UV Pnnnnbshhxhdhhdhhdhhdhhdhdhhdhhdhdhhdhh

Uv spectroscopy Thyroglobulin is a protein made by the thyroid gland
Tbg is transport protein carrying harmones to tissues
Glycogenolysis : glycogen to glucose
Gluconeogenesis : non hexose precursors glycerol,lactate , pyruvate etc to glucose
Postnatal life :after birth
Type 1: pancreas produces little or no insulin
Thyroglobulin is a protein made by the thyroid gland
Tbg is transport protein carrying harmones to tissues
Glycogenolysis : glycogen to glucose
Gluconeogenesis : non hexose precursors glycerol,lactate , pyruvate etc to glucose
Postnatal life :after birth
Type 1: pancreas produces little or no insulin
Thyroglobulin is a protein made by the thyroid gland
Tbg is transport protein carrying harmones to tissues
Glycogenolysis : glycogen to glucose
Gluconeogenesis : non hexose precursors glycerol,lactate , pyruvate etc to glucose
Postnatal life :after birth
Type 1: pancreas produces little or no insulin
Thyroglobulin is a protein made by the thyroid gland
Tbg is transport protein carrying harmones to tissues
Glycogenolysis : glycogen to glucose
Gluconeogenesis : non hexose precursors glycerol,lactate , pyruvate etc to glucose
Postnatal life :after birth
Type 1: pancreas produces little or no insulin
Thyroglobulin is a protein made by the thyroid gland
Tbg is transport protein carrying harmones to tissues
Glycogenolysis : glycogen to glucose
Gluconeogenesis : non hexose precursors glycerol,lactate , pyruvate etc to glucose
Postnatal life :after birth
Type 1: pancreas produces little or no insulin
Thyroglobulin is a protein made by the thyroid gland
Tbg is transport protein carrying harmones to tissues
Glycogenolysis : glycogen to glucose
Gluconeogenesis : non hexose precursors glycerol,lactate , pyruvate etc to glucose
Postnatal life :after birth
Type 1: pancreas produces little or no insulin
Thyroglobulin is a protein made by the thyroid gland
Tbg is transport protein carrying harmones to tissues
Glycogenolysis : glycogen to glucose
Gluconeogenesis : non hexose precursors glycerol,lactate , pyruvate etc to glucose
Postnatal life :after birth
Type 1: pancreas produces little or no insulin
Thyroglobulin is a protein made by the thyroid gland
Tbg is transport protein carrying harmones to tissues
Glycogenolysis : glycogen to glucose
Gluconeogenesis : non hexose precursors glycerol,lactate , pyruvate etc to glucose
Postnatal life :after birth
Type 1: pancreas produces little or no insulin
Thyroglobulin is a protein made by the thyroid gland
Tbg is transport protein carrying harmones to tissues
Glycogenolysis : glycogen to glucose
Gluconeogenesis : non hexose precursors glycerol,lactate , pyruvate etc to glucose
Postnatal life :after birth
Type 1: pancreas produces little or no insulin
Thyroglobulin is a protein made by the thyroid gland
Tbg is transport protein carrying harmones to tissues
Glycogenolysis : glycogen to glucose
Gluconeogenesis : non hexose precursors glycerol,lactate , pyruvate etc to glucose g

1. UVVISIBLESPECTROSCOPY

2. IntroductiontoElectroMagneticRadiation(EMR)
Lightissupposedtotravelinwaves.LightorEMRisaformofenergythatistransmitted
throughspaceataconstantvelocityof3×10
8
�����/������.Theseradiationsaresaidto
havedualnatureexhibitingboth
Wavecharacter
Particlecharacterorcorpusculartheory
Accordingtowavetheory,lighttravelsintheformofwaves.Thiswavemotionconsistsof
oscillatingelectric(E)&magnetic(H)fields(vectors)directedperpendiculartoeachother&
perpendiculartothedirectionofthepropagationofwave(Fig.1).

3. Fig.1:Wavenatureoflight

4. Anemissionspectrumisproducedbytheemissionofradiantenergybyanexcitedatom.Theexcitationof
atomscanbebroughtaboutthermally(byheatingthesubstancestrongly)orelectrically(bypassingelectric
dischargethroughthevapoursofthesubstanceataverylowpressure).Whenanelectricdischargeispassed
throughthevapoursofthesubstance,energyisabsorbed&electronsinthegroundstatearepromotedto
Meta-stablestates.WhenelectronsfromtheMeta-stablestatejumptothelowerenergystate,thensome
energyofdefinitefrequencyisreleasedasradiation.
Ifthisradiationemittedisanalyzedwiththehelpofaspectroscope,anemissionspectrumisobserved.

