CHAPTER 3.

Introduction to radiant
energy
Compiled by-Mohammed Aliye,BSC
(MLT),Msc fellow
 Absorption & transmission

 when some radiant energy passing through a solution,

transparent glass, or semitransparent substances

Some amount of light is transmitted &

Some is absorbed or trapped by the medium.

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 Absorption measurement
Many determinations in clinical chemistry are based

on the measurement of the radiant energy

 Emitted (e.g. Fluorometer)

 Transmitted

 Absorbed (absorption spectrophotometer)

 Reflected (reflectance photometer)

 Refracted (refractometer)

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 Absorption Measurment

Light transmittance
Is defined as the proportion of the incident light

that is transmitted.

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 Absorption Measurement
Transmittance (T) = I/Io

where I = transmitted light

Io = original incident
Usually this ratio is described as a percentage:

 %T = I/ Io x 100%

As the concentration of a compound in solution increases, more

light is absorbed by the solution & less light is transmitted.

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 Light absorbance

The relationship between %T & concentration is

not linear but varies inversely & logarithmically.

As a result it is more convenient to use the concept

of absorbance to avoid the use of logarithmic units.

However the concept of transmittance is important

because only transmitted light can be measured.

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 Light Absorbance
 % T can be related light of absorbance of a solution

by:
Absorbance, A = log10 I0 / I

o A = log10 1 / T

o A = log10 100 / %T

o A = 2 - log10 %T

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 1. Absorption spectrophotometry

Fundamental law of absorption

a. Introduction
 When a radiant energy, Io, passes through a
solution to be analyzed,
 some of the radiant energy will be absorbed
 some of it will be transmitted

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 Absorption Spectrophotometry

The transmitted light, I, is affected by factor such as:

Incident light

Optical path length

Concentration of solution

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 2. Beers- Lambert's law
 It is commonly referred to as Beer’s Law

1. Beer’s law
 It states that concentration of a substance is:
 directly proportional to the amount of light absorbed by
the solution
 Inversely proportional to the logarithm of transmittance
A=C
A = a. c
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 2. Lambert's law

It states that the amount of radiant energy absorbed is

directly proportional to the thickness of the medium

through which the light pass.

A=b
A = a.b

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 3. Beers-Lambert’s law

 A = a.b.c

 It is the combination of the two laws.

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 Beer-Lambert's law indicates direct proportionality
between A and c only if:
Incident radiation is monochromatic

each molecule in solution acts as an independent absorbing

species in solution
Absorption takes place in a solution of uniform cross-section

(a well mixed solution)

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  Limitations of Beer’s law-cause for deviation from the law

 non-monochromatic light
 Elevated concentration

 Solvent absorption

 Transmitted light by other mechanisms

 Non-parallel sides of cuvets

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 Deviations from beer’s law

a. Spectral interference
 The beers-lambert’s law express the linear
relationship b/n the concentration of the sample &
the absorbance value recorded.
 As a result, the linearity is only true under certain
limiting conditions

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 Beers-Lambert’s law

Some amount of radiation will be

reflected from the surface of the sample holder,

absorbed by the material of which the cell is composed or

The solvent may also absorb or reflect radiation.

Io = absorbed + transmitted + others

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 Spectrophotometry

To focus attention on the compound of interest,

elimination of these factors is necessary.

This is done through the use of blank or reference

solution
This blank should be identical to the test sample in all

aspects except the presence of the test substance

Blank reading = Io - other loses

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 Spectrophotometry
 Hence:
 Absorbed = blank – transmitted.

 Types of blank solution

1. reagent blank
2. Sample blank
3. Water saline or air blank

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 Spectrophotometry
1. Reagent blank
 reagent + solvent or
 A solution of reagents with out sample
 Used to correct high absorbance of the reagent

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 Spectrophotometry

2. Sample blank
Sample + diluent

A solution of sample & reagents missing a key

reagents that initiate the rxn or cause formation of

final rxn product.

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 Summary
Radiant energy: radio waves (longest) to gamma rays (shorts)

Properties of Electromagnetic radiation as wave and particle.

Visible light is 350-700 nm.

Interaction of EMR with matter is one of six types:

diffraction, refraction, reflection, dispersion, absorption or

transmission.

Basic law of absorption: Beer-Lambert’s law

which is A = a.b.c
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 Reference
1. Burtis, Carl A., and Ashwood, Edward R. Tietz:
Fundamentals of Clinical Chemistry. WB Saunders,
Co. Philadelphia, 2001.
2. Arneson, W and J Brickell: Clinical Chemistry: A
Laboratory Perspective 1st ed. FA Davis Co.,
Philadelphia, 2007
3. Burtis, Carl A., and Ashwood, Edward R. Tietz:
textbook of Clinical Chemistry. WB Saunders, Co.,
Philadelphia, 1999.

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 Review Questions
What are the four properties of waves?
What are the five types of interactions of EMR with
matter?
What is the formula representing Beer-Lambert’s law?
How is Beer-Lambert’s law used in Clinical
Chemistry?

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 Next Chapter
Chapter 4
Analytical procedures and Instrumentation

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