SIEL Soil Analysis « Center for Applied Isotope Studies (CAIS)

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Soil Analysis

We analyze all types of soils for nitrogen and carbon stable isotopes and total content. We can also analyze soil samples for total nitrogen (TN), total phosphorus (TP) and a variety of metals and offer a variety of digestion and extraction techniques.

Total Nitrogen and Total Carbon

Overview of technique
Micro-Dumas combustion analysis for total carbon and total nitrogen in solid-phase samples (plant tissue, soils, sediments, etc.) is based on transformation to gas phase by extremely rapid and complete flash combustion of the sample material. More details of the process are found below.

Sample prep considerations
Samples must be oven-dried, ball-milled to less than 250 um particle size and weighed (~3 mg, with ug digits significant) into 5 x 5 mm tin capsules before combustion. Poor precision can often be traced to inadequate grinding that leaves fibrous matter or visible granules in the sample. Sample prep for total-C/total-N analysis is found below. Clients may send us unprocessed samples (e.g. litterbag contents) or may provide any of the above processing themselves at a major reduction in price.

Nitrogen-15

Method Summary
Isotope-ratio analysis for 15N in solid-phase samples such as animal tissue starts with transformation to gas phase by extremely rapid and complete flash combustion of the sample material. Ionized combustion product (dinitrogen) is mass-analyzed by means of differing mass/charge ratios among the various isotopic species of N2. More details of the process are found here.

Sample prep considerations
Samples must be oven-dried, ball-milled to less than 250 um particle size and weighed (~3 mg, with ug digits significant) into 5 x 5 mm tin capsules before combustion. Poor precision can often be traced to inadequate grinding that leaves fibrous matter or visible granules in the sample. Sample prep for plant 15N analysis is found below. Clients may send us unprocessed samples (e.g. leaves, stems, roots) or may provide any of the above processing themselves at a major reduction in price.

Carbon-13

Overview of technique
Isotope-ratio analysis for carbon-13 in solid-phase samples such as plant tissue starts with transformation to gas phase by extremely rapid and complete flash combustion of the sample material. Ionized combustion product (carbon dioxide) is mass-analyzed by means of differing mass/charge ratios among the various isotopic species of CO2. A great many more details of the process are available here.

Clients may send us unprocessed samples (e.g. leaves, stems, roots) or may provide any of the above processing themselves at a major reduction in price.

Nitrate/ Ammonium

Overview of technique
This method describes the extraction of nitrogen as nitrate from soil by potassium chloride (KCl) extraction using Soil Science Society of America Methods of Soil Analysis section 33-3 and colorimetric analysis using APHA 4500-NO3 F.

Method Description
A 4-g aliquot of homogenized moist material is shaken for 1 hour with 20 mL of 2 molar KCl at room temperature in plastic centrifuge tubes. The tubes are centrifuged for 10 minutes at 3000 rpm and the supernatant is pipetted into scintillation vials. Nitrate (NO₃-N) is then analyzed by continuous-flow colorimetry. Concentration is calculated by multiplying the colorimetrically-measured N-value by the volume of added KCl extract and divided by the wet weight of the sample. Nitrogen content per dry mass requires additional processing available for a fee upon request.

Reference
Keeney, D. R. and D. W. Nelson. 1987. Nitrogen–Inorganic Forms, sec. 33-3, extraction of exchangeable ammonium, nitrate, and nitrite. pp.648-9. 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.

Detailed soil extraction protocol can be found below.

Phosphate

More information coming soon.

Total Phosphorus

More information coming soon.

Micro-Dumas Overview

Available Analysis

Plant (Total N)
Soil (Total N)
Animal (Total N)

Plant (Total C)
Soil (Total C)
Animal (Total C)

Overview of technique

For total nitrogen and total carbon analysis, biological sample materials in their naturally occurring solid or liquid state must be converted into simple gases N2 and CO2.

For N, the Kjeldahl-Rittenberg procedure (e.g. Hauck 1982) has a long history and a large following. In this wet-chemistry method an acid digestion is used to produce an ammonium salt, which is then oxidized to N2 gas using hypobromite. This procedure is slow and laborious and involves certain hazards such as hot acid fumes. It also requires care to avoid loss of N during transfer between steps.

The alternative dry Micro-Dumas combustion analysis for total carbon and total nitrogen in solid-phase samples (plant tissue, soils, sediments, etc.) is based on transformation to gas phase by extremely rapid and complete flash combustion of the sample material.

In the apparatus shown above, a rotating multiplace sample dropper (a) delivers one sample at a time into the top of a quartz combustion tube (b). This tube contains granulated chromium III oxide combustion catalist and is held at 1200 degrees C. A pulse of pure O2 is admitted with each sample, which is enclosed in an ultrapure tin combustion capsule. Thermal energy from the combustion of the tin and the sample material can generate an instantaneous temperature of as much as 1700 degrees C at the moment of flash combustion. All combustible materials in the sample are burned and the resulting gas-phase combustion products are swept out the bottom of the furnace by a constant stream of nonreactive helium carrier gas.

