ISOTOPE DILUTION ANALYSIS

Isotope Dilution Analysis There are certain analytical methods that can be applied to all fields of tracer
use. Foremost among these isotope dilution analysis (IDA). The method of isotope dilution comprises
the addition of known amounts of isotopically-enriched substance to the analyzed sample. Mixing of the
isotopic standard with the sample effectively "dilutes" the isotopic enrichment of the standard and this
forms the basis for the isotope dilution method. A known quantity of a compound containing an
unknown quantity of a particular element is mixed with a spike (a known weight of a radioactive isotope
of the element). The specific activity (disintegrations per second per kilogram) of the spike is known
precisely, so the isotopic composition of the mixture can be used to calculate the amount of the element
in the sample. A small amount of the mixture is isolated from the sample, weighed, and its specific
activity measured. The concentration of the inactive element in the sample may be estimated by the
dilution of the radiotracer. Isotope dilution analysis can be applied to all elements that have two or
more naturally occurring isotopes (about 80% of all elements), provided that a spike enriched in one of
the isotopes of that element is available.
Direct IDA

In direct IDA, we are faced with the problem of determining the amount of some inactive material A in a
system. Let us define this unknown amount as x grams. To the system containing x grams of inactive A,
we add y grams of active material A* Of known activity D. Thus, we know the specific activity of the
added active material, S1 . That is,

After thoroughly mixing the active material A* with the inactive A in the system, one isolates, not
necessarily quantitatively, and purifies a sample of the mixture of A and A* and measures its specific
activity, S2 . Clearly, conservation of material says that

This is the basic equation of direct isotope dilution analysis. The unknown amount x of material A is
given in terms of the amount y of added labeled material A* and the two measured specific activities S 1
and S2 .
Example Problem:

Let us consider a practical problem to illustrate the use of this technique. A protein hydrolysate is to be
assayed for aspartic acid. Exactly 5.0 mg of aspartic acid, having a specific activity of 0.46 µCi/mg, is
added to the hydrolysate. From the hydrolysate, 0.21 mg of highly purified aspartic acid, having a
specific activity of 0.01 µCi/mg, can be isolated. How much aspartic acid was present in the original
hydrolysate?

Thus, by isolating a small fraction of the added aspartic acid and measuring the diminution in its specific
activity, the aspartic acid content of the original sample can be determined. Note this example involved
a large change in specific activity upon dilution. Poor experimental design or other circumstances may
lead to a small change in specific activity upon dilution. In such cases, the results obtained from IDA
involve a small difference between two large numbers and are quite uncertain.
Inverse IDA
A known quantity of non labelled compound is added to a mixture containing an unknown
amount of labelled compound. In contrast to direct method, this method allows the addition of
relatively large quantity of un-labelled carrier compound. This facilitates isolation and
purification. This is especially useful metabolism studies of labelled drugs.
Advantages
Isotope dilution analysis is a highly sensitive, selective analytical method capable of high precision. It
offers the opportunity to determine the amount of material present in a system without the need for a
quantitative separation of the material from the system. Partial loss of analyte during preparation is
compensated for since physical and chemical interferences are not an issue and will cancel out as they
will affect each isotope identically. Isotope dilution is classified as a method of internal standardization,
because the standard (isotopically-enriched form of analyte) is added directly to the sample. In addition,
unlike traditional analytical methods which rely on signal intensity, isotope dilution employs signal
ratios. It is free of interference from other elements present and its accuracy is governed by the
calibration of the spike solution.
Disadvantages
➢ Generally only applicable to multiple
➢ Isotopic elements
➢ Need an enriched isotope spike for the analyte of interest
➢ Not always available or sometimes at very high cost
➢ Need two interference free isotopes
➢ VERY time consuming