A New and Specific Method for the Detection of NO and 15NO, N2O
Many of the NO detection methods currently in use rely on the chemical derivatization of NO; e.g., the Greiss (nitrite), chemiluminescence (ozonolysis), and electrochemical (redox) methods. Further, many of the data from these methods suffer from contamination of other species that could interfere with the NO signal intensity.
The Richter-Addo and McCann (OU Department of Electrical Engineering) research groups have collaborated over the last few years to develop proof-of-concept for (i) the direct and specific detection of NO and 15NO from chemical and biochemical reactions, and (ii) the simultaneous detection of NO and N2O from a single NO/HNO-generating precursor. We became interested in developing a direct, specific, and accurate method to detect NO from chemical and biochemical reactions. This NO detection method is based on high-resolution tunable diode laser absorption spectroscopy (TDLAS) using a multi-pass Herriott cell. This system provides sensitivities in the 1 ppb range for NO measurements,
Figure 1 (left) shows the second-harmonic spectrum of NO and 15NO, and Figure 1 (middle) shows its application for the detection of NO (top) and 15NO (bottom) released during the chemical oxidation of nitrosyl myoglobin. Figure 1 (right) demonstrates the use of TDLAS for the detection of NO released from the Cu-catalyzed GSNO decomposition as a function of [GSNO] using a constant initial [Cu(II)] of 40 mM without using a nitrogen gas flow. After each run, the solution in the reagent chamber was replaced with a fresh solution of Cu(II) and then the appropriate amount of GSNO was added to result in a renewed release of NO: (1) 100 nM GSNO, (2) 1 mM GSNO, (3) 10 mM GSNO, and (4) 0.1 mM GSNO. Interestingly, this particular Cu(II)-catalyzed NO release reaction is not linearly dependent on [GSNO] because the GS-SG side-product sequesters Cu(II) to form the Cu-GSSG product shown.
Isotopic ratio mass spectrometry has been utilized previously by others for the identification of 15NO and oxidation products such as nitrite/nitrate. In our work, we are able to detect gas phase 15NO in the GS15NO decomposition reaction without the need for mass spectrometry! This detection is specific for 15NO.
Real-time measurement of N2O, and the simultaneous detection of NO and N2O: In an effort to develop methodology that will enable the hitherto-unreported simultaneous detection of NO and N2O, we selected as a prototype the decomposition of the well-known Angeli’s salt (disodium trioxodinitrate, Na2N2O3). Angeli’s salt is known to generate HNO at physiological (and high) pH. However, it also generates NO at low pH.
This pH-dependent decomposition to NO and/or HNO was attractive as a prototypical system, and we reasoned that the results obtained from the simultaneous detection should provide a pH-dependent profile of released NO/N2O under the same experimental reaction conditions. We note that although the decomposition of Angeli's salt had been studied (by following the disappearance of the reagent by UV-vis spectroscopy), no study of the simultaneous generation of NO and N2O had been reported prior to our study.
The real-time measurement of N2O production from the decomposition of Angeli's salt at pH 7.4 is shown in Figure 3 (the real-time measurement of NO was performed similarly). The system was allowed initially to equilibrate at 45 torr with a supplemental nitrogen gas flow. Opening the empty sample chamber to laboratory air while shutting off the supplemental nitrogen gas flow resulted in a spike in N2O detection (labeled (a) in Figure 3). Angeli's salt (2 mg) was then added to the chamber by opening the chamber and introducing the solid; the spike in N2O detection (labeled (b)) is due to the ambient air. Upon addition of the degassed buffer to initiate the aqueous decomposition of the salt, a large N2O response (labeled (c)) became evident which then trailed to near-baseline levels over time.
For the simultaneous measurent of NO and N2O from Angeli's salt decomposition as a function of pH, we performed two general kinds of experiments. First, we established the pattern of NO and N2O generation as a function of pH using real-time measurements; this allowed us to correctly estimate when the NO (or N2O) generation was essentially complete within a given set of reaction conditions. To establish the ratio of NO and N2O production, we performed experiments that allowed us to accumulate the gases generated over the reaction time period (this time period was calculated from the real-time measurements).
It has been reported previously that the rate of Angeli’s salt decomposition in aqueous solution (i.e., rate of disappearance of the salt in solution) is pH independent in the pH 4–8 range. However, there does not appear to be any report on the relative ratios of the N2O and NO gases generated as a function of pH. In order to provide a semi-quantitative measurement regarding the ratio of NO and N2O produced from the aqueous Angeli’s salt decomposition reactions in the pH 2.0–9.4 range, we allowed the decompositions to run for several hours in a sealed vessel to insure completion of the reactions. The headspace was then simultaneously analyzed for NO and N2O after this period using the TDLAS system. The relative ratios of NO and N2O detected from Angeli’s salt decomposition (this work) in the pH 2.0–9.4 range using our TDLAS-based system are shown in Figure 4.
Important Chemical Implications: It is important to note that our development and use of the TDLAS methodology has helped provide new intellectual and substantial information on the decomposition of Angeli's salt as a function of pH. Prior to our work, it had been assumed that the HNO-generation was pH independent in the pH 4.5-8 range. We have shown that this may not necessarily be the case. The NO generation at low pH was confirmed by our studies. However, the HNO/N2O generation is maximum around pH 4, but then drops significantly at higher pHs. In fact, at physiological pH, the amount of N2O detected is less than half of what is generated at pH 4. This result will thus impact several chemists looking to prepare synthetic analogues of Angeli's salt for use as NO/HNO generators.