Atomic absorption Spectrophotometry

Spectrophotometer , types of spectrophotometer, atomic absorption spectrophotometer working principle, instruments of AAS, cathode hollow lamp, chopper, Flame, Burner , total consumption burner, pre-mix burner , Monochromators prism and diffraction grating , read out device, types of AAS, Flame AAS , non flame AAS(Graphite furnace AAS ), applications , interference , atomic emission spectrophotometry different types of flame , principle of emission spectrophotometry, comparision of spectrophotometry.

1. Atomic absorption Spectrophotometry Spectrophotometry is defied as the measurement of the intensity of light at selected wavelength Widely used method of quantitative and qualitative analysis in the chemical and biological sciences Types of spectrophotometry Visible spectrophotometer Ultraviolet-visible spectrophotometer Infrared spectrophotometer Fluorescence spectrophotometer Atomic absorption spectrophotometer. Atomic emission spectrophotometer .

2. Atomic absorption Spectrophotometry Introduction Invention Working Principle of AAS Instrumentation Interferences

3. Introduction The atomic absorption spectrophotometer is used to measure concentration by detecting absorption of electromagnetic radiation by atom rather than by molecules. It is a very common technique for detecting metals and metalloids in sample. Widely used in clinical laboratories to measure elements Such as aluminum, calcium, copper, lead, lithium, magnesium, zinc, and other metals

4. Elements detectable by atomic absorption are highlighted in pink in this periodic table

5. Invention Introduced in 1955 by Alan Walsh in Australia First commercial atomic absorption spectrometer was introduced in 1959 Used for mining, medical treatment &agriculture

6. Principle Atomic absorption is an absorption spectrophotometric technique in which a metallic atom in the sample absorbs light of a specific wavelength. The element is not appreciably excited in the flame, but is merely dissociated from its chemical bonds (atomized) and placed in an unexcited or ground state (neutral atom ). This ground state atom absorbs radiation at a very narrow bandwidth corresponding to its own line spectrum.

7. Principle A hollow cathode lamp with the cathode made of the material to be analyzed is used to produce a wavelength of light specific for the atom. Thus, if the cathode were made of sodium, sodium light at predominantly 589 nm would be emitted by the lamp. When the light from the hollow cathode lamp enters the flame, some of it is absorbed by the ground-state atoms in the flame, resulting in a net decrease in the intensity of the beam from the lamp. This process is referred to as atomic absorption Concentration measurements are usually determined from a working curve after calibrating the instrument with standards of known concentration.

8. Instrument Light source Chopper Atomizer Monochromators Detector Amplifier Read out device

9. ATOMIC ABSORPTION SPECTROPHOTOMETER- BASIC COMPONENT

10. Types of AAS  Flame atomic-absorption spectrophotometer  Graphite-furnace atomic-absorption spectrophotometer

11. Light Source Hollow Cathode Lamps Electrodeless Discharge Lamp

12. Hollow Cathode Lamp Cathode--- in the form of a cylinder, made of the element being studied in the flame Tungsten Anode Filled with an inert gas (neon or argon) sealed in a glass tube Quartz or special glass that allows transmission of the proper wavelength, is used as a window

13. How it works Applying a potential difference(300-500V) between the anode and the cathode leads to the ionization of some gas atoms . These gaseous ions bombard the cathode and eject metal atoms from the cathode in a process called sputtering. Some sputtered atoms are in excited states and emit radiation characteristic of the metal as they fall back to the ground state .