All carbon in the sample is converted CO2 during flash combustion. Nitrogen-bearing combustion products include N2 and various oxides of nitrogen NOx; these pass through a reduction column filled with chopped Cu wire (600 degrees C) in which the nitrogen oxides give up their oxygen to the copper and emerge as N2.

Water vapor from the sample is removed by a gas trap (d) containing magnesium perchlorate. If the samples are being analyzed for nitrogen only, CO2 is removed by a second gas trap containing a CO2 scrubber (sodium hydroxide on silicate carrier granules).

The clean sample gases now pass through a gas chromatograph column (E) to separate the N2 and CO2. N2 elutes from the GC column first, then CO2.

The sample gas pulses and a separate reference stream of helium (f) pass through a detector (g); differences in thermal conductivity between the two streams are displayed as visible peaks and recorded as numerically integrated areas.

Linear regression applied to combustion of known standard materials yields a regression line by means of which peak areas from unknowns are converted into total element values for each sample.

Calibration and reference materials

Elemental analyzers such as ours (NA1500 C/H/N Analyzer, Carlo Erba Strumentazione, Milan) are calibrated by including solid-phase reference materials in the tin capsule stage at the beginning of each run and at fixed intervals thereafter (usually one reference per ten unknowns.)

Ultra-high purity acetanilide is the most frequently used standard material; the total C and total N content of this material can be easily calculated from the chemical formula. In addition, each new lot of standard acetanilide is checked before use on real runs by analyzing samples of National Bureau of Standards NBS1572 Citrus Leaves.

Empty tin-capsule blanks are also included periodically in each batch, and any detectable N or C in these blanks is subtracted from the sample and standard values to give a true zero baseline. Blanks allow correction for traces of C originating from the tin capsules and for the small amount of N2 gas introduced as an impurity in the oxygen pulse.

Sample prep considerations

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.

Bibliography

Hauck, R. D. 1982. Nitrogen-Isotope Ratio Analysis, sec.36-3.2.2, Conversion of total nitrogen to ammonium-nitrogen. pp.744ff. In Methods of Soil Analysis, Part 2, Chemical and Microbiological Properties. American Society of Agronomy, Madison, Wisconsin.

Kirsten, Wolfgang. 1983. Organic Elemental Analysis: Ultramicro, Micro, and Trace Methods. Academic Press/Harcourt Brace Jovanovich, New York.

Sample Collection

Samples for colorimetric analysis

Liquid samples must be free of turbidity and particulate matter. Any such substances must be removed before analysis by filtering or centrifugation.
Strongly colored samples may contribute confounding absorbance at the analytical wavelength.
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]
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.)

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.

Nutrient Extraction From Soils & Sediments

Equipment

100 ml Erlenmeyer flasks & clean rubber stoppers
Whatman #42 filter paper
Long-stemmed funnels and funnel rack
Mechanical shaker box
Polyethelyne scintillation vials with poly-lined caps

2M potassium chloride extraction solution

in 1000 ml volumetric flask, add 150 g KCl 800 ml deionized water (diH2O)
Add stir bar; place on stir plate and mix until dissolved.
Remove stir bar and bring to full 1000 ml volume with diH2O.
Cap with parafilm and invert several times to mix.

Procedure

Place 4 g wet-weight soil into each flask; add 20 ml extraction solution to each. Stopper flasks.
Place flasks in shaker box and shake 1 hour, medium speed.
Filter flask contents through funnels lined with filter paper; catch filtrate in labelled scintillation vials.
Cap vials and refrigerate until ready to analyze.
Analyze extracts by (Alpkem) continuous-flow colorimetry (read more below).

Post-analysis calculations

Calculate soil content from the extract’s nitrate and ammonium values as determined by colorimetric analysis: μg analyte per g dry soil =

1000 μg 0.02 L

(determined value, mg/L) x ———- x ———-

1 mg soil dry weight (gm)L

(The dry weight value of the soil sample is based on percent moisture data for each sample, obtained separately.)

Bibliography
Keeney, D. R. and D. W. Nelson. 1987. Nitrogen–Inorganic Forms, sec. 33-3, extraction of exchangeable ammonium, nitrate, and nitrite. pp.648-9. 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.

Colorimetry Overview

Introduction

The basic principle of continuous flow colorimetry is to eliminate chemical analysis by hand-mixing of reagents in individual items of glassware and to substitute a continuously flowing stream of liquid reagents circulating through a closed system of tubing. Reference and sample solutions are introduced by turns into this flowing stream of color-forming reagents.The stream is segmented into small discrete liquid increments (slugs) by providing frequent bubbles of air or other gas that entirely fill the stream tubing bore. While moving through the system, each of these liquid increments is subjected to carefully controlled conditions specific to a particular assay, such as reagent concentration, incubation temperature, reaction time, and so on, to generate a color reaction.This color reaction conforms to Beer’s law (the light absorbance of a solute at a particular wavelength is a function of its concentration in the solution, so that absorbance measurements can be used to measure concentration.) At the end of its flow path each reacted segment passes through a light absorbance cell where its concentration is read by a colorimeter. The recorded result for each sample and standard thus does not represent a single reacted aliquot but is the sum of the measurements for a large number of liquid-increment subsamples.The color reactions on which the process is based are, with only minor modifications, the same as the ones that have long been accepted for manual colorimetric assays (e.g., Murphy/Riley 1962 for ortho-phosphate.) Automated methods cannot be any more accurate than the manual methods on which they are based, but they are less subject to variability since they eliminate the errors of consistent practice involved in analyzing large batches of samples by hand.

