Chem 3052 Spectroscopy

CHAPTER 2

Absorption Laws (Quantitative Analysis)

Outline:

2.1 Lambert-Beer's Law

2.2 Deviation from Beer's Law
2.3 Errors associated with Beer's Law

Absorption Methods

Quantitative absorption methods require two power measurements: one before a beam has passed
through the medium that contains the analyte (Po) and the other after (P). Two terms, which are
widely used in absorption spectrometry and are related to the ratio of Po and P, are
transmittance and absorbance.

Transmittance

A beam of parallel radiation before and after it has passed through a medium that has a thickness
of b cm and a concentration c of an absorbing species. As a consequence of interactions between
the photons and absorbing atoms or molecules, the power of the beam is attenuated from Po to
P. The transmittance T of the medium is then the fraction of incident radiation transmitted by the
medium:
T = PO /P

Transmittance is often expressed as a percentage or

%T = PO /P x 100%

Absorbance

The absorbance A of a medium is defined by the equation

A = - log T = log Po/P

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Note that, in contrast to transmittance, the absorbance of a medium increases as attenuation of

the beam becomes greater.

Beer's Law

For monochromatic radiation, absorbance is directly proportional to the path length b through the
medium and the concentration c of the absorbing species. These relationships are given by
A = abc
where a is a proportionality constant called the absorptivity. The magnitude of a depends on the
units used for b and c. For solutions of an absorbing species, b is often given m centimeters and c
in grams per liter. Absorptivity then has units of L g-1 cm-1.

When the concentration in Equation 6-33 is expressed in moles per liter and the cell length is in
centimeters, the absorptivity is called the molar absorptivity and is given the special symbol
Thus, when b is in centimeters and c is in moles per liter,
A = bc
where  has the units L mol-1 cm-1.

These equations are expressions of Beer's law, which serves as the basis for quantitative analyses
by both atomic and molecular absorption measurements.

Measurement of Transmittance and Absorbance

A simple instrument called a photometer is used for measuring the transmittance and absorbance
of aqueous solutions with a filtered beam of visible radiation. Here, the radiation from a tungsten
bulb passes through a colored glass filter that restricts the radiation to a limited band of
contiguous wavelengths. The beam then passes through a variable diaphragm that permits
adjustment of the power of the radiation that reaches the transparent cell that contains the
sample. A shutter can be imposed in front of the diaphragm that completely blocks the beam.
With the shutter open, the radiation impinges on a photoelectric transducer that converts the
radiant energy of the beam to a direct current that is measured with a micro-ammeter. The output

of the meter S is described by Equation S = kP +kd . Note that the meter has a linear scale that
extends from 0 to 100.

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To make such an instrument direct reading in percent transmittance, two preliminary adjustments
are made; the 0% T, or dark current, adjustment, and the 100% T adjustment. The 0% T
adjustment is made with the detector screened from the source by closing the mechanical
shutter. Any small dark current in the detector is nulled electrically until the needle of the
detector reads zero.
The 100% T adjustment is made with the shutter open and with the solvent cell in the light
path. Usually, the solvent is contained in a cell that is as nearly as possible identical to the cell
that contains the sample. The 100% T adjustment with this instrument involves varying the
power of the beam by means of the variable diaphragm; in some instruments, the same effect is
realized by varying the radiant output of the source electrically. The radiant power that reaches
the detector is then varied until the meter reads exactly 100. Effectively, this procedure sets Po in
%T = PO /P x 100%
Equation at 100%. When the solvent is replaced by the cell that contains
the sample, the scale then indicates the percent transmittance directly, as shown by the equation

An absorbance scale can also be scribed on the readout device.

When light is absorbed by a sample, the irradiance of the beam of light is decreased.
Irradiance, P, is the energy per second per unit area of the light beam. Light is passed through a
monochromator (a prism, a grating, or even a filter) to select one wavelength. Light of a single
wavelength is said to be monochromatic, which means “one color.” The monochromatic light,
with irradiance, P0, strikes a sample of length b. The irradiance of the beam emerging from the
other side of the sample is P. Some of the light may be absorbed by the sample, so P ≤ P0.

Transmittance, T, is defined as the fraction of the original light that passes through the sample.

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Therefore, T has the range 0 to 1. The percent transmittance is simply 100T and ranges between
0 and 100%. Absorbance is defined as

When no light is absorbed, P = P0 and A=0. If 90% of the light is absorbed, 10% is transmitted
and P = P0/10. This ratio gives A = 1. If only 1% of the light is transmitted, A = 2.

Absorbance is sometimes called optical density. Absorbance is so important because it is directly

proportional to the concentration, c, of the light-absorbing species in the sample.

2.1 Lambert-Beer's Law

Beer’s law: the relationship between a sample’s absorbance and the concentration of the
absorbing species and is given as
A = bc
This equation is the heart of spectrophotometry as applied to analytical chemistry, is called the
Beer-Lambert law, or simply Beer’s law. Absorbance is dimensionless, but some people write
“absorbance units” after absorbance. The concentration of the sample, c, is usually given in units
of moles per liter (M). The path length, b, is commonly expressed in centimeters. The quantity 
(epsilon) is called the molar absorptivity (or extinction coefficient in the older literature) and
has the units M-1 cm-1 to make the product bc dimensionless. Molar absorptivity is the
characteristic of a substance that tells how much light is absorbed at a particular wavelength.

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Example: A 5.00 x 10–4 M solution of an analyte is placed in a sample cell that has a path length of
1.00 cm. When measured at a wavelength of 490 nm, the absorbance of the solution is found to be
0.338. What is the analyte’s molar absorptivity at this wavelength?

