Chapter three

Principles and application of

Spectroscopic Techniques
Outline

 Basic electromagnetic radiation

 UV-visible Spectrometry
 Atomic Spectrometry
 Flourometry
 Nephelometry and Turbidimetry
 Reflectance Spectrometry
 Mass Spectrometry
Basics of Electro Magnetic
Radiation
Chapter Objectives

Upon completion of this chapter the student will be able to:

 Explain different types of electromagnetic radiation

 Describe how radiation interacts with matter
 Discuss Beer-Lambert law of electromagnetic
radiation (EMR)
Outline

 Introduction
 Interaction of radiant energy and matter
 Basic concepts of Absorption vs. transmission
 fundamental laws of absorption
 Application of Beer-Lambert law
Introduction
 Radiation is a form of energy that shows electric and
magnetic field varying periodically and perpendicular to the
direction of propagation: electromagnetic radiation (EMR)
 Quantum theory
 EMR travels as a stream of 'energy packets' – photons
or quanta.
 It travels in the form of wave (zigzag fashion) through an
imaginary line know as axis of propagation.
 It characterized by different features
 Amplitude
 wavelength (λ)
 Frequency (V)
Introduction

 Frequency and wavelength of a radiation are inversely

related.
 V α 1/ λ, using the proportionality constant velocity c, V = c / λ

 Electromagnetic phenomena exhibit:

 energy (E)
 Frequency (ν)
 Wavelength (λ)
 Intensity

 All four terms are interrelated and can be explained either in

terms of wave form or particles termed photons or quanta
Introduction

 Energy of EMR is inversely proportional to the

wavelength and directly proportional to the
frequency, E α V.
 Using the proportionality constant h, (where h is
Planck's constant, 6.62× 10–34 J s, or 6.62 x 10-
27ergs).

E= h V, or E = h[c/λ]
Electromagnetic radiation…

9
Interaction of radiation Vs. matter
 The internal energy of a molecule is due to energy
associated with
 the electrons
 the vibrations of atoms and
 the energy associated with the rotation of various
groups of atoms within the molecules relative to the
other groups
 The exact amount of energy required to produce change
in the molecule from one energy level to another
depends on the wavelength or frequency of the radiation.
Interaction of radiation Vs. matter

 The different phenomenon that occur as a result

of interaction of radiant energy with matter
includes:
 Absorption
 Transmission
 Diffraction or
 Reflection
Absorption and Emission

 Absorption spectrum is observed, if

the energy is derived from
electromagnetic radiation (electron
transition).

 When an electrons falls from a higher to

lower energy level, then one quantum of
energy is emitted from the system,
giving rise to an Emission spectrum.
Concepts of Absorption vs. Transmission

 When an atom, ion, or molecule absorbs a photon, the

added energy results in alteration of state, and the
species is said to be excited
 Excitation may involve one of the following processes:
 Transition of an electron to a higher energy level
 A change in the mode of vibration of the molecule’s
covalent bond
 Alteration of its mode of rotation within the covalent
bonds
Electromagnetic properties

 Understanding of the properties of electromagnetic

radiation and its interaction with matter allows to:

- Recognize the different types of spectra, and

consequently spectroscopic techniques

- The application of these techniques in laboratories for

sample assay and identifications of components
Fundamental Laws of Absorption

When monochromatic light passes through a solution,

 The amount of light absorbed is directly proportional
to the thickness of the cuvet; b, i.e. A α b, (Lambert
Law)
 The amount of light absorbed is directly proportional
to the concentration of the absorbing medium, i.e. A α
c, (Beer’s Law)
Interaction of radiant Cont’d…
Fundamental Laws of Absorption
 To make a linear relationship between the
absorbance and concentration and light path, a
certain proportionality constant defined as
absorptivity, a, is inserted.
o A = abc
 The amount of light absorbed is inversely and
logarithmically related to the intensity of the
transmitted light.
Application of Beer-Lambert law

A= log 1/T = -log T, But T = Is/Io,

A = - log Is/Io
= log Io- log Is .
Is Assume Io is considered as 100 unit,
Io
Therefore
A = 2 - log %T = abc

 When there is no absorber particle inside the

cuvette, Is/Io =1, and %T becomes100%.
 This means no radiant energy of the incident light is
absorbed
Application of Beer-Lambert law

