Atomic Absorption Spectroscopy

(AAS)



Topics to be covered


+Importance of elemental analysis .

+ Introduction to spectroscopy.


+ Atomic Absorption Spectroscopy (AAS
+ Atomic Emission Spectroscopy (AES).

+ Inductively Coupled Plasma Spectroscopy (ICP).

Importance of elemental analysis
Monitoring levels of certain elements in samples ( eg.
pharmaceutical products and standards) to detect
the concentrations of these elements.
homodyalysis
protonialdyalysis
ringer lactate
Monitoring the levels of the toxic elements in
samples ( eg. cosmetics, food supplements, entire
plant or part of it) to ensure its safety.

With these information, we can take steps to approve
or withdraw products from the markets.
Widely used in clinical chemistry and environmental
laboratories.

Elements (Heavy Metals)

The term heavy metal refers to any metallic
chemical element that has a relatively high density
and is toxic or poisonous at low concentrations.
They have a specific gravity that is at least 5 times
the specific gravity of water.
Example: Arsenic 5.7,Cadmium 8.65 , Iron 7.9,
Lead 11.34 Mercury 13.546








Trace elements :
Heavy metals that are nutritionally essential for
a healthy life.
Examples are ( Iron, Manganese, Copper and
Zinc).
Commonly encountered toxic heavy
metals


+ Arsenic



+ Lead


+ Mercury

+ Cadmium



+ Iron



+ Aluminum
Types of samples for analysis

+ Pharmaceutical
product


+ Standards


+ Cosmetics


+Food
supplements





+Entire plant
or part of it


+ Mixture of
known & unknown
herb

Spectrometric methods are a large group of
analytical methods
Spectroscopy is the science that deals with the
interactions of radiation with matter (atomic and
molecular).

The most widely used spectrometric methods
are based on electromagnetic radiation (light,
gamma rays, X-rays, UV, microwave, and radio-
frequency).
Spectroscopy

Electromagnetic Radiation:


+ consists of discrete packets of energy, which
we call photons
+ A photon consists of an oscillating electric
field component, E, and an oscillating
magnetic field component, M.


+ The characteristics of these fields are:

Orthogonal ( perpendicular ) to each other
Orthogonal to the direction of propagation
of the photon





They flip direction as the photon travels
All photons (in a given, non-absorbing
medium) travel at the same
velocity, v.
What is Frequency (v)?
The number of flips, or oscillations, that occur in one
second.
What is A Wavelength ()?
The physical distance in the direction of
propagation over which the electric and magnetic fields
of a photon make one complete oscillation.
Unit: Angstrom, nm, m

Velocity Of Light = 2.99792 x 10
8
m/s

The electromagnetic nature of all photons is the same,
but photons can have different frequencies
The relationship between the light velocity, wavelength,
and frequency is :





The energy, E, of one photon depends on its frequency of
oscillation :


where h is Planck's constant (6.62618x10-34 Js)


v=v

E = hv = hv /
The relationship between the speed of light c ,
wavelength, and frequency is :



When light passes through other media, the velocity
of light +

Since the energy of a photon is fixed, the frequency of
a photon does not change.

Thus for a given frequency of light, the wavelength
must + as the velocity decreases.



The decrease in velocity is quantitated by the
refractive index, n, which is the ratio of c to the
velocity of light in another medium, v:

C=v

n = c / v
Electromagnetic Spectrum:

1- Absorption of Radiation
When radiation passes through a layer of
solid, liquid, or gas, certain frequencies may
be absorbed, a process in which
electromagnetic energy is transferred to the
sample.

Absorption promotes these particles
from their ground state to more higher-
energy excited state.

Tow types of absorption spectra:
Atomic absorption spectrum.
Molecular absorption spectrum.

Beers Law
Many compounds absorb radiation. The
diagram below shows a beam of
monochromatic radiation of radiant power P
0

directed at a sample solution.

Absorption takes place and the beam of
radiation leaving the sample has radiant
power P.

The amount of radiation absorbed may be
measured in a number of ways:

Transmittance, T = P / P
0
% Transmittance, %T = 100 T

Absorbance,
A = log
10
P
0
/ P
A = log
10
1 / T
A = log
10
100 / %T
A = 2 - log
10
%T

The last equation, A = 2 - log
10
%T , is
worth remembering because it allows
you to easily calculate absorbance from
percentage transmittance data.
The relationship between absorbance
and transmittance is illustrated in the
following diagram:

The equation representing the Beers law:
A = b c
Where
A is absorbance (no units, A = log
10
P
0
/ P ).

is the molar absorbtivity (is a measure of
the amount of light absorbed per unit
concentration) with units of L mol
-1
cm
-1
.

b is the path length of the sample that is, the

path length of the cuvette in which the sample
is contained. We will express this
measurement in centimeters.

c is the concentration of the compound in

solution, expressed in mol L
-1
.


