Hotometry: Presenter: DR - Anurag Yadav

PHOTOMETRY II
Presenter: Dr.Anurag Yadav
Moderator: Mr.Arun Kumar

CONTENT :
Nephelometry.
Turbidimetry.
Reflectance photometry.
NEPHELOMETRY AND TURBIDIMETRY
These are the analytical techniques used to measure scattered

light.
Principle of nephelometry intensity of light scattered by a

suspension is measured at 90 degrees angle.
Intensity of scattered light concentration of suspension
Principle of turbidimetry- measurement of decrease in light

transmitted through a turbid solution is measured .
FACTORS INFLUENCING LIGHT SCATTER
1. Particle size
2. Concentration of particles
3. Molecular weight of particles
4. Wavelength dependence
5. Effect of polarization of incident light
6. Distance of observation
LIGHT SCATTERING
3 types
1. Wavelength of light > particle size - RAYLEIGH
light symmetrically scattered around the particle

RAYLEIGH
eg Ig, Albumin
LIGHT SCATTERING
2. Wavelength of light < particle size - MIE THEORY
light appears scattered forward due to destruction out of

phase background scatter- MIE THEORY
Particle size 7000- 40,000nm like in RBC and bacteria .

LIGHT SCATTERING
3. wavelength of light = particle size RAYLEIGH DEBYE
SCATTER
light scattered is more in forward than in backward direction
RAYLEIGH DEBYE SCATTER
RAYLAIGH DEBYE
Application
Light scatter analysis is used for Ag- Ab reactions with size 250
1500nm its Rayleigh Debye scatter and blank scatter by Rayleigh.
Wavelength dependence of light scattering:
Intensity of light scattered is inversely proportional to the
wavelength of incident light.
Scattered light intensity is inversely related to distance from

the particle to detector.
Concentration and molecular weight:

From equation, it direct relationship of light scattering to the
conc & molecular weight of particle.
INSTRUMENTATION OF
NEPHELOMETER
1. Light source- quartz halogen lamp,mercury arc lamps, xenon
lamps,lasers.
Lasers:
stable, collimated intense light beams,
Reduces stray light, background scatter
2. Collimating optics
3. Sample cell
4. Collection optics light scattering optics
- detector filter
- detector(PMD)
INSTRUMENTATION OF
NEPHELOMETER
INSTRUMENTATION OF TURBIDIMETER:
LIMITATIONS
Antigen excess
Ag -Ab reactions are complex and appear to result in a

mixture of aggregate sizes .
Turbidity adding Ag to Ab & then marking the

beginning of antigen excess.
LIMITATIONS
Matrix effects
Particles, solvent and serum macromolecules scatter light.
Lipoprotein and chylomicrons in lipemic samples
background interference
This is avoided by rate measurements with elimination of
initial sample blank
Large particles: suspended dust background interference
Filtering all buffers, diluted antisera before analysis.
APPLICATIONS
Quantify AA , proteins , vitamins , glycogen , and antibiotics
in blood.
Quantification of urine, csf protein( conc is less) by

immunonephalometry
Quantification of urine ALB, ASO, CRP, U.MAU

Immunoturbidimetry
DIFFERENCE BETWEEN
NEPHELOMETRY AND TURBIDIMETRY
Nephalometry Turbidimetry
1. Mercury arc lamp 1. Tu / Du lamp is used

2. Rectangular cuvette used 2. Semi octagonal cuvette
3. Scattered light is measured 3. Light transmitted is
4. Measured at 90 deg measured
5. PMT is detector 4. Measured in straight line
5. Photocell is detector
REFLECTANCE SPECTROPHOTOMETRY
Beam of light is directed at a flat reaction surface & the
reflected light is quantified.
Reaction mixture in a carrier is illuminated with diffuse

light, & the intensity of the reflected light from the
chromogen is compared with the intensity of the light
reflected from a reference surface.
The reflected light intensity is non linear in relation to
conc of analyte.
DR = log ( Ro/Rtest)
Kubelka-Munk or Clapper-Williams transformation

