Flame photometer, Atomic Absorbtion Spectrophotometer

Flame photometer, Atomic Absorbtion Spectrophotometer (AAS)

1. Flame Photometry Atomic Absorption Spectrophotometry Dipesh Tamrakar M.Sc. Clinical Biochemistry 1

2. OVERVIEW Principle, Components and applications of : Flame Photometry Atomic Absorption Spectrophotometry 2

3. Basics: Atom is the smallest particle of an element Bohr’s shell model of sodium atom 3

4. Atomic Spectroscopy Absorption Spectroscopy: AAS Emission Spectroscopy: FES, ICP-AES(OES) Mass Spectrometry 4

5. Flame Emission Spectroscopy: 5

6. 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) 6

7. 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 7

8. 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 c n 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 8

9. Various metals emit a characteristic color of light when heated: ELEMENTS EMISION WAVELENGTH (nm) FLAME COLOR Barium (Ba) 554 Lime green Sodium (Na) 589 Yellow Calcium (Ca) 662 Orange Lithium (Li) 670 Red Potassium 766 Violet 9

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11. COMPONENTS Main Unit: An atomizer Mixing chamber Burner Optical filters Photodetectors Digital displays Air regulator Gas regulator Gas pressure gauge Compressor unit 11

12. As seen in the figure, the flame may be divided into following regions or zones Preheating zones Primary reaction zone (inner zone) Internal zone- max temp, used for flame photometry Secondary reaction zone Structure of flame 12

13. Nebulizers or atomizer: a means of transporting a homogeneous solution (converting into a fine mist) into the flame at a steady rate can be done by passing a gas of high velocity over the upper outlet of capillary tube, the lower end of which is inserted into the sample Liquid is then drawn up into the chamber and dispersed into small droplets The fine mist is then burnt in either laminar flow burner or total consumption burner The aerosol is desolvated , vaporized and atomized in the flame of the burner 13

14. A number of nebulisation methods are available like; Pneumatic nebulisation Ultrasonic nebulisation Electro thermal vaporization Hydride generation(used for certain elements only). Pneumatic Nebulizer is the most commonly used nebulizer 14

15. Burner (source): a flame that can be maintained in a constant form and at a constant temperature * should have ability to evaporate the liquid droplets from the sample solutions * must have capacity to excite the atoms formed and cause them to emit radiant energy a cylinder of compressed gas and two stage pressure regulator are required High pressure tubing must be used to lead the gases to the flame 15

16. Pre-mixed Burner: widely used because uniformity in flame intensity In this energy type of burner , aspirated sample , fuel and oxidant are thoroughly mixed before reaching the burner opening. The fine mist or aerosol of sample solution is produced in a vaporization chamber Larger droplets go to waste while the fine mist enters the flame, thus producing a less noisy single In addition, the path length through the flame of the burner is longer than that of the total consumption burner producing greater absorption and increases the sensitivity of the measurement 16

17. Total consumption burner: Made of 3 concentric tubes: The central tube is a fine capillary tube The sample solution is carried up by this tube directly into flame The fuel gas, the oxidant gas and sample solution is aspirated through a capillary by high pressure of fuel and Oxidant and burnt at the tip of burner In this fuel and oxidant are hydrogen and oxygen gases Entire sample is consumed. 17

18. Pre-mixed burner Total Consumption Burner 18

19. Flame temperature for various gas mixtures Gas mixture Flame temperature, ̊ C Natural gas + air 1840 Propane + air 1924 Hydrogen + air 2115 Acetylene + air 2250 Hydrogen + oxygen 2700 Natural gas + oxygen 2800 Propane + oxygen 2850 Acetylene + oxygen 3110 19

20. Slits: Entrance and exit slits Entrance slit : Cuts out most of the radiation from the surrounding Allows only the radiation from the flame to enter into the monochromator Exit slit : Placed after the monochromator Allows only the selected wavelength to pass through the photodetector 20

21. Monochromators : a means of isolating light of the wavelength to be measured from that of extraneous emissions When kept between the flame and detector, the radiation of the desired wavelength from the flame will be entering the detector and be measured Remaining will be absorbed by the monochromator and not measured Either prisms or diffraction gratings Prism : Quartz material is used for making prism, as quartz is transparent over entire region Grating : it employs a grating which is essentially a series of parallel straight lines cut into a plane surface 21

