NEPHELOMETRY AND TURBIDIMETRY.pptx

NEPHELOMETRY AND
TURBIDIMETRY
BY
E.W.OJONG
PhD CHEMICAL PATHOLOGY

LESSON OBJECTIVES
 Explain the principles of nephelometry and
turbidimetry
Discuss the instrumentation of nephelometry
and turbidimetry
 Describe the applications of nephelometry
and turbidimetry

INTRODUCTION
 When electromagnetic radiation (light) strikes on a
particle in a solution, some of the light will be absorbed by
the particle, some will be transmitted through the solution
and some of the light will be scattered or reflected.
 The amount of light scattered is proportional to the
concentration of insoluble particles.
 Scattered light may be measured by turbidimetry and
nephelometry
 Turbidimetric measurements are made at 180oC from the
incident light beam
 In nephelometry, the intensity of the scattered light is
measured, usually but not necessarily at right angles to the
incident beam

INTRODUCTION
Both turbidimetry and nephelometry are based
on the property of light scattering by particles
dispersed in a solution.
Both methods can be used to determine the
concentration of a particulate solution
However, they differ in the manner of measuring
the scattered radiation
In turbidimetry, measurement is made in the
direction of the incident light
In nephelometry, measurements are made in the
right angle to the incident light

TURBIDIMETRY
 When a beam of monochromatic light is allowed to
pass through a solution, part of the incident radiant
energy is dissipated by absorption, reflection, and
refraction while the remainder is transmitted.
 the power of transmitted light is measured in the
direction of the beam as a function of the
concentration of suspended particles.
 The transmittance (T = I/Io) of the incident light is
measured
 Measurements are made at 18OoC from the incident
light beam

TURBIDIMETRY
The transmittance T is related to the
concentration (C) of suspended material by
the equation;
S = log Io/I = KbC
Where
S = turbidance
K = turbidity coefficient
 b = path length of the solution

TURBIDIMETRY AND COLORIMETRY
Turbidimetry is much similar to colorimetry
because both involve the measurement of the
intensity of light transmitted through a
medium
But these differ in the sense that the light
intensity is decreased by turbidimetry and by
absorption in colorimetry.

APPLICATIONS OF TURBIDIMETRY
 For measuring abundant large particles and
bacterial suspension
Used to measure plasma and urinary
proteins.Calibrators are used to create a
standard curve

NEPHELOMETRY
When a beam of monochromatic light is allowed
to pass through a solution having suspended
particles, the radiant powered of the scattered
beam at 45oC, 90oC, 135oC etc to the incident
beam is measured as a function of the
concentration of suspended particles
 Measurement of the intensity of the scattered
light as a function of the concentration of the
dispersed particles form the basis of
nephelometric analysis

NEPHELOMETRY
If the particle size is larger than the wavelength of
the light source, then most of the light will be
scattered in the forward direction at an angle of
less than 90o to the incident beam. This
phenomenom is known as Mie scatter
Particles that are smaller than the wavelength of
the light source will scatter light in many
directions and equally in the forward and
backward direction. This phenomenon is known
as Raleigh Scatter

NEPHELOMETRY AND FLUORIMETRY
Nephelometry is much similar to fluorimetry
because both involve the measurement of
scattered light
 But the basic difference is that the scattering is
elastic in fluorimetry and inelastic in
nephelometry
Both incident and scattered light are of the same
wavelength in nepehlometry whereas scattered
light measured in fluorimetry is of a longer
wavelenth than the incident light

APPLICATIONS OF NEPHELOMETRY
 Measuring the amount of antigen-antibody complexes. Antigen-
antibody complexes when formed at a high rate, will precipitate out
of solution resulting in a turbid or cloudy appearance.
 Can detect either antigen or antibody.
(a) Endpoint tests allow antigen-antibody reactions to go to
completion. If complexes get too large, they will fall out of solution,
causing a falsely decreased result.
(b) Kinetic tests add andtigens and antibody then measure at a specific
time. The rate of formation must be known and concentration should
be calculated based on standards.
 Measure small particles of low concentrations in body fluids e.g
microalbumin, haptoglobin, ceruloplasmin, immunoglobulins.

