We often see reactions such as these in textbooks to highlight how reaction mechanisms work: enter image description here enter image description here

How are the radioactive substrates required for these mechanisms synthesized in the first place? There seems to be no easy way of just 'substituting' a radioactive atom in place of a non-radioactive one (especially for Benzene-14C, it might be easier to make a radioactive sodium salt using $\ce{^{24}Na}$). Also, Once the substrate is synthesized, how is it separated and purified? I'm assuming that the physical properties of these radioactive substrates would be very similar to their non-radioactive counterparts, which would make it very difficult to do so.


The current methods employed for synthesis fall into the following three categories1:

  • Chemical synthesis
  • Biochemical methods
  • Isotope exchange reactions

Chemical Synthesis:

Most chemical syntheses involving $\ce{^14 C}$ are done with Grignard reagent and $\ce{^14 CO2}$ or $\ce{^14 CN-}$. As an example, Benzene-14 is synthesized as such2:

Benzene-14 Synthesis

Basic preparatory methods such as the above have been worked out long ago. An example of a more complex synthesis would be preparation of 18F-Fluorodeoxyglucose:

Fluorodeoxyglucose synthesis

Biochemical methods

A few examples of these, as mentioned in (1), are:

High yields of [U-14C]sucrose, up to 70% based on $\ce{^14 CO2}$, are obtainable in detached leaves after a few hours photosynthesis using tobacco or Canna indica leaves for example.

DNA labelled either with $\ce{^14 C}$ or with $\ce{^3 H}$ is prepared by feeding E.coli with the correspondingly labelled thymine precursor. Similarly, by feeding [$\ce{^35 S}$]sulphate to E.coli or to yeast cells, isolation and hydrolysis of the proteins yields L.[$\ce{^35S}$]methionine and L-[$\ce{^35 S}$]cystine in radiochemical yields of 30% and 10% respectively.

Another example is the synthesis of C-14 labelled Phenylpyruvic acid3

C-14 Phenylpyruvic acid synth

Isotope exchange reactions

These are fairly common for atoms that can easily be exchanged, such as replacing $\ce{^23 Na}$ for $\ce{^24 Na}$, which was mentioned in the question. Another common mechanism for synthesis of Hydrogen isotope compounds is Hydrogen-Deuterium exchange. A similar mechanism also allows hydrogen-tritium exchange4.

Purification of radiolabelled compounds

This is mainly done through Chromatography1, as the amount of radiolabelled substrate required is generally quite small. High-performance liquid chromatography is used to separate the compounds. The chemical identity of the compounds is confirmed by co-chromatographic behaviour with the unlabelled compound.

For further information, refer to the references (1) and (3), which go into a lot more detail with regards to the synthesis of these compounds.


  1. Anthony Evans, E. “Synthesis of Radiolabelled Compounds.” Journal of Radioanalytical Chemistry, vol. 64, no. 1–2, Mar. 1981, pp. 9–32. doi:10.1007/BF02518337.
  2. Schmid, K., et al. “A Simple Method of Preparing Labeled Benzene.” Advances in Tracer Methodology, edited by Seymour Rothchild, Springer US, 1966, pp. 37–44. doi:10.1007/978-1-4684-8625-4_5.
  3. Atzrodt, Jens, and John Allen. “Synthesis of Radiolabeled Compounds for Clinical Studies.” Drug Discovery and Evaluation: Methods in Clinical Pharmacology, edited by Hans Gerhard Vogel et al., Springer Berlin Heidelberg, 2011, pp. 105–18. doi:10.1007/978-3-540-89891-7_12.
  4. Englander, S. W., and J. J. Englander. “[3] Hydrogen—Tritium Exchange.” Methods in Enzymology, vol. 49, Academic Press, 1978, pp. 24–39. doi:10.1016/S0076-6879(78)49005-6.
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    $\begingroup$ You asked and answered the question at the same time? Hmm? The chemical syntheses of monolabeled benzene do not explain how specifically labeled chlorobenzene, in the question, is prepared. $\endgroup$ – user55119 Sep 4 '20 at 14:44
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    $\begingroup$ @user55119 I was doing some research for the question, and then realized I had done enough research to write an answer :) I'm still open for a better answer, or additions to this answer, though. $\endgroup$ – Aniruddha Deb Sep 4 '20 at 15:52
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    $\begingroup$ Some more papers for you: here, here and here. $\endgroup$ – Nilay Ghosh Sep 4 '20 at 17:47
  • $\begingroup$ For specifically C-labeled toluene and benzoic acid, see: science.sciencemag.org/content/109/2820/35 $\endgroup$ – user55119 Sep 4 '20 at 18:44
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    $\begingroup$ @Karl: My bad! Totally missed the bis-Grignard reagent example. I had actually gone back and read the paper cited previously by me. The work was instrumental in J. D. Roberts landmark proof of the benzyne mechanism. My apologies to Aniruddaha Deb, also. Two upvotes. $\endgroup$ – user55119 Sep 5 '20 at 12:23

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