UV-Visible Spectroscopy, MPAT, Sem-1, 1st Year, M.Pharm

1. UV VISIBLE SPECTROSCOPY
Prepared by:
Kartiki M. Bhandari
M Pharm (Pharmaceutics) 1st year
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2. Content:
 Introduction
 Principle & Theory
 Laws & Limitations
 Instrumentation
 Choice of solvents & Solvent effect
 Advantages & Limitations
 Applications
 References
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3. Introduction:
• Spectroscopy: branch of science concerned with the study of spectra
produced when matter interacts with or emits electromagnetic radiation.
• UV-VIS absorption spectroscopy (electronic spectroscopy):
measurement of the light absorbed at each wavelength of UV-VIS region
of the electromagnetic spectrum (190-800nm).
• Oldest instrumental technique.
• Determines micro & semi-micro quantities of analytes.
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4. 4
A spectrum of electromagnetic radiations
Vacuum

5. Principle & Theory:
• Measurement of spectrum of a sample containing atoms or molecules.
• Spectrum: graph of absorbed or emitted radiation vs frequency(ν) &
wavelength(λ).
• Spectrophotometer: instrument used to measure the spectrum of a
compound.
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6. • At RT, electrons of atoms are in the lowest energy orbital- ground state
(G.S.), which can absorb radiations & transit to a higher energy orbital-
excited state (E.S.)
• The electronic excitation in molecules is possible if-
Energy of absorbed electromagnetic radiation = Energy gap(∆E) between
G.S. & E.S.
Fewer conjugated π bonds= higher ∆E, lower λmax (towards UV region)
More conjugated π bonds= lower ∆E, higher λmax (towards VIS region)
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7. • The transitions that absorbs electromagnetic radiation are transitions
between electronic energy levels.
• When molecule absorbs energy-
Electron from an occupied orbitalUnoccupied orbital with higher energy.
• Most probable transition-
Highest occupied molecular orbital (HOMO) to Lowest unoccupied
molecular orbital (LUMO).
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8. • Not all of the transitions that at first sight appear possible are observed.
• Certain restrictions, called selection rules, must be considered.
• Transition probability:
Allowed= extinction coefficient value ≥ 10⁴ (high intensity absorptions)
Forbidden= extinction coefficient value ≤ 10³ (low intensity absorptions).
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9. Unoccupied
levels
Occupied
levels
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Electronic energy levels & transitions.

10. In various compounds the electrons may undergo several
possible transitions of different energies.
Some of the most important transitions are:
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11. Laws:
• Greater the number & more the effectiveness of molecules to
absorb light, greater the extent of light absorption.
• From these guideline, the following expression, known as the
Beer–Lambert law, may be formulated as-
A = log(Io/I ) = ɛcl
Where,
A= absorbance, Io= intensity of incident light, I= intensity of
transmitted light, c= molar concentration of solute, l (/ b)=
length of sample cell (cm), ɛ (/ a)= molar absorptivity
coefficient. 11

12. Limitations of Beer-Lambert’s law:
• Obeyed when single species gives rise to the observed absorption.
• Law may not be obeyed when-
Different forms of the absorbing molecule are in equilibrium,
Solute & solvent form complexes through some sort of association,
Thermal equilibrium exists between the electronic G.S. & a low-lying
E.S.,
Fluorescent compounds or compounds changed by irradiation are
present.
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13. • Not applicable at higher concentrations (>0.01M).
• Deviation from the direct proportionality between measured
absorbance & concentration frequently occur when l is constant.
• Some of these deviations, called real deviations, are fundamental &
represent real limitations of the law.
• Other limitations are a result of how the absorbance measurements are
made, called as instrumental deviations or a result of chemical
changes that occurs when the concentration changes, called as
chemical deviations.
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14. Schematic representation of single beam UV-VIS
spectrophotometer:
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(https://microbenotes.com/spectrophotometer-principle-
instrumentation-applications/)

