Nephelometry, Turbidimetry

Question 1

Write short notes on the factors affecting light scattering

Answer 1

1. Particle size (circumference) and molecular weight- when a particle is small, the electrical field of the light wave causes all charges on that particle to oscillate in synchrony. Emitted light is in phase and reinforced. Scattered light is emitted in all directions, symmetrically both forwards and backwards. As a particle increases in size, the electrical field exerts its effects unequally across the charges in the particle. Light emitted is not in phase. Reinforcement of radiation occurs in some directions while destructive interference occurs in others. Scattering patterns will be characteristic of particle shape and size.
   Wavelength
   Distance of observation
   Polarisation of incident light
   Particle concentration
   Molecular weight of particle

Question 2

Difference between Rayleigh and Rayleigh-Debye scattering

Answer 2

Rayleigh scattering occurs when particle size is much smaller than wavelength (<1/10 wavelength).

Rayleigh scattering is symmetric, with forward and back scattering equal, with the just scattering at 90 degrees. Rayleigh-Debye scattering is dissymmetric, with more forward scatter than back scatter.

Question 3

Which plasma proteins exhibit Rayleigh scattering at 400nm (visible light)?

Answer 3

Most immunoglobulins
Beta-lipoprotein
Albumin

Question 4

Which plasma proteins exhibit Rayleigh-Debye scattering at 400nm (visible light)?

Answer 4

IgM
Chylomicrons
Aggregating Ig-Ag complexes

Question 5

Instrumentation needed for nephelometry/turbidimetry?

Answer 5

1. Light source
2. Collimating optics:
   2.1 Excitation optics
   2.2 Excitation filter
3. Sample cell
4. Collection optics:
   4.1 Light scattering optics
   4.2 Detection optical filter
   4.3 Detector

Question 6

Light source commonly used for nephelometry and turbidimetry

Answer 6

1. Quartz-halogen lamps
2. Xenon lamps
3. Lasers

Question 7

Why are lasers useful light sources in nephelometry? Give an example

Answer 7

1. High intensity
2. Coherence
   Eg helium-neon lasers at wavelength 633nm are used in nephelometric immunoassays and particle size and shape determinations

Question 8

What are 2 ways of reducing background light scatter from lipaemia?

Answer 8

1. Dilution.
2. Rate blanking - used when analyte concentrations are low, limiting the number of dilutions that can be performed.

Question 9

Describe what is shown in the diagram

Answer 9

Rayleigh scattering:
Scatter intensity diagram for small particles excited by 1) vertically polarised light 2) horizontally polarised light 3) non-polarised light. Intensity of scattered light varies as a function of the angle of observation.
With small particles (size <1/10th wavelength of incident light), back scatter and forward scatter are equal/symmetric and with non-polarised light, intensity at 90 degrees is less than at 180 degrees.

Question 10

Limitations of nephelometry and turbidimetry

Answer 10

1. Antigen excess
2. Matrix effects

Question 11

Generally speaking which is more sensitive: turbidimetry or nephelometry?

Answer 11

Nephelometry