The document provides an overview of UV-visible spectroscopy. It discusses how UV-visible spectroscopy works by measuring absorption or emission of electromagnetic radiation by molecules. It describes the instrumentation used in UV-visible spectroscopy including light sources, sample handling using cuvettes, and detectors. It also covers concepts like chromophores, transitions between molecular orbitals, and selection rules. Applications discussed include analysis of functional groups, determination of structure and configuration of compounds.

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PHARMACAD

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Introduction
 Spectroscopy is the tool for study of atomic & molecular structure.
 UV/visible spectroscopy is also called electronic spectroscopy because the
absorption spectra are a result of the behaviour of electrons in the target
molecules.
 It deals with interaction of electronic radiation with matter involving the
measurement & interpretation of the extension of absorption or emission of
EMR by molecule.
 It provides information about electronic properties of molecules
 Most important consequence of such interaction is the energy is absorbed or
emitted by the matter in discrete amount called as quanta.
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Introduction
 UV radiation starts at blue end of visible light (4000 Å) & ends at
2000 Å.
 It divided into two spectral region-
Near UV region- 2000Å – 4000Å.
Far UV region- below 2000 Å.
 UV-spectroscopy involved with electronic excitation.
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Introduction
 The difference in energy between molecular bonding, non-bonding
and anti-bonding orbitals ranges from 125–650 kJ/mole
 This energy corresponds to EM radiation in the ultraviolet (UV)
region, 200-400 nm, and visible (VIS) regions 400-800 nm of the
spectrum
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5. Characteristics of UV-Vis spectra of Organic Molecules
 Absorb mostly in UV, unless highly conjugated system
 Spectra are broad, usually to broad for qualitative identification
purposes
 The most common detector for HPLC

