Nephelometry

Two useful methods available for measuring the concentration of a solution that contains particles too large for absorption spectroscopy are nephelometry and turbidimetry. These nonabsorptive methods may be suitable for quantitative assays using antigen-antibody complexes or measuring the amount of proteins in fluids.

To understand the principles of nephelometry and turbidimetry, the idea of radiation scattering by particles in solution must first be reviewed. When a collimated light beam strikes a particle in suspension, portions of the light are absorbed, reflected, scattered, and transmitted. Nephelometry is the measurement of the light scattered by a particulate solution. Three types of light scatters occur based on the relative size of the light wavelength by Gauldie, 1981. If the wavelength (A) of light is much larger than the diameter (d) of the particle, where d is less than 0.1 A, the light scatter is symmetric around the particle. Minimum light scatter occurs at 90 degree to the incident beam and was described by Rayleigh by Rayleigh, 1885. If the light wavelength is much smaller than the particle diameter, where d greater 10 A, the light scatters forward owing to the destructive out-of-phase back-scatter, as described by the Mie theory. If the wavelength of light is approximately the same amount as the particle size, more light scatters in the forward direction than in other directions, as defined by the Rayleigh-Debye theory. A common application of nephelometry is the measurement of antigenantibody reactions. Because most antigen-antibody complexes have a diameter of 250 to 1500 nm and the wavelengths used are 320 to 650 nm, the light scatter is essentially of the Rayleigh-Debye type.

Components of a Nephelometer

A typical nephelometer consists of a light source, a collimator, a monochromator, a sample cuvette, stray light trap, and a photodetector. Light scattered by particles is measured at an angle, typically 15 to 90 degree to the beam incident on the cuvette. Light scattering depends on the light wavelength and the particle size. For macromolecules with size close to or larger than the light wavelength, measurement of the forward light scatter increases the sensitivity of nephelometry. Light sources include a mercury-arc lamp, a tungsten-filament lamp, a light-emitting diode, and a laser.
Lasers produce stable, nearly ideal monochromatic light of narrow linewidth. It emits radiant energy that is coherent, parallel, and polarized. A laser beam can be maintained as a very slim cylinder only a few micrometers in cross section by Willard, 1988. A typical helium-neon laser lamp consists of a helium-pumping electrode cathode and a hollow glass laser core surrounded by a laser plasma tube anode. Both the plasma tube and the core are filled with free helium and neon gases. The electrical discharge between the cathode and the anode is confined to the hollow glass core to keep it concentrated for maximum energy transfer. Two mirrors are positioned at the ends of the laser tube. One of them is fully reflective and the other partially transparent. When the electrode is charged, the helium atoms are excited to a higher energy state and then transfer this energy to the neon atoms by collision. In turn, the excited neon atoms emit photons. Photons bounce back and forth between the two end mirrors, stimulating other atoms to emit further photons, resulting in an amplification process. The amplified light eventually emerges as a laser beam through the partially transparent mirror. With the high intensity monochromatic beam, a substantial increase in sensitivity has been seen with lasers over conventional instruments. Disadvantages of laser sources include cost, safety problems, limited availability of wavelengths, and substantial cooling requirements for all but the smallest lasers.

Common Nephelometers in Use

At present, the single most common uses.of nephelometry is the measurement of antigen antibody complexes formed in enzyme immunoassays. This instrument measures the formation rate of insoluble immuno-precipitation products resulting from a specific antigen combining with a specific antibody. One kind of model allows random access, fully automated testing of many proteins, such as apolipoproteins, immunoglobulins, prealbumin, and therapeutic drugs such as theophylline.

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