Natural Abundance Level Research Articles

Long-Term Nitrogen Additions and Nitrogen Saturation in Two Temperate Forests

This article reports responses of two different forest ecosystems to 9 years (1988–96) of chronic nitrogen (N) additions at the Harvard Forest, Petersham, Massachusetts. Ammonium nitrate (NH4NO3) was applied to a pine plantation and a native deciduous broad-leaved (hardwood) forest in six equal monthly doses (May–September) at four rates: control (no fertilizer addition), low N (5 g N m-2 y-1), high N (15 g N m-2 y-1), and low N + sulfur (5 g N m-2 y-1 plus 7.4 g S m-2 y-1). Measurements were made of net N mineralization, net nitrification, N retention, wood production, foliar N content and litter production, soil C and N content, and concentrations of dissolved organic carbon (DOC) and nitrogen (DON) in soil water. In the pine stand, nitrate losses were measured after the first year of additions (1989) in the high N plot and increased again in 1995 and 1996. The hardwood stand showed no significant increases in nitrate leaching until 1995 (high N only), with further increases in 1996. Overall N retention efficiency (percentage of added N retained) over the 9-year period was 97–100% in the control and low N plots of both stands, 96% in the hardwood high N plot, and 85% in the pine high N plot. Storage in aboveground biomass, fine roots, and soil extractable pools accounted for only 16–32% of the added N retained in the amended plots, suggesting that the one major unmeasured pool, soil organic matter, contains the remaining 68–84%. Short-term redistribution of 15N tracer at natural abundance levels showed similar division between plant and soil pools. Direct measurements of changes in total soil C and N pools were inconclusive due to high variation in both stands. Woody biomass production increased in the hardwood high N plot but was significantly reduced in the pine high N plot, relative to controls. A drought-induced increase in foliar litterfall in the pine stand in 1995 is one possible factor leading to a measured increase in N mineralization, nitrification, and nitrate loss in the pine high N plot in 1996.

H and 15N Dynamic Nuclear Polarization Studies of Carbazole

15N NMR experiments, combined with dynamic nuclear polarization (DNP), are reported on carbazole doped with the stable free radical 1,3 bisdiphenylene-2-phenylallyl (BDPA). Doping shortens the nuclear relaxation times and provides paramagnetic centers that can be used to enhance the nuclear signal by means of DNP so that 15N NMR experiments can be done in minutes. The factors were measured in a 1.4 T external field, using both unlabeled and 98% 15N labeled carbazole with doping levels varying between 0.65 and 5.0 wt % BDPA. A doping level of approximately 1 wt % produced optimal results. DNP enhancement factors of 35 and 930 were obtained for 1H and 15N, respectively making it possible to perform 15N DNP NMR experiments at the natural abundance level.

Evidence for an NIH Shift as the Origin of the Apparently Anomalous Distribution of Deuterium in Estragole from Artemisia dracunculus

Feeding experiments of [2-(2)H]- and [4'-(2)H]phenylalanine in Artemisia dracunculus validate the hypothesis that the marked difference in deuterium content at the natural abundance level between the aromatic carbons of estragole (1) is due to an NIH shift during the hydroxylation of the benzene ring.

Theoretical and experimental aspects of enantiomeric differentiation using natural abundance multinuclear nmr spectroscopy in chiral polypeptide liquid crystals

Liquid crystalline organic solutions of poly-γ-benzyl-L-glutamate generate a sufficient differential ordering effect to visualize enantiomers using multinuclear high-resolution NMR spectroscopy at natural isotopic abundance levels. Chiral discrimination can be observed through a difference in the order-sensitive NMR observables, namely: proton–proton, carbon–proton and carbon–carbon residual internuclear dipolar couplings, carbon chemical shift anisotropies, and deuterium quadrupolar splittings. In most cases, the enantiodifferentiation is large enough to allow determation of the enantiomeric excesses satisfactorily. All theoretical considerations and significant experimental parameters that affect the efficiency of this methodology are presented and discussed. The various possible anisotropic NMR techniques provide a very reliable and powerful alternative to the current analytical techniques which operate in the isotropic phase.

