● GENERAL PRESENTATION GUIDELINES

● ATOMIC ABSORPTION SPECTROPHOTOMETRY

● NEPHELOMETRY AND TURBIDIMETRY

BY: DR. MANISHA

GENERAL PRESENTAION GUIDELINES

=> Visual aids

=> All instructional media – previewed

=> Visuals – Simple and minimal wording

-- readable from distance

=> Audience line of vision should not be obstructed

=> Visual material retention time on screen

=> Effective instructors talk to students and not visual aids.

ATOMIC ABSORPTION SPECTROPHOTOMETER

● atoms in the vapor state are subjected to external source of radiation which produces one line or beam of monochromatic light with single wavelength.

● This wavelength is a resonance one for the atoms and that will be absorbed by them.

● A Hollow cathode lamp - with the cathode made of the material to be analyzed is used to produce wavelength of light specific for that material.

● Cathode made of sodium , sodium light predominantly at 589 nm would be emitted by the lamp.

Pretreatment of Sample

=> Sample should be dissolved in a solution (matrix) in order to undergo nebulization

=>chemistry necessary to dissolve the sample in a suitable matrix is very important component of the analytical process

=>reagents used to dissolve samples must not contain substances that interfere with absorption measurement

Delivery Of Sample

Liquid sample delivery system consists of 3 components:

a nebulizer - breaks up the liquid into small droplets (aerosol),

an aerosol modifier- removes large droplets from the stream, and

the flame or atomizer - converts the analyte into free atoms

Nebulization: sample solution is introduced through an orifice into a high velocity gas jet, usually the oxidant,

in either parallel or perpendicular manner

sample stream is converted into a cloud of droplet in the aerosol modifier or spray chamber, combined with the oxidizer/fuel and carried to the burner. Large droplets go to waste and fine mist enter flame.

Atomization

=>convert the analyte within the aerosol into free analyte atoms in the ground state for AAS analysis

=>Two common methods : Flame atomization and electrothermal atomization

Flame atomization: nebulized sample (+ gaseous oxidant/fuel) is carried into a flame where complex set of events occurs

Ex conversion of a metallic element M from a dissolved salt, MX in the sample to free M atoms

aerosol containing MX enter the flame,solvent is evaporated, leaving particles of dry, solid MX (desolvation)

solid MX ==> MX vapor

(volatilization)

portion of MX molecules are dissociated to give free M atoms (Dissociation)

Electrothermal Atomizer

=>Nebulization with flames, wastes the sample and the residence time of free analyte atoms in the flame is short.

=>electrically heated devices viz. graphite furnaces and carbon rod analyzer, are common electrothermal, flameless, atomizer .

=>In electrothermal atomizer, few μl of sample are first evaporated, then ashed in electrically heated graphite and atomized.

=>are used for atomic absorption .

Advantages of Electrothermal

=>High sensitivity, because production of free analyte atoms is more efficient than flame atomizer

=>Ability to handle small volume of sample (5-100 ul)

=>Ability to analyze solid samples directly without pretreatment (in most cases)

=>Low noise from the furnace

=>Can maintain relatively high conc. of free atoms, albeit for only a brief time

Disadvantage

=>much more difficult to use than flame atomizer

=>precision is less because matrix effect from component in sample are much more severe than encountered in flame AAS

=>reproducibility of the results is less

Instrumentationconsist of a radiation source, sample holder, a wavelength selector, a detector, and a signal processor and readout unit

a) Radiation source:

in order for Beer’s law, to be followed, band-width of the source must be narrow relative to the width of an absorption peak (0.002-0.005nm)

as a result nonlinear calibration curves are inevitable when continuous radiation source are used

problem is solved by using the line sources with bandwidths narrower than absorption peaks

Disadvantage: separate lamp source is needed for each element

Hollow Cathode lamps:

when potential is applied, ionization of inert gas occurs and gaseous cation dislodge some of the metal atoms from the cathode surface

=>portion of metal atoms are in excited states and emit their characteristic radiation as they return to the ground state

=>variety of hollow cathode lamps are available commercially

=>some cathode are made up of a mixture of several metals permitting analysis of many elements

b) Sample holder: It is the atomizer cell that contains the gaseous atomized sample

c) Monochromator: to select a desirable wavelength

d) Detector: photomultiplier tube

INTERFERENCE1) SPECTRAL INTERFERENCE:

Absorption by other closely absorbing atomic species, absorption by molecular species, scattering by non volatile salt particles or oxides and background emmission

2) NON SPECTRAL INTERFERENCE:

Are either nonspecific or specific

=> Nonspecific : affect nebulization by altering viscosity , surface tension or density of the analyte solution and consequently sample flow rate.