Relation ship between Concentration Vs. Transmittance and

Concentration Vs. Absorbance.
 UV-VISIBLE
SPECTROPHOTOMETER
Learning Objectives

At the end of this topic, students will be able;

 List the basic components of spectrophotometer
 Describe the instrumentation principle of UV-Visible
spectroscopy
 Explain the measurement principle of
spectrophotometer
 Demonstrate knowledge of how to perform
preventive maintenance
 Demonstrate knowledge of how to calibrate and
validate a new instrument in terms of photometric
linearity and wavelength accuracy
 Outline

UV-Visible Spectrophotometer
 Introduction
 Essential Instrumentation
 Performance verification
 Basic features of manual Vs. automated
spectrophotometers
 Unpacking and priming of instruments
Introduction

 Spectrophotometer is an instrument that measures light

intensity.
 “Spectrophotometery”(“light measurement”): the term is
used to refer to any analytical methods in which the
intensity of light is the sensed and measured quantity.
 There are different kinds of spectrophotometers like UV-
Visible spectroscopy, atomic spectroscopy, reflectance
spectroscopy, fluoresce spectroscopy, etc.
ultraviolet-visible
spectrophotometry
 UV-Visible Spectroscopy is based on two
principles:
1. substances absorb light at unique
wavelengths and
2. the amount of light absorbed is proportional
to the amount of substance that is present
 UV/Vis Spectrum
 Visible wavelengths cover a range from approximately 400 to
800 nm. The longest visible wavelength is red and the
shortest is violet. Other common colours range from indigo to
orange of the spectrum, in order of decreasing wavelength.

 Violet:   400 - 420 nm

 Indigo:   420 - 440 nm
 Blue:   440 - 490 nm
 Green:   490 - 570 nm
 Yellow:   570 - 585 nm
 Orange:  585 - 620 nm
 Red:   620 - 780 nm
UV and UV/Vis Spectrometry

 When an organic molecule is irradiated with electromagnetic

energy, the radiation either passes through the sample or
absorbed, depending on its energy.

 The UV region of the electromagnetic spectrum extends from

the low-wavelength end of the visible region (4 x 10-7 to 10-8
m).
1 nm = 10-9 m UV region of greatest interest is 200 - 400
nm
Essential Instrumentation
(Spectrophotometer)
Basic spectrophotometer components include:
1. Light sources (UV and visible)
2. Wavelength selector (monochromator)
3. Sample containers (cuvettes)
4. Detector
5. Signal processor and readout
Component of a UV/Vis
spectroscopy
Light source

 Tungsten filament lamp is the most common source of

visible lamp
 It is useful for the wavelength region 350-2500 nm
 It is temperature dependent (2870 K)

 Hydrogen and Deuterium lamps

 Both lamps produce outputs in the range of 160-800 nm
 Mercury vapor lamp(line spectrum, 54nm,313nm,365nm
 Light Amplification by Stimulated Emission of Radiation
(LASER): intense, focused, &non divergent beam
Light source

 Tungsten-halogen lamps (quartz-halogen lamps)

 Deuterium lamps use for UV measurements and

tungsten-halogen lamps for UV/Vis measurements
Monochromator

 A monochromator is a set of components used to isolate

a very narrow spectrum of the radiant energy known as
monochromatic radiation.
 All monochromators contain the following component
parts:
 Entrance slit
 Collimating lens
 Dispersing device (filters, prism, grating)
 Focusing lens
 Exit slit
Monochromator
Absorbance Filter
 Narrow-band pass filter can be constructed by
combination of two or more glass filters (sharp-cutoff and
wide band pass filters), but the light source must be
intense enough in order to penetrate the layer of glasses

The net effect is

narrow band width of the
out put spectrum
Prisms

 Prisms separate white light into a continuous spectrum

of light by refraction with shorter wavelength that are
bent, or refracted, more than longer wave lengths as
they pass through the prism
 Prisms in a spectrophotometer are rotated to allow the
wavelength of choice to pass through the exit slit and the
cuvette and onto the photocell.

