A new method for measuring 13carbon abundance in tracer experiments

Abstract

Abstract. A new technique for the precise measurement of 13C‐abundance and concentration is described. It is based on the differences in infra‐red spectra between 12CO2 and 13CO2 and can be applied to gas mixtures or organic materials which have been oxidized to CO2. The gas mixture is first dried and then passed through two infra‐red gas analysers (IRGAs) connected in parallel. The two IRGAs are fitted with different optical filters so they differ in their relative sensitivities to 12CO2 and 13CO2. Once these sensitivities are known then simple algebra allows the concentrations of 12CO2 and 13CO2 to be calculated from the two readings. Two variants of this basic system have been tested. In both, one IRGA was a normal commercial instrument with a narrow band pass interference filter making it highly specific for 12CO2; the second instrument was fitted with either a wide‐band pass filter covering both the 12CO2 and 13CO2 absorption bands, or a narrow band pass filter specific for 13CO2. These variations convey different advantages in operation. The wide‐band system can be easily calibrated using a single natural abundance 12CO2 standard but is only moderately precise at low abundances. It is particularly valuable for continuous monitoring of the relatively high abundance sources used in plant photosynthesis experiments. The narrow‐band system gives high precision but requires a more complex standardization procedure. It is recommended for measurements on low‐abundance samples resulting from tracer experiments. Here, its high sensitivity permits measurements on samples as small as 3 μmole C, thus enabling plant fractions and individual metabolites to be investigated. While the wide‐band system can be manually operated under field conditions, it is necessary for highest precision to use computerized data collection and linearization. These processes are described, as are novel techniques for standardization, the preparation of small quantities of CO2 of known abundance, and the transfer of gas samples from oxidizer to analyser. Determinations by the wide band system of % abundance in standard gas mixtures gave a standard error of ±0.03% but this increased to over ±0.1% for abundances below 20%. Corresponding values for the narrow‐band system were ±0.01% over the whole abundance range an accuracy almost identical to that observed with an organic mass spectrometer. Two pulse‐chase experiment with 13CO2 are described in which the technique was used for studies on growth and metabolism of Lemna minor. The first demonstrated that 13C‐accumulation within the plants matched closely the predictions from the net assimilation rate and measurements of 13C‐abundance in the gas phase. The second revealed the rapid changes in the 13C‐labelling of some plant components during pulse and chase phases. These examples demonstrate the potential of the method for studies in plant physiology and biochemistry. In view of its relative cheapness, ease of maintenance and operation, accuracy, and sensitivity, it is suggested that this new method may encourage a wider use of the safe stable 13C for biological and medical applications.