Uv vis spectroscopy- part-1

UV visible spectroscopy

1. UV Visible Spectroscopy
Part-1
Dr. Priyanka S. Chaudhari

2. UV-Visible absorption spectroscopy
Contents:
Introduction to Spectroscopy
Basic Principle of UV-Vis spectroscopy
Origin & theory of UV spectra
Type of Electronic transitions
Fundamental Laws of absorption
Instrumentation
Applications

4. Basic principles:
•Ultraviolet (UV) and visible radiation comprise only a
small part of the electromagnetic spectrum
•The energy associated with electromagnetic
radiation is defined by the following equation:
E = hν
where E = energy (in joules),
h = Planck’s constant (6.62 ×10-34 Js), and
ν= frequency (in seconds).

5. Electromagneticradiationcanbeconsideredacombination
ofalternatingelectricandmagneticfieldsthattravel
throughspacewithawavemotion.Becauseradiationacts
asawave,itcanbeclassifiedintermsofeitherwavelength
orfrequency,whicharerelatedbythefollowingequation:
C=ν×λ
where n is frequency (in seconds), c is the speed of light
(3 ×108 ms-1), and l is wavelength (in meters).
In UV-visible spectroscopy, wavelength usually is expressed
in nanometers (1 nm = 10
-9
m).
radiation with shorter wavelength = higher energy.
In UV-visible spectroscopy,
The low-wavelength UV light = the highest energy.

8. •Ultraviolet light: 10 to 400 nm
Near UV region: 200-400 nm
Far UV region : below 200 nm
•Visible light: 400 to 800 nm
Ultraviolet/visible spectroscopy involves the
absorption of ultraviolet light by a molecule causing
the promotion of an electron from a ground
electronic state to an excited electronic state.

9. Electronic excitation spectroscopy:
The spectroscopy which utilizes the ultraviolet
(UV) and visible (VIS) range of electromagnetic
radiation, is frequently referred to as Electronic
Spectroscopy.
Photon absorption promotes an electron from
its ground state to an excited state.

11. Origin of UV-visible spectra
•When radiation interacts with matter, a number of
processes can Occur.
•absorption of radiation by matter causes the energy
content of the molecules or atoms to increase.
•The total potential energy of a molecule generally is
represented as the sum of its electronic, vibrational,
and rotational energies
E
total= E
electronic+ E
vibrational+ E
rotational
E
electronic> E
vibrational> E
rotational

13. Fig: Electronic transitions & UV-Visible spectra in molecule

16. Origin of absorption
•Valence electrons can generally be found in one of
three types of electron orbital:
1. single, or σ, bonding orbitals;
2. double or triple bonds (π bonding orbitals);
3. non-bonding orbitals(lone pair electrons).
•Sigma bonding orbitalstend to be lower in energy
than π bonding orbitals, which in turn are lower in
energy than non-bonding orbitals.
•When electromagnetic radiation of the correct
frequency is absorbed, a transition occurs from one
of these orbitalsto an empty orbital, usually an
antibondingorbital, σ* or π*

30. Beer-Lambert Law
•Whenabeamoflightispassedthroughatransparentcell
containingasolutionofanabsorbingsubstance,reduction
oftheintensityofthelightmayoccur.
•Thisdueto:
1.reflectionattheinner&outersurfacesofthecell
2.scatterbyparticlesinthesolution
3.absorptionoflightbymoleculesinthesolution

31. •The reflections at the cell surfaces can be
compensated by a reference cell containing
the solvent only. Scatter may be eliminated by
filtration of the solution, the intensity of light
absorbed is then given by:
I
absorbed= I
0 –I
T

32. When light passes through or is reflected from a sample,
the amount of light absorbed is the difference between the
incident radiation (Io) and the transmitted radiation (I).
The amount of light absorbed is expressed as either
transmittance or absorbance.
Transmittance usually is given in terms of a fraction of 1 or
as a percentage and is defined as follows:

35. •When a beam of light is allowed to pass
through a transparent medium, the rate of
decrease of intensity of radiation with the
thickness of medium is directly proportional
to the intensity of light.
•i.eabsorbance is proportional to the
thickness (pathlength) of the solution.

41. Rememberthattheabsorbanceofasolutionwill
varyastheconcentrationorthesizeofthe
containervaries.
Molarabsorptivitycompensatesforthisby
dividingbyboththeconcentrationandthelength
ofthesolutionthatthelightpassesthrough.
Essentially,itworksoutavalueforwhatthe
absorbancewouldbeunderastandardsetof
conditions-thelighttravelling1cmthrougha
solutionof1moldm
-3
.