Effect of Concomitant18O in13C-Urea on the Urea breath test

Helicobacter pylori infection in stomach has been extensively assessed by the 13C-urea breath test (UBT) with infra-red or mass spectrometer. However, analyses of commercially available 13C-urea for UBT showed about 10 atom% of 18O abundance. It is therefore very important to investigate the 18O-isotope effect on UBT. Using a mass spectrometer, breath samples of UBT were analyzed to investigate the change of mass spectra by the isotopic exchange reaction between 18O of carbon dioxide and 16O of water. As the result, the 18O content of 13C, 18O-carbon dioxide exhaled in UBT reached its natural abundance level immediately after the administration of 13C, 18O-urea. It was concluded that the 18O content of 13C, 18O-urea has no effect on UBT using either infra-red or mass spectroscopy. Copyright © 2000 John Wiley & Sons, Ltd.

Use of carbon and nitrogen isotope ratios in termite research

In this paper, I review carbon and nitrogen isotopic (natural abundance levels) studies of termites. The carbon isotope ratio of CH4 emitted from termites, together with the emission rates of CO2, CH4 and H2, showed several trends corresponding to the kinds of symbiotic microbes and feeding habits. The fraction of methane oxidized in the nest structure was estimated by comparing carbon isotope ratio of CH4 emitted from the nest with that produced by termites in the nest. Symbiotic nitrogen fixation in the gut of termites has been shown to have a significant contribution to the nitrogen economy in some wood‐feeding termites. The carbon isotope ratio distinguishes between C4 from C3 plants, and the fractional contribution of grass in the diet can thereby be estimated. The carbon and nitrogen isotope ratios in termites are discernible among soil‐feeders, fungus cultivators and wood‐feeders. Wood/soil‐interface feeders have intermediate values between wood‐ and soil‐feeders, and thus carbon and nitrogen stable isotope ratios are assumed to characterize the degree of humification of the material consumed by termites. It is suggested that carbon and nitrogen isotope ratios are useful indicators of the functional position of termites in the decomposition process. A similar isotope pattern has been obtained in earthworms, suggesting that isotope signatures might be useful parameters in investigating detritivorous animals in general.

On‐line quantitative determination of 2H/1H isotope ratios in organic and water samples using an elemental analyser coupled to an isotope ratio mass spectrometer

A rapid continuous-flow technique for the quantitative determination of hydrogen isotope ratios in organic materials and water, at natural abundance levels, is described. Samples are pyrolysed in a helium stream at 1080°C over an inert form of carbon. Hydrogen is separated from the other pyrolysis gases by gas chromatography and enters the ion source of the isotope ratio mass spectrometer (IRMS) via a crimp. The 2H/1H ratio of the gas was determined by the simultaneous integration of the m/z 2 (1H1H) and m/z 3 (2H‰1H) ion beams over time. δ2H‰ values of organic and water samples were determined by comparison with organic and water standards, respectively. The precision of the on-line δ2H‰ measurement is <3.3‰. The accuracy of the method was demonstrated by the analysis of a laboratory standard water (NTW), a European Community Bureau Reference material, N,N-tetramethylurea (TMU) and an International Atomic Energy Association polyethylene reference material (IAEA-CH-7). The measured values were NTW -39.4‰, TMU -144.1‰ and IAEA-CH-7 -100.7‰ (reference values are NTW -38.5‰, TMU -147.4‰ and IAEA-CH-7 -100.3‰). No apparent memory effect was observed when measuring samples at the natural abundance level. Calibration of organic measurements with water reference materials did not give satisfactory results, probably owing to differences in their respective pyrolysis pathways. The potential of the technique for food analysis was demonstrated by measuring the δ2H‰ values of vanillin samples derived from natural and synthetic sources and by the analyses of hexamethylenetetramine synthesized from beet, cane and apple juice sugars. © Crown copyright 1998. Reproduced with the permission of the Controller of Her Majesty’s Stationery Office.

On-line quantitative determination of2H/1H isotope ratios in organic and water samples using an elemental analyser coupled to an isotope ratio mass spectrometer