22. Detectors: a means of measuring the intensity of radiation emitted by the flame Should be sensitive to radiation of all the wavelengths that has to be analyzed The flame instability reduces their accuracy so multichannel polychromator is used in some procedures Photomultiplier tubes Photo emissive cell Photo voltaic cell Photo emissive cells or photomultiplier tubes are commonly employed for the purpose. Photovoltaic cell: It has a thin metallic layer coated with silver or gold which act as electrode, also has metal base plate which act as another electrode Two layers are separated by semiconductor layer of selenium, when light radiation falls on selenium layer. This creates potential diff. between the two electrode and cause flow of current. 22

23. Read-out Device: It is capable of displaying the absorption spectrum as well absorbance at specific wavelength The output from the detector is suitably amplified and displayed on a readout device like a meter or a digital display. 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. 23

24. INTERFERENCES: In determining the amount of a particular element present, other elements can also affect the result. Such interference may be of 3 kinds: Spectral interferences : occurs when the emission lines of two elements cannot be resolved or arises from the background of flame itself. They are either too close, or overlap, or occur due to high concentration of salts in the sample Ionic interferences : high temperature flame may cause ionization of some of the metal atoms, e.g. sodium. Ionization interference is 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. The Na + ion possesses an emission spectrum of its own with frequencies, which are different from those of atomic spectrum of the Na atom. 24

25. Chemical interferences: The chemical interferences arise out of the reaction between different interferents and the analyte. Includes: Cation-anion interference: The presence of certain anions, such as oxalate, phosphate, sulfate, in a solution may affect the intensity of radiation emitted by an element. E.g. calcium + phosphate ion forms a stable substance, as Ca3(PO4)2 which does not decompose easily, resulting in the production of lesser atoms. Cation-cation interference: These interferences are neither spectral nor ionic in nature Eg . aluminum interferes with calcium and magnesium. 25

26. Steps of carrying out test Sample preparation: Prepare a 1:500 dilution of the plasma sample in de ionized water. Calibrator Reagents Procedure: Set up the instrument with required filter and adjust the flame as described in instruction manual. Adjust the instrument to zero by DI water and obtain given values of calibrator on the standard and zero reading on the blank again Aspirate sample and note the reading at constant value Aspirate deionized water to remove all traces of sample, which might otherwise block the nebulizer. 26

27. Applications : It is used in the study of electrolyte balance in physiology and in clinical analysis To quantify metal atoms like Na, K, Li, Ca, Mg, Ba in body fluids To measure trace elements like Cu, Fe and Mg in biological samples Lithium estimation is required in some psychiatric disorder where it is used therapeutically To measure the amount of lead in petrol To quantify chromium in steel To determine calcium and magnesium in cement 27

28. Quality control when using a flame photometer Proper sample collection Proper dilution as per reagent supplies Appropriate use and storage of controls Regular cleaning of instrument before and after use Regular maintenance of instrument Recent advances: In recent years, the argon inductively coupled plasma (ICP) torch has become commercially available as an excitation source for emission photometry With this source, argon ions are inductively coupled to radio-frequency generator that serves as the means to excite ions and molecules to energy status that will produce light emission 28

29. Advantages vs disadvantages of FES Advantages Simple quantitative analytical tool Cheaper Easy and convenient method of determination of alkali and alkaline earth metals Selective and sensitive method Disadvantages Inaccurate measurement Special standard solution is required with corresponding emission spectra Difficulty in measuring very high or low concentration samples Limited range of element detection Limited atomization so only used for alkaline and alkaline earth metal Need Perfect control over flame temperature Interference of other elements can not eliminated Complex spectra heavy metals 29