 The angle of deviation depends on the wavelength; red

light deviating the least, and violet the most.
Prism
Diffraction grating
 Diffraction is the change in the direction of light rays due to
their interaction with a device called a diffraction grating

 The gratings act as scattering centers for the rays of light,

with each groove producing diffracted waves of light.
 Radiant energy bend (refract) at each sharp corner give
rise to a fine spectrum.
 The grating is rotated and different parts of the spectrum
are allowed to fall on the photocell
Diffracting grating
Cuvettes

 Cuvette can be round, square or rectangular

 Constructed from glass, silica or plastic
 Square or rectangular cuvette have a constant light
path, the most usual being 1 cm in length
 Glass cuvette are suitable for use between 320 and 950
nm, because Glass cuvette absorbs much UV than silica
cuvette.
 for UV radiation, below 320 nm, silica (quartz) cuvette
are used and they must be clean and free of scratches
Detectors

 Devices that convert light beam into an electric signal that

is proportional to the number of photons striking its
photosensitive surface.
 Photosensors are not equally sensitive to all wavelengths
 Photosensors have a frequency below which they will not
respond.
 This is one of the reasons why one must always reset
100% transmittance after changing the wavelength of a
photometer
Signal Processor/Read Out

 On a fully manual spectrophotometer, the readout

will be in %Transmittance or Absorbance.
 Semi- or automated instruments will have an internal
conversion
 All the necessary information is processed by the
microprocessor of the instrument or software loaded
on a linked PC.
Concept of Spectral Purity
 Spectral purity of monochromator is described in terms of half
bandwidth or band pass; range of wavelengths passed by a
monochromator
 It is the range of wavelengths transmitted at a point equal to
one-half the peak intensity transmitted.
 It is defined as the width in nm.
 It corresponds to 75% of the total radiant energy present in the
emerging beam of light.
Half band width of
filter “a” is greater then
filter “b”, then spectral
purity of “b” is greater
than “a”
Concept of Lamda max (Λmax)

Is that possible to
appreciate, which solution
is concentrated using
human eye, if the two
solutions happened to be
mixed?

 According to the diagram shown above, Solution A appears as

green to human eye, when viewed against white light. This is
because the solution transmits light maximum between 500 and
580nm but absorbs light at other wavelength.
 Solution B on the other hand absorbs light maximum around 580 but
transmits maximum in the blue-violet [400-460nm] and red [700nm]
portion of the spectrum.
 Human eye recognizes this mixture of color as purple.
Concept of lamda max (Λmax)

 Measurement is taken at a wavelength that gives

maximum absorbance improves sensitivity and
specificity. How?
 The wavelength at which there is maximum absorbance
for that particular solution is referred as lamda max,
symbolically represented as “λmax”
Concept of lamda max (Λmax)
 When establishing a new Spectrophotometric procedure,
it is important to record the absorption spectrum of the
material that is being measured in relation to either water
or a reagent blank
 A general rule for selecting optimum wavelength at
which to monitor a Spectrophotometric reaction includes:
 Choose absorption peak with the greatest possible
molar absorptivity.
 Choose a peak that is as far as possible from the
absorption peaks of commonly interfering
chromogens
Relationship b/n Bandwidth and λmax
 Lack of agreement with Beer’s law occurs when a part of
the spectral energy transmitted by the monochromator is
not absorbed at all by the substance being measured; the
case observed in monochromator having wider band
pass.
 The natural bandwidth of an absorbing substance is
defined as the bandwidth of the spectral absorbance
curve at a point equal to one-half the maximum
absorbance.
 As a general rule the spectral bandwidth should not
exceed 10% of the natural bandwidth of the analyte to be
measured
Effect of interference on the
λmax
 In order to improve the precision and accuracy of an
assay, the actual λmax , where there is maximum
absorbance for that analyte, could be modified due to the
presence of interfere from the sample or other analyte.

 Example, Jaffe method. Although the absorbance

maximum of the jaffe reaction is between 490 and
500nm, the reagent blank itself absorbs light strongly
blow 500nm. Hence, 520nm is used as λmax for the
assay.
Bichromatic absorbance
measurement
 Multiple absorbance reading can minimize background
interferences such as matrix effect of sample, finger
prints, dusts, etc.
 Taking measurement at the peak absorbance (primary
wavelength) and another at a point near the base of the
peak absorbance (reference wavelength) provides a
blank reference point for each sample.
 Measuring absorbance at the peak and at two other
wavelengths equidistant from the peak. and subtracting
the average of the two reference absorbance's from the
peak absorbance is the Allen correction .
Bichromatic absorbance
measurement
Schematic Diagram of a Double-Beam
UV-Vis. Spectrophotometer
Double-Beam, Cont’d…
 Single-beam devices prone to two major problems:
 Variations in the light sources intensity and
 Drift in the “dark” or background current originating at
the detector.
 The term double beam is used to describe in which the
incident light and the transmitted light are compared
either by splitting the optical beam in space or by
splitting the beam in time.
 In a dual-beam–in-time photometer

