Transcript Applications of UV-Vis Spectroscopy 1

Applications of UV-Vis
Spectroscopy
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The Molar Absorptivity
As seen earlier: validation of Beer’s law is
dependent on the nature of the molar
absorptivity.
Factors affect the molar absorptivity:
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.
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For Beer’s law to be valid:
The molar absorptivity is supposed to be constant.
The molar absorptivity is a measure of the ability of
an analyte to absorb light at a specified wavelength.
Therefore:
The value of the molar absorptivity is crucial for the
sensitivity of an analysis.
Molar absorptivity usual range:
From zero (for a nonabsorbing species) to 105 (for
highly absorbing species).
For quantitative analysis:
A value of at least 103 is necessary for a reasonable
quantitative analytical determination of an analyte .
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Absorbing Molecular Species
 What happened if an incident beam of radiation
having a suitable wavelength hits a molecule:
 Absorption of a photon takes place and the molecule
becomes excited.
 Excited molecules will lose excitation energy as heat
or photons (luminescence).
 Absorption of UV-Vis radiation is capable of affecting
the excitation of bonding electrons and other valence
electrons.
 Types of electrons that affected by excitation of
electrons in chemical by bonds absorption of UV-Vis
radiation of a suitable wavelength.
(1) ,  and n electrons (mostly organics)
(2) d and f electrons (inorganics/organometallics)
(3) charge-transfer (CT) electrons
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
Molecular UV-Vis Spectroscopy: Theory
Molecular energy levels and absorbance wavelength:
   * and   * transitions:
high-energy, accessible in vacuum UV (max <150 nm). Not usually
observed in molecular UV-Vis.
 n  * and   * transitions:
non-bonding electrons (lone pairs), wavelength (max) in the 150-250 nm
region.
 n  * and   * transitions:
 most common transitions observed in organic molecular UV-Vis,
observed in compounds with lone pairs and multiple bonds with max =
200-600 nm.
Molecules Containing , , and n Electrons
1. Molecules with  Bonds Only
Let us start with a molecule like CH4 and then
expand our discussion to more complex
molecules:
 All bonds in methane are  bonds.
The only transition possible is the -* transition.
 However, the -* transition requires very high
energy which occurs in vacuum UV (125-185 nm).
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It is not wise to think of doing UV measurements on
molecular species in the vacuum UV region (125-185
nm) for five important reasons:
1)The high energy required can cause rupture of the 
bonds and breakdown of the molecule .
2)Air components absorb in vacuum UV which limits the
application of the method .
3)Working in vacuum UV requires special training and
precautions which limit wide application of the method.
4)Special sources and detectors, other than those
described earlier, must be used .
5)All solvents contain  bonds.
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2. Molecules with n Electrons
Electrons in the valence shell that are not used up in
chemical bonds are referred to as nonbonding electrons
(n electrons).
Consider a molecule like ammonia:
The dots on nitrogen is a symbol for two nonbonding
electrons.
Now, the type of transitions observed in this molecule
can be listed as:
-*
n-*
We have seen earlier that the -* transition is not useful
in practical UV-Vis spectroscopy.
The other transition (n-*) is of lower energy and should
be further discussed.
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The absorption wavelength for a n- *transition occurs at
about 185 nm.
Unfortunately, most solvents absorb at this region.
For example:
The most important solvent is, undoubtedly, water:
Has two pairs of nonbonding electrons that will strongly
absorb as a result of the n- *transitions.
This precludes the use of this transition for studies in
aqueous and other solvents with nonbonding electrons.
In summary:
It is also impractical to think of using UV-Vis absorption
spectroscopy to determine analytes based on a n- *
transition .
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3. Molecules with  Bonds.
Absorption of radiation by an alkene, containing a double
bond:
Can result in -* or -* transitions.
We have seen that a -* transition is not useful.
The -* is useful since:
It requires reasonable energy.
Has good absorptivity.
A molecule having , , and n electrons can show all types of
transitions possible in UV-Vis spectroscopy.
For example:
The aldehyde molecule shows all these transitions since it
contains , , and two pairs of n electrons.
Two transitions are possible for the n electrons:
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n-*
n-*
n-* transition:
It is not very useful due to
absorbance from solvents
and other frequently used
additives which have n
electrons.
n-* transition:
Requires very little energy
and seem to be potentially
useful.
However,
unfortunately,
the absorptivity of this
transition is very small
which precludes its use for
sensitive
quantitative
analysis .
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Effect of Solvent Polarity on Absorption Wavelength
The molar absorptivity for a n-* transition:
 Rather small (10-100 L mol-1 cm-1)
 Energy required for transition is affected by solvent
polarity.
