Chapter 2

Absorption Theories and

Laws
 Absorption / Emission laws:
Beer-Lambert Law

The law relates the fraction of radiation absorbed by an analyte (single or mixture) to its concentration.

When a monochromatic radiation with power P0 (or intensity I0) is absorbed by a

sample, its power (or intensity) decreases exponentially to P (or I).

𝑃
𝑇= 𝐴 = −log 𝑇
𝑃0
 𝑃 Where ,
𝑇=
𝑃0 T: transmittance
P0: incident radiant power or I0: incident intensity
𝐴 = −log 𝑇 P: transmitted radiant power or I : transmitted intensity
𝑃 A: absorbance
𝐴 = −𝑙𝑜𝑔 a: absorptivity (cm-1g-1L)
𝑃0
b: path length of the light through the sample (cm)
𝑃0 c: concentration of the analyte (mol L-1) or (g L-1)
𝐴 = 𝑙𝑜𝑔  : molar absorptivity (cm-1mol-1L)
𝑃
𝑃0 𝑃0
𝐴 = 𝑙𝑜𝑔 =𝑎 𝑏 𝑐 𝐴 = 𝑙𝑜𝑔 =𝑏 𝑐
𝑃 𝑃
Remember :

 = 𝑎 x 𝑀𝑤𝑡 𝑤𝑡
number of moles =
𝑀𝑤𝑡
%T = 𝑇 x 100

Have a GO!!:
Derive this math. formula  A = 2 − log %𝑇
 RRRelation between %T and pathlength
Relation between A and pathelength

Path length /
0 0.2 0.4 0.6 0.8 1.0
cm
Draw the graph of %T vs
pathlength and the graph of A vs %T 100 50 25 12.5 6.25 3.125
pathlength
Absorbance 0 0.3 0.6 0.9 1.2 1.5

eeLinear
eeExponential
Example 2.1
A sample in a 1.0 cm cell is determined with a spectrometer to
transmit 85% light at a certain λ. If the absorptivity of this
substance at this λ is 2.0, what is the concentration of the
substance?

Answer: %T = 𝑇 x 100

%T = 85% T = 85/100 = 0.85

A = - log T = - log 0.85 = 0.070
Try to get A from :
A = 2 − log %𝑇

A = a b c = 2.0 cm-1g-1L x 1.0 cm x c

0.070 = 2.0 g-1L x c

c = 0.070/ 2.0 = 0.035 g/L

Example 2.2

Chloroaniline (M.wt = 127.6) in a sample is determined as amine

picrate. A 0.0265 g sample is reacted with picric acid and diluted to 1 L.
The solution exhibits an absorbance of 0.368 in a 1 cm cell. What is the
% chloroaniline in the sample (ε = 1.25 x 104 cm-1mol-1L)?

Answer:

A = εbc
0.368 = 1.25 x 104cm-1mol-1L x 1.00 cm x c
c = 2.94 x 10-5mol/L

M.wt of chloroaniline = 127.6

c = 2.94 10-5 mol/Lx 127.6 g/mole = 3.76 x 10-3g /L
% chloroaniline = (3.76 x 10-3g/ 0.0265 g) x 100 = 14.2%
 See also, Ex. 16.2, 16.3 pages 497-498
(Analytical Chemistry, Gary Christian, 7th edition, 2014)

Example 16.2: A solution containing 1.00 mg iron (as the thiocyanate

complex) in 100 mL was observed to transmit 70.0 % of the incident light
compared to an appropriate blank. (a) What is the absorbance of the
solution at this wavelength? (b) What fraction of light would be
transmitted by a solution of iron four times as concentrated?
Answer: (a) A = 0.15, (b) T = 0.25

Example 16.3: Amines, RNH2, react with picric acid to form amine
picrates, which absorb strongly at 359 nm ( = 1.25 × 104). An unknown
amine (0.1155 g) is dissolved in water and diluted to 100 mL. A 1-mL
aliquot of this is diluted to 250 mL for measurement. If this final solution
exhibits an absorbance of 0.454 at 359 nm using a 1.00-cm cell, what is
the formula weight of the amine?
Answer: M.wt = 127.2 g/mol
 What if the sample is a mixture of absorbing species,
can we still apply Beer Lambert law?

