As shown in Eq.
1, KIEs are related to F, the conversion, and R/R
0, a measure of the change in the ratio of isotopically substituted substrate to unsubstituted substrate (Eqs.
2–
3).
$${\text{F}}=1 - \frac{{{\text{[1]~}}}}{{{{\left[ 1 \right]}_0}}}$$
(2)
$$\frac{{\text{R}}}{{{{\text{R}}_0}}}=\frac{{[1{\text{-}}{d_i}{\text{]~~}}{{\left[ 1 \right]}_0}}}{{{{[1{\text{-}}{d_i}{\text{]}}}_0}\left[ 1 \right]}}$$
(3)
In order to obtain reliable values of the KIE, the error ΔKIE must be low. ΔKIE derives from a combination of the errors related to the measurement of the conversion (ΔF) and of the concentration of the isotopically substituted substrates (ΔR/R
0), as reported by Melander and Saunders (Eqs.
4–
6) [
5].
$$\Delta \text {KIE}_\text{F} = \Delta {\text F}~\frac{{\partial\text{KIE}}}{{\partial {\text F}}} = \frac{{ - \ln \left( {{\text R/\text{R}_{0}} } \right)~~\Delta {\text F}}}{{\left( {1 - {\text F}} \right)\ln ^{2} \left[ {\left( {1 - {\text F}} \right) {\text R/\text{R}_{0}} } \right]}}$$
(4)
$$\Delta \text {KIE}_\text{R} = \Delta \left( {{\text R/\text{R}_{0}} } \right)\frac{{\partial \text {KIE}~~}}{{\partial \left( {\text{R/R}_{0 } } \right)}} = \frac{{ - \ln \left( {1 - {\text F}} \right)~\Delta {\text R/\text{R}}_{0} }}{{\left( { {\text R/\text{R}}_{0} } \right)\ln ^{2} \left[ {\left( {1 - {\text F}} \right) {\text R/\text{R}_{0}} } \right]~}}$$
(5)
$$\Delta {\text {KIE}} = \sqrt {\Delta {\text {KIE}_{{\text F}} ^{2}} + \Delta {\text {KIE}_{{\text R}} ^{2}} }$$
(6)
To evaluate ΔF a “reasonable possible error” of 5% of the conversion has been used to account for several sources of error in the measurement of F that are difficult to quantify, including: imprecision in the NMR integrations due to the vagaries of the spectrometer; comparing inequivalent multiplets; inaccuracy in the measurement of F due to the presence of impurities that could affect the integrations; experimental error arising from weighing both substrate and internal standard; signal to noise error in the NMR instrument. The values of ΔR/R
0 have instead been estimated based on extrapolations from our empirical data
4. The error in R/R
0, ΔR/R
0, is related to the relative error in the
2H NMR integrals for
1-
d (Eq.
7). This relative error is equal to the absolute error in the integral, Δ
I, divided by the magnitude of the integral,
I. The absolute error in an NMR integral, Δ
I, is related to the spectral noise, the number of points in the integral and the total number of points in the spectrum [
20]. Comparing different spectra of the same compound, using the same acquisition parameters (and assuming there are no changes in shim or temperature), should give predictable changes in Δ
I. The number of points in both the integral and spectra should be constant when comparing different spectra of the same compound, provided the same integral width is considered. The relative error in the integral is inversely proportional to the concentration of deuterated substrate in the NMR sample (Eq.
8), and proportional to the square root of the number of scans run. Since the total mass of substrate weighed out for the NMR sample is constant, the total concentration of substrates in the sample is approximately constant (
c, Eq.
9), the concentration of
1-
d
i
in the NMR sample is expressed by Eq.
9. Putting this into Eq.
8 and introducing a scaled constant of proportionality
a gives Eq.
10. We have used our experimental errors to calculate the value of
a
5, then applied Eq.
7 to evaluate ΔR/R
0.
$${{\Delta }}{\text{R/}}{{\text{R}}_{\text{0}}}={\text{R/}}{{\text{R}}_{\text{0}}}\sqrt {{{\left( {\frac{{{{\Delta }}{{{I}}_{1{\text{-}}{d_i}}}}}{{{{{I}}_{1{\text{-}}{d_i}}}}}} \right)}^2}+{{\left( {\frac{{{{\Delta }}{{{I}}_{{{\left( {1{\text{-}}{d_i}} \right)}_0}}}}}{{{{{I}}_{{{\left( {1{\text{-}}{d_i}} \right)}_0}}}}}} \right)}^2}}$$
(7)
$$\frac{{{{\Delta }}{{{I}}_{1{\text{-}}{d_i}}}}}{{{{{I}}_{1{\text{-}}{d_i}}}}}=\frac{{{{\Delta }}[1{\text{-}}{d_i}]}}{{[1{\text{-}}{d_i}]}}~ \propto ~\frac{1}{{{{[1{\text{-}}{d_i}]}_{NMR~sample}}~}}~$$
(8)
$${[1{\text{-}}{d_i}]_{NMR~sample}}=~\frac{{[1{\text{-}}{d_i}]}}{{\left[ 1 \right]}}~ \times {{c}}~$$
(9)
$$\frac{{{{\Delta }}{{{I}}_{1{\text{-}}{d_i}}}}}{{{{{I}}_{1{\text{-}}{d_i}}}}}~ \propto ~\frac{{\left[ 1 \right]}}{{[1{\text{-}}{d_i}] \times {\text{c}}}}=~\frac{{{{a}}\left[ 1 \right]}}{{[1{\text{-}}{d_i}]}}$$
(10)