Spectrophotometer , types of spectrophotometer, atomic absorption spectrophotometer working principle, instruments of AAS, cathode hollow lamp, chopper, Flame, Burner , total consumption burner, pre-mix burner , Monochromators prism and diffraction grating , read out device, types of AAS, Flame AAS , non flame AAS(Graphite furnace AAS ), applications , interference , atomic emission spectrophotometry different types of flame , principle of emission spectrophotometry, comparision of spectrophotometry.

2. Atomic absorption Spectrophotometry
 Spectrophotometry is defied as the measurement of
the intensity of light at selected wavelength
 Widely used method of quantitative and qualitative
analysis in the chemical and biological sciences
 Types of spectrophotometry
 Visible spectrophotometer
 Ultraviolet-visible spectrophotometer
 Infrared spectrophotometer
 Fluorescence spectrophotometer
 Atomic absorption spectrophotometer.
 Atomic emission spectrophotometer .

3. Atomic absorption Spectrophotometry
Introduction
Invention
Working Principle of AAS
Instrumentation
Interferences

4. Introduction
 The atomic absorption spectrophotometer is used to
measure concentration by detecting absorption of
electromagnetic radiation by atom rather than by
molecules.
 It is a very common technique for detecting metals
and metalloids in sample.
 Widely used in clinical laboratories to measure
elements
 Such as aluminum, calcium, copper, lead, lithium, magnesium,
zinc, and other metals

5. Elements detectable by atomic absorption are highlighted in pink in
this periodic table

6. Invention
Introduced in 1955 by Alan Walsh in
Australia
First commercial atomic absorption
spectrometer was introduced in 1959
Used for mining, medical treatment
&agriculture

7. Principle
 Atomic absorption is an absorption
spectrophotometric technique in which a metallic
atom in the sample absorbs light of a specific
wavelength.
 The element is not appreciably excited in the flame,
but is merely dissociated from its chemical bonds
(atomized) and placed in an unexcited or ground state
(neutral atom).
 This ground state atom absorbs radiation at a very
narrow bandwidth corresponding to its own line
spectrum.

8. Principle
 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
 Concentration measurements are usually determined from a
working curve after calibrating the instrument with standards of
known concentration.

9. Instrument
1. Light source
2. Chopper
3. Atomizer
4. Monochromators
5. Detector
6. Amplifier
7. Read out device

10. ATOMIC ABSORPTION SPECTROPHOTOMETER- BASIC COMPONENT

12. Types of AAS
Flame atomic-absorption spectrophotometer
Graphite-furnace atomic-absorption
spectrophotometer

13. Light Source
 Hollow Cathode Lamps
 Electrodeless Discharge Lamp

14. Hollow Cathode Lamp
 Cathode--- in the form of a cylinder, made of the
element being studied in the flame
 Tungsten Anode
 Filled with an inert gas (neon or argon) sealed in a
glass tube
 Quartz or special glass that allows transmission of the
proper wavelength, is used as a window

15. How it works
 Applying a potential difference(300-500V) between the
anode and the cathode leads to the ionization of some
gas atoms .
 These gaseous ions bombard the cathode and eject
metal atoms from the cathode in a process called
sputtering.
 Some sputtered atoms are in excited states and emit
radiation characteristic of the metal as they fall back to
the ground state .

16. LIGHT SOURCES
Electrodeless Discharge Lam
 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.
 Used for difficult to make stable hollow cathode lamp
from certain elements particularly those that are
volatile, such as arsenic, germanium

17. Chopper
 A rotating wheel is interposed between the hollow cathode lamp
and flame .
 It is interposed to break the steady light coming from the lamp
into pulsating light which is used to measure the intensity of light
absorbed by elements without interference by radiation from the
flame itself.
 Pulsating light gives pulsating current in photocell.
 There is also steady current caused by light which is emitted by
flame. But only pulsating current is amplified and recorded

18. Atomizer
 Atomization is separation of particles into individual
molecules and breaking molecules into atoms .This is
done by exposing the analyte to high temperatures in
a flame or graphite furnace
 Atomiser converts the liquid into small droplets which
are easily vaporised.
 Types of Atomisers :-
1.Flame atomizer:-
a.) Total consumption burner
b.) premixed burner
2.Non-flame atomizer(Electrothermal atomizer)

19. Flame atomizer
 Nebulization
• Conversion of the liquid sample to a fine spray
 Desolvation
• Solid atoms are mixed with the gaseous fuel.
 Volatilization
• Solid atoms are converted to a vapor in the flame.
 There are three types of particles that exist in the
flame:
1) Atoms
2) Ions
3) Molecules

20. Nebulization
 Before the liquid sample enters the burner ,it is
converted into droplets this method a formation of
small droplets its called nebulization
 Common method of nebulization is by use of
gas moving at high velocity, called pneumatic
nebulization.

