CM2007
Experiment 4
Ultra-Violet Visible Spectroscopy
The Beer-Lambert Law
Object:
To apply the Beer-Lambert relationship to an aqueous solution containing two
absorbing substances and thus determine their respective concentrations.
Introduction:
The ultraviolet region of the spectrum extends from the lower wavelength limit of
the visible region (400nm2500cm-1) to ca. 1000nm. Absorption of u.v. radiation causes
electronic changes in atoms or molecules, which can be thought of in terms of the
promotion of one electron into an orbital of higher energy. The resultant absorption band
(E=400KJ/mole) is usually broad in molecular species since rotational and vibrational
changes (E=20KJ/mole) compound the electronic transitions.
Absorption in the near ultraviolet (400nm–190nm) by organic molecules is
indicative of the presence of unsaturated groups, e.g. consider a keto group, it contains ,
 and n type orbitals which may be represented by the following energy diagram where in
the normal occupancy of molecular orbitals involving the keto groups when in its ground
electron state is changed on the right to be altered by promotion of one electron into the
orbital.
Absorption of u.v. light can promote the group to an excited state and the energy
required will be characteristic of the active group (chromophore) and its environment.
Ultra-violet/Visible spectra are obtained on the one instrument as a plot of
wavelength () against absorbance (or optical density O.D.). The intensity of the
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CM2007
maximum absorption is related to the probability or ease of the electronic transition
brought about by photons of that wavelength and expressed by max the molar extinction
coefficient e.g. isophorone has an absorption maximum at 2170 nm the molar extinction
coefficient being 1300, and it exhibits the following spectra in ethanol.
Beer-Lambert Law of Light Absorption:
Consider a beam of monochromatic light passing through a portion of solution of length,
L, in which the concentration of the absorbing molecule is C, and the solvent is
transparent to the light.
The combined Beer-Lambert Law states
Log10 I0/I =  C L
where I0 = intensity of incident monochromatic light
I = intensity of transmitted monochromatic light
 = molar extinction coefficient
C = molarity
L = length of cell in cm
Thus: =OD/CL and Log10 I0/I = OD (optical density)
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(1)
CM2007
This Law may also be applied to solutions containing mixtures of more than one type of
absorbing molecule but it is then necessary to allow as in Eqn. 2 for the possibility that
OD at any selected wavelength may have contribution from each component.
OD = (CL)1 + (CL)2 +  (CL)n
(2)
Where D is the optical density at wavelength , of a solution with a concentration Cn
of the nth component whose molar extinction coefficient at  is n).
Restrictions on the Applications of the Beer-Lambert Law:
In principle the Beer-Lambert Law is applicable to all regions of the spectrum
provided:
(1) Monochromatic radiation is used (i.e. a wavelength band, , small compared
to the absorption bandwidth used),
(2) a transparent solvent medium is employed and
(3) no chemical processes affect the absorber molecules.
Procedure:
You are supplied with (1) a solution containing unknown concentration of
methylene blue and methyl orange, (2) a stock solution of methylene blue, (3) a stock
solution of methyl orange and (4) glassware for manipulating solutions.
Use accurate volumetric procedures to make up a 4:1, 3:2, 1:4 and 1:9 mixture of
each stock solution and distilled water. OBTAIN INSTRUCTION FROM A
DEMONSTRATOR BEFORE RUNNING SPECTRA. Spectra are run on one of the
UV-Vis Spectrometers in the Spectroscopy Lab.
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Notes
(1) Handling of cells:
When handling the u.v. cells be careful not to touch the clear glass sides with your
fingers, and insure that the light beam passes through these sides rather than the opaque
sides, which may be handled.
(2) Cleaning of Cells:
The cells may be cleaned with distilled water, rinse sample cell with a little of test
solution before filling it. When working with a series of solutions, it is good practice to
start with the most dilute sample.
Treatment of Results: The objective of the experiment (see above) requires that the OD of
methylene blue in the unknown solution be carefully measured at its characteristic max
and likewise for methyl orange. Compare the spectra of methylene blue (M.B.) with that
for methyl orange (M.O.). It is evident that one substance has an absorption maximum at
a wavelength max(1) at which the other either does not absorb at all or does so only
weekly. Thus, application of equation 1 to OD, of the mixture at max (1) can yield the
concentration of this component. Since the other component has an absorbance maximum
at a wavelength at which component (1) has absorbance as well, equation 2 must be
employed to determine its concentration. It is convenient to apply equation 2 at a
wavelength corresponding to the absorbance maximum of the second component (and
where component (1) does not have an absorbance maximum) we call this max(2).
From the spectra of the single-component solutions read-off the optical densities
of the solutions at max (1) and max (2), and tabulate them as follows:
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CM2007
Table of OD Values:
OD
Concentration
max (1)
max (2)
M.B. (1)
(2)
(3)
(4)
(5)
M.O. (l)
(2)
(3)
(4)
(5)
Plot these data in a graph of OD versus molar concentration for both substances at
max(1)
and max(2)
and from the slopes OD/CL determine the respective molar
extinction coefficient values, (l) and (2), for each of methylene blue and methyl
orange at each relevant values of max. (Note it may well be that the value of  for
component (2) will be negligibly small at max for the other component but this should be
demonstrated to be so rather than simply assuming it to be so. Use these epsilon values
and the OD values of the mixture into equation (2) at relevant values of max to obtain the
required concentration values of methylene blue and methyl orange respectively in the
unknown solution.
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