Nephelometry and Turbidimetry

NEPHELOMETRY
AND
TURBIDIMETRY
MODERATOR PRESENTED BY
DR. PRASHANT NIGAM DR. SRIKRISHNA PAUL
H.O.D &ASSOC. PROF 2ND YEAR P.G STUDENT
DEPT. OF BIOCHEMISTRY DEPT. OF BIOCHEMISTRY
C.I.M.S , Bilaspur. C.I.M.S , Bilaspur.
When electromagnetic radiation strikes a particle in
solution ------
some of the light is absorbed by the particle ,
some will be transmitted through the solution,
and
some will be scattered or reflected
Turbidimetry and nephelometry are methods used to
measure scattered light .
TYNDALL EFFECT -- light scattering by colloid

particles.
FACTORS AFFECTING SCATTERING OF LIGHT IN SOLUTION
 I. Order and rate of mixing of substance within solution.
II. Agitation of solution containing particulate matters.
III. Temperatures of solution as it affect solubility of
component present in it.
IV. Viscosity of medium.
V. Presence or absence of electrolyte or protective colloid.
VI. Concentration of solution.
VII. Number of particulate matter suspended.
VIII. Dimension of particulate matter.
IX. Wavelength of radiating beam.
X. Refractive index of medium.
NEPHELOMETRY -
Detection of light energy scattered or reflected toward a
detector that is not in the direct path of transmitted light
It is concerned with measurement of intensity of scattered
light from a cuvette containing suspended particles in a
solution.
Commonly Nephelometric measurements are often made
arbitrarily at right angle to incident beam.
Ideal nephelometric measurements should be free of stray
light .
However because of stray light generating components in
optical system and in sample cuvette or in sample itself, a
truly dark field situation is difficult to obtain.
Some nephelometers are designed to measure scattered
light at an angle other than 90 degree to take advantage of
increased forward scatter intensity caused by light
scattering from larger particles ( eg. Immune complexes).
TURBIDIMETRY –
Because of light scattering that occurs with turbidity, the
intensity of light reaching the detector at 180 degrees is
reduced. Measurement of this decrease in intensity is called
turbidimetry.
Turbidity –
I = Ioe-bt
Or,
t = (1/b) ln(Io/I)
Where t = turbidity , b = path length of incident light
through the solution of light scattering particles, I =
intensity of transmitted light, Io = intensity of incident light.
Turbidity is measured at 180 degrees from the incident
beam , or in the same manner as absorbance are measured
in spectrophotometer.
CHOICE BETWEEN BOTH THE TECHNIQUES
Choice between nephelo or turbidimetric methods depends

upon following two parameters :
1. Scattered radiation intensity vs incidence radiation intensity:
In high concentration of particulate matter turbidimetry is
choice over nephelometry while just opposite is true for very
dilute solutions containing fewer particulate matter.
2. Size of particulate matters:
Nephelometry is choice in case where particles suspended in
medium are of smaller dimension while in case of particle of
larger dimension turbidimetry is preferred and measurements
for same are done at an angle of 180°.
Turbidimeter –
Turbidimetric measurements are easily performed on
photometers or spectrophotometers and require little
optimization.
The principle concern of turbimetric measurements is
signal to noise ratio(SNR).
Photometric systems with electro optical noise in the
range of ±0.0002 absorbance unit or less are useful for
turbidity measurements.
Nephelometer –
These devices place photo multiplier tube detector at
appropriate angles to the excitation light beam.
The principle concern of light scatter instrumentation
include excitation intensity, wavelength, distance of
detector from sample cuvette, and minimization of
external stray light.
Basic components of a nephelometer include –
light source,
collimating optics,
sample cell,
collection optics which include light scattering optics,
a detector optical filter and a detector.
Light source may be quartz halogen lamp, xenon

lamp and lasers.
APPLICATIONS
1. Determination of particle size present in suspensions.
2. Measurement of atmospheric pollutants.
3. Determining concentration of solute in solution.
4. Growth of bacterial cell in a liquid nutrient medium.
5.Turbidimetry and nephelometry has numerous
applications in water treatment plants, sewage work,
steam generating plant, beverage bottling industry, in pulp
and paper manufacturing, petroleum refining and
pharmaceutical industries
Limitation of light scattering instruments –
antigen excess which decreases turbidity
and
matrix effects due to macromolecules like lipoproteins,
chylomicrons and dust in sample matrix.
Nephelometry Turbidimetry
Suspended particles < 100 mg/l Suspended particles >100 mg/l
Intensity of scattered light is measured Intensity of unscattered light is measured
Low concentration suspension is used High concentration suspension is used
Scattering is uniform Scattering is not uniform
Intensity of scattered light is directly Intensity of scattered light is directly

proportional to the concentration of proportional to the concentration of
suspended particles suspended particles
Measured at 90° Measured at 180°
Detector is photomultiplier tube Detector is photocell

References --
Teitz Clinical Biochemistry and Molecular Diagnosis
 Godkar and Godkar Textbook of Medical Technology
Upadhyay and Upadyhay Biophysical Chemistry
THANK YOU

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NEPHELOMETRY

TYNDALL EFFECT -- light scattering by colloid

Choice between nephelo or turbidimetric methods depends

Light source may be quartz halogen lamp, xenon

Suspended particles < 100 mg/l Suspended particles >100 mg/l

Intensity of scattered light is measured Intensity of unscattered light is measured

Low concentration suspension is used High concentration suspension is used

Scattering is uniform Scattering is not uniform

Intensity of scattered light is directly Intensity of scattered light is directly

Measured at 90° Measured at 180°

Detector is photomultiplier tube Detector is photocell

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