Republic of the Philippines

Laguna State Polytechnic University

San Pablo City Campus
Del Remedio, San Pablo City

COLLEGE OF TEACHER EDUCATION



Subject: Analytical Chemistry Prepared by: Michael Demdam and Arlene Suaze
Topic: Nephelometry Submitted to: Mrs. Clarissa F. Gregana
Course/Yr/Sec: BSED IV-U


Nephelometry

I. DEFINITION
Nephelometry is a technique used in immunology to determine the levels of several blood plasma
proteins.
It is important in quantification of free light chains in diseases such as multiple myeloma. Quantification
is important for disease classification and for disease monitoring once a patient has been treated.
In nephelometry the measurement is made by measuring the light passed
through a sample at an angle.
Nephelometry can be used to detect either antigen or antibody, but it is
usually run with antibody as the reagent and the patient antigen as the
unknown.
End point nephelometry tests are run by allowing the antibody/antigen
reaction to run through to completion (until all of the present reagent
antibodies and the present patient sample antigens that can aggregate
have done so and no more complexes can form).
In kinetic nephelometry, the rate of scatter is measured right after the reagent is added.
II. HISTORY
Richards, Theodore William (1868-1928) was an American chemist. He won the 1914 Nobel Prize in
chemistry for his exact determinations of the atomic weights of chemical elements, including those used to
determine virtually all other atomic weights.
In 1894, Richards invented the nephelometer, a device that uses light dispersion to measure the concentration
of suspended matter in a liquid.
III. TYPES OF INSTRUMENT
Nephelometer - is an instrument for measuring concentration of suspended
particulates in a liquid or gas colloid.
A nephelometer measures suspended particulates by employing a light beam
(source beam) and a light detector set to one side (often 90) of the source
beam. Particle density is then a function of the light reflected into the
detector from the particles.
A nephelometer measures suspended particulates by employing a light beam
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(source beam) and a light detector set to one side (often 90) of the source beam. Particle density is then a
function of the light reflected into the detector from the particles.
The main uses of nephelometers relate to air quality measurement for pollution monitoring, climate
monitoring, and visibility. Airborne particles are commonly biological contaminants, particulate
contaminants, gaseous contaminants, or dust.
IV. OPERATION HOW TO HANDLE
Sensidyne Nephelometer
The Nephelometer measures particulate in the air using a laser engine. laser light is scattered by the particles
in the view volume and detected by a photo detector. This information is the multiplied by a K factor and
displayed on the screen and logged as a sample event.
The Nephelometer logs each sample event into memory; data is transferred to a computer using the
companion software.
A cable is supplied for connection to the USB port on the users computer. Each sample event records a time,
date, serial number, environmental setting and an average concentration.
V. APPLICATION
The main uses of nephelometers relate to air quality measurement for pollution monitoring, climate
monitoring, and visibility. Airborne particles are commonly either biological contaminants, particulate
contaminants, gaseous contaminants, or dust.
Nephelometers are also used for measurement of visibility with simple one-wavelength nephelometers used
throughout the world by many EPAs. Nephelometers, through the measurement of light scattering, can
determine visibility in distance through the application of a conversion factor called Koschmieder's formula.
Koschmieder's formula- A fundamental equation of visual-range theory, relating the apparent luminance of
a distant black object, the apparent luminance of the background sky above the horizon, and the extinction
coefficient of the air layer near the ground.
Nephelometry is a widely used technique for investigating different immunological problems however there
are two different groups of proteins involved in recognition and clearance of pathogens that are regularly
measured; Complement proteins and Immunoglobulins.
Nephelometers are also used in global warming studies, specifically measuring the global radiation balance.
Three wavelength nephelometers fitted with a backscatter shutter can determine the amount of solar radiation
that is reflected back into space through dust and particulate matter.
VI. PRINCIPLES/ LAWS INVLOVED
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 90
o
, 70
o
or 37
o
depending on the angle at which most scattered light are found.
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.
Considerations in turbidimetry and nephelometry
The reaction in turbidimetry & nephelometry does not follow Beer's Law
Therefore, standard curves must be plotted and the concentration of the unknown is determined from the
standard curve.
Because the absorbance is dependent on both number and size of particles, the standard solution which is
used for the standard curve must have similar size in suspension as unknown.
Because some precipitation and settlement of particles may occur with time, in order to obtain good
accuracy it is important to ; a) mix the sample well prior to placing the cuvette in the instrument, and, b)
keep the same time for measurement of every sample throughout the measurement.
Kinetic reactions (measurement of the progress of reaction with time) provides higher degree of accuracy,
sensitivity, precision and less time than end-point reactions (measuring the reaction at the start and finish
of the reaction)
o Additionally in kinetic reactions there is no need for reagent blank since the previous reading is
taken as the base-line for the next reading.
o Kinetic reaction may be taken in 60, 90 or 120 seconds (taking readings at 10 seconds intervals),
whereas endpoint reactions may take much longer time e.g. 15 -120 minutes.
Selection of a wavelength
o If both solution and suspended particles are colorless, then use any wave length in the visible
range
o If the solution is coloured but the particles are not coloured, then use a wave length that gives
minimum absorption for the solution
o If the particles are coloured and the solution is colorless then use a wavelength that gives
maximum absorption with the particles.
o If both solution and particles are coloured then use two wavelengths; one that gives minimum
absorbance for the solution and the other one maximum absorbance for the particles. Subtract the
solution absorbance from the particles absorbance.
VII. IMAGES