High productivity analysis of ¹⁵N and ¹³C in soil/plant research

Abstract

On-line sample preparation and analysis enables faster testing of hypotheses in biological research, particularly in field experiments where many samples must be processed to integrate spatial variability. Soil scientists were first to recognise the need for a fast, easy-to-use ¹⁵N analyser to replace the isotope ratio mass spectrometer (IRMS) and Kjeldahl-Rittenberg sample preparation. Development has since led to a variety of ‘continuous-flow’ methods. Today’s instruments accept solid, liquid or gaseous samples in their naturally occurring form (leaves, soil, algae, soil gas) and process 100–125 samples per day. Automated sample preparation and analysis takes 3–10 minutes per sample.

By 1984, three groups had interfaced an automated nitrogen analyser to an IRMS. Continuous-flow combustion converted N in plant tissue or soil to a pulse of N₂ gas, which was taken to the mass spectrometer by a helium carrier. Throughput increased from 20 to 100 analyses per day and only 5 μg N was required compared with 50 μg N for Kjeldahl-Rittenberg preparation and IRMS analysis. 1984 was also the year in which a gas Chromatograph (GC) was first used on-line with an IRMS for isotopic analysis of individual compounds separated from a mixture (GC-IRMS).

In 1987, a software-controlled Automated Nitrogen Carbon Analyser-Mass Spectrometer (ANCA-MS) was developed for solid and liquid samples. Both ¹⁵N and ¹³C were measured to (SD, n = 5) 0.0002 atom%, enough for tracer studies. Less hardware made it portable, enabling use at remote sites. Software control enabled sub-microgram quantities of N to be measured by moving the O₂ pulse, with its N₂ blank, out of phase with the sample. Software also allows an ‘accelerated mode’ for ¹⁵N, doubling throughput for a small loss in precision.

Several workers have developed micro-diffusion methods for ANCA-MS determination of NH ⁺₄ and NO ^-₃ in KCl extracts. In 1990, a ‘dual isotope’ mode was added for sequential analysis of ¹³C and ¹⁵N from the same sample. In 1992 a new, 120° deflection, mass analyzer was designed to improve accuracy and precision at the high pressures used in CF-IRMS. Its precision of < 0.1‰ ¹³C for > 100 μg C and < 0.3‰ ¹⁵N for > 50 μg N extends ANCA-MS to ‘natural abundance’ applications.

Gaseous samples were first analyzed by CF-IRMS by manually injecting into the carrier flow of an ANCA-MS instrument. The initial interest was for ¹³C breath tests — radiation-free non-invasive tests of metabolic function. Demand for these led to the development in 1989 of an automated GC-IRMS method to separate and measure ¹³C in CO₂ from human breath. The instrument was further developed for isotopes in other gases: ¹³CO₂, ¹⁵N₂, ¹⁵N₂O, and ¹³CH₄ above soil; ¹⁸O from water equilibrated with CO₂ and ¹⁵NO from NO ^-₃ in KCl plant extracts. The ‘dual isotope’ mode allows ¹⁵N to be measured in both N₂ (mass 28) and N₂O (mass 44) separated from the same sample. Recently GC-IRMS has been used to determine ¹⁵N in N₂O produced from NO ^-₂ or NO ^-₃ in KCl soil extracts.

We predict that these simpler, automated instruments and the growth of analytical services will encourage more widespread use of stable isotope techniques by agricultural researchers.