The analytical chemistry technique of nephelometry (from the Greek nephelo: cloud) is used to measure the amount of turbidity or cloudiness in a solution caused by the presence of suspended insoluble particles.
Light is emitted, absorbed (blocked), and dispersed when guided through a turbid solution containing suspended solid particles (reflected off the particles). The size, shape, and concentration of insoluble particles in solution, as well as the incident wavelength of light, all influence the amount of scattered light.
Light scattering theories and principles were first developed at the end of the 19th century and the beginning of the 20th century, primarily by Rayleigh, Mie, and Debye.
Immunoassays for the identification and quantification of serum proteins in the blood, such as immunoglobulins and macromolecules, were the first to use nephelometry in clinical chemistry. These programs are still being used today. They are primarily used to analyze material precipitation, such as drug solubility and protein aggregation, or bacterial growth, using microplate readers.
In this article, we will study Nephelometry, Principles of Nephelometry, rate nephelometry, and nephelometry method in detail.
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Principles of Nephelometry
In liquids, light scattering follows the rules of elastic scattering in particle physics, in which neither particle absorbs any energy during the "collision." The energy of a photon remains unchanged before and after the scattering event. The elastic scattering of large and small particles differs. Light scatters mostly in the forward direction for large particles (forward-angled). Scattering is symmetrically distributed when the particle size is less than 5% of the wavelength of light with which they are struck.
Soluble molecules are usually small in size (in comparison to incident light wavelengths) and scatter almost symmetrically (fig.4A). Precipitates and complexes in solution, on the other hand, are usually larger (closer to the wavelengths of incident light), resulting in mostly forward-angled scatter. Forward scatter is normally measured in nephelometric detection.
To determine the rate nephelometry:
1.Scatter and Concentration of Particles
The following equation connects the strength of scattered light (IS) and the concentration of precipitate (C):
IS = ks * I0 * CIs = ks* I0 * CIS = ks * I
where ks is a constant derived from the system's calibration and I0 is the light source's intensity.
Different variables affect the physical properties of a particle suspension. Since the strength of scattered light is dependent on the coequally concentrated samples of different-sized precipitates, it will have different scattering levels.
Temperature, pH, and reagent concentration, as well as the order of mixing, stirring, and the time between precipitation and detection, both influence the size and shape of the precipitate. All of these various variables must be taken into account in order to achieve repeatable conditions and outcomes through samples and assays.
2. The Wavelength of the Light Source
Wavelength selection is normally unimportant since incident light absorption by particles in suspension is seldom taken into account, as long as the sample does not fluoresce. As a result, if non-fluorescent samples are used, there is no need to specify a wavelength. The wavelength is chosen mainly to reduce possible interferences and has no impact on the strength of the incident light or the scattering itself.
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.
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).
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.
Did You Know?
Difference between nephelometry and turbidimetry:
Nephelometry or turbidimetry used to detect turbidity or cloudiness. Both methods are non-destructive and rely on light scattering induced by a solid particle suspension. While these two words are sometimes used interchangeably, they are not interchangeable.
Turbidimetry is the measurement of the intensity loss of light transmitted through a sample as a result of insoluble particle scattering. Turbidimetry, like absorbance, measures the strength of light transmitted through a sample, specifically its attenuation.
In turbidimetry, a sample containing insoluble particles in solution is illuminated with light of a specific wavelength. The light that passes through the sample is collected by a detector that is positioned in line with the light source. As compared to a reference, the reduction in light transmission is estimated, and the absorbed light is measured in Optical Density (OD) units.
By precisely quantifying the strength of the light scattered by insoluble particles in the sample, nephelometry, on the other hand, calculates the cloudiness of a solution. To prevent interference from possible transmitted light, dispersed light is usually measured at an angle relative to the incident light source. It is analogous to the calculation of fluorescence intensity in this regard.