UV-visible spectroscopy - 2021

ELECTROMAGNETIC RADIATION
THEORY ABOUT UV-VISIBLE SPECTROSCOPY
INSTRUMENT
FACTORS AFFECTING ABSORBTION
Woodward-Fieser Rules
APPLICATIONS

1. Ph.D.AhmedMetwaly
Ultraviolet/visible spectroscopy
ORCID account
Email: [email protected]
1
•Associate Professor of Pharmacognosy , faculty of Pharmacy, Al-Azhar University
•Senior research fellow, Liaoning University of Traditional Chinese Medicine, China (20118-2019)
•Visiting scholar, School of Pharmacy, University of Mississippi, USA (2012-2014)

2. OBJECTIVES
•ELECTROMAGNETIC RADIATION
•THEORY ABOUT UV-VISIBLE SPECTROSCOPY
•INSTRUMENT
•FACTORS AFFECTING ABSORBTION
•Woodward-FieserRules
•APPLICATIONS
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3. Electromagnetic Radiation
Electromagneticradiationconsistofdiscretepacketsofenergy
whicharecalledasphotons.
Aphotonconsistsofanoscillatingelectricfield(E)&an
oscillatingmagneticfield(M)whichareperpendiculartoeach
other.

5. Electromagnetic Radiation
Frequency(ν):
Itisdefinedasthenumberoftimeselectricalfieldradiationoscillates
inonesecond.
TheunitforfrequencyisHertz(Hz).
1 Hz = 1 cycle per second
Wavelength(λ):
Itisthedistancebetweentwonearestpartsofthewaveinthesame
phasei.e.distancebetweentwonearestcrestortroughs.

6. Electromagnetic Radiation
Asphotonissubjectedtoenergy,so
E = hν= h c / λ

7. Electromagnetic Radiation
Thevisiblespectrumconstitutesasmallpartofthetotalradiationspectrum.
Mostoftheradiationthatsurroundsuscannotbeseen,butcanbedetectedby
instruments.

8. Ultraviolet and Visible Spectroscopy:
Theultravioletandvisible(UV/Vis)radiationisthatregionofEMR(electromagnetic
radiation)orspectrumwhosewavelengthsrangefrom10-800nm.
The(UV/Vis)regioncanbedividedintothreetypesofradiationthatare
1.VacuumUV;λ=10-200nm
2.NearUV;λ=200-400nm
3.Visibleradiation;λ=400-800nm

9. Electromagnetic Radiation
Violet400 -420 nm Yellow570 -585 nm
Indigo420 -440 nm Orange585 -620 nm
Blue 440 -490 nm Red 620 -780 nm
Green 490 -570 nm

10. 10
Electronic Transitions

11. 11

12. Thecomonelectronictransitionscangraphicallyshownas:

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14. Principles of Spectroscopy
Theprincipleisbasedonthemeasurementofspectrumofa
samplecontainingatoms/molecules.
Spectrumisagraphofintensityofabsorbedoremitted
radiationbysampleversesfrequency(ν)orwavelength(λ).
Spectrometerisaninstrumentdesigntomeasurethe
spectrumofacompound.

16. Instrumentation
Components of UV-Visible spectrophotometer
Source
Filters & Monochromator
Sample compartment
Detector
Recorder

18. Five Basic Optical Instrument Components
1) Source –A stable source of radiant energy at the desired wavelength (or range).
2) Wavelength Selector –A device that isolates a restricted region of the EM
spectrum used for measurement (monochromators, prisms & filters).
3) Sample Container –A transparent container used to hold the sample (cells,
cuvettes, etc).
4) Detector/Photoelectric Transducer –Converts the radiant energy into a useable
signal (usually electrical).
5) Signal Processor & Readout –Amplifies or attenuates the transduced signal and
sends it to a readout device as a meter, digital readout, chart recorder, computer,
etc.

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24. LIGHT SOURCES
Various UV radiation sources are as follows
a. Deuterium lamp
b. Hydrogen lamp
c. Tungsten lamp
d. Xenon discharge lamp
e. Mercury arc lamp
Various Visible radiation sources are as follow
a. Tungsten lamp
b. Mercury vapour lamp
c. Carbonone lamp
SAMPLE COMPARTMENT

25. SUMMARY
Types of source, sample holder and detector for various EM region
REGION SOURCE
SAMPLE
HOLDER
DETECTOR
Ultraviolet Deuterium lamp Quartz/Fused
silica
Phototube, PM
tube, diode array
Visible Tungstenlamp Glass/Quartz Phototube, PM
tube, diode array

26. Beer Lamberts Law:
Beer Lamberts Law:
A = εb c
A=absorbance
ε=molar absorbtivitywith units of L /mol.cm
b=path length of the sample (cuvette)
c =Concentration of the compound in solution, expressed
in mol /L

28. Chromophore
Chromophore: covalently unsaturated groups responsible for electronic
absorption
e.g. NO
2, N=O, C=O, C=N, C≡N, C=C, C=S, etc
Auxochrome
Thefunctionalgroupsattachedtoachromophorewhichmodifiestheabilityofthe
chromophoretoabsorblight,alteringthewavelengthorintensityofabsorption.
OR
The functional group with non-bonding electrons that does not absorb radiation in
near UV region but when attached to a chromophore alters the wavelength &
intensity of absorption.

