Carbon and Nitrogen Analysis of Environmental Samples

Environmental Science

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Overview

Source: Laboratories of Margaret Workman and Kimberly Frye - Depaul University

Elemental Analysis is a method used to determine elemental composition of a material. In environmental samples such as soils, scientists are particularly interested in the amounts of two ecologically important elements, nitrogen and carbon. Elemental analysis by the flash combustion technique works by oxidizing the sample with a catalyst through combustion in a high-temperature chamber. The products of combustion are then reduced to N2 and CO2 and detected with a thermal conductivity detector.

Unlike other methods for total nitrogen determination (Kjeldahl method) and total carbon determination (Walkley-Black, Heanes or Leco methods), the flash combustion technique does not use toxic chemicals and is therefore much safer to use.

This video will demonstrate combustion-based elemental analysis using the Flash EA 1112 instrument from Thermo Fisher Scientific.

Cite this Video

JoVE Science Education Database. Environmental Science. Carbon and Nitrogen Analysis of Environmental Samples. JoVE, Cambridge, MA, (2017).

Principles

The soil samples are placed in a tin disc and dropped into the oxidation reactor via an autosampler where it is burned in an oxygen environment at greater than 900 °C in the presence of an oxidation catalyst. The carbon in the sample is converted to carbon dioxide and the nitrogen is converted to nitrogen gas and some nitrogen oxides.

C + O2 → CO2
4 N + x O2 → N2 + 2 NOx

Helium gas carries these products into a second reaction tube filled with copper that reduces the nitrogen oxides to nitrogen gas and removes excess oxygen. This is completed at 680 °C.

NOx + Cu → N2 + CuO
O2 + Cu → CuO

The gas stream then flows through a filter filled with magnesium perchlorate to remove any water vapor before the stream reaches the gas chromatograph column.

The N2 will exit the gas chromatography column first at about 110 s, and then the CO2 will exit at about 190 s. Using a standard curve created using aspartic acid, the %N and %C in the soil sample can be determined.

Procedure

1. Preparation of Soil Samples

  1. Dry soil samples at 60 °C for 48 h.
  2. Pass the soil through a 2 mm x 2 mm sieve.
  3. Put approximately 5 g of the soil into the ball mill grinder and grind for 2 min. It is important to get a homogeneous sample since your sample size will be very small.
  4. Put milled soil into a small container and store in a desiccator until ready to use.

2. Setting up the Instrument Parameters

  1. Turn on the Flash EA 1112 instrument in the back by flipping the switch up.
  2. Turn on the computer.
  3. Double click on the “Eager 300” icon to start the software program that runs the instrument.
  4. Double click on the “NC Soils” icon to open the method that runs the instrument setup for soils.
  5. Heat up the instrument by opening up the “Edit Elemental Analyzer Parameters” and clicking on the “Send” button. The parameters should be as follows (See Figures 1–3):
    a. Temperatures:  Left = 900 °C, Right = 680 °C, Oven = 50 °C
    b. Gas flow:  Carrier = 130 mL/min, Oxygen = 250 mL/min, Reference = 100 mL/min
    c. Cycle Runtime = 360 s
    d. Sampling Delay = 12 s
    e. Oxygen Injection End = 5 s
    f. Detector = Filament On
  6. Create a sample table by clicking on “Edit Sample Table” and then “Fill Sample Table”. Change the filename to today’s date. Input the number of samples you plan to run, including the standards and blanks. Then click “Replace” to replace the last sample table that was created with your new sample table.

3. Creating a Standard Curve

  1. Using forceps, remove one tin disc from the pack and mold it into a cup shape using the special sealing device. Avoid touching the tin disc with your fingers to avoid transferring oils from your fingertips. (See Figures 4–5)
  2. Using forceps, place the tin disc on the microbalance and zero the balance.
  3. Using forceps, remove the tin disc from the microbalance and using a microspatula, place approximately 1 mg of aspartic acid standard into the tin disc.
  4. Weigh the tin disc with the aspartic acid standard on the microbalance. Enter this weight into the data table in the Eager 300 software on the computer.
  5. Seal up the tin disc with the forceps so that none of the aspartic acid standard will spill out of it. Place the tin package into the autosampler. (See Figure 6)
  6. Repeat steps 3.1 – 3.5, using approximately 5 mg of aspartic acid standard.
  7. Repeat steps 3.1 – 3.5, using approximately 7.5 mg of aspartic acid standard.
  8. Repeat steps 3.1 – 3.5, using approximately 10 mg of aspartic acid standard.

