NEPHELOMETRY AND TURBIDIMETRY.pptx

Turbidimetry and nephelometry

1. NEPHELOMETRY AND TURBIDIMETRY BY E.W.OJONG PhD CHEMICAL PATHOLOGY

2. LESSON OBJECTIVES Explain the principles of nephelometry and turbidimetry Discuss the instrumentation of nephelometry and turbidimetry Describe the applications of nephelometry and turbidimetry

3. INTRODUCTION

4. LIGHT SCATTERING PHENOMENOM

5. INTRODUCTION When electromagnetic radiation (light) strikes on a particle in a solution, some of the light will be absorbed by the particle, some will be transmitted through the solution and some of the light will be scattered or reflected. The amount of light scattered is proportional to the concentration of insoluble particles. Scattered light may be measured by turbidimetry and nephelometry Turbidimetric measurements are made at 180oC from the incident light beam In nephelometry, the intensity of the scattered light is measured, usually but not necessarily at right angles to the incident beam

6. INTRODUCTION

7. INTRODUCTION

8. LAW OF PHOTOMETRY

9. INTRODUCTION

10. INTRODUCTION

11. INTRODUCTION

12. INTRODUCTION Both turbidimetry and nephelometry are based on the property of light scattering by particles dispersed in a solution. Both methods can be used to determine the concentration of a particulate solution However, they differ in the manner of measuring the scattered radiation In turbidimetry, measurement is made in the direction of the incident light In nephelometry, measurements are made in the right angle to the incident light

13. TURBIDIMETRY When a beam of monochromatic light is allowed to pass through a solution, part of the incident radiant energy is dissipated by absorption, reflection, and refraction while the remainder is transmitted. the power of transmitted light is measured in the direction of the beam as a function of the concentration of suspended particles. The transmittance (T = I/Io) of the incident light is measured Measurements are made at 18OoC from the incident light beam

14. TURBIDIMETRY The transmittance T is related to the concentration (C) of suspended material by the equation; S = log Io/I = KbC Where S = turbidance K = turbidity coefficient b = path length of the solution

15. TURBIDIMETRY

16. TURBIDIMETRY

17. TURBIDIMETRY

18. TURBIDIMETRY AND COLORIMETRY Turbidimetry is much similar to colorimetry because both involve the measurement of the intensity of light transmitted through a medium But these differ in the sense that the light intensity is decreased by turbidimetry and by absorption in colorimetry .

19. TURBIDIMETRY AND COLORIMETRY

20. APPLICATIONS OF TURBIDIMETRY For measuring abundant large particles and bacterial suspension Used to measure plasma and urinary proteins.Calibrators are used to create a standard curve

21. NEPHELOMETRY When a beam of monochromatic light is allowed to pass through a solution having suspended particles, the radiant powered of the scattered beam at 45oC, 90oC, 135oC etc to the incident beam is measured as a function of the concentration of suspended particles Measurement of the intensity of the scattered light as a function of the concentration of the dispersed particles form the basis of nephelometric analysis

22. NEPHELOMETRY

23. NEPHELOMETRY

24. NEPHELOMETRY

25. NEPHELOMETRY If the particle size is larger than the wavelength of the light source, then most of the light will be scattered in the forward direction at an angle of less than 90o to the incident beam. This phenomenom is known as Mie scatter Particles that are smaller than the wavelength of the light source will scatter light in many directions and equally in the forward and backward direction. This phenomenon is known as Raleigh Scatter

26. NEPHELOMETRY

27. NEPHELOMETRY

28. NEPHELOMETRY: WORKING

29. NEPHELOMETRY: WORKING

30. NEPHELOMETRY AND FLUORIMETRY Nephelometry is much similar to fluorimetry because both involve the measurement of scattered light But the basic difference is that the scattering is elastic in fluorimetry and inelastic in nephelometry Both incident and scattered light are of the same wavelength in nepehlometry whereas scattered light measured in fluorimetry is of a longer wavelenth than the incident light