Beers law tells us that absorbance depends
on the total quantity of the absorbing
compound in the light path through the
cuvette. If we plot absorbance against
concentration, we get a straight line passing
through the origin (0,0).

The working curves are used to

1.Determine the concentration of an
unknown sample.

2.To calibrate the linearity of an
analytical instrument.
What are the Processes by which a molecule
can absorb radiation?

1- Rotational transition:
The molecule rotate about various axes, the
energy of rotation being at definite energy
levels, so the molecule may absorb radiation
and be raised to a higher rotational energy level.




2- Vibrational transition:

The atoms or group of atoms within a
molecule vibrate relative to
each other. The molecule may then absorb a
discrete amount of energy
and be raised to a higher vibrational energy
level..
3- Electronic transition:

The electrons of molecule may be raised to a
higher electron Energy.






The three types of internal energy are
quantized

Type of Radiation Frequency Range (Hz) Wavelength Range Type of Transition
gamma-rays 10
20
-10
24
<1 pm nuclear
X-rays 10
17
-10
20
1 nm-1 pm inner electron
Ultraviolet 10
15
-10
17
400 nm-1 nm outer electron
Visible 4-7.5x10
14
750 nm-400 nm outer electron
near-infrared 1x10
14
-4x10
14
2.5 m-750 nm
outer electron molecular
vibrations
Infrared 10
13
-10
14
25 m-2.5 m molecular vibrations
Microwaves 3x10
11
-10
13
1 mm-25 m
molecular rotations,
electron spin flips*
radio waves <3x10
11
>1 mm nuclear spin flips*
Rotational transitions: low energy E [long (microwave or
far-infrared)]
Vibrational transitions: takes place at high energy E [ near,
far infrared region]
Electronic transitions: takes place at higher energy E
[visible and U.V region]


Cont. Introduction to Spectroscopy
TRANSITION SPECTRUM TECHNIQUE MAIN USE
Electronic
Transitions
UV-vis UV-vis spectroscopy

Atomic Absorption
Spectroscopy
Quantitative Analysis

Quantitative Analysis
Vibrational
Transitions
IR IR Spectroscopy Functional groups
Structural
Identification
Spin Orientation Radio NMR Structure
Identification
+ Which molecules or atoms can absorb radiation?

+ Molecules:
For absorption to occur there must be change in the dipole
moment (polarity) of the molecule.
i.e polar covalent bond in which a pair of electrons is
shared unequally.
eg: of a molecule that can not exhibit a dipole
moment.
N N Can not exhibit a dipole and will not absorb in the I.R region.
eg. of a molecule that can exhibit a dipole moment.
O= C =O Unsymmetrical diatomic molecule, does have a permanent dipole
and so will absorb light.
OC:O Vibration mode symmetry and no dipole moment
O:C:O By induced dipole dipole moment and the molecule can absorb I.R radiation.
K
L
M
N
O
P
+ Atoms:
Incase of atoms only electronic transition occurs.
Molecular absorption spectra Atomic absorption spectra
The outer most electrons occupy o,
t or n electronic energy in the
ground state.
1- The outer most electrons
occupy one of the atomic orbitals
and have its energy levels [K, L,
M, N, ......
s, s,p s,p,d s,p,d,f ]
Upon excitation electrons
raised to t* or o* energy.
levels
2- Upon excitation electrons are
promoted to any permissible higher
atomic energy levels
Since there are bonds, there are
vibrational and rotational energy
levels in both the ground and
excited states
3-Since there are no bonds there
are no vibrational or rotational
energy levels in either the ground or
excited state.
The analytical wavelength is the

max
.
4- The analytical wavelength is the
resonance wavelength of the
analyte
The spectra are in the form
of bands due to the presence
of very close, superimposed
and unresolved vibrational and
rotational energy levels in the
the excited state.
5- The spectra are line form.
Atomic Absorption Spectroscopy (AAS)
AAS was employed in the 1950s
Used for qualitative and quantitative detection.
Its used for the determination of the presence and
concentrations of metals in liquid samples.
Metals that can be detected include Fe, Cu, Al, Pb,
Ca, Zn, Cd and many more.
Concentrations range is in the low mg/L (ppm)
range.
Elements that are highlighted in
pink are detectable by AAS
:
the AAS instrument

