ATOMIC ABSORPTION SPECTROSCOPY and PROTEIN DETERMINATION with BIURET METHOD THEORY
ATOMIC ABSORPTION SPECTROSCOPY
Spectroscopy is the use of the absorption, emission, or scattering of electromagnetic radiation by atoms or molecules (or atomic or molecular ions) to qualitatively or quantitatively study the atoms or molecules, or to study physical processes. The interaction of radiation with matter can cause redirection of the radiation and/or transitions between the energy levels of the atoms or molecules. A transition from a lower level to a higher level with transfer of energy from the radiation field to the atom or molecule is called absorption. A transition from a higher level to a lower level is called emission if energy is transfered to the radiation field, or nonradiative decay if no radiation is emitted. Redirection of light due to its interaction with matter is called scattering, and may or may not occur with transfer of energy, i.e., the scattered radiation has a slightly different or the same wavelength. When atoms or molecules absorb light, the incoming energy excites a quantized structure to a higher energy level. The type of excitation depends on the wavelength of the light. Electrons are promoted to higher orbitals by ultraviolet or visible light, vibrations are excited by infrared light, and rotations are excited by microwaves.Atomic-absorption (AA) spectroscopy uses the absorption of light to measure the concentration of gas-phase atoms. Since samples are usually liquids or solids, the analyte atoms or ions must be vaporized in a flame or graphite furnace. The atoms absorb ultraviolet or visible light and make transitions to higher electronic energy levels. The analyte concentration is determined from the amount of absorption. Applying the Beer-Lambert law directly in AA spectroscopy is difficult due to variations in the atomization efficiency from the sample matrix, and nonuniformity of concentration and path length of analyte atoms (in graphite furnace AA). Concentration measurements are usually determined from a working curve after calibrating the instrument with standards of known concentration.