3500K)
Theiodinereactswithgaseoustungsten,formedbysublimation,
producingthevolatilecompoundWI
2.
WhenmoleculesofWI
2hitthefilamenttheydecompose,
redepositingtungstenbackonthefilament.
Thelifetimeofatungsten/halogenlampisapproximatelydouble
thatofanordinarytungstenfilamentlamp.
Tungsten/halogenlampsareveryefficient,andtheiroutput
extendswellintotheultra-violet.Theyareusedinmanymodern
spectrophotometers.

12. •Drawback-it emits the major portion of its radiant energy in
the near IR region of the spectrum i. e. only about 15%of
radiant energy falls within the visible region at an operating
temperature 2725.
•If temperature is increased above 2850 this increase the total
energy output & shifts the wavelength of maximum intensity to
shorter wavelength.
•At high temperature life of lamp is too much reduced.

13. 2. Hydrogen Lamp(UV region 160 -375 nm):
Mostcommonsourceofultravioletradiation.
AcontinuumspectruminUVregionisproducedbyelectrical
excitationofhydrogenatlowpresssure.
Quartzwindowsmustbeemployedsinceglassabsorbs
stronglyatwavelengthlessthanabout350nm.

14. 2.

15. 3. Deuterium discharge lamp (160 -375 nm)
The electrical excitation of deuterium or hydrogen at low pressure
produces a continuous UV spectrum.
Formation of an excited molecular species, which breaks up to give
two atomic species and an ultraviolet photon.(dissociation &
photoemission)
D
2+ electrical energy -D
2
*
-D' + D'' + hv
Both deuterium and hydrogen lamps emit radiation in the range 160 -
375 nm. Quartz windows must be used in these lamps, and quartz
cuvettes must be used, because glass absorbs radiation of
wavelengths less than 350 nm.
Deuterium lamps give five times greater light intensity than hydrogen
lamps.

16. Halogen lamps
•Thehalogenlampreducesunevenevaporationof
thefilamentandeliminatesdarkeningofthe
envelopebyfillingthelampwithahalogengasat
lowpressure,ratherthananinertgas.
•Thehalogencycleincreasesthelifetimeofthe
bulbandpreventsitsdarkeningbyredepositing
tungstenfromtheinsideofthebulbbackontothe
filament.
•Thehalogenlampcanoperateitsfilamentata
highertemperaturethanastandardgasfilledlamp
ofsimilarpowerwithoutlossofoperatinglife.

17. 4. Xenon Arc Lamp (160-2000nm)
A xenon arc lampis a
specialized type of gas
discharge lamp, light
produced by passing
electricity through ionized
xenongas at high pressure.

19. 5

20. A mercury-vapor lampis a gas discharge lampthat uses an
electric arc through vaporized mercuryto produce light. The arc
discharge is generally confined to a small fused quartz arc tube
mounted within a larger borosilicate glass bulb.
Mercury vapor lamps are more energy efficientthan
incandescentand most fluorescent lights.
Their other advantages are a long bulb lifetime in the range of
24,000 hours and a high intensity, clear white light output.

22. Lamp Construction:
Attheheartofthelampisanarctubewhichisfabricatedfromquartz,
withatungstenelectrodedisposedateitherend.
Thetubecontainsafewmilligramsofmercuryandaround25-50torr
ofpureargonasabuffergastocarrythedischargewhilethelamp
warmsup,producingheattovaporisethemercuryandbringitintothe
discharge.
Anauxiliarystartingelectrodeisplacednexttooneofthemain
electrodestofacilitatelampignition.
Whenthelampisfirstenergisedthefullopencircuitvoltageisapplied
acrossthearctube.Thedistancebetweentheelectrodesissolargethat
theresultingvoltagegradientisnothighenoughtocauseionisationof
thegasfilling.

