Light Scattering Phenomenon: The blue color of the sky and the red color of the sun at sunset result from scattering of light of small dust particles, H2O molecules and other gases in the atmosphere. The efficiency with which light is scattered depends on its wavelength(λ). The sky is blue because violet and blue light are scattered to a greater extent than other longer wavelengths. A clear cloudless day-time sky is blue because molecules in the air scatter blue light from the sun more than they scatter red light. When we look towards the sun at sunset, we see red and orange colours because the blue light has been scattered out and away from the line of sight. Scattered radiation: • Radiate scattering- second major spectral method of analysis. • In this technique some radiation that passes through a sample strikes particles of the analyte and is scattered in a different direction. • A detector is used to measure either the intensity of the scattered radiation or the decreased intensity of the incident radiation • Depending on the scattering mechanism, the method can be employed for either qualitative or quantitative analysis. For chemical analysis three forms of radiative scattering are important – viz. Tyndall, Raman, and Rayleigh scattering. Tyndall Scattering occurs when the dimensions of the particles that are causing the scattering are larger than the wavelength of the scattered radiation. It is caused by reflection of the incident radiation from the surfaces of the particles, reflection from the interior walls of the particles, and refraction and diffraction of the radiation as it passes through the particles. Scattering of light  - by particles in a colloid or suspension. The longer-wavelength light is more transmitted while the shorter- wavelength light is more reflected via scattering Nephelometry & Turbidimetry: When electromagnetic radiation (light) strikes a particle in 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 particle. In Nephelometry, the intensity of the scattered light is measured. In Turbidimetry, the intensity of light transmitted through the medium, the unscattered light, is measured. Light scattering is the physical phenomenon resulting from the interaction of light with a particles in solution Turbidimetry is involved with measuring the amount of transmitted light (and calculating the absorbed light) by particles in suspension to determine the concentration of the substance in question. Amount of absorbed light, and therefore, concentration is dependent on ; 1) number of particles, and 2) size of particles. • Measurements are made using light spectrophotometers Factors affecting on scattering of light: Concentration of particles Particle size Wavelength Distance of

1. NEPHELOMETRY AND
TURBIDIMETRY
Proff. Priyanka shah
M.Pharm(QA)
KPGU
1

2. Light Scattering Phenomenon:
• The blue color of the sky and the red color of the sun at sunset result from scattering of light of small dust particles,
H2O molecules and other gases in the atmosphere.
• The efficiency with which light is scattered depends on its wavelength(λ).
• The sky is blue because violet and blue light are scattered to a greater extent than other longer wavelengths.
• A clear cloudless day-time sky is blue because molecules in the air scatter blue light from the sun more than they scatter red
light.
• When we look towards the sun at sunset, we see red and orange colours because the blue light has been scattered out and
away from the line of sight.
Scattered radiation:
• Radiate scattering- second major spectral method of analysis.
• In this technique some radiation that passes through a sample strikes particles of the analyte and is scattered in a different
direction.
• A detector is used to measure either the intensity of the scattered radiation or the decreased intensity of the incident radiation
• Depending on the scattering mechanism, the method can be employed for either qualitative or quantitative analysis.
2

3. For chemical analysis three forms of radiative scattering are important – viz.
Tyndall,
Raman, and
Rayleigh scattering.
• Tyndall Scattering occurs when the dimensions of the particles that are causing the scattering are larger than the
wavelength of the scattered radiation.
• It is caused by reflection of the incident radiation from the surfaces of the particles,
• reflection from the interior walls of the particles, and refraction and diffraction of the radiation as it passes through the
particles.
• Scattering of light
• - by particles in a colloid or suspension.
• The longer-wavelength light is more transmitted while the shorter-
wavelength light is more reflected via scattering.
3

4. Raman and Rayleigh scattering occur when the dimensions of the scattering particles are less than 5 percent of the
wavelength of the incident radiation.
• Both Rayleigh and Raman scattering are caused by the effect on the analyte of the fluctuating electromagnetic field
that is associated with the passing incident radiation.
The fluctuating field induces an electric dipole (separation of charges equal in size but opposite in sign) within the
scattering particles that oscillates at the same frequency as the incident radiation.
The oscillating dipole behaves as a point source of emitted radiation.
4