14. LIGHT SOURCES Electrodeless Discharge Lam Consists of an evacuated tube in which the metal of interest is placed. The tube is filled with argon at low pressure and sealed off. Used for difficult to make stable hollow cathode lamp from certain elements particularly those that are volatile, such as arsenic, germanium

15. Chopper A rotating wheel is interposed between the hollow cathode lamp and flame . It is interposed to break the steady light coming from the lamp into pulsating light which is used to measure the intensity of light absorbed by elements without interference by radiation from the flame itself. Pulsating light gives pulsating current in photocell. There is also steady current caused by light which is emitted by flame. But only pulsating current is amplified and recorded

16. Atomizer Atomization is separation of particles into individual molecules and breaking molecules into atoms .This is done by exposing the analyte to high temperatures in a flame or graphite furnace Atomiser converts the liquid into small droplets which are easily vaporised . Types of Atomisers :- 1.Flame atomizer:- a.) Total consumption burner b.) premixed burner 2.Non-flame atomizer( Electrothermal atomizer )

17. Flame atomizer Nebulization Conversion of the liquid sample to a fine spray Desolvation Solid atoms are mixed with the gaseous fuel. Volatilization Solid atoms are converted to a vapor in the flame. There are three types of particles that exist in the flame: 1) Atoms 2) Ions 3) Molecules

18. Nebulization Before the liquid sample enters the burner ,it is converted into droplets this method a formation of small droplets its called nebulization Common method of nebulization is by use of gas moving at high velocity, called pneumatic nebulization.

19. Total consumption burner In this whole sample is atomized into the flame, hence named as total consumption burner. In this burner, the sample solution, the fuel, and oxidizing gases are passed through separate passages to meet at the opening of the base of flame. Then the flame breaks the sample in liquid form into the droplets which are evaporated and burns. Leaving the residue which is reduced to atoms. Fuel used – H 2 /acetylene Oxidant – O 2

20. Premixed burner It is most widely used because of uniformity in flame intensity. In this the sample solution ,fuel and oxidant are mixed before they reach the tip. The fine droplets get carried out along with the fuel gas at outlet, the large drops of sample get collected in chamber and are drained out. Advantages Non-turbulent Noiseless Stable Disadvantages Only 5% sample reaches to the flame Rest 95 % is wasted.

21. Flame temperature for various gas mixtures Fuel Oxidant Temperature c Natural gas Air 1700 - 1900 Natural gas Oxygen 2700 - 2800 Hydrogen Air 2000 - 2100 Hydrogen Oxygen 2550 - 2700 Acetylene Air 2100 – 2400 (Most common) Acetylene Oxygen 3050 - 3150 Acetylene Nitrous oxide 2600 - 2800 Selection of flame type depends on the volatilization temperature of the atom of interest.

22. Flame Structure Different region or zone in flame are: Preheating zones Primary reaction zone (inner zone) Internal zonal region- max temp, Secondary reaction zone Interzonal region is the hottest part of the flame and best for atomic absorption. Oxidation of the atoms occurs in the secondary combustion zone where the atoms will form molecular oxides and are dispersed into the surroundings.

23. Copper calcium Potassium Cobalt

24. Flame AAS Advantages Short analysis time possible Good precision Easy to use Cheap Limitation Sensitivity Dynamic range Requires flammable gases Unattended operation is not possible because of flammable gases Must not contain excessive amounts of dissolved solids

25. Non flame atomizer (Electro Thermal Atomizer ) The graphite furnace is an electro thermal atomizer system that can produce temperatures as high as 3,000°C. The heated graphite furnace provides the thermal energy to break chemical bonds within the sample held in a graphite tube, and produce free ground state atoms. The ground-state atoms are capable of absorbing energy, in the form of light, and are elevated to an excited state. The amount of light energy absorbed increases as the concentration of the selected element increases

26. Graphite furnace technique Uses a graphite coated furnace to vaporize the sample. ln GFAAS sample, samples are deposited in a small graphite coated tube which can then be heated to vaporize and atomize the analytes . The graphite tubes are heated using a high current power supply.

