Cebu Doctors’ University

College of Arts and Sciences

Physical Sciences Department

MODULE 4.2 – THE RELATIONSHIP BETWEEN ABSORPTION OF RADIATION AND

CONCENTRATION OF ANALYTE
Compiled by: Joselito R. Tumulak Jr., RChT, MS (cand.)
Analytical Chemistry Professor

UNIT OUTLINE
Topic Page
I. Absorption of Radiation
A. The Absorption Process
B. Transmittance and Absorbance
II. The Beer-Lambert’s Law
A. Relationship Between Absorbance and Concentration
B. Limitations of the Beer’s Law

I. ABSORPTION OF RADIATION

A. The Absorption Process

• When a beam of electromagnetic radiation passes through sample, much of the radiation is
transmitted without loss of intensity. However, at selected frequencies, the radiation’s intensity is
attenuated. This process of attenuation is called absorption.
• Requirements for Analytes to Absorb Radiation:
1. There must be a mechanism by which the radiation’s electric field or magnetic field interacts
with the analyte.
a. For ultraviolet and visible radiation, this interaction involves the electronic energy
of valence electrons.
b. For infrared radiation, a chemical bond’s vibrational energy is altered.
2. The energy of the electromagnetic radiation must exactly equal the difference in energy, ∆E,
between two of the analyte’s energy states.

The figure on the right is a good representation

of absorbance. The thick lines labeled E0 and E1
represent the analyte’s ground (lowest)
electronic state and its first electronic excited
state. Superimposed on each electronic energy
level is a series of lines representing vibrational
energy levels. The picture illustrates than
vibrational transitions require less energy than
electronic transitions. Moreover, if each arrow
represents electromagnetic radiation beam at
different frequencies, only the last beam causes
an electronic transition.

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B. Transmittance and Absorbance
• The attenuation of electromagnetic radiation as it
passes through a sample is described quantitatively by two
separate, but related terms: transmittance and
absorbance.
• Transmittance is defined as the ratio of the
electromagnetic radiation’s power exiting the sample, P, to
that incident on the sample from the source, P0,
𝑃
𝑇=
𝑃!
• Multiplying the transmittance by 100 gives the percent
transmittance (%T), which varies between 100% (no
absorption) and 0% (complete absorption). All methods of
detection, whether the human eye or a modern
photoelectric transducer, measure the transmittance of
electromagnetic radiation.
• An alternative method for expressing the attenuation of electromagnetic radiation is absorbance,
A, which is defined as:
𝑃 𝑃!
𝐴 = − log 𝑇 = − log = log
𝑃! 𝑃
Absorbance is the more common unit for expressing the attenuation of radiation because, as shown
in the next section, it is a linear function of the analyte’s concentration.

• Sample calculation:
1. What is the absorbance when a sample has a percent transmittance of 50%?

Solution:

%𝑇 50
%T = T × 100 → T = = = 𝟎. 𝟓𝟎
100 100

𝐴 = − log 𝑇 = − log 0.50 = 𝟎. 𝟑𝟎𝟏

II. THE BEER-LAMBERT’S LAW

A. Relationship Between Absorbance and Concentration

• According to the Beer-Lambert’s law (or simply Beer’s law), absorbance is directly proportional to
the concentration of the absorbing species, c, and to the path length, b, of the absorbing medium.
This is expressed as

𝑃!
𝐴 = log = 𝑎𝑏𝑐
𝑃
In the equation, a is the proportionality constant called absorptivity. Because absorbance is a
unitless quantity, the absorptivity must have units that cancel the units of b and c.
o If, for example, c has the units of g/L and b has the units of cm, absorptivity has the units of
L/g∙cm.
• When we express the concentration in in moles per liter and b in cm, the proportionality constant is
called the molar absorptivity and is given the symbol 𝜀 (unit is L/mol∙cm). Thus,

𝐴 = 𝜀𝑏𝑐

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Using Beer’s Law
• Beer’s law can be used in:
1. Calculating molar absorptivities of species if the concentration is known.
2. Calculating concentration using measured absorbance if absorptivity and path length are
known.
§ However, absorptivities, are functions of such variables as solvent, solution
composition, and temperature. It is never a good idea to depend on literature values
for quantitative work. Hence, a standard solution of the analyte in the same solvent
and at a similar temperature is used to obtain the absorptivity at the time of the
analysis. Most often, we use a series of standard solutions of the analyte to construct
a calibration curve, or working curve, of A versus c or to obtain a linear regression
equation.