5. Wavelength: It is the distance between the adjacent crests or troughs in a particular wave. It is denoted by ‘λ’
(lambda). It can be expressed in Angstrom or nanometer (nm) or millimicrons(mμ) or centimeter (cm) or
micrometer (μm).
1 nm = 10
-9
m = 10
-3
μm
Nanometer is frequently used in UV-Visible technique.
Wave Number: It is the reciprocal of wavelength & it is expressed in per centimeter; or it is defined as the total
number of waves which can pass through a space of 1 cm. It is expressed as
‘ū (nu bar)’. It is frequently used in IR technique.
Characteristics/Units of wave (Fig.2):

7. QuantumtheorydescribestheEMRasconsistingofastreamofenergypackets,calledPhotonsorQuanta,which
travelinthedirectionofpropagationofthebeamwiththevelocityoflight.
Thus,duringemissionorabsorptionoflightbychemicalspecies,theenergychangestakeplaceonlydiscretely
alwaysasintegralmultiplesofsmallunitsofenergyi.e.photon.
Theenergyofthephotonisproportionaltothefrequencyofradiation,i.e.Eαν,or,E=hν,Where,h=Plank’s
constant=6.626x10
-27
erg.sec.
Theenergyofaphotoniscalledquantumofenergy&thisdependsonlyonthefrequencybutnotontheintensityof
radiation.
Thewavesarecharacterizedbytheirwavelengthsorfrequenciesorwavenumbers.
TheenergycarriedbyanEMRisdirectlyproportionaltothefrequency.
Alltypesofradiationstravelwiththesamevelocity&nomediumisrequiredfortheirpropagation.Theycantravel
throughvacuumalso.
Whenvisiblelight(agroupofEMR)ispassedthroughaprism,itissplitupintosevencolourswhichcorrespondto
definitewavelengths.Thisphenomenoniscalled‘dispersion’.
Quantum theory of EMR

8. Thewordspectroscopyisderivedfromspectrumwhichmeansabandofdifferentcoloursformeddueto
differenceinwavelengthandskopinmeansexaminationorevaluation.Thus,spectroscopyisthebranchof
sciencethatdealswiththeexaminationorevaluationofspectrum.
Itisdefinedastheinteractionbetweenthematter&EMR.Itdealswithemissionaswellasabsorption
spectra.
Itisusedtomeasuretheenergydifferencebetweenvariousmolecularenergylevels&todeterminetheatomic
&molecularstructures.Theinstrumentsusedinsuchstudiesarecalledspectrophotometer.
IfEMR(ofcertainwavelengthrange)arepassedthroughthesubstanceunderanalysisforsometimes,then
radiationsofcertainwavelengthsareabsorbedbythesubstance.Thewavelengthswhichareabsorbed
characterizesomeParticularfunctionalgroupspresentinthecompoundorthecompounditself.This
darkpatternoflineswhichcorrespondstothewavelengthsabsorbsiscalledAbsorptionspectrum.After
absorption,thetransmittedlightisanalyzedbythespectrometerrelativetotheincidentlightofagiven
frequency.Theabsorbedenergymayheatupthesampleorisre-emitted.
Spectroscopy

9. Wavelength: It is the distance between the adjacent crests or troughs in a particular wave. It is denoted by ‘λ’
(lambda). It can be expressed in Angstrom or nanometer (nm) or millimicrons(mμ) or centimeter (cm) or
micrometer (μm). 1 nm = 10
-9
m = 10
-3
μm. Nanometer is frequently used in UV-Visible technique.
Wave Number: It is the reciprocal of wavelength & it is expressed in per centimeter; or it is defined as the
total number of waves which can pass through a space of 1 cm. It is expressed as ‘ū (nu bar)’. It is frequently
used in IR technique.
Characteristics/Units of wave (Fig.2)

10. Electromagneticspectrum:
WhenamoleculeabsorbsEMR,itcanundergovarioustypesofexcitation.This
excitation maybe
•Electronicexcitation
•Rotationexcitation
•Excitationleadingtoa changeinnuclearspin
•Excitationresultingin bond deformation&so on.

11. Iftheenergyavailableapproachestheionizationpotentialofthemolecule,anelectronmay
beejected&ionization mayoccur.
Sinceeachmodeofexcitationrequiresaspecificquantityofenergy,thedifferent
absorptions appearin differentregionsof the electromagneticspectrum.
Thevariousregionsofelectromagneticspectrumaresetout(Table:1&Fig.3)&arelabeled
eitheraccordingtothewavelength/waveno.rangeused,oraccordingtothetypeofthe
molecularenergylevelsinvolved,e.g.UV(electronic)spectra,IR(vibrational)spectraor
RF(NMR)spectra.

13. Table-1:Variousregionsofelectromagneticspectrum
Typeof
Radiation Wavelength Typeofmolecularspectrum
RF > 100 mm NMR (Spin orientation)
Microwave 1–100mm Rotational
FarIR 50μm–1mm Vibrationalfundamentalorrotational
MidIR 2.5μm–50μm Vibrationalfundamental
NearIR 780nm–2.5μm Vibrational(overtones)
Visible 380nm–780nm Electronic(valenceorbital)
NearUV 200nm–380nm Electronic(valenceorbital)
VacuumUV 10nm–200nm Electronic(valenceorbital)
X-rays 10pm–10nm Electronic (coreorbitals)
Gammarays 10
-10
cm Mossbauereffect(Nucleartransitions)
excitedstatesofnuclei
Cosmicrays 10
-12
cm

15. Fig.2:Characteristicsofwave

16. Electromagneticspectrum:
Theelectromagneticspectrum,formostspectroscopicpurposes,isconsideredtobe
consisting ofregionof radiantenergyranging fromwavelengths of 10 mto 1 x12
-12
cm.
WhenamoleculeabsorbsEMR,itcanundergovarioustypesofexcitation.This
excitation maybe
Electronicexcitation,
Rotationexcitation,
Excitationleadingtoa changeinnuclearspin,
Excitationresultingin bond deformation&so on.