 In the design of a dual-beam-in-space photometer

Performance Verification of
spectrophotometer
 Photometer accuracy check
 Absorption cell check
 Wavelength accuracy check
 Linearity check
 Stray light
Wavelength Accuracy
 Ensures that the radiant energy being emitted from the
exit slit of the monochromator is the same as that
specified by the wavelength selector
 Should be checked whenever a new lamp is installed
and then checked routinely
 Method: Cobalt chloride (peak at 510 nm), didymium
filter (peak at 585 nm),
Photometric Linearity
 Ensures that a linear relationship exists between the
radiant energy absorbed and the instrument readout
(abs vs. concentration),
 ensures a true Beer’s law relationship between
absorbance and sample concentration
 permits the use of a standard curve as used in many
assays
 Factors affecting linearity include (1) the detector, (2) the
sample’s response to Beer’s law (3) wide band pass, (4)
stray light, and (5) molecular fluorescence of the sample.
 Linearity is checked with various dilutions of cobalt
chloride or other solutions known to follow Beer’s law
Photometric Accuracy

 Photometric accuracy means that the

absorbance displayed on the instrument is
the true absorbance of the solution SPECTRAL
SPECTRAL
BANDWIDTH
BANDWIDTH
(sometimes
(sometimes
 Checks for changes in band pass and called
called NOMINAL
NOMINAL
BANDPASS)
BANDPASS)

amount of light energy falling on the

INTENSITY
INTENSITY
TOTAL
TOTAL BAND
BAND OFOF
WAVELENGTHS
WAVELENGTHS
photocell PASSED
PASSED
(sometimes
(sometimes called
called
EFFECTIVE
EFFECTIVE
 Photometric accuracy can either be done BANDPASS)
BANDPASS)

using standard neutral-density glass

500
500 nm 520
520 nm
filters that have known absorbance at nm nm

particular wavelengths or solution such WAVELENGTH

WAVELENGTH

Potassium dichromate, cobalt ammonium

sulfate, and potassium nitrate can be used.
 Procedure of using a glass
filter

1. Adjust the spectrophotometer to read an absorbance of 0

at 280 nm and 500 nm (air blanking)
2. Check the filter for surface contamination; wipe clean with
an optical lens tissue.
3. Place the filter in sample holder.
4. Read and record the absorbance value at 280 nm and 500
nm.
5. The absorbance at each of these wavelengths should
be within the expected limits established for the
spectrophotometer.
Absorption cells check
 The most common cause for significant differences
between absorption cells is scratch or dirty windows
 To test cleanliness and quality of the reaction cell,
 The quartz cell is filled with reagent grade water and
an absorbance reading against air is taken in the
visible region (at 650 nm) and in the ultraviolet region
(240 nm).
 The acceptable absorbance must be ≤ 0.093 for each
of these wavelengths.
 Keep record of all data from the performance checks
to see on trends in order to trace early on
deterioration of sample holders.
Stray Light
 Stray light, in general terms, is radiation of wavelengths
outside the narrow band nominally transmitted by the
monochromator
 To detect the presence of stray light, different solutions
can be used. For example a 50g/L aqueous solution of
sodium nitrite should show essentially 0% T when read
against water over the range of 300 to 385nm.
Basics features Manual vs.
Automated Spectrophotometer

Semi-automated Chemistry Manual

Analyzer Spectrophotometer
Basics features

 Manual Method:
 Slower turn-around time (TAT)
 Requires more hands-on Technologist time
 Less precision and accuracy in Quality Control
 Increased safety risk to Techs (biological and reagent)
 Automated Method:
 Less handling of biological specimens by Techs
 Quicker TAT
 Greater precision and accuracy in Quality Control
 Can run and report many more test results in shorter period of
time
How to Set up a New Instrument

