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Chapter 14

APPLICATION OF ULTRAVIOLET/VISIBLE
MOLECULAR ABSORPTION SPECTROMETRY
Absorption measurements based upon ultraviolet
and visible radiation find widespread application
for the identification and determination of
myriad inorganic and organic species. Molecular
ultraviolet/visible absorption methods are
perhaps the most widely used of all quantitative
analysis techniques in chemical and clinical
laboratories throughout the world.

THE MAGNITUDE OF MOLAR


ABSORPTIVITIES
Molar absorptivities range from zero up to a
maximum on the order of 105 are observed. The
magnitude of depends upon the probability for
an energy-absorbing transition to occur. Peaks
having molar absorptivities less than about 10 3
are classified as being of low intensity. They
result from so-called forbidden transitions,
which have probabilities of occurrence that are
less than 0.01.

ABSORBING SPECIES
The absorption of ultraviolet or visible radiation
by a molecular species M can be considered to
be a two-step process, excitation
M + h
M*
The lifetime of the excited species is brief (10 -8
to 10-9 s). Relaxation involves conversion of the
excitation energy to heat.
M*
M + heat
The absorption of ultraviolet or visible radiation
generally results from excitation of bonding
electrons.

Electronic Transitions
There are three types of electronic
transitions. The three include transitions
involving:
(1) , , and n electrons
(2) d and f electrons
(3) charge transfer electrons.

Types of Absorbing Electrons


The electrons that contribute to absorption by a
molecule are: (1) those that participate directly
in bond formation between atoms; (2)
nonbonding or unshared outer electrons that are
largely localized about such atoms as oxygen,
the halogens, sulfur, and nitrogen. The
molecular orbitals associated with single bonds
are designated as sigma () orbitals, and the
corresponding electrons are electrons.

Types of Absorbing Electrons


The double bond in a molecule contains
two types of molecular orbitals: a sigma
() orbital and a pi () molecular orbital. Pi
orbitals are formed by the parallel overlap
of atomic p orbitals. In addition to and
electrons, many compounds contain
nonbonding electrons. These unshared
electrons are designated by the symbol n.

Energy
The energies for the various types of
molecular orbitals differ significantly. The
energy level of a nonbonding electron lies
between the energy levels of the bonding and
the antibonding and orbitals. Electronic
transitions among certain of the energy levels
can be brought about by the absorption of
radiation. Four types of transitions are
possible:
*, n *, n *, and *.

Antibonding

Antibonding
n *

*
*

n *

Nonbonding

Bonding

Bonding

Energy

* Transition
An electron in a bonding orbital of a
molecule is excited to the corresponding
antibonding orbital by the absorption of
radiation. The energy required to induce a
* transition is large. Methane can
undergo only * transitions, exhibits
an absorption maximum at 125 nm.
Absorption maxima due to *
transitions are never observed in the
ordinarily accessible ultraviolet region.

n * Transitions
Saturated compounds containing atoms
with unshared electrons are capable of n
* transitions. These transitions require
less energy than the * type and can
be brought about by radiation in the region
of between 150 and 250 nm, with most
absorption peaks appearing below 200 nm.
The molar absorptivities are low to
intermediate in magnitude and range
between 100 and 3000 L cm-1 mol -1.

n * and * Transitions
Most applications of absorption spectroscopy are
based upon transitions for n or electrons to the
* excited state because the energies required for
these processes bring the absorption peaks into an
experimentally convenient spectral region (200 to
700 nm). Both transitions require the presence of
an unsaturated functional group to provide the
orbitals. The molar absorptivities for peaks
associated with excitation to the n, * state are
generally low and ordinarily range from 10 and
100 L cm-1 mol -1; values for * transitions,
on the other hand, normally fall in the range
between 1000 and 10,000.

Effect of Conjugation of Chromophores


electrons are considered to be further
delocalized by conjugation; the orbitals involve
four (or more) atomic centers. The effect of this
delocalization is to lower the energy level of the
* orbital and give it less antibonding character.
Absorption maxima are shifted to longer
wavelengths as a consequence. Conjugation of
chromophores, has a profound effect on spectral
properties. 1,3-butadiene, CH2=CHCH=CH2, has
a strong absorption band that is displaced to a
longer wavelength by 20 nm compared with the
corresponding peak for an unconjugated diene.

Absorption Involving d and f Electrons


Most transition-metal ions absorb in the
ultraviolet or visible region of the
spectrum. For the lanthanide and actinide
series, the absorption process results from
electronic transition of 4f and 5f electrons;
for elements of the first and second
transition-metal series, the 3d and 4d
electrons are responsible.

