UV/VISIBLE SPECTROSCOPY
UV/VIS
Spectroscopy
What does a
UV/Vis Spectrometer
Measure?
UV/VIS
Spectroscopy
Transmittance (T) is defined as the amount of
light passing through the sample solution (P)
divided by the amount of incident radiation (Po)
T = P/Po
In practice Po is estimated by a “blank”
which accounts for reflection and
scattering losses.
UV/VIS
Spectroscopy
As the radiation is
absorbed in the sample,
the total intensity of
radiation is reduced
as it travels through the
sample. This results in a
non-linear relationship
between transmittance
and concentration.
UV/VIS
Spectroscopy
Absorbance (A) is based
on the amount of light
absorbed by the solution
and is defined as the log
of the inverse of the
transmittance.
A = log10(1/T)
UV/VIS
Spectroscopy
Within limits, the relationship
between absorbance and
concentration is linear.
UV/VIS
Spectroscopy
Beer’s law or the Beer-Lambert law
A = bc
 is the molar absorptivity with units of (L/mol-cm)
b is the path length of the sample ie. the inside
cross section of the sample cuvette (cm)
c is the concentration of the compound in solution
in (mol/L)
UV/VIS
Spectroscopy
Increased interaction between the molecules
can affect absorbance at high concentrations.
Electrolytes can also alter the molar absorptivity
of the analyte by electostatic interactions.
Molar absorptivity is also altered by the refractive
index of the solution.
UV/VIS
Spectroscopy
When an analyte associates or dissociates in
solution producing products with different
absorption spectra, deviations from Beer’s law
can be observed if the equilibrium shifts at
different concentrations.
Hindicator
H+ +
Indicator-
UV/VIS
Spectroscopy
Other causes of deviation from Beer’s law:
Radiation is not monochromatic
Stray radiation
Absorbance readings less than 0.10 and higher
than 1.5 can contain significant error and should
be avoided.
UV/VIS
Spectroscopy
Absorbance
Quantification of Two Compounds
Spectra of Compound 1
Spectra of Compound 2
Wavelength
UV/VIS
Spectroscopy
Quantification of Two Compounds
The method to determine the concentrations of
two compounds (a & b) in a mixture involves the
simultaneously solution of the following two
equations:
At wavelength 1 A1 = ea1ca + eb1cb
At wavelength 2 A2 = ea2ca + eb2cb
UV/VIS
Spectroscopy
A = bc
Quantification of Two Compounds
At wavelength 1 A1 = ea1ca + eb1cb
At wavelength 2 A2 = ea2ca + eb2cb
Where ea1, eb1, ea2, eb2 are the molar
absorptivities for the two compounds at
the two wavelengths which are
determined from standard solutions and ca and cb are the concentrations of
the two unknown compounds.
UV/VIS
Spectroscopy
Quantification of Two Compounds
EXAMPLE
Sample
Compound a
Compound b
Absorptivity
at λ465
11636
17949
Absorptivity at
λ540
26579
2667
Absorbance of the mixture at λ465 = 0.870
at λ540 = 0.362
UV/VIS
Spectroscopy
Quantification of two compounds
Set up equations:
(1)
(2)
0.870 = 11636ca + 17949cb
0.362 = 26579ca + 2667cb
multiply equation (2) by 6.73, giving
equation (4):
(3)
(4)
0.870 = 11636ca + 17949cb
2.436 = 178877ca + 17949cb
UV/VIS
Spectroscopy
Quantification of two compounds
Set up equations:
(3)
(4)
0.870 = 11,636ca + 17,949cb
2.436 = 178,877ca + 17,949cb
Subtract (3) from (4), giving equation (5):
(5)
1.566 = 167,240ca
UV/VIS
Spectroscopy
Quantification of Two Compounds
(5)
1.566 = 16724ca
Solve for ca:
(6) ca = 9.36 x 10-6 moles/L = concentration of
compound a
Substitute value from (6) into equation (1) and
solve for cb:
0.870 = 11636(9.36 x 10-6) + 17949cb
0.870 = 0.109 + 17949cb
0.760 = 17949cb
cb = 4.24 x 10-5 moles/L = concentration of
compound b