APPLICATION OF
UV/VIS
SPECTROSCOPY
Dr Tahir Mahmood
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APPLICATION OF UV/VIS SPECTROSCOPY
CONTENTS:
Detection of extent of conjugation
Detection of functional groups
Determination of the purity of a substance
Identification of unknown compound
Determination of configurations of geometrical isomers
Quantitative analysis
Dissociation constants of acids and bases
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APPLICATION OF UV/VIS SPECTROSCOPY
Detection of extent of conjugation
• The extent of conjugation in the Dienes, Trienes and
Polyenes can be detected with the help of UV spectroscopy.
• The presence of conjugate double bond, decreases the
energy difference between HOMO and LUMO of resulting
diene. As the number of conjugated double bonds is
increased, the gap between highest occupied molecular
orbital (HOMO) and lowest unoccupied molecular orbital
(LUMO) is lowered.
• Therefore, the increase in size of the conjugated system
gradually shifts the absorption maximum (λmax) to longer
wavelength and also increases the absorption. This figure
shows the change in energy of MO on conjugation.
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APPLICATION OF UV/VIS SPECTROSCOPY
Detection of extent of conjugation
Therefore, increase in size of the conjugated system gradually shifts the absorption
maximum (λmax) to longer wavelength and also increases the absorption.
Example:
The effect of increasing conjugation on the absorption spectrum:
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APPLICATION OF UV/VIS SPECTROSCOPY
Detection of extent of conjugation
The effect of increasing conjugation on the absorption spectrum when groups
containing n electrons are conjugated with a π electron group; e.g;
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APPLICATION OF UV/VIS SPECTROSCOPY
Detection of extent of conjugation
Aromatic systems, which contain p electrons, absorb strongly in the ultraviolet:
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APPLICATION OF UV/VIS SPECTROSCOPY
Detection of functional group:
• UV spectroscopy is used to detect the presence or absence of chromophore in the
compound.
• The absence of a band in a particular band can be seen as an evidence for the
absence of a particular functional groups.
• If the spectrum of a compound comes out to be transparent above 200 nm than it
confirms the absence of Conjugation, Carbonyl groups, Benzene or aromatic
compounds and Bromo or Iodo atoms.
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APPLICATION OF UV/VIS SPECTROSCOPY
Detection of functional group:
• Some of the most important chromophore (functional) groups are:
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APPLICATION OF UV/VIS SPECTROSCOPY
Detection of functional group:
Example
The spectrum of functional group benzene λmax 254 and Tolune λmax 261
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APPLICATION OF UV/VIS SPECTROSCOPY
Determination of the purity of substance:
• Purity of a substance can also be determined with the help of UV spectroscopy.
• The absorption of the sample solution is compared with the absorption of the
reference solution. The intensity of the absorption can be used for the relative
calculation of the purity of the sample substance.
• 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 255 nm.
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APPLICATION OF UV/VIS SPECTROSCOPY
Determination of the purity of substance:
The impurities in cyclohexane is also detected by UV spectroscopy.
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APPLICATION OF UV/VIS SPECTROSCOPY
Identification of unknown compounds
• An unknown compound can be identified with the help of UV spectroscopy.
• The spectrum of unknown compound is compared with the spectrum of a
reference compound and if both the spectrums coincide then it confirms the
identification of the unknown substance.
• UV spectroscopy is useful in the structure elucidation of organic molecules,
the presence or absence of unsaturation, the presence of hetero atoms etc.
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.
• There are many examples where the structure of an organic compound can be
determined only by UV spectroscopy.
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APPLICATION OF UV/VIS SPECTROSCOPY
Identification of unknown compounds
For example:
• The following two isomeric compounds can be
distinguished on the basis of their UV spectra.
Compound I contain a conjugated diene system,
which shows an absorption maximum at 232 nm;
the calculated value is also 232 nm. Compound II
absorbs near 175 nrn, the value for a single
double bond, because its two double bonds are
not conjugated.
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APPLICATION OF UV/VIS SPECTROSCOPY
Determination of configuration of geometric isomers:
• It is observed that cis-alkenes absorb at different wavelength than the trans-alkenes.