A rapid continuous-flow technique for the quantitative determination of hydrogen isotope ratios in organic materials and water, at natural abundance levels, is described. Samples are pyrolysed in a helium stream at 1080°C over an inert form of carbon. Hydrogen is separated from the other pyrolysis gases by gas chromatography and enters the ion source of the isotope ratio mass spectrometer (IRMS) via a crimp. The 2 H/ 1 H ratio of the gas was determined by the simultaneous integration of the m/z 2 ( 1 H 1 H) and m/z 3 ( 2 H‰ 1 H) ion beams over time. δ 2 H‰ values of organic and water samples were determined by comparison with organic and water standards, respectively. The precision of the on-line δ 2 H‰ measurement is <3.3‰. The accuracy of the method was demonstrated by the analysis of a laboratory standard water (NTW), a European Community Bureau Reference material, N,N-tetramethylurea (TMU) and an International Atomic Energy Association polyethylene reference material (IAEA-CH-7). The measured values were NTW -39.4‰, TMU - 144.1‰ and IAEA-CH-7 - 100.7‰ (reference values are NTW -38.5‰, TMU - 147.4‰ and IAEA-CH-7 - 100.3‰). No apparent memory effect was observed when measuring samples at the natural abundance level. Calibration of organic measurements with water reference materials did not give satisfactory results, probably owing to differences in their respective pyrolysis pathways. The potential of the technique for food analysis was demonstrated by measuring the δ 2 H‰ values of vanillin samples derived from natural and synthetic sources and by the analyses of hexamethylenetetramine synthesized from beet, cane and apple juice sugars. © Crown copyright 1998. Reproduced with the permission of the Controller of Her Majesty's Stationery Office.

On-Line Analysis of Stable Isotopes of Nitrogen in NH3, NO, and NO2 at Natural Abundance Levels

Methods were developed for the on-line analysis of stable isotopes of nitrogen (at natural abundance levels) in NH(3), NO and NO(2) in order to study the contribution of these trace gases to nitrogen cycling in ecosystems. Standard methods for the combustion of organic substances to N(2) (for the determination of the (15)N/(14)N ratios by mass spectrometry) failed to satisfactorily oxidize or reduce the respective trace gases. The following oxidation or reduction techniques ensured quantitative conversion of the trace gases to N(2) and analytical precision close to the internal precision of the instrument (precision ±0.15‰): (1) oxidation of NH(3) to N(2) at 1150 °C on a NiO surface (which needs a reoxidation before each NH(3) analysis; measuring range 11.7-58.8 nmol of NH(3), precision ±0.30‰); (2) reduction of NO to N(2) at 1150 °C on a Ni surface (measuring range 33.3-133.3 nmol of NO, precision ±0.28‰); (3) reduction of NO(2) to form NO at 420 °C on a Mo surface followed by further treatment as for NO (measuring range 26.0-129.9 nmol of NO(2), precision ±0.90‰). This last technique was developed due to the poor chromatographic properties of NO(2).

Utilization of nitrogen derived from seabird guano by terrestrial and marine plants at St. Paul, Pribilof Islands, Bering Sea, Alaska

Dense, conspicuous colonies of seabirds and pinnipeds breed on ocean islands throughout the world. Such colonies have been shown to have local impacts on prey populations, but whether or not they affect nutrient cycling has been debated. We determined the natural abundance levels of the stable isotopes (C and N) of primary producers, seabirds and other consumers at and near St. Paul Island, Pribilof Islands, Bering Sea, in summer 1993. Marine primary producers (phytoplankton, as particulate organic matter, and kelp) collected near seabird colonies were ca. 6.5‰ enriched in both 15N and 13C relative to those collected further from shore. Terrestrial plants collected near the seabird colonies were enriched in 15N (δ15N ca. 22‰) compared with conspecifics collected away from the colonies (δ15N ca. 11‰). The trend towards higher δ15N values in marine and terrestrial plants near bird colonies is consistent with their uptake of ornithogenic N. This 15N-enrichment of plants using ornithogenic N can be attributed to a combination of two processes: trophic enrichment, and volatilization of ammonia produced during degradation of terrestrially deposited guano. Seabird breeding colonies at St. Paul Island appear to be significant sources of recycled nitrogen for terrestrial plants in the vicinity of colonies and for phytoplankton in the nearshore zone.

Oxygen Isotope Analysis of Cellulose: An Interlaboratory Comparison

Oxygen isotope analysis of tree ring cellulose is hindered by the lack of a fast and reliable technique to measure δ18O at natural abundance levels. Most of the existing off-line techniques are not conducive to rapid generation of oxygen isotope data. Further, a comparison between oxygen isotope values of a cellulose sample measured using the different off-line techniques resulted in a discrepancy of 1.3‰. This discrepancy is a result of inconsistencies inherent to these techniques and the lack of a reliable cellulose oxygen isotope standard. We addressed these problems by analyzing a number of cellulose samples by three different analytical procedures. These include two relatively well established off-line methods (pyrolysis in nickel tubes and decomposition using mercury(II) chloride) and a recently proposed on-line method based on pyrolysis in the presence of glassy carbon in an elemental analyzer and the subsequent isotope analysis of carbon monoxide. We were successful in improving the reproducibility of the on-line method (σn-1 ≈0.2‰) to the extent that natural variations can be resolved and found a good agreement between the on-line and off-line methods, although the cellulose samples extracted from tree rings are subject to more scatter than the commercial cellulose samples. The on-line method may even allow determination of δ13C and δ18O of a cellulose sample in a single run (from 29/28 and 30/28 ratios of CO).