The simple diagram for the AAS
1. We set the instrument
at certain wavelength
suitable for a certain
element


2. The element in
the sample will be
atomized by heat


3. A beam of UV light will
be focused on the sample


5. The monochromator
isolates the line of
interest
4. The element in the sample
will absorb some of the light,
thus reducing its intensity

6. The detector
measures the change in
intensity

7. A computer data system
converts the change in
intensity into an absorbance
Hollow Cathode Lamp Laser
1.Light source
The disadvantage of both the HCL and laser is that they
have narrow-band light sources and so only one element is
measurable at a time.

2. Laser:
They are intense enough to excite atoms to higher
energy levels. This allows AA and atomic fluorescence
measurements in a single instrument.





Flame Graphite Furnace Hydride
2. Atomiser
Monochromator:
used to isolate the absorption line from
background light due to interferences.
3.Light Separation
Note:
Simple dedicated AA instruments often replace the
monochromator with a bandpass interference filter.




Photomultiplier tubes
4.Detectors

The technique requires a liquid sample to be
aspirated, aerosolized, and mixed with
combustible gases, such as acetylene and air
or acetylene and nitrous oxide.
The mixture is ignited in a flame whose
temperature ranges from 2100 to 2800 C.

A. Flame Atomic Absorption Spectroscopy:
1. Nebulizer:
1. mixes acetylene (the fuel)
and oxidant
(air or nitrous oxide).
2. A negative pressure is formed at the
end of the small diameter, plastic
nebulizer tube (aspiration).
3. The result is a heterogeneous mixture
of gases (fuel + oxidant) and suspended
aerosol (finely dispersed sample).
4. The mixture flows
immediately into the burner
head.
5. It burns as a smooth,
laminar flame evenly
distributed along a narrow
slot.
6. Liquid sample not flowing
into the flame collects in the
waste.
Note:
When do we use NO2 ?

The process of lighting the AAS flame involves:
turning on first the fuel then the oxidant and then
lighting the flame with the instrument's auto
ignition system.

The flame breaks down the analyte's matrix create
the elemental form of the analyte atom.



2. Ignition:
During combustion, atoms of the element of
Interest in the sample are reduced to
free,

unexcited ground state atoms, which absorb
light at characteristic wavelengths,
as shown in the figure.


1. Aspirating a solution containing the element

2. Adjusting the fuel/oxidant mix until the
maximum light absorbance is achieved.

3. Careful control of the fuel/air mixture is
important because each element's response
depends on that mix in the burning flame.

Optimization is accomplished by :
Consists of a cathode and an anode. The cathode is
made of the element of interest




1. A large voltage across the anode and cathode will
cause the inert gas to ionize.
2. The inert gas ions will then be accelerated into the
cathode, sputtering off atoms from the cathode.
3. Both the inert gas and the sputtered cathode
atoms will in turn be excited by collisions with each
other.
3. Lamp (Hollow Cathode Lamb):
4- When these excited atoms decay to lower
energy levels they emit a few spectral
lines characteristic of the element of
interest.
5- The light is emitted directionally through
the lamp's window, a window made of a
glass transparent in the UV and visible
wavelengths.
6- The light can then be detected and a
spectrum can be determined.

4. Monochromator
:
The light passes from the HCL through the element in
the sample to the monochromator.
Its function is:
It isolates the specific light of the element of
interest from the other background lights and
transfers it to the photomultiplier tube (detector).
5. Photomultiplier Tube (PMT)
:
Before an analyte is aspirated, a measured signal is
generated by the PMT as light from the HCL passes
through the flame. When analyte atoms are present in
the flame--while the sample is aspirated--some of that
light is absorbed by those atoms. This causes a
decrease in PMT signal that is proportional to the
amount of analyte
.


PMT
The PMT detects the amount of reduction of
the light intensity due to absorption by the
analyte, and this can be directly related to
the amount of the element in the sample.

The PMT converts the light signal into an
electrical
signal and a computer system translates
it into
absorbance.
Photomultiplier Tube
e
-
Light
Dynode
Dynode
Dynode
Photocathode
Current
Convert light energy to electrical energy

Different flames can be achieved using different
mixtures of gases, depending on the desired
temperature and burning velocity.

Some elements can only be converted to atoms at high
temperatures.

Some metals form oxides that do not re-dissociate into
atoms. To inhibit their formation, conditions of the
flame may be modified to achieve a reducing, non-
oxidizing flame.


