Exploring the Practical Limits of Natural Abundance 13C Kinetic Isotope Effect Measurements
Year: 2026
Type: preprint
Abstract: The use of quantitative 13 C KIE methodology at natural abundance developed by Daniel Singleton, commonly known as the ‘Singleton Method’, to determine kinetic isotope effects (KIEs) provides a powerful tool to probe the fine details of reaction mechanisms. Modern advances in NMR technology have led to improved sensitivity of 13 C NMR spectroscopy, enabling the measurement of more precise 13 C NMR integrations used to determine isotopic enhancement in natural abundance methodology. Herein, we present a systematic experimental examination of the precision limits of both inter- and intramolecular natural abundance 13 C KIE measurements using modern NMR instrumentation. Employing the Baeyer–Villiger oxidation of cyclohexanone as a model system, we measured intermolecular 13 C KIEs across fractional conversions ranging from 15% to 80%. Nonlinear weighted fitting and a 95% prediction interval are provided as guidance in determining the accuracy of measurements across the range of fractional conversions. Statistical analysis shows that relatively accurate measurements can be obtained at lower fractional conversions provided careful attention is given to signal-to-noise, calibration of fractional conversion, and replication of measurements. The requirement of calibrating fractional conversion measurements is emphasized and, in this case, accounted for a difference of nearly 0.4% in the measured 13 C KIE. No significant difference in measured KIEs was found between 400 and 600 MHz instruments. Intramolecular 13 C KIEs measured across 20%–99% conversion yielded a consistent KIE (range 1.044-1.048), confirming independence from fractional conversion. These results establish quantitative benchmarks for precision and accuracy of natural abundance 13 C KIE measurements and expand the practical range of conditions under which reliable mechanistic data can be obtained.
Source: ChemRxiv
Institution: Binghamton University
Language: English
Topic: Chemical Reaction Mechanisms
Subfield: Organic Chemistry
Field: Chemistry
Domain: Physical Sciences
Open Access status: green
OpenAlex ID: W7155391258