Yes! Under condition!!

Beer’s law is also applied to solutions containing more than one kind of absorbing
substance. Provided that there are no interactions among the various species,

What is the formula!!

the total absorbance is the sum of the individual
absorbances.

Atotal = A1 + A2 + … An
= 1bc1 + 2bc2 + … + nbcn

where 1, 2,…n are the absorbing components

 Example 2.3: Problem on absorption in mixtures

Potassium dichromate and potassium permanganate have overlapping

absorption spectra in 1M H2SO4. K2Cr2O7 has an absorption maximum at
440 nm, and KMnO4 has a band at 545 nm. A mixture is analyzed by
measuring the absorbance at these two wavelengths with the following
results: A440 = 0.405, A545 = 0.712 in a 1-cm cell. The absorbances of pure
solutions of K2Cr2O7 (1.00 x 10-3 M) and KMnO4 (2.00 x 10-4 M) in 1 M H2SO4
using the same cell gave the following results: ACr440 = 0.374, ACr545 = 0.009,
AMn440 = 0.019, AMn545 = 0.475. Calculate the concentrations of dichromate
and permanganate in the sample solution.
Step 1 : from the data of pure solution, calculate the ɛ K2Cr2O7 at the two given λ

Pure K2Cr2O7 A K2Cr2O7 = ɛ c b

At 440nm 0.374 = ɛ x 1.00 x 10-3 ɛ = 374

At 545 nm 0.009 = ɛ x 1.00 x 10-3 ɛ=9

Step 2 : from the data of pure solution, calculate the ɛ KMNO4 at the two given λ

Pure KMnO4 A KMnO4 = ɛ c b

At 440nm 0.019 = ɛ x 2.00 x 10-4 ɛ = 95

At 545 nm 0.475 = ɛ x 2.00 x 10-4 ɛ = 2375

Step 3 : from the data of the mixture, calculate the concentrations at the two λ

A mixture = (ɛ c b) K2Cr2O7 + (ɛ c b) KMnO4

At 440nm 0.405 = 374 x cK2Cr2O7 + 95 x cKMnO4 X9

At 545 nm 0.712 = 9 x cK2Cr2O7 + 2375 x cKMnO4 X 374

Step 4 : solve the two equations algebraically

A mixture = (ɛ c b) K2Cr2O7 + (ɛ c b) KMnO4

3.645 = 3366 x cK2Cr2O7 + 855 x cKMnO4
-
266.288 = 3366 x cK2Cr2O7 + 888250 x cKMnO4

262.643 = 887395 x cKMnO4

CKMnO4 = 2.95 x 10-4 M CK2Cr2O7 = 1.01 x 10-3 M
 Example 2.4: Problem on absorption in mixtures

A solution containing 0.002 M K2Cr2O7 and 0.003 M KMnO4 has

absorbance (A) of 0.675 at 390 nm. Another solution containing
0.005 M KMnO4 was found to have transmittance (T) of 0.187 at
390 nm. What is the transmittance of a solution containing
0.001 M K2Cr2O7 and 0.002 M KMnO4 at the same wavelength
(b of the cell = 1.00 cm).

(Ans. ɛ Mn= 145.63, ɛ cr = 119.05, A = 0.410, T = 0.389)

Types of spectrophotometer:

1) Single beam spectrophotometer

The sample and the reference are measured separately (one after the other).
A problem could arise if 1) the two cells are not matched
2) electricity is fluctuating
 2) Double beam spectrophotometer

The sample and the reference are measured simultaneously.

