page_title: Kinetic isotope effect
section_title: Experiments
hierarchical_section_title: Kinetic isotope effect / Experiments
caption_attribution_description: English: Reaction energy profile for when the C-H bond cleavage occurs at a product-determining step after the RDS
context_page_description: In physical organic chemistry, a kinetic isotope effect is the change in the reaction rate of a chemical reaction when one of the atoms in the reactants is replaced by one of its isotopes. Formally, it is the ratio of rate constants for the reactions involving the light and the heavy isotopically substituted reactants:
  This change in reaction rate is a quantum mechanical effect that primarily results from heavier isotopologues having lower vibrational frequencies compared to their lighter counterparts. In most cases, this implies a greater energetic input needed for heavier isotopologues to reach the transition state, and consequently, a slower reaction rate. The study of kinetic isotope effects can help the elucidation of the reaction mechanism of certain chemical reactions and is occasionally exploited in drug development to improve unfavorable pharmacokinetics by protecting metabolically-vulnerable C-H bonds.
context_section_description: Simmons and Hartwig refer to the following three cases as the main types of kinetic isotope effect experiments involving C-H bond functionalization:
  A) KIE determined from absolute rates of two parallel reactions
  In this experiment, the rate constants for the normal substrate and its isotopically labeled analogue are determined independently, and the KIE is obtained as a ratio of the two. The accuracy of the measured KIE is severely limited by the accuracy with which each of these rate constants can be measured. Furthermore, reproducing the exact conditions in the two parallel reactions can be very challenging. Nevertheless, a measurement of a large kinetic isotope effect through direct comparison of rate constants is indicative that C-H bond cleavage occurs at the rate-determining step. (A smaller value could indicate an isotope effect due to a pre-equilibrium, so that the C-H bond cleavage occurs somewhere before the rate-determining step.)
  B) KIE determined from an intermolecular competition
  In this type of experiment, the same substrates that are used in Experiment A are employed, but they are allowed in to react in the same container, instead of two separate containers. The kinetic isotope effect from this experiment is determined by the relative amount of products formed from C-H versus C-D functionalization (or it can be inferred from the relative amounts of unreacted starting materials). It is necessary to quench the reaction before it goes to completion to observe the kinetic isotope effect (see the Evaluation section below).  Generally, the reaction is halted at low conversion (~5 to 10% conversion) or a large excess (> 5 equiv.) of the isotopic mixture is used.  This experiment type ensures that both C-H and C-D bond functionalizations occur under exactly the same conditions, and the ratio of products from C-H and C-D bond functionalizations can be measured with much greater precision than the rate constants in Experiment A. Moreover, only a single measurement of product concentrations from a single sample is required. However, an observed kinetic isotope effect from this experiment is more difficult to interpret, since it may either mean that C-H bond cleavage occurs during the rate-determining step or at a product-determining step ensuing the rate-determining step. The absence of a kinetic isotope effect, at least according to Simmons and Hartwig, is nonetheless indicative of the C-H bond cleavage not occurring during the rate-determining step.
  C) KIE determined from an intramolecular competition
  This type of experiment is analogous to Experiment B, except this time there is an intramolecular competition for the C-H or C-D bond functionalization. In most cases, the substrate possesses a directing group (DG) between the C-H and C-D bonds. Calculation of the kinetic isotope effect from this experiment and its interpretation follow the same considerations as that of Experiment B.  However, the results of Experiments B and C will differ if the irreversible binding of the isotope-containing substrate takes place in Experiment B prior to the cleavage of the C-H or C-D bond.  In such a scenario, an isotope effect may be observed in Experiment C (where choice of the isotope can take place even after substrate binding) but not in Experiment B (since the choice of whether C-H or C-D bond cleaves is already made as soon as the substrate binds irreversibly).  In contrast to Experiment B, the reaction does not need to be halted at low consumption of isotopic starting material to obtain an accurate kH/kD, since the ratio of H and D in the starting material is 1:1, regardless of the extent of conversion.
  One non-C-H activation example of different isotope effects being observed in the case of intermolecular (Experiment B) and intramolecular (Experiment C) competition is the photolysis of diphenyldiazomethane in the presence of t-butylamine.  To explain this result, the formation of diphenylcarbene, followed by irreversible nucleophilic attack by t-butylamine was proposed.  Because there is little isotopic difference in the
caption: Reaction energy profile for when the C-H bond cleavage occurs at a product-determining step after the RDS
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