17. Iftheenergyavailableapproachestheionizationpotentialofthemolecule,anelectronmay
beejected&ionization mayoccur.
Sinceeachmodeofexcitationrequiresaspecificquantityofenergy,thedifferent
absorptions appearin differentregionsof the electromagneticspectrum.
Thevariousregionsofelectromagneticspectrumaresetout(Table:1&Fig.3)&arelabeled
eitheraccordingtothewavelength/waveno.rangeused,oraccordingtothetypeofthe
molecularenergylevelsinvolved,e.g.UV(electronic)spectra,IR(vibrational)spectraor
RF(NMR)spectra.

18. Table-1:Variousregionsofelectromagneticspectrum
Typeof
Radiation Wavelength Waveno. Typeofmolecularspectrum
RF > 100 mm < 3 x 109 Hz NMR (Spin orientation)
Microwave 1–100mm 10–0.1cm
-1
Rotational
FarIR 50μm–1mm 200–10cm
-1
Vibrationalfundamentalorrotational
MidIR 2.5μm–50μm 4000–667cm
-1
Vibrationalfundamental
NearIR 780nm–2.5μm (13–4)x10
3
cm
-1
Vibrational(overtones)
Visible 380nm–780nm (2.6–1.3)x10
4
cm
-1
Electronic(valenceorbital)
NearUV 200nm–380nm (5–2.6)x10
4
cm
-1
Electronic(valenceorbital)
VacuumUV 10nm–200nm (10
2
–5)x 10
4
cm
-1
Electronic(valenceorbital)
X-rays 10pm–10nm 10
9
-10
6
cm
-1
Electronic (coreorbitals)
Gammarays 10
-10
cm
10
10
cm-1
Mossbauereffect(Nucleartransitions)
excitedstatesofnuclei
Cosmicrays 10
-12
cm
10
12
cm-1

19. UVwavelengthregionrangesfrom200nm–to380nm,whereasVisiblewavelengthregion
rangesfrom380nmto780nm.
ItisalsoknownasElectronicspectroscopysinceitinvolvesthepromotionofelectrons(σ,π,n-
electrons)fromthegroundstatetothehigherenergystate.
Itveryusefultomeasurethenumberofconjugateddoublebonds&alsoaromaticconjugation
withinthevariousmolecules.
Itdistinguishesbetweenconjugated&non-conjugatedsystems,α,β-unsaturatedcarbonyl
compoundsfromβ,γ-analogues,homoannular&heteroannularconjugateddienes,etc.
UV-Visible Spectroscopy

20. Ultraviolet(UV)andvisibleradiationareasmallpartoftheelectromagneticspectrum,whichincludes
otherformsofradiationsuchasradio,infrared(IR),cosmic,andXrays.

21. Whenradiationinteractswithmatter,severalprocessescanoccur,includingreflection,scattering,
absorbance,fluorescence/phosphorescence(absorptionandre-emission),andphotochemicalreactions
(absorbanceandbondbreaking).Typically,whenmeasuringsamplestodeterminetheirUV-visible
spectrum,absorbanceismeasured.Becauselightisaformofenergy,absorptionoflightbymatter
causestheenergycontentofthemolecules(oratoms)inthemattertoincrease.Thetotalpotentialenergy
ofamoleculeisrepresentedasthesumofitselectronic,vibrational,androtationalenergies:
UV-visible spectra
E
total= E
electronic+ E
vibrational+ E
rotational
Theamountofenergyamoleculepossessesineachformisnotacontinuumbutaseriesofdiscretelevels
orstates.Thedifferencesinenergyamongthedifferentstatesareintheorder:
E
electronic> E
vibrational> E
rotational

23. Thesetransitionsresultinverynarrowabsorbancebandsatwavelengthshighlycharacteristicofthe
differenceinenergylevelsoftheabsorbingspecies.Thisisalsotrueforatoms,asdepictedinFigure3.
Figure3.Incidentlightofaspecificwavelengthcausesexcitationofelectronsinanatom.Thetypeof
atomandtheenergylevelstheelectronismovingbetweendeterminesthewavelengthofthelight
thatisabsorbed.Transitionscanbebetweenmorethanoneenergylevel,withmoreenergyi.e.lower
wavelengthsoflight,requiredtomovetheelectronfurtherfromthenucleus.

