INSTRUMENTAL METHODS IN ANALYTICAL CHEMISTRY

PHOTOCOLORIMETRY AND SPECTROPHOTOMETRY

1. Introduction:
The variation of the colour of a system with change in concentration of some component
forms the basis of what the chemist commonly terms colorimetric analysis. The colour is usually
due to the formation of a coloured compound by the addition of an appropriate reagent, or it may be
inherent in the desired constituent itself. The intensity of the colour may then be compared with that
obtained by treating a known amount of the substance in the same manner.
Colorimetry is concerned with the determination of the concentration of a substance by
measurement of the relative absorption of light with respect to a known concentration of the
substance. In visual colorimetry, natural or artificial white light is generally used as a light source,
and determinations are usually made with a simple instrument termed a colorimeter, or colour
comparator. When the eye is replaced by a photoelectric ce11 (thus largely eliminating the errors
due to the persona1 characteristics of each observer) the instrument is termed a photoelectric
colorimeter. The latter is usually employed with light contained within a comparatively narrow
range of wavelengths furnished by passing white light through filters, i.e. materials in the form of
plates of coloured glass, gelatin, etc., transmitting only a limited spectral region: the name 'filter
photometer' is sometimes applied to such an instrument.
In spectrophotometric analysis a source of radiation is used that extends into the ultraviolet
region of the spectrum. From this, definite wavelengths of radiation are chosen possessing a
bandwidth of less than 1 nm. This process necessitates the use of a more complicated and
consequently more expensive instrument. The instrument employed for this purpose is a
spectrophotometer.
An optical spectrometer is an instrument possessing an optical system which can produce
dispersion of incident electromagnetic radiation, and with which measurements can be made of the
quantity of transmitted radiation at selected wavelengths of the spectral range. A photometer is a
device for measuring the intensity of transmitted radiation or a function of this quantity. When
combined in the spectrophotometer, the spectrometer and photometer are employed conjointly to
produce a signal corresponding to the difference between the transmitted radiation of a reference
material and that of a sample at selected wavelength. The chief advantage of colorimetric and
spectrophotometric methods is that they provide a simple means for determining minute quantities
of substances. The upper limit of colorimetric methods is, in general, the determination of
constituents which are present in quantities of less than 1 or 2 per cent. The development of
inexpensive photoelectric colorimeters has placed this branch of instrumental chemical analysis
within the means of even the smallest teaching institution.

THE UNIVERSITY OF DODOMA, COURSE CODE: CH 215 1

INSTRUCTOR: DR. VEGI MAHESWARA RAO
 INSTRUMENTAL METHODS IN ANALYTICAL CHEMISTRY

2. Theory of Spectrophotometry and Colorimetry:

When light (monochromatic or heterogeneous) falls upon a homogeneous medium, a portion
of the incident light is reflected, a portion is absorbed within the medium, and the remainder is
transmitted. If the intensity of the incident light is expressed by Io, that of the absorbed light by Ia,
that of the transmitted light by It, and that of the reflected light by Ir, then:

For air-glass interfaces arising from the use of glass cells, it may be stated that about 4 per cent of
the incident light is reflected. Ir is usually eliminated by the use of a control, such as a comparison
cell, hence:

Credit for investigating the change of absorption of light with the thickness of the medium is
frequently given to Lambert. Beer later applied similar experiments to solutions of different
concentrations. The two separate laws governing absorption are usually known as Lambert's Law
and Beer's Law. In the combined form they are referred to as the Beer-Lambert Law.
2.1 Lambert's Law:
This law states that when monochromatic light passes through a transparent medium, the rate of
decrease in intensity with the thickness of the medium is proportional to the intensity of the light.
This is equivalent to stating that the intensity of the emitted light decreases exponentially as the
thickness of the absorbing medium increases arithmetically. We may express the law by the
differential equation:

where I is the intensity of the incident light of wavelength λ, l is the thickness of the medium, and k
is a proportionality factor. Integrating this equation and putting I = I0 when l = 0, we obtain:

or stated in other terms,

where I0 is the intensity of the incident light falling upon an absorbing medium of thickness l, It is
the intensity of the transmitted light, and k is a constant for the wavelength and the absorbing
medium used. The ratio It/I0 is the fraction of the incident light transmitted by a thickness 1 of the
medium and is termed the transmittance T. Its reciprocal I0/It is the opacity, and the absorbance A of
the medium.
2.2 Beer's Law:
We have so far considered the light absorption and the light transmission for monochromatic light
as a function of the thickness of the absorbing layer only. In quantitative analysis, however, we are
THE UNIVERSITY OF DODOMA, COURSE CODE: CH 215 2
INSTRUCTOR: DR. VEGI MAHESWARA RAO
 INSTRUMENTAL METHODS IN ANALYTICAL CHEMISTRY
mainly concerned with solutions. Beer studied the effect of concentration of the coloured
constituent in solution upon the light transmission or absorption. He found the same relation
between transmission and concentration as Lambert had discovered between transmission and
thickness of the layer. i.e. the intensity of a beam of monochromatic light decreases exponentially as
the concentration of the absorbing substance increases arithmetically. This may be written in the
form:

where c is the concentration, and k1 is a constants.

Combining Lamberts law and Beers law:
I
A  log( 0 )  cl
It
This is the fundamental equation of colorimetry and spectrophotometry, and is often spoken of as
the Beer-Lambert Law. Here ε is called molar absorption coefficient.
2.3 Deviations from Beer-Lambert Law:
The Beer-Lambert Law is rigorously obeyed when a single species gives rise to the
observed absorption. The law may not be obeyed, however
i) When the concentration of sample is very high and electrostatic interactions between
particles occur.
ii) When different forms of the absorbing molecule are in equilibrium.
iii) When solute and solvent form complexes through some sort of association.
iv) When fluorescent compounds or compounds changed by irradiation are present.
v) When the light is scattered.
vi) When the light is not monochromatic.

3. Energy levels and transitions:

When continuous radiation passes through a transparent material, a portion of the radiation
may be absorbed. If that occurs, the residual radiation, when it is passed through a prism, yields a
spectrum with gaps in it, called an absorption spectrum. As a result of energy absorption, atoms or
molecules pass from a state of low energy (the initial, or ground state) to a state of higher energy the
excited state. The Figure 1 depicts this excitation process, which is quantized.

Figure 1: Electronic transition.

THE UNIVERSITY OF DODOMA, COURSE CODE: CH 215 3
INSTRUCTOR: DR. VEGI MAHESWARA RAO
 INSTRUMENTAL METHODS IN ANALYTICAL CHEMISTRY
The electromagnetic radiation that is absorbed has energy exactly equal to the energy
difference between the excited and ground states. In the case of ultraviolet and visible spectroscopy,
the transitions that result in the absorption of electromagnetic radiation in this region of the
spectrum are transitions between electronic energy levels. As a molecule absorbs energy, an
electron is promoted from an occupied orbital to an unoccupied orbital of greater potential energy.
Generally, the most probable transition is from the highest occupied molecular orbital (HOMO) to
the lowest unoccupied molecular orbital (LUMO). The energy differences between electronic levels
in most molecules vary from 125 to 650 kJ/mol. For most molecules, the lowest-energy occupied
molecular orbitals are the σ orbitals, which correspond to σ bonds. The π orbitals lie at somewhat
higher energy levels, and orbitals that hold unshared pairs, the nonbonding (n) orbitals, lie at even
higher energies. The unoccupied, or antibonding orbitals π* and σ * are the orbitals of highest
energy. The Figure 2 shows a typical progression of electronic energy levels and the corresponding
electronic transitions.

Figure 2: Molecular electronic energy levels and corresponding electronic transitions.