15. Instrumentation:
• The UV-VIS spectrophotometer consists of a light source, a
monochromator, & a detector.
• Light source is usually a deuterium lamp, which emits the
electromagnetic radiation in the UV region.
• 2nd light source is a tungsten lamp, which is used for the
wavelengths in VIS region.
• Monochromator is a diffraction grating; which spreads the
beam of light into its component wavelengths.
• Slits focuses the desired wavelength on sample cell.
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16. • The light passing through sample cell reach the detector.
• The detector records intensity of the transmitted light (I) .
• The detector is generally a photomultiplier tube (PMT).
• In the modern instruments, photodiodes are also used.
• In double-beam instrument, light emitted by source split
into 2 beams, sample beam & reference beam.
• When there is no sample cell in the reference beam, the
detected light is taken to be equal to the intensity of light
entering the sample (Io).
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17. • Sample cell must be constructed of a material that is
transparent to the electromagnetic radiation being
used.
• For spectra in visible region, cells composed of glass or
plastic are generally suitable.
• For the measurements in the UV region, glass & plastic
cannot be used; because they absorb the UV radiation.
• Instead, cells made of quartz must be used since it does
not absorb radiation in this region.
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18. • Instrument design is quite suitable for measurement at only one
wavelength.
• If complete spectrum is desired, this type of instrument has some
deficiencies.
• A mechanical system is required to rotate the monochromator &
provide a scan of all desired wavelengths, but this type of system
operates slowly, & hence requires considerable time to record a
spectrum.
• Therefore, a modern improvement is done on the traditional
spectrophotometer, forming diode-array spectrophotometer.
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19. • Series of photodiode detectors placed side by side on silicon
crystal.
• Each diode records a narrow band of spectrum.
• Diodes are connected so that entire spectrum is recorded at
once.
• No moving parts & can record spectra too quickly.
• Furthermore, its output can be passed to a computer, which
can process the information & provide variety of useful output
formats.
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20. Double beam UV-VIS Spectrophotometer:
• In double beam instrument, two beams are formed by “U”
shaped mirror called as beam splitter or beam chopper.
• Beam chopper is a device consisting of a circular disc.
• 1/3rd disc is opaque, 1/3rd is transparent & 1/3rd is
mirrored.
• It splits monochromatic beam of light into two beams of
equal intensities.
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21. Schematic representation of double beam
UV-VIS spectrophotometer:
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22. Single beam UV-VIS
spectrophotometer:
Double beam UV-VIS
spectrophotometer:
Calibration should be done with blank every
time.
Calibration is done only in the beginning.
Radiant energy intensity changes with
fluctuation of voltage.
Permit large degree of inherent
compensation for intensity fluctuations.
Measures total amount of transmitted light
reaching detector.
Measures percentage of light absorbed by the
sample.
Cannot compare blank & sample together. Blank & sample can be compared together in
2 different paths.
Radiant energy wavelength has to be
adjusted every time.
Scanning can be done over a wide
wavelength range.
Tedious & time consuming. Non tedious & fast.
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23. Choice of solvents & Solvent effect:
• The first criterion for a good solvent is that it should not
absorb UV radiation in the same region as the
substance whose spectrum is being determined.
• The solvent cutoff is the wavelength below which the
solvent itself absorbs the radiation.
• So, solvent cutoff point must be taken into consideration
while selecting the solvent for analysis of any compound.
• Solvents that do not contain conjugated systems are most
suitable for this purpose.
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24. Lists some common UV spectroscopy solvents & their cutoff
points or minimum regions of transparency.
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25. • The second criterion for a good solvent is its effect on the
fine structure of an absorption band.
• A nonpolar solvent does not form the hydrogen bond with the
solute, & the spectrum of the solute closely approximates the
spectrum that would be produced in a gaseous state, in which
fine structure is often observed.
• In a polar solvent, the hydrogen bonding forms solute–
solvent complex, & the fine structure may disappear as show
in next figure-
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26. 26
Formation of solute-solvent complex in polar solvent Ethanol
leading to disappearing fine structure

