Forensic Biology & Its biological significance.pdf
Atomic Absorption Spectroscopy (AAS)
1. ATOMIC ABSORPTION
SPECTROSCOPY
Dr. V.U. Barge Madam Name of Student.: Parijat S. Suryawanshi
(Guide) Dept.: Quality Assurance Technique
Year .: First Year M.Pharmacy Sem-1
Subject.: Modern Pharmaceutical
Analytical Techniques
Parijat
Suryawanshi
2. INTRODUCTION
In the mid-1950's by Alan Walsh , atomic absorption spectroscopy has proved itself to be
the most powerful instrumental technique for the quantitative determination of trace
metals in liquids.
Atomic absorption spectroscopy is a method of elemental analysis. It is particularly useful
for determining trace metals in liquids and is almost independent of the molecular form of
the metal in the sample.
Example, we can determine the total cadmium content of a water sample-it does not
matter whether the cadmium exists as a chloride, nitrate, sulfate, or other salt.
The method is very sensitive and can detect different metals in concentrations as low as
and frequently lower than 1ppm and the comparative ease with which quantitative
results can be obtained.
By this technique, the determinations can be made in the presence of many other
elements. It means that it becomes unnecessary to separate the teat element from the
other elements present in the sample and thus it saves a great deal of time and in the
process eliminates several sources of error.
As atomic absorption spectroscopy does not demand sample preparation, it is an ideal
tool for non chemist also, e.g., the engineer, biologist or clinician are interested only in the
significance of the results.
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3. PRINCIPLE
Energy absorbed by ground state atom in gaseous forms the atomic absorption
spectroscopy.
Following are simplified version of events :-
I. A solution containing metallic species is introduced into a flame.
II. The vapour of metallic species will be obtained.
III. Some of the metal atoms may be raised to an energy level sufficiently high to emit the
characteristic radiation. (a phenomenon that is utilized in the familiar technique of
emission flame photometry). But a large percentage of the metal atoms will remain in the
non-emitting ground state.
IV. These ground state atoms of a particular element are receptive of light radiation of their
own specific resonance wavelength (in general, the same wavelength as they would
emit if excited).
V. When a light of this wavelength is allowed to pass through a flame having atoms of the
metallic species, part of that light will be absorbed.
VI. Absorption will be proportional to the density of the atoms in the flame. Thus in AAS, one
determines the amount of light absorbed.
VII. Once this value of it atom is known, the concentration of the metallic element can be
known because the absorption is proportional to the density of the atoms in the flame.
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4. VIII. The total amount of light absorbed may be given by the expression as follows:
Total amount of radiation absorbed by atom at particular frequency ( )
=
𝝅𝒆²
𝒎𝒄
𝑵𝒇 …………………………… (1)
where, e = is the charge on the electron
m = mass of atom
c = the speed of light
N = the total number of atoms that can absorb at frequency v in the light path
f = the oscillator strength or ability for each atom to absorb at frequency,( )
As π, e, m and c are constants, equation (1) can be simplified to the following expression:
Total amount of radiation absorbed = constant x N x f …………….... (2)
From above equation we can conclude that, it follows that the absorption by atom is
independent of the wavelength of absorption and the temperature of the atoms.
These two features provide atomic absorption spectroscopy distinct advantages over flame
emission spectroscopy.
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7. Radiation Source
Ideal Characteristics :-
I. It should emit stable, intense radiation of the element to be determined.
II. The resonance spectral lines should be narrow as compared with the width of the
absorption lines to be measured.
III. There should be no general background or other extraneous lines emitting within the
band pass of the monochromator.
IV. The problem of using such narrow spectral lines has been solved by adopting a hollow
cathode lamp as the radiation source.
A. Hollow Cathode Lamp :-
The cathode consists of a hollow cup. In the cup is the element which is to be determined,
(in this case sodium). The anode is a tungsten wire.
The two electrodes are housed in a tube containing an inert gas.
The lamp window is constructed of either quartz, silica, or glass. The exact material
depends upon the wavelength which is to be transmitted.
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8. I. When a dc voltage of 300-500 V applied to between two electrodes cathode and
anode. Current in milli-ampere range arises.
II. The inert gas is charged at the anode.
III. Charged gas is attracted at high velocity to the cathode
IV. Impact with the cathode vaporised some of the Na-atom
V. These are excited and upon returning to ground
state give rise to Na-emission spectrum.
VII. Thus, the emission spectrum produced by a hollow
a lamp is a sharp line spectrum of the cathode material
and the filled gas.
