Polychromatic light

A beam of light containing several wavelengths , e.g. white light

Monochromatic light

A beam of light containing radiation of only one discrete wavelength

Theory of light absorption :

When a monochromatic light

having intensity (Io) is allowed to
pass through absorbing
medium ;

Some is absorbed (I a), reflected (I r), transmitted

(It), refracted (If) and scattered (Is).
Is = zero for clear solution, while If and Ir may be canceled by
means of control cuvette containing the solvent in which the
substance to be anaylsed is dissolved

Therefore, under experimental conditions.

Io = Ia + It or Ia = Io - I t
The diagram below shows a beam of monochromatic
radiation of radiant power Po, directed at a sample solution.
Absorption takes place and the beam of radiation leaving
the sample has radiant power P.

Transmittance % Transmittance
T = I t / Io %T = 100 x T

Absorbance

A = log10 I0 / It

A = log10 1 / T

A = log10 100 / %T A = 2 - log10 %T

The relationship between absorbance and transmittance is
illustrated in the following diagram:

So, if all the light passes through a solution without any

absorption, then absorbance is zero, and percent
transmittance is 100%. If all the light is absorbed, then
percent transmittance is zero, and absorption is infinite.
Path length /
0 0.2 0.4 0.6 0.8 1.0
cm
%T 100 50 25 12.5 6.25 3.125
Absorbance 0 0.3 0.6 0.9 1.2 1.5
DERIVATION OF BEER
LAMBERT’S LAW
 INTRODUCTION:
Bouger, Lambert and Beer are the three
important persons who made contributions to the
field of spectrophotometer and colorimetry.

Bouger - Investigated range of absorption of light

with thickness of medium.
Lambert - Extended concepts developed by Bouger.
Beer - Applied Lambert’s law to solutions of
different concentrations.
Two laws governing absorption of radiation are….
1. Beer’s law.
2. Lambert’s law.
These two laws are applicable under the following
conditions
a) Io = Ia + It
Where
Io - intensity of incident radiation
It - intensity of transmitted radiation
Ia - intensity of absorbed light
No reflection / scattering of light take place.
 Beer’s law :states that when a beam of
monochromatic radiation passes through
a transparent , homogenous medium ,
intensity of beam of monochromatic light
decreases exponentially with increase in
concentration of absorbing substance.
When a monochromatic light is passed through
a sample solution of varying concentrations and
uniform path length ‘b’ of the absorption
species.

I It

b
Where I is the intensity of incident radiation
It is the intensity of transmitted light

According to law:

- dI /dc α I

Upon introducing the concept of proportionality,

-dI /dc = KI
On rearranging
-dI /I = kdc

- ∫ I = kc + b
1
(On integration , b is constant integration)
c- concentration
k-proportionality constant
When concentration =0, there is no absorbance . Hence( I=I 0)

substitution in equation (1)

- ∫ I0 =k×0+b

- ∫ I0 =b

u
Substituting the value of b in equation (1)

- ∫ I0 = kc –∫I0

∫ I0- ∫ I = kc

∫I0/I= kc (since log A-log B=log A/B)

I0/I=e kc (removing natural logarithm)

I/ I0=e -kc (making inverse on both sides)

2
I= I0 e -kc
(Equation of Beer,s law)
 LAMBERT’S LAW:
It states that when a beam of light is allowed to pass
through a transparent medium, the rate of decrease of
intensity with thickness of medium is directly
proportional to intensity of light.

(OR)
When a beam of monochromatic radiation passes
through homogenous uniform absorbing medium of
constant concentration, intensity of transmitted
radiation decreases logarithmically as path length of
radiation or thickness of absorber increases
arithmetically.
 i.e. –dI/dt α I
This equation can be simplified, similar to equation 2 to
get the following equation by (replacing ‘c’ with ‘t’ )

I = I0 e-kt 3

Combining Eq 2 and 3 to get

I = I0 e-kct

I = IO 10-kct (converting natural

logarithm to base 10 and k =k ×0.4343)

I/ IO =10-kct (rearranging terms)

 IO /I =10kct (inverse on both side )

log IO /I =kct 4
(taking log on both side)

 Transmittance:
When a beam of radiation passes through an
absorbing medium, fraction of radiation transmitted
out of absorber is known as transmittance.
T= I/Io
 Absorbance:
When a beam of radiation passes through
absorbing medium, fraction of radiation absorbed
by the absorber is known as absorbance.
A= log 1/T

A= -log(T)

5
A= log Io/I

using equation 4 and 5 ,since A= log Io/I and

log Io/I = kct
 A=kct(instead of k we can use є)
A= єct (Mathematical equation on
Beers Lamberts law)
Where A=absorbance or extinction coefficient
є =molecular extinction coefficent
c= concentration of drug (mmol/lit)
t =path length (normally 10mmor 1cm)

Molar absorptivity is specific coefficient for a

concentration of 1 mole/ cm3 and a path length of
1cm.
A=abc
A= absorbance
a= absorptivity
b= pathlength
c= concentration
 1.2
1
y = 0.02x
0.8
0.6
A