• The two isomers can be distinguished with each other when one of the isomers has
non-coplanar structure due to steric hindrances.
• The cis-isomer suffers distortion and absorbs at lower wavelength as compared to
trans-isomer.
• If the steric effect hinders the coplanarity of a system, delocalization of pi electrons
will be hindered; and as a consequence, the length of conjugation will decrease.
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APPLICATION OF UV/VIS SPECTROSCOPY
Determination of configuration of geometric isomers:
For example:
As trans-cinnamic acid absorbs at a longer wavelength (λrnax
= 272 nm, ԑrnax = 15,900) than cis-cinnamic acid (λrnax = 268
nm, ԑrnax = 10,700). This is because the former has a coplanar
structure and delocalization of π electrons occurs completely
across the molecule while in cis isomer the delocalization is
hindered because it loses coplanarity owing to the overlap of
ortho hydrogen of the phenyl group and the carboxylic group
as shown in figure;
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APPLICATION OF UV/VIS SPECTROSCOPY
Quantitative Analysis:
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:
𝑰𝟎
𝟏
𝑨 = 𝒍𝒐𝒈 = 𝒍𝒐𝒈 = − 𝒍𝒐𝒈 𝑻 = 𝒂𝒃𝒄 = ∈ 𝒃𝒄
𝑰𝒕
𝑻
Where “A” is absorbance of the sample, ε is extinction co-efficient (molar
absorptivity), 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.
• calibration curve method
• simultaneous multicomponent method
• difference spectrophotometric method
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• derivative spectrophotometric method
APPLICATION OF UV/VIS SPECTROSCOPY
Quantitative Analysis:
For example:
The molar absorptivity of methyl vinyl ketone dissolved in hexane is 14,000 M-1
cm-1 at 219 nm. The solvent in which the sample is dissolved is reported because
molar absorptivity is not exactly the same in all solvents. Therefore, the UV
spectrum of methyl vinyl ketone in hexane would be reported as λmax = 219 nm
(ԑmax = 14,000, hexane).
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APPLICATION OF UV/VIS SPECTROSCOPY
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 spectra.
• Qualitative analysis through spectrophotometric methods achieve fast and
accurate result using only small sample quantity.
• Qualitative analysis has proven highly useful in major forms of organic
compounds and helps to ensure patient health and safety.
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APPLICATION OF UV/VIS SPECTROSCOPY
Qualitative Analysis:
For example:
UV absorption spectroscopy is generally used for characterizing aromatic compounds
and aromatic olefins.
One of the most common medication used around the word is paracetamol. Qualitative
analysis assures that the measurement process of active ingredient is precise and free of
impure compounds. The result from these methods provide the data that can be used
for the quality control and consistency in the product formation. Having precise and
accurate result assures that the proper dosage and measurements are used for the
intended application.
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APPLICATION OF UV/VIS SPECTROSCOPY
Dissociation constants of acids and bases:
• The pKa of a compound can be determined by UV spectroscopy if either the
acidic form or the basic form of the compound absorbs UV radiation.
𝑨
𝒑𝑯 = 𝒑𝑲𝒂 + 𝒍𝒐𝒈
𝑯𝑨
𝐅𝐫𝐨𝐦 𝐭𝐡𝐞 𝐚𝐛𝐨𝐯𝐞 𝐞𝐪𝐮𝐚𝐭𝐢𝐨𝐧, 𝐭𝐡𝐞 pKa value can be calculated if the ratio of [A-] /
[HA] is known at a particular pH and the ratio of [A-] / [HA] can be determined
spectrophotometrically from the graph plotted between absorbance and
wavelength at different pH values.
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APPLICATION OF UV/VIS SPECTROSCOPY
Dissociation constants of acids and bases:
For example:
The phenolate ion has a λmax at 287 nm. If the absorbance at 287 nm is
monitored as a function of pH, the pKa of phenol can be ascertained by
determining the pH at which exactly one-half the increase in absorbance has
occurred. At this pH, half of the phenol has been converted into phenolate ion,
so this pH is equal to the pKa of the compound.
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