24. Principle
Sincetheenergylevelsofamoleculearequantized,theenergyrequiredtobringabouttheexcitationisafixed
quantity.Thus,theEMRwithonlyaparticularvalueoffrequencywillbeabletocauseexcitation.Ifthe
substanceisexposedtoradiationofsomedifferentvalueoffrequency,energywillnotbeabsorbed&thus,light
radiationwillnotsufferanylossinintensity.
WhenUVorVisibleradiationispassedthroughasubstance,absorptionofenergyresultsinthepromotionof
electronfromthegroundelectronicstatetotheexcitedelectronicstate.Theamountofabsorptionofenergy
dependsuponwavelengthoftheradiation&thestructureofcompound
Duringtheprocessofabsorption,alargenumberofphoton-moleculecollisionsarepossiblebutonlythose
collisionswillcauseabsorptionofenergyinwhichtheenergyofthephotonmatchestheenergydifference
betweentheground&theexcitedelectronicstateofthemolecule.Theabsorptionofenergyisquantized.
Thewavelengthatwhichmaximumabsorptionofradiationtakesplaceiscalledλ
maxThisλ
maxischaracteristic
oruniqueforeverysubstance&thisisaqualitativeaspect,usefulinidentifyingthesubstance(Fig.4).

26. Purpose to measure UV/VIS spectra
There are various main reasons to measure UV/VIS spectra
UV/VISspectraallowcomponentspresentinthesamplesolutiontobeidentified.Moreprecisely,
thepositionand,tosomeextent,theprofileoftheabsorptionpeaksallowspecificcompoundstobe
identified.Forexample,organiccompoundscanbeidentifiedbytheirspectra,orsolventpuritycan
beeasilycheckedbyUV/VISspectroscopy
Absorptionpeakscanbeusedtoquantifytheinvestigatedsample.Forexample,thesample
concentrationcanbecalculatedfromtheabsorbancevalueofthepeak:
Basedontherelationshipbetweenabsorbanceandsampleconcentration,UV/VISspectroscopy
isappliedasaquantitativeanalyticaltechniqueinmarketsegmentssuchase.g.WaterTesting,Food
andBeverages,Pharmaceutical,ChemicalandBiotechIndustry.

27. Thepositionofthepeaksinthespectrumrevealsinformationaboutthemolecularstructureofthe
sample.Forexample,specificfunctionalgroupsofamolecularstructure,suchascarbon-oxygen,
C=O,orcarbon-carbondoublebonds,C=C,absorbatspecificcharacteristicwavelengths.
Thespectrummayrevealspecificphysicalpropertiesofthesamplemolecules.Forinstance,fromthe
UV/VISspectrumitispossibleto:–calculatetheextinctioncoefficientofthesample–calculatethe
meltingpointofproteinsandnucleicacidsbymeasuringtheUV/VISspectraatdifferent
temperatures–determinetherateofareactionbymonitoringtheabsorptionspectraasafunctionof
time(alsoknownaskineticmeasurements).
Finally,positionandprofileofthepeaksinthespectrumcangiveinformationaboutthemicroscopic
environmentofthesamplemolecules.Asanexample,thepresenceofimpuritiesorothersolventsin
thesamplesolutionhasaneffectonpositionandoftheprofileofthepeaks.Inotherwords,thepeaks
maybebroaderorhaveshiftedduetoimpurities.

28. Electronic transitions
WhenthemoleculeabsorbsUVorvisiblelight,itselectrongetspromotedfromthegroundstate
tothehigherenergystate.
Inthegroundstate,thespinsoftheelectronsineachmolecularorbitalareessentiallypaired.
Thehigherenergystatesaredesignatedashighenergymolecularorbitals&alsocalledasanti-
bondingorbitals.
Thehigherprobabletransitionduetoabsorptionofquantizedenergyinvolvesthepromotionofone
electronfromthehighestoccupiedmolecularorbital(HOMO)tothelowestavailableunfilled
molecularorbital(LUMO).
Thehigheristheenergygap,theloweristhewavelengthofthelightabsorbed

30. Types of Electronic Transitions
Accordingtothemolecularorbitaltheory,whenamoleculeisexcitedbytheabsorptionofenergy
(UVorvisiblelight),itselectronsarepromotedfromabondingorbitaltoananti-bondingorbital.
Thereareseveraltypesofelectronictransitionsavailabletoamoleculeincluding:
Theanti-bondingorbitalwhichisassociatedwiththeexcitationofσ-electroniscalledσ*-anti-bonding
orbital.σ→σ*transitiontakesplacewhenσ-electronpromotedtoanti-bonding(σ*)orbital.
Whenanon-bondingelectron(η)getspromotedtoananti-bondingsigmaorbital(σ*),thenitrepresents
η→σ*transition.
Similarly,π→π*transitionrepresentsthepromotionofπ-electronstoananti-bondingπ-orbital,(π*-
orbital)
Similarly,whenaη-electron(non-bonding)ispromotedtoananti-bondingπ-orbital,itrepresentsη→π*
transition.