0.4
0.2
0
0.0 20.0 40.0 60.0
concentration (uM)
 According to Beers Lamberts law plot
of absorbance versus concentration is
expected to be a direct
proportionality
 This is not always true; there are
certain limitations.
 The law does not hold for all species under
every condition.
 Many a times instead of a straight line, a
curvature in the plot may be observed as
shown in Figure.
 The upward curvature, curve (a), is known
as positive deviation and the downward
curvature, curve(c), as negative
deviation.
Small change in concentration produces a
greater change in absorbance

Positive deviation

Negative deviation

Large change in concentration produces a

smaller change in absorbance
Deviations in Beer’s law can be
classified into:
 True deviation
 Chemical deviation
 Instrumental deviation
True deviations are related to the
concentration of the absorbing substance.
 Beer’s law holds good only for dilute
solutions with concentration in range of
10-6 M to 10-7 M.
Chemical deviations arise if absorbing species
undergo
changes such as :-
 Association
 Complex formation
 Dissociation
 Hydrogen bonding
 Hydrolysis
 Ionization or polymerisation
Association
 Metylene blue at concentration of 10-
5
M exists as monomer
 10-3 M exists as dimer or trimer
Dissociation
Potassium dichromate in high
concentration exists as orange solution
Dichromate ions are dissociate in to
chromate ions is yellow colored

dichromate ion chromate ion

Stray radiation:

 It is the radiation from the instrument

striking the detector that is outside the
nominal wavelength band chosen for the
determination which is the result of
scattering and reflection of the surface
of gratings, lenses, filters.

 The wave length of stray radiation often

differ greatly from that of the principal
radiation and in addition, the radiation
may not have passed through the
sample.
Polychromatic radiations:
 Which occurs if the absorber's absorption
coefficient alpha varies over the wavelength
interval of light passing through the sample.
 Strict adherence to Beer’s law is observed
only with truly monochromatic radiation. This
sort of radiation is only approached in
specialized line emission sources.
 All monochromators, regardless of quality and
size, have a finite resolving power and
therefore minimum instrumental bandwidth.
Mismatched cells:

 Unequal light path lengths across the light

beam

 In most laboratory applications, the

samples are measured in square sample
cells ,to insure a constant path length for
all photons.

 When round sample cells are used, the

light beam passing through the sample is
restricted to the central region of the
sample tube in order to minimize this
effect
Other deviations:
 There are other potential sources of
deviation that are not so serious under the
conditions of typical laboratory applications
of absorption spectrophotometry, or
because they can be avoided by proper
experiment design.

 These are:
1. Fluorescence of the sample

2. Light-scattering by the sample matrix,

especially in turbid samples

3. Width of slit Is not proper

4. unequal light path lengths across the light
beam.

5. unequal absorber concentration across the

light beam.

6. shifts in chemical equilibrium involving the

absorber as a function of concentration.

7. changes in pH as a function of concentration.

8. if the light intensity is extremely high (like a

focused laser), it's possible to observe non-
linear optical effects, which are a
fundamental failure of the Beer-Lambert
Law.
 Stray radiations and Polychromatic radiations
results in concave- down curvature of Beers
law.

 (4)&(5) are easily avoided by proper

experiment and instrument design (square
cuvettes, well-mixed solutions).
 (6)&(7) can be avoided by buffering the
solutions to constant pH and adjusting the
concentration of reagents.
 (8) never occurs in standard laboratory
instruments with conventional light sources.
Limitations of the Beer-Lambert law
The linearity of the beer-lambert law is limited by
chemical and instrumental factors. Causes of nonlinearity
include:

1. Deviations in absorptivity coefficients at high concentrations

(>0.01M) due to electrostatic interactions between molecules
in close proximity

2. Scattering of light due to particulates in the sample

3. Fluorescence or phosphorescence of the sample

4. Changes in refractive index at high analyte concentration

5. Shifts in chemical equilibria as a function of concentration

6. Non-monochromatic radiation, deviations can be minimized by

using a relatively flat part of the absorption spectrum such as
the maximum of an absorption band

7. Stray light
 It is not applicable for suspensions.
 It is not valid if absorbing material undergo
coagulation.
 Only monochromatic radiations should be
used.
 The measured absorbance value should be in
the range lie between 0.7 to 0.2. for better
results.
 It is applicable only at low concentrations.
Applications:
1. Clinical, inorganic, organic, biochemical
applications.
2. Very high sensitivity. Normally (analyte)
<0.01M.
3. Low detection limit.
4. Reasonable selectivity-few interferences
in most methods.
5. Good precision.
6. Convenient-rapid performed, minimal
cost.
7. To determine the Po43- in aqueous samples.
Method is applicable for marine water.
8. Estimation of water and waste water by
ascorbic acid method. The principal
involved is Mo-blue via ascorbic acid reduction
in presence of potassium antimony tartrate.
At pH 3-11 range HPO4 & H2PO4 are colorless.