Effect of presence of a polar solvent on n-* :
 Nonbonding electrons will interact with protic
solvents to form hydrogen bonds.
 The solvation of n electrons is the result.
So:
 lowers the energy of the orbitals holding the n
electrons.
 Partial stabilization of the polar * is also observed
but to a much lower extent than the n electrons.
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Overall Conclusion (hypsochromic shift or blue shift):
 A net increase in energy required for a n-* transition
is thus observed in protic solvents; like water or
alcohols.
 Therefore, an increase in energy will reflect a
decrease in transition wavelength.
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Effect of presence of a polar solvent on -* :
 The -* transition is affected in an opposite manner with solvent
polarity.
 In presence of a polar solvent, the more polar * orbital will be more
stabilized than the  orbital leading to a net decrease in the
transition energy.
Overall Conclusion: (bathochromic shift (red shift)):
This results in an increase in transition wavelength.
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Spectral nomenclature of shifts
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Molecular UV-Vis Spectroscopy: Terminology
 UV-Vis Terminology
– Chromophore:
group
a UV-Visible absorbing functional
– Bathochromic shift (red shift):
to longer
wavelengths
– Auxochrome: a substituent on a chromophore that
causes a red shift
– Hypsochromic shift (blue shift):
to shorter
wavelengths
– Hyperchromic shift: to greater absorbance
– Hypochromic shift: to lesser absorbance
Conclusions on Electronic Transitions
A -* and a n-* transition :
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.
 Is seldom used in quantitative UV-Vis spectroscopy.
The -* transition:
The most frequently used transition is for the following reasons:
1. The molar absorptivity for the -* transition is high allowing
sensitive determinations.
2. The energy required is moderate, far less than dissociation
energy.
3. In presence of the most convenient solvent (water), the
energy required for a -* transition is usually smaller.
It is therefore primitive that an analyte to be determined by UVVis absorption spectroscopy be of unsaturated nature (or!!).
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Applications of UV-Vis
Spectroscopy
Lecture 26
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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.
Auxochromes:
Is a functional group that does not absorb by
itself (like -NH2 and –OH), but its presence in a
molecule can increase absorption and usually
results in a red-shift.
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The human eye sees the complementary color to that
which is absorbed
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Chromophore
Example
Excitation
λmax ,nm
ε
Solvent
C=C
Ethene
π g π*
171
15,000
hexane
C≡C
1-Hexyne
π g π*
180
10,000
hexane
C=O
Ethanal
n g π*
π g π*
290
180
15
10,000
hexane
hexane
C-X X=Br
X=I
Methyl
bromide
Methyl
Iodide
n g σ*
n g σ*
205
255
200
360
hexane
hexane
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Effect of Conjugation of Chromophores:
 As conjugation is increased in a molecule, more
delocalization (stability) of the  electrons results.
 The effect of this delocalization is to decrease the *
molecular orbital.
 The result is a decrease in transition energy from -* and
thus a red or bathochromic shift.
 The molar absorptivity will increase in this case and better
quantitative analysis will be achieved.
 In cases of introduction of more unconjugated double
bonds, the molar absorptivity will increase as well
depending on the number of the double bonds.
For example:
At 185 nm,1-hexene has a molar absorptivity of about 10,000
L mol-1 cm-1
But hexa-1,4-diene has a molar absorptivity of twice as much
as 1-hexene.
 However, when the double bonds are conjugated as in hexa1,3-diene the molar absorptivity is about 21,000 L mol-1 cm-1.
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Rule of thumb for conjugation
If greater then one single bond apart:
-e are relatively additive (hyperchromic shift)
-  constant
CH3CH2CH2CH=CH2
max= 184 emax = ~10,000
CH2=CHCH2CH2CH=CH2 max=185 emax = ~20,000
If conjugated
- shifts to higher ’s (red shift)
H2C=CHCH=CH2
max=217 emax = ~21,000
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Effect of Aromaticity of Chromophores
Aaromaticity results in extraordinarily high degree of
delocalization of electrons and thus stabilization of the p*.
 If we assume a molar absorptivity of about 10,000 L mol-1
cm-1 for each double bond.
 We expect the sum of the three double bonds in benzene to
be just above 30,000 L mol-1 cm-1 (at 185 nm).
 But actually the value is about 60,000 L mol-1 cm-1 due to
increased delocalization as a result of aromaticity.
It is therefore advantageous to use UV-Vis
absorption spectroscopy for determination of
compounds having aromatic character.
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Absorption by Inorganic Groups
Inorganic groups containing double bonds absorb in
the UV-Vis region.
The most transitions are a result of n-* transitions:
In nitrate (313 nm),
Carbonate (217 nm),
Nitrite (280 and 360 nm)
Azide (230 nm)
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Absorption Involving d and f Orbitals:
 Many transition metals have colored solutions and are also
colored in the solid state.