27. Graphite furnace technique

28. Graphite Furnace AAS Atomizer Advantages Small sample sizes ( as low as 0.5 uL ) Very little or no sample preparation is needed High sensitivity due to entire sample is atomized at one time free atoms remain in the optical path longer Reduced sample volume Ultra trace analysis possible

29. Graphite Furnace AAS Atomizer Limitation Very slow Fewer elements can be analyzed Poorer precision More chemical interferences Method development requires skill Standard additions calibration required more frequently (compared to flame AA) Expensive consumables (graphite tubes)

30. Monochromators Important part in an AA spectrophotometer. It is used to separate out all of the thousands of lines. Without a good monochromator , detection limits are severely compromised. A monochromator is used to select the specific wavelength of light which is absorbed by the sample, and to exclude other wavelengths. The selection of the specific light allows the determination of the selected element in the presence of others. They are of two types: 1) Prism 2) Diffraction Grating

31. Grating monochromator :- it consists of a series of parallel straight lines cut into a plane surface Prism monochromator :- Quartz material is used for making prism, as quartz is transparent over entire region

32. Detector The light selected by the monochromator is directed onto a detector that is typically a photomultiplier tube , whose function is to convert the light signal into an electrical signal proportional to the light intensity. The processing of electrical signal is fulfilled by a signal amplifier. The signal could be displayed for readout , or further fed into a data station for printout by the requested format

33. DETECTOR Photomultiplier Tubes Components Made of a glass vacuum tube Photocathode Several dynodes One anode

34. How it works

35. Photodiodes. Photodiodes are solid-state photodetectors that are fabricated from photosensitive semiconductor materials such as (1) silicon, (2) gallium arsenide, (3) indium antimonide , (4) indium arsenide, (5) lead selenide, and (6) lead sulfide. These materials absorb light over a characteristic wavelength range (e.g., 250 nm to 1100 nm for silicon). Capable of measuring light at a multitude of wavelengths. .

36. Read-out Device The output from the detector is suitably amplified and displayed on a readout device like a meter or a digital display. It is capable of displaying the absorption spectrum as well absorbance at specific wavelength Nowadays the instruments have microprocessor controlled electronics that provides outputs compatible with the printers and computers Thereby minimizing the possibility of operator error in transferring data.

37. Operational of AAS A pulsed hollow cathode lamp and a tuned amplifier are incorporated into most AA instruments. Operationally , the power to the hollow cathode lamp is pulsed, so that light is emitted by the lamp at a certain number of pulses per second. On the other hand, all of the light originating from the flame is continuous. When light leaves the flame, it is composed of pulsed , unabsorbed light from the lamp and a small amount of nonplused flame spectrum and sample emission light. The detector senses all light, but the amplifier is electrically tuned to the pulsed signals and can subtract the background light measured when the lamp is off and the total light that includes both lamp and flame background light. In this way, the electronics, in conjunction with the monochromator , discriminates between the flame background emission and the sample atomic absorption.

38. Calibration Curve A calibration curve is used to determine the unknown concentration of an element in a solution.

39. Applıcatıons The are many applications for atomic absorption: Clinical analysis Analyzing metals in biological fluids such as blood and urine. Environmental analysis Monitoring our environment – e g finding out the levels of various elements in rivers, seawater, drinking water, air, and petrol.

40. Applıcatıons Pharmaceuticals . In some pharmaceutical manufacturing processes, minute quantities of a catalyst used in the process (usually a metal) are sometimes present in the final product. By using AAS the amount of catalyst present can be determined. Industry : Many raw materials are examined and AAS is widely used to check that the major elements are present and that toxic impurities are lower than specified Ex. in concrete, where calcium is a major constituent, the lead level should be low because it is toxic.

41. Applıcatıons Mining : By using AAS the amount of metals such as gold in rocks can be determined to see whether it is worth mining the rocks to extract the gold . Trace elements in food analysis Trace element analysis of cosmetics Trace element analysis of hair

42. Interference Interference is a phenomenon in which two waves superimpose to form a resultant wave of greater or lower amplitude. Interference decrease the intensity of absorption of light . Types of interferences 1) Spectral interference 2) Non –Spectral interference Non specific Specific

43. Spectral interferences Include absorption by other closely absorbing atomic species, absorption by molecular species, scattering by nonvolatile salt particles or oxides, and background emission (which can be electronically fitered ). Absorption by other atomic species usually is not a problem because of the extremely narrow bandwidth (0.01 nm) used in the absorption measurements.

