Isotope dilution

Basic principle of isotope dilution
Adding of an isotopically altered standard to the sample changes the natural isotopic composition of the analyte. By measuring the resulting isotopic composition, it is possible to calculate the amount of the analyte present in the sample.

Isotope dilution analysis is a method of determining the quantity of chemical substances. In its most simple conception, 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. Isotope dilution is classified as a method of internal standardisation, 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. Owing to both of these advantages, the method of isotope dilution is regarded among chemistry measurement methods of the highest metrological standing.[1]

Early history

The Hungarian chemist George de Hevesy was awarded the Nobel Prize in Chemistry for development of radiotracer method, which is a forerunner of isotope dilution

Analytical application of the radiotracer method is forerunner of isotope dilution. This method was developed in the early 20th century by George de Hevesy for which he was awarded the Nobel Prize in Chemistry for 1943.

An early application of isotope dilution in the form of radiotracer method was determination of the solubility of lead sulphide and lead chromate in 1913 by George de Hevesy and Friedrich Adolf Paneth.[2] In the 1930s, US biochemist David Rittenberg pioneered the use of isotope dilution in biochemistry enabling detailed studies of cell metabolism.[3]

Tutorial example

Tutorial illustration of isotope dilution analysis with fish counting in lakes

Isotope dilution can be effectively explained using mark and recapture method from biology - a method commonly used in ecology to estimate the population size of fish.

Isotope dilution can be likened to Lincoln-Petersen method. Assume that the number of fish in a pond is to be determined. Five labeled fish are added to the pond during the first visit (nB = 5). On the second visit, a number of fish is captured and one observes that the ratio of native-to-labeled is 10:1. From here, we can estimate the original number of fish in the pond, nA:

This is a simplified view of isotope dilution yet it illustrates the salient features of isotope dilution. A more complex situation arises when the distinction between labeled and unlabeled fish becomes fuzzy. This can occur, for example, when the lake already contains a small number of labeled fish from the previous field experiments. In such situation, the following expression can be employed:

where RA is the ratio of the native-to-labeled fish in lake, RB is the ratio of the native-to-labeled fish in the lot of nB marked fish which are added to the pond, and RAB is the ratio of the native-to-labeled fish captured during the second visit.


Isotope dilution is almost exclusively employed with mass spectrometry in applications where high-accuracy is demanded. For example, all National Metrology Institutes rely significantly on isotope dilution when producing certified reference materials. In addition to high-precision analysis, isotope dilution is applied when low recovery of the analyte is encountered. In addition to the use of stable isotopes, radioactive isotopes can be employed in isotope dilution which is often encountered in biomedical applications, for example, in estimating the volume of blood.

Single dilution method

Isotope dilution notation
Name Symbol
Analyte A
Isotopic standard (Spike) B
Analyte + Spike AB

Consider a natural analyte rich in isotope iA (denoted as A), and the same analyte, enriched in isotope jA (denoted as B). Then, the obtained mixture is analyzed for the isotopic composition of the analyte, RAB = n(iA)AB/n(jA)AB. If the amount of the isotopically-enriched substance (nB) is known, the amount of substance in the sample (nA) can be obtained:[4]

Here, RA is the isotope amount ratio of the natural analyte, RA = n(iA)A/n(jA)A, RB is the isotope amount ratio of the isotopically-enriched analyte, RB = n(iA)B/n(jA)B, RAB is the isotope amount ratio of the resulting mixture, x(jA)A is the isotopic abundance of the minor isotope in the natural analyte, and x(jA)B is the isotopic abundance of the major isotope in the isotopically-enriched analyte.

For elements with only two stable isotopes, such as boron, chlorine, or silver, the above single dilution equation simplifies to the following:

In a typical gas chromatography analysis, isotopic dilution can decrease the uncertainty of the measurement results from 5% to 1%. It can also be used in mass spectrometry (commonly referred to as isotopic dilution mass spectrometry or IDMS), in which the isotopic ratio can be determined with precision typically better than 0.25%.[5]

Optimum composition of the blend

In a simplified manner, the uncertainty of the measurement results is largely determined from the measurement of RAB:

From here, we obtain the relative uncertainty of nA, ur(nA) = u(nA)/nA:

Lowest relative uncertainty of nA corresponds to the condition when the first derivative with respect to RAB equals zero. In addition, it is common in mass spectrometry that u(RAB)/RAB is constant and therefore we can replace u(RAB) with RAB. These ideas combine to give

Solving this equation leads to the optimum composition of the blend AB, i.e., the geometric mean between the isotopic compositions of standard (A) and spike (B):

This simplified equation was first proposed by De Bievre and Debus numerically[4] and later by Riepe and Kaiser analytically.[6] It has been noted that this simple expression is only a general approximation and it does not hold, for example, in the presence of Poisson statistics[7] or in the presence of strong isotope signal ratio correlation.[8]

Double dilution method

Single dilution method requires the knowledge of the isotopic composition of the isotopically-enriched analyte (RB) and the amount of the enriched analyte added (nB). Both of these variables are hard to establish since isotopically-enriched substances are generally available in small quantities of questionable purity. As a result, before isotope dilution is performed on the sample, the amount of the enriched analyte is ascertained beforehand using isotope dilution. This preparatory step is called the reverse isotope dilution and it involves a standard of natural isotopic-composition analyte (denoted as A*). First proposed in the 1940s[9] and further developed in the 1950s,[10] reverse isotope dilution remains an effective means of characterizing a labeled material.

