Detailed applications of uv-visible spectroscopy with examples

1. APPLICATIONS OF UV-VISIBLE
SPECTROSCOPY
Presented by:
Aditya Sharma
M.S. (Pharm)
Pharmaceutical Analysis
NIPER Guwahati

2. 1. Detection of Impurities:
• UV absorption spectroscopy is one of the best methods for
determination of impurities in organic molecules.
• Additional peaks can be observed due to impurities in the sample and
it can be compared with that of standard raw material.
• By also measuring the absorbance at specific wavelength, the
impurities can be detected.
• Example: Benzene appears as a common impurity in cyclohexane. Its
presence can be easily detected by its absorption at 255nm.

4. 2. Structure Elucidation of Organic
Compounds:
• UV spectroscopy is useful in the structure elucidation of organic
molecules, the presence or absence of unsaturation, the presence of
hetero atoms like S, O, N or halogens can be determined.
• From the location of peaks and combination of peaks, it can be
concluded that whether the compound is saturated or unsaturated,
hetero atoms are present or not, etc.

6. 3. Detection of Functional Group:
• The technique is applied to detect the presence or absence of the chromophore.
• The absence of a band at a particular wavelength may be regarded as an
evidence for the absence of a particular group in the compound.
• If the spectrum is transparent above 200nm, it shows the absence of:
a. Conjugation
b. A carbonyl group (aldehydes and ketones)
c. Benzene or aromatic compounds
d. Bromo or iodo atoms
• An isolated double bond or some other atoms or groups may be present.
• It means that no definite conclusion can be drawn if the molecule absorbs below
200nm.

8. 4. Identification of Compounds in Different
Solvents:
• Sometimes the structure of the compound changes with the change
in solvent.
• Example: Chloral hydrate shows an absorption maximum at 290nm in
hexane while the absorption disappears in the aqueous solution.
Clearly, the compound a carbonyl group in hexane solution and its
structure is CCl4.CHO.H2O whereas in aqueous solution it is present as
CCl3.CH(OH)2.

9. 5. Determination of Configurations of
Geometrical Isomers:
• The results of absorption show that cis- alkenes absorb at different
wavelengths as compared to their corresponding trans-isomers.
• The distinction becomes possible when one of the isomers is forced
to be non-coplanar by steric hindrance. Thus, cis forms suffer
distortion and absorption occur at lower wavelength.
Cis-stillbene
λmax=283nm Trans-stillbene
λmax=295.5nm

10. 6. Molecular Weight Determination:
• Molecular weights of compounds can be measured spectrophotometrically
by preparing the suitable derivatives of these compounds.
• If we want to determine the molecular weight of amine then it is converted
to amine picrate. The known concentration of amine picrate is dissolved in
a litre of solution and its optical density is measured at lambda max
380nm. After this the concentration of the solution in gram moles per liter
can be calculated by using the formula:
• C= log (I0/I) / €max x l
• “C” can be calculated using above equation, the weight “w” of amine
picrate is known. From “C” and “w”, molecular weight of amine picrate can
be calculated. And the molecular weight of picrate can be calculated using
the molecular weight of amine picrate.

11. 7. Quantitative Analysis:
a. Using E1CM
1% values.
b. E1CM
1% not available, but raw material is available.
c. Single standard or direct comparison method.
d. Calibration curve method or multiple standard method.
• UV absorption spectroscopy can be used for the quantitative determination of compounds that
absorb UV radiation. This determination is based on Beer’s law which is as follows.
A = log I0 / It = log 1/ T = – log T = abc = εbc
Where ε is extinction co-efficient, c is concentration, and b is the length of the cell that is used in
UV spectrophotometer.
Other methods for quantitative analysis are as follows:
a. calibration curve method
b. simultaneous multicomponent method
c. difference spectrophotometric method
d. derivative spectrophotometric method

12. 8. Qualitative analysis:
• 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
aromatic compounds and aromatic olefins.

13. 9. Preference over to tautomeric form:
• Consider 2-hydroxy pyridine which exists in equilibrium with its
tautomeric form, pyridone-2. The spectra of these two compounds
were found to favour pyridone-2 which is an ᾳ, β-unsaturated ketone
and clearly, the equilibrium is shifted towards the right, i.e., pyridone-
2.

14. 10. Determination of Strength of Hydrogen
Bonding:
• Solvents like water, acetone forms a hydrogen bond with the n-
electron of the carbonyl oxygen.
• Due to this the energy of n-electron in the ground state is lowered
depending on the strength of hydrogen bond.
• Thus n→π* transition of carbonyl compound is shifted towards
shorter wavelength. Hence, by measuring the λmax of the carbonyl
compound in a non-polar and polar protic solvent, the strength of
hydrogen bond can be determined.

15. Example:
n→π* transition of acetone in hexane (at 279 nm) and that in water at
264.5 nm. Blue shift by 14.5nm corresponds to an energy of kcal/mol.
The strength of hydrogen bond between water and acetone is 5kcal/mol.
It is in fair agreement with the known strength of hydrogen bond.

16. 12. Chemical Kinetics:
• Kinetics of reaction can also be studied using UV spectroscopy. The
UV radiation is passed through the reaction cell and the absorbance
changes can be observed.

17. 13. As HPLC Detector:
• A UV/Vis spectrophotometer may be used as a detector for HPLC. The
presence of an analyte gives a response which can be assumed to be
proportional to the concentration.
• For more accurate results, the instrument's response to the analyte in
the unknown should be compared with the response to a standard; as
in the case of calibration curve.

18. 14. Examination of Polynuclear Hydrocarbons:
• Benzene and polynuclear hydrocarbons have characteristic spectra in
the UV and Visible region.
• Thus, the identification of the polynuclear hydrocarbons can be made
by comparison with the spectra of known polynuclear compounds.
• The presence of substituents on the ring, generally, shifts the
absorption maximum to longer wavelength.