Nephelometry and Turbidimetry.pptx

this is a slide of Nephelometry and Turbidimetry One of Spectroscopic technique

1. Nephelometry and Turbidimetry Theory, Principle, Instrumentation & Applications Sewagegnehu Getachew January 19, 2023

2. At the end of this presentation you will except to:- January 19, 2023 Differentiate between turbidimetry and nephelometry Discuss the principles of turbidimetry and nephelometry Explain the factors that affect the measurement Describe the pharmaceutical application of turbidimetry and nephelometry 2

3. Introductory Why is the sky blue during the day and R ed color of the sun at the sun set? January 19, 2023 3

4. Introductory This is due to scattering of light by small particles of dust, molecules of water, and other gases in the atmosphere. The sky is blue because violet and blue light are scattered to a greater extent than other longer wavelengths of light. For the same reason, the sun appears to be red when observed at sunset because red light is less efficiently scattered and, therefore, transmitted to a greater extent than other wavelengths of light January 19, 2023 4

5. Turbidimetry and Nephelometry are techniques in which an incident source of radiation is elastically scattered by a suspension of colloidal particles. If incoming radiant energy gets impinged, upon particles suspended in a medium which is having a RI different from that of the suspended particles, the light which strikes the particles is transmitted at angles other than 1 80° from the incident light. The light is said to be scattered . Nephelometry involves the direct measurement of the scattered light, whereas t urbidimetry involves the measurement of the decrease in the intensity of t ransmitted light in a suspension (i.e. that which is not scattered) January 19, 2023 5

6. Principle of turbidimetric analysis When light is allowed to pass through a suspension, the part of the incident radiant energy is dissipated by absorption, reflection and refraction while the reminder is transmitted. Measurement of the intensity of the transmitted light as function of the concentration of the suspended particles forms basis of turbidimetric analysis. January 19, 2023 6

7. Turbidimetry and Colorimetry Turbidimetry is much similar to colorimetry because both involve measurement of the intensity of light transmitted through a medium. But differ in that the light intensity is ↓ ed by scattering in tur bidimetry and by absorption in colorimetry. Due to this reason both techniques may use similar or even identical apparatus. 7 Colorimetry is measurement of the wavelength and the intensity of EMR in the visible region of the spectrum

8. Principle of nephelometric analysis At this time the light is also allowed to pass directly through the sample solution having suspended particles. The amount of radiation scattered by particles is measured at an angle (usually 90 ° ) to the incident beam. Thus nephelometry is measurement of intensity of the scattered light as a function of the concentration of the dispersed phase. January 19, 2023 Figure 2 8

9. Nephelometry and Fluorimetry Nephelometry is much similar to fluorometric method because both involve the measurement of scattered light. But the basic difference is that the scattering is elastic in fluorimetry and inelastic in nephelometry . It means that both incident and scattered light are of the same wavelength in nephelometry whereas the scattered light is measured in fluorimetry is of longer wavelength than the incident length. 9 Fluorimetry  measures the fluorescence at a specific emission WL after induction with a light pulse of a specific excitation WL

10. Nephelometry and Fluorimetry Fluorimetry Nephelometry Similarity Emergent radiation measured at 90° Similarity Emergent radiation measured at 90° Difference Emitted radiation measured Emitted radiation longer wavelength than incident light Difference scattered radiation measured Emitted radiation same wavelength as that of than incident light 10 elastic scattering, radiation is absorbed by the analyte and re-emitted without a change in the radiation’s energy. When the radiation is re-emitted with a change in energy, the scattering is said to be inelastic

11. January 19, 2023 11  There are two types of light scattering: elastic scattering, in which the wavelength of the scattered light and incident light are the same; and inelastic light scattering, in which the wavelength of the scattered light and incident light are different. Only the first type of light scattering (elastic) is relevant to nephelometry and turbidimetry. United States Pharmacopeia (2022). General Chapter, 〈855〉 Nephelometry and Turbidimetry. USP-NF. Rockville, MD: United States Pharmacopeia .

12. January 19, 2023 Elastic scattering is divided into two types: Rayleigh, or small-particle scattering, and large-particle scattering. Rayleigh scattering occurs when the scattering particles largest dimension is less than 5% of the radiation’s wavelength. The intensity of the scattered radiation is symmetrically distributed and is proportional to its frequency to the fourth power (v4), accounting for the greater scattering of blue light compared with red light. For larger particles , the distribution of scattered light increases in the forward direction and decreases in the backward direction as the result of constructive and destructive interferences. 12

13. Reflection and Scattering Both reflection and scattering phenomena are very important in turbidimetry and nephelometry . If light is allowed to pass through a solution having suspended particles, Reflection will take place when the dimensions of suspended particles are larger than the wavelength of incident light. Scattering will take place if the dimensions of suspended particles are smaller than the incident wavelength. January 19, 2023 13

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15. Factors affecting measurement Concentration of particles Particle geometry (size, shape) I ncident wavelength Refractive index January 19, 2023 15

16. 1. Concentration In turbidimetry , one measures the transmitted of a primary beam of radiation as: T = It / I According to Beer’s law: S = log I / It = kbc k is the proportionality constant known as turbidity coefficient k depends on the particle size and shape, wavelength and refractive indexes of the suspended and suspending media. I t inversely proportional to the concentration It is good only for small particles where Rayleigh’s scattering is the main mechanism of attenuation and for dilute suspensions where multiple scattering is unlikely. A working curve can be prepared by plotting S vs known concentration of scattering material and then the unknown concentration are observed from this curve by knowing their S value from the turbidimeter January 19, 2023 16

