this is a slide of Nephelometry and Turbidimetry One of Spectroscopic technique

1. University of Gondar
College of Veterinary Medicine and Animal Science
Department of Veterinary Pharmacy
Nephelometry and Turbidimetry
Theory, Principle, Instrumentation & Applications
Sewagegnehu Getachew
January 19, 2023

2. At the end of this presentation you will
except to:-
January 19, 2023
Differentiate between turbidimetry and nephelometry
Discuss the principles of turbidimetry and nephelometry
Explain the factors that affect the measurement
Describe the pharmaceutical application of turbidimetry and nephelometry
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3. Introductory
Why is the sky blue during the day
and
Red color of the sun at the sun set?
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4. Introductory
• This is due to scattering of light by small particles of dust, molecules of
water, and other gases in the atmosphere.
• The sky is blue because violet and blue light are scattered to a greater
extent than other longer wavelengths of light.
• For the same reason, the sun appears to be red when observed at
sunset
• because red light is less efficiently scattered and, therefore,
transmitted to a greater extent than other wavelengths of light
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5. Turbidimetry and Nephelometry
• are techniques in which an incident source of radiation is elastically
scattered by a suspension of colloidal particles.
• If incoming radiant energy gets impinged, upon particles suspended
in a medium which is having a RI different from that of the suspended
particles,
• the light which strikes the particles is transmitted at angles other
than 180° from the incident light. The light is said to be scattered.
• Nephelometry involves the direct measurement of the scattered light,
• whereas turbidimetry involves the measurement of the decrease in
the intensity of transmitted light in a suspension
• (i.e. that which is not scattered)
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6. Principle of turbidimetric analysis
• When light is allowed to pass through a suspension, the part of the
incident radiant energy is dissipated by absorption, reflection and
refraction while the reminder is transmitted.
• Measurement of the intensity of the transmitted light as function of the
concentration of the suspended particles forms basis of turbidimetric
analysis.
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7. Turbidimetry and Colorimetry
• Turbidimetry is much similar to colorimetry
• because both involve measurement of the intensity of light
transmitted through a medium.
• But differ in that the light intensity is ↓ed by scattering in turbidimetry
and by absorption in colorimetry.
• Due to this reason both techniques may use similar or even
identical apparatus.
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Colorimetry is measurement of the wavelength and the intensity of EMR in the visible region of the spectrum

8. Principle of nephelometric analysis
• At this time the light is also allowed to pass directly through the
sample solution having suspended particles.
• The amount of radiation scattered by particles is measured at an angle
(usually 90° ) to the incident beam.
• Thus nephelometry is measurement of intensity of the scattered light
as a function of the concentration of the dispersed phase.
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Figure 2
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9. Nephelometry and Fluorimetry
Fluorimetry Nephelometry
Similarity
Emergent radiation measured at 90°
Similarity
Emergent radiation measured at 90°
Difference
• Emitted radiation measured
• Emitted radiation longer wavelength
than incident light
Difference
• scattered radiation measured
• Emitted radiation same wavelength
as that of than incident light
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elastic scattering, radiation is absorbed by the analyte and re-emitted without a change in the radiation’s energy.
When the radiation is re-emitted with a change in energy, the scattering is said to be inelastic

