MP101

Atomic Absorption Spectrophotometry

Trainer: Chandramouli R

INTRODUCTION

necessary to measure the metal content of different kinds of samples in the pharmaceutical industry samples- products that have metals, Ca, Mg added for their therapeutic value or biological samples, such as blood, urine, and tissue

PRINCIPLE OF OPERATION FOR ATOMIC ABSORPTION SPECTROPHOTOMETRY

ground state of an atom - electronic state- all the electrons are in their most stable configuration or orbitals When light is absorbed by an atom, one or more of the electrons is excited to a higher energy orbital. energy or the wavelength of the energy that is needed to cause the electrons in the atoms to shift from the ground state to an excited state

absorbance of light by the free, ground-state atoms of the element of interest

Principle...

each element absorbs at very discrete wavelengths, the lamp used for analysis of a particular metal emits light only at the desired wavelengths and is specific for that element two kinds of lamps hollow cathode lamp (HCL) and the electrodeless discharge lamp (EDL). These lamps contain a deposit of the metal of interest in an inert gas atmosphere and then use either a charged anode/cathode or a radio frequency to excite the metal atoms

excited atoms relax, they emit the absorbed energy as light energy at the characteristic wavelength(s) for that particular metal. This light energy is focused through the sample chamber onto the monochromator and detector

The concentration of the analyte in the sample chamber is proportional to the amount of light absorbed and, under the proper experimental conditions, follows Beers law A = abc, where A is the absorbance, a is the absorption coefficient of the analyte, b is the cell path length, and c is the concentration of the analyte

SAMPLE INTRODUCTION TECHNIQUES FOR AAS

There are four primary methods:

1. flame (aspiration of sample solution) 2. cold vapor (or chemical vaporization) 3. hydride generation (or chemical vaporization) 4. Graphite furnace (or electrothermal atomization) All methods common goal - to get the analyte atoms into the light path as free,ground-state atoms.

aspirating the sample solution into a burner chamber, where it is mixed with a fuel gas and an oxidant gas. mixture is then burned in a specially designed burner head The light beam is directed lengthway down the burner, and the absorption of the analyte atoms in the flame is measured.

Flame atomization assembly equipped with spray chamber and slot burner

Fuels and Oxidants Used for Flame Combustion

most commonly used gas mixtures are air with acetylene and nitrous oxide with acetylene

Electrothermal Atomizers - graphite furnace

consists of a cylindrical graphite tube approximately 13 cm in length, and 38 mm in diameter graphite tube housed in an assembly that seals the ends of the tube with optically transparent windows assembly also allows for the passage of a continuous stream of inert gas, protecting the graphite tube from oxidation, and removing the gaseous products produced during atomization

Samples between 5 and 50 mL are injected into the graphite tube through a small-diameter hole located at the top of the tube.

Atomization achieved in three stages. sample is dried using a current that raises the temperature of the graphite tube to about 110 C desolvation solid residue Ashing - temperature is increased to 3501200 C - organic material in the sample is converted to CO2 and H2O, and volatile inorganic materials are vaporized the final stage the sample is atomized by rapidly increasing the temperature to 20003000 C.

Quantitative Applications

flame or electrothermal atomization widely used for the analysis of trace metals in a variety of sample matrices Determination in diverse samples - blood, urine, muscle, tissue, hair, milk, plasma, serum , CSF et al. Developing a quantitative atomic absorption method requires several considerations - method of atomization, selecting the wavelength and slit width, preparing the sample for analysis, minimizing spectral and chemical interferences, and selecting a method of standardization

Qualitative applications

Preparing the Sample

Flame and electrothermal atomization require that the sample be in a liquid or solution form. Samples in solid form are prepared for analysis by dissolving in an appropriate solvent. When the sample is not soluble, it may be digested, either on a hot plate or by microwave, using HNO3, H2SO4, or HClO4. the analyte may be extracted via a Soxhlet extraction. Liquid samples may be analyzed directly or may be diluted or extracted if the matrix is incompatible with the method of atomization. Serum samples, for instance, may be difficult to aspirate when using flame atomization and may produce unacceptably high background absorbances when using electrothermal atomization. A liquidliquid extraction using an organic solvent containing a chelating agent is frequently used to concentrate analytes. SPE current widely adopted method