Set up
Calibrate
Validate
Start maintenance
Initial Instrument Setup

 Pre-installation Steps

 Out-of-the box steps

 Save paperwork
 Save operating manual

 Establish a working
procedure following
operating manual
Pre-installation Requirements
 Check for the following items:
 Proper electrical
wiring/voltage

 Temperature controlled area

 Adequate distilled/deionized
water supply

 Instrument fits well on bench

and with workflow
Pre-Installation Requirements

 Check for the following items:

 Proper waste disposal capability
 Adequate air circulation / flow around instrument
 Dust free environment (cover instrument when not in
use)
 Stable bench top
Out of box

 The instrument should be checked for the

following items before installation
 Visual damage
 Loose parts or connection
 All parts and accessories
 Computer boards are properly seated
Before Instrument Validation

 Permit instrument to stabilise/equilibrate

 Let all components reach proper temperature
 Set in any parameters that may be required
 Ranges
 Temperatures
Initial Instrument Calibration
 Follow manufacturers recommendation for reagent and
control preparation
 Place reagents on instrument as required
 Place calibrator(s) on instrument according to procedure
 Run prepared calibrator(s) on both automated and
manual methods
Instrument Maintenance

 Train users to do routine maintenance

 It includes for example, systematic and routine
cleaning, simple adjustment or replacement of
instrument and equipment parts and may be
done on a daily, weekly or monthly basis
 Periodic maintenance may be done by expert
Review Questions,

1. What is the main difference between single

beam and double beam photometers
2. List some of the approaches of evaluating
performance of photometers
3. Describe and explain the major components of
spectrophotometers
4. Explain the relation between spectral purity,
linearity range, and half bandwidth.
 Review Questions, Cont’d…
5. Verify the linearity range of the two instruments (all
the readings were done at 550nm),[HINT: Sketch on
a linear graph paper and see the linear and non
linear range.]

Adjusted Absorbance of 0.00 0.25 0.50 0.75 1.00 1.25 1.50

the instruments
Observed absorbance (o.d) 0.09 0.34 0.59 0.84 1.09 1.26 1.43
of Spectrophotometer “A”
Observed absorbance (o.d) 0.1 0.35 0.6 0.84 1.05 1.25 1.43
of Spectrophotometer “B”
 ATOMIC
SPECTROPHOTOMETRY
Learning objective
At the end of this topic students should be able to:
 List the different types of atomic spectrometers
 Explain basic components of atomic spectrometer
 Explain the measurement principle of atomic
spectrometers
 Discuss common problems encountered in atomic

spectrometer
Topic Outline
1. Introduction
2. Atomic absorption Spectrophotometry
3. Flame emission Spectrophotometry
4. Atomic fluorescent Spectrophotometry
Atomic Spectroscopy
Introduction
 The study of electromagnetic spectrum of elements is
called Atomic Spectroscopy.
 Atomic Spectroscopy is the determination of
elemental composition by its electromagnetic or mass
spectrum.
 Electrons exist in different defined energy levels within
an atom
 The energy absorbed to move an electron to a more
energetic level or the energy emitted as the electrons
move to a less energy level is equal. This packet of
energy is called photon.
ATOMIC SPECTROSCOPY, Cont’d…

Atomic Spectroscopy has yielded three techniques

 Atomic Absorption
 Atomic Emission
 Atomic Fluorescence
Atomic Absorption
Spectrophotometry (AAS)
 Like simple spectrophotometer, measure light to
determine the concentration of analyte
 In the clinical laboratory, used for the
determination of calcium, magnesium, lithium,
lead, copper, zinc and other metals
AAS
Principle
 Vaporized ground state atoms suspended in a flame will
absorb radiation at a very narrowly defined wavelength
characteristic wavelength of light originates from a
hollow cathode lamp
 The amount of absorbed light is directly proportional to
the concentration of atoms in the flame
 The absorption bands are in the order of 0.001 to
0.01nm in width, thus atomic absorption spectroscopy is
a highly specific analytical technique.
Functional parts
 Hollow cathode lamp
 Nebulizer or Atomizer
 Monochromator
 Photomultiplier
 Signal processor
 Light block
Schematic diagram of AAS
Common technical problems

 Chemical interference
 Ionization interference
 Emission interference
 chopper
Flame Emission Spectrophotometry