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9. B. Electrodeless Discharge Lamp :-
It is difficult to make stable hollow cathodes from certain elements, particularly those that
are volatile, such as arsenic, germanium, or selenium. An alternative light source has
been developed in the electrodeless discharge lamp (EDL).
It 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.
The sealed tube is then placed in a microwave discharge
cavity.
Under these conditions the argon becomes a plasma
and causes excitation of the metal sealed inside the tube.
The emission from the metal is that of its spectrum,
including the resonance line.
The intensity of these lamps is very high,
and they have been made quite stable in recent years.
Fig: EDL with vacuum jackets;(a)Dismountable (b)Permanent
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10. CHOPPER
A rotating wheel is interposed between the hollow cathode lamp and the flame.
This rotating wheel is known as chopper and is interposed to break the steady light from
the lamp into an intermittent or pulsating light.
This gives a pulsating current in the photocell.
The absorption of light will be measured without interference from the light emitted by
the flame itself.
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11. Atomisers
In order to achieve absorption of atoms, it becomes necessary to reduce the sample
to the atomic state. This is done by -
(a) Flame Atomiser
(b) Non-Flame Atomiser
Flame Atomiser :-
The most common way is to use a flame which is used for converting the liquid sample
into the gaseous state and also for conversion of the molecular entities into an atomic
vapour.
There are two types of burners in common use, which is as follows :-
I. Total Consumption Burner :-
In this type of burner the sample solution, the fuel and oxidizing gases are passed through
separate passages to meet at the opening of the base of the flame.
As the sample containing metallic element to be estimated is a liquid, the flame breaks
up the liquid sample into droplets which are then evaporated or burnt, leaving the
residue which is further reduced to atom.
Total consumption burners do use oxygen, with hydrogen or acetylene, and give very hot
flames
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12. This burner is noisy and hard to use.
The efficiency of this burner is not very good.
II. Premixed burner :-
In this burner a mixture of the sample (liquid)
and premixed gases (C,H,+02) is used.
The premix burner is very suitable for the
atomic absorption studies of metals of
groups I A, I B and II B,
together with Ga, In ,Ti ,Pb ,Te , Mn ,Ni ,Pd.
Long path provides sensitivity.
Quiet operation. Little tendency to clog.
Disadvantage :-
1. Rate of sample induction is low
2. Possibility of explosion in mixing chamber.
3. Selective evaporation of mixed solvent
can lead to analytical error.
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13. Carbon Atomiser :-
In 1961 B.V. L'vov built an atomizer using a carbon rod heated with an electrical
discharge. (Fig.: High–temperature furnace as designed and used by L’vov)
This system was orders of magnitude more sensitive than flame atomizers, but was difficult
to control enough to provide quantitative data.
The method was later modified by workers , two typical commercial atomiser are made
with process used is similar.(Fig.: (A) Heated graphite tube atomiser ; (B) Carbon Filament
atom reservoir)
I. Sample loaded of the order of 2-30 µl.
II. It is then warmed gently to remove the solvent.
III. The temperature is then increased under controlled conditions to ash the sample and
remove most of the organic material present.
IV. Finally the sample is heated rapidly to very high temperatures to cause atomization. The
free atoms are vaporized from the carbon atomizer into the optical light path, where
their absorption is measured.
Atomisation process is extremely fast and must be rigidly controlled.
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15. L’vov Platform :-
The precision of the carbon atomizer has been improved by the use of the L'vov platform.
In this system a carbon platform is inserted into the standard atomizer.
During the ashing and atomization step the metal atoms tend to condense on the
platform, which is cooler than the electrically heated furnace.
After a short delay the platform becomes by radiation from the inside of the furnace and
its temperature rises.
At the increased temperatures the condensed metal atoms are revaporized and entered
into the light path. The reproducibility of the procedure is improved by the L'vov platform.
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16. BACKGROUND CORRECTION
Most of the spectral interferences and all other types of interferences result in the
attenuation of AAS signal to some extent.
In general the attenuation varies from negligible to several percent depending upon the
matrix.
This signal is known more commonly as background absorption which can be easily
estimated by aspiring a closely matching reference solution which does not contain the
analyte.
The absorbance of the reference (or blank as it is known) must be subtracted from that of
the calibration standards as well as the samples.
Alternately radiation from a deuterium lamp can be measured at the resonance
wavelength to determine the background absorption.
When the intensity of the signal from the deuterium source lamp is subtracted from the
intensity of the element cathode lamp, the intensity due to the sample alone is obtained.
