PHR312 –Pharmaceutical Analysis-II

Class 6
UV and Visible Spectroscopy
Class Outlines

Spectrum

Sample compartment

Selection of wavelength

Solvent & Temperature Effect

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 Instrumentation : Sample compartment (cells)
For UV-Visible spectroscopy, a liquid sample is usually
contained in a cell called a cuvette.
Glass is suitable for visible but not for UV spectroscopy because it
absorbs UV radiation. Quartz can be used in UV as well as in visible
spectroscopy
Long pathlength 1 cm pathlength cuvet
Common UV-vis Spectroscopic Cell
Opaque
Face

Transparent
Face

1 cm 1 cm
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Shazid _Pharmaceutical Analysis pathlength (b) 3
 Instrumentation : Spectrum
Instrumentation and Spectra
The Spectrum
1. The y-axis of the spectrum is in absorbance, A

2. From the spectrometers point of view, absorbance is the inverse of

transmittance: A = log10 (I0/I) = 1/T

3. From an experimental point of view, three other considerations must be

made:
i. a longer path length, l through the sample will cause more
UV light to be absorbed – linear effect

ii. the greater the concentration, c of the sample, the more UV

light will be absorbed – linear effect

iii.some electronic transitions are more effective at the

absorption of photon than others – molar absorptivity, e
this may vary by orders of magnitude…
4. These effects are combined into the Beer-Lambert Law: A = e c l
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Instrumentation : Selection of Wavelength
Absorbance measurements are always carried out at fixed wavelength (using
monochromatic light). When a wavelength is chosen for quantitative analysis,
three factors should be considered

1. Wavelength should be chosen to give the highest possible sensitivity.

This can be achieved by selecting max or in general the wavelengths at
which the absorptivity is relatively high.

λmax

max - wavelength where maximum absorbance occurs

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Instrumentation : Selection of Wavelength
By performing the analysis at such wavelengths, it will be sure that
the lowest sample concentration can be measured with fair accuracy.

For example, the lowest sample concentration (10-5 M) can be

measured with good accuracy at max, while at other wavelength (1), it may
not be detected at all.
10-2 M
10-3 M

Absorbance
10-4 M
5x10-5 M
10-5 M

max 1
wavelength
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Instrumentation : Selection of Wavelength
2. It is preferable to choose the wavelength at which the absorbance will not
significantly change if the wavelength is slightly changed, i.e., A /  is minimum.

Example: At a wavelength corresponding to broad horizontal band on the spectrum

(band A), the radiation is mainly absorbed to the same extent (A /  zero).

However on a steep portion of the spectrum (band B), the absorbance will change
greatly if the wavelength is changed (A /  is large) . Thus on repeating the
absorbance measurements, you might get different readings and the precision of the
measurements will be poor.
Band A
A Broad horizontal
bands
Band B
Absorbance

A shoulder

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  m wavelength
Instrumentation : Selection of Wavelength
3. If the solution contains more than absorbing species, the wavelength
should be chosen, whenever possible, in region at which the other species
does not absorb radiation or its absorbance is minimum. By this way, the
second species does not interfere in the determination.

sample
Absorbance

Interfering
species

m wavelength

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UV and Visible Spectroscopy: Applications

1. Detection of Impurities
2. Structure elucidation of organic compounds.
3. Quantitative analysis
4. Qualitative analysis
5. As HPLC (High Performance Liquid Chromatography) detector

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Solvent effects

1) If the group is more polar in the ground state than in

the excited state >then increasing polarity of the solvent >
absorption is shifted to lower wavelength.

Because, n -> π* transition, the n state is much more

easily stabilized by polar solvent effects (H-bonds and
association)

2) If the group is more polar in the excited state > then

absorption is shifted to longer wavelength >with increasing
polarity.

Because, the non-bonding electrons in the excited state.

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Solvent effects

Summary: Increase in polarity of the solvent generally shi

fts n -> π* and n -> σ* bands to shorter wavelengths and
π -> π* band to longer wavelengths.

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Solvent effects
O

n → π* Hypsochromic shift
4-methyl-3-penten-2-one
Methanol Heptane
λmax
λmax

λmax λmax

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Temperature effects

Dodecapentaenoic acid

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UV and Visible Spectroscopy: Definition

Spectroscopy
Spectroscopy involves the interaction between EMR (light) and the substance
under study.
Absorbance
Absorbance is a measure of the absorption of radiation by a sample.
A=Ɛcl =log (I0/I)
In UV-vis spectrum
Sharp peaks are seldom observed, but broad bands (peaks) are observed. The
reason is that vibrational and rotational effects are superimposed on electronic
transitions.
Effects of UV-vis radiation on compound
When a substance under study is subjected to the action of UV and Visible light,
then it cause changes in the electronic energy levels within the molecule.
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Woodward-Fieser rules
The more the conjugation of the π-electron system within the molecule, the higher the
wavelength of light it can absorb. Robert Burns Woodward and Louis Fieser put down
a set of rules which allows one to calculate the wavelength of maximum absorption
(λmax) for a molecule empirically.

These sets of rules to calculate the wavelength of maximum absorption or λmax of a

compound in the ultraviolet-visible spectrum, based empirically have been called the
Woodward-Fieser rules or Woodward’s-rules.

This quantification is referred to as the Woodward-Fieser Rules which we will apply to

various types of double bonds:
1. Alicyclic dienes or, dienes contained in an open chain system
2. Homo-annular conjugated double bonds
3. Hetero-annular conjugated double bonds
4. Exocyclic and Endocyclic conjugated double bonds

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