CHAPTER 3.

Introduction to radiant energy

 Objective

At the end of this chapter the student will be

able to:
• Definition of terms
• Discuss radiant energy
• Describe properties of EMR
• Explain about interaction of EMR with matter
• Discuss basic law of absorption: Beer-Lambert’s law
 Outline of radiant energy lecture
• Introduction to radiant energy
• Properties of EMR
• Interaction of EMR with matter
• Electromagnetic spectrum
• Absorption measurements: Beer-Lambert’s
law, stray light
 3.Introduction to radiant energy

Electromagnetic radiation
• Radiation showing electric & magnetic
characterstics in the form of waves or photons is
termed as electromagnetic radiation

• It travels at approx. 3 x 105 km/s in the vacuum of

space.

• In materials which are transparent to

electromagnetic radiation, the velocity is slightly
less than the velocity in a vacuum.
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Dual Nature of EMR Energy
• Energy transfers in the physical world either by
waves or particles

• In general, electromagnetic radiation behaves:

as a wave when moving through space,
as a particle when it interacts with matter

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Wave Properties
• Wave is the way of transferring an energy from one
place to another
• Consists of discrete packets of energy or quanta
called photons
• It Can be described by:
1. Velocity (c )
2. Amplitude
3. Wave length (λ)
4. Frequency (ν)

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1. Amplitude
 The height the wave crest or troughs from the
baseline
 Governs brightness of light

2. Wavelength
 distance between two wave crests or troughs
 One full wave cycle (crest top to next crest
top).

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3. Frequency
how fast it oscillates (goes up & down)
measured in cycles (remember crest to crest)
per second.

The number of wave crests per second, that

is the number of wave crests that passes by a
given point in one second.
1cycle/second = 1 Hz (hertz) = 1s-1.

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4. Speed of Light (velocity)
 Speed of the wave

 For example;

– water - few meters per second

– Sound wave – 340 m/sec

– Light - 3 x 108 meters/second

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Relationship between c, λ & ν.

 The longer the wavelength the lower the frequency,

or the shorter the wavelength the higher the
frequency.

 This relationship is expressed in the formula

ν = c/λ where: - ν - frequency of light in cycle/sec.

- c - speed of EM wave in vacuum
- λ - wave length in cm

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  E = hv

 Where h = planck’s constant (6.62x10-27

erg.sec)
ν = frequency
E = energy

 E = h c /λ

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Interaction of EMR with matter

• In order to use photometric instruments

correctly & to be able to develop & modify
spectroscopic techniques it is necessary to
understand the principle of interaction of
radiation with matter.

• The only way to observe electromagnetic

radiation is by its interaction with matter.

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• It involves:
 Diffraction

 Reflection

 Refraction

 Dispersion

 Absorption & transmission

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1. Diffraction
Is the change of direction of the EM beam when
it strikes the edge of an opaque body or it
passes through a small hole

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2. Refraction
• bending of light as it passes through materials of
different optical density

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3. Reflection
 When radiation falls on silver coated glasses,
the beam of the radiation returns towards the
source of radiation

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4. Dispersion
• is the change in refractive index with a change in
wavelength

• The velocity of light in a materials & its refractive

index depends on the wavelength of the light.

• This causes the light to be refracted by different

amounts according to the wavelength (or color).

• This gives rise to the colors seen through a

prism.
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• Rainbows are caused by dispersion of light
inside the raindrop & total internal reflection of
light from the back of raindrops.

• The following is a chart giving the index of

refraction for various wavelengths of light in
glass

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The electromagnetic Spectrum
• Spectrum is an ordered arrangement of radiant
energy according to the wavelength.

A. Continuous spectrum
• A spectrum which is composed of visible lights
of all wavelengths are called Continuous
spectrum

• It is a continuous spectrum because one color

fades into another.
• E.g. sun light or light from ordinary
incandescent bulb

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 Component energies of the electromagnetic
spectrum

a. Radio waves
 The longest- from a few meters to longer than the
size of the earth.

 They can travel long distance in the atmosphere

b. Microwaves
 The wavelength is from about 1 millimeter to 1 meter.

 Used in communication, radar & cooking

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 c. Infra red

 Wavelength from 10-3 to 10-6 (micron)

 Ranges from approx 12,500 – 50 cm-1
• Used in toxicology and molecular structure
determination
• 4000 to 1000 cm -1 – used for the analysis of
organic compounds

• 1000 to 400 cm -1 - is used for the analysis of

inorganic compounds

• 12,500 to 4000 cm -1 is not helpful for such

analysis.
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 d. Visible

• It is a very small portion of the total EM spectrum

visible to human eye

• It ranges from 700nm (at red light) to 400nm (violet)

• the visible color of a solution corresponds to the

wavelength of the light that are transmitted, not
absorbed, by the solution.

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• The different colors have different
wavelengths & frequencies.