31. Theenergyrequiredforvarioustransitionsobeythefollowingorder:
σ→σ*>η→σ*>π→π*>η→π*

32. Let us consider various transitions involved in UV spectroscopy:
a.σ→σ*transitions
Itisahighenergyprocesssinceα-bondsareverystrong.
Itisobservedwithsaturatedcompounds(especiallyhydrocarbons),inwhichallthevalenceshells
electronsareinvolvedintheformationofsigmabondsdonotshowabsorptioninthenormalUV
region,i.e.120nm–180nm.e.g.methane,ethane,propane,cyclopropane,etc.
Itrequiresradiationofveryshortwavelength.

33. b. η → σ* transitions
It occurs in saturated compounds containing one hetero atom with unshared pair of electrons
(η-electrons).
E.g. saturated halides, alcohols, ethers, amine, etc.
It requires comparatively less energy than that required for σ → σ* transitions.
In saturated alkyl halides, the energy required for such a transition decreases with the increase in size
of the halogen atom (or decrease in the electro-negativity of the atom)

34. c. π → π* transitions
This type transitions occur in the unsaturated centers of the molecule; i.e. in compounds
containing double or triple bonds & also in aromatics.
Absorption usually occurs within the region of ordinary UV-spectrophotometer.
The excitation of π-electron requires smaller energy & hence, transition of this type occurs at longer
wavelength.
A π-electron of a double bond is excited to π*-orbital. E.g. alkenes, alkynes, carbonyl compounds,
cyanides, azo compounds, etc.
This transition requires still lesser energy as compared to η→σ* transition.

35. d.η→π*transition
Inthistype,anelectronofunsharedelectronpaironheteroatomgetsexcitedtoπ*-antibonding
orbital.
Thistypeoftransitionrequiresleastamountofenergyoutofallthetransitions,&henceoccursat
longerwavelength.
Insimplecases,itisquiteeasytotellwhetherthetransitionisη→π*orπ→π*sincetheexcitation
coefficientfortheformerisquitelowascomparedtothatoflater.
Theexactelectronicstructureofthemoleculesintheexcitedstateisnotknownbuttheelectronic
transitioninvolvestheredistributionofelectronswithinthemolecule.E.g.aldehydes,ketones,nitro
compounds,etc.containbothη→π*&π→π*transitions.

36. InUVspectroscopy,thesampleisirradiatedwiththebroadspectrumoftheUVradiationIfa
particularelectronictransitionmatchestheenergyofacertainbandofUV,itwillbeabsorbed
TheremainingUVlightpassesthroughthesampleandisobservedFromthisresidualradiationa
spectrumisobtainedwith“gaps”atthesediscreteenergies–thisiscalledanabsorptionspectrum
The Spectroscopic Process

37. Fromthemolecularorbitaldiagram,thereareseveralpossibleelectronictransitionsthatcanoccur,each
ofadifferentrelativeenergy
Observed electronic transitions

38. Observed electronic transitions
AlthoughtheUVspectrumextendsbelow100nm(highenergy),oxygenintheatmosphereisnot
transparentbelow200nm
SpecialequipmenttostudyvacuumorfarUVisrequired
RoutineorganicUVspectraaretypicallycollectedfrom200-700nm

39. PRESENTATION OF SPECTRA
Theultraviolet–visiblespectrumisgenerallyrecordedasaplotofabsorbanceversuswavelength.

40. PRINCIPLE
Basic principle of spectroscopyis the Beer-Lambert’s law
1.2.1 BEER LAW
•Beer's law stated that absorbanceis proportional to the concentrationsof the material sample.
1.2.2 LAMBERT LAW
•Lambert's law stated that absorbanceof a material is directly proportional to its thickness(path
length).

41. ThemodernderivationoftheBeer–Lambertlawcombinesthetwolawsandcorrelatestheabsorbance
toboththeconcentrationsandthethicknessofthematerial.

42. formostUVspectrometers,lwouldremain constant(standardcellsaretypically1cmin pathlength)
ConcentrationC istypicallyvarieddependingonthestrengthof absorptionobserved
molarabsorptivitiesεvarybyordersof magnitude:
A =ε C.L
Themolarabsorptivity(formerlycalledtheextinctioncoefficient)ofacompoundisaconstantthatis
characteristicofthecompoundataparticularwavelength

43. Transmittance (T)is the fraction of incident light which is transmitted.
In other words, it’s the amount of light that “successfully” passes through the substance and comes
out the other side.
It is defined as T = I/Io,
where I = transmitted light (“output”) and Io = incident light (“input”).
%T =(I/Io) x 100.