 It is a photometer with a carefully controlled flame used

to measure the concentration of alkali and alkaline earth
metal group (i.e. Sodium, potassium, and lithium), which
are relatively easy to be excited in a flame.
 These metals, when given sufficient heat energy, a
specific amount heat energy is absorbed by an orbital
electron, which results in ejection of specific amount of
radiant energy during returning to ground state.
Principle

 The intensity of the characteristic wavelengths of

radiant energy produced by the atoms in the flame is
directly proportional to the number of atoms excited in
the flame, which is directly proportional to the
concentration of the substance of interest in the
sample.
 Emission is directly proportional to concentration over
a wide range, except, ionization reduces emission,
and self-absorption can reduce the emission.
Functional Parts

 Fuel and air supply

 The atomizer
 The burner/flame assembly
 Filters
 Photo detectors and signal processing display

 Excitation occurs in the hot portion or cone of

flame
Essential component of Flame
Emission Photometer
Common technical problems in
flame spectroscopy
 the intensity of a spectral line is very sensitive to
changes in flame temperature
 Spectral interferences may arise from the close
proximity of other emission lines or bands to the
analyte line or by overlap with it.
 Self-absorption
Atomic Fluorescence Spectrophotometry

Measurement principle

 Like atomic absorption, ground state atoms created in a

flame are excited by focusing a high-intensity broad
spectrum beam of light into the atomic vapor
 Like emission spectrophotometer, the emission resulting
from the decay of the atoms excited by the source of
light is measured instead of looking at the amount of light
absorbed in the process
Atomic Fluorescence
Spectrophotometry
 Like atomic absorption, ground state atoms
created in a flame are excited by focusing a high-
intensity broad spectrum beam of light into the
atomic vapor
 Like emission spectrophotometer, the emission
resulting from the decay of the atoms excited by
the source of light is measured instead of looking
at the amount of light absorbed in the process
Atomic Fluorescence
Spectrophotometry
 The radiation is emitted in all directions and may
be monitored from angles other than in a direct
line with radiation from the irradiating source.
 This ensures that the detector will not respond
to the primary absorption process or to
unabsorbed radiation from the lamp.
Review Questions

1. What is the basic difference between UV-Visible

spectroscopy and Atomic spectroscopy?
2. Explain the common technical problems in atomic
spectroscopy.
3. Discuss the essential components of Atomic emission
and atomic absorption.
FLUOROMETRY
Learning Objectives

At the end of this topic students should be able to:

 Describe the basic principle of fluorometry
 Explain the optical property of fluorescence dyes
 Discuss the instrumentation and measurement principle
of fluorometer
Topic Outline
 Introduction
 Principle of Fluorescence Emission
 Measurement principle
 Functional parts of the instrument
 Common technical problems in Fluorometry
FLUOROMETRY

 A Fluorometer is a photometer that measures

the light emitted (relatively long wavelength) by a
substance that has been previously excited by a
source of short wavelength radiation.
 The main advantage of this technique is its
sensitivity; measurements of nanogram (10-9)
quantities are possible.
Principle of Fluorescence
Emission
 When light is transmitted, there is no loss of
energy, scatted, there is no change of energy;
the incident light and the transmitted light is the
same before and after it interacts with matter.
 But when light is absorbed by the matter, the
absorber molecule gets excited and
spontaneously returned back to the ground
state.
Principle of Fluorescence
Emission

 Relaxation from excited electronic states to the ground

state is mostly occurred by:
 Vibrational relaxation (Radiationless processes):
where the excess energy is as heat by collisions
between solute and solvent molecules.
 Photoluminescence: in which radiation of a longer
wavelength than that originally absorbed is re-emitted
after about 10–8 seconds or longer
 Fluorescence and phosphorescence are two
distinct forms of photoluminescence
Principle of Fluorescence
Emission
 The intensity of fluorescent emission is dependent on a
quantum efficiency factor or yield, ɸ F, which can vary
between zero (no fluorescence) and unity (all excited
molecules relax by fluorescence).
 It describes the efficiency of the energy conversion
process that takes place when a molecule absorbs
energy and then re-emits it as light.
 It is determined by a number of structural factors,
including the presence and positions of hetero-atoms
in the molecule, and the solvent used.
Measurement principle
 There is a linear relation between the concentration, C,
of a fluorescent analyte and the intensity of emission, IF,
i.e. derivation of Beer-Lambert’s law.
IF = 2.303 IOɸF(εbC)
Where:
IO = intensity of excitation or incident light
IF = fluorescence intensity
ɸF = quantum yield, the number of photons emitted divided by the
number of photons absorbed.
ε = the absorptivity coefficient, a constant given for a particular
substance
b = the path length through the sample
C = the concentration of the substance
Measurement principle