29. Factors affecting UV/Vis absorption:
The position and intensity of an absorption band of a chromophore may be modified by
substituent groups attached to the chromophore, type of solvent, degree of conjugation and
stereochemicaleffect.
Bathochromic (Red) shift: The shift of absorption to a longer wavelength.
Hypsochromic(Blue)shift: The shift of absorption to a shorter wavelength.
Hyperchromiceffect: The increase in absorption intensity.
Hypochromiceffect: The decrease in absorption intensity.

30. Application of UV/vis spectroscopy:
Chrysophanol, λmax (EtOH) nm: 224, 254, 288, 432,
By addition of one drop of 5% NaOH, a bathochromicshiftwill occurs; 232, 250,
330, 500.
Ferulicacid, λmax (MeOH) nm: 230, 279.
Vanillicacid, λmax (MeOH) nm: 252, 291, 328.
Iridoidsand secoiridoids, λmax (MeOH) nm: 236; characteristics for enol-ether
system conjugated with carbonyl group (-OCO-C=CH-O-).
UV Aspect of Flavonoids:

31. •Whenabsorptionmaxima(λ
max)ofa
compoundshiftstolongerwavelength,itis
knownasbathochromicshiftorredshift.
•Theeffectisduetopresenceofanauxochrome
orbythechangeofsolvent.
•e.g.Anauxochromegrouplike–OH,-OCH
3
causesabsorptionofcompoundatlonger
wavelength.
•Bathochromic Shift (Red Shift)1

32. •Inalkalinemedium,p-nitrophenolshowsred
shift.Becausenegativelychargedoxygen
delocalizesmoreeffectivelythantheunshared
pairofelectron.
p-nitrophenol
λ
max=255nm λ
max=265nm
•Bathochromic Shift (Red Shift)1OH
N
+
O
-
O
OH
-
Alkaline
medium
O
-
N
+
O
-
O

33. •Whenabsorptionmaxima(λ
max)ofa
compoundshiftstoshorterwavelength,itis
knownashypsochromicshiftorblueshift.
•Theeffectisduetopresenceofangroup
causesremovalofconjugationorbythe
changeofsolvent.
•Hypsochromic Shift (Blue Shift)2

34. •Anilineshowsblueshiftinacidicmedium,it
losesconjugation.
Aniline
λ
max=280nm λ
max=265nm
•Hypsochromic Shift (Blue Shift)2NH
2
H
+
Acidic
medium
NH
3
+
Cl
-

35. •Whenabsorptionintensity(ε)ofacompoundis
increased,itisknownashyperchromicshift.
•Ifauxochromeintroducestothecompound,theintensity
ofabsorptionincreases.
Pyridine 2methylpyridine
λ
max=257nm λ
max=260nm
ε=2750 ε=3560
•Hyperchromic Effect3N N CH
3

36. •Whenabsorptionintensity(ε)ofacompoundis
decreased,itisknownashypochromicshift.
Naphthalene 2-methylnaphthalene
ε=19000 ε=10250CH
3
•Hypochromic Effect4

37. Wavelength ( λ)
Absorbance ( A )
Shifts and Effects
Hyperchromic shift
Hypochromic shift
Red
shift
Blue
shift
λ
max

38. Alkenes
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39. conjugations
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40. Woodward-FieserRules
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1. Conjugated dienes
2. Conjugated dienones
3. Aromatic systems

41. Dienes
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Acyclic butadiene = 217 nm

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43. Cyclic Dienes
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49. Enones
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51. APPLICATIONS
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•Detection of Impurities. ...
•Structure elucidation of organic compounds. ...
•Quantitative analysis. ...
•Qualitative analysis. ...
•HPLC
•Chemical kinetics. ...

52. SUMMARY
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•ELECTROMAGNETIC RADIATION
•THEORY ABOUT UV-VISIBLE SPECTROSCOPY
•INSTRUMENT
•FACTORS AFFECTING ABSORBTION
•Woodward-FieserRules
•APPLICATIONS

53. Ph.D.Ahmed M.Metwaly
Associateprofessor,Pharmacognosy
department,
FacultyofPharmacy,Al-Azhar University.
[email protected]
Email: [email protected] 53