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1. Atomic absorption (AA)
spectrophotometry
Prepared and Presented by :
Amany Mohamed Elshamy
Department of Medical
Laboratories
Badr Academy

2. Outlines
Introduction
Definition
Applications
Principle
Limitations
Summary
Questions

3. Atomic Spectroscopy
Atomizer
Principle
Tool
Atomic
spectroscopy
Absorption
Flame
Graphite
furnace
Emission Flame
Graphite
furnace
technique is
more
sensitive
than the
flame
method.

4. Introduction
• Atomic absorption (AA)
spectrophotometry is an optical
technique in which an element in
the sample is excited and the
radiant energy produced is
measured as the element returns
to its lower energy level.

5. Definition
• Atomic absorption (AA)
spectrophotometry An emission
technique in which an element in a
sample is dissociated from its
chemical bonds (atomized) and
placed in an unexcited or ground
state (neutral atom); the atom at low
energy is able to absorb radiation
and the radiant energy given o as the
element returns to its ground state is
measured.

6. Application
• The atomic absorption
spectrophotometer is
used to measure
concentration by
detecting the absorption
of electromagnetic
radiation by atoms rather
than by molecules.

7. Applications
• Atomic absorption (AA)
spectrophotometry is used widely in
clinical laboratories to measure elements
including
(1)Aluminum
(2) Calcium
(3) Copper
(4) Lead
(5) Lithium
(6) magnesium
(7) zinc

8. Components
• Basic components of an atomic absorption spectrophotometer.

9. Components
• Hollow-cathode lamp —» chopper —»
burner head for flame —>
monochromator —> detector —» readout
device

12. Main
functions for
each
component
• Hollow-cathode lamp : source of light to
produce a specific single beam of
wavelength.
• Chopper : to chop the light leaving the
source.
• Flame : for the sample Atomization.

13. Main
functions for
each
component
• Monochromator: to separate the
spectral line of interest from others
spectral lines with different
wavelengths emitted by the hollow-
cathode lamp.
• Detector: measures the amount of
absorption in the sample and
compares it to a reference with a
known concentration of the element in
question to determine its
concentration in the sample

14. Principle
• Principle: Ground-state atoms absorb
light at defined wavelengths.
a. Line spectrum refers to the wavelengths
at which an atom absorbs light; each metal
exhibits a specific line spectrum.
b. The sample is atomized in a flame
where the atoms of the metal to be
quantified are maintained at ground state.

15. Principle
c. Then a beam of light from a hollow-
cathode lamp (HCL) is passed through a
chopper to the flame.
d. The ground-state atoms in the flame
absorb the same wavelengths of light from
the HCL as the atoms emit when excited.

16. Principle
• e. The light produced in the HCL passes
through a chopper and then to the flame, and
the light is absorbed by the metal in the
sample. The light not absorbed will be read
by the photomultiplier tube (the detector).
• The light not absorbed by the atoms is
measured as a decrease in light intensity
by the detector, The detector
(photomultiplier tube) will selectively read
the pulsed light from the chopper that passes
through the flame and will not detect any
light emitted by the excited atoms when they
return to ground state.

17. Principle
f. The difference in the amount of light leaving
the HCL and the amount of light measured by
the detector is indirectly proportional to the
concentration of the metal analyte in the
sample.
• When a ground state atom absorbs light
energy, an excited atom is produced. The
excited atom then returns to the ground state,
emitting light of the same energy as it
absorbed.
• The flame sample thus contains a dynamic
population of ground state and excited atoms,
both absorbing and emitting radiant energy.

19. 1. Light
source
Electrodeless discharge lamps are a
relatively new light source for atomic
absorption spectrophotometers.
A bulb is filled with argon and the element
to be tested.
A radiofrequency generator around the bulb
supplies the energy to excite the element,
causing a characteristic emission spectrum
of the element.

20. Hollow-cathode lamp
Light Source:
• Hollow-cathode lamp (HCL) consists of
an evacuated gas-tight chamber
containing an anode, a cylindrical cathode
of metal being analyzed, and an inert gas,
such as helium or argon.

21. Hollow-cathode lamp (HCL)

22. Hollow-cathode lamp (HCL)

23. Principle of the Hollow-cathode lamp
• Applied voltage causes ionization of the gas, and these excited ions are
attracted to the cathode, where they collide with the metal coating on
the cathode, knocking off atoms and causing atomic electrons to
become excited. When the electrons of the metal atoms from the
cathode return to ground state, the characteristic light energy of that
metal is emitted.
• Vaporized metal atoms from the sample can be found in the flame. The
flame serves as the sample cuvette in this instrument.

24. Principle of the Hollow-cathode lamp
• The emitted energy from the flame will go in all directions, and it will
be a steady emission. Because the purpose of the instrument is to
measure the amount of light absorbed, the light detector must be able
to distinguish between the light beam emitted by the hollow-cathode
lamp and that emitted by excited atoms in the flame.
• To do this, the hollow-cathode light beam is modulated by inserting a
mechanical rotating chopper between the light and the flame or by
pulsing the electric supply to the lamp.

