Nephelometry and Turbidimetry
Nephelometry and Turbidimetry
Objectives
At the end of the session the student will be able to
• Identify Nephelometry and Turbidimetry as important methods of determination of solid pharmaceutical substances
• Distinguish and compare Nephelometry and turbidimetry
• Compare and differentiate between Nephelometry and Fluorimetry
• Interpret the data obtained in Nephelometric determinations
Nephelometry:
• Nephelometry is concerned with measurement of scattered light from a cuvette containing suspended particles.
• 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 scattered light.
• Detectors may be placed at 90o, 70o or 37o depending on the angle at which most scattered light are found.
Turbidimetry
• Turbidimetry (the name being derived from turbidity) is the process of measuring the loss of intensity of transmitted light due to the scattering effect of particles suspended in it.
CHOICE OF THE METHOD:
Depends upon the amount of light scattered by suspended particles present in solution.
• TURBIDIMETRY: high conc.Suspensions
• NEPHELOMETRY: low conc. Suspensions more accurate results
Difference between Nephlometry and Turbidimetry
| Nephlometry | Turbidimetry | |
| Definition | The measurement of the intensity of scattered light at right angles to the direction of the incident light as a function of the concentration of the dispersed phase, It is most sensitive for very dilute suspensions (100 mg/L). |
Light passing through a medium with dispersed particles, so the intensity of light transmitted is measured. |
| Instrument used | Nephelometer | spectrophotometer |
| Type of light measured | Scattered light | Transmitted light |
| Arrangement of photometer | Measure the light scattered at right angle to the direction of the propagation of light from the source.
It could be movable detectors which allow operator to vary the angle of detection |
Made in the same direction as the propagation of the light from the source.
|
| Clinical uses | Ag-Ab reaction, immuno complex reaction, ppts, lipoproteins | Ag-Ab reaction, immuno complex reaction, ppts, liver dysfunction, protein in urine or CSF |
INSTRUMENTATION:
Turbidimeters
1- Light source:
• Tungsten: Its relatively low intensity makes it less useful for samples with low light scattering.
• Alternatives are: quartz halogen lamp, xenon lamp and laser which have higher intensities than tungsten lamp.
2- Lens assembly:
• Light enters the sample holder through lens assembly.
3- Monochromator:
• there is provision for the insertion of filter between the sample and source of light
4- Detector (photo –cell):
• It is shielded to minimize interference from stray light.
5-Read out device:
• Light intensity is converted to an electrical signal by the detector.
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.
• The standard solution which is used for the standard curve must have similar size in suspension as unknown.
• 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) provide higher degree of accuracy, sensitivity, precision and less time than end-point reactions (measuring the reaction at the start and finish of the reaction)
Selection of a wavelength
• If both solution and suspended particles are colorless, then use any wave length in the visible range
• If the solution is coloured but the particles are not coloured, then use a wave length that gives minimum absorption for the solution
• If the particles are coloured and the solution is colorless then use a wavelength that gives maximum absorption with the particles
• 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.
Clinical Applications of turbidimetry
• Determination of the concentration of total protein in biological fluids such as urine and CSF which contain small quantities of protein (mg/L quantities) using trichloroacetic acid
• Determination of amylase activity using starch as substrate. The decrease in turbidity is directly proportional to amylase activity.
• Determination of lipase activity using triglycerides as substrate. The decrease in turbidity is directly proportional to lipase activity.
Clinical applications of nephelometry.
• Widely used to determine concentrations of unknowns where there is antigen-antibody reactions such as
• Determination of immuno globulins (total, IgG, IgE, IgM, IgA) in serum and other biological fluids
• Determination of the concentrations of individual serum proteins; hemoglobin, heptoglobin, transferring c-reactive protein, !1-antitrypsin, albumin (using antibodies specific for each protein)
• Determination of the size and number of particles (laser-nephelometer}
Summary
• Nephelometry and Turbidimetry are based on scattering of light
• They are used determine the solid dispersions
• Concentration ,shape and size of the particles determine the choice of the method
• In Nephelometry ,the intensity of light scattered is measured
• In Turbidimetry the transmitted light after scattering is measured.
• The stability of many dispersions is uncertain and hence a reference standard dispersion of formazine is used.
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