NEPHELOMETRY AND

TURBIDIMETRY

BASIC CONCEPTS, INSTRUMENTATION AND

APPLICATIONBy

Dr. Basil, B – MBBS (Nigeria),Department of Chemical Pathology/Metabolic Medicine,

Benue State University Teaching Hospital, Makurdi.September 2014.

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.

BASIC CONCEPTS:• The light incident on the sample is scattered in all

direction and the scattered light is at thesamewavelength 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.

• 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)

NEPHELOMETRY

TURBIDIMETRY

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

• 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.

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

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 nephelometricintensity

– 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

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