21. Total consumption burner
 In this whole sample is atomized into the flame, hence
named as total consumption burner.
 In this burner, the sample solution, the fuel, and oxidizing
gases are passed through separate passages to meet at the
opening of the base of flame.
 Then the flame breaks the sample in liquid form into the
droplets which are evaporated and burns. Leaving the
residue which is reduced to atoms.
 Fuel used – H2 /acetylene
 Oxidant – O2

22. Premixed burner
 It is most widely used because of uniformity in flame intensity.
 In this the sample solution ,fuel and oxidant are mixed before
they reach the tip.
 The fine droplets get carried out along with the fuel gas at
outlet, the large drops of sample get collected in chamber and
are drained out.
 Advantages
 Non-turbulent
 Noiseless
 Stable
 Disadvantages
 Only 5% sample reaches to the flame
 Rest 95% is wasted.

23. Flame temperature for various gas mixtures
Fuel Oxidant Temperature 0c
Natural gas Air 1700 - 1900
Natural gas Oxygen 2700 - 2800
Hydrogen Air 2000 - 2100
Hydrogen Oxygen 2550 - 2700
Acetylene Air 2100 – 2400 (Most common)
Acetylene Oxygen 3050 - 3150
Acetylene Nitrous oxide 2600 - 2800
Selection of flame type depends on the volatilization
temperature of the atom of interest.

24. Flame Structure
 Different region or zone in flame are:
1. Preheating zones
2. Primary reaction zone (inner zone)
3. Internal zonal region- max temp,
4. Secondary reaction zone
 Interzonal region is the hottest part of the flame and
best for atomic absorption.
 Oxidation of the atoms occurs in the secondary
combustion zone where the atoms will form molecular
oxides and are dispersed into the surroundings.

25. Copper
calciumPotassium
Cobalt

26. Flame AAS
Advantages
 Short analysis time possible
 Good precision
 Easy to use
 Cheap
Limitation
• Sensitivity
• Dynamic range
• Requires flammable gases
• Unattended operation is not possible because of flammable
gases
• Must not contain excessive amounts of dissolved solids

27. Non flame atomizer (Electro Thermal
Atomizer )
 The graphite furnace is an electro thermal atomizer
system that can produce temperatures as high as
3,000°C.
 The heated graphite furnace provides the thermal
energy to break chemical bonds within the sample
held in a graphite tube, and produce free ground state
atoms.
 The ground-state atoms are capable of absorbing
energy, in the form of light, and are elevated to an
excited state.
 The amount of light energy absorbed increases as the
concentration of the selected element increases

28. Graphite furnace technique
Uses a graphite coated furnace to vaporize the
sample.
ln GFAAS sample, samples are deposited in a
small graphite coated tube which can then be
heated to vaporize and atomize the analytes.
The graphite tubes are heated using a high
current power supply.

29. Graphite furnace technique

30. Graphite Furnace AAS Atomizer
Advantages
 Small sample sizes ( as low as 0.5 uL)
 Very little or no sample preparation is needed
 High sensitivity due to
 entire sample is atomized at one time
 free atoms remain in the optical path longer
 Reduced sample volume
 Ultra trace analysis possible

31. Graphite Furnace AAS Atomizer
Limitation
•Very slow
•Fewer elements can be analyzed
•Poorer precision
•More chemical interferences
•Method development requires skill
•Standard additions calibration required more
frequently (compared to flame AA)
•Expensive consumables (graphite tubes)

32. Monochromators
 Important part in an AA spectrophotometer.
 It is used to separate out all of the thousands of
lines. Without a good monochromator, detection
limits are severely compromised.
 A monochromator is used to select the specific
wavelength of light which is absorbed by the
sample, and to exclude other wavelengths. The
selection of the specific light allows the
determination of the selected element in the
presence of others.
 They are of two types:
1) Prism
2) Diffraction Grating

33. Grating monochromator :- it
consists of a series of parallel
straight lines cut into a plane
surface
Prism monochromator :- Quartz
material is used for making prism,
as quartz is transparent over entire
region

34. Detector
 The light selected by the monochromator is directed
onto a detector that is typically a photomultiplier tube
, whose function is to convert the light signal into an
electrical signal proportional to the light intensity.
 The processing of electrical signal is fulfilled by a signal
amplifier.
 The signal could be displayed for readout , or further
fed into a data station for printout by the requested
format

35. DETECTOR
Photomultiplier Tubes
 Components
 Made of a glass vacuum tube
 Photocathode
 Several dynodes
 One anode

36. How it works

37. Photodiodes.
 Photodiodes are solid-state photodetectors that are
fabricated from photosensitive semiconductor materials
such as (1) silicon, (2) gallium arsenide, (3) indium
antimonide, (4) indium arsenide, (5) lead selenide, and (6)
lead sulfide.
 These materials absorb light over a characteristic
wavelength range (e.g., 250 nm to 1100 nm for silicon).
 Capable of measuring light at a multitude of
wavelengths.
.