31. APPLICATIONS OF NEPHELOMETRY Measuring the amount of antigen-antibody complexes. Antigen-antibody complexes when formed at a high rate, will precipitate out of solution resulting in a turbid or cloudy appearance. Can detect either antigen or antibody. (a) Endpoint tests allow antigen-antibody reactions to go to completion. If complexes get too large, they will fall out of solution, causing a falsely decreased result. (b) Kinetic tests add andtigens and antibody then measure at a specific time. The rate of formation must be known and concentration should be calculated based on standards. Measure small particles of low concentrations in body fluids e.g microalbumin , haptoglobin , ceruloplasmin , immunoglobulins .

32. INSTRUMENTATION OF TURBIDIMETRY AND NEPHELOMETRY Much of the theory and equipment used in colorimetry apply with little modification The basic components of the instruments include Radiation source Sample cell Detector Readout device The instruments for both methods are similar. The only difference is with the detectors. One uses the photovoltaic cell while the other uses phototube

33. INSTRUMENTATION OF TURBIDIMETRY AND NEPHELOMETRY LIGHT SOURCE White light or monochromatic light is more advantageous to minimize absorption and sample heating Monochromatic light also obtains a uniform scatter Short wavelengths are used to increase the efficiency of scattering Mercury arc or laser beam Tungsten lamp is used for determination of concentration

34. INSTRUMENTATION OF TURBIDIMETRY AND NEPHELOMETRY CELLS Cylindrical cells, with flat faces where the entering and exiting beams are to be passed to minimize reflections and multiple scattering from the cell walls In general, cells with a rectangular cross section is preferred where measurements are to be made at 90oC Octagonal faces will allow measurements to be made at 0o, 45o, 90o, 135o to the primary beam Walls through which light beams are not to pass are painted dull black to absorb unwanted radiation and minimize stray radiation Reagents must be free of any particles, cuvettes must be free of any scratches.

35. INSTRUMENTATION OF TURBIDIMETRY AND NEPHELOMETRY

36. INSTRUMENTATION OF TURBIDIMETRY AND NEPHELOMETRY

37. INSTRUMENTATION OF TURBIDIMETRY AND NEPHELOMETRY DETECTORS In nephelometry, a sensitive photomultiplier tube acts as a detector because the intensity of scattered radiation is very small. Usually, the detector is fixed at 90o to the primary beam. In some nephelometers , the detector is mounted on a circular disc which allows measurements at many angles i.e at 0o and from 30o to 135o In turbidimetry, ordinary detectors such as phototubes and photovoltaic cells are used.

38. CHOICE BETWEEN TURBIDIMETRY AND NEPHELOMETRY The choice between the two methods depends upon the fraction of light scattered by the suspension. When scattering is less due to small concentration of dispersed phase, nephelometry is preferred. In this case it is possible to measure accurately the small amount of light scattered by the suspended particles. When the scattering is intense due to a high concentration of suspended particles (dispersed phase), turbidimetry is preferred. In this case it is possible to measure accurately the small amount of transmitted light.

39. CHOICE BETWEEN TURBIDIMETRY AND NEPHELOMETRY

40. COMPARISON OF TURBIDIMETRY AND NEPHELOMETRY

41. COMPARISON OF TURBIDIMETRY AND NEPHELOMETRY NEPHELOMETRY TURBIDIMETRY Specialized instrument Simple spectrophotometer More sensitive Less sensitive Not affected by size and concentration Affected by size and concentration Measures light which is scattered Measures light which passes through Source of radiation: Mercury arc lamp/high pressure xenon lamp Tungsten lamp/Mercury lamp/laser Radiation detection device: Photomultiplier tube Photovoltaic cell Cell/sample holder (glass or plastic): Semi octagonal cell (45o, 90o, 180o Cylindrical cell/rectangular cell with flat faces on both sides