4. Loading the Autosampler with Soil Samples

  1. Using forceps, remove one tin disc from the pack and mold it into a cup shape using the sealing device. You should not touch the tin with your fingers to avoid transferring oils from your fingertips.
  2. Using forceps, place the tin disc on the microbalance and zero the balance.
  3. Remove the tin disc from the microbalance and place approximately 50 mg of the homogenized soil into the tin disc using a microspatula.
  4. Weigh the tin disc with soil sample on the microbalance. Enter this weight into the data table in the Eager 300 software on the computer.
  5. Seal up the tin disc using the forceps so that the soil is contained. Transfer the tin package to the autosampler tray.
  6. Repeat steps 4.1 – 4.5 for all of your samples. It is recommended to run triplicate trials of each sample. A triplicate experiment is considered a good rule of thumb to rule out experimental errors.

5. Running the Samples

  1. When the appropriate temperatures have been reached on the instrument, the green “Temperature Ready” light will turn on. At the bottom of the screen on the computer, it will also say “Ready for Analysis”.
  2. Before starting your sample run, click on “File” and “Save Method” to save the data you just input. It is recommended that you save the method with your last name and the date.
  3. To begin the run, click on the green arrow and push “Start Now”.
  4. It will take approximately 6 min per sample to run.
  5. After the run is complete, you can see the results by clicking on “Recalculation” then “Summarize Results”.

Figure 1
Figure 1. Flash EA 1112 parameters setup screen 1.

Figure 2
Figure 2. Flash EA 1112 parameters setup screen 2.

Figure 3
Figure 3. Flash EA 1112 parameters setup screen 3.

Figure 4
Figure 4. Removing a tin disc with forceps.

Figure 5
Figure 5. The tin disc molded into a cup shape using the sealing device.

Figure 6
Figure 6. The tin package being placed into the autosampler.

Analyzing the amounts of the carbon and nitrogen in environmental samples - a process known as "elemental analysis" - provides important insight into the ecological properties of the environment.

Carbon and nitrogen are two of the most important elements for life. Carbon is the foundation of organic compounds that form the basis of all living things, and is particularly useful as a measure for molecules such as carbohydrates, the primary energy source for organisms. On the other hand, nitrogen is found in molecules such as nucleic and amino acids. These serve, respectively, as genetic material and as the building blocks of the proteins used by organisms for structure and function.

Because these different classes of organic molecules have different biological roles, organisms require them at different amounts. For example, microorganisms in soil typically require food sources with a C:N ratio of 24:1. Because different plant residues have different C:N ratios that range from 13:1, such as alfalfa, to 57:1, as in corn, they will be decomposed by microbes at different rates and to different extents, in turn affecting how nutrients are returned to the soil.

This video will introduce the principles of analyzing carbon and nitrogen elemental composition; a protocol for performing elemental analysis on soil samples; and finally, some applications of this analysis method to environmental research.

Elemental analysis can be performed in a number of ways, such as the use of specific chemical reactions, often involving strong acids, resulting in characteristic products that can be detected. A major improvement in elemental analysis methodology was the development of the flash combustion technique, which removed the need for using dangerous chemicals, greatly simplified and sped up the process, and allowed for automation.

The basis of flash combustion-based elemental analysis is to oxidize the sample in an "oxidation chamber", by burning it in the presence of oxygen at high temperatures of around 1,000 °C in the presence of a catalyst, which speed up the reaction. This converts the carbon in the sample into carbon dioxide gas, and the nitrogen into nitrogen oxide and nitrogen gases. An inert "carrier gas" such as helium is then used to transport these combustion products to a "reduction chamber" with copper filling, where the nitrogen oxides are further converted into nitrogen gas. Excess water vapor is removed from the gas mixture by filtration with a desiccant such as magnesium perchlorate.