 The transition metals have some of their d orbitals empty
where a d-d transition can occur.
 The d-d transitions require excitation energy in the UV-Vis
region.
 The direct interaction of the d electrons with ligands around
the transition metal results in a spectrum of broad band
nature.
 On the other hand, inner transition elements show transitions
by absorption of UV-Vis radiation (f-f transitions).
 Since the electrons in the f orbitals are far inside the metal
orbitals and are screened by electrons in orbitals of higher
principal quantum numbers, f-f electronic transitions will not
be affected by the nature of ligands or solvent around the
inner transition metals.
 Therefore, the spectra of inner transition metals have narrow
bands.
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The crystal field theory:
 Usually used to explain splitting of the d orbital energy so
that a transition from a lower energy d orbital electron can
be excited to a higher energy d orbital.
 The theory will be described for a transition metal with six
ligands or molecules of water around it.
 An octahedral (only this case will be discussed)
arrangement of these ligands is most appropriate where
ligands will be located at the z axis and at the x and y axis
(will repel electronic cloud in the dz 2and dx-2y 2orbitals and
thus will make these two orbitals to have higher energies).
 The other four ligands will be arranged in between axis
(dxy ,dxz ,and dyz )which will increase their energies but to a
lower extent.
 The net result is the splitting of the degenerate d orbital
into two groups of d orbitals of different energies (the
energy difference is referred to as D).
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Transition metal ions
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Charge Transfer Absorption:
 When a ligand permanently donates an electron to a
metal ion, a charge transfer is said to take place.
 The net outcome of the process is an oxidation
reduction phenomenon occurring within the complex.
 An example is the reaction of Fe3+ with thiocyanate
where the product is an excited species with neutral
thiocyanate and Fe3+.
 In less common situations, the transfer of electrons can
take place from the electron deficient metal ion to the
ligand.
 An example is the Fe2+ or Cu+ complexes with 1,10phenanthroline where Fe2+ and Cu+ metal ions donate
electrons to 1,10-phenanthroline.
 The complex will then have Fe3+ and Cu2+ ions. Charge
transfer complexes are of special interest their molar
absorptivities are unusually high; allowing very
sensitive determinations.
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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.
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.
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Applications of UV-Vis
Spectroscopy
Lecture 27
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Solvents
 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
remove the fine spectral details.
So, a non polar solvent like hexane should be used:
 To obtain the fine spectral details which really needed
(as in qualitative analysis).
Addition conditions of solvents:
1. The solvent must be optically clear (does not absorb
incident radiation).
2. Well dissolve the sample.
3. Chemically pure.
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Quantitative Analysis
The basis for quantitative analysis in the UV-Vis relies on
Beer’s 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.
3. The relative standard deviation occurs within 1-3%
which reflects good precision.
4. Easy to perform and convenient.
5. Can be used for quantitative analysis in liquid
chromatographic separations.
6. Non absorbing species can also be determined if they
are derivatized with an absorbing species as the case of
40metal ions when complexed to ligands.
Procedural Details
1. 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 more than an absorption maximum is available, the
wavelength far from the instrument extremes should
be preferred
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
4. If interferences are present, the wavelength that is far
away from interferences should be selected
5. Working in the visible region should be preferred
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2. 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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3. 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 standars should be made or the
analyte concentration must be adjusted to occur on the
calibration plot.
 We have seen earlier that it is not allowed to theoretically
extrapolate or interpolate a calibration plot.
 It should also be appreciated that the composition of
standard solutions must approximate that of the sample
solution.
 In cases where the sample composition is not clear, the
method of standard addition should be used.
 The slope of the linear calibration plot is the molar
absorptivity when the path length is 1.00 cm. Larger slopes
mean higher sensitivities.
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4. Standard Addition method
 The use of this method provides better correlations
between absorbances of standards and sample due to
constant matrix effects.
 The method involves addition of the same sample volume to
a set of tubes or containers.
 Variable volumes of a standard are added to the tube set
followed by completion to a specific volume.
 Now, all tubes contain the same amount of sample but
different concentrations of analyte.
 A plot is then made for the volume of standard and
absorbance.
 This plot will have an intercept (b) with the y axis and a
slope equals m.
 The concentration of the analyte can be determined by the
relation:
Cx = bCs/mVx
Where, Cx and Vx are concentration and volume of analyte and
Cs is the concentration of standard.
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Conc. Of Un (if Ab. is
taken versus conc.
of standard
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Cx = bCs/mVx
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Two points calibrations:
One can only use two points to get the analyte
concentration using the relation:
Cx = AxCsVs/(At –Ax)Vx
Where:
Ax is the absorbance of the analyte solution brought to a
final volume Vt .