38. Read-out Device
 The output from the detector is suitably amplified and displayed
on a readout device like a meter or a digital display.
 It is capable of displaying the absorption spectrum as well
absorbance at specific wavelength
 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.

39. Operational of AAS
A pulsed hollow cathode lamp 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 nonplused 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.

40. Calibration Curve
 A calibration curve is used to determine the unknown
concentration of an element in a solution.

41. Applıcatıons
The are many applications for atomic
absorption:
Clinical analysis
Analyzing metals in biological fluids such as
blood and urine.
Environmental analysis
Monitoring our environment – e g finding out the
levels of various elements in rivers, seawater,
drinking water, air, and petrol.

42. Applıcatıons
 Pharmaceuticals.
In some pharmaceutical manufacturing processes,
minute quantities of a catalyst used in the process
(usually a metal) are sometimes present in the final
product. By using AAS the amount of catalyst present
can be determined.
 Industry :
Many raw materials are examined and AAS is widely
used to check that the major elements are present
and that toxic impurities are lower than specified
Ex. in concrete, where calcium is a major constituent,
the lead level should be low because it is toxic.

43. Applıcatıons
Mining:
By using AAS the amount of metals such as
gold in rocks can be determined to see whether
it is worth mining the rocks to extract the gold .
Trace elements in food analysis
Trace element analysis of cosmetics
Trace element analysis of hair

44. Interference
 Interference is a phenomenon in which two waves
superimpose to form a resultant wave of greater or
lower amplitude.
 Interference decrease the intensity of absorption of
light .
Types of interferences
1) Spectral interference
2) Non –Spectral interference
 Non specific
 Specific

45. Spectral interferences
 Include absorption by other closely absorbing
atomic species,
 absorption by molecular species,
 scattering by nonvolatile salt particles or oxides,
 and background emission (which can be
electronically fitered).
 Absorption by other atomic species usually is not
a problem
 because of the extremely narrow bandwidth (0.01 nm)
used in the absorption measurements.

46. Non spectral interference
Nonspecific interferences
 Affect nebulization
by altering the viscosity, surface tension, or density of
the analyte solution, and consequently the sample
flow rate
Specific interferences
 Also called chemical interferences
because they are more analyte dependent.

47. Non Spectral Interference
Solute volatilization interference
 refers to the situation in which the contaminant forms
nonvolatile species with the analyte.
 Example :
 phosphate interference in the determination of calcium that is
caused by the formation of calcium–phosphate complexes.
 The phosphate interference is overcome by adding a cation,
usually lanthanum or strontium; the cation competes with
calcium for the phosphate.
Dissociation interferences
 affect the degree of dissociation of the analyte.
 Analytes that form oxides or hydroxides are especially
susceptible to dissociation interferences.

48. Non Spectral Interference
Ionization interference
 occurs when the presence of an easily ionized element,
 such as K, affects the degree of ionization of the analyte, which leads to
changes in the analyte signal.
 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.
Excitation interference,
 Analyte atoms are excited in the atomizer, with subsequent
emission at the absorption wavelength.
 This type of interference is more pronounced at higher
temperatures.

49. Background 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..
 Th intense magnetic field splits the degenerate (i.e., of equal
energy) atomic energy levels into two components that are
polarized parallel and perpendicular to the magnetic field,
respectively

50. Background correction

52. Atomic Emission Spectrophotometry

53. Flame photometry
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)

54. Principle
 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

55. Mechanism
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

56. Atomic Emission Spectrophotometry
 Various metals emit a characteristic color of light when
heated

57. AAS Vs Flame photometry
 Atomic absorption
 Hollow cathode lamp as source of
light
 Most of the atoms are in the ground
state and are able to absorb light
emitted by the cathode lamp.
 approximately 100 times more
sensitive than flame emission
methods.
 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

58.  Atomic absorption
depends upon the
number of ground state
atoms .
 It measures the
radiation absorbed by
the ground state atoms.
 The temperature in
the atomizer is adjusted
to atomize the analyte
atoms in the ground
state only.
 Atomic emission depends
upon the number of
excited atoms .
 It measures the
radiation emitted by
the excited atoms
 The temperature in the
atomizer is big enough
to atomize the analyte
atoms and excite them
to a higher energy level

59. References
 Tietz Textbook of CLINICAL CHEMISTRY MOLECULAR
DIAGNOSTICS, Carl A. Burtis, Ph.D., Edward R. Ashwood,
M.D,David E. Bruns, M.D, Fifth edition
 Methods in clinical chemistry , Kaplan L.A, Pesce A J
 Concepts, Instrumentation and Techniques in Atomic
Absorption Spectrophotometry Richard D. Beaty and Jack D.
Kerber, 2nd edition.
 Spectrophotometry and Spectrofluorimetry,Michael. G. Gore
 https://www.hitachihightech.com/global/products/science/tech/a
na/aa/basic/course8.html
 http://vlab.amrita.edu/?sub=2&brch=193&sim=1351&cnt=1
 Internet sources