RADIOCHEMICAL TECHNIQUES

These techniques are labor intensive and generate liquid waste due to the chemical separations that are
involved. Further more, detection limits tend to be high for long-lived isotopes using radiochemical
methods.

activity is induced in one or more elements of the sample by

Activation irradiation with suitable radiation or particles.
analysis Most commonly thermal neutrons from a nuclear reactor source
is used
Method of radiation or analysis

Radioactivity is introduced into sample by adding a measured

Tracer method
amount of a radioactive species.

Most important-quantitative method-weighed quantity of

radioactively tagged analyte having known activity. Mixture is
Isotope dilution homogenized. Fraction of compound of interest is isolated and
purified
analysis is based upon activity of the isolated fraction

Isotopes
All isotopes of an element have the same atomic number and their chemical behavior is very similar
Example: Tritium can subsitute for hydrogen. Iodine-125 or Iodine-131 can substitute for Iodine-126 in
chemical reactions.
Radioactive isotopes have unstable nuclei that spontaneously change to form more stable nuclei. As a result,
either new isotopes or new elements are produced.

Radioactive decay
Radioactive isotopes (radionuclides), undergo spontaneous disintegration, which ultimately leads to stable
isotopes. Radioactive decay of isotopes occurs with the emission of electromagnetic radiation in the form of
X-ray or gamma ray.
Accompanying this emission is the formation of electrons, positrons, and the helium nucleus or fusion in
which a nucleus breaks up into smaller nuclei.

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 common radioactive process encountered with heavier isotopes
Alpha particle is helium nucleus having a mass of and charge +2
Alpha decay particles lose their energy due to collisions as they pass through matter
ultimately converted into helium atoms by capturing two electrons from
sourrounding
Radioactive decay

Negatron
formation
Positron
Beta decay radioactive process-atomic number changes but mass number stays same
formation

Electron capture

produced by nuclear relaxations

emitted due to excited nucleus returning to ground state in one or more
Gamma
quantized steps with release of monoenergetic gamma rays
decay
expect from their source-they are indistinguishable from X-rays of same
energy

Advantages of Radiochemical Techniques

Characterized by good accuracy
Ability to be adapted to a wide number of applications
Minimize or even eliminate the need for separations that are required in other analytical methods

Measurement of Radioactivity
1. Ionization Method (Geiger-Muller tube method)
Invented by Hans Geiger in 1908 and developed by Walther Muller in 1928.
Detects the low level and radiation which are produced from radioactive sources.
2. Liquid Scintillation method
It is process in which energy of fast moving electrons is transferred with the help of
solvent that produces scintillant molecules.
Polash proposed this method which is mainly used for the determination of C-14.
Liquid used in this method is benzene or mixture of benzene and toluene.
Liquid is first converted into Carbon dioxide and reacted with lithium to produce
lithium carbide and flourescene is detected with photomultiplier tube

3. Radiometric method
Based upon principle, it is classified as
a. Isotope dilution method
introduced by Havesy and Hobbie in 1932. Used for determination of inorganic
compounds. Addition of radiotracer enable the sample to be detected by this
method.
Small amount of radiotracer is added to the solvent. Organic solvent containing
suitable reagent is added. Solution is stirred well and radiotracer is then
extracted. Aliquot of the organic phase is separated. Organic phase is
evaporated to dryness. Sample is then counted by Geiger-Muller counter
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 Ideal features are: must be pure, should have chemical identity, uniform
distribution, should have long-half life.
Advantages are: Highly sensitive, small amount can be easily handled, less
expensive, instrumentation is very simple.
Applications: elemental analysis of geological, biological, and environmental
samples; determination of vitamins, antibiotics, aminoacids, and hormones;
study of interaction of elements in disease and determination of total volume of
the blood in human body.
Following types are:
I. Direct isotopic dilution analysis
Known amount of the labelled isotope is added to the sample which
contains unlabeled compound.
II. Inverse isotopic dilution analysis
Known amount of non-labelled compound is added to the sample
containing unknown amount of labelled compound.
b. Radiometric titrations
This method is based on the determination of radioactivity by the addition of
radioactive tracer. End point is determined by sudden change in radioactivity.
Its limitation is that it requires phase separation. Example: determination of
halides using Ag-110 as a tracer

