Optical Techniques sims 2021

Optical Techniques
Course: Clinical Laboratory Principle (SIMS-443)
ZA School of Medical Technology
1
Dr. Ali Raza
Senior Lecturer
SIMS-SIUT

Optical Techniques
 Introduction
Electromagnetic Spectrum
Beer-Lambert Law
Calculate the concentration of a solution
 Nucleic acid calculation
 Applications
Photometry
Spectrophotometer
Flame Emission Spectrophotometry
Atomic Absorption Spectrophotometry
Fluorometry
Luminometry 2

Optical Techniques
Optics is the branch of physics which involves the
behavior and properties of light”
 Interactions with matter
 Construction of instruments.
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Optical Technique
•Radiant Energy :
 Energy transmitted in wave motion
 Travel through space
For example:
Heat from the sun, which is located very far from the earth via radiation.
Electromagnetic waves
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The Electromagnetic Spectrum
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Optical Technique
The EM Radiation interact with biomolecule
 Reflected
 Absorbed
Scattered
Emitted
 Transmitted
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Optical Technique
Example:
The EM Radiation interact with biomolecule
 Reflected
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Absorption:
Examples:
Spectrophotometry
 Photometry
 X-ray spectroscopy
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Scattering
Turbidity:
 The cloudiness of a solution caused by
suspended particles that scatter light
 light scattered related to the concentration
and sizes and shapes of the particles.
Turbidimetry:
Performed through use of an instrument
(spectrophotometer) measures the ratio of the
intensity of the light transmitted through
dispersion to the intensity of the incident light.
10

Emission
 When the atoms move to a lower energy state.
 Release of energy, usually in the form of a photon,
which is a tiny energy packet.
 This energy emits from the atom in the form of
radiation, generally away from the atom.
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Examples of Emission:
Luminescence: the emission of light or
radiant energy when an electron returns
from an excited or higher energy level to a
lower energy level.
Example:
Fluorescence
Phosphorescence
Chemiluminescence:
The emission of light by molecules in excited
states produced by a chemical reaction,
Example: Fireflies
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Relationship Between Transmittance
and Absorbance
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Relationship Between Transmittance and Absorbance

When an incident light beam with intensity I0. passes through a
square cell containing a solution of a compound that absorbs light
of a specific wavelength, λ
• The intensity of the transmitted light beam Is is less than lo,
•The transmitted light (T) is defined as
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 Some of the incident light may be reflected by
• Surface of the cell
• Absorbed by the cell wall or Solvent
These factors are eliminated by Reference cell
Reference Cell:
•Identical to the sample cell
•Except that the compound of interest (sample) is omitted from
the solvent in the reference cell.
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Transmittance
The Transmittance (T) through this Reference cell is
T = IR
Io
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Light Transmittance of sample versus reference = Is / IR.
• Transmittance for the compound in solution is defined as
T= Is
IR
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•In practice the Reference cell is inserted and the instrument
adjusted to an arbitrary scale reading of 100 ( 100% T)
•Non-Absorbing samples: Transparent
• Io = I;
•Io is incidence light and l is transmitted light
•T= 100%
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Transmittance
•Opaque samples
• Io = 0;
•Io is incidence light
•T= 0%
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Absorbance (A)
 The capacity of a substance to absorb radiation
Molar absorptivity (Ɛ): A constant for a one molar
solution of a given compound at a given wavelength
and a 1cm path length under prescribed condition of
solvent, temperature and ph.
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Relationship Between Transmittance (T) and Absorbance (A)
Expressed as the logarithm (log) of the reciprocal of
transmittance (T)
 The amount of light absorbed (A) as the incident light
passes through the sample is equivalent to
Abs (A)= log (1/ T)
= -log T
= -log Is
IR
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T= Is
IR