10. Reflection and Scattering
• Both reflection and scattering phenomena are very important in
turbidimetry and nephelometry.
• If light is allowed to pass through a solution having suspended
particles,
Reflection will take place when the dimensions of suspended
particles are larger than the wavelength of incident light.
Scattering will take place if the dimensions of suspended particles
are smaller than the incident wavelength.
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11. Factors affecting measurement
1. Concentration of particles
2. Particle geometry (size, shape)
3. Incident wavelength
4. Refractive index
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12. 1. Concentration
 In turbidimetry, one measures the transmitted of a primary beam of radiation as:
T = It/I0
 According to Beer’s law:
S = log I0/It = kbc
k is the proportionality constant known as turbidity coefficient
 k depends on the particle size and shape, wavelength and refractive indexes of
suspended and suspending media.
 It inversely proportional to the concentration
 It is good only for small particles where Rayleigh’s scattering is the main
mechanism of attenuation and for dilute suspensions where multiple scattering is
unlikely.
 A working curve can be prepared by plotting S vs known concentration of
scattering material and then the unknown concentration are observed from
this curve by knowing their S value from the turbidimeter
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13. Cont …
 In nephelometry, one cannot relate the scattered intensity to the
concentration by any sample theoretical equation.
 The reason for this is that the scattered intensity in nephelometry depends
upon a number of complicated factors;
• like the properties of the scattering suspension and the angle and
geometry of measuring instrument
 The best equation will be that one which will relate the Is to the C suspended
particles by the approximate empirical equation such as:
IS = kSI0C
Where: KS is the empirical constant for a particular system
I0 is the intensity of incident light and
IS is the scattered of light intensity
IS directly proportional to the concentration
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14. Cont …
 Whenever quantitative analysis is to be carried out in nephelometry,
• a working curve is obtained by plotting the concentration of
suspended particles vs Is/I0 under carefully controlled conditions.
 But in most of the cases log(Is/I0) is plotted vs C to confirm with the
more usual spectrophotometric and turbidimetric practices
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15. 2. Particle geometry:
 In both techniques the most critical factor is the control of particle size and
shape.
 The ideal situation is that when all samples and calibration solution with
which they are compared should possess the same distribution of small
medium and large particles.
 One should prepare samples and standards under identical conditions.
 The conditions include concentration of reactants, temperature, agitation,
pH, the presence of non-reactants and order of mixing of reactants.
 If one does not maintain these conditions one may get different sizes
particles which may introduce major error in turbidimetry and
nephelometry
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16. 3. Wavelength
 If the sample solution is colorless, one must use the incident light of the
same color.
 On the other hand if clear solutions are having dark particles, light in the
infrared region must be used where there is maximum absorption.
 In nephelometry, absorption is much less of a problem. For
nonfluorescent sample white light is generally used as a convenience.
 Shorter wavelength scatter to greater extent while longer wavelength
scatter to lesser extent.
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17. 4. RI difference:
 Best results are obtained when there is an appreciable RI difference
between the particle and its surroundings medium.
 Sometimes it is advantageous to change solvents in order to increase the
refractive index references.
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18. Choice between Turbidimetry and
Nephelometry
• It is determined by two principal factors.
i. The intensity of the transmitted or scattered radiation relative to the
intensity of radiation from the source.
• When the solution contains a small concentration of scattering
particles, the intensity of the transmitted radiation It, will be very
similar to the intensity of the radiation source, I0.
• Thus, nephelometry is a more appropriate choice for samples
containing few scattering particles.
• On the other hand, turbidimetry is a better choice for samples
containing a high concentration of scattering particles.
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19. Cont ..
ii. The second consideration is the size of the scattering particles.
• For nephelometry, the intensity of scattered radiation at 90° will be
greatest if the particles are small enough that Rayleigh scattering is in
effect.
For larger particles scattering intensity is diminished at 90°. When
using an ultraviolet or visible source of radiation, the optimum particle
size should be 0.1– 1 µm.
• The size of the scattering particles is less important for turbidimetry, in
which the signal is the relative decrease in transmitted radiation.
• In fact, turbidimetric measurements are still feasible even when the
size of the scattering particles results in an increase in reflection and
refraction.
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20. Recapping
is method in which the decrease in transmitted radiation due
to scattering is measured and used for high concentration suspensions.
Nephelometry is the measurement of the transmitted light by the suspended
particles at right angles to the incident beam. True/
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Turbidimetry
False
transmitted

21. Instrumentation
is very similar to spectrophotometer devices.
1, Sources
• One may use white light in nephelometers but it is advantageous to use
monochromatic radiation.
 Similarly, monochromatic radiation is used in turbidimeters to
minimize absorption.
• In either case it is necessary to use sources providing high intensity
monochromatic radiation and wherever possible short wavelengths are
used to increase the efficiency of Rayleigh scattering.
• Mercury lamp and Tungsten lamp may be used
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22. 2, Filters/Monochromater:
Filters will convert the polychromatic light to monochromatic light.
Generally filters are used for this purpose. Fitters are of two types:
 Absorption filters
 Interference filters
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absorption filters that absorb unwanted wavelengths and
interference filters that remove selected wavelengths by internal destructive interference and reflection

