Atomic absorption spectrophotometry

clinical chemistry

1. ATOMIC ABSORPTION SPECTROPHOTOMETRY BASIC CONCEPTS, INSTRUMENTATION, OPERATION, AND APPLICATION

2. INTRODUCTION : Introduced by Alan Walsh(1950) It is a method of elemental analysis used widely in clinical laboratories Useful for determining trace metals like Aluminum, Calcium, Copper, Lead, Lithium, Magnesium and Zinc in liquid or biological samples. Highly sensitive and can detect metals in concentrations lower than 1ppm

3. BASIC CONCEPTS: This technique basically uses the principle that free atoms (gas) generated in an atomizer can absorb radiation at specific frequency . AA is an emission technique in which an element in the sample is excited and the radiant energy given off is measured as the element returns to its lower energy level as in FES. However, the element is not appreciably excited in the flame, but it is merely dissociated from its chemical bonds ( atomised ) and placed in an unexcited ground state (neutral atom)

4. These ground state atom of particular element are receptive of light radiation of their own specific resonance wavelength (monochromatic). Thus, when a light of this wavelength is passed through a flame having atom of metallic species, part of the light will be absorbed and the absorption will be proportional to the density of atom in the flame. The analyte concentration is calculated from the amount of absorption .

5. Differences btw Atomic Absorption Spectrophotometry (AAS) and Flame Emission Spectrophotometry: AAS Amount of light absorbed by ground state atom is measured Absorption intensity does not depend upon temperature Beers law is obeyed over wide range of concentration FES Amount of light emitted by the excited atom is measured Absorption intensity is greatly influenced by temperature variation Beer’s law is not obeyed

6. AAS AES (FES)

7. INSTRUMENTATION: The basic requirements are : a light source; a sample cell; and a means of specific light measurement . Components of AA Spectrophotometer. Single beam AAS:

8. TYPES of ASS: Single beam AAS: Light source is placed ahead of flame with mechanical chopper between light source and flame Has low stability Double beam AAS: Beam from hollow cathod lamp is divided into two part one passes through flame while other bypass it Double beam ASS:

9. Light source: Radiation source: are of two types Hollow cathode lamp- emits spectral line of that metal which is used in the cathod (HCL) Electrodeless discharge lamp (EDL) A hollow-cathode lamp are made of the metal of the substance to be analyzed (different for each metal analysis). The anode is made of Tungsten Each HCL will have a particular current for optimum performance. In general, higher currents will produce brighter emission and less baseline noise. As the current continues to increase, lamp life may shorten and spectral line broadening may occur, resulting in a reduction in sensitivity and linear working range. If an alloy is used, it results in a multi-element lamp

10. Limitations of HCL: A finite lifetime – due to depletion of the analyte element from the cathode Adsorption of fill gas atoms onto the inner surfaces of the lamp – the primary cause for lamp failure Some cathode materials can slowly evolve hydrogen when heated – a background continuum emission contaminates the purity of the line spectrum of the element, resulting in a reduction of atomic absorption sensitivity and poor calibration linearity . Chopper: It is a rotating wheel interposed between the hollow cathode lamp and flame. Gives pulsating current in the photocell

11. Atomiser : Flame atomiser : total consumption burner and premixed burner Non flame atomiser (carbon rod or “graphite furnace) Temp of the rod is raised to dry, char, and atomise the sample in a chamber More sensitive than the conventional flame methods Permits determination of trace metals in small samples of blood or tissue Zeeman correction used to correct for background absorption by placing the analyte in a strong magnetic field which splits the degenerate atomic energy into two components that are polarised parallel and perpendicularly and interacts differently with polarized light .

12. Two absorption measurement are taken at different polarizer setting. one measures both analyte and background absorption, A t ,the other measures only background absorptions. Thus, the same light source at the same wavelength is used to measure the total and the background absortion . The difference btw the two is the corrected absorbance. Nebulization of liquid sample: method of formation of small droplet from the liquid sample Nebulization is done by use of a gas moving at high velocity Monochromator : Function of monochromator is to select given absorbing line from spectral line emitted from hollow cathode Common monochromator are prism and gratings

13. Detctor : Photomultiplier tube is most suitable. The intensity of the light is fairly low, so a photomultiplier tube (PMT) is used to boost the signal intensity Has good stability if used with stable power supply A detector (a special type of transducer) is used to generate voltage from the impingement of electrons generated by the photomultiplier tube Amplifier: It amplifies electric current Lock in amplifier are preferred to achieve excellent signal to noise ratio(measure of signal strength relative to background noise) Read out device: Digital read out device are used

14. Advantages of AAS: Technique is specific because the atom of particular element can only absorb radiation of their own characteristic wavelength Is independent of flame temperature Disadvantages of AAS: Separate lamp for each element to be determined is required Technique cannot be used successfully for estimation of element like Mo , Si etc because these element give rise to oxide in the flame Predominant anion affect the signal to negotiable degree

15. OPERATION: A meter is adjusted to zero absorbance when blank solution is sprayed on the flame, and rest of the light passes on to the photomultiplier tube. Then the light solution containing absorbing species is introduced a part of light is absorbed, resulting in a decrease in light intensity falling on a photomultiplier tube .

16. INTERFERENCES IN AAS: Spectral Interferences: include Absorption from other closely absorbing atomic species, usually not a problem because of the extremely narrow band-width used Absorption and scattering by molecular species, usually problematic at lower atomising temperatures Scattering by non-volatile salt particles and oxides Background emission (which can be electronically filtered)

17. Non-spectral Interferences: could be specific or non-specific Non-specific interferences: affect the nebulization by altering the viscosity, surface tension, or density of the analyte solution and consequently the sample flow rate Some contaminants also decrease the desolvation and atomization efficiency by lowering the atomizer temperature Specific interferences : are also called chemical interferences because they are more analyte dependent Solute volatilization interference: refers to the situation when the contaminant forms non-volatile species with the analyte

18. Dissociation interferences: affects the degree of dissociation of the analyte . Analytes that form oxides and hydroxides are esp susceptible to dissociation interferences Ionization interferences: occurs when the presence of an easily ionized element, such as K, affects the degree of ionization of the analyte which leads to changes in the analyte signal . Excitation interferences: occur when the analyte atoms are excited in the atomizer with a subsequent emission at the absorption wavelength . This is more pronounced at higher temperatures .

19. APPLICATION OF AAS: Qualitative and quantitative analysis Determination of metallic elements in biological system Determination of metallic element in food industry Determination of Ca, Mg, Na, K in serum Determination of lead in petrol

20. REFERENCES: Shriver and Atkins’ Inorganic Chemistry, Fifth Edition Concepts, Instrumentation and Techniques in Atomic Absorption Spectrophotometry by Richard D. Beaty and Jack D. Kerber An elementary overview of elemental analysis by thermo elemental