Beer's Law
Absorbance is directly proportional to concentration of solution
in which light has to passed.
Abs= ε c
ε = Molar extinction co-efficient
C= concentration of solution
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Lambert’s Law
“The loss of light intensity when it propagates in a medium is directly
proportional to intensity and path length.”
Absorbance is proportional to length of sample in which light passed
Abs= log 10 (1/ T) α l
L = Path length
Abs= ε l
ε = Molar extinction co-efficient
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Beer-Lambert Law
Combining both
Abs= log 10 (1/ T) α c l
Abs= ε c l
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Beer-Lambert Law
Mathematically, Beer's law is expressed as
A = ɛ x l x c
A = abc
A = Absorbance = the amount of light absorbed by the
sample for a given wavelength
a = ɛ is the molar absorptivity
b = the distance that the light travels through the solution,
Light path in centimeters
c = Concentration of the absorbing compound (grams per liter)
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Beer-Lamberts law
Application
Generally, it can be used to determine
 concentrations of a particular substance,
 determine the molar absorptivity of a substance.
Spectrophotometry
Example:
Determination of bilirubin in blood plasma samples.

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Beer-Lamberts law
Application
Absorption spectra: a linear calibration curve of the absorbance
versus concentration

Beer‘s law follow if the following conditions are met:
• Incident radiation on the substance of interest is
monochromatic.
• The solvent absorption is insignificant, compared with
the solute absorbance.
• The solute concentration is within given limits.
•An optical interferant is not present.
• A chemical reaction does not occur between the molecule of
interest and another solute or solvent molecule.
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Deviations to the law
The Beer-Lambert law maintains linearity under specific
conditions only.
The law will make inaccurate measurements at high
concentrations
because the molecules of the analyte exhibit stronger
intermolecular and electrostatics interactions which is due to
the lesser amount of space between molecules.
 This can change the molar absorptivity of the analyte.
Not only does high concentrations change molar absorptivity,
but it also changes the refractive index of the solution causing
departures from the Beer-Lambert law.

What is a Spectrometer?
Any instrument that is used to measure the variation of a
physical characteristic over a given range; i.e. a spectrum.
 Mass Spectrometer : Mass-to-charge ratio Spectrum
 NMR Spectrometer : Variation of nuclear resonant
frequencies
 Optical Spectrometer: Change in the absorption and
emission of light with wavelength
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Spectrophotometers are classified as being either
Single- Beam
Double-Beam.
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Nucleic acid concentration
Absorbance = A 260 = 0.52
We know that
1 Abs 260 = 1 OD= 50mg/ml
0.52 Abs 260 = ? mg/ml
Dilution:
1:20, 1:10, etc.
Formula:
Concentration: Abs 260 x molar cofficient x dilution F
= 0.52 x 50 x 20 = mg/ml

Optical Techniques
Photometry
Spectrophotometer
Flame Emission Spectrophotometery
Atomic Absorption Spectrophotometery
Fluorometry
Luminometry
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Photometer and Spectrophotometer
 Devices used to measure intensity of light
emitted by,
passed through
reflected by a substance
Photometry:
The measurement of the luminous intensity of light or the
amount of luminous light falling on a surface from such a source.
 Defined originally as the process used to measure light intensity
independent of wavelength.
 Use Photometers filters
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Light Meter

