High productivity analysis of15N and13C in soil/plant research

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-use¹⁵N 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 5µg N was required compared with 50µg 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. Both¹⁵N and¹³C 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 submicrogram 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 for¹⁵N, doubling throughput for a small loss in precision.Several workers have developed micro-diffusion methods for ANCA-MS determination of NH₄⁺ and NO₃^- in KCl extracts. In 1990, a dual isotope mode was added for sequential analysis of¹³C and¹⁵N 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 < 0.1¹³C for > 100µg C and < 0.3¹⁵N for > 50µg 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 for¹³C 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 measure¹³C in CO₂ from human breath. The instrument was further developed for isotopes in other gases:¹³CO₂,¹⁵N₂,¹⁵N₂O, and¹³CH₄ above soil;¹⁸O from water equilibrated with CO₂ and¹⁵NO from NO₃^- in KCl plant extracts. The dual isotope mode allows¹⁵N to be measured in both N₂ (mass 28) and N₂O (mass 44) separated from the same sample. Recently GC-IRMS has been used to determine¹⁵N in N₂O produced from NO₂^- or NO₃^- 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.