Applications of uv spectroscopy, by Dr. Umesh Kumar Sharma and Anu Mathew.

1. APPLICATIONS OF
UV- VISIBLE
SPECTROSCOPY
1
By: Dr. Umesh Kumar Sharma
& Anu Mathew
Department Of Pharmaceutics,
Mar Dioscorus College Of Pharmacy,
Alathara, Sreekaryam, Thiruvananthapuram

2. 2
• Absorption of light in the UV / visible part of the spectrum.
• Transitions that result in absorption of the electromagnetic
radiation in this region.
• The most probable transition from highest occupied molecular
orbital to lower occupied molecular orbital.
• UV spectroscopy is routinely used in analytical chemistry for
quantitative determination of different analytics such as
transition metal ions, highly conjugated organic compounds and
biological macromolecules.

3. QUALITATIVE APPLICATIONS
3
• UV absorption spectroscopy can characterize those types of
compounds which absorbs UV radiation.
• Identification is done by comparing the absorption spectrum
with the spectra of known compounds.
• UV absorption spectroscopy is generally used for characterizing
the aromatic compounds & conjugated olefins.

4. 4
• Detection of conjugation.
Conjugation may be between,
a. Between two/more C=C bonds or C≡C bonds.
b. Between C=C or C=O bonds,
c. Between double bonds and aromatic ring.
Presence of an aromatic ring & the number & the locations of
substituents attached to the carbons of the conjugated system.
Eg: No. of bonds λmax
Ethylene (CH2=CH2 ) 1 174nm
Acetone (CH3-CO-CH3) 2 217nm

5. 5
Structure of inorganic complexes
 Used to distinguish between cis and trans isomers.
 Geometrical isomerism-easily distinguished from visible spectra
Eg: [Co(en)2F2]NO3: Cis form - violet color, Trans form - green color.
 Planar tetrahedral equilibrium
* Planar form- as temp↑ intensity of spectra ↓ .
* Tetrahedral form- as temp ↑ intensity ↑ .
Equilibrium shift from planar to tetrahedral complex as ↑ temp.
Eg: Schiff base

6. 6
Detection of functional groups
 Detect the presence or absence of a functional group in a compound.
 Absence of a band at particular wavelength regarded as an evidence for absence of
particular group.
 a) λmax for CH3-COOH 208nm
CH3-CO-CH3 189nm
b) Carbonyl group absorption band between 280-290nm
Aromatic ring absorption band at 260nm.

7. 7
Detection of impurities
 The bands due to impurities are very
intense
 The organic compounds can be
classified into saturated compounds
having little absorption & unsaturated
compounds having strong absorption
bands.
 Eg: The common impurity in
cyclohexane is benzene. Its presence
can be easily detected by its
absorption at 255nm.

8. 8
Determination of pKa value of indicator acid or base :
• Let us consider an acid HA. It undergoes dissociation in water to form H3O+ & A-.
HA+ H2O H3O+ + A-
• A- conjugated base. The acid dissociation Ka is defined by the expression
Ka =[H3O]+[A] [HA]
On taking logarithm on both sides
-log Ka = log [H3O+]-log [A-]/[HA]
pKa = pH –log [A]/[HA]
pKa = pH+ log [HA]/[A]
• The ratio [HA]/[A] can be determined by spectrophotometer from the graph
plotted between absorbance & wavelength at different pH values.

9. 9
Quantitative applications
• Used to determine quantity of compounds
• Used in Pharmaceutical research, chemical
research, biochemistry, chemical analysis &
industrial processing
Measurement of unknown concentration by:
a. Calibration curve method
The solution of most of the drug obey beer-
lambert’s law up-to certain limit
By making serial solution from a stock solution &
calibration graph is drawn, concentration along x
axis and absorbance along y axis.
Eg: beer-lambert’s limit for cetirizine is 2-16μg/ml

10. 10
b) Sensitivity
• UV absorption analysis is frequently quite sensitive.
• It determines compounds of concentration >1ppm
• The sensitivity determines the lowest concentration & that can be determined
quantitatively by this method.
Molecular weight determination
 Can be measured spectrophotometicaly by preparing the suitable derivative of
these compounds.
Eg: To determine the molecular weight of amine, it is converted to amine picrate.
Then known concentration of amine picrate is dissolved in a liter of solution & its
optical density is measured at 380nm.

