absorption-spectroscopy csir net exam help

Absorption spectrometry

1. Absorption Spectroscopy
By
Srinivas Acharya
Lecturer, Env. Science

4. Visible& near-UVregion wavelength (nm)
Microwave&radiowaveregionfrequency (Hz)
Infaredregion wavenumber (cm
-1
)
Far-UV, x-ray, g-ray energy (DE=hn)

5. Absorption & Emission
Rapid process(10
-15
s)

6. Absorption & Emission

7. Radiation-Induced Transition
•Absorption
•Stimulated emission
•Spontaneous emission  nn
n
c
I

8. UV-Visible Spectroscopy
•Ultraviolet-visible spectroscopy involves
the absorption of ultraviolet/visible light by
a molecule causing the promotion of an
electron from a ground electronic state to an
excited electronic state.
•Ultraviolet/Visible light:
wavelengths (l) between 190 and 800 nm

9. UV-visible spectrum
The two main properties of an
absorbance peak are:
1.Absorption wavelength
l
max
2.Absorption intensity
A
max
Housecroft and Sharpe, p. 466

10. Beer-Lambert Law
Beer-Lambert Law:
log(I
0/I) =ebc
e=A/cb
A =ebc
A=ec (whenbis 1 cm)
I
0= intensity of incident light
I= intensity of transmitted light
e= molar absoptivity coefficient in cm
2
mol
-1
c= concentration in mol L
-1
b= pathlength of absorbing
solution in cm
-1
A = absorbance = log(I
o/I)
0.1
cm
ℓ

11. Beer-Lambert Law
•AAbsorbance or optical density (OD)
•eabsorptivity; M
-1
cm
-1
•cconcentration; M
•TtransmittanceTbl
I
I
A
t
loglog
0
 e

12. Transmittance, Absorbance, and Cell
Pathlength

13. Deviations from the Beer-Lambert Law
The Beer-Lambert law assumes that
all molecules contribute to the
absorption and that no absorbing
molecule is in the shadow of another
Low
c
High
c

14. Sample Concentrations
Solution too concentrated Diluted five-fold

15. UV-visible spectrum of 4-nitroanaline
Solvent: Ethanol
Concentration: 15.4 mg L
-1
Pathlength: 1 cmNH
2
NO
2
Molecular mass = 138
Harwood and Claridge, p. 18

16. UV-visible spectrum of 4-nitroanaline
1.Determine the absorption maxima (l
max) and absorption intensities
(A) from the spectrum:
l
max= 227 nm, A
227= 1.55l
max= 375 nm, A
375= 1.75
2. Calculate the concentration of the compound:
(1.54 x 10
-2
g L
-1
)/(138 g/mol) = 1.12 x 10
-4
mol L
-1
3.Determine the molar absorptivity coefficients (e) from the Beer-
Lambert Law: e= A/cℓ
e
227= 1.55/(1.0 cm x 1.12 x 10
-4
mol L
-1
) = 13,900 mol
-1
L cm
-1
e
375= 1.75/(1.0 cm x 1.12 x 10
-4
mol L
-1
) = 15,700 mol
-1
L cm
-1

17. Molar absorptivities (e)
Molarabsoptivitiesareverylargeforstronglyabsorbing
chromophores(e>10,000)andverysmalliftheabsorptionisweak(e
=10to100).Themagnitudeofereflectsboththesizeofthe
chromophoreandtheprobabilitythatlightofagivenwavelengthwill
beabsorbedwhenitstrikesthechromophore.Ageneralequation
statingthisrelationshipmaybewrittenasfollows:
e= 0.87 x 10
20
P x a
where Pis the transition probability (0 to 1)
ais the chromophore area in cm
2
The transition probability depends on a number of factors including
where the transition is an “allowed” transition or a “forbidden”
transition

18. UV-visible spectroscopy definitions
chromophore Any group of atoms or part of a molecule that absorbs
light whether or not a color is thereby produced.
auxochrome A group which extends the conjugation of a chromophore
by sharing of nonbonding electrons.
bathochromic shiftThe shift of absorption to a longer wavelength.
hypsochromic shift The shift of absorption to a shorter wavelength.
hyperchromic effectAn increase in absorption intensity.
hypochromic effectA decrease in absorption intensity.

21. How to impress your friends and family!
Making Molecular Orbitals
Antibonding
Bonding
In this case, the energies of the A.O.’s are identical

22. Absorption and Emission of Photons

23. Absorption and Emission
Emission
Absorption:A transition from a lower level to a higher level with transfer of
energy from the radiation field to an absorber, atom, molecule, or solid.
Emission: A transition from a higher level to a lower level with transfer of
energy from the emitter to the radiation field. If no radiation is emitted, the
transition from higher to lower energy levels is called nonradiative decay.
Absorption

24. Singlet and Triplet Excited States

25. Absorption and emission pathways
McGarvey and Gaillard, Basic Photochemistry

26. Selection Rules
In electronic spectroscopy there are three selection rules which
determine whether or not transitions are formally allowed:
1.Spin selection rule: DS = 0
allowed transitions: singlet singletor triplet triplet
forbidden transitions: singlet triplet or triplet singlet
Changes in spin multiplicity are forbidden

27. Selection rules
2.Laporte selection rule: there must be a change in the parity
(symmetry) of the complex
Laporte-allowed transitions: gu
Laporte-forbidden transitions: ggoru u
gstands for gerade–compound with a center of symmetry
ustands for ungerade–compound without a center of symmetry
3.Selection rule of Dℓ = ±1 (ℓ is the azimuthal or orbital quantum
number, where ℓ = 0 (s orbital), 1 (p orbital), 2 (d orbital), etc.)
allowed transitions: s p, pd, d f, etc.
forbidden transitions: s s, d d, p f, etc.