5. Nephelometry & Turbidimetry:
When electromagnetic radiation (light) strikes a particle in 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 particle.
In Nephelometry, the intensity of the scattered light is measured.
In Turbidimetry, the intensity of light transmitted through the medium, the unscattered light, is measured. Light scattering is
the physical phenomenon resulting from the interaction of light with a particles in solution
Turbidimetry is involved with measuring the amount of transmitted light (and calculating the absorbed light) by
particles in suspension to determine the concentration of the substance in question.
Amount of absorbed light, and therefore, concentration is dependent on ;
1) number of particles, and
2) size of particles.
• Measurements are made using light spectrophotometers
5

6. Factors affecting on scattering of light:
• Concentration of particles
• Particle size
• Wavelength
• Distance of observation
• MW of particle
6

7. Concentration of particles :Turbidimetry
Concentration of particles:
At low concentration of particles for scattering of light Beers Lamberts law is applicable.
S=Log10
Io/It
S=KtC=-logT
Turbidance is directly proportional to concentration
i.e. S α C
Where
S = Turbidance
IO= Intensity of incident light
It=Intensity of transmitted radiation
T=Turbidance
C=Concentration of solution
K=constant depend on linearity of light
7

8. Concentration of particles :Nephelometry
In Nephelometry an equation that describe the relation between the
intensity of scattered radiation , intensity of incident radiation , and
concentration of particles
Is= Ks x IO x C
Where
IO= Intensity of incident light
Is=Intensity of scattered radiation
Ks= It is constant which depend on suspended particle and suspension
medium.
C=Concentration of solution
8

9. Particle Size
The fraction of light scattered at any angle depends upon size and shape of
particles.
The amount of scattering (S) α proportional to square of effective radius of
the particle.
To control the particle size and shape, sample solutions and standards must
be prepared under identical conditions.
Following care must be taken:
i) Concentrations of two ions forming ppt.
ii) Ratio of concentration of the solutions.
iii) Order of mixing of ppt.
iv) Temperature at which suspension is prepared.
9

10. 10

11. 11

12. Wavelength
• The intensity of scattered radiation depends upon wavelength of the
incident light.
• Shorter wavelength are scattered to greater extent than the longer
one.
• Wavelength of light is chosen in such a way that analyte solution
does not absorbs strongly.
• Turbimetric & Nephelometric measurements are carried using white
light
12

13. Molecular Weight
Direct relationship exist
• Distance of Observation:
Light scattering decrease by the distance (r)2 from the light scattering
particles to the detector
S 1/ ∞ r2
13

14. Nephelometry.
Principle
• Nephelometry is concerned with measurement of scattered light from a
cuvette containing suspended particles in a solution.
• The components of a nephelometer are the same as a light
spectrophotometer except that the detector is placed at a specific angle from
the incident light.
• The detector is a photomultiplier tube placed at a position to detect forward
scattered light.
• Detectors may be placed at 90o, 70o or 37o depending on the angle at which
most scattered light are found.
14

15. • Since the amount of scattered light is far greater than the
transmitted light in a turbid suspension, nephelometry
offers higher sensitivity than turbidimetry.
• The amount of scattered light depends on the size and
number of particles in suspension.
• For most clinical applications, the light source is a tungsten
lamp giving light in the visible region
• For higher sensitivity and for applications that determine
the size and number of particles in suspension, laser light
nephelometers is used
15

16. Turbidimetry and Nephelometry
When particles are suspended in a solution in a cuvette, they make the solution unclear (turbid).
Incident light entering the cuvette will be subjected to three reactions;
1- some of the light will be absorbed (blocked) by the particles
2- some will be transmitted through the cuvette
3- some will be scattered in various directions.
Turbid metric + nephlometeric analysis:
When part of incident radiant energy is dissipated by absorption, reflection, and refraction, while the remainder is
transmit light as a function of the concentration of the dispersed phase is the basis of turbid metric analysis
16