6. Introduction
The Spectroscopic Process
 In UV spectroscopy, the sample is irradiated with the broad spectrum
of the UV radiation
 If a particular electronic transition matches the energy of a certain band
of UV, it will be absorbed
 The remaining UV light passes through the sample and is observed
 From this residual radiation a spectrum is obtained with “gaps” at these
discrete energies – this is called an absorption spectrum
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7.  According to MO concept when molecule irradiated with UV-VIS
radiation the transfer of electron takes place from HOMO level to
LUMO level (valency shell MO’s) .
 During these electron transfers, the molecule absorbs energy and
absorbed energy converted into UV-VIS peaks/ bands .
 The transfer of electrons from HOMO to LUMO (BMO-ABMO) is
called electronic excitations or transitions.
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Molecular Orbitals
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8. Graphical Representation of MOs
Energy
s
n
Atomic orbital
Atomic orbital
s
Molecular orbitals
Occupied levels
Unoccupied levels
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All possible excitations
1)
2)
3)
4)
5)
6)
*
*
*
*
n
n
*
*
Types of MOs
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10. Observed electronic transitions
 From the molecular orbital diagram, there are several possible electronic
transitions that can occur, each of a different relative energy:
Energy
s
s
n
s
s
n
n
s
s
alkanes
carbonyls
unsaturated cmpds.
O, N, S, halogens
carbonyls
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11. Observed electronic transitions
 Special equipment to study vacuum or far UV is required
 Routine organic UV spectra are typically collected from 200-700 nm
 This limits the transitions that can be observed:
n
n
s s
s
s
carbonyls
alkanes 150 nm
carbonyls 170 nm
unsaturated cmpds.180 nm √ - if conjugated!
O, N, S, halogens 190 nm
300 nm √
Energy
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12. Instrumentation
sample
reference
detector
I0
I0 I0
I
1. The construction of a traditional UV-VIS spectrometer is very similar
to an IR, as similar functions – sample handling, irradiation, detection
and output are required
2. Here is a simple schematic that covers most modern UV
spectrometers:
log(I0/I) = A
200 700
,nm
monochromator/
beam splitter optics
UV-VIS sources
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13. Instrumentation
3. Two sources are required to scan the entire UV-VIS band:
 Deuterium lamp – covers the UV – 200–330
 Tungsten lamp – covers 330–700
4. As with the dispersive IR, the lamps illuminate the entire band of UV
or visible light; the monochromator (grating or prism) gradually
changes the small bands of radiation sent to the beam splitter
5. The beam splitter sends a separate band to a cell containing the
sample solution and a reference solution
6. The detector measures the difference between the transmitted light
through the sample (I) vs. the incident light (I0) and sends this
information to the recorder
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14. Instrumentation
sample
Polychromator
– entrance slit and dispersion device
7. As with dispersive IR, time is required to cover the entire UV-VIS
band due to the mechanism of changing wavelengths
8. A recent improvement is the diode-array spectrophotometer - here a
prism (dispersion device) breaks apart the full spectrum transmitted
through the sample
9. Each individual band of UV is detected by a individual diodes on a
silicon wafer simultaneously – the obvious limitation is the size of the
diode, so some loss of resolution over traditional instruments is
observed
Diode array
UV-VIS sources
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15. Instrumentation
Instrumentation – Sample Handling
1. Virtually all UV spectra are recorded solution-phase
2. Cells can be made of plastic, glass or quartz
3. Only quartz is transparent in the full 200-700 nm range; plastic and
glass are only suitable for visible spectra
4.Concentration (we will cover shortly) is empirically determined A
typical sample cell (commonly called a cuvet):
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16. Instrumentation
Instrumentation – Sample Handling
5. Solvents must be transparent in the region to be observed; the
wavelength where a solvent is no longer transparent is referred to as
the cutoff
6. Since spectra are only obtained up to 200 nm, solvents typically only
need to lack conjugated π systems or carbonyls
Common solvents and cutoffs:
acetonitrile 190
chloroform 240
cyclohexane 195
1,4-dioxane 215
95% ethanol 205
n-hexane 201
methanol 205
isooctane 195
water 190
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17. Instrumentation and Spectra
Instrumentation – Sample Handling
7. Additionally solvents must preserve the fine structure (where it is
actually observed in UV!) where possible
8. H-bonding further complicates the effect of vibrational and rotational
energy levels on electronic transitions.
9. The more non-polar the solvent, the better (this is not always possible)
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18.  Transitions are faster due to parallel
arrangement of π and π* orbital's.
 Interactions / overlapping is more
irrespective of energy gap.
 Transitions slow due to
perpendicular arrangement of
n and π* orbitals.
 These transitions may be due to
vibrations or twisting of the
bonds.
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 Allowed excitations are more probable and faster
 Forbidden excitations (less probable)
Electronic Excitations
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19. *** Selection Rules
 Not all transitions that are possible are observed
 For an electron to transition, certain quantum mechanical constraints
apply – these are called “selection rules”
 For example, an electron cannot change its spin quantum number
during a transition – these are “forbidden”
Other examples include:
 the number of electrons that can be excited at one time
 symmetry properties of the molecule
 symmetry of the electronic states
 To further complicate matters, “forbidden” transitions are sometimes
observed (albeit at low intensity) due to other factors
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20. *** Selection Rules
1) The symmetry allowed excitations are high probable excitations.
Ex: π—π*
2) Symmetry forbidden excitations are low probable excitations
Ex: n—π*
3) Excitations can takes place among BMO to ABMO and NBMO to
ABMO’S.
4) During electronic transition spin inversion is forbidden.
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21. 5) During electronic transitions change in multiplicity is forbidden.
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*** Selection Rules
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22. 6) Change in position of nuclei of bond during electronic transition is forbidden
(Frank Condon Principle).
(There is no change in the internuclear distance of the molecule during the
excitation process. This is known as “Franck-Condon principle”.)
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*** Selection Rules
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23. Representation of UV spectrum diagram
 Each UV-band is characterized with its intensity and its position.
 Є or logЄ α Intensity.
 Forbidden transitions UV band are low intense bands. Є <100
 Allowed transitions UV bands are high intense bands Є>10000 (10,000,
50,000, 100,000)
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24. Є = Molar absorptivity or Molar extension coefficient FromBeer’s-
Lamberts law
A = Є. c. l
Where A = Absorbance ( no units)
c = concentration (moles/lit)
l = length of solution or thickness of sol. or length of the tube/cell
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Representation of UV spectrum diagram
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25. Q: Certain sample solutions concentration is 0.001 gms/100 ml. M.Wt is 424,
l = 1 cm, A = 0. 3025. Calculate Є ?
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Problems
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26. Q: For a solution of camphor in hexane in a 10 cm cell absorbance at 295 nm
was found to be 2.52. What is the concentration of Camphor ? Molar
extenction coefficient is 14.
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Problems
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27. Important Terms
200 to 800 nm
• a) Chromophore:-
• Any group which is absorbing energy from UV-VIS range of radiation
called as Chromophore.
• b)Auxochrome:-
• The group which can not absorb radiation 200 to 800 nm range is the
auxochrome. Auxochrome shift UV band towards higher λ side (right). It
is called Bathochromic shift .
• Ex:- lone pair electron groups and –Ve charged groups.
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28. • c) Bathochromic Shift:-
• Movement of UV peak towards higher λ side (right side ) is called the
Bathochromic shift (red shift).
• d) Hypsochromic shift:-
• Movement of UV peak or band towards lower λ side (left side) is the
Hypsochromic shift (Blue Shift).
• e) Hyperchromic shift :-
• Increase in intensity of UV peak is hyperchromic shift (more Є or logЄ value)
• f) Hypochromic shift:-
• Decrease in intensity of UV peak of band (lower Є or logЄ value).
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Important Terms
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29. Absorption & Intensity Shifts
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Theoretical prediction of λmax of conjugated π-system
 ***Woodward – fieser rules (Empirical rules)
 Eg: 1,3-butadiene system, c=c-c=c
 Parent acyclic diene 217 nm (base values, π-π*)
 Parent hetero annular diene 215 nm ( ,, )
 Parent homo annular dienes 253 nm ( ,, )
 Increments for the substituents
1) Alkyl group or ring residues +5
2) For each exocyclic double bond +5
3)
4)
5)
Double bond with extending conjugation +30
On diene if halogen present +5
On diene if alkoxy group present +6
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31. Limitation:-
 These rules are valid for unsaturated system which is having less than four
double bonds in conjugation.
 1)Acyclic C=C-C=C 217 nm
 2) Hetero annular (two double bonds not in the same ring)
 3) Homo annular (two double bonds with conjugation in same ring system)
 4) If in the same molecule homo and hetero dienes present, homo diene is
the base value (higher value)
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32. Q: Calculate the Absorption Maximum?
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33. Q: Calculate the Absorption Maximum? NIPER
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34. Q: Calculate the Absorption Maximum? NIPER
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35. Applications of UV-Visible Spectroscopy
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Why should we learn this stuff?
After all, nobody solves structures with UV any longer!
 Many organic molecules have chromophores that absorb UV
 UV absorbance is about 1000 x easier to detect per mole than NMR
 Still used in following reactions where the chromophore changes. Useful
because timescale is so fast, and sensitivity so high. Kinetics, esp. in
biochemistry, enzymology.
 Most quantitative Analytical chemistry in organic chemistry is conducted
using HPLC with UV detectors
 One wavelength may not be the best for all compound in a mixture.
 Affects quantitative interpretation of HPLC peak heights