Segmented vs. laminar flow

The principal problem of continuous-flow methods is obtaining sufficient mixing of liquids within the tubing system. Aerial photographs show strikingly that when two rivers whose waters are different colors join, the waters derived from each tributary can maintain their separate identity for miles downstream with very little mixing. The following illustration shows a similar mixing problem when two reagent streams A and B are brought together at low velocity at a tubing junction: lamin02a A second mixing difficulty can be caused by laminar flow of liquid within the tubing. As shown schematically in the next illustration, the effect of wall friction and fluid viscosity on flow rate at different distances from the center of a tube is to produce a condition in which the flow rate is faster in the center and slower at the outer edge of the tube. If part of the fluid races ahead and part lags behind, this can lead to contamination from one sample to the next. lamin03a One solution is to increase fluid velocity in the system. Complete mixing can result if the two fluid streams are brought together at high velocity, giving sufficient friction at the wall of the tubing to produce turbulent flow. One may increase fluid velocity by a) increasing reagent and sample flow rate or b) decreasing tube bore diameter. The former leads to greater use of reagents and may be unusable because there is not enough sample. The latter increases the likelihood of obstruction in the tubing by precipitates or by particulate matter from the sample.A different approach is to keep the flow rate relatively low and the tube bore diameter safely larger than any particulate obstructions that are likely to occur, and to introduce bore-filling gas bubbles into the stream. As shown below, each small aliquot of liquid between two bubbles is well mixed by turbulence due to wall friction, and laminar flow and cross-contamination between samples is prevented by complete separation between each pair of liquid slugs. The bubbles continually clean the system by wiping the walls of the tubing and driving forward any stationary liquid film that might contaminate following samples. lamin04a Standard materials and calibrants

Calibration of continuous-flow apparatus is achieved by including standard solutions of known analyte concentration in each batch of samples analysed. A sequence of four or five such standards at the beginning of a run gives absorbance data for these known concentrations from which one can calculate a regression curve to generate concentration values from sample absorbances that fall between the standard values. Within-run drift correction is achieved by including check and recalibrant standards every ten or twenty samples.For typical analyses performed in this lab (for nitrate, ammonium, ortho-phosphate, total nitrogen and total phosphorus) 1000ppm standard stocks are made from appropriate dry reagents (KNO3, NaNO2, (NH4)2SO4, and KH2PO4.) Working standards made from these stocks are checked by analyzing Environmental Protection Agency certified Nutrient-1 quality control solutions of known analyte values. Additionally, digests for total nitrogen and total phosphorus are checked by digesting and analyzing E.P.A. Nutrient-2 QC solutions formulated to challenge digestion techniques.

Sample preparation and handlingSamples must be free of turbidity and particulate matter. Any such substances must be removed before analysis by filtering or centrifugation.Strongly colored samples may contribute confounding absorbance at the analytical wavelength.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; this list is available for reference here.

Bibliography

Coakley, W. A. 1981. Handbook of Automated Analysis: Continuous Flow Techniques. Marcel Dekker, Inc., New York.

Furman, W. B. 1976. Continuous Flow Analysis: Theory and Practice. Marcel Dekker, Inc., New York.

Murphy, J. and J. Riley. 1962. A modified single solution for the determination of phosphate in natural waters. Anal. Chim. Acta v.27 p.31-36.

U. S. Environmental Protection Agency. 1983. Nitrogen, Nitrate-Nitrite. Method 353.2 (Colorimetric, Automated, Cadmium Reduction). pp.353-2.1 — 353-2.5. In Methods for Chemical Analysis of Water and Wastes, EPA-600/ 4-79-020. U.S.E.P.A., Cincinnati, Ohio, USA.

U. S. Environmental Protection Agency. 1983. Nitrogen, Ammonia. Method 350.1 (Colorimetric, Automated Phenate). pp.350-1.1 — 350-1.4. In Methods for Chemical Analysis of Water and Wastes, EPA-600/ 4-79-020. U.S.E.P.A., Cincinnati, Ohio, USA.

U. S. Environmental Protection Agency. 1983. Phosphorus, All Forms. Method 365.1 (Colorimetric, Automated, Ascorbic Acid). pp.365-1.1 — 365-1.7. In Methods for Chemical Analysis of Water and Wastes, EPA-600/ 4-79-020. U.S.E.P.A., Cincinnati, Ohio, 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.