Spectrophotometery

2nd stage
Presented By
MSC. Anwar Qasim
MSC. Abbas Talib
 Many determinations performed in the clinical
chemistry laboratories are based upon measurement
of radiant energy, transmitted, or absorbed reflected
under controlled condition (e.g. temperature, pH,
reagents concentrations).
Light :
It is the term which is used to describe
radiant with wavelength available to
human eye.
Wavelength (nm) :
It is distance between two peaks as the
light travels in a wave like manner .
 Human eye responds to radiant energy
with wavelengths ranging between 400
and 750 nm.

 Absorbance: The amount of light that is

absorbed by a solution and is expressed
on optical density (O.D) or (A).
 Bear – Lamberts Law :

Beer’s law was defined as the direct relationship of the

absorbance and concentration of a solution(c) also , Beer’s
law states that when monochromatic light passes through a
colored solution the amount of light transmitted decreases
with the increase in concentration of the colored substance
i.e. the absorbance or optical density increases (↑↑) with the
concentration of the substance.

A= a . b . C (Beer- Lambert’s law)… 1

a = absorptive constant
to calculate the concentration of unknown solution(CU) by comparison to standard

a = As / b . Cs ................................ 2

a = Au / b.Cu...................................3
AS AU
= 4
b. CS b . CU

The light path b remains constant in a given method of analysis with a fixed
cuvette size equal 1cm

Au
CU = . CS 5
As

Equation 5 is the formula to calculate the concentration of unknown solution (CU) colorimetry
 Spectrophotometer
 It is apparatus which is used to measure the absorbed light by
colored solution when monochromatic light passed through it.
 parts of photoelectric colorimeter
1. A Source of Light: Tungsten lamp is the source of visible light in the
colorimeters (Spectrophotometers)
(a) Tungsten lamp (400–700 nm).
(b) Deuterium lamp – U.V. (200–400 nm).

2. Monochromator(Specific wave length of light) obtained by:

(i) Selective filters.
(ii) Prisms.
(iii) Diffraction by a grating
2. Slit: This is to allow a narrow beam of selected monochromatic light to
pass through the samplesolution.

3. Cuvette: The glass container to keep the test solution

4. Photocells: Which convert quanta of radiation (light) to electrical energy,

which may be amplified, detected and recorded.