23. Howeverthesamevoltageisalsoappliedbetweenoneelectrode
andtheauxiliaryviaasmallresistor.Thegapbetweenthese
electrodesismuchsmaller,andthevoltagegradientissufficiently
highthationisationwilloccur.Asmalldischargestrikes,theseries
resistorof10-30kWlimitingthecurrentflowtoaboutone
thousandthofthenormallampcurrent.Oncefreeelectrons,ions
andphotonshavebeenproducedinthearctubeitisthenvery
easytostrikeadischargeacrossthemainelectrodes.

24. Oncethelamphaswarmedupandthemercuryisfullyvaporised,
thedischargeoperatesinunsaturatedmercuryvapourata
pressurevaryingfrom18barforthesmallesttypes,to2barforthe
largest.Thearcemitsthecharacteristicgreen,yellowandviolet
mercurylinesandtheretherearealsoconsiderableamountsof
invisiblelong-waveultravioletat365nmalongwithabroadrange
ofshorterwavelengths.Intheearliestlampshavingclearouter
bulbsthisinvisibleUVradiationwascompletelywasted.Todaythe
outerbulbisinternallycoatedwithathinlayerofafluorescent
phosphorwhichconvertstheUVintovisiblelight,generallyatthe
redendofthespectrumwherethemercuryoutputislacking,to
improvethecolourrenderingpropertiesofthislightsource.

25. Theouterenvelopeisfilledwithanitrogenorargon-nitrogen
mixture,oroccasionallywithcarbondioxide,bothtoprevent
oxidationofthearctubesealsandtoslowtherateof
deteriorationofcertainphosphors.

26. Mercury vapor lamps are widely used to light both indoor and
outdoor areas such as gymnasiums, factories, department
stores, banks, highways, parks, and sports fields.
Mercury vapor lamps consist of an inner arc discharge tube
constructed of quartz surrounded by an outer hard borasilicate
glass envelope.

27. 2. Collimating system:
•The radiation is collimated by lenses, mirrors & slits.
•A perfect parabolic mirror will bring parallel rays to a focus at a
single point.
•Materials used for lensesmust be transparent to the radiation
being used. Absorbance less than 0.2 % at the wavelength used.
•Ordinary silicate glass is suitable material for visible region.
•Below 300 nm quartz or fused silica is used
•Prism:
from glass- visible
from quartz or fused silica-UV visible region
2. Collimating system:

28. •Mirrors are used to reflect, focus or collimate light
beams in specrtophotometer.
•Front surfaces mirrors are used to minimise light
losses.
•To reduce scatttering by reflections, glass surfaces are
coated with magnesium fluoride in a thin film.
•Slit width is an important device in resolving
polychromatic radiation into its individual wavelength

29. 2. Collimating system:
•The purpose of employing devices is to resolve wide band of
polychromatic radiation into a narrow band of monochromatic
radiation.
•For spectroscopic analysis radiation that consists of a limited,
narrow, continuous group of wavelengths called as band is required.
•A narrow bandwidth enhances the sensitivity of absorbance
measurements.
•Ideally wavelength selector produce radiation of a single
wavelength, but no one approaches this ideal. Instead a band is
formed.
3. Monochromator(Wavelength selector):

30. Two types of wavelength selectors:
a)Filters
b)Monochromators

31. a) Filters:
•Used for isolation of narrow band of radiant energy of desired
spectral region.
•It allows transmission of only limited wavelength region, while
absorbing most of the radiation of other wavelengths.
i) Absorption filter -restricted to visible region of the spectrum
ii) Interference filter -ultraviolet, visible, infrared

32. i) Absorption filter
•Work by selective absorption of unwanted wavelength.
•Less expensive
•Widely used for band selection in the visible region
•It consists of colored glass or dye suspended in gelatin &
sandwiched between glass plates.
•It has effective bandwidth that range from 30-250nm