In all compounds other than alkanes, the electrons may undergo several possible transitions
of different energies. Some of the most important transitions are shown in Figure 3.

Figure 3: Comparison of energies of different electronic transitions.

3.1 Selection Rule:
Not all of the transitions that at first sight appear possible are observed. Certain restrictions,
called selection rules, must be considered. One important selection rule states that transitions that
involve a change in the spin quantum number of an electron during the transition are not allowed to
take place; they are called "forbidden" transitions. Transitions that are formally forbidden by the

THE UNIVERSITY OF DODOMA, COURSE CODE: CH 215 4

INSTRUCTOR: DR. VEGI MAHESWARA RAO
 INSTRUMENTAL METHODS IN ANALYTICAL CHEMISTRY
selection rules are often not observed. However, theoretical treatments are rather approximate, and
in certain cases forbidden transitions are observed, although the intensity of the absorption tends to
be much lower than for transitions that are allowed by the selection rules. The n   * transition is
the most common type of forbidden transition.

4. Origin of U. V. Band Structure:

For an atom that absorbs in the ultraviolet, the absorption spectrum sometimes consists of
very sharp lines, as would be expected for a quantized process occurring between two discrete
energy levels and this is called as line spectra.
For molecules, however, the UV absorption usually occurs over a wide range of
wavelengths because molecules normally have many excited modes of vibration and rotation at
room temperature. In fact, the vibration of molecules cannot be completely "frozen out" even at
absolute zero. Consequently, a collection of molecules generally has its members in many states of
vibrational and rotational excitation. The energy levels for these states are quite closely spaced,
corresponding to energy differences considerably smaller than those of electronic levels. The
rotational and vibrational levels are thus "superimposed" on the electronic levels. Hence in a
molecule the electronic transitions are accompanied by vibrational transitions and the vibrational
transitions are accompanied by rotational energy transitions. Therefore a molecule undergoes the
electronic, vibrational and rotational energy transitions simultaneously as shown in Figure 4.
Because there are so many possible transitions, each differing from the others by only a slight
amount, each electronic transition consists of a vast number of lines spaced so closely that the
spectrophotometer cannot resolve them. Rather, the instrument traces an "envelope" over the entire
pattern. What is observed from these types of combined transitions is that the UV spectrum of a
molecule usually consists of a broad band of absorption centered near the wavelength of the major
transition.
Etotal = E electronic + E vibrational + E rotational

Figure 4: Transitions responsible for band structure.

THE UNIVERSITY OF DODOMA, COURSE CODE: CH 215 5

INSTRUCTOR: DR. VEGI MAHESWARA RAO
 INSTRUMENTAL METHODS IN ANALYTICAL CHEMISTRY

In Figure 4 actually each vibrational energy level again consists of so many closely spaced
rotational energy levels which are not shown in the figure.

