Applications of UV-Vis
Spectroscopy
1
Molar Absorptivity
We have seen earlier that validation of Beer’s 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
2
Molar absorptivities
e = 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
3
The molar absorptivity, however, is supposed
to be constant for Beer’s law to be valid. 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. The value of the molar absorptivity
can usually 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
4
Visible Absorption Spectra
5
Molecules Containing p, s, and n
Electrons
1. Molecules with s Bonds Only
Let us start with a molecule like CH4 and then expand our
discussion to more complex molecules:
All bonds in methane are s bonds and the only transition
possible is the s-s* transition. However, the s-s*
transition requires very high energy which occurs in
vacuum UV. 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:
6
1. The high energy required can cause rupture of
the s 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 s bonds
7
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 line on nitrogen is a symbol for two
nonbonding electrons. Now, the type of
transitions observed in this molecule can be
listed as:
a.
s-s*
b.
n-s*
8
We have seen earlier that the s-s* transition is not
useful in practical UV-Vis spectroscopy but the other
transition (n-s*) is of lower energy and should be
further discussed. The absorption wavelength for a
n-s* transition occurs at about 185 nm where,
unfortunately, most solvents absorb. For example,
the most important solvent is, undoubtedly, water
which has two pairs of nonbonding electrons that
will strongly absorb as a result of the n-s*
transitions; which 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-s*
transition.
9
3. Molecules with p Bonds
Absorption of radiation by an alkene, containing a
double bond, can result in s-s* or p-p* transitions.
We have seen that a s-s* transition is not useful but
on the other hand, the p-p* turned out to be very
useful since it requires reasonable energy and has
good absorptivity. A molecule having s, p, and n
electrons can show all types of transitions possible
in UV-Vis spectroscopy. For example, an aldehyde
molecule shows all these transitions since it
contains s, p, and two pairs of n electrons.
•
10
MO Diagram for
Formaldehyde
(CH2O)
H
C
O
H
s=
11
p=
n=
12
We have seen that a n-s* is not very useful
due to absorbance from solvents and
other frequently used additives which have
n electrons. The n-p* 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.
13
Singlet vs. triplet
• In these diagrams, one electron has been excited
(promoted) from the n to p* energy levels (non-bonding to
anti-bonding).
• One is a Singlet excited state, the other is a Triplet.
14
Effect of Solvent Polarity on
Absorption Wavelength
The molar absorptivity for a n-p* transition is
rather small (10-100 L mol-1 cm-1) and the
energy required for transition is affected by
solvent polarity. In presence of a polar
solvent, nonbonding electrons will interact
with protic solvents to form hydrogen bonds.
The solvation of n electrons is the result;
which lowers the energy of the orbitals
holding the n electrons. Partial stabilization
of the polar p* is also observed but to a much
lower extent than the n electrons.
15
A net increase in energy required for a n-p*
transition is thus observed in protic
solvents; like water or alcohols. Therefore,
an increase in energy will reflect a decrease
in transition wavelength, or what is called
hypsochromic shift or blue shift.
On the other hand, a p-p* transition is affected
in an opposite manner with solvent polarity.
In presence of a polar solvent, the more polar
p* orbital will be more stabilized than the p
orbital leading to a net decrease in the
transition energy. This results in an increase
in transition wavelength or what is called a
bathochromic shift (red shift).
16
Spectral nomenclature of shifts
17
Optically clear solvent concept
18
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 s-s* and a n-s* are not useful for
reasons discussed earlier. The n-p* transition
requires low energy but the molar
absorptivity is also low and transition energy
will increase in presence of polar solvents.
The n-p* transition is seldom used in
quantitative UV-Vis spectroscopy.
19
The most frequently used transition is the p-p*
transition for the following reasons:
a.
The molar absorptivity for the p-p*
transition is high allowing sensitive
determinations.
b.
The energy required is moderate, far less
than dissociation energy.
c.
In presence of the most convenient
solvent (water), the energy required for a pp* transition is usually smaller.
