clinical chemistry

1. NEPHELOMETRY
AND
TURBIDIMETRY
BASIC CONCEPTS,
INSTRUMENTATION AND
APPLICATION
By
Dr. Basil, B – MBBS (Nigeria),
Department of Chemical Pathology/Metabolic Medicine,
Benue State University Teaching Hospital, Makurdi.
September 2014.

2. INTRODUCTION:
• When electromagnetic radiation (light) strikes a
particle in solution, some of the light will be
absorbed by the particle, some will be transmitted
through the solution and some of the light will be
scattered or reflected.
• Nephelometry and Turbidimetry are analytical
techniques used to measure scattered light
• The amount of light scattered is proportional to
the concentration of insoluble particle.
• Tyndall Effect: Scattering of light- by particles in a
colloid or suspension.

4. BASIC CONCEPTS:
• The light incident on the sample is scattered in all
direction and the scattered light is at thesame
wavelength as the incident light
• In turbidimetry, the intensity of light transmitted
through the medium, the unscattered light, is
measured at 180o from the incident light beam.
• In Nephelometry, the intensity of the scattered light is
measured, usually, but not necessarily, at right angles
to the incident light beam.

5. • The two techniques differs only in the manner of
measuring the scattered radiation
• Turbidity can be measured on most routine analysers
by a spectrophotometer (absorbed light)
– Reduced sensitivity and precision.
– Extent of light scattering increases as wavelength increases
• The intensity of scattered light is normally measured
by Nephelometer. Fluorometers are often used to
perform Nephelometric measurements
• The choice of method used is dependent upon the
amount of light scattered by suspended particles
present in solution.
– highly concentrated suspensions – turbidimetry
– Low concentration – nephelometry (more accurate results)

6. NEPHELOMETRY
TURBIDIMETRY

7. TYNDALL EFFECT:
• Scattering of light- by particles in a colloid or suspension.
• Light scattering is the physical phenomenon resulting from
the interaction of light with a particles in solution.
• the longer-wavelength light is more transmitted while
the shorter-wavelength light is more reflected via
scattering.
Factors that influence Light Scattering:
• Particle size:
• Wavelength: the intensity of light scattering is inversely proportional
to the wavelenght of the incident light.
• Distance of observation: scattered light intensity is inversely
proportional to the distant from the light scattering particles to the
detector

8. • Concentration of particles: is directly proportional to light
scattering intensity
• Molecular weight of particles: directly proportional to light
scattering intensity
• Polarization of incident light:
– The total light scattered by small particles is less when excited by
polarized light than by nonpolarized light
– Light scattering intensity from small particles excited by
nonpolarized light shows symmetric angular dependence of light
scattering about the 90 degrees axis
– For larger particles, it is dissymmetrical and the dissymmetry
increases even further as the particle size increases
– The dissymmetry and the change in angular dependence of light
scattering with change in the size of the particle is very useful for
characterization and differentiation of various classes of
macromolecules and cells.

10. INSTRUMENTATION:
• The principal concern of light scatter instrumentation
are:
– Excitation intensity
– Wavelength
– Distance of the detector from the sample cuvet
– Minimization of external stray light
• The basic components of the Nephelometer include:
– Light source
– Collimating optics: including Light scattering optics,
detector optical filter and a detector
• Operationally, the optical components used in
turbidimeters and nephelometers are similar to those
used in fluorometers and photometers

12. LIMITATIONS OF LIGHT SCATTERING MEASUREMENTS:
• Antigen Excess:
– As turbidity increases during addition of antigen to
antibodies, the signal increases to a maximum value and
then decreases
– The point at which the decrease begins marks the
beginning of the phase of antigen excess
• Matrix Effects:
– Particles, solvents and all serum macromolecules scatter
light.
– Lipoproteins and chylomicrons in lipemic serum provide
the highest background turbidity and nephelometric
intensity
– To minimize this, rate measurements are employed where
the initial sample blank is eliminated
– Large particles, like dust, that cause background scatter can
be filtered before analysis commenced

13. APPLICATION:
• Analysis of water: clarity, conc. of ions
• Determination of CO2
• Determination of inorganic substances:
– Sulphate – barium chloride
– Ammonia – Nesslers reagent
– Phosphorus – Strychine molybedate
• Biochemical Analysis
• Quantitative Analysis – (ppm level)
• Miscellaneous: water treatment plants, sewage work,
refineries, paper industry
• Atmospheric pollution: Smokes & fogs
• Determination of molecular Weight of high polymers
• Phase titration