42. COMPARISON OF TURBIDIMETRY AND NEPHELOMETRY

43. PRINCIPLE AND THEORY OF NEPHELOMETRY AND TURBIDIMETRY

44. FACTORS AFFECTING THE SCATTERING OF LIGHT

45. FACTORS AFFECTING THE SCATTERING OF LIGHT CONCENTRATION OF PARTICLES (TURBIDIMETRY) At low concentration of particles for scattering of light, Beer-Lambert’s Law is applicable. S = log(Io/It) S = KtC = - logT Turbidence (S) is directly proportional to concentration (C) Io, intensity of incident light It, intensity of transmitted light T, turbidance C, concentration Kt , Constant which depends on the linearity of light

46. FACTORS AFFECTING THE SCATTERING OF LIGHT CONCENTRATION OF PARTICLES (TURBIDIMETRY) Is = Ks x Io x C Io, intensity of incident light Is, intensity of scattered light Ks, Constant which depends on suspended particle and suspended medium C, concentration

47. FACTORS AFFECTING THE SCATTERING OF LIGHT PARTICLE GEOMETRY Controlling particle size and shape is the most critical factor in turbidimetry and nephelometry The fraction of the light scattered at any angle depends to a large extent upon the size and shape of the solid particles responsible for scattering. Hence it is important to control the particle size and shape. The amount of scattering (S) is proportional to the square of the effective radius of the particle The factors/conditions which influence particle size during precipitation like concentration of reagents, time allowed for particle growth, rate and order of mixing of reagents, time, temperature, presence of non-reactants, pH, and ionic strength also affect turbidimetric and nephelometric assays

48. FACTORS AFFECTING THE SCATTERING OF LIGHT PARTICLE GEOMETRY If the size of the suspended particles is of the same order or smaller than the wavelength of the incident light, scattering will be dominant In nephelometry, scattering pattern of secondary rays in space should be such that it has maximum intensity at 90oC. The scattering efficiency falls if the particle size is too small or too large. For measurements in the uv and visible regions, the optimum size should be 0.1 – 1.0nm

49. FACTORS AFFECTING THE SCATTERING OF LIGHT PARTICLE GEOMETRY In turbidimetry, particles larger than the wavelength of incident light do not pose complications because measurements depend on the total radiation removed from the primary beam irrespective of the mechanism by which it is removed, or the angle through which it undergoes deviation. The only problem is that the absorbance does not vary linearly with concentration (deviation from Beer’s Law) and this leads to inadequate measurements

50. FACTORS AFFECTING THE SCATTERING OF LIGHT PARTICLE GEOMETRY Ideally, the sample solution and the standard solutions should have the same distribution of small, medium and large particles To control particle size and shape, the sample solution and the standard must be prepared under identical conditions since different particle sizes may produce erratic results.

51. FACTORS AFFECTING THE SCATTERING OF LIGHT WAVELENGTH The intensity of scattered radiation depends on the wavelength of the incident light. The wavelength of incident light plays an important role Shorter wavelengths are scattered to a greater extent than longer wavelengths Turbidimetry: Radiation or selected wavelength of should not strongly be absorbed by the suspension medium/ coloured solution i.e. colour of the filter used for selection of wavelength should be the same as coloured solution. Nephelometry: Absorption is much less a problem in nephelometry so white light is generally used for convenience.

52. WAVELENGTH

53. FACTORS AFFECTING THE SCATTERING OF LIGHT MOLECULAR WEIGHT OF PARTICLES A direct relationship exists

54. FACTORS AFFECTING THE SCATTERING OF LIGHT DISTANCE OF OBSERVATION Light scattering decreases by the distance (r)2 from the light scattering particles to the detector S proportional to 1/r2

55. FACTORS AFFECTING THE SCATTERING OF LIGHT REFRACTIVE INDEX In both techniques, satisfactory results are obtained when the refractive index difference between the refractive indices of the particle and solvent is appreciable (can be achieved by a change in solvent)