Optical Spectrometer
 Goal: Measure the interaction (absorption, reflection,
scattering) of electromagnetic radiation with a sample or the
emission (fluorescence, phosphorescence) of electromagnetic
radiation from a sample.
 Optical spectrometers are concerned with electromagnetic
radiation that falls within the optical region of the
electromagnetic spectrum. i.e light spanning the ultraviolet
(180-390nm), Visible (390-780nm) and Infrared (780- 12,000nm
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Spectrophotometer
Spectrophotometry:
•The measurement of the intensity of light at selected wavelengths.
• Use prisms or Gratings
Ultraviolet (UV) radiation = <380 nm, The 180 to 390nm region of
the electromagnetic spectrum.
Visible light radiation = The 390 to 780 nm region of the
electromagnetic spectrum that is visible to the human eye.
Infrared (IR) Radiation: The 770 to 12,000nm region of the
electromagnetic spectrum.
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Spectrophotometer Components
1. Light source: Tungsten light bulb, Quartz-halogen lamp, laser
Mercury-vapour lamp, hydrogen lamp,
Deuterium lamp
2. Slit
3. Monochromator: Filters, Prisms and diffraction gratings, Fiber
optics
4. Cuvets: Glass, Silica (quartz) and plastics
5. A Photodetector: Photomultiplier tube(PMT)
6. A Readout device
7. A computer.
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Flame Emission Spectrophotometry
flame atomic emission spectrometry (FAES).
A necessary tool in the field of analytical chemistry.
Used to determine the concentration of certain metal ions
like
– Sodium,
– Potassium,
– lithium,
– calcium,
– cesium etc.
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Principle of Flame photometer
• Compounds of the alkali and alkaline earth metals
(Group II) dissociate into atoms when introduced into
the flame.
• Some of these atoms further get excited to even
higher levels. But these atoms are not stable at
higher levels.
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• These atoms emit radiations when returning back to
the ground state. These radiations generally lie in the
visible region of the spectrum.
• Each of the alkali and alkaline earth metals has a
specific wavelength.
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• These colors are characteristic of the metal
atoms that are present as cations in solution.
• The light intensity of the characteristic
wavelength produced by each of the atoms is
directly proportional to the number of atoms
that are emitting energy, which in turn is
directly proportional to the concentration of the
substance of interest in the sample.
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• It is based on the measurement of the emitted light
intensity when a metal is introduced into the flame.
• The wavelength of the colour gives information about the
element and the colour of the flame gives information
about the amount of the element present in the sample
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https://www.studyandscore.com/studymaterial-detail/flame-photometer-principle-components-working-procedure-applications-advantages-and-disadvantages

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Branch of Atomic Absorption Spectroscopy. Flame photometer
Desolvation:
Desolvation involves drying a sample in a solution. The metal particles in
the solvent are dehydrated by the flame and thus solvent is evaporated.
Vaporization: The metal particles in the sample are also dehydrated. This
also led to the evaporation of the solvent.
Atomization: Atomization is the separation of all atoms in a chemical
substance. The metal ions in the sample are reduced to metal atoms by
the flame.
Excitation: The electrostatic force of attraction between the electrons and
nucleus of the atom helps them to absorb a particular amount of energy.
The atoms then jump to the higher energy state when excited.
Emission: Since the higher energy state is unstable the atoms jump back
to the ground state or low energy state to gain stability. This jumping of
atoms emits radiation with characteristic wavelength. The radiation is
measured by the photo detector.

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 Limitation of the Flame Photometer
 The element is not appreciably excited in the flame, but is
merely dissociated from its chemical bonds (atomized) and
placed in an unexcited or ground state (Neutral atom)
 This unexcited atom is capable to absorbing radiation at a
specific wavelength.
 For example: if the cathode lamp of Na was made, sodium light
(589nm) will be emitted by the lamp.
 When light enters in the flame, some of it is absorbed by
ground state atoms in the flame.
 Results in a net decrease in the intensity of the lamp beam

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Atomic Absorption Spectrophotometry (AAS)
 Analysis technique for rapid trace metal analysis
It is based on element specific wavelength light
absorption by ground state atoms in the flame or
electro thermal graphite furnace.

50
 When the salt soln. is put into the flame
first solvent is vaporized the tiny particles
of solute molecules are obtained which
on further heating in the flame are
converted into gaseous molecules the
molecules dissociate into atoms.
 Some of the atoms are exited unexcited
atoms will absorb their characteristic
radiation.
 A.A.S. is concerned with this part of light
(radiations) which are absorbed by the
unexcited atoms present in the ground
state.

