analytical chemistry

1. 1
UV-Vis Spectroscopy

2. 2
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

3. 3
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

4. 4
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

5. 5
Molecules Containing p, s, and n Electrons
1. Molecules with s Bonds Only
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. 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,, must be used
5. All solvents contain s bonds

7. 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. 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 . 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 is 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. 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. 10

11. 11
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.

12. 12
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).

13. 13
Spectral nomenclature of shifts

14. 14
Optically clear solvent concept

15. 15
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.

16. 16
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 p-p* transition is usually smaller.
Conclusions on Electronic Transitions

17. 17
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.

18. 18
Examples of
UV-Visible Absorptions
LOW!

19. 19
UV-Visible Absorption Chromophores

20. 20
Typical UV Absorption Spectra
Chromophores?

21. 21
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.

22. 22
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.

23. 23
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=CH2 lmax=217 emax = ~21,000
Rule of thumb for conjugation

24. 24
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
hexa-1,3-diene the molar absorptivity is about 21,000 L mol-1
cm-1.

25. 25
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.

26. 26
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)

27. 27
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).

28. 28
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.

29. 29
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 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.

30. 30

31. 31
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+.

32. 32
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.

33. 33
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 liberate 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

34. 34
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

35. 35
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.