34. ii) Interference filter:
•Narrowbandwidthsareobtained.
•Rejectionofunwantedradiationbyselectivereflection.
Construction:
•Itconsistsofatransparentdielectric(Magnesiumfluoride)thatoccupies
thespacebetweentwosemitransparentmetallicfilms.Thisarrayis
sandwichedbetweentwoplatesofglassorothertransparentmaterial.
•Thethicknessofdielectriclayercarefullycontrolled&determinesthe
wavelengthofthetransmittedradiation.
Working:
•Whenbeamofradiationstrikesthisarray,fractionpassesthroughthefirst
metalliclayerwhiletheremainderisreflected.
•Aportionthatispassedundergosimilarpartitionwhenitstrikesthe
secondmetallicfilm

35. •Interference filters are more energy efficient & yield
more pure radiation

36. b)Monochromator:
Amonochromator("singlecolor")isusedtoselectthewavelengthatwhichan
absorptionmeasurementistobemade.
Usedtodispersetheradiationaccordingtowavelength.
Infact,itisnotpracticallypossibletoobtainonewavelength,butrathera
narrowrangeofwavelengths,whichdefinesthespectralresolutionofthe
spectrometer.

37. •Essential components of monochromator are:
•Entrance slit-sharply defines the incoming beam of
heterochromatic radiation
•Dispersing element-disperses heterochromatic radiation into
its component wavelength.
a) Prism b) Diffraction grating
•Exit slit-that allows nominal wavelength together with a band
of wavelength on either side of it.

39. Prism:
•Prism can be used to disperse ultraviolet, visible & IR
radiation. The material used for their construction differs
depending upon the wavelength region.
•Made from glass, quartz or silica
•Glass: used in visible portion, has dispersing
power about 3 times that of quartz or silica
•Quartz or fused silica: Ultraviolet

41. •Theeffectiveseparationofwavelengthdependsondispersivepowerof
theprismmaterial&apicalangleoftheprism.Formostsatisfactorywork,
itis60
o
•Twotypesofmountingoftheprisms;
•Cornutype:whichallowsthelightbeamtopassthroughtheprismin
cornutype,hasapicalangleofprism60
o
Itisadjustedsuchthaton
rotationtheemerginglightisallowedtofallonexitslit.
•Littrowtype:apicalangleofprism30
o
Itsonesurfaceisaluminisedwhichreflectslightbacktopassthroughprism
&toemergeonthesamesideoflightsource.i.elightdoesnotpass
throughtheprismontheotherside.
•In both types of prism, two surfaces of prisms must be very carefully
polished & cleaned.

42. Diffraction Gratings:
•Morerefinednatureofdispersionoflightisobtained.
•Theseconsistsoflargenumberofparallellinesorgroovesabout15000to
30,000perinchareruledonhighlypolishedsurfaceofaluminium.
•Thelinesorgroovesactasscatteringcentresforlightbeamimpingingon
it.
•Lightraysdispersed&exitslitseparateraysofdesiredwavelength.
•Resolvingpowerofgratingdependsonthenumberoflinesruledperinch
onthesurface&increaseswithincreasingnumberoflines.
•Betterresolvingpowerthanprism

44. Grating

46. •Gratingsaredifficulttoprepare,originalgratingsare
expensive.
•Soreplicagratingpreparedfrommastergrating;
•Thisisdonebycoatingthemastergratingwithafilmofepoxy
resin.
•Whentheepoxyresinhashardened&setthereplicaistaken
out,itssurfacemadereflectivebyaluminizing.
•Replicagratingsarelessexpensive

47. Advantage of gratings are:
1.It provides a light of narrow wavelength.
2.There is no loss of energy due to absorption by the
material.
3.Are sturdy & are less affected by water vapours
4.Give linear dispersion but suffer from overlap of
spectral orders.

48. Monochromator
Czerny-Turner design

50. Itiseasiertoachievehighspectralresolutionwithgratings,
buttheyhavethedisadvantageofhavingmorethanone
orderofdiffraction.
Thismeansthatifthemonochromatorissetto600nm
forexample,thenitwillalsopass300nm(secondorder)
radiation.Thisproblemiseasilyovercomebytheuseof
filterstoremovetheunwantedradiation.

