Quantitative Analysis of UVVis Spectroscopy

Applications of Absorbance
Measurement to Qualitative Analysis
As seen earlier, the broad band absorption
spectra obtained in UV-Vis absorption
spectroscopy is usually featureless and
lacks details that can be used in
qualitative analysis. Therefore, this
technique is mainly a quantitative
technique.
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Plotting Spectral Data

A plot of either the absorbance or
%transmittance against wavelength can
be made. However, the most common
practice is to plot the absorbance versus
wavelength.

Quantitative Analysis
The basis for quantitative analysis in the UVVis relies on Beers law.
Several characteristics of quantitative
measurements using UV-Vis absorption
spectroscopy can be rationalized:
1. Applicability to all types of analytes as far as
they absorb in the UV-Vis region.
2. Moderate sensitivities

3. The relative standard deviation occurs

within 1-3% which reflects good
precision.
4. Easy to perform and convenient.
5. Non absorbing species can also be
determined if they are derivatized with
an absorbing species as the case of
metal ions when complexed to
ligands.

Procedural Details
Selection of Wavelength
The first step in a successful determination
is to find the suitable wavelength for the
analysis. This is accomplished by plotting
the absorbance/wavelength curve.
However, the following points should also
be considered:

1. If an interferences are present, the

wavelength that is far away from
interferences should be selected
2. A wavelength at the maximum of a broad
peak should be preferred to another of a
sharp peak
3. The peak with a maximum peak height is
preferred

An analyst should use his experience and

knowledge to work for the best bargain of
the abovementioned points. Several
factors affect the location of the
wavelength and the absorbance and thus
must be considered. These include the
nature of solvent, the pH of solution,
electrolyte concentration, interferences, as
well as temperature.
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Cleaning and Handling the Cell

First, one should appreciate the use of good quality
matched cells that are free from wearing, etching,
and scratches. In addition, cleaning procedures of
external and internal cell surfaces are also
important. A suggested cleaning procedure
involves moistening a lens paper with methanol
and wiping the external surface, then leaving the
cell to evaporate. The interior of the cell is first
washed with water followed by methanol and the
solvent is also allowed to evaporate. Disposable
polypropylene cuvettes are incompatible with non
polar solvents and formulations having these
solvents should be avoided; or large errors will be
encountered.
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Calibration Curves

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Standard Addition method

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Analysis of Mixtures of
Absorbing Substances

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Analysis of Mixtures of
Absorbing Substances
When the sample solution contains more
than one absorbing species, the
absorbance of the solution will be the sum
of all absorbances:
At = A1 + A2 + A3 + .

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When only two absorbing species are present, the

solution is formidable and is executed by finding
the absorbance of the solution at two wavelength
(wavelength maximum for each analyte):
A = xbcx + ybcy
A = xbcx + ybcy

(1)
(2)

x, x, y, y can be determined from standards

of analytes x and y at
* values obtained are inserted in equations 1 and
2 where two equations in two unknowns can be
easily solved.
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