42. •Lambert’slawshowsthatthereislogarithmic
relationshipbetweenthetransmittance&length
ofthepath.Beer’sobservedasimilar
relationshipbetweentransmittance&the
concentrationofthesolution.
•Beer’slawisdefinedasfollows:theintensityofa
beamofmonochromaticradiationdecreases
exponentiallywiththeincreaseinconcentration
oftheabsorbingsubstancearithmetically.
•Moresimplyitisstatedasabsorptionis
proportionaltoconcentration

43. Beer’s law tells us that absorption is proportional
to the number of absorbing molecules –ie to the
concentration of absorbing molecules (this is only
true for dilute solutions) –
Lambert’s law tells us that the fraction of radiation
absorbed is independent of the intensity of the
radiation.
Combining these two laws, we can derive the
Beer-Lambert Law:
A = log10(Io/I) = log10(100/T) = kcL

44. A = log10(Io/I) = log10(100/T) = kcL
where Io = the intensity of the incident radiation
I = the intensity of the transmitted radiation
ε = a constant for each absorbing material, known
as the molar absorption coefficient (called the molar
extinction coefficient in older texts) and having the units
mol-1 dm3 cm-1, but by convention the units
are not quoted
ι = the path length of the absorbing solution in cm
c = the concentration of the absorbing species in
mol dm-3

47. Theimportanceofconcentration
Theproportionofthelightabsorbedwilldependonhow
manymoleculesitinteractswith.
Supposeyouhavegotastronglycolouredorganicdye.Ifitis
inareasonablyconcentratedsolution,itwillhaveavery
highabsorbancebecausetherearelotsofmoleculesto
interactwiththelight.
However,indilutesolution,itmaybeverydifficulttosee
thatitiscolouredatall.Theabsorbanceisgoingtobevery
low.

48. Lambert's Law states that each layer of equal thickness of an
absorbing medium absorbs an equal fraction of the radiant
energy that traverses it.
The fraction of radiant energy transmitted by a given
thickness of the absorbing medium is independent of the
intensity of the incident radiation, but the radiation does
not alter the physical or chemical state of the medium.
If the intensity of the incident radiation is Io and that of the
transmitted light is l, then the fraction transmitted is:
l/lo = T
The percentage transmission is
%T = l/lo x 100

49. •The Beer-Lambert Law states that the concentration of a
substance in solution is directly proportional to the 'absorbance
', A, of
the solution.
•Absorbance A = constant x concentration x cell length
•The law is only true for monochromatic light, that is light of a
single wavelength or narrow band of wavelengths, and provided
that the physical or chemical state of the substance does not
change with concentration.
•When monochromatic radiation passes through a homogeneous
solution in a cell, the intensity of the emitted radiation depends
upon the thickness (l) and the concentration (C) of the solution.
•Io is the intensity of the incident radiation and I is the intensity
of the transmitted radiation. The ratio I/Io is called
transmittance.
•This is sometimes expressed as a percentage and referred to as
%transmittance.

50. Mathematically, absorbance is related to percentage
transmittance T by the expression:
A = log10(Io/I) = log10(100/T) = kcL
where L is the length of the radiation path through the
sample, c is the concentration of absorbing molecules in
that path, and k is the extinction coefficient -a constant
dependent only on the nature of the molecule and the
wavelength of the radiation.

51. If, in the expression A = kcl, c is expressed in molar-1 and l
in m, then k is replaced by the symbol τ and is called the
molar absorption coefficient.
The units of τ are mol-1m2. τ was formerly called the molar
extinction coefficient and concentrations were
often expressed as mol l-1, mol dm-3 or M and the cell
length in cm to give units mol-1lcm-1, mol-1dm3cm-1 and
M-1cm-1
respectively. C Sometimes is expressed in g dm-3(gl-1) and l
in cm. In this case, k is replaced by A (sometimes E). A is
known as the specific absorption coefficient.

53. Specific absorbance:
•Absorbance of a specified concentration in a
cell of specified pathlength
•i.e A(1%, 1cm), which is the absorbance of a
1g/100ml (1%w/v) solution in a 1cm cell.
A= A
1%
1cmbc

54. •Deviations or
limitations to
Beer’s Law

55. Deviations from Beer’s Law:
•In Beer’s law it states that if we plot
absorbance A against concentration C a
straight line passing through the origin is
obtained, but usually deviation from linear
relationship between concentration &
absorbance & an apparent failure of beer law

56. There are two types of deviation
•Positive deviation
When a small change in concentration produces
a greater change in absorbance
•Negative deviation
When a larger change in concentration produces
a greater change in absorbance

