BS- Biochemistry

1. Techniques in Biochemistry

2. Introduction
 Atomic absorption spectrometry (AAS) is an analytical
technique that measures the concentrations of
elements.
 Atomic absorption is so sensitive that it can measure
down to parts per billion of a gram (μg/dm–3) in a
sample.
 The technique makes use of the wavelengths of light
specifically absorbed by an element.
 They correspond to the energies needed to promote
electrons from one energy level to another, higher,
energy level.

3. Process of Absorption
Spectrometre

5. INSTRUMENTATION
 Sharp-line radiation source (usually a hollow-cathode
lamp)
 A solution nebulizer and burner (an electrically heated
furnace)
 Mono-chromator
 Photomultiplier
 Recording system

6. SHARP-LINE SOURCES
 The absorption line width for the ground state atoms
may be from 0.001-0.01nm. So the light sourced must
emit radiation of a line width less than that of the
absorption line width of the element being
determined.
 This is achieved by vapour discharge lamps for certain
easily excited elements, e.g. sodium and potassium.

7. HOLLOW CATHODE LAMP
 When a current flows between the anode and cathode
in this lamps, metal atoms are sputtered from the
cathode cup, and collisions occur with the filler gas.
 A number of metal atoms become excited and give off
their characteristic radiation.

8. SAMPLE VAPORIZATION BY FLAME
 The production of an homogeneous atomic vapour
from a sample is achieved by aspirating a solution into
a flame or evaporating small volumes in an electrically
heated tube furnace or from the surface of a carbon
rod. In all cases, the thermal energy supplied must :
(a) Evaporate the solvent
(b) Dissociate the remaining solids into their
constituent atoms without causing appreciable
ionization.

9. Monochromator
 Some elements have a single emission line (principal
line). But several elements have more than one
emission line (secondary line).
 Hence it is necessary to isolate required absorption
line from a radiation source by a grating
monochromator.

10. DETECTOR AND READOUT DEVICE
 The intensity of radiation absorbed by elements, in the
UV or visible region(190-900nm) can be detected
using photometric detector like photomultiplier tube.
 The readout device is capable of displaying the
absorption spectrum as well as the absorbance at a
specified wavelength.

12. INTERFERENCES IN ATOMIC
ABSORPTION SPECTROSCOPY
 Interferences in atomic absorption measurements can
arise from spectral chemical and physical sources.
 SPECIAL INTERFACE resulting from the overlap of
absorption lines is rare because of the simplicity and
the sharpness of the lines.
 However , broadband by molecular species can lead to
significant background interface.
 Correction for this may be made by matrix matching
of samples and standards , or by use of a standard
addition method.

13. Advantages
 Solutions, slurries and solid samples can be analysed.
 Much more efficient atomization
 Greater sensitivity
 Smaller quantities of sample (typically 5 – 50 μL)
 Provides a reducing environment for easily oxidized

14. DISADVANTAGES
 Expensive
 Low precision
 Low sample throughput
 Requires high level of operator skill
 Sample must be in solution or at least volatile
 Individual source lamps required for each element

15. Applications
 Clinical analysis. Analysing 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, petrol
and drinks such as wine, beer and fruit drinks.
 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.

16.  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 – eg in concrete, where calcium is a major
constituent, the lead level should be low because it is
toxic.
 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.

18. The End