The chemistry of soil organic nitrogen: a review

1. From the data presented herein it is possible to deduce the following distribution of total N in humic substances and soils: proteinaceous materials (proteins, peptides, and amino acids) – ca. 40%; amino sugars – 5–6%; heterocyclic N compounds (including purines and pyrimidines) – ca. 35%; NH3–19%; approximately 1/4 of the NH3 is fixed NH4 +. Thus, proteinaceous materials and heterocyclics appear to be major soil N components. 2. Natural 15N abundance levels in soils and humic materials are so low that direct analysis by 15N NMR is very difficult or impossible. To overcome this difficulty, the soil or humic material is incubated with 15N-enriched fertilizer. Even incubation in the laboratory for up to 630 days does not produce the same types of 15N compounds that are formed in soils and humic materials over hundreds or thousands of years. For example, very few 15N-labelled heterocyclics are detected by 15N NMR. Does this mean that heterocyclics are not present? Or are the heterocyclics that are present not labelled under these experimental conditions and therefore not detected by the 15N NMR spectrometer ? Another possibility is that a large number of N heterocyclics occur in soils, but each type occurs in very low concentrations. Until the sensitivity is improved, 15N NMR will not provide results that can be compared with data obtained from the same soil and humic material samples by chemical methods and mass spectroscopy. 3. What is most important with respect to agricultural is that all major N forms in soils are available to organisms and are sources of NH3 or NH4 + for plant roots and microbes. Naturally, some of the NH3 will enter the N cycle. 4. From chemical and pyrolysis-mass spectrometric analyses it appears that N heterocylics are significant components of the SOM, rather than degradation products of other molecules due to pyrolysis. The arguments in favor of N heterocyclics as genuine SOM components are the following: a) Some N-heterocyclics originate from biological precursors of SOM, such as proteinaceous materials, carbohydrates, chlorophyll, nucleic acids, and alkaloids, which enter the soil system as plant residues or remains of animals. b) In aquatic humic substances and dissolved organic matter (DOM) at considerably lower pyrolysis temperatures (200 to 300°C), free and substituted N-heterocyclics such as pyrroles, pyrrolidines, pyridines, pyranes, and pyrazoles, have been identified by analytical pyrolysis (Schulten et al 1997b). c) Their presence in humic substances and soils was also detected without pyrolysis by gel chromatography – GC/MS after reductive acetylation (Schnitzer and Spiteller 1986), by X-ray photoelectron spectroscopy (Patience et al. 1992), and also by spectroscopic, chromatographic, chemical, and isotopic methods (Ikan et al. 1992). 5. While we can see light at the end of the tunnel as far as soil-N is concerned, further research is needed to identify additional N-containing compounds such as N- heterocyclics, to determine whether these are present in the soil or humic materials in the form in which they were identified or whether they originate from more complex structures. If the latter is correct, then we need to isolate these complex N-molecules and attempt to identify them.

N-Isotopic Composition in Human Plasma Protein Amino Acids at Natural Abundance Level and After a Single [15N2]urea Administration Measured by GC-C-IRMS

The GC-C-IRMS technique represents a new and useful tool to study the 15N abundance in amino acids for metabolic studies. Here we present 15N analysis data for 13 human plasma protein amino acids at natural abundance and in slightly enriched samples using GC-C-IRMS analysis of N-pivaloyl-i-propyl esters of amino acids. A mean precision of ± 0.71‰ Δ15N or ±0.77‰ Δ15N was determined for all amino acids analysed at natural abundance values and for the slightly enriched samples (< 100‰ δ15N), respectively. However, for individual amino acids at natural abundance derivatized and injected in duplicate the standard deviation ranged from 0.24 to 1.49‰. The lowest natural 15N abundance was found for threonine (about - 5‰ δ15N) and the highest for proline and glutamic acid (about 15‰ δ15N). A standard deviation (SD) range of±0.71 (glutamic acid) to ± 4.35 ‰ δ15N (phenylalanine) (mean SD = ± 1.at δ15N) was obtained in protein amino acids in a group of 7 volunteers at natural abundance level. A single oral [15N2]urea bolus was administreted to one healthy volunteer to demonstrate the feasibility of the GC-C-IRMS technique to trace human nitrogen kinetics in plasma protein amino acids. Small but significant differences in 15N abundance were found between amino acids, presumably due to their differential participation in transamination reactions.