5. Instrumentation of UV-Vis. Spectrometer:

There are two important types of spectroscopic instruments.
5.1 Single beam Instrument
5.2 Double beam Instrument
5.1 Single Beam Instrument:
A typical single beam spectrophotmeter is shown in the Figure 5. The light source is usually
a deuterium lamp, which emits electromagnetic radiation in the ultraviolet region of the spectrum. A
second light source, a tungsten lamp, is used for wavelengths in the visible region of the spectrum.
A filter, diffraction grating or a monochromator for wavelength selection, its role is to spread the
beam of light into its component wavelengths. A system of slits focuses the desired wavelength on
the sample cell. Matched cells that can be placed alternately in the radiation beam. The sample cell
must be constructed of a material that is transparent to the electromagnetic radiation being used in
the experiment. For spectra in the visible range of the spectrum, cells composed of glass or plastic
are generally suitable. For measurements in the ultraviolet region of the spectrum, however, glass
and plastic cannot be used because they absorb ultraviolet radiation. Instead, cells made of quartz
must be used since quartz does not absorb radiation in this region. The instrument design just
described is quite suitable for measurement at only one wavelength. The light that passes through
the sample cell reaches the detector, which records the intensity of the transmitted light. The
detector is generally a photovoltaic cell, photomultiplier tube, although in modem instruments
photodiodes are also used. An amplifier is used to amplify the signal, and a readout device used for
display the signal. Normally, a single-beam instrument requires a stabilized voltage supply to avoid
errors resulting from changes in the beam intensity during the time required to make the 100%T
measurement and determine %T for the analyte.
Single-beam instruments vary widely in their complexity and performance characteristics.
The simplest and least expensive consists of a battery-operated tungsten bulb as the source, a set of
glass filters for wavelength selection, test tubes for sample holders, and photovoltaic cells as
transducer and an analog meter as the readout device. At the other extreme are sophisticated,
computer-controlled instruments with a range of 200 to 1000 nm or more. These
spectrophotometers have interchangeable tungsten and deuterium lamp sources, use rectangular
silica cells, and are equipped with a high-resolution grating monochromator with variable slits.
Photomultiplier tubes are used as transducers, and the output is often digitized, processed, and
stored in a computer so that it can be printed or plotted in several forms.
THE UNIVERSITY OF DODOMA, COURSE CODE: CH 215 6
INSTRUCTOR: DR. VEGI MAHESWARA RAO
 INSTRUMENTAL METHODS IN ANALYTICAL CHEMISTRY

Figure 5: Block diagram of single beam spectrophotometer.

5.2 Double Beam Instrument:
Many modern photometers and spectrophotometers are based on a double-beam design. A
double-beam-in-space instrument in which two beams are formed in space by a V-shape mirror
called a beam splitter as shown in Figure 6. One beam passes through the reference solution to a
photo detector, and the second simultaneously traverses the sample to a second, matched detector.
The two outputs are amplified, and their ratio (or the logarithm of their ratio) is determined
electronically or by a computer and displayed by the readout device. With manual instruments, the
measurement is a two-step operation involving first the zero adjustment with a shutter in place
between selector and beam splitter. In the second step, the shutter is opened and the transmittance or
absorbance is displayed directly.

Figure 6: Block diagram of double beam spectrophotometer.

Double-beam instruments offer the advantage that they compensate for all but the most
short-term fluctuations in the radiant output of the source as well as for drift in the transducer and
amplifier. They also compensate for wide variations in source intensity with wavelength. The
double-beam design lends itself well to the continuous recording of transmittance or absorbance
spectra.

6. Presentation of Spectra:
The ultraviolet-visible spectrum is generally recorded as a plot of absorbance versus
wavelength. It is customary to then replot the data with either ε or logε plotted on the ordinate and
wavelength plotted on the abscissa. The Figure 7 indicates spectrum of benzoic acid, is typical of
the manner in which spectra are displayed. However, very few electronic spectra are reproduced in

THE UNIVERSITY OF DODOMA, COURSE CODE: CH 215 7

INSTRUCTOR: DR. VEGI MAHESWARA RAO
 INSTRUMENTAL METHODS IN ANALYTICAL CHEMISTRY
the scientific literature; most are described by indications of the wavelength maxima and
absorptivities of the principal absorption peaks. For benzoic acid, a typical description might be
λmax =230 nm logε = 4.2
272 nm 3.1
282 nm 2.9

Figure 7: UV-Visible spectrum of benzoic acid.

7. Quantitative Analysis by Absorption Measurements:
Absorption spectroscopy based on ultraviolet and visible radiation is one of the most useful
tools available to the scientist for quantitative analysis.
Important characteristics of spectrophotometric and photocolorimetric methods are:
i. wide applicability to both organic and inorganic systems
ii. typical detection limits of 10-4 to 10-5 M (in some cases, certain modifications can lead to
lower limits of detection)
iii. moderate to high selectivity
iv. good accuracy
v. ease and convenience of data acquisition.