Absorption by Lanthanide and Actinide Ions


The ions of most lanthanide and actinide
elements absorb in the ultraviolet and visible
regions. Their spectra consist of narrow, welldefined, and characteristic absorption peaks.
The transitions responsible for absorption by
elements of the lanthanide series appear to
involve the various energy levels of 4f
electrons, while it is the 5f electrons of the
actinide series that interact with radiation.

Absorption by Elements of the First and


Second Transition-Metal Series
The ions and complexes of the first two
transition series tend to absorb visible radiation
in one if not all of their oxidation states. The
absorption bands are often broad and are
strongly influenced by chemical environmental
factors. The spectral characteristics of transition
metals involve electronic transitions among the
various energy levels of d orbitals.

Charge-Transfer Absorption
Species that exhibit charge-transfer absorption are of
particular
importance
because
their
molar
absorptivities are very large (max > 10,000). Thus,
these complexes provide a highly sensitive means for
detecting and determining absorbing species.
Complexes exhibit charge transfer absorption are
called charge-transfer complexes. In order for a
complex to exhibit a charge-transfer spectrum, it is
necessary for one of its components to have electrondonor characteristics and for the other component to
have electron-acceptor properties. Absorption of
radiation then involves transfer of an electron from the
donor to an orbital that is largely associated with the
acceptor.

APPLICATION OF ABSORPTION
MEASREMENT TO QUALITATIVE
ANALYSIS
Methods of Plotting Spectral Data: Several
different types of spectral plots are encountered
in qualitative molecular spectroscopy. The
ordinate
is
most
commonly
percent
transmittance, absorbance, log absorbance, or
molar absorptivity. The abscissa is usually
wavelength or wavenumber, although frequency
is occasionally employed.

Solvents: In choosing a solvent, consideration


must be given not only to its transparence, but
also to its possible effects upon the absorbing
system. Polar solvents such as water, alcohols,
esters, and ketones tend to obliterate spectral fine
structure arising from vibrational effects; spectra
that approach those of the gas phase are more
likely to be observed in nonpolar solvents such as
hydrocarbons. In addition, the positions of
absorption maxima are influenced by the nature
of the solvent. Clearly, the same solvent must be
used when comparing absorption spectra for
identification purposes.

QUANTITATIVE ANALYSIS BY
ABSORPTION MEASUREMENTS

Absorption spectroscopy is one of the most


useful and widely used tools available to the
chemist for quantitative analysis. Important
characteristics of spectrophotometric method
include: (1) wide applicability to both organic
and inorganic systems, (2) typical sensitivities
of 10-4 to 10-5 M, (3) moderate to high
selectivity, (4) good accuracy, (5) ease and
convenience of data acquisition.

Scope: The applications of quantitative,


ultraviolet/visible absorption methods not only
are numerous, but also touch upon every field in
which quantitative chemical information is
required. It has been estimated that in the field
of health alone, 95% of all quantitative
determinations
are
performed
by
ultraviolet/visible spectrophotometry and this
number represents well over 3,000,000 daily
tests carried out in the United States.

Procedural Details: The first steps in a


photometric or spectrophotometric analysis
involve the establishment of working conditions
and the preparation of a calibration curve
relating concentration to absorbance.
Selection of Wavelength: Spectrophotometric
absorbance measurements are ordinarily made
at a wavelength corresponding to an absorption
peak because the change in absorbance per unit
of concentration is greatest at this point; the
maximum sensitivity is thus realized.

Variables That Influence absorbance: Common


variables that influence the absorption spectrum of
a substance include the nature of the solvent, the
pH of the solution, the temperature, electrolyte
concentrations, and the presence of interfering
substances.
Cleaning and Handling of Cells: Accurate
spectrophotometric analysis requires the use of
good-quality, matched cells. These should be
regularly calibrated against one another to detect
differences that can arise from scratches, etching,
and wear.

Determination of the Relationship Between


Absorbance and Concentration: It is necessary
to prepare a calibration curve from a series of
standard solutions that bracket the concentration
range expected for the samples. Ideally,
calibration standards should approximate the
composition of the samples to be analyzed not
only with respect to the analyte concentration but
also with regard to the concentrations of the
other species in the sample matrix in order to
minimize the effects of various components of
the sample on the measured absorbance.

The Standard Addition Method: The standard


addition method involves adding one or more
increments of a standard solution to sample
aliquots of the same size. Each solution is then
diluted to a fixed volume before measuring its
absorbance. It is possible to perform a standard
addition analysis using only two increments of
sample. Here, a single addition of Vs mL of
standard would be added to one of the two
samples. This approach is based upon the
equation:
A 1c sV s

cx

A 2 A 1 V