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Spectroscopy
Electromagnetic Spectrum
Gamma Rays: < 0.01 nm
Hard X-rays: 0.01 0.1 nm
Soft X-rays: 0.1 10 nm
10 180 nmVac. UV:
380 700 nmVisible:
180 380 nmNear UV:
700 1400 nmNear IR:
Mid IR: 1400 15000 nm
Far IR:
15 1000 m
Microwave: 1 10 mm
Radio waves: > 10 mm
/http://nationalatlas.gov/articles/mapping/IMAGES
Molar Absorptivity
We have seen earlier that validation of Beers law is
dependent on the nature of the molar absorptivity. It
was found that the molar absorptivity is influenced by:
1. The wavelength of radiation
2. The refractive index and is thus indirectly related to
concentration
3. Electrostatic interactions taking place in solution; and
thus electronic distribution
4. In rare cases, like methylene blue, the molar
absorptivity is directly dependent on concentration
Molar absorptivities
= 8.7 x 10 19 P A
A: cross section of molecule in
cm2 (~10-15)
P: Probability of the electronic
transition (0-1)
P>0.1-1 allowable transitions
P<0.01 forbidden transitions
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MO Diagram for
Formaldehyde
(CH2O)
H
=
12
=
n=
13
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Conclusions on Electronic
Transitions
There are four different types of electronic
transitions which can take place in
molecules when they absorb UV-Vis
radiation. A * and a n-* are not useful for
reasons discussed earlier. The n-* transition
requires low energy but the molar
absorptivity is also low and transition energy
will increase in presence of polar solvents.
The n-* transition is seldom used in
quantitative UV-Vis spectroscopy.
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Organic Chromophores
Molecules having unsaturated bonds or free
nonbonding electrons that can absorb
radiation of relatively low energy are called
chromophores. Examples include alkenes,
alkynes, ketones, aldehydes, phenyl and
other aromatic species, etc.
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Examples of
UV-Visible Absorptions
LOW!
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UV Absorption Spectroscopy
Transmittance, T = I / I0
Absorbance, A = -log T
Beers Law
A = .L.C
= absorption coefficient
L = path length
C = concentration of analyte
Example Absorption
Spectra shown here
Image:
http://www.cem.msu.edu/~reusch/VirtualText/Spectrpy/spectro.
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Auxochrome is a functional
group that does not absorb in
UV region but
has the effect of shifting
chromophore peaks to longer
wavelength as well
As increasing their intensity.
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max = ~10,000
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Terbium Spectrum
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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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Solvents
We have seen earlier that solvent polarity
affects the absorptivity of the analyte
molecules; due to change in transition
energies. Usually, polar solvents are used
when possible. However, polar solvents like
water or alcohol tend to oliberate the fine
spectral details. Therefore, in cases where
the fine spectral details are really needed (as
in qualitative analysis) a non polar solvent
like hexane should be used. In addition, the
solvent must be optically clear (does not
absorb incident radiation), well dissolve the
sample, and chemically pure
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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 in the range from 10 -4
to 10-6 with possibility to extend this range
under certain conditions
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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:
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Calibration Curves
Usually, a plot of the absorbance of a series of
standards is plotted versus the
concentration. The absorbance of the
unknown is then determined and the
prepared calibration plot is used for the
determination of the analyte concentration.
If the absorbance of the analyte was
located outside the calibration plot, more
standards should be made or the analyte
concentration must be adjusted to occur on
the calibration plot.
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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 + .
The different constituents can be determined if
we build equations equal to the number of
unknowns. However, this procedure, if
manually performed, is impractical due to
lengthy and difficult math involved.
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Derivative
spectroscopy
is excellent
for
determination
of multi
components
in a sample, if
they can be
.resolved
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Applications of Derivative
Spectra
1. Better qualitative analysis and identification of
the number of absorbing species in a sample
2. Accurate determination of max
3. Obtaining spectra in solutions with high
scattering was possible using dual wavelength
instruments
4. Spectral resolution of multi component systems
by measurement at two wavelengths; where the
interferent has identical molar absorptivity while
the analyte does not, can result in good
exclusion of interferences.
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Photometric Titrations
In cases where an analyte reacts with a
reagent so that the analyte, the reagent or
the product absorbs UV-Vis radiation, the
technique can be used for determination
of the analyte by a photometric titration
reaction. Photometric titrations are similar
to conventional visual titrations but
following the course of a photometric
titration occurs with the aid of a UV-Vis
detector, rather than the naked eye.
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Instrumentation
A conventional photometer or
spectrophotometer can be adapted to
performing photometric titrations where
the analyte is placed in the sample cell
which contains a small magnet and is
located on the top of a magnetic stirrer.
The wavelength is selected and the titrant
is added, from a dark burette, gradually
and the absorbance is recorded.
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