44. Non spectral interference Nonspecific interferences A ffect nebulization by altering the viscosity, surface tension, or density of the analyte solution, and consequently the sample flow rate Specific interferences Also called chemical interferences because they are more analyte dependent.

45. Non Spectral I nterference Solute volatilization interference refers to the situation in which the contaminant forms nonvolatile species with the analyte . Example : phosphate interference in the determination of calcium that is caused by the formation of calcium–phosphate complexes. The phosphate interference is overcome by adding a cation, usually lanthanum or strontium; the cation competes with calcium for the phosphate. Dissociation interferences affect the degree of dissociation of the analyte . Analytes that form oxides or hydroxides are especially susceptible to dissociation interferences.

46. Non Spectral Interference Ionization interference 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. Controlled by adding a relatively high concentration of an element that is easily ionized to maintain a more consistent concentration of ions in the flame and to suppress ionization of the analyte . Excitation interference , Analyte atoms are excited in the atomizer, with subsequent emission at the absorption wavelength. This type of interference is more pronounced at higher temperatures.

47. Back ground correction In Zeeman background correction, the light source or the atomizer is placed in a strong magnetic field. In practice, because Zeeman correction requires special lamps, the analyte is placed in the magnetic field .. Th intense magnetic field splits the degenerate (i.e., of equal energy) atomic energy levels into two components that are polarized parallel and perpendicular to the magnetic field, respectively

48. Back ground correction

49. Atomic Emission Spectrophotometry

50. Flame photometry Also called Flame Atomic Emission Spectrometry A photoelectric flame photometer: an instrument used in determination of electrolytes such as metal ions: sodium , potassium, calcium, lithium Flame photometry is based on measurement of intensity of the light emitted when a metal is introduced into flame: The wavelength of color tells what the element is (qualitative) The color intensity tells us how much of the element present (quantitative)

51. Principle Basic principle: matter absorbs light at the same wavelength at which it emits light When a metal salt solution is burned, the metal provides a colored flame and each metal ion gives a different colored flame Simple flame tests can be used to test for the absence or presence of metal ion

52. Mechanism Flame photometry employs a variety of fuels mainly air, oxygen as oxidant The temperature of the flame depends on fuel-oxidant ratio The intensity of the light emitted could be described by the Scheibe Lomakin Equation I = k x cn where , I = intensity of emitted light c = concentration of element k = proportionality constant n~1 (at the linear part of the calibration curve) then I = k x c The intensity of emitted light is directly related to the concentration of the sample

53. Atomic Emission Spectrophotometry Various metals emit a characteristic color of light when heated

54. AAS Vs Flame photometry Atomic absorption Hollow cathode lamp as source of light Most of the atoms are in the ground state and are able to absorb light emitted by the cathode lamp. approximately 100 times more sensitive than flame emission methods . Unique specificity of the wavelength from the hollow cathode lamp, these methods are highly specific for the element being measured. Atomic Emission Burning flame as source of light Once atom is excited and upon reaching ground state emits specific wavelength light as the property of various elements Less sensitive Less specific

55. Atomic absorption depends upon the number of ground state atoms . It measures the radiation absorbed by the ground state atoms. The temperature in the atomizer is adjusted to atomize the analyte atoms in the ground state only. Atomic emission depends upon the number of excited atoms . It measures the radiation emitted by the excited atoms The temperature in the atomizer is big enough to atomize the analyte atoms and excite them to a higher energy level

56. References Tietz Textbook of CLINICAL CHEMISTRY MOLECULAR DIAGNOSTICS , Carl A. Burtis , Ph.D., Edward R. Ashwood , M.D,David E. Bruns , M.D, Fifth edition Methods in clinical chemistry , Kaplan L.A, Pesce A J Concepts, Instrumentation and Techniques in Atomic Absorption Spectrophotometry Richard D. Beaty and Jack D. Kerber, 2 nd edition. Spectrophotometry and Spectrofluorimetry , Michael. G. Gore https:// www.hitachihightech.com/global/products/science/tech/ana/aa/basic/course8.html http://vlab.amrita.edu/?sub=2&brch=193&sim=1351&cnt=1 Internet sources