At is the total absorbance of the solution (final volume is
Vt) containing same amount of analyte and a volume
of the standard (Vs).
 However, this procedure assumes that both points
occur at the linear portion of the calibration plot.
 In case where this is not true, an error will be
encountered.
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5. 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 especially when more
than two components are present.
Remember that we need to calculate n2 values for e where n is
the number of components.
For two unknowns y and X: (4 e values are needed)
A’ = ex’bcx + ey’bcy
(1)
A” = ex”bcx + ey”bcy
(2)
ex’, ex”, ey’, ey” can be determined from standards of analytes x
and y at ’, ”
and values obtained are inserted in equations 1 and 2 where
two equations in two unknowns can be easily solved.
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6. Derivative UV-Vis Absorption Spectroscopy
In this technique:
 DA/D is plotted against the wavelength.
 The derivative spectrum contains sharper peaks where
better location of peaks and wavelengths maxima can be
achieved.
 Not only the first derivative of the absorbance spectrum
can be obtained but up to the fourth derivative is
possible.
 However, as the differentiation order increases, the
noise increases as well and if a lower derivative is fine,
going to higher derivatives is a waste of time and effort.
 Derivative spectroscopy is excellent for determination of
multi components in a sample, if they can be resolved.
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Obtaining a Derivative Spectrum
 Derivative spectra can either be recorded on time or by
manipulation
of
data
obtained
in
an
absorbance/wavelength spectrum.
 The later is easy and can be made by simple electronic or
mathematic operations; by taking DA for a fixed D (few
nm) and plotting the data versus wavelength.
 However, the most common on line instrumental
recording of derivative spectra involves the use of a dual
wavelength instrument with two monochromator
operated at the same speed but with a lag of few nm from
each other.
 A chopper will sequentially pass the beams from both
monochromators and thus their difference divided by the
constant nm lag value is recorded versus the average
wavelength.
 The following schematic is helpful to visualize how this is
accomplished:
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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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Photometric Titration Curves:
 A plot of absorbance versus titrant volume is
called a photometric titration curve.
 The titration curve is supposed to consist of
two linear lines intersecting in a point
corresponding to the end point of the reaction.
 The absorbance reading should be corrected
for volume where as a titrant is added to the
reaction mixture the absorbance will change.
 Several shapes of photometric titration curves
can be identified depending on the reaction.
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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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Advantages and Applications of Photometric Titrations
1. Usually, photometric titrations are more accurate than visual
titrations.
2. Photometric titrations are faster than visual titrations as only few
points at the beginning and end of the titration is necessary.
Extrapolation of the straight lines will intersect at the end point.
3. Titration reactions that are slow at the end point can not be
performed by visual titrations but are well suited for photometric
titrations.
Only few points at the beginning and end of the titration, well away
from the equivalence point where the reaction is slow, are
necessary.
Extrapolation of the straight lines will intersect at the end point.
Therefore, dilute solutions or weak asids and bases can be also
titrated photometrically.
4. Mixtures can be easily titrated using photometric titrations if
absorbance is measured at the absorption wavelength of one of
the two analytes. In addition, if both analytes give colored
products but with different molar absorptivities, they can be
determined photometrically.
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For example:
Figure a below shows the EDTA titration of Bi+3 and Cu+3 at the
wavelength of the Cu-EDTA where the Bi-EDTA does not absorb.
Figure b shows a hypothetical reaction where two products absorb
incident radiation.
The first substance (x) reacts first and followed by the second one
which has a lower molar absorptivity.
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Photoacoustic Spectroscopy (PAS)
 UV-Vis spectra of solid, semisolid, and turbid samples
can be obtained by photoacoustic spectroscopy,
although PAS is more important in infrared absorption
spectroscopy.
 The technique of photoacoustic spectroscopy involves
subjecting the sample in a photoacoustic cell
(containing a gas) to a chopped beam of radiation.
 If the sample absorbs the incident wavelength, as the
chopper rotates, the sample emits the absorbed energy
as heat.
 Periodic absorption and reemission causes periodic
fluctuations in the gas temperature and thus pressure.
 If the chopper rotates in a rate that occurs in the
acoustical range, the fluctuations in pressure can be
detected by a sensitive microphone.
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Applications of Photoacoustic Spectroscopy
1. Recording UV-Vis spectra of solids and
turbid solutions. Samples as complex as
whole blood were tested by PAS.
2. Study of the absorption characteristics of
polymers, paints archeological samples.
3. Spectra of samples on surfaces, like silica
thin layer chromatographic plates, can be
recorded.
3. In the IR region, qualitative analysis is
possible to a variety of samples.
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