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Applications
• Analysis of clinical samples, which often involves the determination of presence of
metals in fluids and tissues, whether for toxicological investigation or for therapeutic
indications.
• Most of these studies are carried out on urine, although determinations are also
made in whole blood, blood serum, hair, biological tissues, or saliva
https://www.sciencedirect.com/topics/chemical-engineering/atomic-absorption-spectrometry

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Applications
• Determination of even small amounts of metals
(lead, mercury, calcium, magnesium, etc.)
 Environmental studies:
Drinking water, ocean water, soil
 Food industry.
 Pharmaceutical industry.

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http://zeiss-campus.magnet.fsu.edu/articles/basics/fluorescence.html
 luminescence family of processes in which susceptible
molecules emit light from electronically excited states created
by
• either a physical (Absorption of light)
• Mechanical (friction),
• Chemical mechanism.
Photoluminescence Generation of luminescence through excitation of a molecule
by ultraviolet or visible light photons is a phenomenon termed photoluminescence,
 depending upon the electronic configuration of the excited state and the emission
pathway divided into two categories
 Fluorescence
 Phosphorescence

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Luminescence Energy level
 Relationship between absorption,
fluorescence and phosphorescence
 Each molecules contains a series of
closely spaced energy levels
 Absorption of light causes the transition
of an electron from ground state to a
number of possible vibrational levels
 It returns to its original energy state by
 Fluorescence
 Phosphorescence
 Radiationless vibrational equilibrium
 Radiationless crossover to a triplet state
 Quenching of the excited singlet state

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Fluorescence:
Fluorescence is the property of
some atoms and molecules to
absorb light at a particular
wavelength and to subsequently
emit light of longer wavelength
after a brief interval, termed the
fluorescence lifetime.

58
Phosphorescence
 The property of some atoms and molecules to absorb
light at a particular wavelength and to subsequently emit
light of longer wavelength, but with a much longer
excited state lifetime.
 Phosphorimetry: The measurement of phosphorescence
 Emission of phosphorescence light is longer than the
decay time of fluorescence

Fluorometry
 When a beam of light is incident on certain
substances they emit visible light or radiations.
This is known as fluorescence.
 Fluorescence starts immediately after the
absorption of light and stops as soon as the
incident light is cut off.
 The substances showing this phenomenon are
known as flourescent substances.
 The measurement of fluorescence is called.
Fluorometry
60

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 Quantify biological analytes as a
function of fluorescence.
 Requires the sample to be bound to a
specific fluorescent agent
 An extensive range of nucleic acid,
cell function dyes, and fluorescent
proteins are commercially available
worldwide.
.
Fluorometry

62
 Fluorophores absorb light of a distinct excitation
wavelength and emit, or fluoresce, light of reduced
energy thus a longer wavelength.
 This behavior can be modified so that the fluorescent
reagents are restricted from emitting light unless
bound to a specific molecule, such as dsDNA.
Fluorometry

Fluorometry
 Fluorophore:
(Fluorochrome)
 Fluorescent chemical
compound that can
re-emit light upon
light excitation.
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Fluorometry
Before fluorescence, some loss of the
excitation energy occurs
The emitted fluorescence light is of less
energy but longer wavelength than
excitation light
Stokes shift:
The difference between the maximum
wavelength of the excitation light and the
maximum wavelength of the emitted
fluorescence light

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Flow cytometer
Cytometry refers to the measurement of physical or chemical
characteristics of cells.
Flow cytometry is a process in which measurements are made
while the cells pass through the measuring apparatus in a fluid
stream
Cells are labelled with different specific fluorescent labels such as
B-phycoerythrin , fluorescein isothiocyanate, rhodamine-6G and
dye-labeled antibodies
Most flow cytometers incorporate two or more fluorescence
emission detection system for multiple fluorescent labels can be
used

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Luminometry
Luminescence in which the excitation event is caused
by chemical, biochemical or electrochemical reaction
and not by photo illumination.
Instrument: Luminometers
Chemiluminescence
Bioluminescence
Electrochemiluminescence

72
Chemiluminescence (CL) :
 Emission of light when an electron returns from an
excited or higher energy levels to a lower energy levels
 The excitation event is caused by a chemical reaction
 Such as oxidation of an organic compounds (luminol,
isoluminol) by an oxidant (hydrogen peroxide,
hyporchlorite or oxygen)
 Light is emitted from excited product form in the
oxidation

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Chemiluminescence (CL) :
Reaction occurs in the presence of
 Enzymes (alkaline phosphatase, horseradish peroxidase)
 Metal ions (Cu and Fe)

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Application: Chemiluminescence Immunoassays (CLIA)
• Assay that combine Chemiluminescence technique with
immunochemical reactions.
• Utilize chemical probes which could generate light emission through
chemical reaction to label the antibody.