Correlating molecular markers with physiological expression in Hordeum, a developing approach using stable isotopes

summaryThis paper describes a developing approach for correlating molecular markers with phenotypic expression in barley, and research examples are given. This approach is intended to elucidate the connection between denned parts of the barley genome and their physiological expressions. To illustrate our approach (up to the point of marker correlation), the materials, methods and results of an experiment are presented. Briefly, the natural abundance levels of carbon and nitrogen isotopes (δ13C δ15and N) serve as the primary screening tools for assessing barley genotypes for their responses to imposed abiotic stresses. The isotopic responses are then correlated with molecular markers as the first step in identifying the mechanisms relating genetic variation with phenotypic expression.A well‐described mechanistic model exists for interpreting plant δ13C, and this enables partitioning of the physiological processes contributing to the whole plant δ13C. Although plant δ15N appears to be equally well correlated with genotype and environment, there is no mechanistic model explaining this isotopic signature. Hence, δ15N is used empirically and in parallel with attempts to understand its underlying mechanism(s).

Preparative Scale Mass Spectrometry: A Brief History of the Calutron

Calutrons were developed in the laboratory of E. O. Lawrence at the University of California at Berkeley. They were a modification of the cyclotrons he had invented and used in his Noble Prize winning investigations of the atomic nucleus. At the time their construction was undertaken, calutrons represented the only certain means of preparing enriched uranium isotopes for the construction of a fission bomb. The effort was successful enough that every atom of the 42 kg of 235U used in the first uranium bomb had passed through at least one stage of calutron separation. At peak production, the first stage separators, α tanks, yielded an aggregate 258-g/d 235U enriched to about 10 at. % from its natural abundance level of 0.72 at. %. The second stage separators, β tanks, used the 10 at. % material as feedstock and produced a total 204-g/d 235U enriched to at least 80 at. %. The latter, weapons grade, material was used in fission bombs. Under typical operating conditions, each α tank operated at a uranium beam intensity at the collectors of approximately 20 mA and each β tank at a beam intensity of approximately 215 mA at the collectors. Bulk separation of isotopes for bomb production ceased in 1945. Since that time calutrons have been used to separate stable isotopes, but on a more limited scale than wartime weapons production. Stable isotope separations since 1960 have taken place using one modified beta tank.

Natural abundance-level measurement of the nitrogen isotopic composition of oceanic nitrate: an adaptation of the ammonia diffusion method

Abstract We have adapted the “ammonia diffusion” method of nitrate extraction for natural-abundance level nitrogen isotopic measurement of oceanic nitrate. The method involves: (1) sample concentration (by boiling or evaporation); (2) conversion of nitrate to ammonia using Devarda's alloy; and (3) the gas-phase diffusion of ammonia onto an acidified glass fiber disk which is sandwiched between two porous Teflon membranes. We have investigated the conditions necessary to effect complete ammonia recovery from natural seawater samples and the use of Devarda's alloy under these conditions. In addition, we have characterized the blanks in this method and designed a protocol to minimize them. Here, we report our protocol for nitrate extraction from seawater and provide an explanation of the protocol based on our method development work. To demonstrate the performance of the method, we present nitrate nitrogen isotopic data from nitrate standard additions to Sargasso Sea surface water and from several Southern Ocean depth profiles. The nitrate extraction method gives highly reproducible, complete recovery of nitrate and a standard deviation for isotopic analysis of The blanks inherent in the extraction procedure are from Devarda's alloy and seawater dissolved organic nitrogen (“DON”). The N blank of the Devarda's alloy reagent depends on brand and lot number. The Devarda's alloy which we are currently using results in a blank of ~ 0.4 nmol N per 100 ml of seawater (effectively 0.4 μM). An isotopic correction is made for this blank. For standard incubation conditions, stored Woods Hole seawater (with ~ 10 μM DON) gives a ~ 0.6 μM DON blank, while stored Sargasso Sea (with ~ 6 μm DON) surface water gives a DON blank of 0.3–0.5 μM. The DON blank appears to cause the ≤ 0.3%. difference between the measured and actual isotopic composition of nitrate added to Sargasso Sea surface water at the 5 μM nitrate level. We discuss several ways to lower the DON blank for samples in which the DON concentration is high relative to the nitrate concentration. The nitrogen isotopic data from several Southern Ocean profiles, in conjunction with the other results presented in this paper, demonstrate the consistency of the data produced by the ammonia diffusion method. The ammonia diffusion-based protocol is more reliable and allows for better precision than the nitrate reduction/ammonia distillation method ( Cline and Kaplan, 1975 ) in our hands. While the samples have an incubation time of 4 days or longer, we find that the diffusion method allows for higher throughput than the distillation method because samples can be run conveniently in large batches.

