Instrumentation

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
Electrons
n = 1 (K)
Lowest
energy level
n = 2 (L)
n = 3 (M)
Highest
energy level
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
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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
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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
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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
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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
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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
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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
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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
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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

23. Chopper

24.  During the other half the beam is reflected and
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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
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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
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Flame Atomic Absorption Spectrometric
Determination of Trace Amounts of Silver after
Solid-Phase Extraction with 2Mercaptobenzothiazole 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
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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

Biophysical Chemistry
Principles & Techniques,
Himalaya Publishing House ,
Edition : 6th (2012),
By Avinash Upadhyay, Kakoli
Upadhyay, Nirmalendu Nath,
Chapter : 8th Spectrophotometry,
Pages : 242-247.

Practical Biochemistry
Principles & Techniques,
Cambridge low-price editions,
Edition:5th,
Edited By Keith Wilson & John
Walker,
Chapter: Spectroscopic
Techniques,
Pages : 486-490.

Principles of Instrumental
Analysis,
A Harcourt Publishers,
Edition : 5th,
By Skoog,Holler,Nieman,
Chapter : 9th : Atomic
Absorption,
Pages : 206-225.

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 : 135-148.
 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
 http://www.hindawi.com/journals/chem/2013/465825/

31. Thank You