75
Application: Chemiluminescence Immunoassays (CLIA)
• Because of its high sensitivity, good specificity, wide range of
applications, simple equipment and wide linear range.
 Life science
 Clinical diagnosis
 Environmental monitoring
 Food safety and
 Pharmaceutical analysis

76
Chemiluminescence
CLIA have different label systems according to the
difference of physical chemistry mechanism of the light
emission.
1. Enzyme Catalyzed Light Emission Reaction
2. Label Chemical Directly Involved in the Light Emission
Reaction

77
Enzyme Catalyzed Light Emission Reaction
 Utilizes enzymes to label antibody
 Horseradish peroxidase (HRP) Alkaline phosphatase (AP)
 It is an enzyme linked immunoassay that uses luminescent chemical as substrate instead of
chromogen.
 The most widely used enzymes are horseradish peroxidase (HRP) and alkaline phosphatase
(AP), each has its own luminescent substrates.
HRP-Luminol system:
 Luminol is a very common chemiluminescent substrate used for detection of HRP.
 HRP catalyzes the decomposition of luminol in the presence of peroxide to produce an
excited state intermediate.
 Flashes of visible light (maximum at 425nm) is emitted on decay of the singlet intermediate.

78
Label Chemical Directly Involved in the Light Emission Reaction
This kind of chemical with special structure can transfer to an excited state
through chemical reaction. Photons would be released when the chemical fell to
ground state from the excited state.
• The typical chemical is acridinium ester and its derivatives.
• Exposure of an acridinium ester label to an alkaline hydrogen peroxide
solution triggers a flash of light.
• A subsequent development has been the acridinium sulfonamide ester
labels.
• It is also triggered by alkaline hydrogen peroxide to emit a flash of light.
• The light emission mechanism of acridinium ester

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 Acridinium labeled compounds have 100 times stronger
chemiluminescence intensity compared to luminol labeled ones,
 acridinium esters have the dominant feature that they do not
lose the luminescence efficiency even after binding to antigen or
antibody

80
Bioluminescence
 Chemiluminescence found in
biological systems.
 An enzyme photoprotein increases
the efficiency of the luminescence
reaction.
 Luciferase
 Aequorin
 Sparkling Squid,
 luminous mushrooms,
 Radiant bacteria,
 Glowing fish.
foxfire mushroom

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Electrochemical luminescence
Redox Reaction Mediated Light Emission Reaction
 This system utilizes Ruthenium tris-bipyridine (bpy) as label, involves reaction
of Ru(bpy)3
3+ and Ru(bpy)3
+ to produce an excited state of Ru(bpy)3
2+, a stable
species which decays to the ground state by emitting an 620 nm orange
emission.
 Ru(bpy)3
3+ and Ru(bpy)3
+ can be electrogenerated from Ru(bpy)3
2+ by
reduction at approximately -1.3 V, and oxidation at approximately + 1.3 V.
 Ultrahigh sensitivity and specificity.

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Electrochemical luminescence
 Reactive species are produce the
Chemiluminescent reaction are
electrochemically generated from
stable precursor at the surface of an
electrode.
Ruthenium tris (bipyridyl) chelate is
Chemiluminescent label.
Generated at an electrode via
oxidation-reduction

83
Reference:
Tietz Fundamentals of Clinical Chemistry,
Sixth Edition. Optical Techniques, Chapter 4,
pg. 63-83

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