clinical chemistry

1. ATOMIC ABSORPTION
SPECTROPHOTOMETRY
BASIC CONCEPTS,
INSTRUMENTATION,
OPERATION, AND APPLICATION

2. INTRODUCTION:
• Introduced by Alan Walsh(1950)
• It is a method of elemental analysis used widely in
clinical laboratories
• Useful for determining trace metals like Aluminum,
Calcium, Copper, Lead, Lithium, Magnesium and
Zinc in liquid or biological samples.
• Highly sensitive and can detect metals in
concentrations lower than 1ppm

3. BASIC CONCEPTS:
• This technique basically uses the principle that
free atoms (gas) generated in an atomizer can
absorb radiation at specific frequency.
• AA is an emission technique in which an element
in the sample is excited and the radiant energy
given off is measured as the element returns to its
lower energy level as in FES.
• However, the element is not appreciably excited in
the flame, but it is merely dissociated from its
chemical bonds (atomised) and placed in an
unexcited ground state (neutral atom)

4. • These ground state atom of particular element
are receptive of light radiation of their own
specific resonance wavelength (monochromatic).
• Thus, when a light of this wavelength is passed
through a flame having atom of metallic species,
part of the light will be absorbed and the
absorption will be proportional to the density of
atom in the flame.
• The analyte concentration is calculated from the
amount of absorption .

5. Differences btw Atomic Absorption
Spectrophotometry (AAS) and Flame Emission
Spectrophotometry:
AAS
• Amount of light absorbed
by ground state atom is
measured
• Absorption intensity does
not depend upon
temperature
• Beers law is obeyed over
wide range of
concentration
FES
• Amount of light emitted
by the excited atom is
measured
• Absorption intensity is
greatly influenced by
temperature variation
• Beer’s law is not obeyed

6. AAS
AES (FES)

7. INSTRUMENTATION:
• The basic requirements are:
– a light source;
– a sample cell; and
– a means of specific light measurement.
Components of AA Spectrophotometer.
Single beam AAS:

8. TYPES of ASS:
• Single beam AAS:
– Light source is placed ahead of flame with mechanical
chopper between light source and flame
– Has low stability
• Double beam AAS:
– Beam from hollow cathod lamp is divided into two part one
passes through flame while other bypass it
Double beam ASS:

9. Light source:
• Radiation source: are of two types
• Hollow cathode lamp- emits spectral line of that metal which is
used in the cathod (HCL)
• Electrodeless discharge lamp (EDL)
• A hollow-cathode lamp are made of the metal of the
substance to be analyzed (different for each metal
analysis). The anode is made of Tungsten
• Each HCL will have a particular current for optimum
performance.
• In general, higher currents will produce brighter
emission and less baseline noise.
• As the current continues to increase, lamp life may
shorten and spectral line broadening may occur,
resulting in a reduction in sensitivity and linear
working range.
• If an alloy is used, it results in a multi-element lamp

10. Limitations of HCL:
• A finite lifetime – due to depletion of the analyte
element from the cathode
• Adsorption of fill gas atoms onto the inner surfaces of
the lamp – the primary cause for lamp failure
• Some cathode materials can slowly evolve hydrogen
when heated – a background continuum emission
contaminates the purity of the line spectrum of the
element, resulting in a reduction of atomic absorption
sensitivity and poor calibration linearity.
Chopper:
• It is a rotating wheel interposed between the hollow
cathode lamp and flame.
• Gives pulsating current in the photocell

11. Atomiser:
• Flame atomiser: total consumption burner and
premixed burner
• Non flame atomiser (carbon rod or “graphite furnace)
– Temp of the rod is raised to dry, char, and atomise the
sample in a chamber
– More sensitive than the conventional flame methods
– Permits determination of trace metals in small samples of
blood or tissue
– Zeeman correction used to correct for background
absorption by placing the analyte in a strong magnetic field
which splits the degenerate atomic energy into two
components that are polarised parallel and perpendicularly
and interacts differently with polarized light.

12. – Two absorption measurement are taken at different
polarizer setting. one measures both analyte and
background absorption, At,the other measures only
background absorptions.
– Thus, the same light source at the same wavelength is used
to measure the total and the background absortion. The
difference btw the two is the corrected absorbance.
Nebulization of liquid sample:
• method of formation of small droplet from the liquid
sample
• Nebulization is done by use of a gas moving at high
velocity
Monochromator:
• Function of monochromator is to select given
absorbing line from spectral line emitted from hollow
cathode
• Common monochromator are prism and gratings

13. Detctor:
• Photomultiplier tube is most suitable. The intensity of
the light is fairly low, so a photomultiplier tube (PMT)
is used to boost the signal intensity
• Has good stability if used with stable power supply
• A detector (a special type of transducer) is used to
generate voltage from the impingement of electrons
generated by the photomultiplier tube
Amplifier:
• It amplifies electric current
• Lock in amplifier are preferred to achieve excellent
signal to noise ratio(measure of signal strength
relative to background noise)
Read out device:
• Digital read out device are used

14. Advantages of AAS:
• Technique is specific because the atom of
particular element can only absorb radiation of
their own characteristic wavelength
• Is independent of flame temperature
Disadvantages of AAS:
• Separate lamp for each element to be determined
is required
• Technique cannot be used successfully for
estimation of element like Mo, Si etc because
these element give rise to oxide in the flame
• Predominant anion affect the signal to negotiable
degree

15. OPERATION:
• A meter is adjusted to zero absorbance when
blank solution is sprayed on the flame, and rest
of the light passes on to the photomultiplier
tube.
• Then the light solution containing absorbing
species is introduced a part of light is absorbed,
resulting in a decrease in light intensity falling on
a photomultiplier tube.

16. INTERFERENCES IN AAS:
Spectral Interferences: include
• Absorption from other closely absorbing atomic
species, usually not a problem because of the
extremely narrow band-width used
• Absorption and scattering by molecular species,
usually problematic at lower atomising
temperatures
• Scattering by non-volatile salt particles and oxides
• Background emission (which can be electronically
filtered)

17. Non-spectral Interferences: could be specific or non-specific
• Non-specific interferences: affect the nebulization by
altering the viscosity, surface tension, or density of the
analyte solution and consequently the sample flow
rate
• Some contaminants also decrease the desolvation and
atomization efficiency by lowering the atomizer
temperature
• Specific interferences: are also called chemical
interferences because they are more analyte
dependent
– Solute volatilization interference: refers to the situation
when the contaminant forms non-volatile species with the
analyte

18. – Dissociation interferences: affects the degree of
dissociation of the analyte. Analytes that form oxides
and hydroxides are esp susceptible to dissociation
interferences
– Ionization interferences: 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 .
– Excitation interferences: occur when the analyte atoms
are excited in the atomizer with a subsequent
emission at the absorption wavelength . This is more
pronounced at higher temperatures.

19. APPLICATION OF AAS:
• Qualitative and quantitative analysis
• Determination of metallic elements in biological
system
• Determination of metallic element in food industry
• Determination of Ca, Mg, Na, K in serum
• Determination of lead in petrol

20. REFERENCES:
• Shriver and Atkins’ Inorganic Chemistry, Fifth
Edition
• Concepts, Instrumentation and Techniques in
Atomic Absorption Spectrophotometry by
Richard D. Beaty and Jack D. Kerber
• An elementary overview of elemental analysis
by thermo elemental