turbidimetry n nephelometry

Turbidimetryand

Nephelometry

Turbidimetry and Nephelometry

Turbidimetry and Nephelometry , methods for determining the amount of cloudiness, or turbidity, in a solution based upon measurement of the effect of this turbidity upon the transmission and scattering of light

Principles

Light scattering is the physical phenomenon resulting from the interaction of light with a particle(s) in solution. Dependent on :• Particle size • Wavelength • Distance of observation, • Concentration of particles • MW of particles

Principles

• TurbidimetryAt higher concentration of a suspension, scattering is not uniform and light is scattered in all direction. This technique requires a special measuring instrument, where the detector is set at an angle to the incident light beam.• NephelometryAt low concentration of a suspension, there is a uniform scattering. This method of measurement is carried out using an absorptiometer (photometer)

a. Turbidimetry• In turbidimetry, the intensity of light

transmitted through the sample is measured,

• Turbidimetry is the measurement of the decrease in the intensity of light as it passes through a solution of particles,

• For High concentration,• Scattering not uniform,• Intensity of transmitted light

measured at 180o

• Turbidimetry is measured using instruments such as photometers or calorimeters.

• Turbidimetry is the technology used to protein assays such as Apolipoproteins, Lp(a), CRP, RF, ASO, C3, C4, Immunoglobulins etc..

• Turbidimetry is generally done at wavelengths around 630 nm to minimize absorbance by the medium because most bacterial growth media are brown or brownish-yellow.

What are These Particles?

• Suspended particles may include– Silt– Clay– Algae and Other

Microorganisms– Organic Matter– Other Minute Particles

What for Turbidity?

• A measure of suspended solids–Particles not in true

solution• A measure of relative

water clarity• An indicator of water

quality

What is Not Turbidity?

• Turbidity is NOT:– Able to identify the type

of particles– A chemical property – A spectrophotometric

type of analysis – A direct measure of total

suspended solids (TSS)

TURBIDIMETER

TURBIDIMETER

Turbid creek water caused by heavy rains.

b. Nephelometry

• In nephelometry the intensity of the scattered light is measured

• For low concentration, • Uniform scattering, • Intensity of scatted light proportional

to concentration measured at 90o.

• Nephelometry can be used to detect either antigen or antibody, but it is usually run with antibody as the reagent and the patient antigen as the unknown

• in the Immunology Medical Lab, two types of tests can be run: "end point nephelometry" and "kinetic (rate) nephelometry"

• End point nephelometry tests are run by allowing the antibody/antigen reaction to run through to completion.

• Unfortunately, the large particles will fall out of the solution and cause a false scatter reading, thus kinetic nephelometry was devised

• In kinetic nephelometry, the rate of scatter is measured right after the reagent is added. As long as the reagent is constant the rate of change can be seen as directly related to the amount of antigen present.

Nephelometer

This is a diagram of a basic 90 degree nephelometer. Light from a tungsten lamp, passes through a lens. This light passes through the sample, where particles scatter the incident light. The amount of scattered light is measured by a photodetector, 90 degrees from the incident light. The calibration in the instrument is then able to convert the amount of scattered light into a turbidity reading (NTU).

Nephelometers – Optical System – Additional Detectors

0.5 NTU

Lamp LensSample Cell

90o Detector

Transmitted Light Detector

Back Scatter

Forward Scatter

NEPHELOMETER

A nephelometer at the Kosan, Cheju Island, South Korea NOAA facility

Factors Affecting Measurements

Concentration Turbidimetry : S = log I/Io = kbcS = turbidence due to scatteringK = turbidity constantb = path lengthC = concentration of suspended material

Nephelometry : Is = Ks Io CIs = scattered intensityKs= emperical constantIo = Incident intensity

Items should be considered:

1. The relative concentration of ingredients must be controlled.

2. The relative ratios of reactants must be constant.3. The manner, order of addition and rate of mixing

must be constant.4. The nature of other solutions present must be

considered and, if necessary, protective colloids added to avoid protein precipitation.

5. the temperature must be maintained constant.6. Constant time lapse before reading

TURBIDIMETRY NEPHELOMETRY

is the measurement of unscattered light. This method of measurement is carried out using an absorptiometer (photometer)

is the measurement of scattered light. This technique requires a special measuring instrument, where the detector is set at an angle to the incident light beam.

Instrument used is a Turbidymeter Instrument used is a Nephelometer

the light passing through a sample is measured

the light Scattered by the suspendedparticles in a sample is measured

DEFFERENCES

Sources of Error

• Stray Light - Excess light in the system (from any source) contributing to a high turbidity measurement– Sample Cells– Gas Bubbles

• Improper Calibration

Stray Light - How to minimise

• Stray light allows more light to reach detector = false high turbidity reading– Keep sample compartment

and optics clean

Applications of Turbidimetry and Nephelometry

• Analysis of waterclarity concentration of ions

• Determination of CO2• Determination of Inorganic

substances• Biochemical analysis• Quantitative analysis (ppm level)• Phase titration

• Commonly used in immunoassayo Rayleigh-Debye light scattero Use spectrophotometer in

turbidimetryo Light scatter in nephelometry may

be measured by end• Point or kinetic methodo Detect AgAb complex particles

(diameter = 250-1500 nm)

Applications of Turbidimetry and Nephelometry

INDUSTRIAL :• Metals and Mining • Chemical Processing • Pulp and Paper • Power • Food and Bevera• Pharmaceuticals • Refining

Applications of Turbidimetry and Nephelometry