Utilization of this principle has led to development of background correctors.
The ray from a deuterium or hydrogen lamp are pulsed alternatively with those from the
element cathode lamp.
Continuous corrected absorbance values are obtained on read-out device.
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18. NEBULIZATIONS OF THE LIQUID SAMPLE
The formation of small droplets from the liquid sample is called nebulisation.
Before the liquid sample enters the burner, it is first of all converted into small droplets.
A common method of nebulisation is by use of a gas moving at high velocity, called
pneumatic nebulisation.
The Beckman total consumption burner is commonly used in atomic absorption
measurements.
In the burner, a back pressure of about
250 torr occurs at the tip of the burner
due to the high velocity of the aspirating
gas as it emerges from the office.
As the liquid is drawn up the capillary,
it is broken into droplets by the high
velocity gas stream.
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19. MONOCHROMATOR
In AAS, the most common monochromators are prisms and gratings.
The function of a monochromator is to select a given absorbing line from spectral lines
emitted from the hollow cathode. In some cases large dispersion and high resolving
monochromators are advantageous for resolving spectra.
Commercially packaged atomic absorption instrumentation commonly includes a
monochromator of about 4 m focal length with a linear reciprocal dispersion in the range
16-35 ºA/mm.
DETECTORS
For AAS, the photomultiplier tube is most suitable.
It has good stability if used with a stable power supply. It works satisfactorily and enables to
compare intense in a satisfactory manner.
In the photomultipliers tube, there is an evacuated envelope which contains a photocathode, a
series of electrodes called dynodes, and an anode.
PHOTON Strikes e- is dislodged Photon is accelerated Liberation Of 2 or more e- from
on photon cathode to Dynode I e- from Dynode I Dynode I to
Dynode II
Amplifier & Received Thus current multiplied Liberation of more e-
Read-Out by Anode at each dynode
system
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21. AMPLIFIER
The electric current from the photomultiplier detector is fed to the amplifier which
amplifies the electric current many times.
Generally, Lock-in amplifiers are preferred which provide a very narrow frequency band
pass and help to achieve an excellent signal-to-noise ratio.
Read-Out Device
In most of the atomic absorption measurements, chart recorders are used as read-out devices.
A chart recorder is a potentiometer using a servo motor to move the recording pen. The
displacement is directly proportional to the input voltage.
In. some atomic absorption measurements, digital read-out devices are the used.
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22. SAMPLING TECHNIQUES
Solid samples :-
In general, solid samples must first be dissolved and the solution then analyzed.
Liquids :-
Frequently liquids can be analyzed.
Typical samples that have been analyzed directly include blood, urine, electroplating
solutions, petroleum products, wines, and pollutants in water.
A calibration curve should always be prepared from a solution of the same solvent as the
sample.
If the samples are too concentrated, they may be diluted prior to analysis. If they are too
dilute, they may be evaporated down or concentrated by, solvent extraction.
Gas sample :-
In conventional samples the metal components must first be collected from gas samples
by absorption or by trapping in a solution. The absorbant or solution may then be
analyzed. Metals in air samples have been analyzed by atomic absorption after first
trapping the metal component in a suitable solvent.
Hydride Analysis - Some metals are first converted to a gaseous hydride. These are
introduced directly into the atomizer and analyzed the normal way using special
equipment. Metals commonly analyzed this way include arsenic, selenium, and tellurium.
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23. INSTRUMENTS
Single Beam atomic Absorption Spectrometer :-
1. In single beam equipment, a light source is placed ahead of the flame with a
mechanical chopper between the light source and the flame.
2. When there is no sample in the flame, the output of the detector-amplifier unit is
arranged to give full deflection.
3. After this the sample solution is sprayed into the flame and the output of the detector
amplifier is recorded.
4. Disadvantage :- Low stability.
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24. Double Beam Atomic absorption Spectrometer :
1. To get increased stability one can use double beam atomic absorption spectrometer.
2. The chopped beam from the hollow cathode lamp is divided into two parts, one part
goes through the flame while, the other part bypasses it.
3. After recombination, the two beams pass through a monochromator to a detector and
readout system.
4. Advantages :-
There is no effect of lamp drift.
There is no change in detector
sensitivity with time.
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25. DETECTION LIMIT & SENSITIVITY
The sensitivity in atomic absorption may be defined as the concentration of element
present in the sample solution which produces 1% absorption.
The "sensitivity for 1 percent absorption" is a theoretical number and will vary with
efficiency of the lamp, atomiser, flame system, monochromator, and photomultiplier.