INSTRUMENTATION OF TURBIDIMETRY
AND NEPHELOMETRY
 Much of the theory and equipment used in colorimetry
apply with little modification
The basic components of the instruments include
 Radiation source
 Sample cell
 Detector
 Readout device
The instruments for both methods are similar. The only
difference is with the detectors. One uses the
photovoltaic cell while the other uses phototube

AND NEPHELOMETRY
LIGHT SOURCE
 White light or monochromatic light is more
advantageous to minimize absorption and sample
heating
 Monochromatic light also obtains a uniform scatter
 Short wavelengths are used to increase the efficiency
of scattering
 Mercury arc or laser beam
 Tungsten lamp is used for determination of
concentration

AND NEPHELOMETRY
CELLS
 Cylindrical cells, with flat faces where the entering and
exiting beams are to be passed to minimize reflections and
multiple scattering from the cell walls
 In general, cells with a rectangular cross section is preferred
where measurements are to be made at 90oC
 Octagonal faces will allow measurements to be made at 0o,
45o, 90o, 135o to the primary beam
 Walls through which light beams are not to pass are
painted dull black to absorb unwanted radiation and
minimize stray radiation
 Reagents must be free of any particles, cuvettes must be
free of any scratches.

INSTRUMENTATION OF
TURBIDIMETRY AND NEPHELOMETRY

AND NEPHELOMETRY
DETECTORS
In nephelometry, a sensitive photomultiplier tube
acts as a detector because the intensity of
scattered radiation is very small. Usually, the
detector is fixed at 90o to the primary beam. In
some nephelometers, the detector is mounted on
a circular disc which allows measurements at
many angles i.e at 0o and from 30o to 135o
In turbidimetry, ordinary detectors such as
phototubes and photovoltaic cells are used.

CHOICE BETWEEN TURBIDIMETRY AND
NEPHELOMETRY
 The choice between the two methods depends upon
the fraction of light scattered by the suspension. When
scattering is less due to small concentration of
dispersed phase, nephelometry is preferred. In this
case it is possible to measure accurately the small
amount of light scattered by the suspended particles.
 When the scattering is intense due to a high
concentration of suspended particles (dispersed
phase), turbidimetry is preferred. In this case it is
possible to measure accurately the small amount of
transmitted light.

CHOICE BETWEEN TURBIDIMETRY
AND NEPHELOMETRY

COMPARISON OF TURBIDIMETRY AND
NEPHELOMETRY

COMPARISON OF TURBIDIMETRY AND
NEPHELOMETRY
NEPHELOMETRY TURBIDIMETRY
Specialized instrument Simple spectrophotometer
More sensitive Less sensitive
Not affected by size and concentration Affected by size and concentration
Measures light which is scattered Measures light which passes through
Source of radiation: Mercury arc
lamp/high pressure xenon lamp
Tungsten lamp/Mercury lamp/laser
Radiation detection device:
Photomultiplier tube
Photovoltaic cell
Cell/sample holder (glass or plastic):
Semi octagonal cell (45o, 90o, 180o
Cylindrical cell/rectangular cell with
flat faces on both sides

PRINCIPLE AND THEORY OF
NEPHELOMETRY AND TURBIDIMETRY

FACTORS AFFECTING THE SCATTERING
OF LIGHT

OF LIGHT
CONCENTRATION OF PARTICLES (TURBIDIMETRY)
 At low concentration of particles for scattering of light,
Beer-Lambert’s Law is applicable.
 S = log(Io/It)
 S = KtC = -logT
 Turbidence (S) is directly proportional to concentration (C)
 Io, intensity of incident light
 It, intensity of transmitted light
 T, turbidance
 C, concentration
 Kt, Constant which depends on the linearity of light

OF LIGHT
CONCENTRATION OF PARTICLES (TURBIDIMETRY)
Is = Ks x Io x C
Io, intensity of incident light
Is, intensity of scattered light
Ks, Constant which depends on suspended
particle and suspended medium
C, concentration

OF LIGHT
PARTICLE GEOMETRY
 Controlling particle size and shape is the most critical factor in
turbidimetry and nephelometry
 The fraction of the light scattered at any angle depends to a
large extent upon the size and shape of the solid particles
responsible for scattering. Hence it is important to control the
particle size and shape.
 The amount of scattering (S) is proportional to the square of
the effective radius of the particle
 The factors/conditions which influence particle size during
precipitation like concentration of reagents, time allowed for
particle growth, rate and order of mixing of reagents, time,
temperature, presence of non-reactants, pH, and ionic strength
also affect turbidimetric and nephelometric assays

OF LIGHT
PARTICLE GEOMETRY
If the size of the suspended particles is of the
same order or smaller than the wavelength of
the incident light, scattering will be dominant
In nephelometry, scattering pattern of secondary
rays in space should be such that it has maximum
intensity at 90oC. The scattering efficiency falls if
the particle size is too small or too large. For
measurements in the uv and visible regions, the
optimum size should be 0.1 – 1.0nm

OF LIGHT
PARTICLE GEOMETRY
In turbidimetry, particles larger than the wavelength
of incident light do not pose complications because
measurements depend on the total radiation
removed from the primary beam irrespective of the
mechanism by which it is removed, or the angle
through which it undergoes deviation. The only
problem is that the absorbance does not vary
linearly with concentration (deviation from Beer’s
Law) and this leads to inadequate measurements

OF LIGHT
PARTICLE GEOMETRY
Ideally, the sample solution and the standard
solutions should have the same distribution of
small, medium and large particles
To control particle size and shape, the sample
solution and the standard must be prepared
under identical conditions since different particle
sizes may produce erratic results.