c. Radiochromatography
This method is combination of paper or TLC with IEC or GC by using
radioactive tracer. The spots are detected by Geiger-Muller counter or
Scintillation counter.
Asbestos paper is spotted with radiotracer and radiotracer is placed in glass
chamber containing the dilute HCl and all the paper for the development of spot
for 1 hour. Paper is dried in drying chamber. Cut the paper along with line and
place in counter chamber for 50sec.
d. Radio immuno assay

Radio Immuno Assay (RIA)

Rosalyn Yallow and Soloman Berson developed this method in 1958 using 131I as radiotracer. It involves
the separation of proteins by antigen-antibody complex.

Principle: Addition of known quantity of the antigen which is labelled with I-131 radiotracer. Then it is
mixed with the sample and reagent antibody. Labelled antigen is combined with antibody to form antigen-
antibody complex.

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Requirements for RIA:
PREPARATION AND CHARACTERIZATION OF ANTIGEN: These are prepared by the synthesis or
by isolation from the natural sources.
TREATMENT OF ANTIGEN WITH RADIOACTIVE I-131: This can be done by tagging procedure.
PREPARATION OF SPECIFIC ANTIBODY: Antibodies are prepared by injecting the antigens into the
blood stream which produces specific antibody. These are extracted from the serum.
ASSAY AND SEPARATION OF COMPLEXES: This step is carried out by the attachment of antibodies
to the micro titre well surface. Addition of antigen binds to the active sits of the antibody. Unbound
antigens are removed by washing of the well. Centrifugation can also be used to remove the unbound
antigen.

Advantages:
Highly specific
Highly sensitive
Applications:
Detection of
o Narcotic drugs
o Hormones
o Neurotransmitters
o Drug poisoning
o Steroids
Screening for hepatitis virus
Cancer diagnosis
Analysis of insulin

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Enzyme Linked Immuno Sorbent Assay (ELISA)
ELISA was developed by Engvall and Pearlmann in 1971. Its principle is similar to that of RIA except the
use of radioactive labelled compound.

ELISA

Sandwich type Indirect type Direct type

ELISA ELISA ELISA

Sandwich type
In this method, two antibodies are required, one is known as capture antibody and second is called detection
antibody. In this method, most commonly used enzymes are Alkaline phosphatases and Horse radish
peroxidase. Substrate used in this method is chromogen (colored compound).
Proceudre/method:
1. Titre wells are coated with suitable antibody and sample is added.
2. Incubate to allow antigen-antibody reaction
3. Wells are washed with buffer to remove unbound antigen
4. Labelled antibody is added with enzyme followed by incubation to bind antigens with labelled
antibodies
5. After that, substrate is added to produce color

Indirect type ELISA

Procedure/method:
1. Antigen to be tested is added to each titre well of ELISA plate
2. Solution of non-reacting protein is added to block the surface
3. To this, antibodies are added which are bound to the antibody containing enzyme.
4. Substrate is added which changes color of the solution that is proportional to the antigen present in
sample.

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Competitive type ELISA
Procedure/method:
1. Tire wells are coated with antibodies
2. Sample containing antigen and antigen labelled enzyme is added
3. Titre wells are incubated to complete the antigen-antibody reaction
4. Substrate is added and again incubated for color production
5. Resulting product is measured for color intensity which is inversely proportional to the antigen in
sample

Advantages for ELISA

Very sensitive even for nano-gram levels.
Values are reproducible
Used for drug monitoring
Applications of ELISA
Screening of contaminated food with HIV-1 and HIV-2
Measurement of toxins in contaminated food
Detection of allergens in food and house dust
Determination of hormonal levels
Detection of infections
o Syphilis
o Taxoplasma gondii
o Chlamudia
Detection of narcotic drugs