Beam splitter is used to split the radiation into two beams.
 Methods of Calibration
(application of Beer’s law – quantitative analysis):

1- Calibration Curve (Standard Curve)

- Prepare series of different concentrations of

standard solution
- Adjust the instrument at certain wavelength
(using Monochromatic light)
- Measure absorbance of each in identical
containers
- Plot the measured absorbance vs. concentration
- Create a standard curve
- Measure the absorbance of an unknown
solution and determine the concentration from
the standard curve
2-Standard Addition Method

Standard addition must be used whenever the matrix (refers to the components of a
sample other than the analyte ) may change the analytical sensitivity of the method as
example (blood sample). In other words, the slope of the working curve for standards
made with distilled water is different from the same working curve.
Prepare the Standards
The concentration and volume of the stock solution added should be chosen to
increase the concentration of the unknown by about 30% in each succeeding flask.

Slope = (A2-A1)/(C2-C1) = Ax / Cx
Example 2.5: Problem on standard addition method

(Ans. Cx = 2.23 ppm)

 Deviation from Beer’s Law:

A- If Beer’s Law is obeyed:

If Beer’s law is obeyed, a plot of absorbance as a function of concentration

is linear with a slope of εb ( or ab) and the plot goes through the origin.

𝐴=𝑎 𝑏 𝑐

𝐴= 𝑏 𝑐
 What are the
types of
deviation from
B- If Beer’s Law is not obeyed: Beer’s law?

The measured absorbance can be either larger or smaller than that predicted
by using Beer’s law:

Positive deviation: Negative deviation:

•If the actual absorbance is
•If the actual absorbance is
less than that predicted using
greater than that predicted using
Beer’s law.
Beer’s law.
 Factors of deviation: Problems and
the solutions

A- Instrumental Deviations:
1- Fluctuations in the electric current :
cause deviation in single-beam instruments by changing the intensity of the radiation
that is emitted by the EMR source and by changing the response of the detector
(negative deviation or positive deviation)
Give reason: Double beam is
preferred than the single beam
2- Stray radiation:
it can result when EMR is reflected from the walls of the cell and the walls of the cavity
within the instrument into which the cell is placed. Or it is any detected light that is
NOT absorbed by the sample. (Stray light is the most common cause of negative
deviation (why?!))

Stray radiation can be minimized or eliminated by painting the cell compartment with
flat black paint and by carefully sealing the door so that radiation from outside the
compartment is prevented from entering.
3- The use of polychromatic radiation
can be minimized by using narrow bandwidth of radiation and by selecting a wavelength on the
spectral peak using monochromator of suitable resolution power (R)

4- Inaccurate response of the detector, when the intensity of the EMR that strikes the detector is
either very large or very small.

i.e: When the concentration of the absorbing species is large, the intensity of radiation that strikes
the detector is v. small.
When the concentration of the absorbing species is small, the intensity P of the EMR that strikes
the detector is nearly identical to the intensity P0
It can be minimized by restricting absorbance measurements to the concentration range in which
the absorbance (A) varies from about 0.2 to 1.

5- Mismatched cells: non uniform cell thickness can affect a quantitative analysis.
 B- Chemical deviations:

Any chemical reaction that alters the concentration of an absorbing species

can result in a deviation from Beer’s law

•A negative deviation occurs if the concentration of the assayed substance

(analyte) is decreased because of the chemical reaction. As well, the product does
not absorb radiation at the wavelength at which the measurement is made.

•A positive deviation occurs if a product of the chemical reaction absorbs more

strongly than the assayed substance.
 Examples:

1-Association-dissociation reactions:

Copper (II) forms a complex with chloride.

The complex absorbs radiation at 438 nm:

Cu2+ + 4Cl- CuCl42-

Neither the copper ion nor the Cl- ions absorb radiation at that wavelength.

▪ Upon addition of another reactant (cation):

According to Le Chatelier’s principle, the equilibrium position is shifted to the
left, (WHY?!) then the concentration of the absorbing species decreases leading
to a –ve deviation from Beer’s law.

