Isotope ratio mass spectrometry
Gas chromatograph subsystem:
Mass separation subsystem:
All carbon in tha sample is converted by combustion to CO2. Nitrogen-bearing combustion products include N2 gas and various oxides of nitrogen NOx. The gasified sample passes from the combustion tube into another furnace (c) containing fine copper granules at 650 degrees C. In this furnace all molecules of NOx give up their oxygen to the hot copper and emerge as pure N2.
Water vapor from the sample is removed by a gas trap (d) containing magnesium perchlorate. If the samples are being analyzed for nitrogen isotopes only, CO2 is removed by a second gas trap containing a CO2 scrubber (sodium hydroxide on silicate carrier granules).
HeV where H = magnetic field strength e = ion charge V = velocity.
Counteracting this is the ion's centrifugal force
mV^2/r where m = mass V = velocity r = radius of curvature.
Solving, we obtain the radius r of each ion's path:
mV^2r = HeV mV r = ---- eH
Or, to paraphrase in English, light ions are deflected more and heavier ones less. The result is that the ion beam is divided into its component masses.
The separated masses finally reach collectors (Faraday cups) at (h), placed so as to receive masses 28, 29, and 30 for dinitrogen or 44, 45, and 46 for carbon dioxide. Here the impact of the ions is translated into a recordable signal that is collected for data processing and analysis. The absolute levels of the signals depend on the amount of gas which is admitted into the ion source. The critical value is the ratio of the signals falling into the different collector cups.
To evaluate very small differences in isotopic composition between samples and standards, the isotope ratios of both are determined by mass spectrometry. For example, in 13C analysis the sample is converted to CO2 gas before analysis. Various combinations of the isotopes of C and O can create molecules of the masses shown below but, as will be evident from summing the percentages of the total CO2, only the mass ratios 45/44 and 46/44 need be determined. Though absolute mass ratios are measured, the principle interest lies in the difference between the sample and standard ratios.
Composition Mass Percent of total CO2 in sample 12C 16O 16O 44 98.43 12C 16O 17O 45 0.04 12C 16O 18O 46 0.20 12C 17O 16O 45 0.04 12C 17O 17O 46 0.00 12C 17O 18O 47 0.00 12C 18O 16O 46 0.20 12C 18O 17O 47 0.00 12C 18O 18O 48 0.00 13C 16O 16O 45 1.09 13C 16O 17O 46 0.00 13C 16O 18O 47 0.00 13C 17O 16O 46 0.00 13C 17O 17O 47 0.00 13C 17O 18O 48 0.00 13C 18O 16O 47 0.00 13C 18O 17O 48 0.00 13C 18O 18O 49 0.00
1) Solid samples must be oven-dried (80 degrees C, 24 hours). Freeze-drying must be used if the samples contain forms of N such as ammonia that would lost in oven-drying.
2) Dried samples are ground to talcum powder consistency (250 um or less) using a ball mill (e.g. Spex Industries 8000) before being sealed into 5 x 9 mm tin capsules. Thorough sample homogenization in the grinder stage is required, to make certain that the tiny subsample taken for analysis (e.g. ~2 mg for 13C in leaves) is representative of the total sample. Poor precision can often be traced to inadequate grinding that leaves fibrous matter or visible granules in the sample. Wiley mills do not do an acceptable job of grinding for this application. (Nor does that favorite of low-budget improvization, the home coffee mill.)
3) Combustion capsule formation is critical for successful analytical runs. Please refer to the detailed [sample encapsulation] instructions for further information.
4) All mass spectrometers show a confounding effect of sample size on determined isotope ratio. Samples are therefore weighed after grinding to achieve a uniform sample size. (If total C or N values are desired in addition to 13C/15N, these weights are recorded and used in the data analysis.)
For isotopic analysis, sample size and total element content have a major effect on the analysis. The size of the subsample weighed out depends upon the density of the material as well as its N and C content. An ideal soil or plant sample intended for natural abundance isotopic analysis at this lab would contain 200 micrograms total N and 800 micrograms total C. It may not, of course, be possible to provide both these ideal amounts in one sample. The lower limits for reliable day-to-day operation of our instrument at natural abundance isotope levels appear to be ~25 micrograms total N and ~200 micrograms total C. For best precision and accuracy a preliminary analysis for total element content should be performed so that standards may be closely matched to samples.
Another sample-size constraint is related to the absolute amount of material that can be completely combusted in micro-Dumas apparatus. The maximum burnable total C content is around 2500 micrograms (e.g., for NBS 1572 citrus leaf standard, which is 43.27% C, this works out to a maximum sample size of 5.8 mg of ground leaf.) If, for example, a given sample is so poor in N that it must contain > 2500 ug C in order to achieve 25 ug N then it is essentially not analyzable for 15N. In addition, for element-poor soil samples the sheer bulk of the sample becomes significant. Soil samples of over 50 mg are very difficult to analyze due to rapid ash buildup in the furnace.
5)Nitrogen diffusion samples frequently fall at the lower end of the acceptable range of total N content. There is a small-sample mode available for samples containing less than 50 ug total N and no C. In this mode the oxygen pulse added to improve combustion, which as mentioned earlier contains a trace N impurity, is injected between samples rather than with them. Enough oxygen for a small sample is retained by the combustion catalyst, and this retained oxygen is of course free of the N2 impurity. Small-sample mode removes the need for baseline blanks and the variability these blanks add to the analytical process. However, in order to use this mode of operation we must know ahead of time that a given sample set will consist exclusively of low-N, no-C samples.
6) Liquid samples may be analyzed either by freeze-drying directly into a tin capsule or pipetting onto an inert absorbant substrate.