It is therefore primitive that an analyte to be
determined by UV-Vis absorption
spectroscopy be of unsaturated nature.
20
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.
21
Examples of
UV-Visible Absorptions
LOW!
22
UV-Visible Absorption Chromophores
23
Typical UV Absorption Spectra
Chromophores?
24
The effects of substitution
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.
25
Effect of Conjugation and
Aromaticity of Chromophores
As conjugation is increased in a molecule,
more delocalization (stability) of the p
electrons results. The effect of this
delocalization is to decrease the p*
molecular orbital. The result is a decrease
in transition energy from p-p* and thus a
red or bathochromic shift. The molar
absorptivity will increase in this case and
better quantitative analysis will be
achieved.
26
Rule of thumb for conjugation
If greater then one single bond apart
- e are relatively additive (hyperchromic
shift)
- l constant
CH3CH2CH2CH=CH2 lmax= 184
emax = ~10,000
CH2=CHCH2CH2CH=CH2 lmax=185 emax = ~20,000
If conjugated
- shifts to higher l’s (red shift)
H2C=CHCH=CH
2
27
lmax=217 emax = ~21,000
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,1hexene has a molar absorptivity of
about 10,000 L mol-1 cm-1 but hexa-1,4diene has a molar absorptivity of twice
as much as 1-hexene. However, when
the double bonds are conjugated as in
hexa-1,3-diene the molar absorptivity is
about 21,000 L mol-1 cm-1.
28
On the other hand, aromaticity 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.
29
Absorption by Inorganic Groups
Inorganic groups containing double bonds
absorb in the UV-Vis region. The most
transitions are a result of n-p* transitions
as in nitrate (313 nm), carbonate (217
nm), nitrite (280 and 360 nm) and azide
(230 nm)
30
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).
31
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.
32
33
Terbium Spectrum
34
The crystal field theory is 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 dz2 and
dx2-y2 orbitals 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.
35
36
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+.
37
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 1,10-phenanthroline
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.
38
39
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.
40
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.
41
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
42
Quantitative Analysis
The basis for quantitative analysis in the UVVis 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
43
3. The relative standard deviation occurs
within 1-3% which reflects good
precision.
4. Easy to perform and convenient.
Can be used for quantitative analysis in
liquid chromatographic separations.
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.
44
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:
45
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 an interferences are present, the
wavelength that is far away from
interferences should be selected
5. Working in the visible region should be
preferred
46
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.
47
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.
48
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.
49
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.
50
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.
51
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.
One can only use two points to get the analyte
concentration using the relation:
Cx = AxCsVs/(At –Ax)Vx
52
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.
53
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):
Al’ = ex’bcx + ey’bcy
(1)
Al” = ex”bcx + ey”bcy
(2)
ex’, ex”, ey’, ey” can be determined from
standards of analytes x and y at l’, l” and
values obtained are inserted in equations 1
and 2 where two equations in two unknowns
can be easily solved.
54
Derivative UV-Vis Absorption
Spectroscopy
In derivative spectroscopy, DA/Dl 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
55
56
57
Derivative
spectroscopy
is excellent
for
determination
of multi
components
in a sample, if
they can be
resolved.
58
Obtaining a Derivative Spectrum
Derivative spectra cab 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 Dl (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.
59
60
Applications of Derivative
Spectra
1. Better qualitative analysis and identification of
the number of absorbing species in a sample
2. Accurate determination of lmax
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.
61
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.
62
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. Consider
addition of analyte to a reagent so that a product is
formed according to the reaction:
63
Analyte + Reagent = Product
64
65
It should be pointed out here that the lines
shown are what is supposed to be.
However, as the equivalence point is
approached concentrations become
smaller that the reaction is not fast enough
and the end point is usually not sharp.
This is not a problem as extrapolation of
the linear portions will result in a very
sharp intersection indicating the end point.
66
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.
67
68
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.
69
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.
70
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. For example,
figure a below shows the EDTA titration of
Bi3+ and Cu2+ at the wavelength of the CuEDTA 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 (y).
71
72
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.
73
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.
74
75
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.
76