PRINCIPLES OF

SPECTROPHOTOMETRY
 Pierre Bouguer
Born: 16 February 1698 in Le Croisic, France
Died: 15 August 1758 in Paris, France

 "father of photometry"

Bouguer's law: This law expresses the relationship between

the absorption of radiant energy and the absorbing medium.
In a medium of uniform transparency the light remaining in a
collimated beam is an exponential function of the length of the
path in the medium.
Johann Heinrich Lambert (1728 - 1777)
26. August 1728 Mülhausen (Mulhouse)
† 25. September 1777 Berlin
• When EMR is allowed to pass through a liquid medium
containing chemical species:

• A part of the incident light is reflected

• A part is absorbed by the medium

• The remainder is allowed to transmit as such

I0 = Ir + Ia + It
The value of Ir can be neglected for glass interfaces, so

I0 = Ia + It

If any absorption of EMR by the medium, the intensity of emergent

light is less than that of incident light.
Beer’s law
The intensity of a beam of monochromatic radiation decreases
exponentially with the increase in the concentration of absorbing
species arithmetically.
dI
 I
dc
dI
  kI
dc
Where k is a proportionality constant
By rearranging the terms

dI
  kdc
I
On integration

dI
   k  dc
I
 ln I  kc  b (1)

Where b is a proportionality constant

When concentration C = 0, there is no absorption. Hence I= I0 substituting I
value in equation (1)

 ln I 0  k (0)  b
 ln I 0  b
Substitution of the value of b in equation (1) gives

 ln I  kc  ln I 0

ln I 0
 ln I  kc

ln I 0
 kc
I
Removing natural logarithm of the equation ln I 0  kc
I

I 0
 e
kc

I
Making inverse on both sides of the equation

I  kc
e
I 0
 kc
I  I 0 .e
Equation for Beer’s Law
The rate of decrease of intensity of monochromatic light with
thickness or pathlength of the absorbing medium is directly
proportional to the intensity of incident light

dI  kt
 I I  I 0 .e
dt Equation for Lambert’s law

 k .c.t
I  I 0 .e
Equation for Beer-Lambert’s law
  kct
I  I 0 .10
By rearranging
I  kct
 10
I 0

Taking inverse on both sides

I  10kct
0
I
Taking log on both sides
log I 0
 kct
I
A  kct
Where
A= Absorbance
K= Absorptivity constant
c= concentration of solution
t= pathlength or thickness of the absorbing medium
Absorbance :
It is the logarithm to the base 10 of the reciprocal transmittance
1
A  log
10 T

A  2  log %T
1%
Specific absorbance: ( ) Units: dl g-1 cm-1
A1cm
The absorbance of 1 g/100 ml or 1% w/v solution measured in a 1 cm cell.
Now Beer-Lambert’s law can be written as

A A 1%
1cm
.C.t
Here Concentration is expressed in %w/v
Molar absorptivity (ε): Units: litre mol-1 cm-1

The absorbance of 1mol/litre solution measured in a 1 cm cell.

 .C.t
Now Beer-Lambert’s law can be written as

A
Where ε = molar extinction coefficient Or molar absorptivity

ε values less than 100 are weakly absorbing species

ε values more than 10,000 are weakly absorbing species
Many absorbing drugs have a ε values between 103.5-104.5
litre.mol-1.cm-1
The relationship between
1%
A1cm & 

1% mol.wt.
  A1cm 
10
 • Absorbance: The reciprocal of the common
logarithm of transmittance
1
A  log
T
A  2  log %T

Transmittance:
It is the ratio of the radiant power transmitted by the sample
(IT) to the radiant power incident on the sample (I0)

T  I T

I 0
Transmittance

I0 I

b
Absorbance & Beer’s Law

Increasing absorbance
External Standard and the
Calibration Curve