7.1 Applications to Absorbing Species:

Different chromophores present in different organic compounds will absorb at different λmax.
Spectrophotometric determination of any organic compound containing one or more chromophore
groups is potentially feasible. A number of inorganic species also absorb UV-visible radiation and
are thus susceptible to direct determination. We have noted that many ions of the transition metals
are colored in solution and can thus be determined by spectrophotometric measurement. In addition;
a number of other species-show characteristic absorption bands, including nitrite, nitrate, and
chromate ions, the oxides of nitrogen, the elemental halogens, and ozone.

7.2 Applications to Non-absorbing Species:

Numerous reagents react selectively with non-absorbing species to yield products that
absorb strongly in the ultraviolet or visible regions. The successful application of such reagents to

THE UNIVERSITY OF DODOMA, COURSE CODE: CH 215 8

INSTRUCTOR: DR. VEGI MAHESWARA RAO
 INSTRUMENTAL METHODS IN ANALYTICAL CHEMISTRY
quantitative analysis usually requires that the color-forming reaction be forced to near completion.
If the amount of product is limited by the analyte, the absorbance of the product is proportional to
the analyte concentration. Color forming reagents are frequently employed as well for the
determination of absorbing species, such as transition-metal ions. The molar absorptivity of the
product is frequently orders of magnitude greater than that of the species before reaction. A host of
complexing agents are used to determine inorganic species. Typical inorganic reagents include
thiocyanate ion for iron, cobalt, and molybdenum; hydrogen peroxide for titanium, vanadium, and
chromium; and iodide ion for bismuth, palladium, and tellurium. Of even more importance, are
organic chelating agents that form stable, colored complexes with cations. Common examples
include diethyldithiocarbamate for the determination of copper, diphenylthiocarbazone for lead,
1,10- phenanthrolene for iron, and dimethylglyoxime for nickel. In the application of the last
reaction to the photometric determination of nickel, an aqueous solution of the cation is extracted
with a solution of the chelating agent in an immiscible organic liquid. The absorbance of the
resulting bright red organic layer serves as a measure of the concentration of the metal.

7.3 The procedural details:

Step 1: Selection of wavelength:
A first step in any photocolorimetric or spectrophotometric analysis is the development of
conditions that yield a reproducible relationship (preferably linear) between absorbance and analyte
concentration. For highest sensitivity, spectrophotometric absorbance measurements are ordinarily
made at a wavelength corresponding to an absorption maximum (λmax), because the change in
absorbance per unit of concentration is greatest at this point. In addition, the absorbance is nearly
constant with wavelength at an absorption maximum, which leads to close adherence to Beer's law.
Finally, small uncertainties that arise from failing to reproduce precisely the wavelength setting of
the instrument have less influence at an absorption maximum.
Step 2: 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, high electrolyte concentrations, and the
presence of interfering substances. The effects of these variables must be known and conditions for
the analysis must be chosen such that the absorbance will not be materially influenced by small,
uncontrolled variations in their magnitudes.
Step 3: 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 difference that can result from
scratches, etching, and wear. It is equally important to use proper cell-cleaning and drying
techniques. Erickson and Surles recommend the following cleaning sequence for the outside
THE UNIVERSITY OF DODOMA, COURSE CODE: CH 215 9
INSTRUCTOR: DR. VEGI MAHESWARA RAO
 INSTRUMENTAL METHODS IN ANALYTICAL CHEMISTRY
windows of cells. Prior to measurement, the cell surfaces should be cleaned with lens paper soaked
in spectrograde methanol. The methanol is then allowed to evaporate, leaving the cell surfaces free
of contaminants. Erickson and Surles showed that this method was superior to the usual procedure
of wiping the cell surfaces with a dry lens paper, which can leave lint and a film on the surface.

