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Sample Collection and Processing

Last revised July 22, 1997
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* [Mass spectrometry samples]
* [Plant/soil total C, total N samples]
* [Samples for colorimetry]
* [Samples for atomic absorption spectrophotometry]

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(Icon) Samples for stable isotope analysis

Biological materials analyzed for stable isotope content include leaves, roots, soil, plasma and other solid and liquid substances. Before samples can be analyzed by they must be converted into the simple gases N2 or CO2. The micro-Dumas combustion elemental analyzer used here as a front end for the mass spectrometer requires some care in preliminary sample preparation (though considerably less than wet-chemistry techniques such as Kjeldahl-Rittenberg.)

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 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.


(Icon) Micro-Dumas samples for total C and total N

1) Soil and plant samples must be oven-dried (80 degrees C, 24 hours). Freeze-drying must be used if the samples (e.g. poultry litter) 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. 2-4 mg for leaf material) 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) Samples are weighed to the microgram level after grinding, and these weights are recorded and used in the data analysis. The size of the subsample weighed out depends upon the density of the material as well as its N and C content. Typical sample weights are 2-4 mg for plant tissue, 10 mg for straw and 25-30 mg for soil.

4) 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.) 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) Combustion capsule formation is critical for successful analytical runs. Please refer to the detailed [sample encapsulation] instructions for further information.


(Icon) Samples for colorimetric analysis

1) Liquid samples must be free of turbidity and particulate matter. Any such substances must be removed before analysis by filtering or centrifugation.

2) Strongly colored samples may contribute confounding absorbance at the analytical wavelength.

3) Water samples which cannot be analyzed immediately after collection must be preserved for shipment. The E.P.A. publication Methods for Chemical Analysis of Water and Wastes lists acceptable preservation methods and holding times for many analytes; you may refer to it here if you wish:

* [E.P.A. sample preservation guidelines]

4) For many purposes, 20 ml polyethylene scintillation vials with poly-lined caps are cheap and acceptable collection containers. Such a container provides enough sample for the full range of colorimetric analyses most often performed here (nitrate-N, ammonium-N, orthophosphate-P and persulfate digests for total N and total P.)


(Icon) Samples for atomic absorption spectrophotometry

Plant digests: The [dry ash/double-acid extraction] procedure calls for an amount of material that yields 0.5 gm. after drying and grinding.

Soil extracts: The [double-acid extraction] procedure calls for an amount of sample soil that yields 5 gm. (~4 ml. volume) after drying and passing through a 2 mm sieve. Investigators should consult the standard literature for considerations and cautions regarding soil sample collection, processing, and storage.

Water content partitioning: Several different analyte partitions (dissolved, suspended, total) may be of interest. Differing treatments of each sample partition are detailed in the U.S. E.P.A.'s discussion of [Content partitioning] of water samples.

To allow for repeat measurements of dissolved element, the investigator should collect 20 ml of sample for each desired analyte. If a digest for total element is desired, considerably more sample must be collected. Please refer to the E.P.A.'s particulars for [Total element digests] of water samples. The investigator should also refer to the E.P.A.'s table of recommended [Sample collection/preservation] procedures for specific details of aquatic sampling.


Bibliography

Allen, S. E., et al. 1974.
Chemical Analysis of Ecological Materials. John Wiley and Sons, New York.

James, D. W. and K. L. Wells. 1990.
Soil sample collection and handling. pp.25-44. In R. L. Westerman, ed., Soil Testing and Plant Analysis. Third ed. Soil Science Society of America, Madison, WI.

Peterson, R. G. and L. D. Calvin. 1986.
Sampling. pp.33-51. In A. L. Page et al., eds., Methods of Soil Analysis: Part 2, Chemical and Microbiological Properties. Agronomy, A Series of Monographs, no.9 pt.2, Soil Science Society of America, Madison, Wisconsin USA.

U. S. Environmental Protection Agency. 1983.
Sample preservation. pp. xv-xx. In Methods for Chemical Analysis of Water and Wastes, EPA-600/4-79-020. U.S.E.P.A., Cincinnati, Ohio, USA.


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