56. CHOICE BETWEEN TURBIDIMETRY AND NEPHELOMETRY REFRACTIVE INDEX In both techniques, satisfactory results are obtained when the refractive index difference between the refractive indices of the particle and solvent is appreciable (can be achieved by a change in solvent)

57. APPLICATIONS OF TURBIDIMETRY AND NEPHELOMETRY Analysis of water: Clarity, concentration of ions, purity, impurities Determination of CO2: This method involves bubbling of gas through an alkaline solution of barium salt and then analysing the BaCO3 suspension by turbidimetry or nephelometry

58. APPLICATIONS OF TURBIDIMETRY AND NEPHELOMETRY Determination of inorganic substances: Sulphate-Barium chloride: The sulphate determination can be used to estimate total sulphur in coke, coal, oils, plastics, rubbers etc. To determine sulphur, it is first converted to sulphate which is then shaken with sodium chloride solution and excess of solid barium chloride to get a suspension of barium sulphate. Finally this suspension is subjected to turbidimetry or nephelometry and the concentration of the suspension is gotten from a calibration curve. Ammonia- Nesslers reagent Phosphorus –Strychine Molybedate

59. APPLICATIONS OF TURBIDIMETRY AND NEPHELOMETRY Air and water pollution: Turbidimetry and nephelometry are used for continuous monitoring of air and water pollution. Water is monitored for turbidity and air is monitored for dust and smoke Biochemical analysis: Turbidimetry is used to measure the amount of growth of a test bacteria in a liquid nutrient medium. It is also used to find out the amount of amino acids, vitamins and antibiotics. Nephelometry is used to determine protein, yeast, glycogen, alpha and beta globulin in blood Quantitative analysis (ppm level)

60. APPLICATIONS OF TURBIDIMETRY AND NEPHELOMETRY Miscellaneous: Water treatment plant, sewage work, refineries, paper industry Atmospheric pollution: Smokes and fog Determination of molecular weight of high polymers Organic Analysis: In food and beverages, turbidimeters are used for analyzing turbidity in sugar products and clarity of citrus juices Used in the determination of benzene percentage in alcohol

61. APPLICATIONS OF TURBIDIMETRY AND NEPHELOMETRY Turbidimetric Titration: These are titrations ( of reactions in which insoluble products are formed) involving a turbidimeter during which turbidance values are recorded after each addition of titrant. When all analytes get precipitated, turbidance becomes constant. Turbidance Vs volume of titrant added is plotted Abrupt change in slope indicated end point of titration.

62. APPLICATIONS OF TURBIDIMETRY AND NEPHELOMETRY End point is determined from the plot of turbidance against volume of titrant added. E.g. Na2SO4 Vs BaCl2, NaCl Vs AgNO3, KF Vs CaCl2

63. APPLICATIONS OF TURBIDIMETRY AND NEPHELOMETRY Phase Titration: A mixture of two immiscible liquids is titrated against a third liquid which is miscible with one of the two liquids but not with the other e.g. water is added to ethanol-benzene mixture. The addition of a sufficient amount of the third liquid produces a turbidity due to the separation of a separate phase e.g. water produces a slight turbidity because it is immiscible in benzene. The appearance of turbidity marks the end-point of the phase titration

64. APPLICATIONS OF TURBIDIMETRY AND NEPHELOMETRY Phase Titration: Another example: Water and Pyridine Vs Chloroform. Chloroform is added as a titrant Causing separation of phase with turbidity

65. APPLICATIONS OF TURBIDIMETRY AND NEPHELOMETRY Phase Titration: Another example: Water and Pyridine Vs Chloroform. Chloroform is added as a titrant Causing separation of phase with turbidity

66. APPLICATIONS OF TURBIDIMETRY AND NEPHELOMETRY Determination of Molecular Weight of Polymers(Macromolecules)

67. APPLICATIONS OF TURBIDIMETRY AND NEPHELOMETRY Determination of Molecular Weight of Polymers(Macromolecules)