Solution

Solving equation 10.5 for e and making appropriate substitutions gives

2.2 Deviation from Beer's Law

According to Beer’s law, a calibration curve of absorbance versus the concentration of analyte in
a series of standard solutions should be a straight line with an intercept of 0 and a slope of ab or
eb. In many cases, however, calibration curves are found to be nonlinear. Deviations from
linearity are divided into three categories: fundamental, chemical, and instrumental.

13B-2 Limitations to Beer's Law

Few exceptions are found to the generalization that absorbance is linearly related to path length.
On the other hand, deviations from the direct proportionality between the measured absorbance
and concentration frequently occur when b is constant. Some of these deviations, called real
deviations, are fundamental and represent real limitations of the law. Others are a result of how
the absorbance measurements are made (instrumental deviations) or a result of chemical
changes that occur when the concentration changes (chemical deviations).

Real Limitations to Beer's Law

Beer's law describes the absorption behavior of media containing relatively low analyte
concentrations; in this sense, it is a limiting law. At high concentrations (usually >0.01 M), the
extent of solute-solvent interactions, solute-solute interactions, or hydrogen bonding can affect
the analyte environment and its absorptivity. For example, at high concentrations, the average
distances between the molecules or ions responsible for absorption are diminished to the point

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where each particle affects the charge distribution of its neighbors. These solute-solute
interactions can alter the ability of the analyte species to absorb a given wavelength of radiation.
Because the extent of interaction depends on concentration, deviations from the linear
relationship between absorbance and concentration occur. A similar effect is sometimes
encountered in media containing low absorber concentrations but high concentrations of other
species, particularly electrolytes. The proximity of ions to the absorber alters the molar
absorptivity of the latter by electrostatic interactions; the effect is lessened by dilution.

Although the effect of molecular interactions is ordinarily not significant at concentrations below
0.01 M, some exceptions occur among certain large organic ions or molecules. For example, the
molar absorptivity at 436 nm for the cation of methylene blue in aqueous solutions is reported to
increase by 88% as the dye concentration is increased from 10 -5 to 10-2 M; even below 10-6 M,
strict adherence to Beer's law is not observed.
Deviations from Beer's law also arise because absorptivity depends on the refractive index of
the medium. Thus, if concentration changes cause significant alterations in the refractive index n
of a solution, departures from Beer's law are observed. A correction for this effect can be made
by substituting the quantity n/(n2 + 2)2 for  in Equation A = bc= log (PO /P). In general, this
correction is never very large and is rarely significant at concentrations less than 0.01 M.

Apparent Chemical Deviations

Apparent deviations from Beer's law arise when an analyte dissociates, associates, or reacts with
a solvent to produce a product with a different absorption spectrum than the analyte. A common
example of this behavior is found with aqueous solutions of acid-base indicators. For example,
the color change associated with a typical indicator Hln arises from shifts in the equilibrium
HIn H+ + In-
Color 1 Color 2

Example: Solutions containing various concentrations of the acidic indicator HIn with Ka = 1.42
x 10-5 were prepared in 0.1 M HCI and 0.1 M NaOH. In both media, plots of absorbance at either
430 nm or 570 nm versus the total indicator concentration are nonlinear. However, in both
media, Beer's law is obeyed at 430 nm and 570 nm for the individual species HIn and In -. Hence,

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if we knew the equilibrium concentrations of HIn and In -, we could compensate for the
dissociation of HIn. Usually, though, the individual concentrations are unknown and only the
total concentration ctotal = [HIn] + [In-] is known.

We now calculate the absorbance for a solution with ctotal = 2.00 x 10-5 M. The magnitude of the
acid dissociation constant suggests that, for all practical purposes, the indicator is entirely in the
undissociated HIn form in the HCl solution and completely dissociated as In - in NaOH. The
molar absorptivities at 430 and 570 nm of the weak acid H1n and its conjugate base In - were
determined by measurements of strongly acidic and strongly basic solutions of the indicator. The
results were

What are the absorbances (1.00-cm cell) of unbuffered solutions of the indicator ranging in
concentration from 2.00 x 10-5 M to 16.00 x 10-5 M?
Solution:
First, we find the concentration of HIn and In- in the unbuffered 2 x 10-5 M solution. Here,
HIn H+ + In-
-
H+ In / HIn
and Ka = 1.42 x 10-5 =
From the equation for the dissociation reaction, we know that [H +] = [In-]. Furthermore, the mass
balance expression for the indicator tells us that [In-] + [HIn] = 2.00 x 10-5 M. Substitution of
these relationships into the Ka expression yields
❑

Rearrangement yields the quadratic expression [In-] 2 + (1.42 x 10-5[In-] - (2.84 x 10-10) = 0
The positive solution to this equation is [In-] = 1.12 x 10-5 M and [HIn] = (2.00 x 10-5) - (1.12 x
10-5)
= 0.88 x 10-5 M
We are now able to calculate the absorbance at the two wavelengths. For 430 nm, we can
substitute into Equation Atotal = A1 + A2 + ... + An = 1bc + 2bc + ... + nbc
A = In- b[In-] + HInb[HIn]
A430 = (2.06 x 104 x 1.00 x 1.12 x 10-5) + (6.30 x 102 x 1.00 x 0.88 x 10-5) = 0.236

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Similarly at 570 nm,

A570 = (9.61 x 102 x 1.00 x 1.12 x 10-5) + (7.12 x 103 x 1.00 x 0.88 X 10-5) = 0.073

Instrumental Deviations due to Polychromatic Radiation

Beer's law strictly applies only when measurements are made with monochromatic source
radiation. In practice, polychromatic sources that have a continuous distribution of wavelengths
are used in conjunction with a grating or with a filter to isolate a nearly symmetric band of
wavelengths surrounding the wavelength to be employed. Assuming that Beer's law applies
strictly for each wavelength, we may write for λ'

2.3 Errors associated with Beer's Law

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