Measurement of oxygen isotope ratios (¹⁸O/¹⁶O) of aqueous O₂ in small samples by gas chromatography/isotope ratio mass spectrometry

Abstract

The successful quantification of (18) O-kinetic isotope effects associated with enzymatic and chemical reduction of dissolved O2 illustrates how the proposed method can be applied for studying enzymatic O2 activation mechanisms in a variety of (bio)chemical processes.

Characterization of Substrate, Cosubstrate, and Product Isotope Effects Associated With Enzymatic Oxygenations of Organic Compounds Based on Compound-Specific Isotope Analysis

“…Oxygen isotope effects for the activation of O 2 by NBDO can be obtained from the analysis of O isotope fractionation in aqueous O 2 . Pati, Bolotin, et al (2016) developed a procedure for "limited turnover assays" with purified enzyme component similar to the one described in the first example for substrate isotope fractionation ( Figure 6, right-hand side). The major differences between enzyme assay for co-substrate vs. substrate isotope analysis are that (i) vials need to be filled with solution without headspace, (ii) one 10-mL reactor is sacrificed for every measurement of O isotope composition (no replicate measurements possible), and (iii) O 2 concentrations are monitored continuously during the reaction with an optical micro-sensor.…”

“…Finally, the isotope ratios of a series of gases and organic vapors is accessible by continuousflow isotope ratio mass spectrometry without the use of chemical conversion interfaces. This approach is chosen here for measurement of 18 O/ 16 O ratios of O 2 as shown in Figure 8b because O 2 isotoplogues can be ionized and measured directly in a isotope ratio mass spectrometer (Pati, Bolotin, et al, 2016). The same strategy is pursued, for example, for analysis of halogen isotopes in organic compounds where isotopologue ratios are determined in fragments of the target analyte (Bernstein et al, 2011;Elsner & Hunkeler, 2008;Zakon et al, 2016).…”

“…Because atmospheric O 2 has a constant isotopic composition (Barkan & Luz, 2005), small amounts of contamination from ambient O 2 can be corrected for with a blank sample subtraction. With this approach, accurate δ 18 O-values can be determined for aqueous O 2 concentrations ranging from 20 − 250 µM (0.6 − 8 mg/L) (Pati, Bolotin, et al, 2016).…”

“…with N 2 gas and automated procedures can be programmed with an autosampler. Setting the injector temperature up to 200°C will ensure a better reproducibility and precision of measured isotope ratios compared to having the injector operated at ambient temperature (Pati, Bolotin, et al, 2016). Separation of O 2 from other gases, in particular N 2 is achieved by means of gas chromatography with a molecular sieve PLOT column (5Å, 30m x 0.32µm I.D.)…”

“…One O 2 reference gas was used for calibrating isotope signatures of O 2 and the use of a second isotope standard with a different O isotope signature could improve accuracy of measurements. The procedure for correcting for ambient O 2 contamination is described in detail in Pati, Bolotin, et al (2016) and is performed according to eq. 16.…”