25. Nebulizer
Nebulization: to convert a
solution sample into a
droplets by spraying then
mixing (fuel + oxidants) then
introducing to the flame.
"Nebulizer"

26. Atomization
Atomizer: The elements which needs
to be analysed needs to be in the
atomic state.
( liquid phase to gas phase)
Here comes the role of atomizer.
• Atomization: It breaks down the
molecules into the atoms by
exposing the analyte to the high
temperatures in the flame.

27. The
analyzed
sample
The analyzed sample must
contain the reduced metal in
the atomic vaporized state.
Commonly, this is done by
using the heat of a flame to
break the chemical bonds and
form free, unexcited atoms.

28. Flame
• The flame is the sample cell in
this instrument, rather than a
cuvette.
• The sample, in solution, is
aspirated as a spray into a
chamber, where it is mixed with
air and fuel. This mixture passes
through baffles, where large
drops fall and are drained off.
Only fine droplets reach the
flame.

29. Flame
• The burner is a long, narrow
slit, to permit a longer path
length for absorption of incident
radiation. Light from the
hollow-cathode lamp passes
through the sample of ground
state atoms in the flame.
• The flame converts samples into
free ground state atoms that can
be excited.

30. Monochromator
• The monochromator is used
to isolate the desired
emission line from other
lamp emission lines. In
addition, it serves to protect
the photodetector from
excessive light emanating
from flame emissions.

31. Monochromator
• Generally, most of the instruments are equipped with
two gratings with the goal to cover a wavelength range
from 189 to 851 nm which is used in atomic absorption

32. Detector
• The light selected by the
monochromator is directed onto a
detector that is typically a
photomultiplier (PMT) tube , whose
function is to convert the light signal
into an electrical signal proportional to
the light intensity.

33. Detector
• The signal could
be displayed for
readout , or
further fed into a
data station for
printout by the
requested format.

34. Advantages of
AA
• Atomic absorption spectrophotometry
is sensitive and precise.
• It is routinely used to measure
concentration of trace metals that are
not easily excited.
• It is generally more sensitive than flame
emission because the vast majority of
atoms produced in the usual propane or
air-acetylene flame remain in the
ground state available for light
absorption.
• It is accurate, precise, and specific.

35. Limitations
1.The inability of the flame to dissociate
samples into free atoms.
2. Problem is the ionization of atoms following
dissociation by the flame, which can be
decreased by reducing the flame temperature.
3. Due to the enhancement of light absorption
by atoms in organic solvents or formation of
solid droplets as the solvent evaporates in the
flame, can be another source of error.

36. Limitations
Interferences include
(1) absorption by other closely
absorbing atomic species,
(2) absorption by molecular
species,
(3) scattering by nonvolatile
salt particles or oxides,
(4) background emission.

37. Limitations Interferences affect nebulization by
altering the
(1) viscosity
(2) surface tension
or
(3) density of the analyte solution

38. Limitations An example: in phosphate interference
in the determination of calcium that is
caused by the formation of calcium-
phosphate complexes.
• This may be overcome by adding
cations that compete with calcium for
phosphate. Routinely, lanthanum or
strontium is added to samples to form
stable complexes with phosphate.

39. Questions
• What quality incorporated into a spectrophotometer can
sometimes improve the linearity of a chemistry procedure?
• A. Flow-through cuvette
• B. Wider bandwidth
• C. Narrower bandwidth
• D. Chopper

40. Questions
• When a pH-sensitive glass electrode is not actively in use, in
what type of solution should it be kept?
• A. Tap water
• B. Physiologic saline solution
• C. The medium recommended by the manufacturer
• D. A buffer solution of alkaline pH

41. • In spectrophotometry, which of the following is a mathematical
expression of the relationship between absorbance and
transmittance?
• A. A = abc
• B. Au _ As C~~C ^U ^5
• C. A = 2 - log %T
• D. A = log %T

42. • In spectrophotometric analysis, what is the purpose of the reagent
blank?
• A. Correct for interfering chromogens
• B. Correct for lipemia
• C. Correct for protein
• D. Correct for color contribution of the reagents

43. • What is the function of the flame in atomic absorption
spectroscopy?
• A. Absorb the energy emitted from the metal analyte in returning
to ground state
• B. Supply the thermal energy needed to excite the metal analyte
• C. Bring the metal analyte to its ground state
• D. Supply the light that is absorbed by the metal analyte

44. • Most atomic absorption spectrophotometers incorporate a beam
chopper and a tuned amplifier. The purpose of these components
is to avoid errors that would be caused by
• A. Variations in flame temperature
• B. Deterioration of the hollow-cathode lamp
• C. Stray light from the hollow-cathode lamp
• D. Measurement of light emitted by the analyte

45. • In assaying an analyte with a single-beam atomic absoiption
spectrophotometer, what is the instrument actually measuring?
• A. Intensity of light emitted by the analyte on its return to the
ground state
• B. Intensity of light that the analyte absorbs from the hollow-
cathode lamp
• C. Intensity of light that the analyte absorbs from the flame
• D. Intensity of the beam from the hollowcathode lamp after it has
passed through the analyte-containing flame

46. • Which of the following isolates light within a narrow region of
the spectrum?
• A. Photomultiplier tube
• B. Monochromator
• C. Photovoltaic cell
• D. Detector