Flame photometer, Atomic Absorbtion Spectrophotometer (AAS)

1. Dipesh Tamrakar
M.Sc. Clinical Biochemistry
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2. Principle, Components and applications of :
Flame Photometry
Atomic Absorption Spectrophotometry
2

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

4. Absorption Spectroscopy:
AAS
Emission Spectroscopy:
FES, ICP-AES(OES)
Mass Spectrometry
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5. Flame Emission Spectroscopy:
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6.  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.  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
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8.  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
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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
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10. 10

11. 1. Main Unit:
a. An atomizer
b. Mixing chamber
c. Burner
d. Optical filters
e. Photodetectors
f. Digital displays
g. Air regulator
h. Gas regulator
i. Gas pressure gauge
2. Compressor unit
11

12.  As seen in the figure, the flame may be
divided into following regions or zones
1. Preheating zones
2. Primary reaction zone (inner zone)
3. Internal zone- max temp, used for
flame photometry
4. Secondary reaction zone
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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
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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
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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
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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.
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18. Pre-mixed burner Total Consumption Burner
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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
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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
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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
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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.
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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.
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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.
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26.  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.
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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
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28.  Quality control when using a flame photometer
1. Proper sample collection
2. Proper dilution as per reagent supplies
3. Appropriate use and storage of controls
4. Regular cleaning of instrument before and after use
5. 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
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29. 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
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30. Atomic Absorption Spectroscopy:
AAS
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31. Elements detectable by AA are highlighted in pink
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32.  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.
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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.
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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.

35. 35

36.  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
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37. HOLLOW CATHODE LAMP
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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.
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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..
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41.  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;
1. Simultaneous multicomponent analysis
2. Determination of metallic elements in biological materials
3. Determination of metallic elements in food industry
4. Determination of calcium, magnesium, sodium and potassium in blood,
serum
5. Determination of lead in petrol
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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.
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43. 1. 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
2. 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.  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
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