11. 11
Charge transfer transitions
Eg: when iodine & benzene are brought together in a 1:1 mole ratio in heptane, a new
absorption band is observed, which is not observable in the spectra of individual
components i.e. benzene & iodine.
• According to mulliken, this new band arises due to the absorption of radiation by a
molecular complex formed between benzene & iodine.
This can be represented as D+A ( DA)complex where D is the benzene.
DA D+A-
(covalent bond) (ionic bond)
The total wave function of the ground state may be put as
Ψ0= Ψ(DA)+λΨ(D+A-)
λ = measure of charge transfer

12. 12
Tautomeric equilibrium
 Determine the percentage of various keto & enol forms present in a tautomeric
equilibrium.
Eg: ethyl acetoacetate
CH3COCH2COOCH5 CH3C(OH)=CHCOOC2H5
The keto form has λmax 275nm & ε=16. this has only the weak n→ Π* band of the
isolated carbonyl group.
The enol form has λmax 244nm & ε=16000. from the strength of the 244nm band can
measure the proportions of tautomers present in the ethyl acetoacetate.

13. 13
Structure of chloral
This compound can have any one of the following structures
H
CCl3-C-OH CCl3-CHO
OH
(I) (II)
When the UV absorption spectrum of this compound is recorded in hexane, it shows a
band at 290nm. On the other hand the UV spectrum of chloral in aqueous solution does
not show any band. This confirms that chloral hydrate has structure I rather than II.

14. 14
Chemical kinetics
 In order to determine the kinetics of a
reaction the change in the concentration of
either a reactant or a product with time is
measured.
 Based on the fact that one of the reactants /
products exhibiting suitable absorption in the
UV region is not overlapped by absorption
due to other species present.
 In this method , two solutions are entering
through X & Y. Then, these are allowed to
pass through reaction chamber B & the flow
of the mixed solutions is stoppered by piston
D. The absorbance of any species which
absorbs in the UV region is measured at C
with a UV spectrophotometer.
Photomultipier is used as a detector whose
output is displayed on the screen with a time
base.

15. 15
Multicomponent analysis
 Absorbance of the sample is the sum of the absorbance of the individual components.
 The selectivity and accuracy of spectrophotometric analysis of samples containing
interfering substances can be improved by derivative and difference spectroscopy.
Derivative spectroscopy
 The change in absorbance with respect to wavelength is recorded.
 1st and 2nd derivative spectrum is recorded & characteristic peak for individual
components can be identified & quantified, using caliberation curve of pure substance.
Difference spectroscopy
 Useful to quantify a substance when interfering species are present.
 Absorbance difference between 2 forms of same drug is measured.
 Achieved by using pH manipulation using a pair of buffers.
 Used to quantify drugs in biological fluids.

16. 16
Photometric titrations :
 Plot of absorbance as a function of volume of titrant
 Usual titrimetric disadvantages can be overcome by spectrophotometric titrations
using spectrophotometer which determines the end point.
Method :
Titration vessel is kept directly in light path of the instrument
Titrant added
Absorbance measured
Plot of absorbance v/s volume of titrant

17. 17
• If titration reaction completes, the titration curve shows 2 straight lines intersecting
at equivalence point.
• If reaction is incomplete gives a curvature at equivalence point region, extrapolation
of two line segments of titration curve gives equivalence point.
a) Characteristic of a case where only titrant absorbs:
Eg: Titration of Arsenic (As) with bromate - bromide mixture.
 Absorbance are taken at wavelength were bromine absorbs. At the initial period,
absorbance will not be changed since only As remains in solution.
 As titration starts and titrant added ,volume increases no change in absorbance.
• After completion of As, bromine alone in vessel, increases the absorbance due to its
color.
The extrapolation of 2 lines gives the equivalence point.

18. 18
b) Only product of the reaction absorbs :
Eg: Titration of Cu I with EDTA of 745nm. This wavelength is selected because at this
position only EDTA –Cu complex possess greater absorbance compared to copper solution
alone.
c) Only the analyte absorbs and product do not absorb :
Eg: Titration of p-toluidine with per-chloric acid at 290nm. This wavelength is selected
because p-toluidine absorbs at this wavelength where as perchloric acid has no absorbance in
that region. As soon as whole qty. of p -toluidine has reacted, the absorbance will become
constant at equivalence point.
d) When a colored analyte is converted into colorless compound by a colored
titrant.
When titrant is added, color of analyte starts fading & form colorless product. After
equivalence point absorbance again rises due to the color of the titrant alone.
a.

19. 19
.
a. Characteristic of a case where only titrant absorbs b) Only product of the reaction absorbs
c) Only the analyte absorbs and product do not absorb
d) When a colored analyte is converted into
colorless compound by a colored titrant.

20. 20
References
• Instrumental methods of chemical analysis
by Gurudeep R Chatwal, Page no. 2.177 - 2.182.
• Principles of instrumental analysis,
by Skoog, Page no. 375-379.
• Organic spectroscopy
by William kemp, Page no. 261-267.
• Pharmatutor.com
• wikipedia

21. 21
THANK YOU
For Feedback / Comments
Write to umeshpanditjp@gmail.com