These techniques are closely related to colorimetry

Both the techniques are based on the scattering of light by non transparent particles in a suspended in a solution.

The two techniques differs only in the manner of measuring the scattered radiation

When the light is allowed to pass through a suspension, the part of the incident radiant energy is dissipated by absorption,reflection,and refraction while the remainder is transmitted.

Tyndall effect

Light scattering by particles in a colloid or particles in a fine suspension.

the longer-wavelength light is more transmitted while the shorter-wavelength light is more reflected via scattering.

PRINCIPLE

NEPHELOMETRY

measurement of the light scattered by suspended particles at right angles(900) (perpendicular) to the incident beam.

TURBIDIMETRY

measurement of the light transmitted by suspended particles to the incident beam.

TURBIDIMETRY & COLORIMETRY

Measurement of the intensity of light transmitted through a medium

Light intensity is decreased

NEPHELOMETRY & FLUORIMETRY

Measurement of scattered light at 900

both incident & scattered light are same wavelength -NEPHELOMETRY

scattered light wavelength is longer than the incident light - FLUORIMETRY

CHOICE OF THE METHOD

depends upon the amount of light scattered by suspended particles present in solution.

TURBIDIMETRY - high concn. Suspensions

NEPHELOMETRY – low concn. Suspensions

more accurate results

THEORY

REFLECTION VS SCATTERING

If the dimensions of the suspended particles larger than the wave length of the incident light - REFLECTION

If the dimensions of the suspended particles smaller(same order) than the wave length of the incident light - SCATTERING

NEPHELOMETRY

suspended particles < incident light wave length :

smaller particles undergo scattering – secondary rays - maximum intensity at 900

- most of the instruments measured at this angle

suspended particles > incident light wave length :larger particles undergo reflection – small fraction of light get deviated maximum intensity at < 900

5-200 / 450

NEPHELOMETRY

suspended particles should neither be too large nor too small otherwise the scattering efficiency falls off.

optimum particle size should be 0.1- 1 micro meters.

TURBIDIMETRY

suspended particles > incident light wave length :

larger particles undergo reflection- measuring transmitted radiation

larger particles- absorbance vs concn – not linear

measurements can not be accurate

Factors affecting measurements

The amount of radiation removed or deviated from the primary radiation beam depends on the following factors

A.Concentration:

TURBIDIMETRY

IT=Transmittance = Io

Beer’s law

Io S = log = kbc I

S= turbidence due to scatteringK = proportionality constant / Turbidity constantb = path lengthC = concentration of suspended material

NEPHELOMETRY

Is = Ks Io c

Is = scattered intensity

Ks = empirical constant

Io = incident intensity

c = concentration of the scattered material

Working curve

C Vs Is / Io

log Io / Is Vs C

B.Particle geometry

Control of particle size & shape - most critical factor

Same distribution

Conditions – concn. Of reactants,

temp,

agitation,

pH,

order of mixing,

time allowed for particle growth

C.Incident light wave length

TURBIDIMETRY

It is an imp factor

Select a wave length- sample solution does not absorb strongly

If the sample solution is colorless – use the incident light of the same color

If clear solutions having dark particles – light in red / IR

NEPHELOMETRY

Absorption is much less- white light is generally used

D.Refractve index difference

Appreciable RI differences between particles & surrounding medium – best results

Change solvents in order to increase the RI differences

INSTRUMENTATION

SOURCES

FILTERS/MONOCHROMATORS

CELLS

DETECTORS

SOURCES

White light – nephelometers

Mercury arc

Tungsten lamp

FILTERS / MONOCHROMATORS

mono chromatic radiation

CELLS

cylindrical cells - flat faces

to minimize reflections & multiple scatterings

cell with a rectangular cross section is preferred

semi octagonal faces

octagonal faces- 00,450,900,1350

NEPHELOMETER

light source

sensitive micro-ammeter

Filter wheel with a series of colour filters

annular photocell

reflector to collect the scattered light

test tube

metal test tube cover to exclude extraneous light.

The test solution (sample) is placed in a test tube (F) that has been duly rested on a light source (A)

The scattered light caused by the particles in a turbid or cloudy solution is immediately directed by the reflector (E) on to an annular photocell (D).

A series of standard colour filters are usually provided in the form of a filter-wheel (C) so as to facilitate analysis of coloured solutions ;

Taking care that the filter chosen must be similar to colour to that of the solution.

The current generated after passing through the photocell (i.e., light energy is being converted to electrical energy) is recorded by a sensitive micro-ammeter (B).

The test tube is provided with a metallic cover (G) to get rid of any extraneous light.

Usually a nephelometer is provided with zero-setting controls, sensitivity adjusting device and a set of previously matched test tubes.

TURBIDIMETER

Either visual or photoelectric colorimeters may be satisfactorily employed as turbidimeters.

However, the use of the blue filter normally enhances the sensitivity appreciably.

It has been observed that the light transmitted by a turbid solution does not normally obey the Beer-Lambert Law accurately and precisely.

Therefore, as an usual practice it is advisable to construct a ‘calibration curve’ by employing several standard solutions.

The concentration of the unknown solution may be read off directly from the above calibration curve as is done in the case of colorimetric assays

APPLICATIONS