The sensitivity is generally expressed in terms of µg/ml for 1% absorbance. The sensitivity
for one percent absorbance is determined by employing the following expression.
𝑪𝟏% =
𝑪𝟎.𝟏 ×𝟎.𝟎𝟎𝟒𝟒
𝟎.𝟏
where 𝐶1% is the concentration that gives rise to 1 % absorption
𝐶0.1 is the concentration that gives rise to an absorbance of 0.1
The detection limit may be defined as the concentration (µg/ml) of an element which
results in the shifting of absorbance signal to an amount that equals to the peak-to-peak
noise of the base line.
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26. INTERFERENCES
A. Spectral interferences :-
This type of interference may be caused by overlapping of any radiation with that of
characteristic radiation of the test element to he estimated.
Examples :- Serious overlapping is the manganese triplet (4031, 4033 and 4035 A), the
gallium line (4033 A) and potassium doublet (4044, 40478).
This type of overlapping can be overcome by selecting other spectral lines or by prior
chemical separation.
If the spectral interference is due to sample matrices or flame components, one can
overcome this by working with AC amplifiers tuned to the frequency at which the source
is chopped or modulated.
B. Chemical, ionization and bulk (Matrix) interference :-
If compounds or complex ions of the element being determined incompletely dissociate
into their atoms, low results will occur.
The more concentrated the solution the greater will be the deviation from the correct
value. This incomplete dissociation is a chemical interference.
It might be removed by the use of a higher flame temperature. In situations where a
hotter flame cannot be utilized, chemical means are suggested.
Example, aluminium and magnesium form a thermally stable mixed oxide.
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27. C. Ionization interference :-
It arises in due to the flame temperature is too high.
When this occurs, a number of the vaporized atoms become ionized by the flame. The
resulting ions absorb at a different wavelength than the vaporized atoms, the new
wavelength will not be selected by the monochromator, and low values result.
The interference is usually minimized by the addition of a more easily ionizable element.
Example, ionization interference of calcium may be corrected for by the addition of large
quantities of sodium or potassium salts to the solution.
D. Matrix or Bulk interference :-
A change in the viscosity of the solution caused by either the change in a solvent or a
change in concentration may result in a matrix or bulk interference.
An increase in concentration results in an increase in viscosity, a slower flow through the
burner, and a corresponding decrease in absorbance.
The viscosity changes would also cause corresponding intensity changes in emission
measurements.
Both ionization and bulk interferences are greatly affected by burner design.
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28. E. Solvent interferences :-
In general, metals in aqueous solutions given lower absorbance regarding than the same
concentration of such metals in an organic solvent.
If the solvent is an organic solvent, such as acetone, alcohol, ether, or a hydrocarbon,
the solvent not only evaporates rapidly, but may also burn, thus increasing the flame
temperature.
The atomization process is more efficient. More free atoms are produced from this system.
An higher absorption signal is registered from organic solvents than from aqueous
solutions, even though the metal concentration in the two solution is equal.
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29. F. Dissociation of metal compounds :-
When metals like La, AI and Ti are aspirated into the flame, metal atoms are not obtained
but extremely stable refractory oxides are obtained.
Thus, the atomic absorption studies with these elements become complicated.
For such elements, nitrous oxide-acetylene flames are used which could dissociate these
metal oxides to enable analysis of these elements by atomic absorption spectrometry.
G. Role of solvent :-
Another potential source of interference is the solvent.
In general, metals in aqueous solutions yield lower absorbance readings than the same
concentration of such metals when present in an organic solvent.
The main reason for this is that metal is more difficult to atomize from an aqueous solution
than from an organic solution.
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30. APPLICATION
The technique is already a firmly established procedure in analytical chemistry, ceramics,
mineralogy, biochemistry , water supplies , metallurgy and soil analysis.
1. Qualitative Analysis :-
In atomic absorption spectroscopy, a different hollow cathode lamp is to be used for
each element to be tested. It means that an element which is used in the construction of
cathode of the hollow cathode lamp, can be detected only.
As qualitative analysis involves the checking of one element at a time, it means that the
process is very laborious. Therefore, atomic absorption spectroscopy is hardly used in
practice for this purpose.
2. Quantitative Analysis :-
The technique of quantitative analysis is based on the determination of the amount of
radiation absorbed by the sample.
As the efficiency of producing atoms from a sample cannot be known, it means that N
cannot be used to calculate the concentration of the element in the sample. In
practice, quantitative measurements are generally based on calibration curves.
