1. Flame Spectrophotometry & Atomic Absorption Spectrometry
- Sailee Gurav MSc Biochemistry Part 1

2. ATOMS Atom : Smallest particle of an element.
Bohr’s shell model: SODIUM atom
n = 1 (K) Lowest energy level
n = 2 (L)
n = 3 (M) Highest energy level
Electrons
Nucleus
Shells
Nucleus- protons (+ve) and neutrons (neutral).
Electrons- (-ve) charged particle.
Shells- consists of subshells.

3. Flame Spectrophotometry
Also known as Flame emission /Flame photometry /Atomic emission spectroscopy.
Study of Radiant Energy
A flame by its heat can raise atoms from lower energy to an excited state of higher energy.
Emission through Radiation.
Determination of radiant energy.

4. Cont….
Spectrometer lines constitutes the emission spectrum of atom obtained.
Intensity of lines measured by photoelectric cell is qualitative and quantitative analysis.

5. Principle Process
Solution containing a metallic salt is aspirated into a flame
Evaporating solvent leaving the solid,
Dissociating solid by vaporization by gaseous atoms,
Raising atoms of the metal to higher energy level by heat of the flame,
Emitting energy in the form of radiation.
For e.g.:-
Orange color is imparted to the flame by calcium compounds.

6. Colors imparted to flame by various compounds

7. Instrumentation Basic components of a flame emission spectrophotometer

8. Nebulizers or atomizer

9. Nebulizers or atomizer :Samples before they can get into the flame must be converted into a fine spray i.e nebulized. The fine mist is then burnt in either laminar flow burner or total consumption burner.
The aerosol is desolvated ,vaporised and atomised in the flame of the burner.
In this some of the atoms are raised to a higher energy level. When these excited atoms fall to the ground state radiation is emitted.
The emitted radiation passes through a monochromator which selects a given emission line & isolates this line from other lines.
The intensity of the line thus selected is determined by a detecor photocell.
The output of the detector is amplified & read on a meter.

10. Burner : There are 2 types of burner in use.
Laminar flow the fine mist or aerosol of sample solution is produced in a vaporisation chamber.
The larger droplets of liquid formed fall out of the gas stream & are allowed to flow out to a waste.
The fine aerosol is mixed with the fuel gas and oxidant gas and sent to the burner head where it burns producing a flame.
Total consumption burner is 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 and the oxidant gas are sent to the burner head seperately and they mix only at the tip of the burner .It is simple to manufacture allows a totally representative sample to reach the flame and its free from hazards of explosion.

11. Total consumption burner

12. Flame Photometers
Monochromators : In sophisticated instruments prisms or sometimes diffraction gratings are used.However for routine analysis of such elements as calcium,sodium,potassium a simple filter might suffice.
Photocells: These are the usual detectors in a flame photometer. Unfortunately the flame instability reduces their accuracy.Therfore a multi channel polychromator is used in some routine procedures to allow measurement of up to six elements simultaneously.

13. Application
Flame photometry is useful for the determination of alkali and alkaline earth metals.
It is used in the study of electrolyte balance in physiology and in clinical analysis.
Used in determination of lead in petrol.
Used in the study of equilibrium constants involving in ion exchange resins.
Used in determination of calcium and magnesium in cement.

14. Atomic Absorption Spectrophotometry
AAS is a method of analysis based on absorption of radiation by atoms.
When a solution of a metallic salt is aspirated into a flame metal atoms in gaseous state are obtained.
In flame only small fraction of atoms are thermally excited.
When a beam of light is made to pass through the flame the dispersed atoms in the ground state absorb a part of the incident radiation much like a solution absorbing radiation passing through it.
Each element absorbs radiation that are characteristic to the element.

15. Thus if the sample solution contains sodium salt then the source of light must be sodium metal.
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.
Each element absorbs radiation that is characteristic of the element.
Therefore a separate lamp source is needed for each element.
Most commonly used source of light is hollow cathode lamp.

16. Hollow cathode lamp

17. 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 ionised.
The ions collide with the cathode surface and dislodge metal atoms from the surface.
Some of the metal atoms are in suffieciently 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.

18. Electrothermal atomiser Graphite furnace

19. Electrothermal atomiser
Electrically heated graphite rods are sometimes used instead of high temperature of flame to produce atoms from the experimental sample.This is called non flame technique.
Also called as graphite furnace.
The atomiser consists of a graphite tube about 50mm in length and about 10mm in internal diameter.
The tube is surrounded by a metal jacket through which water is circulated.
The tube is so arranged that the ray of light passes along the axis of the tube which is seperated from the metal jacket by a gas space.
Argon is generally circulated in the gas space.

20. The solution of the experimental sample is introduced by means of a micro pipette through a detachable window in the outer jacket and then into the graphite tube.
The graphite tube is carefully heated electrically to remove the solvent from the solution .
The current is then increased to first ash the sample and then to vaporise it to form metal atoms in the gaseous state.
These atomisers are quiet sensitive because the whole of the sample is atomised and atoms remain in the optical beam for about one second.

