RADIOCHEMICAL

TECHNIQUES
RADIOCHEMISTRY:
• Radiochemical analysis, a branch of analytical chemistry, holds a
unique place in scientific investigations. It involves the study and
measurement of radioactive substances and their properties.
• Radiochemistry is defined as “the chemical study of radioactive
elements, both natural and artificial, and their use in the study of
chemical processes”.
• Radiochemical methods of analysis take advantage of the instability of
some elemental isotopes, which decay through the release of alpha
particles, beta particles, gamma rays, and/or X-rays, often provide for
a selective analysis for one analyte in a complex mixture of other
species without the need for a prior separation.
RADIOCHEMICAL METHODS:
• Radiochemical methods are primarily concerned with the study of
radioactivity in naturally occurring radioactive materials and in other
materials in which radionuclides and their compounds are produced
by irradiation.
• Radiochemical methods are characterized by good accuracy and their
ability to be adapted to a wide number of applications. Another
advantage to this method is that they minimize or even eliminate the
need for separations that are required in other analytical methods.
TYPES OF RADIOCHEMICAL
METHODS:
• Radiochemical method of analysis is of five different types:
1. Isotope dilution analysis IDA
• i. Direct IDA
• ii. Indirect IDA
2. Neutron activation analysis
3. Radiometric analysis
4. Radio Immune Assay
5. Autoradiography
 1-Isotopes Dilution Analysis (IDA):
• Isotopic dilution was introduced by Von Havesy and Hofer in 1934. It
involves the preparation of the analyte in a radioactive form.
• Isotope Dilution Analysis (IDA) is a quantitative analytical technique
used to determine the concentration of an element or a compound in
a sample by adding a known quantity of an isotopically enriched form
of the element or compound (the "spike") and measuring the
resulting isotopic ratios.
• This method is highly accurate and precise, making it valuable in
various fields such as chemistry, biochemistry, environmental science,
and pharmacology.
• DIRECT ISOTOPES DILUTION ANALYSIS:
• A known amount of compound tagged or labeled with radioactive
compound (O-18 or a hydrogen with H-2) is added to the unknown
labeled compound and both are thoroughly mixed.
• After treatment to ensure homogeneity between the labeled and
unlabeled species, a portion is recovered as a chemically pure
substance.
• The pure substance is weighed and its radioactivity measured. The
extent of the dilution of the radioactive sample may be then
calculated and related to the amount of the radioactive substance in
the original sample.
• Data Analysis:
• The isotopic ratios in the resulting mixture are measured using mass
spectrometry or another suitable analytical technique.
• Use the isotopic ratio data to calculate the original concentration of the
analyte in the sample. The calculation typically involves the following
equation:

• Where:
• 𝐶sample= Concentration of the analyte in the sample.
• 𝐶spike = Concentration of the analyte in the spike.
• R initial​= Isotopic ratio in the sample before adding the spike.
• 𝑅 final= Isotopic ratio in the sample after adding the spike.
• 𝑊initial​= Weight or volume of the sample.
• 𝑊final = Weight or volume of the spike added.
• Where:
• W = Weight of the unlabeled compound to be determined
• Wo = Weight of added labeled compound.
• S = Specific activity of isolated pure compound
• So = Specific activity of added labeled compound.
 Inverse / Reverse IDA:
• The inverse method in isotope dilution analysis (IDA) is a variation of
the traditional IDA approach.
• In the inverse method, a known amount of the natural sample is
added to a solution containing a known concentration of the
isotopically enriched element. This approach is often used when the
analyte concentration in the sample is too low for direct
measurement.
• Instead of adding the spike to the sample, a precisely measured
amount of the natural sample is added to a solution containing a
known concentration of the isotopically enriched spike.
Applications of IDA:

• Environmental Analysis: Determining trace metal concentrations in

water, soil, and air samples.
• Clinical Chemistry: Measuring levels of drugs, hormones, and
metabolites in biological fluids.
• Food and Beverage Testing: Analyzing nutrient and contaminant
levels in food products.
• Pharmaceuticals: Quantifying active ingredients and impurities in
drug formulations.
2-Neutron activation analysis NAA:
• NAA is a sensitive analytical technique useful for performing both
qualitative and quantitative multi-element analysis of major, minor
and trace elements in the samples from almost every conceivable
field of scientific or technical interest.
• NAA offers sensitivities that are superior to those attainable by other
methods, on the order of parts per billion or better.
• Because of its accuracy and reliability, NAA is generally recognized as
the “referee method” of choice.
• It is based on the process of neutron activation, where samples are
irradiated with neutrons, causing some of the nuclei in the sample to
capture neutrons and become radioactive. The resulting radioactive
isotopes decay, emitting gamma rays that are characteristic of the
specific elements in the sample. By measuring these gamma rays, one
can identify and quantify the elements present.
Key Steps in Neutron Activation Analysis:
• Sample Preparation: The sample to be analyzed is prepared, typically by cleaning and weighing
• Neutron Irradiation: The sample is exposed to a source of neutrons, usually in a nuclear reactor or
a neutron generator. During irradiation, stable isotopes in the sample capture neutrons and
become radioactive isotopes.

• Decay Period: After irradiation, the sample is allowed to decay for a period to reduce the activity
of short-lived isotopes and to allow for the decay of longer-lived isotopes. This period is called the
"cooling" or "decay" time.

• Gamma Ray Spectroscopy: The radioactive sample emits gamma rays, which are detected using a
gamma-ray spectrometer. Each element emits gamma rays with specific energies, allowing for the
identification of the elements present in the sample.

• Data Analysis: The intensity of the gamma rays is measured and analyzed to determine the
concentration of each element in the sample. This involves comparing the observed gamma-ray
spectra to known standards.
 3-Radiometric Assay:
• A radiometric assay is a technique used to measure the concentration of
radioactively labeled substances within a sample. This type of assay takes
advantage of the radioactive decay properties of isotopes, which emit radiation
that can be detected and quantified.
• The use of a radioactive reagent of known activity to isolate the analyte from the
other components of the sample.
• The activity of the product is directly proportional to the amount of analyte.
• In this analysis the radioactive substance is used indirectly to determine the
quantity of an inactive compound.
• Example: Chromate has been determined by precipitating it with radioactive Ag
(Ag-111) of a known activity. Determining the activity of the precipitate of
Ag2CrO4 allows for the determination of the amount of chromate.
 4-Radio Immune Assay:

• Radioimmuno assay is a highly sensitive method to determine an

antigen’s concentration in a sample.
• This technique was introduced by Rosalyn Yalow and Solomon Berson
for the detection of insulin in human blood.
• Principle:
• Antigens and antibodies bind specifically to form the Ag-Ab complex. The antigen
can be labeled or conjugated with radioisotopes. The unlabeled antigens from the
sample compete with radiolabeled antigens to bind on paratopes of specific
antibodies. The unlabeled antigens replace labeled antigens that are already linked
with the antibodies. The unlabeled antigens when bind with antibodies, increases
the amount of free radiolabeled antigens in the solution. Hence the concentration
of free labeled antigens is directly proportional to the bound unlabeled antigens.

• It involves a combination of three principles.

• An immune reaction i.e. antigen, antibody binding.

• A competitive binding or competitive displacement reaction. (It gives specificity)
• Measurement of radio emission. (It gives sensitivity)
Measurement of radio emission:

• Once the incubation is over, then washings are done to remove any unbound antigens. Then
radio emission of the antigen antibody complex is taken, the gamma rays from radio labeled
antigen are measured.
• This method is based upon the completion between radio-labeled antigen (Ag*) and unlabeled
antigens (Ag) for binding to a specific antibody (Ab) serum.
• 𝐴𝑔∗ + 𝐴𝑔+𝐴𝑏 ⇋[𝐴𝑔∗𝐴𝑏]+ [𝐴𝑔 𝐴𝑏]+ [𝐴𝑔∗ +𝐴𝑔]
• Where,
• Ag* = Radio-labeled antigen
• Ag = Antigen
• Ab = Antibody
• [Ag*Ab] = Radioactive antigen-antibody complex
• [Ag Ab] = Non-reactive
• [Ag* + Ag] = un-reacted
• The complex is separated and its radioactivity is measured.
• The method can also be useful for quantitative analysis of various drugs, steroids, and hormones.
 5-Autoradiography
• Autoradiography is a technique used to visualize the distribution and
localization of radioactively labeled molecules in biological samples,
such as tissues, cells, or gels.
• This method combines the principles of radioactivity and
photographic imaging to create an image that shows the presence
and distribution of radioactive substances.
• By combining the sensitivity of radioactive detection with the spatial
resolution of photographic imaging, autoradiography provides
valuable insights into molecular localization and dynamics in a wide
range of scientific fields.