57. LIMITATIONSOFTHEBEER-LAMBERTLAW
ThelinearityoftheBeer-Lambertlawislimitedbychemicaland
instrumentalfactors.
Causesofnonlinearityinclude:
·Deviationsinabsorptivitycoefficientsathighconcentrations
(>0.01M)duetoelectrostaticinteractionsbetweenmoleculesin
closeproximity
·scatteringoflightduetoparticulatesinthesample
·fluoresecenceorphosphorescenceofthesample
·changesinrefractiveindexathighanalyteconcentration
·shiftsinchemicalequilibriaasafunctionofconcentration
·non-monochromaticradiation,deviationscanbeminimizedby
usingarelativelylatpartoftheabsorptionspectrumsuchasthe
maximumofanabsorptionband
·straylight

58. Deviationmayoccurduetopresenceofimpuritiesthat
fluoresceorabsorbattheabsorptionwavelength.This
interferenceintroducesanerrorinthemeasurement
ofabsorptionofradiationpenetratingthesample.
Deviationmayoccurifmonochromaticlightisnot
used.
Ifwidthofslitisnotproper&thereforeitallows
undesirableradiationtofallonthedetector.These
undesirableradiationmightbeabsorbedbyimpurities
presentinthesample.
Ifthesolutionspeciesundergoespolymerisation

59. •Example:
•benzylalcoholinCCl
4inhighconcentration
existinpolymericform,dissociationofthis
polymerincreaseswithdilution,duetothis
change,absorbancewillchange.
•Beerslawcannotbeappliedtothe
suspensionbutcanbeestimated
colorimetricallyafterpreparingareference
curvewithknownconcentration

60. •Real deviation to Beer’s Law:
•Apparent deviation:
Instrumental deviation:stray radiation,
improper slit width, fluctuation in single
beam& when monochromatic light is not used
can influence the deviation
Chemical deviation: factors like association,
dissociation, ionization, faulty development of
color, refractive index at high concentration

61. Real deviation:
•Beer’sLawissuccessfulindescribingtheabsorption
behaviourofmediacontaininglowanalyte
concentration,inthissenseitislimitingLaw.
•Athighconcentration(usually>0.01M)theaverage
distancebetweenthemoleculesresponsiblefor
absorptionisdiminished,becauseeachmoleculeaffects
thechargedistributionofitsneighbours.This
interactionaltertheabilityofmoleculetoabsorba
givenwavelengthofradiation.
•Theextentofinteractiondependsuponconcentration,
•Sodeviationsfromthelinearrelationshipbetween
absorbance&concentration.

62. •A similar effect is occur in media containing
low absorber concentrations but high
concentration of other species (electrolyte)
•Deviation arise because molar absorptivity
depends upon Refractive index of the
medium.

63. Instrumental deviation:

64. Deviationsduetopolychromaticradiation:
StrictadherencetoBeer'slawisobservedonlywithtruly
monochromaticradiation.
Beerslawrequiredmonochromaticradiationbecauseabsorptivityis
aconstantatasinglewavelength&varieswithachangein
wavelength.
Thepossibilityoferrorduetopracticalimpossibilityofobtaining
monochromaticradiation.Soitisminimizedbytheselectionofa
spectralregionwhereachangeiinaabsorptivitywithachangein
wavelengthisverysmall.
Monochromatorsareusedtoisolateportionsoftheoutputfrom
continuumlightsources,henceatrulymonochromaticradiation
neverexistsandcanonlybeapproximated,i.e.byusingavery
narrowexitslitonthemonochromator.

66. Deviationsinpresenceofstrayradiation:
Theradiationexitingfromamonochromatoris
contaminatedwithsmallamountsofscatteredorstray
radiation,whichreachestheexitslitasaresultof
scattering&reflectionsfromvariousinternalsurfaces.
Strayradiationdiffersgreatlyinwavelengthfromthatof
principalradiation.
Athighconcentration&atlongerpathlength,stray
radiationcausesignificantdeviationsfromlinear
relationshipbetweenabsorbance&pathlength.

68. Chemical deviation:
•it arises when analyte dissociates, associate
or reacts with a solvent to produce a product
having a different absorption spectrum from
the analyte.

69. •A chromophore(literally color-bearing) group is a functional group,
not conjugated with another group, which exhibits a
•characteristic absorption spectrum in the ultraviolet or visible
region. Some of the more important chromophoricgroups are:
•If any of the simple chromophoresis conjugated with another (of
the same type or different type) a multiple chromophoreis
•formed having a new absorption band which is more intense and at
a longer wavelength that the strong bands of the simple
•chromophores.
•This displacement of an absorption maximum towards a longer
wavelength (i.e. from blue to red) is termed a bathochromicshift.
•The displacement of an absorption maximum from the red to
ultraviolet is termed a hypsochromicshift.