Gases used in the FAAS:
Max. flame speed (cm/s) Max. temp. (
o
C)
Air-Coal gas 55 1840
Air-propane 82 1925
Air-hydrogen 320 2050
Air-50% oxygen-acetylene 160 2300
Oxygen-nitrogen-acetylene 640 2815
Oxygen-acetylene 1130 3060
Oxygen-cyanogen 140 4640
Nitrous oxide-acetylene 180 2955
Nitric oxide-acetylene 90 3095
Nitrogen dioxyde-hydrogen 150 2660
Nitrous oxide-hydrogen 390 2650
Table of the characteristics of various flames.


(GFAAS) Graphite Furnace Atomic Absorption
Spectroscopy
(ETAAS) Electro thermal Atomic Absorption
Spectrometry
B. Graphite Furnace Atom Absorption
Spectroscopy:


1. The source of light (lamp)
emits light with a wavelength
specific to the element of
interest
3. Samples are deposited in the graphite tube
heated to vaporize and atomize the analyte atoms
absorb ultraviolet or visible light and make
transitions to higher electronic energy levels.
2. A controlled voltage is applied at the
ends of the graphite tube, which is
heated rapidly to high temperatures (up
to 2600C).
5. The monochromator isolates the light of the
element of interest from the background lights
to the PMT. The PMT tube measures the
change intensity.
4. The graphite tube is
permanently flushed with argon
while it is in operation
Small sample
Formation of stable Thermal oxide (Al, Mo, Ti)
Residual elements
Biological samples
Effective atomization
Sample stay for Long time in graphite tube>>>high
sensetivety
Suitable for solid samples
Ionic interferase rather than chemical or physical
due to high temp.
-Free mercury atoms can exist at room temperature
can be analysed using atomic absorption without
employing flame and graphite furnace techniques at
high temperatures.

Mercury Cold Vapor
-Mercury is reduced in solution using stannous
chloride or sodium borohydride in a closed
system.
-The reaction quantitatively releases mercury
(from the sample solution) and is carried by a
stream of air or argon through a quartz sample
sell placed in the light path of an AA
instrument for analysis.

The detection limit for mercury by this cold
vapor technique is approximately 0.02 mg/L.

Hydride Generation Atomic Absorption
Spectroscopy (HGAASC.):

Its used for elements that are forming volatile
hydrides (e.g. As, Se, pb, Sb, Te, Sn, Bi)
(HAAS) is very useful in case of interferences, poor
reproducibility, and poor detection limits.
3. The monochromator isolates analytical
lines' photons passing through the optical
cell
and removes the scattered light of other
wavelengths from the optical cell
4. The PMT detects the amount of reduction of
the light intensity due to absorption by the
analyte and can be directly related to the
amount of the element in the sample

1. The HCL emits the light with a
wavelength characteristic to the
element of interest
2. The Light passes from the HCL
through the optical cell to the
monochromator then to the detector
sample flow in the (HGAAS)
1. The metal oxyanions reacts with Sodium
Borohydride and HCl and produces a
volatile hydride: H2Te, H2Se, H3As, H3Sb,
etc.
2. The peristaltic pump and the flowing reagents along with
the tubing of specific lengths controls the time from reagent
mixing and separation of
the volatile hydride from the
liquid and sending the sample to the optical cell.
3. The liquid mixture flows
into a gas/liquid separator where
the hydride and some gaseous hydrogen are
purged (via a high purity inert gas) into the optical cell
via a gas transfer line
4. In the optical cell the flame is ignited automatically by the
air/C
2
H
4
and the gaseous
metal hydride form decomposes into the elemental form which
can absorb the
HCL's beam. The light passes to the Mon. and then to the PMT
Gas liquid separator
Optical cell
peristaltic
pump
Mixing
reaction
coil
Volatile Hydride

AAS Interferences


Any factor that affects the ground state population
of the analyte element.
Factors that may affect the ability of the
instrument to read this parameter.

A) Spectral interferences: due to radiation
overlapping than of the light source.

B) Formation of compounds that do not dissociate in
the flame.
The most common example is the formation of
calcium and strontium phosphates.

C) Ionization of the analyte reduces the
signal.
This is commonly happens to barium, calcium,
strontium, sodium and potassium.

D) Matrix interferences: due to differences
between surface tension and viscosity of test
solutions and standards
.
E) Broadening of a spectral line

1. Doppler effect
2. Lorentz effect
3. Quenching effect
4. Self absorption or self-reversal effect