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Practical Applications
• Pharmacy Practice
– Ultraquin (psoriasis med. Needs UV.Act.
– Pregnancy tests (colorimetric assays)
– Blood glucose tests, Bilichek
– ELISA’s
• Pharmaceutics
– pH titrations, purity measurement
– concentration measurement

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Pharmaceutical Applications
 On Line Analysis of Vitamin A and Coloring Dyes for the Pharmaceutical
Industry
 Determination of Urinary Total Protein Output
 Analysis of total barbiturates
 Comparison of two physical light blocking agents for sunscreen lotions
 Determination of acetylsalicylic acid in aspirin using Total Fluorescence
Spectroscopy
 Automated determination of the uniformity of dosage in Quinine Sulfate
tablets using a Fibre Optics Autosampler
 Determining Cytochrome P450 by UV-Vis Spectrophotometry
 Light Transmittance of Plastic Pharmaceutical Containers

39. Applications of UV-Visible Spectroscopy
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 Detection of functional groups
 Estimation of extent of conjugation
 Distinction in conjugated and non-conjugated compounds
 Identification of an unknown compound
 Examination of Polynuclear hydrocarbons
 Elucidation of the structure of Vitamins A and K
 Preference over two tautomeric forms
 Identification of compound in different solvents
 Determination of configurations of Geometrical isomers
 Distinguishes between Equatorial and Axial conformations
 Determination of strength of H-bonding
 Hindered rotation and conformational analysis

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UV vs. IR vs. NMR
 UV has broad peaks relative to IR & NMR
 UV has less information than IR & NMR
 UV spectra are easier to collect
 UV spectra are faster to collect
 UV spectrometers are cheaper
 UV spectra require only nanograms of material or chemicals

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