29. s ands* orbitals

30. p and p* orbitals

31. Electronic Transitions: pp*
McGarvey and Gaillard, Basic Photochemistry
The pp* transition involves orbitals that
have significant overlap, and the probability is
near 1.0 as they are “symmetry allowed”.

32. p p* transitions -Triple bonds
Organic compounds with -C≡C-or -C≡N groups, or transition
metals complexed by C≡N
-
or C≡O ligands, usually have “low-
lying” p* orbitals

33. Electronic Transitions: n p*
McGarvey and Gaillard, Basic Photochemistry
The n-orbitals do not overlap at all well with the
p* orbital, so the probability of this excitation is
small. The eof the np* transition is about 10
3
times smaller than efor the pp* transition as
it is “symmetry forbidden”.

35. Molecular Orbitals in H
2
The next-lowest energy orbital is unoccupied. As it lies above the highest
atomic orbital, we refer to it as an anti-bonding orbital.
Look also at the shape of the lobes:
The anti-bonding orbitalhas a nodebetween the
two nuclei.
Where the bonding orbital has an electron density
build-upbetween the nuclei, the anti-bonding
orbital would have a reduced
electron density (y
2
).
This orbital is called the Lowest
Unoccupied Molecular Orbital
(LUMO)
This orbital is called the Highest
Occupied Molecular Orbital
(HOMO)
1s
2s
R= 
(H)

0.735
Å
(H
2)
s
s
*

36. Lycopene from Tomatoes
http://www.purdue.edu/UNS/html4ever/020617.Handa.lycopene.html

37. Chlorophyll
B-carotene
hemoglobin

38. Quantitative Analysis
•A plot of absorption versus wavelength is the absorption spectrum 
   
 
 



























NMNM
MM
NMNM
NN
NM
NM
NMNMNMtotal
AA
l
AA
l
NMl
NMl
NMlNlMlAAA
1221
21
1221
12
222
111
12
21
1
N
1
M
so
A
A
h wavelengt2under smeasurment
systemN and Mcomponent -for two
llll
ll
llll
ll
lll
lll
lllllll
eeee
ee
eeee
ee
ee
ee
eeee

39. Solutions containing the amino acids tryptophan and tyrosine can be
analyzed under alkaline conditions (0.1 M KOH) from their different uv
spectra. The extinction coefficients under these conditions at 240 nm
and 280 nm are
•Molar absorptivity Ɛ
try=11300 mol
-1
cm
2
and Ɛ
tyr=5380 mol
-1
cm
2
A 10-mg smaple of the protein glucagon is hydrolyzed to its constituent
amino acids and diluter to 100 mL in 0.1 M KOH. The absorbance of
this solution (1 cm path) was 0.717 at 240 nm and 0.239 at 280 nm.
Estimate the content of tryptophan and tyrosine in mol (g protein)
-1
  
  

  
  
Mtyr
Mtyr
5
5
1081.2
19601500538011300
717.01500239.011300
1085.5
19601500538011300
239.01960717.05380











40. Isosbestic points
Isosbestic wavelength is the wavelengthat which two or more
components have the same extinction coefficient. The
occurrence of two or more isosbestics in the spectra of a series
of solutions of the same total concentration demonstrates the
presence of two and only two components absorbing in that
spectra region.

41. Isosbestic points 
 NMl
NlMl
iso
isoiso


e
eel
isoA : isosbestic iso
NM
eee
ll 
Inspectroscopy,anisosbesticpointisaspecificwavelength,
wavenumberorfrequencyatwhichthetotalabsorbanceofa
sampledoesnotchangeduringachemicalreactionora
physicalchangeofthesample.

42. UV spectrum of BSA
UV spectrum of DNA
from E. coli

43. UV Absorption of amino acid

44. Effect of Secondary structure

45. Origin of Spectroscopic Changes
1.Change in local charge distribution
2.Change in dielectric constant
3.Change in bonding interaction
4.Change in dynamic coupling between different parts of
the molecule

48. 11

51. Human Eye
Retina
Retina
Outer segment
Light sensitive
protein
http://www2.mrc-lmb.cam.ac.uk/groups/GS/eye.html

52. Rhodopsinis a protein
in the membrane of the
photoreceptor cell in the
retina of the eye. It
catalyses the only light
sensitive step in vision.
The 11-cis-retinal
chromophore lies in a
pocket of the protein
and is isomerised to all-
trans retinal when light
is absorbed. The
isomerisation of retinal
leads to a change of the
shape of rhodopsin
which triggers a
cascade of reactions
which lead to a nerve
impulse which is
transmitted to the brain
by the optical nerve
http://www2.mrc-lmb.cam.ac.uk/groups/GS/rmovie.html
1BRD

53. 1BRD
1BM1