Some contaminants decrease desolvation and atomization by lowering atomizer temperature

=>Specific: are also called chemical interference because they are more analyte dependent

-Solute volatalization intereference

-Dissociation interference

-Ionization intereference

-Excitation intereference

NEPHELOMETER AND TURBIDIMETER

• When electromagnetic radiation (light) strikes a particle in 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 particle. Turbiditimetry and nephelometry are based on principle of light scattering.

=>turbidimetry- measurement of the intensity of light transmitted through the suspension, to determine the concentration of substance of interest.

=> Nephelometry – measurement of scattered light.

• Turbidometric measurements are made at 180o 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 light beam.

=>Turbidity can be measured on most routine analysers by a spectrophotometer (absorbed light)

-Reduced sensitivity and precision. =>The intensity of scattered light is normally measured by

Nephelometer.

Light scattering is the physical phenomenon resulting from the interaction of light with a particles in solution.

Dependent on :

=>Particle size

=>Wavelength

=>Distance of observation,

=>Concentration of particles

=>MW of particles

FACTORS AFFECTING LIGHT SCATTERING

1) Particle size

-When the particle size is much smaller than the wavelength then scattered light from such particles are in phase and reinforce each other.

-As particle size increases scattered light no more remain in phase . Some of the light reinforce each other and some cancel each other. The scattered light pattern from such large particles are characteristic of size and shape of the particle.

2) Wavelength Dependence Of Light Scattering

- Intensity of light scattering increases with decrease in wavelength of incident light.

- Light intensity decreases as distance from light scattering to detector increases.

3) Concentration and Molecular wt. Of particle:

-Intensity of light scattered increases with increase in concentration and MW of particles.

=>For Larger particles, symmetry of scattered light around 90o is lost. eg. Immunoglobulin of M class,Chylomicrons and Ag / Ab complexes.

This is Rayleigh Debye Scattering.

=> For even larger particles,eg. RBCs and bacteria they show complex angular dependence of light scattering. Produce scattered light in narrow angular region in forward direction. This is Mie scattering

MEASUREMENT OF SCATTERED LIGHT

Turbidimetry: t = 1/b log Io/I

Nephelometry :

INSTRUMENTATION

The basic instrument contains

=>Light Source:Tungsten,Quartz-Halogen lamp,xenon lamp and laser.

=>Filters

=>Sample cells

=>Detectors – Photomultiplier tube.

CELLS

• cylindrical cells - flat faces to minimize reflections & multiple scatterings

CLINICAL UTILITYTURBIDIMETRY

=> Determination of :

- Total Protein in body fluids such as urine and csf containing small quantities of protein using trichloroacetic acid.

- Lipase activity using triglycerides as substrate. Decrease in turbidity is directly proportional to the lipase activity.

NEPHELOMETRY

=> Determination of

-Immunoglobulins ( Total, IgG,IgM,IgE )

-Serum proteins eg. Haptoglobin, Transferrin, albumin.(using Antibodies specific for each protein)

-Size and no. Of particles ( Laser nephelometry)

LIMITATIONS

=> Antigen excess: As turbidity increases during addition of Ag to Abs, signal increases to maximum and then decreases, this marks the beginning of Ag excess.

=> Matrix effects: eg. Lipemic sample.

Ans to the problem: sample blanking.

=> Doesn't follow beer's law.

=> Because some precipitation of particles may occur with time – mix sample well before placing it in instrument. And – keep the measurement time same for all samples.

=> Kinetic reactions provide higher degree of accuracy , sensitivity and precision.

CONSIDERATIONS

=>Selection of wavelength:

- If solution and suspended particles are colorless ---> use any wavelength in visible region.

- If solution is colored and particles colorless ---> use wavelength that give minimum absorption for solution.

- If particles colored and solution colorless ----> use wavelength that give maximum absorption for particle.

-If both are colored---> use wavelength that give minimum absorption for solution and other one that give maximum absorption for particle.then subtract the solution abs from particle abs.

Things which still eludes me

● Zeeman correction● Effect of Polarised light on scattering by

particle.