Calibration Curves :- The first job in quantitative analysis is the preparation of calibration
curve. In order to prepare this curve, the read-out device should be adjusted to 100%
transmittance with a blank and 0% transmittance when no radiant energy is entering the
monochromator slit.
Absorbance = Slope x Concentration
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31. This parameters that influence a quantitative determination and can be controlled by
analyst are as follows :-
i. Flame system :- Optimum and uniform flame conditions can be obtained by selecting
the height of the flame, setting the oxidising gas to a fixed flow rate and regulating the
flow of a fuel so that peak absorption is obtained when a suitable standard solution is
sprayed into the flame.
ii. Wavelength :- The choice of wavelength is also an important parameter in the
determination of any element. In most of the cases it is limited to the selection of the
most absorbing line, although in certain cases the choice requires more consideration.
iii. Solution :- The constitution of standard and test solutions can be adjusted to increase the
sensitivity .
3. Simultaneous Multicomponent Analysis :-
If a multi-element emission source is available, one can do simultaneous multicomponent
analysis. Mitchell (1973) described a multi-element atomic absorption system using a
multi-element hollow cathode source and a vidiocon detection system.
Using a spectral region from 2320 to 3281A, Mitchell detected eight elements (Zn, Cd, Ni,
Co, Fe, Mn, Cu, and Ag) simultaneously. In the near future such a system may well be
developed to provide quantitative results.
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32. 4. Determination of Metallic Elements in Biological Materials :-
The atomic absorption spectroscopy is becoming a very important tool for the
determination of trace metals in biological materials.
5. Determination of Metallic Elements in Food Industry :-
Copper, zinc, and nickel are the most common toxic elements of interest to food analyst.
For solid foodstuffs the most common procedure is to extract the trace metals by
digestion with the dilute sulphuric acid or with nitric acid or with 50% hydrogen peroxide.
6. Determination of Calcium, Magnesium, Sodium and Potassium in Blood Serum :-
The determination of these elements in blood serum is of vital importance in diagnosing
many pathological conditions, diabetes, and primary aldosterionism.
Atomic absorption method is regarded as the most suitable method for determination of
all these elements in blood serum.
The determination of sodium and potassium in blood serum can also be measured by
atomic absorption spectrophotometer by operating it in the emission mode. By emission,
both elements can be readily determined in sera at dilutions of 50 or 100: 1.
7. Determination of Lead in Petrol :-
In petrol the two antiknocking additives are tetraethyl and tetramethyl lead.
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33. ADVANTAGES
I. The atomic absorption technique is specific because the atoms of a particular element
can only absorb radiation of their own characteristic wavelength.
II. Atomic absorption spectroscopy is independent of flame temperature.
III. Detection of nonmetals by AAS :- Atomic absorption cannot be used for direct
determination of nonmetal but use of capillary discharge lamp, iodine has been
measured directly.
IV. However nonmetals has been determined by indirect method. E.g. – chlorides can be
precipitated as silver chloride; from the subsequent determination of silver, the chloride
can be calculated.
DISADVANTAGES
I. A separate lamp for each element to be determined is requited.
II. This technique cannot be used very successfully for estimation of elements like Al, Ti, W, Mo, Si ,
etc., because these elements give rise to oxides in the flame. However, the estimations can be
carried out under modified conditions.
III. In aqueous solutions, the predominant anion affects the signal to a negotiable degree.
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34. The fundamental difference between emission
spectroscopy and absorption spectroscopy may be
defined as :
I. For emission to occur, a number of atoms must be in the excited
state.
II. For atomic absorption to occur, a number of atoms must be in
ground state.
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35. DIFFERENCE BETWEEN AAS vs FES
Sr.
No.
Atomic Absorption Spectroscopy Flame Emission Spectroscopy
1. Amount of light absorbed by ground state
atoms is measured.
Amount of light emitted by excited atom is
measured.
2. Absorption intensity and signal response
does not depend upon temperature.
Absorption intensity and signal response
greatly influenced by temperature
variation.
3. Beer’s law is obeyed. Beer’s law is not obeyed.
4. Intensity of absorbed radiation is directly
proportional to number of atoms in ground
state.
Intensity of emitted radiation is directly
proportional to number of atoms in excited
state.
5. Absorption intensity vs concentration of
analyte is much linear.
Relation between emission intensity vs
concentration is not much linear.
6. Atomization flame is used. Atomization & excitation flame is used.
7. Absorbance vs concentration data is
obtained.
Intensity vs concentration data is
obtained.
8. Useful for more than 70 metals. Limited to alkali & alkali earth metals.
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