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Instrumental Anlaysis
CHE 331
Dr. Rahni
EXPERIMENT 1: Simultaneous UV Analysis of a Three-Component Mixture
Objectives
This experiment introduces the simultaneous analysis of a mixture of components
that have similar properties. Mixtures of three isomers of cresol will be analyzed using an
ultraviolet spectrophotometer. From the UV spectra, each component and an unknown
mixture will be determined.
Reference
1. D.T. Sawyer, W.R. Heineman, J.M Beebe, “Chemistry Experiments for
Instrumental Methods,” John Wiley & Sons, Canada, 1984
Theory
There are instances when the presence of one species in a sample does not
influence the measurement of another species in the same sample; i.e., they do not
interfere. In this experiment three analytes are present and neither affects the lightabsorbing properties of the other. Thus, the absorption of light by the components of the
sample solution is additive; that is, the total absorption of light at any given wavelength is
just the sum of the absorbances the three substances would show if measured individually
under the same conditions. Indeed, this experiment starts with demonstrating that the
spectrum of the analyte mixture is the sum of the spectra of the three components
measured separately. Because the boiling points of these three compounds are nearly the
same, a separation of a mixture of the three into its pure components is impractical. One
could simply measure the absorbances at those three wavelengths to determine the
concentration of the individual analytes directly. However, in most cases this does not
hold strictly true, and analytes absorb, if only weakly, across the spectrum. Fortunately,
by choosing wavelengths where the absorption of is strong and weak, and vice versa, it is
still possible to determine their concentrations because the absorbances are additive.
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This experimental technique relies upon the absorption of ultraviolet light. Atoms
and/or molecules in the sample absorb incident energy, enter an excited state, and may
dissipate their absorbed energy via thermal, radiant, or chemical processes. Beer’s law is
a simple formulation relating the absorbance of incident energy to the particular chemical
species, the concentration of that species in solution, and the distance the incident energy
must travel through the solution as shown in figure 1.
Figure 1
where A is the absorbance of the solution, ε is the molar absorptivity (L/molcm, specific
to the chemical species and wavelength of light used), b is the cell path length (cm), and c
is the concentration of absorbing species (mol/L). Traditionally, measurements using
Beer’s law have been made at particular wavelengths of light where there exists a linear
relationship between concentration and absorbance as shown in figure 2. Usually the
pathlength, b, is known; thus, we can solve these simultaneous equations for the
concentrations of different species if the absorptivity coefficients of both species at both
wavelengths are known. The absorptivity coefficients of each cresol isomer will be
determined at different wavelength by Beer’s Law plots. The absorbance of the sample
mixture will then be obtained for the same wavelengths; solution of simultaneous
equations will thus enable us to
Figure 2
obtain estimates of the cresol
concentrations.
Beer’s law is valid
simultaneously for all absorbers in
a solution. Let’s imagine that we
have three analyte species, a, b,
and c, present in a solution. The
absorbance due to the three
individual species will usually add to give the total absorbance of the solution. The
absorbance at any wavelength is due to the absorbance of each species:
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Absorbance at wavelength λ= abs. by species a + abs. by species b + abs. by species c
Beer’s Law applies to each absorbing species independently; thus, we may express the
absorbance at any wavelength as
Abs  aAbC A  aBbCB  aC bCC
where A is the absorptivity of species a at wavelength λ. This additive property of
absorbance can determine the concentrations of both components of a mixture by
measuring the absorbance at three different wavelengths within the range of the scan.
The absorbance of “unknown” sample(s) at three different wavelengths; the
concentration of the three analytes, a, b and c, are determined by solving the following
equations:
A1  k1a [a ]  k1b [b]  k1c [c]
A2  k2a [a ]  k2b [b]  k2c [c]
A3  k3a [a ]  k3b [b]  k3c [c]
Where A1 , A2 , and A3 are the three absorbance measurements at λ1 ,λ2 , and λ3
respectively. The k values, the products of absorptivity and pathlength, must be
determined by separate calibration (i.e., Beer’s Law plots). From these equations, [a],
[b], and [c] in terms of the k's, and A1 , A2 , and A3 and can be solved for the
concentrations.
Ortho-cresol, meta- cresol, and para- cresol are structural isomers. The names of
the three compounds indicate which of the hydrogens on the benzene ring portion of the
molecule have been replaced. They are obtained from coal tar or petroleum. The mixture
of cresols obtained from coal tar is called cresylic acid, an important technical product
used as a disinfectant and in the manufacture of resins and tricresyl phosphate. Cresols
are useful as raw materials for various chemical products, disinfectants and synthetic
resins. The isomer o-cresol is a starting material for the herbicides 4,6-dinitro-o-cresol
(DNOC) and 2-methyl-4-chlorophenoxyacetic acid (MCPA). The isomers m-cresol and
p-cresol are used in phenol-formaldehyde resins and are converted to tricresyl phosphate
(a plasticizer and gasoline additive) and to di-tert-butylcresols (antioxidants called BHT).
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o-cresol
m-cresol
p-cresol
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Materials / Chemicals Required
Chemicals
Diluted stock solutions of o-, m-, p-cresol will be provided. Their exact concentrations
will be provided by the instructor.
Instrument and Equipment
Instrumentation
This experiment will be conducted using a Perkin Elmer lambda 2 UV-Visible
Spectrophotometer. The instrument has a wavelength range of 190-1100 nm with a
resolution of 2 nm. The optical system (shown below) consists of two lamps, arranged so
that the emission from the tungsten lamp shines through the deuterium lamp. The lamps
emit light at 190-800 nm and 370-1100 nm, respectively.
Procedure
I.
First, determine the suitable wavelengths for the analysis by obtaining an
absorption spectrum for m-cresol, p-cresol, and o-cresol separately.
A. Prepare the following solutions:
1.
Pipet 5, 10, 15 and 20 mL of the 0.05 g/L stock solution of
p-cresol into a 25-mL volumetric flask and diluting to the
mark. Mix well.
2.
Repeat for m-cresol and o-cresol.
3.
Follow the SOP for operation of the UVspectrophotometer.
4.
Obtain the spectra for both solutions from 240 nm to 300
nm (or as narrow a range as possible).
5.
Generate a Beer's Law (A vs. C) for all species
II.
Next, prepare mixtures of the following solutions:
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Mixtures
#1
p-15
#2
m-15
#3
o-15
III.
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o-cresol
5 ml
5 ml
15 ml
m-cresol
5 ml
15 ml
5 ml
p-cresol
15 ml
5 ml
5 ml
Finally, obtain unknown(s) from the instructor. This solution contains a
mixture of the cresol isomers. Obtain its absorption spectrum, recording the
absorbance at the wavelengths of interest.