UV-Vis Absorption
Spectroscopy
Lecture 22
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Measurement of Transmittance and
Absorbance:
The power of the beam transmitted by the analyte
solution is usually compared with the power of the
beam transmitted by an identical cell containing
only solvent. An experimental transmittance and
absorbance are then obtained with the equations.
P0 and P refers to the power of radiation after it has
passed through the solvent and the analyte.
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Beer’s law and mixtures
• Each analyte present in the solution absorbs
light!
• The magnitude of the absorption depends on
its e
• A total = A1+A2+…+An
• A total = e1bc1+e2bc2+…+enbcn
• If e1 = e2 = en then simultaneous determination
is impossible
• Need to measure A at nl’s (get 2n e’s) to solve
for the concentration of species in the mixture
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Limitations to Beer’s Law
• Real limitations
• Chemical deviations
• Instrumental deviations
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1.
a.
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Real Limitations
Beer’s law is good for dilute analyte
solutions only. High concentrations (>0.01M)
will cause a negative error since as the
distance between molecules become smaller
the charge distribution will be affected which
alter the molecules ability to absorb a
specific wavelength. The same phenomenon
is also observed for solutions with high
electrolyte concentration, even at low analyte
concentration. The molar absorptivity is
altered due to electrostatic interactions.
b.
In the derivation of Beer’s law we have
introduced a constant (e). However, e is
dependent on the refractive index and the
refractive index is a function of
concentration. Therefore, e will be
concentration dependent. However, the
refractive index changes very slightly for
dilute solutions and thus we can practically
assume that e is constant.
c.
In rare cases, the molar absorptivity
changes widely with concentration, even at
dilute solutions. Therefore, Beer’s law is
never a linear relation for such compounds,
like methylene blue.
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2.
Chemical Deviations
This factor is an important one which
largely affects linearity in Beer’s law. It
originates when an analyte dissociates,
associates, or reacts in the solvent, or
one of matrix constituents. For
example, an acid base indicator when
dissolved in water will partially
dissociate according to its acid
dissociation constant:
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Chemical deviations from Beer’s law for
unbuffered solutions of the indicator Hln.
Note that there are positive deviations at 430
nm and negative deviations at 570 nm. At 430
nm, the absorbance is primarily due to the
ionized In- form of the indicator and is
proportional to the fraction ionized, which
varies nonlinearly with the total indicator
concentration. At 570 nm, the absorbance is
due principally to the undissociated acid Hln,
which increases nonlinearly with the total
concentration
.
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Calculated Absorbance Data for Various Indicator
Concentrations
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3.
a.
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Instrumental Deviations
Beer’s law is good for monochromatic
light only since e is wavelength dependent. It
is enough to assume a dichromatic beam
passing through a sample to appreciate the
need for a monochromatic light. Assume that
the radiant power of incident radiation is Po
and Po’ while transmitted power is P and P’.
The absorbance of solution can be written
as:
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The effect of polychromatic radiation on
Beer’s law. In the spectrum at the top,
the absorptivity of the analyte is nearly
constant over Band A from the source.
Note in the Beer’s law plot at the
bottom that using Band A gives a linear
relationship. In the spectrum, Band B
corresponds to a region where the
absorptivity shows substantial changes.
In the lower plot, note the dramatic
deviation from Beer’s law that results.
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Therefore, the linearity between
absorbance and concentration breaks
down if incident radiation was
polychromatic. In most cases with UVVis spectroscopy, the effect small
changes in wavelengths is insignificant
since e differs only slightly; especially
at the wavelength maximum.
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b.
Stray Radiation
Stray radiation resulting from scattering or
various reflections in the instrument will
reach the detector without passing through
the sample. The problem can be severe in
cases of high absorbance or when the
wavelengths of stray radiation is in such a
range where the detector is highly sensitive
as well as at wavelengths extremes of an
instrument. The absorbance recorded can be
represented by the relation:
A = log (Po + Ps)/(P + Ps)
Where; Ps is the radiant power of stray
radiation.
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