▪ If the concentration of Cl- is increased from 0.01 to 1 M, the reaction shifts

to the right, causing increased amount of the absorbing complex to be
formed (+ve deviation).
2- Acid-Base reactions:

p-nitrophenol

In an unbuffered solution, changes in concentration result in a

shift in the equilibrium position and a deviation from Beer’s law.
If the solution is buffered, the pH of the solution will be fixed
and the ratio of p-nitrophenol to p-nitrophenolate will be
constant.

G. R. HA H+ + A-

In any system containing a weak acid-base couple, the system should be buffered
3- Polymerization reactions:
The monomeric and polymeric forms of the absorbing species normally do not have
identical spectra e.g. the indicator methylene blue (MeBL):

MeBL = (MeBL)2 = (MeBL)3

At concentration up to 1 x 10-5M methylene blue exists in monomeric form, which has

an absorption maximum at ~ λ = 660 nm.
At concentration between 1 x 10-5M and 5 x 10-3M a mixture of monomer and dimer is
present in solution (absorption maximum at ~ λ = 610 nm).
At concentration between5 x 10-3M and 0.1M a mixture of dimer and trimer is present
in solution (absorption maximum at ~ λ = 580 nm).
.

G. R.

For methylene blue Beer’s law is obeyed only at concentration below about 1 x 10-5M.
At this concentration, the monomer of MeBL is the only form in solution
 4- Isobestic point:
A wavelength at which more than one absorbing species have identical absorptivities is
an isobestic point.
To avoid deviations from Beer’s law, we should choose a unique wavelength for the analysis at which all of
the possible absorbing species have the same absorptivity i.e working at the isobestic point.

For example, the different colored forms of an indicator in equilibrium (e.g. the red and yellow forms of
methyl orange) often exhibit an isobestic point, supporting evidence that two two colored species participate
in the equilibrium.
C- Temperature changes:
can cause shifts in chemical equilibria which can alter the absorption at a specific
wavelength. The deviation can be avoided by maintaining the cell at constant
temperature.

D- Presence of luminescing substance:

A substance that luminescence at the wavelength of the absorbance measurement

emits radiation that can strike the detector, causing a negative deviation from
Beer’s law. (HOW to solve this interference?!!)

E- Solvent effect:
The solvent can affect both the location of an absorbance maximum and the absorptivity of
the compound. (solution: using of double beam instrument where both sample cell and
reference cells contain the same solvent)
 Home work:
The problem Type of deviation from solution
Beer’s law
1- Fluctuations in the electric current : …………………………….. ……………………
2-Stray radiation: …………………… ……………………
3- polychromatic radiation …………………… ……………………
4- Inaccurate response of the detector …………………… ……………………
A- high sample concentration
B- very low sample concentration
5- Mismatched cells: …………………… ……………………

6- association reaction Cu2+ + 4Cl- ↔ CuCl42- …………………… ……………………

A-when adding cation
B- when adding Cl-
7- Acid base reactions (example – in acidic – in basic ) …………………… ……………………
8- MeBL = (MeBL)2 = (MeBL)3 at conc > 1 x 10-5 M …………………… ……………………
9- Presence of luminescing substance …………………… ……………………
 Problems on Chapter 2:
(Analytical Chemistry, Gary Christian, 7th edition, 2014 pages 543-544)

[35] A compound of formula weight 280 absorbed 65.0% of the

radiation at a certain wavelength in a 2-cm cell at a concentration of
15.0 μg/mL. Calculate its molar absorptivity at the wavelength.
Ans. Absorbed 65.0 % means %T = 100-65% = 35%

get A from : A = 2 − log %𝑇

A= 2 − log 35 = 0.4559
A=abc
0.4559 = a x 2.0 cm x (15.0 μg/mL x 10-6 g x 103 /L)

a = 15.197 cm-1 g-1L

 = a x M.wt
 = 15.197 x 280 = 4.25 x 103 cm-1 mol-1L
Problems on Chapter 2:
(Analytical Chemistry, Gary Christian, 7th edition, 2014 pages 543-544)

BEER’S LAW : problems no.

33, 34, 36, 37, 38, 39 page 543

Mixtures: problems no.

45, 46 page 544

Answers: page 813