27. • The third criterion for a good solvent is its ability to influence the
wavelength of UV light that will be absorbed via stabilization of either the
G.S. or E.S of the molecule.
• Polar solvents do not form hydrogen bonds readily with E.S. as with G.S.
which increases the energies of electronic transitions in the molecules.
• Therefore, polar solvents shift the nπ* transition to shorter wavelength.
• Whereas, in some cases the polar solvent form stronger hydrogen bond
with E.S. than with G.S. which decreases the energy of electronic transition.
• Hence, polar solvents shift the ππ* transition to longer wavelength.
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28. 28
Shift of wavelength of acetone due to effect of polar solvents

29. Advantages: Limitations:
Rapid technique with simple instrumental
design used for atoms, ions, molecules;
coloured & colourless solutions of
compounds.
Limited to UV-VIS absorbing compounds.
Covers broad spectrum of wavelength
(190-800nm).
Any fluctuation in the intensity of radiation
source affects the absorbance.
Since the choice of wavelength can be very
specific, one particular species can be
selected & analysed in a complex sample.
Solutions with higher concentration requires
dilutions, thereby leading to complex
calculations.
UV-VIS technique is non-destructive to the
sample & has a high sensitivity for detecting
organic compounds.
Compounds with similar λmax values may
show overlapping in spectra leading to
difficulties in estimation.
Less interferences than other techniques. Requirement of skilled analyst.
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30. Applications:
• Qualitative as well as quantitative analysis.
• Detection of functional groups.
• Detection of extent of conjugation in a compound.
• Detection of impurities.
• Identification of unknown compounds.
• Determination of configurations of geometrical isomers.
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31. • Determination of molecular weight.
• Determination of dissolution constant of acids & bases.
• Determination of percentage of various keto & enol forms
present in tautomeric equilibrium.
• To study chemical kinetics of reaction.
• Detection of drugs with chromophoric group.
• Simultaneous estimation of mixture of two or more drugs.
Applications:
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32. UV spectra of Paracetamol (λmax= 243nm)
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33. UV spectra of simultaneous estimation of Caffeine & Sodium Benzoate
(λmax of caffeine= 273nm & λmax of sod. benzoate= 224nm)
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34. References:
Books:
• Pavia DL, Lampman GM, Kriz GS, Vyvyan JA. Introduction to
spectroscopy. Edn 5, Nelson Education; 2014
• Skoog DA, Holler FJ, Crouch SR. Principles of instrumental
analysis. Edn 6, Thomson Brooks/Cole; 2007
• Chatwal GR, Instrumental methods of chemical analysis. Edn 5,
Himalaya publishing house, Mumbai, pp 2.177-2.182
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35. Journal Articles:
• Khan S, Newport D, Le Calvé S. Gas Detection Using Portable Deep-UV
Absorption. Sensors. 2019;19(23):5210
• Braga MS, Gomes OF, Jaimes RF, Braga ER, Borysow W, Salcedo WJ.
Multispectral colorimetric portable system for detecting metal ions in
liquid media. In2019 4th International Symposium on Instrumentation
Systems, Circuits and Transducers (INSCIT) 2019 Aug 26 (pp. 1-6). IEEE.
References:
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36. References:
Journal Articles:
• Manu B, Mahamood S, Vittal H, Shrihari S. A novel catalytic route to
degrade paracetamol by Fenton process. IJRCE. 2011 Jul;1:157.
• Pahade AR, Gandhi SV, Tapale SR. Chemometric-assisted UV
Spectrophotometric and RP-HPLC Methods for the Simultaneous
Determination of Caffeine and Sodium Benzoate in Synthetic Mixture.
Current Trends in Biotechnology and Pharmacy. 2017 Jul 1;11(3):309-
15.
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37. Thank You!
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