OF LIGHT
WAVELENGTH
 The intensity of scattered radiation depends on the wavelength
of the incident light.
 The wavelength of incident light plays an important role
 Shorter wavelengths are scattered to a greater extent than
longer wavelengths
 Turbidimetry: Radiation or selected wavelength of should not
strongly be absorbed by the suspension medium/coloured
solution i.e. colour of the filter used for selection of
wavelength should be the same as coloured solution.
 Nephelometry: Absorption is much less a problem in
nephelometry so white light is generally used for convenience.

OF LIGHT
MOLECULAR WEIGHT OF PARTICLES
A direct relationship exists

OF LIGHT
DISTANCE OF OBSERVATION
Light scattering decreases by the distance (r)2
from the light scattering particles to the detector
 S proportional to 1/r2

OF LIGHT
REFRACTIVE INDEX
In both techniques, satisfactory results are
obtained when the refractive index difference
between the refractive indices of the particle and
solvent is appreciable (can be achieved by a
change in solvent)

CHOICE BETWEEN TURBIDIMETRY AND
NEPHELOMETRY
REFRACTIVE INDEX
In both techniques, satisfactory results are
obtained when the refractive index difference
between the refractive indices of the particle and
solvent is appreciable (can be achieved by a
change in solvent)

APPLICATIONS OF TURBIDIMETRY AND
NEPHELOMETRY
Analysis of water: Clarity, concentration of ions,
purity, impurities
 Determination of CO2: This method involves
bubbling of gas through an alkaline solution of
barium salt and then analysing the BaCO3
suspension by turbidimetry or nephelometry

NEPHELOMETRY
Determination of inorganic substances:
 Sulphate-Barium chloride: The sulphate
determination can be used to estimate total sulphur
in coke, coal, oils, plastics, rubbers etc. To determine
sulphur, it is first converted to sulphate which is then
shaken with sodium chloride solution and excess of
solid barium chloride to get a suspension of barium
sulphate. Finally this suspension is subjected to
turbidimetry or nephelometry and the
concentration of the suspension is gotten from a
calibration curve.
 Ammonia-Nesslers reagent
 Phosphorus –Strychine Molybedate

NEPHELOMETRY
Air and water pollution: Turbidimetry and
nephelometry are used for continuous monitoring of
air and water pollution. Water is monitored for
turbidity and air is monitored for dust and smoke
Biochemical analysis: Turbidimetry is used to
measure the amount of growth of a test bacteria in
a liquid nutrient medium. It is also used to find out
the amount of amino acids, vitamins and antibiotics.
Nephelometry is used to determine protein, yeast,
glycogen, alpha and beta globulin in blood
 Quantitative analysis (ppm level)

NEPHELOMETRY
Miscellaneous: Water treatment plant, sewage
work, refineries, paper industry
Atmospheric pollution: Smokes and fog
 Determination of molecular weight of high
polymers
Organic Analysis: In food and beverages,
turbidimeters are used for analyzing turbidity in
sugar products and clarity of citrus juices
Used in the determination of benzene
percentage in alcohol

NEPHELOMETRY
Turbidimetric Titration: These are titrations (of
reactions in which insoluble products are
formed) involving a turbidimeter during which
turbidance values are recorded after each
addition of titrant. When all analytes get
precipitated, turbidance becomes constant.
Turbidance Vs volume of titrant added is plotted
Abrupt change in slope indicated end point of
titration.

NEPHELOMETRY
End point is determined from the plot of turbidance
against volume of titrant added.
E.g. Na2SO4 Vs BaCl2, NaCl Vs AgNO3, KF Vs CaCl2

NEPHELOMETRY
Phase Titration: A mixture of two immiscible
liquids is titrated against a third liquid which is
miscible with one of the two liquids but not with
the other e.g. water is added to ethanol-benzene
mixture. The addition of a sufficient amount of
the third liquid produces a turbidity due to the
separation of a separate phase e.g. water
produces a slight turbidity because it is
immiscible in benzene. The appearance of
turbidity marks the end-point of the phase
titration

NEPHELOMETRY
Phase Titration: Another example: Water and
Pyridine Vs Chloroform.
Chloroform is added as a titrant
Causing separation of phase with
turbidity

NEPHELOMETRY
 Determination of Molecular Weight of
Polymers(Macromolecules)

NEPHELOMETRY AND TURBIDIMETRY.pptx

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