51. Atypicalmonochromatordesignisshownbelow-
•Itconsistsofthediffractiongrating(dispersingelement),slits,and
sphericalmirrors.Scanningisaccomplishedbyrotatingthegrating.
•Asthegratingisslowlyrotated,lightofdifferentwavelengthswillemerge
fromtheexitslitandpassthroughthesampletothedetector.Thusthe
spectrumisobtainedsequentiallyasthegratingisrotatedtoselectthe
wavelengthandthedetectorobservesthetransmittedradiationintensity.
Thespectralresolutionofthespectrometercanbevariedbychangingthe
sizeoftheslits

52. Slits: The slits of a monochromator play important role in
determining the monochromator’s performance characteristics
& quality.
i) Entrance slit –The main function is to provide a narrow source
of light so there should be no overlapping of
monochromatic images.
ii) Exit slit-from this exit slit selects a narrow band of dispered
spectrum for observation by the detector.
Both slits have equal width.

53. Sample Holder: Cells
•Thecellsorcuvettesortesttubesareusedforhandlingthe
liquidsamples.
•Thecellsshouldfulfill3mainconditions-
1.Theymustbeuniforminconstruction,thethicknessmust
beconstant(1cm)&surfacesfacingtheincidentlightmust
beopticallyflat.
2.Thematerialofconstructionshouldbeinerttosolvents.
3.Theymusttransmitlightofdesiredwavelength.
Thecellsmayberectangularorcylindricalinshape.
Avarietyofsamplecellsareavailableforuv&visible
wavelengthregion.

55. Choice of cells depend upon
1.Transmission characteristics at desired wavelength:
Pyrex transmits in 320-2500nm range
Silica from 170-2500 nm
1.The path length, shape, size
2.The relative expense.

56. •Forstudyintheultravioletregion-quartzorfusedsilica.(below350nm)
•Silicateglassescanbeusedintheregionbetween350to2000nm
•Plasticcontainersinvisibleregion.
•Therectangulartypesare1cmor4cmininternaldiameter.
•Largersizecellsarepreparedfromgoodqualityglasswithquartzwindow.
Microcellsareemployedforsmallsamples.
•Thecuvetteswithlidareusedforhandlingvolatiletypesolvents&
solutions.
•The sample cell contains a solution of the substance you are testing -
usually very dilute. The solvent is chosen so that it doesn't absorb any
significant amount of light in the wavelength range we are interested in
(200 -800 nm).
•The reference cell just contains the pure solvent.

57. •Thebestcellshavewindowsthatarenormaltothedirectionofbeamin
ordertominimizereflectionlosses.
•Whendouble-beaminstrumentationisusedtwocellsareneeded,onefor
reference&oneforsample.
•Matchedcellsareusedformostaccuratework.
•Thesearecellsinwhichabsorptionofeachoneisequaltoabsorptionof
other.Aremanufacturedatonetime,&theirrespectivesabsorptivities
aremeasured.
•Thesurfacesofabsorptioncellsmustbekeptclean.Nofingerprintsshould
bepresentoncells.
•Cleaningofcellscanbecarriedoutbywashingwithdistilledwaterorwith
dilutealcohol,acetoneordetergentsolutions.

58. •Thequalityofabsorbancedatadependentuponthewaythe
matchescellsareused&maintained.
•Fingerprints,greaseorotherdepositsonthewallsalterthe
transmissioncharacteristicsofacell.Socleaningbefore&after
usedismust.
•Atalltimeswhencellsarenotinuseditshouldbekeptclean&dry.
Anysampleleftinacellwilltendtodryout,causeastainoncell
walls&thiswillleadtoanalyticalerror.Destructionofcells.
•Matchedcellsneverbedriedbyheatinginovenorflame
•Maycausephysicaldamageorachangeinpathlength.