 If IO and b are constant and the absorbance (ε bC) is

small, i.e. the analyte concentration is low, then,
IF = KC
 The equation indicates that fluorescence intensity is
directly proportional to the concentration of the
fluorophor.
Functional parts of the instrument

 Fluorometer has a set of filters or a

monochromator before and after the cell to
isolate the emitted light.
 The fluorescence emission, which is emitted
from the sample equally in all directions, is
measured at 90° to the direction of the excitation
beam.
 This prevents incident, scattered and reflected
radiation from reaching the detector.
Functional parts of the
instrument…
Functional parts of the
instrument
1. Light source 7. Signal voltage amplifier
2. focusing lens 8. display
3. primary or excitation 9. reference beam
monochromator
photosens
4. sample cuvet
10. source supply
5. secondary or emission
monochromator 11. light block
6. photomultiplier tube
Common technical problems

 Quenching effects
 Molecular collusion
 Decrease in pH of the sample
 Photodecomposition
Review Questions
1. Describe the basic principle of fluorometry
2. Explain the optical property of fluorescence
dyes
3. Discuss the instrumentation and measurement
principle of fluorometer
 Fluorescence: Emission of a photon when the
analyte returns to a lower-energy state with the
same spin as the higher-energy state
 Phosphorescence: Emission of a photon when
the analyte returns to a lower-energy state with
the opposite spin as the higher-energy state.
NEPHELOMETRY AND
TURBIDIMETRY
Learning Objectives
At the end of the topic students shall be able to:
 Discuss the instrumentation principle of turbidimetry and
nephelometry
 Explain the measurement principle of nephelometry and
turbidimetry
Topic Outline

 Basic concepts of light scattering

 Instrumentation and principle of measurement
turbidimetric and nephelometric techniques
 Important remarks of measurement
Basic concepts of light
scattering

 When parallel or non divergent beam of light

strikes a particle in suspension,
 Reflected
 Scattered
 Absorbed or transmitted
Basic concepts of light
scattering

 If the wavelength, λ, of light is much larger than the

size the particle (d<0.1 λ), the light is symmetrically
scattered around the particle
 If the wavelength of light is approximately equal to
the size of the particles, more light appears in a
forward direction than in the backward direction
 If the wavelength of the incident light is much
smaller than the size of the particle (d > 10 λ), most
of the light appears to be scattered forward
Turbidimetry and Nephelometry

 Turbidimetry: A method in which the decrease

in transmitted radiation due to scattering is
measured.
 Nephelometry: A method in which the intensity
of scattered radiation is measured at an angle of
90° to the source.
Nephelometry

 It is a photometer designed to measure the light

scattered by a colloidal or colloidal like suspension (e.g.
Antigen-Antibody complex).
 Common nephelometers measure scattered light at right
angle to the incident light.
 The ideal nephelometric instrument would be free of
stray light; neither light scatter nor another signal would
be seen by the detector when no particles are present in
the solution.
Turbidimetry

 It measures the reduction in the light

transmission caused by particle formation, and it
quantifies the residual light transmitted.
 The intensity of incident light beam is encountered
with is scattering, reflectance, and absorption by the
particle in the sample
 The instrumentation is similar to simple manual or
automated spectrophotometer
Nephelometry and Turbidimetry
Selection of Nephelometry &
Turbidimetry
 Choosing between turbidimetry and nephelometry is
determined by two principal factors:
 intensity of the transmitted or scattered radiation
relative to the intensity of radiation from the
source
 the size of the scattering particles
 Turbidimetry is a better choice for samples containing a
high concentration of scattering particles
 Nephelometry is a more appropriate choice for samples
containing few scattering particles.
Limitation of light-scattering
measurements
 Antigen excess and
 Matrix effects
Review Questions

1. Discuss the instrumentation principle of turbidimetrty

and nephelometry
2. Explain the measurement principle of nephelometry
and turbidimetry