44. Principles and instrumentation for UV-Vis-IR
Ultraviolet(UV)spectroscopyisanimportantphysicaltoolwhichexploitslightinultraviolet,visible,and
nearinfraredrangeofelectromagneticspectrum.
Beer-Lambertlawestablishesalinearrelationshipbetweenabsorbance,concentrationofabsorbers(or
absorbingspecies)inthesolutionandthepathlength.
Therefore,UV-Visspectroscopycanbeemployedfordeterminingtheconcentrationoftheabsorbingspecies,
forafixedpathlength.Thisisaverysimple,versatile,fast,accurateandcosteffectivetechnique.
Instrumentemployedforultraviolet−visible(orUV-Vis)spectroscopyiscalledUV−Vis−NIR
Spectrophotometer.Thiscanbeusedtoanalyzeliquids,gasesandsolidsbyusingradiativeenergy
correspondingtofarandnearultraviolet(UV),visible(Vis)andnearinfrared(NIR)regionsofelectromagnetic
spectrum.Consequently,predeterminedwavelengthsintheseregionshavebeendefinedas:UV:300-400nm;
Vis:400-765nm;andNIR:765-3200nm.

45. INSTRUMENTATION
Source of light.
Monochromator.
Sample solution in cuvette.
Photo detector.
Readout device.

46. Source of Light
PartoftheUVandVisibleradiationsourceisTungstenlamp.
UVradiationsourceisDeuteriumorHydrogenlamp.
Rangeofwavelength200-400nm.
Tungsten lamp
Deuterium lamp

47. MONOCHROMATOR
It is a device that breaks the polychromatic radiation into component wavelengths.

48. The monochromatorunit consists of :
Entrance slit: defines narrow beam of radiation from source.
Collimating mirror:(polished surface) collimates the lights.
Diffraction grating or Prism (make of quartz): disperses the light into specific wavelength.
Focusing mirror: captures the dispersed light & sharpens the same to the sample via exit slit
Exit slit: allows the corrected wavelength of light to the sample .

49. Sample solution in cuvette
liquid sample is usually contained in a cell called a cuvette.
Fingerprints and droplets of water disrupt light rays during measurement.
Cuvette from Quartz can be used in UV as well as in visible spectroscopy.
Cuvette from Glass is suitable for visible but not for UV spectroscopy because it absorbs UV radiation.
Sample solution in cuvette

50. PHOTO DETECTOR
Aphotodetectorisasemiconductordevicewhichconvertslightenergytoelectricalenergy.
ItconsistsofasimpleP-Njunctiondiodeandisdesignedtoworkinreversebiasedcondition.The
photonsapproachingthediodeareabsorbedbythephotodiodeandcurrentisgenerated.4Seefigure
11.

51. READOUT DEVICE
Digital screen to record an uvspectrograph with absorbance against the wavelength.
TYPES of SPECTROPHOTOMETER

52. Althoughtheabsorptionofultravioletradiationresultsfromtheexcitationofelectronsfromground
toexcitedstates,thenucleithattheelectronsholdtogetherinbondsplayanimportantrolein
determiningwhichwavelengthsofradiationareabsorbed.
Thenucleideterminethestrengthwithwhichtheelectronsareboundandthusinfluencetheenergy
spacingbetweengroundandexcitedstates.Hence,thecharacteristicenergyofatransitionandthe
wavelengthofradiationabsorbedarepropertiesofagroupofatomsratherthanofelectrons
themselves.
Thegroupofatomsproducingsuchanabsorptioniscalledachromophore.Asstructuralchanges
occurinachromophore,theexactenergyandintensityoftheabsorptionareexpectedtochange
accordingly.
The Chromophore
Terms used in Spectroscopy

53. Allthosecompoundswhichabsorblightofwavelengthbetween400-800nmappearcoloredtothe
humaneye.Exactcolordependsuponthewavelengthoflightabsorbedbythecompound.
Originally,achromophorewasconsideredanysystemorunsaturatedatoms/groups,whichis
responsibleforimpartingcolortothecompound.
E.g.Nitrogroupgivesyellowcolor,arylconjugatedazogroupgivesazodyescolor.
Chromophore/chromogenicagent
Chromophoreisdefinedasanyisolatedcovalentlybondedgroupthatshowsacharacteristic
absorptionintheultravioletorthevisibleregion,OR,
Chromophoreisdefinedasanygroupwhichexhibitsabsorptionofelectromagneticradiationsin
thevisibleorultravioletregion.
AchromophoreisthatpartofamoleculethatabsorbsUVorvisiblelight

54. The absorption occurs irrespective of the fact whether color is produced or not. Some important
chromophores are
Ethylenic, acetylenic, carbonyls, acids, esters, nitrile groups, etc.
A carbonyl isolated group does not produce any color in the UV spectroscopy by absorbing light
Chromophores -
in which the group contains π-electrons & they undergo π→π* transitions, e.g. ethylenes, acetylenes,
etc.
which contain both π-electrons & η-electrons, such chromophores undergo two types of transitions
i.e. π→π* & η→π*; e.g. carbonyls, nitriles, azo compounds, nitro compound, etc.

55. Auxochrome
An auxochromecan be defined as any group which does not itself acts as a chromophore but whose
presence brings about a shift of the absorption band towards the red end of the spectrum (longer
wavelength).
These are covalently saturated groups with lone pair of electrons.
The absorption at longer wavelength is due to the combination of a chromophore & an auxochrome
to give rise to another chromophore.
An auxochromicgroup is called as color enhancing group.
Auxochromicgroup do not show characteristic absorption above 200 nm. Some common
auxochromicgroups are –OH, -OR, _NH
2, -NHR, -NR
2, -SH, etc.