equation used to convert the data into linear format.
INSTRUMENTATION :
Components are same as
Absorbance photometry.
except that the
geometry of the system is
modified so that the light
source & the detector are
on one side of the sample.
USES:
Used as quantitative measurement of surface
reactions such as dipstick or Dry film chemistry
system.
REFERENCE
Clinical chemistry: Kaplan
Clinical chemistry: TIETZ
IMMUNONEPHELOMETRY
Principle
o Ag +Ab form small aggregates that scatter light turbid
appearance
o These agg to form large matrix as seen in immunoppt assays
like double diff or radial immunodiff .
o Light scatter intensity amt of ppt in Ab excess
o Agg from primary reaction seconds to minutes
o Secondary reaction- takes hours
o Light scatter assay measure early 2nd order reaction bet Ag

and Ab
Agg formation enhanced by addition of solu polyethylene glycol
of conc 2% to 4%
IMMUNONEPHELOMETRY
Monoclonal Ab reagents
Polyclonal Ab need monitoring of titre specificity
and affinity .
This is overcome by use of monoclonal Ab
IMMUNONEPHELOMETRY
Sample req and preparation serum urine CSF
Reagents
Instrumentation
Common pitfalls
Ab excess
high background scatter
interference by coloured solu
Mixing insufficient
Limitations
Diff to determine if ppt is in Ag or Ab excess
LIGHT SCATTER INHIBITION
IMMUNOASSAY
NINIA
1ST described for progesterone by CAMBIASO ET AL 1974
Principle ppt from antihapten is inhibited by adding free
hapten
Used for rapid analysis of drugs in mg/l like phenytoin ,
phenobarbital , theophylline .
LIGHT SCATTER INHIBITION
IMMUNOASSAY
Sample req and prep serum
Reagents
Instrumentation
Common pitfalls
Reaction should be in antigen excess.

ADDITIONAL ASSAY
MODIFICATION
1. Particle enhanced light scatter
Type of agglutination procedure
Ag or Ab coupled with inert carrier particles like
polystyrene latex beads
Fast signal transmission and economy of reagents
Eg latex fixation test for detection of RF
2. Monoclonal Ab reagents
Polyclonal Ab need monitoring of titre specificity and
affinity .
This is overcome by use of monoclonal Ab .

Hotometry: Presenter: DR - Anurag Yadav

Uploaded by

Copyright:

Available Formats

Hotometry: Presenter: DR - Anurag Yadav

Uploaded by

Document Information

Original Description:

Original Title

Copyright

Available Formats

Share this document

Share or Embed Document

Sharing Options

Did you find this document useful?

Is this content inappropriate?

Copyright:

Available Formats

Hotometry: Presenter: DR - Anurag Yadav

Uploaded by

Copyright:

Available Formats

PHOTOMETRY II

Presenter: Dr.Anurag Yadav

Moderator: Mr.Arun Kumar

These are the analytical techniques used to measure scattered

Principle of nephelometry intensity of light scattered by a

Intensity of scattered light concentration of suspension

Principle of turbidimetry- measurement of decrease in light

3. Molecular weight of particles

5. Effect of polarization of incident light

1. Wavelength of light > particle size - RAYLEIGH

light symmetrically scattered around the particle

light appears scattered forward due to destruction out of

Particle size 7000- 40,000nm like in RBC and bacteria .

Scattered light intensity is inversely related to distance from

Concentration and molecular weight:

Ag -Ab reactions are complex and appear to result in a

Turbidity adding Ag to Ab & then marking the

Quantification of urine, csf protein( conc is less) by

Quantification of urine ALB, ASO, CRP, U.MAU

1. Mercury arc lamp 1. Tu / Du lamp is used

Reaction mixture in a carrier is illuminated with diffuse

Kubelka-Munk or Clapper-Williams transformation

o Agg from primary reaction seconds to minutes

o Secondary reaction- takes hours

o Light scatter assay measure early 2nd order reaction bet Ag

Reaction should be in antigen excess.

You might also like