30. Atomic Absorption Spectroscopy: AAS 30

31. Atomic absorption spectroscopy (AAS) Elements detectable by AA are highlighted in pink 31

32. Atomic absorption spectrophotometer Atomic absorption (AA) spectrophotometry is used widely in clinical laboratories to measure elements such as aluminum, calcium, copper, lead, lithium, magnesium, zinc, and other metals. Atomic absorption is an absorption spectrophotometric technique in which a metallic atom in the sample absorbs light of a specific wavelength. Used to measure concentration by detecting absorption of electromagnetic radiation by atoms rather than by molecules The absorption of radiation by atoms also follows Beer-Lamberts law i.e absorbance is directly proportional to the concentration of atoms in the flame and to the path length in the flame. 32

33. Each element absorbs radiation that are characteristic to the element. Therefore a separate lamp source is needed for each element. Most commonly used source of light is hollow cathode lamp. 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. 33

34. 34 Light source – hollow cathode lamp. Each element has its own unique lamp. Atomic cell – flame (gas mixture) or graphite furnance (accepts solutions, slurries, or even solids). Detector – photomultiplier .

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36. Hollow cathode lamp It consists of a tungsten anode and a hollow cylindrical cathode sealed in a glass tube containing an inert gas such as argon or neon at a low pressure. The cathode is made of the same metal as the one under consideration. When a high potential is applied across the electrode the inert gas is ionized. The ions collide with the cathode surface and dislodge metal atoms from the surface. Some of the metal atoms are in sufficiently excited state to emit their characteristic radiation. This appears as a glow inside the hollow cathode space. Such cathodes allows the analysis of more than one element. An argon-filled lamp produces a blue to purple glow during operation, and the neon produces a reddish-orange glow inside the hollow cathode lamp 36

37. HOLLOW CATHODE LAMP 37

38. Quartz or special glass that allows transmission of the proper wavelength, is used as a window. A current is applied between the two electrodes inside the hollow cathode lamp, and metal is sputtered from the cathode into the gases inside the glass envelope. When the metal atoms collide with the neon or argon gases, they lose energy and emit their characteristic radiation. Calcium has a sharp, intense, analytical emission line at 422.7 nm, which is used most frequently for calcium analysis. In an ideal interference-free system, only calcium atoms absorb the calcium light from the hollow cathode as it passes through the flame. 38

39. A electric beam chopper 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 non-pulsed 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. 39

40. In flameless AA techniques (carbon rod or “graphite furnace”), the sample is placed in a depression on a carbon rod in an enclosed chamber. In successive steps, the temperature of the rod is raised to dry, char, and finally atomize the sample into the gas phase in the chamber. The atomized element then absorbs energy from the corresponding hollow cathode lamp. This approach is more sensitive than conventional flame methods and permits determination of trace metals in small samples of blood or tissue. With flameless AA, a novel approach used to correct for background absorption is called the Zeeman 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.. 40

41. APPLICATIONS OF AAS: It is routinely used to measure concentration of trace metals that are not easily excited. Determination of even small amounts of metals (lead, mercury, calcium, magnesium, etc AAS has a various applications in every branch of chemical analysis; Simultaneous multicomponent analysis Determination of metallic elements in biological materials Determination of metallic elements in food industry Determination of calcium, magnesium, sodium and potassium in blood, serum Determination of lead in petrol 41

42. Advantages; Sensitive, accurate, precise, and specific In general, Atomic absorption methods are approximately 100 times more sensitive than flame emission methods. These methods are also highly specific for the element being measured. Disadvantage; inability of the flame to dissociate samples into free atoms. 42

43. Interferences in AAs Spectral interference Occurs when absorption by other closely absorbing atomic species It is generally not a problem as resolved by narrow bandwidth measurement Problematic at lower atomizing temperature Nonspectral interference Either Nonspecific or specific Viscosity, surface tension, density of analyte, flow rate alteration and sample contamination causes non specific interference Specific or chemical interferences Solute volatization interference Dissociation interferences Ionization interferences Excitation interference 43

44. AAS Vs flame photometry Atomic absorption Hollow cathode lamp as source of light Only a small fraction of the sample in the flame contributes emission energy and only a fraction of this is transmitted to the detector. Hence, most of the atoms are in the ground state and are able to absorb light emitted by the cathode lamp. In general, AA methods are approximately 100 times more sensitive than flame emission methods. In addition, owing to the 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 44

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