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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