• Sample calculation:
1. A 7.25 × 10"# M solution of potassium permanganate has a transmittance of 44.1% when
measured in a 2.10-cm cell at a wavelength of 525 nm. Calculate (a) the absorbance of this
solution and (b) the molar absorptivity of KMnO4.

Solution:

(a) 𝐴 = − log 𝑇 = − log 0.441 = 0.356

! $.&'( 4
(b) 𝐴 = 𝜀𝑏𝑐 → 𝜀 = = = 2.34 × 10&
"# (*.+$ #-)(/.*' × +$$% -12/4) -12∙#-

Applying Beer’s Law to Mixtures

• Beer’s law also applies to solutions containing more than one kind of absorbing substance. Provided
that there is no interaction among the various species, the total absorbance for a multicomponent
system at a single wavelength is the sum of the individual absorbances. In other words,

𝐴&'&() = 𝐴* + 𝐴+ + ⋯ + 𝐴, = 𝜀* 𝑏𝑐* + 𝜀+ 𝑏𝑐+ + ⋯ + 𝜀, 𝑏𝑐,

where the subscripts refer to the absorbing species 1, 2, …, n.

• Sample calculation:
1. The molar absorptivity for Co and Ni complexes with 2,3-quinoxaline dithiol are 36,400 and 5,520
at 510 m, respectively. At 656 m, their molar absorptivities are 1,240 and 17,500. A 0.425-g
sample was dissolved and diluted to 50.0 mL. A 25.00-mL aliquot was treated to eliminate
interferences, and after addition of the coloring reagent, the volume was adjusted to 50.00 mL.
This had an absorbance of 0.446 at 510 m and 0.326 at 656 nm in a 1.00-cm cell. Calculate the
molarity of Co and Ni.

Solution:

*Absorbance at 510 nm:

𝐴(#*! ,/) = 𝐴* + 𝐴+ = 𝜀* 𝑏𝑐* + 𝜀+ 𝑏𝑐+ = (36400)(1 𝑐𝑚)(𝐶1' ) + (5520)(1 𝑐𝑚)(𝐶23 ) = 0.446
= (𝟑𝟔𝟒𝟎𝟎)(𝑪𝑪𝒐 ) + (𝟓𝟓𝟐𝟎)(𝑪𝑵𝒊 ) = 𝟎. 𝟒𝟒𝟔 (1)

*Absorbance at 656 nm:
𝐴(8#8 ,/) = 𝐴* + 𝐴+ = 𝜀* 𝑏𝑐* + 𝜀+ 𝑏𝑐+ = (1240)(1 𝑐𝑚)(𝐶1' ) + (17500)(1 𝑐𝑚)(𝐶23 ) = 0.326
= (1240)(𝐶1' ) + (17500)(𝐶23 ) = 0.326
𝟎. 𝟑𝟐𝟔 − (𝟏𝟕𝟓𝟎𝟎) (𝑪𝑵𝒊 )
∴ 𝑪𝒄𝒐 = (𝟐)
𝟏𝟐𝟒𝟎
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 *Substituting (2) to (1) to solve the concentration of Ni:
(36400)(𝐶1' ) + (5520)(𝐶23 ) = 0.446

0.326 − (17500) (𝐶23 )