56. Theeffectoftheauxochromeisduetoitsabilitytoextendtheconjugationofachromophorebythesharing
ofnon-bondingelectrons.Thus,anewchromophoreresultswhichhasadifferentvalueoftheabsorption
maximumaswellastheextinctioncoefficient.
E.g.Incaseofanilineabsorptionmaximumtakesplaceat280nmbecausethepairofelectronsonnitrogen
atomisinconjugationwiththeπbondsystemofthebenzenering.
(-NH
2)aminogroupisanauxochrome.Itinvolvestheshiftofabsorptionmaximumtowardslongerwavelength.
Allauxochromicgroupscontainnon-bondingelectrons.Duetothis,thereisextensionofconjugationofthe
chromophorebysharingthenon-bondingelectrons

57. Consider trans—Azobenzeneand trans-p-ethoxyazobenzene
The presence of —OC
2H
5group (an auxochrome) increases the value of λ
max as well as ε
max

58. Spectral shifts/Absorption and Intensity Shifts
Changesinchemicalstructureortheenvironmentleadtochangesintheabsorptionspectrumof
moleculesandmaterials.Thereareseveraltermsthatarecommonlyusedtodescribetheseshifts,
Bathochromic effect,
Hypsochromiceffect,
Hyperchromiceffect,
Hypochromic effect

59. a.Bathochromicshiftn(redshift)—ashifttolowerenergyorlongerwavelength
Theeffect,byvirtueofwhichtheabsorptionmaximumisshiftedtowardslongerwavelengthdueto
thepresenceofanauxochromeorbythechangeofsolvent,iscalledasBathochromicshiftorRed
shift.
Theabsorptionsoftwoormorechromophoreswhichareseparatedbymorethanonebondare
usuallyadditive,butwhenchromophoresareconjugated,i.e.separatedbyasinglebond,
pronouncedeffectsareproduced.Themaximumabsorptionisshiftedtolongerwavelengths.

60. Forexample,
Benzeneshowsλ
max256nmandanilineshowsλ
max280nm.Thus,thereisabathochromicshiftof24
nmintheλ
maxofbenzeneduetothepresenceoftheauxochromeNH
2.
Similarly,abathochromicshiftofntoπ*bandisobservedincarbonylcompoundsondecreasing
solventpolarity,e.g.λ
maxofacetoneisat264.5nminwaterascomparedto279nminhexane.

62. b.Hypsochromicshift(blueshift)—ashifttohigherenergyorshorterwavelength.
Itisaneffectbyvirtueofwhichtheabsorptionmaximumisshiftedtowardsshorterwavelength.
TheabsorptionshiftedtowardsshorterwavelengthiscalledBlueshiftorHypsochromicshift.
Itmaybecausedbytheremovalofconjugation&alsobychangingthepolarityofthesolvent.

63. E.g.anilinehasλ
max280nm,becausethepairofelectronsonnitrogenatomisinconjugationwiththeπ-
bondsystemofthebenzenering.Butinacidicmedium,anilinebehavesasC
6H
5-NH
3
+
(aniliniumion)
astheelectronpairisnolongerpresent&henceconjugationisremoved,whichcausesblueshift&
absorptionoccursatshorterwavelength

64. c. Hyperchromicshift: an increase in intensity.
It is an effect due to which the intensity of absorption maximum increases, i.e. ε
maxincreases.
E.g. Pyridine => λ
max= 257 nm, ε
max= 2750
2-methyl pyridine => λ
max= 262 nm, ε
max= 3560
The introduction of an auxochromeusually increases intensity of absorption.

65. d. Hypochromic shift:
It is an effect due to which the intensity of absorption maximum decreases i.e. extinction
coefficient, ε
maxdecreases.
The introduction of group which distorts the geometry of the molecule causes hypochromic effect.
E.g. Biphenyl => λ
max= 250 nm, ε
max= 19000
2-methyl biphenyl => λ
max= 237 nm, ε
max= 10250
It is due to the distortion caused by the methyl group in 2-methyl biphenyl.

66. Choice of Solvent used and effect of solvent on λmax:
A solvent is a liquid that dissolves another solid, liquid, or gaseous solute, resulting in a solution at
specified temperature.
The solvent use should be high purity, generally referred to as ‘spectrograde’.
A good solvent should be transparent over the desired range of wavelengths. Usually solvents which
do not contain conjugated system are most suitable for running the UV spectrum.
Commonly used solvents are water, 95% ethanol, n-hexane, cyclohexane.
A solvent should be chosen so that it does not react chemically with the sample.
Solvents can be broadly classified into two categories:
Polar
Non-Polar

