Chapter 14
Applications of UV-Vis Molecular Absorption Spectrometry
The 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:
a. The wavelength of radiation
b. The refractive index and is thus indirectly related to concentration
c. Electrostatic interactions taking place in solution; and thus electronic
distribution
d. In rare cases, like methylene blue, the molar absorptivity is directly dependent
on concentration
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.
Absorbing Molecular Species
When 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. Therefore, excitation of electrons in chemical bonds (π and σ)
or nonbonding electrons (n) is the result of absorption of UV-Vis radiation of a
suitable wavelength. Absorption will thus be dependent on the availability of π and σ
bonds or n electrons that can absorb incident radiation.
Molecules Containing π, σ, and n Electrons
1. Molecules with σ Bonds Only
Let us start with a molecule like CH4 and then expand our discussion to more
complex molecules:
All bonds in methane are σ bonds and the only transition possible is the σ-σ*
transition. However, the σ-σ* 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:
a. The high energy required can cause rupture of the σ bonds and breakdown of
the molecule
b. Air components absorb in vacuum UV which limits the application of the
method
c. Working in vacuum UV requires special training and precautions which limit
wide application of the method.
d. Special sources and detectors, other than those described earlier, must be used
e. All solvents contain σ bonds
Therefore, if the compound under consideration has σ bonds only, UV-Vis will not be
the right technique for chemical analysis.
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:
N
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. σ−σ*
b. n−σ*
We have seen earlier that the σ−σ* transition is not useful in practical UV-Vis
spectroscopy but the other transition (n−σ*) is of lower energy and should be further
discussed. The absorption wavelength for a n−σ* 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-σ* 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-σ* transition.
3. Molecules with π Bonds
Absorption of radiation by an alkene, containing a double bond, can result in σ−σ* or
π−π* transitions. We have seen that a σ−σ* transition is not useful but on the other
hand, the π−π* turned out to be very useful since it requires reasonable energy and
has good absorptivity. A molecule having σ, π, 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 σ, π, and two pairs of n electrons.
O
Two transitions are possible for the n electrons:
a. n−σ*
b. n−π*
Energy
σ
π
π∗
n
∗
σ−σ∗
π−π∗
n−σ∗
n−π∗
We have seen that a n-σ* is not very useful due to absorbance from solvents and other
frequently used additives which have n electrons. The n-π* 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.
Effect of Solvent Polarity on Absorption Wavelength
The molar absorptivity for a n-π* 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 π* is also observed
but to a much lower extent than the n electrons. A net increase in energy required for
a n−π* 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 π−π* transition is affected in an opposite manner with solvent
polarity. In presence of a polar solvent, the more polar π* orbital will be more
stabilized than the π 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).
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 σ−σ* and a n−σ* are not useful for
reasons discussed earlier. The n−π* transition requires low energy but the molar
absorptivity is also low and transition energy will increase in presence of polar
solvents. The n−π* transition is seldom used in quantitative UV-Vis spectroscopy.
The most frequently used transition is the π−π* transition for the following reasons:
a. The molar absorptivity for the π−π* 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
π−π* transition is usually smaller.
It is therefore primitive that an analyte to be determined by UV-Vis absorption
spectroscopy be of unsaturated nature.
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.
Effect of Conjugation and Aromaticity of Chromophores
As conjugation is increased in a molecule, more delocalization (stability) of the π
electrons results. The effect of this delocalization is to decrease the π* molecular
orbital. The result is a decrease in transition energy from π−π* 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 hexa-1,3-diene the
molar absorptivity is about 21,000 L mol-1 cm-1. On the other hand, aromaticity results
in extraordinarily high degree of delocalization of electrons and thus stabilization of
the π*. 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.
Absorption by Inorganic Groups
Inorganic groups containing double bonds absorb in the UV-Vis region. The most
transitions are a result of n-π* transitions as in nitrate (313 nm), carbonate (217 nm),
nitrite (280 and 360 nm) and azide (230 nm)
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.
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. 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 ∆).
This can be schematically represented as:
dz2, dx2-y2
E
∆
dxy, dxz, dyz
No Ligand Field
Isolated d
Orbital
In Presence
of Ligands