17. Cont … In nephelometry , one cannot relate the scattered intensity to the concentration by any sample theoretical equation. The reason for this is that the scattered intensity in nephelometry depends upon a number of complicated factors; like the properties of the scattering suspension and the angle and geometry of measuring instrument The best equation will be that one which will relate the Is to the C suspended particles by the approximate empirical equation such as: I S = k S I C Where: K S is the empirical constant for a particular system I is the intensity of incident light and I S is the scattered of light intensity I S directly proportional to the concentration January 19, 2023 17

18. Cont … Whenever quantitative analysis is to be carried out in nephelometry, a working curve is obtained by plotting the concentration of suspended particles vs Is/I0 under carefully controlled conditions. But in most of the cases log(Is/I0) is plotted vs C to confirm with the more usual spectrophotometric and turbidimetric practices January 19, 2023 18

19. 2. Particle geometry: In both techniques the most critical factor is the control of particle size and shape. The ideal situation is that when all samples and calibration solution with which they are compared should possess the same distribution of small medium and large particles. One should prepare samples and standards under identical conditions. The conditions include concentration of reactants, temperature, agitation, pH, the presence of non-reactants and order of mixing of reactants. If one does not maintain these conditions one may get different sizes particles which may introduce major error in turbidimetry and nephelometry January 19, 2023 19

20. 3. Wavelength If the sample solution is colorless, one must use the incident light of the same color. On the other hand if clear solutions are having dark particles, light in the infrared region must be used where there is maximum absorption. In nephelometry, absorption is much less of a problem. For nonfluorescent sample white light is generally used as a convenience. Shorter wavelength scatter to greater extent while longer wavelength scatter to lesser extent. January 19, 2023 20

21. 4. RI difference: Best results are obtained when there is an appreciable RI difference between the particle and its surroundings medium. Sometimes it is advantageous to change solvents in order to increase the refractive index references. January 19, 2023 21

22. Choice between Turbidimetry and Nephelometry It is determined by two principal factors. The intensity of the transmitted or scattered radiation relative to the intensity of radiation from the source. When the solution contains a small concentration of scattering particles, the intensity of the transmitted radiation I t , will be very similar to the intensity of the radiation source, I0. Thus, nephelometry is a more appropriate choice for samples containing few scattering particles. On the other hand, turbidimetry is a better choice for samples containing a high concentration of scattering particles. January 19, 2023 22

23. Cont .. The second consideration is the size of the scattering particles. For nephelometry, the intensity of scattered radiation at 90° will be greatest if the particles are small enough that Rayleigh scattering is in effect. For larger particles scattering intensity is diminished at 90° . When using an ultraviolet or visible source of radiation, the optimum particle size should be 0.1– 1 µm . The size of the scattering particles is less important for turbidimetry, in which the signal is the relative decrease in transmitted radiation. In fact, turbidimetric measurements are still feasible even when the size of the scattering particles results in an increase in reflection and refraction. January 19, 2023 23

24. Recapping is method in which the decrease in transmitted radiation due to scattering is measured and used for high concentration suspensions. Nephelometry is the measurement of the transmitted light by the suspended particles at right angles to the incident beam. True / January 19, 2023 24 Turbidimetry False transmitted

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26. Instrumentation is very similar to spectrophotometer devices. 1, Sources One may use white light in nephelometers but it is advantageous to use monochromatic radiation. Similarly, monochromatic radiation is used in turbidimeters to minimize absorption. In either case it is necessary to use sources providing high intensity monochromatic radiation and wherever possible short wavelengths are used to increase the efficiency of Rayleigh scattering. M ercury lamp and Tungsten lamp may be used January 19, 2023 26

27. 2, Filters/ Monochromater : Filters will convert the polychromatic light to monochromatic light. Generally filters are used for this purpose. Fitters are of two types: Absorption filters Interference filters 27 absorption  filters that absorb unwanted wavelengths and  interference  filters that remove selected wavelengths by internal destructive interference and reflection

28. 3, Sample cells Although we can use cylindrical cells, they must have flat faces where the entering and exiting beams are to be passed. This is to minimize reflections and multiple scattering from the cell walls. In general, a cell with a rectangular cross-section is preferred. Where measurements are to be made at angles other than 90°, Semi-octagonal cells are widely used. 28

29. Cont ... Generally walls through which light beams are not pass are painted a dull black to absorb unwanted radiation and minimize stray radiation. In experimental cells, a blackened curved horn is frequently affixed to the wall directly opposite the entering beam to trap all the beam which is not scattered. Alternatively, one can put a light trap for this purpose in the cell of the chamber in which the cell is located. January 19, 2023 29

30. 4, Detectors Most commonly used detectors in the nephelometry and turbidimetry are photomultiplier tubes. In nephelometers photomultiplier tubes should be as detectors because the intensity of scattered radiation is usually very small. In most of the nephelometers, detector is generally fixed at 90° to the primary beam but for maximum versatility and sensitivity is desirable to vary the detector angle which is generally close to primary beam. In some nephelometers, the detector is mounted on circular disc which allows measurement at many angles. In turbidimeters, ordinary detectors such as phototubes may be used. January 19, 2023 30

31. 5, Turbidimetres In most turbidimeters, ordinary calorimeters (or) spectrophotometers may be used . Others like Du Pont model 430 can also be used which is more sensitive to low concentration of suspended particle than ordinary turbidimetry. January 19, 2023 Du Pont Model 430 turbimeter 31

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33. 5, Nephlometer Ordinary fluorimeters are generally used for nephelometric measurements. In some cases spectrophotometers can be employed as nephelometer. The photo-multiplier tube detector is used as a receiver which is mounted on a turnable and may be positioned at any desired angles from 0° to 180° relative to the exit beam. January 19, 2023 33

34. Generally sensitivity of the method is improved by the following: Addition of water-soluble polymers Gives greater stability of the immune complex Reduced reaction concentrations Increased sensitivity January 19, 2023 34