(36400) L M + (5520)(𝐶23 ) = 0.446
1240
36400
[0.326 − (17500)(𝐶23 )] + (5520)(𝐶23 ) = 0.446
1240

9.5696774 − (513709.677)(𝐶23 ) + (5520)(𝐶23 ) = 0.446

−(513709.677)(𝐶23 ) + (5520)(𝐶23 ) = 0.446 − 9.5696774

(−508189.677)(𝐶23 ) = 0.446 − 9.5696774

0.446 − 9.5696774
(𝐶23 ) = = 𝟏. 𝟖𝟎 × 𝟏𝟎"𝟓 𝑴
−508189.677

* Solve for the concentration of Co using (2):

0.326 − (17500) (𝐶23 ) 0.326 − (17500) (1.80 × 10"# )
𝐶;' = = = 𝟖. 𝟖𝟕 × 𝟏𝟎"𝟔 𝑴
1240 1240

Selecting Wavelength to Use in Absorption Spectroscopy

• To help us easily understand this, let’s consider a question: “Why is red solution red?”
o For example, an aqueous solution of the complex Fe(SCN)2+ is not red because the complex
adds red radiation to the solvent. Instead, it absorbs green from the incoming white radiation
and transmits the red component. Thus, in a colorimetric determination of iron based on its
thiocyanate complex, the maximum change in absorbance with concentration occurs with
green radiation; the absorbance change with red radiation is negligible. In general, then, the
radiation used for a colorimetric analysis should be the complementary color of the analyte
solution. The following table shows this relationship for various parts of the visible spectrum.

The Visible Spectrum

Wavelength Region Absorbed Complementary Color
Color of Light Absorbed
(nm) Transmitted
400 – 435 Violet Yellow-green
435 – 480 Blue Yellow
480 – 490 Blue-green Orange
490 – 500 Green-blue Red
500 – 560 Green Purple
560 – 580 Yellow green Violet
580 – 595 Yellow Blue
595 – 650 Orange Blue-green
650 – 750 Red Green-blue

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B. Limitations of the 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 εb. In many
cases, however, calibration curves are found to be
nonlinear. Deviations from linearity are divided into three
categories: fundamental, chemical, and instrumental.

Fundamental Limitations to Beer’s Law

• Beer’s law is a limiting law that is valid only for low

concentrations of analyte. There are two contributions to
this fundamental limitation to Beer’s law:
o At higher concentrations the individual particles
of analyte no longer behave independently of one another. The resulting interaction between
particles of analyte may change the value of ε.
o A second contribution is that the absorptivity, a, and molar absorptivity, ε, depend on the
sample’s refractive index. Since the refractive index varies with the analyte’s concentration,
the values of a and ε will change. For sufficiently low concentrations of analyte, the refractive
index remains essentially constant, and the calibration curve is linear.

Chemical Limitations to Beer’s Law

• Chemical deviations from Beer’s law can occur when the absorbing species is involved in an
equilibrium reaction. From previous lesson, we already established that both reactants and
products coexist in an equilibrium system. Beer's Law assumes that the analyte of interest is the
only species contributing to the absorbance. In complex samples, such as a system in equilibrium,
there may be other substances present that can interfere with the measurement, causing
inaccuracies.
• Beer's Law also assumes that the solvent used does not affect the absorption of light by the analyte.
However, changes in the solvent properties, such as refractive index or pH, can influence the
absorption characteristics and lead to deviations from Beer's Law.

Instrumental Limitations to Beer’s Law

• There are two principal instrumental limitations to Beer’s law:
1. The first limitation is that Beer’s law is strictly valid for purely monochromatic radiation; that
is, for radiation consisting of only one wavelength. Recent technology has developed
wavelength selector, but still, it passes radiation with a small, but finite effective bandwidth.
Using polychromatic radiation always gives a negative deviation from Beer’s law but is
minimized if the value of ε is essentially constant over the wavelength range passed by the
wavelength selector.
2. Stray radiation is the second contribution to instrumental deviations from Beer’s law. Stray
radiation arises from imperfections within the wavelength selector that allows extraneous
light to “leak” into the instrument.

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