Biochemical instrumental analysis - 5
CLS 332
Dr. Maha Al-Sedik
2015
What happens to the laser light as it strikes
the particles.
Light
scattering
Light scattering:
 It occurs when a particle deflects incident laser light.
 Factors that affect light scattering are the cell size, nucleus, and
any granular material inside the cell. Cell shape and surface
topography also contribute to the total light scatter.
Types of light scattering:
 Forward-scattered light (FSC).
 Side-scattered light (SSC).
Forward-scattered light (FSC):
 FSC is proportional to cell surface area or size.
 FSC is detected at the same axis of the incident laser beam.
 FSC provides a suitable method of detecting particles greater than
a given size independent of their fluorescence.
Side scattered light (SSC):
 SSC is proportional to cell granularity or internal complexity.
 SSC is collected at any other axis other than the light extension.
When particles are suspended in a solution in a cuvette,
they make the solution unclear (turbid).
Incident light entering the cuvette will be subjected to three
reactions;
 1- some of the light will be absorbed by the particles.
 2- some will be transmitted through the cuvette.
 3- some will be scattered in various directions.
Incident light entering the
cuvette
Absorbed (blocked) by the
particles
Transmitted through the
cuvette
Scattered in various
directions
What is the difference between clear and turbid
solution?
Turbidity:
The cloudiness of a fluid caused by large numbers of
individual particles that are generally invisible to the naked eye,
similar to smoke in air.
Nephelometry and turbidemetry
Turbidimetry
Principle of turbidemetry:
 Light is scattered by particles in the solution.
 Turbidemetry measures the intensity of transmitted light. the
transmitted light intensity is inversely proportional to the particle
concentration.
 Factors affecting transmitted light are:
a) number of particles.
b) size of particles.
Clinical Applications:
 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.
http://www.youtube.com/watch?v=Tyh2XLuUHlU
http://www.youtube.com/watch?v=YI7wZOJfPlw
Nephelometry
Principle
 Nephelometry is concerned with measurement of scattered light
from a cuvette containing suspended particles in a solution.
 The amount of scattered light depends on the size and number
of particles in suspension.
COMPONENTS:
The components of a nephelometer are the same as a light
spectrophotometer except that the detector is placed not as the
same angle from the incident light.
Components of nephlometer:
 Light source
 Monochromator
 Cuvet
 Photo detector .... Not at the same extension of the light source.
 Signal processor
 Record
Location of the detectors:
Clinical Applications:
 Determination of immunoglobulins (total, IgG, IgE, IgM, IgA) in
serum and other biological fluids.
 Determination of the concentrations of individual serum proteins
eg. albumin (using antibodies specific for each protein) in
microalbuminurea.
Do not forget
Since the amount of scattered light
is far greater than the transmitted
light in a turbid
suspension, nephelometry offers
higher sensitivity than turbidimetry.