23. 3, Sample cells
• Although we can use cylindrical cells, they must have flat faces where
the entering and exiting beams are to be passed.
 This is to minimize reflections and multiple scattering from the cell
walls.
• In general, a cell with a rectangular cross-section is preferred.
Where measurements are to be made at angles other than 90°,
Semi-octagonal cells are widely used.
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24. Cont ...
• Generally walls through which light beams are not pass are painted a
dull black to absorb unwanted radiation and minimize stray radiation.
• In experimental cells, a blackened curved horn is frequently affixed to
the wall directly opposite the entering beam to trap all the beam which
is not scattered.
• Alternatively, one can put a light trap for this purpose in the cell of the
chamber in which the cell is located.
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25. 4, Detectors
• Most commonly used detectors in the nephelometry and turbidimetry
are photomultiplier tubes.
• In nephelometers photomultiplier tubes should be as detectors
because the intensity of scattered radiation is usually very small.
• In most of the nephelometers, detector is generally fixed at 90° to the
primary beam
› but for maximum versatility and sensitivity is desirable to vary
the detector angle which is generally close to primary beam.
• In some nephelometers, the detector is mounted on circular disc which
allows measurement at many angles.
• In turbidimeters, ordinary detectors such as phototubes may be used.
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26. 5, Turbidimetres
• In most turbidimeters, ordinary calorimeters (or) spectrophotometers may be used.
• Others like Du Pont model 430 can also be used which is more sensitive to low
concentration of suspended particle than ordinary turbidimetry.
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Du Pont Model 430 turbimeter
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27. 5, Nephlometer
• Ordinary fluorimeters are generally used for nephelometric
measurements. In some cases spectrophotometers can be employed as
nephelometer.
• The photo-multiplier tube detector is used as a receiver which is
mounted on a turnable and may be positioned at any desired angles
from 0° to 180° relative to the exit beam.
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28. • Generally sensitivity of the method is improved by the following:
 Addition of water-soluble polymers
 Gives greater stability of the immune complex
 Reduced reaction concentrations
 Increased sensitivity
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29. Applications of nephelometry and
turbidimetry
oTurbidimetry and nephelometry are widely used to determine the
clarity of water, beverages, and food products. The following are some
applications of nephelometry and turbidimetry.
oOrganic Analysis – in food and beverages, turbidimeter is used for
analysis of turbidity in sugar products, and clarity of citrus juices.
Another interesting application is the determination of benzene in
alcohol by dilution with water to make an immiscible suspension.
oBiochemical Analysis – an important application of turbidimetry is to
measure the amount of growth of a test bacterium in liquid nutrient
medium. It is also used to find out the amount of amino acids,
vitamins and antibiotics.
oNephelometry has been used for the determination of yeasts, glycogen
and of beta and gamma globulin in blood serum and plasma.
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30. Applications of nephelometry and
turbidimetry
• Used in the determination of sulphate as barium sulphate. Eg: Carbonate
as BaCO3, Chloride as AgCl, Fluoride as CaF2
• Phosphorus can be estimated as a concentration of 1 part in more than
300 million parts of water as a precipitate with strychnine molybdate
reagent (mainly used in water treatment plants).
• Used in the determination of turbidity in the sugar products.
• Used in the determination of benzene percentage in alcohol.
• Used in the monitoring of the air and water pollution.
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31. Summary
Spectroscopic measurements involve the scattering of light by a particulate form
of the analyte.
In turbidimetry, the decrease in the radiation’s transmittance through the sample
is measured and related to the analyte’s concentration through Beer’s law.
In nephelometry we measure the intensity of scattered radiation, which varies
linearly with the analyte’s concentration.
Both are analytical methods which have wide range applications from the simple
detection of the pollution to determination body constituents hence these play a
vital role not only in the analysis of the compounds but also in the clinical analysis.
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33. References
• P. D. Chaithanya Sudha (2013) Pharmaceutical Analysis: PEARSON
• David H. (2000) Modern analytical chemistry: McGraw-Hill
• G.R. Chatwal S.K. Anand (2011) Instrumental method of chemical analysis:
Himalaya publishing book
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Thanks for Your Attention
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