• The rest of the EM spectrum is not visible

to the human eye

• Source of visible light:

o tungsten lamp.
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 wavelength Color of Color of light /solution
in nm light reflected/complementary
absorbed
350-430 Violet Yellow
430-475 Blue Orange
475-495 Blue-green Red -Orange
495-505 Blue-green Orange - Red
505-555 Green red
555-575 Yellow- Violet red
green
575-600 Yellow Violet
600-650 Orange Blue
650-700 Red green
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Note:
• A substance that absorbs green light at 500 nm
reflects or transmits all other lights or
wavelengths & appears as purple.

• To measure the concentration of a blue solution,

light at about 590 nm is passed through the
solution

• The amount of yellow light absorbed varies

directly as the concentration of the absorbing
substance in solution
28
 The absorbed color is the complementary of the
transmitted color.

 Thus to make absorption measurement, one

must use the wavelength at which a colored
solution absorbs light.

 For example, a red solution absorbs green light

& transmitted red light. Therefore, a red solution
should be measured at 490 to 550nm

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• In photometer using filter used as a monochromator,
the filter chosen is usually complementary to the color
of the solution to be measured.
 blue solution – yellow filter

 Yellow solution –blue filter

 Red solution – blue green filter

 Blue green solution – red filter

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 e. UV Light

• It ranges from 400 to 100 nm

• It is dangerous to tissue & cells (common sun burns)

• It is obtained by energy transition in the valence

electrons of the molecules.

• Widely used in the quantitative & qualitative

determination of clinical chemistry tests.

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• Source:

– discharge tube containing hydrogen or

deuterium at reduced pressure.

– High pressure mercury & xenon arc lamps.

• c + d + e = light

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 f. X- ray

• It ranges from 100 to 0.1 nm

• High frequency, high energy waves that can

penetrate several centimeters into most solid
matter.

• Used in radiological diagnosis

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 g. Gamma rays

 it ranges from 0.1 to less than 10-16

 It is the highest energy ray in the EM

spectrum

 It is generally produced in nuclear reactions &

not as common in nature,

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B. Line/atomic emission spectrum

• a spectrum with only certain colors. (NOT

continuous like sunlight)

• Samples of elements emit light when they are

vaporized (heated) or electricity passes through
them.

• Every element has a unique line

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• The wavelength of the line are characteristics of
a particular element

• It can be used for qualitative identification &

quantitative determination of elements in an
unknown mixture

• For example, flame photometer, atomic

absorption spectrophotometer.

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 5. 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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Light transmittance

• Is defined as the proportion of the incident light

that is transmitted.

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• 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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• % 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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Relationship between absorbance & transmittance

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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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• The transmitted light, I, is affected by factor such
as:
o Incident light

o Optical path length

o 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:
o directly proportional to the amount of light
absorbed by the solution &
o inversely proportional to the logarithm of
transmittance
o A=C
o 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 a 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.

• However, the relationship is only an

experimental one & not a fundamental law of
nature.
• As a result, the linearity is only true under
certain limiting conditions

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• 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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 • 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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 • Hence:
• Absorbed = blank – transmitted.

• Types of blank solution

1. reagent blank

2. Sample blank

3. Water saline or air blank

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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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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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 Io IR - reference cell

Io Is – sample

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 o T = Is/ IR

o A = log 1/T

o A = log 1/ Is/ IR

o A = log IR / Is

o A = - log Is / IR

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b. Stray light
• Quantitative radiation rely on radiation that reach the
detector passing through the sample

• But it is impractical, because it is difficult to design

instrument which are capable of effectively eliminating
all extraneous radiation

• Much of these unwanted radiation arises from the

scattering of the incident radiation by irregularities in
surfaces (by faults in manufacturers) or scratches

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• Such light, stray light, results in a deviation from
Beer’s law &

• The effect is that absorbance measurements are lower

than they should be.

• It is possible to asses the proportion of stray light by

measuring the amount of radiation transmitted by
samples which are optically opaque at the wavelength
to be assessed but which transmit radiation of other
wavelengths

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• The instrument is set to zero %T with blocking
light path & 100% transmittance with a reagent
blank in the normal way & opaque substance
introduced into the sample compartment.

• The amount of light transmitted by the sample,

measured in percentage transmittance is quoted
as the stray light at a specified wavelength.

12/23/22
 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
 Reference
1. Burtis, Carl A., and Ashwood, Edward R.. Tietz:
Fundamentals of Clinical Chemistry. Philadelphia, 2001.
2. Arneson, W and J Brickell: Clinical Chemistry: A
Laboratory Perspective 1st ed. 2007 FA Davis

3. Burtis, Carl A., and Ashwood, Edward R.. Tietz:

textbook of Clinical Chemistry. Philadelphia, 1999.
 Next Chapter

Chapter 4
•
Analytical procedures and
Instrumentation