A species-specific bacterial productivity method using immunomagnetic separation and radiotracer experiments

A species-specific bacterial productivity assay, combining radiotracer methods with immunomagnetic bead separation, was developed and tested in the marine environment. The capture method was optimized using cultures of the marine denitrifying strain, Pseudomonas stutzeri (ATCC 14 405). Immunocapture was optimal at a bead to target cell ratio of 10:1 using an indirect antibody technique, in which the target cell is first incubated with specific (primary) polyclonal antiserum and then with the secondary antibody-coated beads. Primary antibody concentration was less important than target cell concentration in determining the efficiency of target cell recovery. Reproducible recovery efficiencies of 75% could be obtained using cultures, but at natural seawater abundance levels, efficiency was much lower, around 20%. Estimates of total heterotrophic bacterial production and P. stutzeri production, based on radiotracer incorporation, were obtained for seawater samples from Monterey Bay, CA. To measure species-specific production, samples were incubated with radiotracers (methyl-[ 3H]-thymidine and [ 14C]-leucine), fixed, and concentrated. After separating P. stutzeri cells from the bacterial assemblage using immunomagnetic separation, target cell fraction radioactivity was measured. P. stutzeri abundance, estimated by immunofluorescence, represented less than 0.1% of the total bacterial abundance, whereas radiotracer incorporation by the target fraction represented 1–3% of the total assemblage tracer incorporation.

Use of nitrous oxide as a purge gas for automated nitrogen isotope analysis by the Rittenberg technique

An apparatus that operates with an isotope-ratio mass spectrometer to automatically perform nitrogen isotope analyses by the Rittenberg technique was modified to permit the use of nitrous oxide (N2O) instead of Freon (CCl2F2 or CHClF2) for the purging of air prior to hypobromite oxidation of ammonium-N to N2 in a plastic microplate. Analytical performance was unaffected by the modifications. Up to 768 samples can be processed in a single loading, at a rate of 6 to 12 samples/h. Within the range of 0.2 to 20 atom % 15N, isotope-ratio analyses of 50 to 200 μg of N using the automated Rittenberg apparatus (ARA) with a double-collector mass spectrometer were accurate to within 0.7%, as compared to manual Rittenberg analyses of 1 mg of N using the same mass spectrometer with a dual-inlet system. Automated analyses of 20μg of N were accurate to within 2%, and automated analyses of 10 μg of N were accurate to within 7%. The relative standard deviation for measurements at the natural abundance level (10 analyses, 20-200 μg of N) was < 0.04 %.

Concomitant oxygen‐18 enrichment in commercial carbon‐13 labelled urea

By mass spectroscopy, 50-fold oxygen-18 enrichment over natural abundance was observed in commercial 13 C-urea (99 atom % 13 C) synthesized from 13 C-carbon monoxide that had been 13 C-enriched by cryogenic distillation. In contrast, 13 C-urea synthesized from 13 C-potassium cyanide (a 13 C-labelled compound having no oxygen atom) showed the natural abundance level of oxygen-18.

Nitrogen-15 NMR studies of hydrogen bonding and proton transfer in complexes of pyridineN-oxides with dichloroacetic acid in CDCl3

The 15N NMR chemical shifts of eight substituted pyridine N-oxides (B) and their complexes (AHB) with methanol and dichloroacetic and trifluoromethanesulfonic acids were measured in chloroform-d at the natural abundance level. The measured chemical shifts are strongly affected by substituents, hydrogen bond strength and protonation. The plot of the relative chemical shifts, Δδ(15N) = δ(AHB) − δ(B), against ΔpKa gives a titration curve that reflects the variation of the hydrogen bond strength and the proton moves from the acid to the N-oxide. According to previous IR data, in the complexes from the inversion region (ΔpKa = 1·26), the proton is either delocalized (B…H…A) or more likely the lifetime is so short (⩽10−13 s) that the method does not recognize the B…H-A and B+H…A− species. Protonation of pyridine N-oxides shifts the nitrogen signal ca 50 ppm to lower frequencies compared with the ca 123 ppm for pyridines.