Dnyanasadhana College, Thane. Department of Chemistry T. Y. B. Sc Dnyanasadhana College, Thane. Department of Chemistry T.Y.B.Sc. Analytical Chemistry TURBIDIMETRY AND NEPHELOMETRY Dr.G.R.Bhagure

Theory, Instrumentation, Application

Theory Origin of Scattering A focused, monochromatic beam of radiation of wavelength l, passing through a medium containing particles whose largest dimensions are less than l is observed to scatter in all directions. For example, visible radiation of 500 nm is scattered by particles as large as 750 nm in the longest dimension.

Rayleigh, or small-particle scattering large-particle scattering Elastic scattering Rayleigh, or small-particle scattering large-particle scattering 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. Rayleigh scattering occurs when the scattering particles largest dimension is less than 5% of the radiation’s wavelength.

Interaction of Light with Particles  Small molecules  Light scattered symmetrically  

Interaction of Light with Particles Big molecules  Light scattered unsymmetrically  

Turbidimetry and Nephelometry Turbidimetry and Nephelometry are two related techniques in which an incident source of radiation is elastically scattered by a suspension of colloidal particles. In turbidimetry, the detector is placed in line with the radiation source, and the

Decrease in the radiation’s transmitted power is measured. In Nephelometry, scattered radiation is measured at an angle of 90° to the radiation source. The similarity of the measurement of Turbidimetry to absorbance, and of Nephelometry to fluorescence, is evident in the block instrumental designs shown in Figure 10.54. In fact, turbidity can be measured using a UV/Vis spectrophotometer, such as a Spectronic- 20, whereas a spectrofluorometer is suitable for Nephelometry.

Turbidimetry A method in which the decrease in transmitted radiation due to scattering is measured. Nephelometry :A method in which the intensity of scattered radiation is measured at an angle of 90° to the source.

Applications Turbidimetry and nephelometry Applications Turbidimetry and nephelometry are widely used to determine the clarity of water, beverages, and food products. For example, the turbidity of water is determined using nephelometry by comparing the sample’s scattering to that of a set of standards. The primary standard for measuring turbidity is formazin (Figure 10.55), which is an easily prepared, stable polymer suspension.26 Formazin prepared by mixing a 1 g/100 mL solution of hydrazine sulfate, N2H4•H2SO4, with a 10-g/100 mL solution of hexamethylenetetramine produces a suspension that is defined as 4000 nephelometric turbidity units (NTU). A set of standards with NTUs between 0 and 40 is prepared and used to construct a calibration curve. This method is readily adapted to the analysis of the clarity of orange juice, beer, and maple syrup.

A number of inorganic cations and anions can be determined by precipitating them under well-defined conditions and measuring the transmittance or scattering of radiation from the precipitated particles. The transmittance or scattering, as given by equation 10.36 or 10.37 is proportional to the concentration of the scattering particles, which, in turn, is related by the stoichiometry of the precipitation reaction to the analyte’s concentration. Examples of analytes that have been determined in this way are listed in Table 10.15. The turbidimetric determination of SO4 2– in water following its precipitation as BaSO4 is described in Method

TURBIDIMETRY & NEPHELOMETRY  Light interact with particle cause light scattering   Wavelength of scattered light is the same as of the incident light Detect by turbidimeter  (detector is parallel to light source) or nephelometer (detector is aligned at an angle 70-75 °)

commonly used in immunoassay   detect AgAb complex particles (diameter = 250-1500 nm) •Rayleigh-Debye light scatter  use spectrophotometer in turbidimetry  Light scatter in Nephelometry may be measured by end point or kinetic method

APPLICATIONS: • Immunoglobulins, • Specific proteins: Haptoglobin, Transferrin, Alpha 1 AT, Lipoproteins, Albumin • Coagulation factors: Antithrombin III • Theraputic drugs Formation of immune complexes

Interaction of Light with Particles  Small molecules  Light scattered symmetrically  

Turbidimeter visible light Filter Photocell Detector Read Out Device Sample Cell visible light Filter

Nephelometer visible light Light Trap Photocell Detector Sample Cell Graduated Disc Collimating Lens Light Trap visible light Photocell Detector Sample Cell Read Out Device