17. Nephlometer
Definition the measurement of the intensity of scattered light at right angles to the direction of the incident lig
Instrument used Nephlometery machine
Type of light measured Scattered light
Arrangement of photometer measure the light scattered at right angle to the direction of the propagation of light from the source
It could be movable detectors which allow operator to vary the angle of detection
Clinical uses Ag-Ab rxn, immunocomplex rxn,ppts, lipoprotein
17

18. Instrumentation
18

19. Radiation source
Tungston filament lamp or
Mercury arc lamp
can be used as source of radiation.
Sample cell:
The cells made from glass or plastic are used for study.
Rectangular cell are used in Turbidimeter and
Semi octagonal sample cell are used in Nephelometer .
19

20. Sample Cell in Turbidimeter Sample Cell in Nephelometer
Detector:
Photocell can be used as detector
20

21. CHOICE OF THE METHOD:
Choice Of The Method depends upon the amount of light scattered by suspended particles present in solution.
TURBIDIMETRY - high concentrated suspensions.
NEPHELOMETRY - low concentrated suspensions - more accurate results
21

22. APPLICATIONS
Quantitative analysis of Inorganic species
Air and water pollution
Turbidimetric titrations
Phase titrations
Determination of Molecular weight
22

23. Quantitative analysis of Inorganic species
In gravimetric analysis certain ppt. particles are very small in size so
they are difficult to filter .
quantitative analysis of such ppt. compound can be carried out by
calibration curve method.
Examples
SO4-2 as BaSO4.
CO3-2 as BaCO3.
Cl as AgCl
Ca+2 as Calcium oxalate
23

24. Air and water pollution
• Air and water pollution Air Pollution: particulate matter in air ,dust,
smoke can be monitored by the Nephelometry and Turbidimetry.
• Water Pollution: Turbidity of water can be monitored continuously by
Nephelometry and Turbidimetry.Ex. Water treatment plants, sewage
work, refineries, paper industry .
• Determination of Phosphorus at trace levels(1 parts in more than
300 million part) by precipitating it by strchine –molybdate reagent.
• Determination of trace levels of ammonia: Ammonia can be
detected at concentration of 1 parts in 160 million by using Nessler’s
reagent
24

25. Turbidimetric titrations
• The Turbidimetric titrations are those in which turbidance is used to
locate end point using a titrant that gives precipitate .
• Ex. Titration of Ba+2 against H2SO4 solutions of concentration up to 10-
5 to 10-6 M can be determined.
Phase titrations:
• The Turbidimetry can be used for titration of two liquids with third
liquid which is miscible with only one of two liquids in mixture.
Ex. Mix. Of Ethanol and benzene , water is miscible with ethanol but
not with benzene.
• Water pyridine mixture titrated with CHCl3
25

26. 5.Determination of Molecular weight:
Molecular wt. of polymers such as polyvinyl alcohol can be determined.
Following relationship holds between turbidity and Mol.wt.
lim Hc/T =1/M
where H is constant for given polymer.
C is concentration of polymer in g/cm3.
26

27. Biochemical analysis
Turbidimetry can be used to measure the growth of bacteria in a liquid nutrient medium.
It is also used to determine the amount of amino acids, vitamins and antibiotics.
Clinical applications
• Widely used to determine concentrations of unknowns where there is antigen- antibody reactions such as Determination of
immunoglobulin's (total, IgG, IgE, IgM, IgA) in serum and other biological fluids
• Determination of the concentrations of individual serum proteins; hemoglobin, haptoglobin, transferring, c-reactive
protein,1-antitrypsin, albumin (using antibodies specific for each protein)
• Determination of the size and number of particles.
Analysis of Water:
For determination of clarity and for determination the concentration of various ions by adding selective
precipatants,nephloturbidimeter is applied.
Determination of inorganic substance:
Inorganic elements like phosphorus ammonia,sulphate,chloride,carbonate,fluoride,cynide,calcium, zinc etc can be estimated by
precipitating them using precipitants.
27