Photometry ii anu
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- 1. PHOTOMETRY II Presenter: Dr.Anurag Yadav Moderator: Mr.Arun Kumar
- 2. CONTENT : Nephelometry. Turbidimetry. Reflectance photometry.
- 3. 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 .
- 4. 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
- 5. LIGHT SCATTERING 3 types 1. Wavelength of light > particle size - RAYLEIGH light symmetrically scattered around the particle RAYLEIGH eg Ig, Albumin
- 6. 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 .
- 7. LIGHT SCATTERING 3. wavelength of light = particle size RAYLEIGH DEBYE SCATTER light scattered is more in forward than in backward direction RAYLEIGH DEBYE SCATTER Application Light scatter analysis is used for Ag- Ab reactions with size 250 1500nm its Rayleigh Debye scatter and blank scatter by Rayleigh. RAYLAIGH DEBYE
- 8. 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.
- 9. 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)
- 10. INSTRUMENTATION OF NEPHELOMETER
- 11. INSTRUMENTATION OF TURBIDIMETER:
- 12. 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.
- 13. 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.
- 14. 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
- 15. DIFFERENCE BETWEEN NEPHELOMETRY AND TURBIDIMETRY 1. Mercury arc lamp 2. Rectangular cuvette used 3. Scattered light is measured 4. Measured at 90 deg 5. PMT is detector 1. Tu / Du lamp is used 2. Semi octagonal cuvette 3. Light transmitted is measured 4. Measured in straight line 5. Photocell is detector Nephalometry Turbidimetry
- 16. 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.
- 17. 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.
- 18. 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.
- 19. USES: Used as quantitative measurement of surface reactions such as dipstick or Dry film chemistry system.
- 20. REFERENCE Clinical chemistry: Kaplan Clinical chemistry: TIETZ
- 21. 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%
- 22. IMMUNONEPHELOMETRY Monoclonal Ab reagents Polyclonal Ab need monitoring of titre specificity and affinity . This is overcome by use of monoclonal Ab
- 23. 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
- 24. 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 .
- 25. LIGHT SCATTER INHIBITION IMMUNOASSAY Sample req and prep serum Reagents Instrumentation Common pitfalls Reaction should be in antigen excess.
- 26. 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 .