Isotope dilution notation
Name Symbol
Analyte A
Natural standard A*
Isotopic standard (Spike) B
Analyte + Spike AB
Standard + Spike A*B

Reverse isotope dilution analysis of the enriched analyte:

Isotope dilution analysis of the analyte:

Since isotopic composition of A and A* are identical, combining these two expressions eliminates the need to measure the amount of the added enriched standard (nB):

Double dilution method can be designed such that the isotopic composition of the two blends, A+B and A*+B, is identical, i.e., RAB = RA*B. This condition of exact-matching double isotope dilution simplifies the above equation significantly:[11]

Triple dilution method

To avoid contamination of the mass spectrometer with the isotopically-enriched spike, an additional blend of the primary standard (A*) and the spike (B) can be measured instead of measuring the enriched spike (B) directly. This approach was first put forward in the 1970s and developed in 2002.[12]

Calculations using calibration curve

Many analysts do not employ analytical equations for isotope dilution analysis. Instead, they rely on building calibration curve from the mixtures of natural primary standard (A*) and the isotopically-enriched analyte (spike, B). The calibration curves are obtained by plotting the isotope ratio in the prepared blends against the ratio of sample mass to the mass of spike solutions in each blend. Isotope dilution calibration graphs sometimes show non-linear relationship and in practice polynomial fitting is often performed to empirically describe such curves.[13]

When calibration plots are markedly nonlinear, one can bypass the empirical polynomial fitting and employ the ratio of two linear functions (known as Padé approximant) which is shown to describe the curvature of isotope dilution curves exactly.[14]

See also


  1. M.J.T. Milton; R. I. Wielgosz (2000). "Uncertainty in SI-traceable measurements of amount of substance by isotope dilution mass spectrometry". Metrologia. 37 (3): 199–206. doi:10.1088/0026-1394/37/3/3.
  2. G. V. Hevesy; F. Paneth (1913). "Die Löslichkeit des Bleisulfids und Bleichromats". Z. anorg. allgem. Chem. 82 (1): 323–328. doi:10.1002/zaac.19130820125.
  3. Isotope dilution Biographical Memoirs of the National Academy of Sciences
  4. 1 2 P. J. De Bievre; G. H. Debus (1965). "Precision mass spectrometric isotope dilution analysis". Nucl. Instr. Meth. 32 (2): 224–228. doi:10.1016/0029-554X(65)90516-1.
  5. EPA publication SW-846, "Test Methods for Evaluating Solid Waste, Physical/Chemical Methods", available at See Method 6800, "Elemental and Speciated Isotope Dilution Mass Spectrometry", available at
  6. W. Riepe; W. Kaiser (1966). "Massenspektrometrische Spurenanalyse von Calcium, Strontium und Barium in Natriumazid durch Isotopenverdünnungstechnik". Anal. Bioanal. Chem. 223 (5): 321–335. doi:10.1007/BF00513462.
  7. R. Hoelzl; C. Hoelzl; L. Kotz; L. Fabry (1998). "The optimal amount of isotopic spike solution for ultratrace analysis by isotope dilution mass spectrometry". Accred. Qual. Assur. 3 (5): 185–188. doi:10.1007/s007690050219.
  8. J. Meija; Z. Mester (2007). "Signal correlation in isotope ratio measurements with mass spectrometry: Effects on uncertainty propagation". Spectrochim. Acta B. 62 (11): 1278–1284. doi:10.1007/BF00513462.
  9. K. Bloch; H.S. Anker (1948). "An Extension of the Isotope Dilution Method". Science. 107 (2774): 228. doi:10.1126/science.107.2774.228.
  10. C. Rosenblum (1957). "Principles of Isotope Dilution Assays". Anal. Chem. 29 (12): 1740–1744. doi:10.1021/ac60132a021.
  11. A. Henrion (1994). "Reduction of systematic errors in quantitative analysis by isotope dilution mass spectrometry (IDMS): an iterative method". Fres. J. Anal. Chem. 350 (12): 657–658. doi:10.1007/BF00323658.
  12. M.J.T. Milton; J.A. Wang (2002). "High Accuracy Method for Isotope Dilution Mass Spectrometry with Application to the Measurement of Carbon Dioxide". Int. J. Mass Spectrom. 218: 63–73. doi:10.1016/S1387-3806(02)00663-2.
  13. J.A. Jonckheere; A.P. De Leenheer; H.L. Steyaert (1983). "Statistical evaluation of calibration curve nonlinearity in isotope dilution gas chromatography/mass spectrometry". Anal. Chem. 55: 153–155. doi:10.1021/ac00252a042.
  14. E. Pagliano; Z. Mester; J. Meija (2015). "Calibration graphs in isotope dilution mass spectrometry". Anal. Chim. Acta. doi:10.1016/j.aca.2015.09.020.

Further reading

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