Step 4: Determining the Relationship between Absorbance and Concentration:

The method of external standards is most often used to establish the absorbance versus
concentration relationship. After deciding on the conditions for the analysis, the calibration curve is
prepared from a series of standard solutions that bracket the concentration range expected for the
samples. Seldom, if ever, is it safe to assume adherence to Beer's law and use only a single standard
to determine the molar absorptivity. It is never a good idea to base the results of an analysis on a
literature value for the molar absorptivity. 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. This can minimize
the effects of various components of the sample on the measured absorbance. For example, the
absorbance of many colored complexes of metal ions is decreased to a varying degree in the
presence of sulfate and phosphate ions because these anions can form colorless complexes with
metal ions. The desired reaction is often less complete as a consequence, and lowered, absorbances
are the result. The matrix effect of sulfate and phosphate can often be counteracted by introducing
into the standard amounts of the two species that approximate the amounts found in the samples.
Unfortunately, matrix matching is often impossible or quite difficult when complex materials such
as soils, minerals, and tissues are being analyzed. When this is the case, the standard-addition
method is often helpful in counteracting matrix effects that affect the slope of the calibration curve.
However, the standard-addition method does not compensate for extraneous absorbing species
unless they are present at the same concentration in the blank solution. Draw the calibration graph
as shown in Figure 8, which the graph between the concentrations of standards and their
absorbances obtained from instrument.
0.8

0.7 Unknown absorbance

0.6
Absorbance

0.5

0.4 Unknown concentration

0.3

0.2

0.02 0.04 0.06 0.08 0.10 0.12 0.14

Concentration

Figure 8: Calibration graph.

THE UNIVERSITY OF DODOMA, COURSE CODE: CH 215 10
INSTRUCTOR: DR. VEGI MAHESWARA RAO
 INSTRUMENTAL METHODS IN ANALYTICAL CHEMISTRY

Step 5: Unknown concentration determination from calibration graph:

After drawing the calibration graph, take the value of absorbance for sample and indicate
that value on the calibration graph. By using extrapolation method take the concentration of the
unknown corresponding to the absorbance of the unknown.
7.4 Solvents used for the analysis:
The choice of the solvent to be used in ultraviolet spectroscopy is quite important. The first
criterion for a good solvent is that it should not absorb ultraviolet radiation in the same region as the
substance whose spectrum is being determined. Usually solvents that do not contain conjugated
systems are most suitable for this purpose, although they vary regarding the shortest wavelength at
which they remain transparent to ultraviolet radiation. The Table 1 lists some common ultraviolet
spectroscopy solvents and their cutoff points or minimum regions of transparency. Of the solvents
listed in Table 1, water, 95% ethanol, and hexane are most commonly used. Each is transparent in
the regions of the ultraviolet spectrum in which interesting absorption peaks from sample molecules
are likely to occur. A second criterion for a good solvent is its effect on the fine structure of an
absorption band. A non-polar solvent does not hydrogen bond with the solute, and the spectrum of
the solute closely approximates the spectrum that would be produced in the gaseous state, in which
fine structure is often observed. In a polar solvent, the hydrogen bonding forms a solute-solvent
complex, and the fine structure may disappear. A third criterion for a good solvent is its ability to
influence the wavelength of ultraviolet light that will be absorbed via stabilization of either the
ground or the excited state. Polar solvents do not form hydrogen bonds as readily with the excited
states of polar molecules as with their ground states, and these polar solvents increase the energies
of electronic transitions in the molecules. Polar solvents shift transitions of the n   * type to
shorter wavelengths. On the other hand, in some cases the excited states may form stronger
hydrogen bonds than the corresponding ground states. In such a case, a polar solvent shifts an
absorption to longer wavelength since the energy of the electronic transition is decreased. Polar
solvents shift transitions of the    * type to longer wavelengths.
Table 1: Cutoff points of different solvents used for UV-Vis specrophtometric analysis.

THE UNIVERSITY OF DODOMA, COURSE CODE: CH 215 11

INSTRUCTOR: DR. VEGI MAHESWARA RAO