The nephelometry involves measurement of the radiation caused by particles (in solution or suspension), thereby measuring the power of the scattered radiation at an angle to the direction of the incident radiation.
When a suspended particle is hit by a beam of light, there is a portion of the light that is reflected, another portion is absorbed, another is deflected, and the rest is transmitted. This is why when the light hits a transparent medium in which there is a suspension of solid particles, the suspension appears cloudy.
What is nephelometry?
Dispersion of radiation by particles in solution
At the moment in which a beam of light hits the particles of a substance in suspension, the direction of propagation of the beam changes its direction. This effect depends on the following aspects:
- Particle dimensions (size and shape).
- Suspension characteristics (concentration).
- Wavelength and intensity of light.
- Incident light distance.
- Detection angle.
- Refractive index of the medium.
The nephelometer is an instrument used to measure suspended particles in a liquid sample or in a gas. So a photocell placed at a 90 ° angle to a light source detects radiation from particles present in the suspension.
Also, the light reflected by the particles towards the photocell depends on the density of the particles. Diagram 1 presents the basic components that make up a nephelometer:
A. Radiation source
In nephelometry it is of vital importance to have a radiation source with a high light output. There are different types, ranging from xenon lamps and mercury vapor lamps, tungsten halogen lamps, laser radiation, among others.
B. Monochromator system
This system is located between the radiation source and the cuvette, so that in this way radiation with different wavelengths compared to the desired radiation is avoided on the cuvette.
Otherwise, fluorescence reactions or heating effects in the solution would cause measurement deviations.
C. Reading cuvette
It is a generally prismatic or cylindrical container, and it can have different sizes. In this is the solution under study.
The detector is located at a specific distance (generally very close to the cuvette) and is in charge of detecting the radiation scattered by the particles in the suspension.
Generally it is an electronic machine that receives, converts and processes data, which in this case are the measurements obtained from the study carried out.
Every measurement is subject to a percentage of error, which is mainly given by:
- Contaminated cells : in the cells, any agent external to the solution under study, whether inside or outside the cell, reduces the radiant light on the way to the detector (defective cells, dust adhering to the walls of the cell).
- Interferences : the presence of a microbial contaminant or turbidity disperses the radiant energy, increasing the intensity of the dispersion.
- Fluorescent compounds : these are those compounds that, when excited by incident radiation, cause erroneous and high scattering density readings.
- Reagent Storage : Inadequate system temperature could cause adverse study conditions and could lead to the presence of cloudy or precipitated reagents.
- Fluctuations in electrical power : to prevent incident radiation from being a source of error, voltage stabilizers are recommended for uniform radiation.
Since the radiant power of the radiation detected is directly proportional to the mass concentration of the particles, nephelometric studies have -in theory- a higher metrological sensitivity than other similar methods (such as turbidimetry).
In addition, this technique requires dilute solutions. This allows both absorption and reflection phenomena to be minimized.
Nephelometric studies occupy a very important position in clinical laboratories. Applications range from the determination of acute phase, complement and coagulation proteins and immunoglobulins.
Immune complex detection
When a biological sample contains an antigen of interest, it is mixed (in a buffer solution) with an antibody to form an immune complex.
Nephelometry measures the amount of light that is scattered by the antigen-antibody reaction (Ag-Ac), and in this way immune complexes are detected.
This study can be carried out by two methods:
End Point Nephelometry:
This technique can be used for end-point analysis, in which the antibody of the biological sample studied is incubated for twenty-four hours.
The Ag-Ac complex is measured using a nephelometer and the amount of scattered light is compared with the same measurement carried out before complex formation.
In this method, the rate of complex formation is continuously monitored. The reaction rate depends on the concentration of the antigen in the sample. Here the measurements are taken as a function of time, so the first measurement is taken at time “zero” (t = 0).
Kinetic nephelometry is the most widely used technique, since the study can be carried out in 1 hour, compared to the long period of time of the end-point method. The dispersion ratio is measured just after adding the reagent.
Therefore, as long as the reagent is constant, the amount of antigen present is considered directly proportional to the rate of change.
Nephelometry is generally used in water chemical quality analysis, to determine clarity and to control treatment processes.
It is also used to measure air pollution, in which the concentration of the particles is determined from the dispersion that they produce in an incident light.