beers and lamberts law, colorimeter, nephlometry and turbidimetry

1. LAMBERT BEER᾿S LAW,
COLORIMETRY,
NEPHLOMETRY AND
TURBIDIMETRY
PRESENTED BY:
AKANSHA SINGH

2. Beer’s law Lambert’s law
The measurement of color
intensity of a colored solution by
photometry is governed by two
laws

3. BEER’S LAW
•The concentration of a
substance is directly
proportional to the
amount of light
absorbed or inversely
proportional to the
logarithm of the
transmitted light

4. Beer’s law
Beer’s law

5. LAMBERT’S LAW
•When a ray of
monochromatic light
passes through an
absorbing medium its
intensity decreases
exponentially as the
length of the light path
through light absorbing
material increases

7. Colorimeter is a
instrument used for the
measurement of
colored substance in
solution.
The instrument is
operative in the visible
range of the
electromagnetic
spectrum

9.  Light Source – Tungsten Lamp emits a light in
the visible range (400-760 nm).
 Slit - it allows only a beam of light to pass
through. it prevents any unwanted light.
 Condensing Lenses - Light after passing
through slit falls on condenser lens which
gives a parallel beam of light.

10.  Filter -
Wavelength Colour absorbed Colour transmitted
400-450 Violet Green-yellow
450-500 Blue Yellow
500-570 Green Red
570-590 Yellow Blue
590-620 Orange Green-blue
620-760 Red Green

11.  Cuvette – the monochromatic light from the filter
passes through the colored solution placed in
cuvette. It is made up of a special
glass/plastic/silica/quartz material.
 Photocell (Detector) – are photosensitive elements
which convert light energy into electrical energy.
 Read Out Device – the electrical signal generated in
Photocell is measured by galvanometer, which
displays percent transmission and optical density.

12.  Used in hospitals and laboratories for estimation of
biochemical samples like Plasma, Serum, CSF,
Urine.
 Also used to quantitative estimation of serum
components such as: glucose, proteins and etc.
 To determine the test for water quality, by
screening for chemicals such as chloride, fluoride,
cyanide, iron, zinc etc.

13.  It is inexpensive.
 Very well applicable for quantitative
analysis of colored compounds.
 Easily transportable.

14.  one wavelength at a time and have a fixed
number of wavelengths that can be used.
 We cannot set specific wavelength.
 Similar colors from interfering substances
can produce errors in results.

16. It is involved with measuring the amount of
transmitted light by particles in suspension to
determine the concentration of the
substance.
Amount of absorbed light, concentration is
dependent on:
• Number of particles
• Size of particles

17. • Determination of total protein in biological
fluids such as urine & CSF.
• Determination of amylase activities 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.

18. Nephlometry is concerned with measurement
of scattered light from the cuvette containing
suspended particles in a solution.
The components of nephelometry are the same
as a light spectrophotometer except that the
detectors placed at a specific angle from the
incident light.

19. Widely used to determine concentrations of
unknowns where there is antigen-antibody reactions
such as:-
• Determine of immunoglobulin's (total IgG, IgE,
IgM, IgA) in serum and other biological fluids.
• Determine of the concentrations of individual
serum proteins, haemoglobin, C-reactive protein
and albumin.
• Determine size and number of particles.

20. • Very rapid procedures
• Simplicity in measurement
• They are accurate

21. • They require high power supply
• Turbidimetry is used for high
concentration
• Nephelometery is used for low
concentration

22. TURBIDIMETRY NEPHELOMETRY
Measures reduction in
intensity of transmitted light
at 180º due to the formation
of immune complex.
Can be performed on most
spectrophotometers.
Sensitivity competitive with
nephelometric for small
immune complexes such as
serum protein.
More precise for measuring
large immune complexes.
 Measures scattering of light
at an angle (usually 90º)
away from the incident light
due to the formation of
immune complexes.
 Requires dedicated
nephelometers.
 Sensitive for measuring
small immune complexes
such as serum proteins.
 More precise for measuring
small immune complexes.

23.  Blanking and reading can be
performed in the same
measuring cuvette.
 Provides better precision due
to slower reaction kinetics as
blanking of immunochemical
reaction can be monitored in
a single cuvette.
 In a solution containing
particles in suspension the
incident light beam is
diminished due to scattering.
Blanking has to be performed
in separate measuring
cuvette.
Because of the fast reaction
kinetics it is difficult to
obtain a sample and a
reagent sample and reagent
blank in case of
nephelometry.
It is the measurement of
scattered light.