Nephelometry

Nephelometry is derived from the Greek word nephew, which means cloud. In analytical chemistry, it is used to measure the amount of turbidity or cloudiness in a solution due to the presence of suspended insoluble particles. 

When the light gets directed through a turbid solution that contains suspended solid particles, the light gets transmitted, absorbed, and scattered. Based on the size, shape, and concentration of the insoluble particles in solution and the incident wavelength of light, the amount of light is scattered.

Principle of Nephelometry

Scattering of light in liquids follows the rules of elastic scattering of particles, where no energy is absorbed by either particle during the “collision”. The energy of a photon before and after the scattering remains the same. Elastic scattering differs in large and small particles. For large particles, the light gets scattered in the forward direction (forward-angled). 

Soluble molecules are small in size and scatter in a symmetrical way. Whereas, precipitates and complexes are larger in size and produce a forward-angled scatter. Nephelometric detection primarily concentrates on measuring forward scatter. 

Scatter and the Concentration of Particles

The relativity of intensity of scattered light (IS) and the concentration of the precipitate (C) is:

IS = kS * I0 * C

where,

kS is a constant 

I0 is the intensity of light.

There are different variables that influence the physical properties of the suspension of particles. Despite scattering being related to the concentration of solid particles in solution, the intensity of the scattered light depends on the size and shape of the particle. Equally concentrated samples which contain precipitates of differing sizes show different levels of scattering. 

Temperature, pH, and reagent concentration, order of mixing, stirring, the interval between the formation of precipitate and detection affects the size and shape of the precipitate. 

The Wavelength of the Light Source 

Wavelength selection is considered irrelevant because the incident light absorption by the suspended particles is generally not considered, and does not induce fluorescence of the sample. If non-fluorescent samples are used, there is wavelength selection has no need. The choice of the wavelength is to minimize the potential interferences and rather affect the incident light intensity.

Nephelometry Instrumentation: How is it Detected?

• While fluorometers may be used to detect nephelometry, the angular dependence of scattering prompted the creation of specialized instruments. Nephelometers are turbidimeters with detectors positioned at an angle to the incident beam and are the standard instrument for measuring low turbidity values. The centration of solid particles in solution is also dependent on their size and shape. The strength of scattered light is measured by a nephelometer, which is a dedicated standalone instrument. The light that is transmitted is not observed.

• A light source, light-scattering optics, and a detector are the essential components of a nephelometer. A beam of light is produced by the light source and guided through the sample. Light sources include halogen and xenon lamps, as well as lasers. Due to their sensitivity, high intensity, and coherent nature (emitted photons are "in step" with each other), laser nephelometry is usually the most popular option. Since the wavelengths of the incoming and outgoing signals are similar, no optical selection is needed.

• A detector is mounted on the opposite side of the light source, at an angle to the incoming light beam. Depending on its location, it senses differences in forward-angled scatter or side scatter. Detectors can be mounted at angles of 30°, 70°, or 90° depending on the amount of scattering that can be obtained.

• Nephelometry may be used as a kinetic or endpoint calculation. After a reaction enters equilibrium or at a predetermined time point, endpoint measurements calculate the maximum light scattering. Kinetic detection (multiple readings over time) can be used in the precipitation process and usually yields more knowledge about the reaction.

Nephelometry Uses

Immunonephelometry has been used in clinical laboratories to analyze immunoassays since the 1970s. It was first used to detect the formation and precipitation of immune complexes (antigen-antibody), and it is still used today for that purpose. Immunonephelometry is also used in high-volume automated coagulometers to assess serum protein concentrations, including immunoglobulin. Multiple-assay coagulation profiles are possible with these instruments, which measure coagulation factors in blood samples.

Nephelometry is primarily used in pharmaceutical laboratories to determine the solubility of drugs or compounds. It's also a promising method for quantifying microbial growth, and it's widely used to count the cells in microorganism suspensions like yeast (e.g. S. cerevisiae).

Microplate-Based Nephelometry

Since it can be used in high-throughput compound solubility screenings, microplate-based nephelometry is a valuable method for the pharmaceutical industry. It can also be used to study microbial growth and protein binding kinetics, as well as calculate calcification tendency in body fluids, rheumatoid factors in serum, antigen-antibody binding, and several other items.

High-throughput screening is an effective tool for drug development in the pharmaceutical industry. In this step, determining the validity of the pharmacological findings and selecting promising compounds requires assessing solubility. Drug availability, composition, dosing, and absorption are all influenced by solubility.

Drug Solubility Assays

The speed of the assay and the ease of handling are both advantages of this method. Pipetting is all that is needed in microplate-based nephelometric assays; no filtration or phase separation of the solution from the undissolved residue is required. Furthermore, there is no need for a liquid transfer phase because the assay setup and measurement can both be done in the same microplate. Finally, it can be used to calculate both the soluble concentration of a compound and the point at which a solute starts to precipitate.

The observed signal is usually linear for up to three orders of magnitude of particle concentration, with a detection limit of about 20 mmol/L for kinetics.