Abstract: In chemical reaction kinetics studies, kinetic isotope effects (KIE) provide critical information for understanding reaction pathways and mechanisms. While hydrogen/deuterium isotope effects have been extensively studied, increasing attention is being paid to isotope effects involving carbon and other heavier atoms. A significant breakthrough occurred in 1995 when Singleton developed a high-precision NMR-based method for measuring ¹²C/¹³C KIE values. This innovative approach eliminates the need for expensive and complex isotope labeling procedures. By utilizing samples with natural ¹³C abundance and high-resolution NMR spectroscopy, the method enables direct measurement of ¹²C/¹³C KIE values with remarkable precision. This article comprehensively describes the theoretical foundations, experimental protocols, and practical applications of this method, emphasizing its importance and advantages in mechanistic and kinetic studies of chemical reactions.

Key words: Kinetic isotope effect (KIE), Nuclear magnetic resonance (NMR) technology, Natural abundance, ¹³C NMR isotope effect experiment, Chemical reaction mechanism