67. Thesolventexertsaprofoundinfluenceonthequalityandshapeofspectrum.
Polarityplaysanimportantroleinthepositionandintensityofabsorptionmaximumofa
particularchromophore.
E.g.Incaseofnon-polarsolventse.g.Iodinesolution(purplecolor)theabsorptionmaximaoccurat
almostthesamewavelengthasiniodinevapour(518nm),
whereasincaseofpolarsolvents,abrownishcolorisobtainedinsteadofpurplecolor,becausethe
absorptionoccursatshorterwavelengths.
Solvent effects

68. Theabsorptionmaximumforthepolarcompoundsisusuallyshiftedwiththechangeinthe
polarityofthesolvents.
Case1
Ifthechromophoreinvolvedinthetransitionismorepolarinitsgroundstatethaninitsexcited
state,thenthegroundstateismorestabilizedthantheexcitedstatebyamorepolarsolventdueto
solvation.
Chromophoreswithn→π*transitionsexhibitsuchbehavior.
Thesolventmoleculesareorientedaroundthesolute(chromophore)moleculestofitwiththeground
statechargedistributionofthesolutemolecules.
Hydrogenbondingorpolarsolventsinteractmorestronglywithunsharedelectronpairsofthe
groundstatemolecule.
Onexcitation,thechargedistributioninsuchsystemschangesmarkedlyandtherefore,thesolvent
moleculeswouldnothavepositionandorientationtointeractwiththeexcitedstatechargedistribution

69. Thus,thegroundstateofsuchsolutemoleculesismorestabilizedthantheexcitedstate.Thiswidens
theenergygapbetweenthegroundandexcitedstateswithincreasingpolarityofthesolvents
(Fig.9).
Therefore,moreenergyisrequiredforthen→π*kindofelectronictransitionwithincreasing
solventpolarity.Thisresultsintheshiftofspectralpeakpositionstowardsshorterwavelength.
Case2
Ontheotherhand,iftheexcitedstateofthechromophoreismorepolarwithrespecttothe
groundstate,thentheexcitedstatewillbemoresolvatedandmorestabilizedbyamorepolar
solvent.Thiskindofpropertyisobservedinthecasechromophoreswithπ→π*transitions.

71. Thisfavorsstrongerinteractionoftheexcitedstatemoleculewithmorepolarorhydrogen
bondingsolventsandtherebystabilizingtheexcitedstatemorethanthegroundstate.
Thisdecreasestheenergygapbetweentheexcitedandthegroundstateswithincreasingsolvent
polarity(Fig.9)whichresultsinshiftofabsorptionpeakpositionstowardslongerwavelengths.
Wemayrecallthatashiftoftheabsorptionpeakposition(λmax)towardsshorterwavelengths
iscalledablueshiftorhypsochromiceffect.
Ontheotherhand,ashiftoftheλmaxtowardslongerwavelengthistermedastheredshiftor
bathochromiceffect.
Whenthereisanincreaseintheabsorptionintensity,(i.e.,absorbance)theeffectistermedas
hyperchromiceffect.
Ifthereisadecreaseintheabsorptionintensity,theeffectistermedashypochromiceffect.

72. Inshort,π*orbitalsaremorestabilizedbyhydrogenbondingwithpolarsolventslikewater&
alcohol.Itisduetogreaterpolarityofπ*orbitalscomparedtoπ-orbital.Thus,smallenergywillbe
requiredforsuchatransition&absorptionshowsaredshift.
Ifthegroup(carbonyl)ismorepolaringroundstatethanintheexcitedstate,thenincreasingpolarity
ofthesolventstabilizesthenon-bondingelectroninthegroundstateduetohydrogenbonding.Thus,
absorptionisshiftedtoshorterwavelength.
Ifthegroupismorepolarinexcitedstate,thenabsorptionisshiftedtolongerwavelengthwith
increaseinpolarityofthesolventwhichhelpsinstabilizingthenon-bondingelectronsintheexcited
state.
Increaseinpolarityofsolventsshiftsη→π*&η→σ*toshorterwavelength
Increaseinpolarityofsolventsshiftsπ→π*tolongerwavelength.

74. Applications of Electronic Spectroscopy in Predicting Absorption Maxima of Organic Molecules
Conjugated Dienes, Trienesand Polyenes
ThepresenceofconjugatedoublebonddecreasestheenergydifferencebetweenHOMOandLUMOof
resultingdiene.Thefigure6showsthechangeinenergyofMOonconjugation.Asaresult,the
radiationsoflongerwavelengthareabsorbed.Theconjugationnotonlyresultsinbathochromicshift
(longerwavelength)butalsoincreasestheintensityofabsorption.Asthenumberofconjugateddouble
bondsisincreased,thegapbetweenhighestoccupiedmolecularorbital(HOMO)andlowestunoccupied
molecularorbital(LUMO)isprogressivelylowered.Therefore,theincreaseinsizeoftheconjugated
systemgraduallyshiftstheabsorptionmaximum(λmax)tolongerwavelengthandalsoincreasesthe
absorption.Forexample,ethyleneabsorbsa175nm(ε=1000)andtheconjugationinbutadienegives
astrongabsorptionatlongerwavelengthat230nmandwithhigherintensity(ε=>1000)