21. Schematic arrangement of a typical atomic absorption spectrophotometer

22. Atomic absorption spectrophotometry
A hollow cathode lamp supplies the necessary radiation.
A suitable line from the radiation is selected for the analysis.
This line is usually the most intense line in the emission spectrum and represents a transition from an excited to the ground state.
It is also the correct frequency absorption by atoms in the ground state in the flame. Such a line is called a resonance line.
The flame is also emitting source and the photo tube responds to radiation from the flame as well as from the hollow Cathode lamp and will create an interference in absorption measurements.
This problem is corrected by beam chopper . A chopper is a rotating mirror of such shape that permits the beam to pass through during one half of its period of rotation.

23. Chopper

24. During the other half the beam is reflected and not allowed to pass.
The result is that an intermittent pulsating beam is obtained.
Such a beam produces an alternating current in the photomultiplier tube.
The radiation from the flame is continous and will produce a direct current in the photo tube.
This direct current is not amplified.
The amplifier is tuned to amplify only the alternating current coming from the chopper.

25. A flame is produced by burning a fuel gas like acetylene or hydrogen in the presence of an oxidant which is usually oxygen.
A pre-mix or laminar type burner is generally used.
The sample solution is aspirated inot the flame by means of the nebuliser.
The beam passes through the flame and ground state metal atoms in the flame absorb the radiation.
The transmitted radiation is sent to the grating monochromator which allows only the resonance radiation to reach the photomultiplier tube .
The photo tube produces an electric current which amplified by the tuned amplifier.
The magnitude of the current is proportional to the intensity of the light incident on the phototube.
The current is read on a readout device which is usually caliberated to read transmittance or absorbance or both.
As in spectrophotometry distilled deionised water or the experimental blank is sprayed into flame and the transmittance ia adjsuted to 100% or absorbance zero.
The absorbance of the sample solution is then found by spraying the solution into the flame.

26. Applications of Atomic Absorption Spectroscopy
Water analysis (e.g. Ca, Mg, Fe, Si, Al, Ba content)
Food analysis
Analysis of animal feedstuffs (e.g. Mn, Fe, Cu, Cr, Se,Zn)
Analysis of additives in lubricating oils and greases (Ba,Ca, Na, Li, Zn, Mg)
Analysis of soils
Clinical analysis (blood samples: whole blood, plasma,serum; Ca, Mg, Li, Na, K, Fe)

27. Current Research
Flame Atomic Absorption Spectrometric Determination of Trace Amounts of Silver after Solid-Phase Extraction with 2- Mercaptobenzothiazole Immobilized on Microcrystalline Naphthalene
A simple and sensitive solid-phase extraction procedure combined with flame atomic was designed for the extraction and determination of trace amounts of silver absorption spectrometry .
A column of immobilized 2-mercaptobenzothiazole on microcrystalline naphthalene was used as the sorbent.
Silver was quantitatively retained on the column in the pH range of 0.5–6.0.
After extraction, the solid mass consisting of silver complex and naphthalene was dissolved out of the column with 5.0 mL of dimethylformamide, and the analyte was determined by flame atomic absorption spectrometry.

28. Current Research
Under the optimum experimental conditions, the adsorption capacity was found to be 1.18 mg of silver per gram of the sorbent. 
A sample volume of 800 ml resulted in a preconcentration factor of 160. 
The relative standard deviation obtained for ten replicate determinations at a concentration of 0.8 µg L−1 was 1.4%, and the limit of detection was 0.02 µg L−1.
The method was successfully applied to the determination of silver in radiology film, waste water, and natural water samples.
The accuracy was examined by recovery experiments, independent analysis by electrothermal atomic absorption spectrometry, and analysis of two certified reference materials.

29. References Principles of Instrumental Analysis, Biophysical Chemistry
A Harcourt Publishers,
Edition : 5th,
By Skoog,Holler,Nieman,
Chapter : 9th : Atomic Absorption,
Pages :
Biophysical Chemistry
Principles & Techniques,
Himalaya Publishing House ,
Edition : 6th (2012),
By Avinash Upadhyay, Kakoli Upadhyay, Nirmalendu Nath,
Chapter : 8th Spectrophotometry,
Pages :
Practical Biochemistry
Cambridge low-price editions,
Edition:5th,
Edited By Keith Wilson & John Walker,
Chapter: Spectroscopic Techniques,
Pages :

30. References College Analytical Chemistry, Himalaya Publishing House,
Edition : 19th (2011), By K.B.Baliga,S.A.Zaveri,Y.V.Ghalsasi,S.S.Mangaonkar,Deepak Teckchandani,Padma Sathe,
Chapter : 4th : Optical Methods,
Pages :
Current Research : Journal of Chemistry Volume 2013 (2013), Article ID 465825, 6 pages, by Farid Shakerian, Ali Mohammad Haji Shabani, Shayessteh Dadfarnia, and Mahdieh Shabani ,Department of Chemistry, Faculty of Science, Yazd University, Yazd,Iran. Received 16 March 2013; Accepted 7 May 2013
Academic Editor: Esteban P. Urriolabeitia

31. Thank You