The flash combustion products can then be separated by gas chromatography, during which the gas molecules pass through tubing, called a column, containing a thin coating of liquid or polymer. The gases repeatedly dissolve and vaporize from this substrate as they pass through the column, at rates that are dependent on how strongly the molecules interact with the substrate and the carrier gas. A species that spends more time dissolved in the substrate will travel more slowly through the column, thus allowing the gases to be differentiated.

Once they exit the column, the gases can be identified by, for example, detecting how well they conduct heat, a property known as thermal conductivity. By plotting the time it takes each gas to travel through the coil, scientists obtain a "chromatogram" with peaks that represent each gas. By calculating the detected amounts of carbon dioxide and nitrogen gases using the area under the respective peaks, the C:N ratio in the original sample can then be deduced.

Now that you understand the principles of carbon and nitrogen elemental analysis using the flash combustion method, let's go through a protocol for performing this using an automated elemental analyzer.

To prepare the soil samples for analysis, first, dry the samples in a 60 °C oven for 48 h. Then, pass the dried soil through a 2 x 2-mm sieve, and discard any soil particle that doesn't pass through. Next, use a ball mill grinder to grind approximately 5 g of the soil for 2 min to make a homogeneous powder. Put the milled soil into a small container such as a polyethylene vial, and store it in a desiccator until ready to use.

Set the analysis parameters on the elemental analyzer according to manufacturer's instructions. These include the temperatures of the oxidation furnace, the reduction furnace, and the gas chromatography oven, the flow rate of the carrier gas, the oxygen injection rate, the flow rate of the reference gas, the cycle run time, the delay between sample drop and oxygen injection into the oxidation chamber, and the duration of oxygen injection.

In order to quantitatively determine the composition of the sample, a standard curve is first created using different amounts of a compound of known composition, such as aspartic acid.

To do this, first use forceps to remove a tin sample-holding disc from a pack and mold it into a cup shape using the specialized sealing device. Avoid touching the tin disc with your fingers, as that could lead to the transfer of oils onto the disc.

Now, place the tin cup on a microbalance, and set the tare mass. Remove the tin cup, then use a microspatula to place approximately 1 mg of the aspartic acid standard into the cup. Weigh the cup and record the mass. Then, seal the tin cup, and place it into the autosampler, which will automatically deliver each sample into the reaction chamber.

Repeat the above steps for several amounts of the standard. Then, place all standards into the autosampler.

Dispense and weigh the soil samples in tin cups similarly as the standards, using approximately 50 mg of each homogenized soil sample. Prepare each sample in triplicate.

Once all samples are placed into the autosampler, and the appropriate temperatures have been reached in the instrument, set the measurements to run. The instrument software will produce a chromatogram for each standard and sample.

Depending on the parameters used, the peak for nitrogen gas should be at about 110 s on the chromatogram, while the carbon dioxide peak is detected at around 190 s. Standard curves are generated with aspartic acid, which has a carbon to nitrogen ratio of 4 to 1. With this knowledge, along with the concentration of each standard, the area under each peak can be used to calculate the amount of nitrogen and carbon in each sample.

Based on the mass of the original sample, the percent-nitrogen and percent-carbon of each sample can be calculated. In this demonstration, the C:N ratio of this soil sample was found to be approximately 13:1, lower than the ratio of 14.25:1 usually found for soil under open woodlands and indicative of woods dominated by the invasive European buckthorn trees.

Carbon and nitrogen content analysis can be applied to a variety of environmental samples in addition to soil, and has wide applications in environmental research.

In this example, researchers collected water samples from various marine habitats, such as coral reefs. To understand the availability of organic nutrients to marine microbial communities, various chemical parameters were measured, including carbon and nitrogen elemental analysis. Levels of dissolved organic carbon were directly measured from the water sample, while particulate organic matter was filtered from the water and analyzed.

Elemental analysis can also be used to monitor nutrient loss in runoff from the irrigation of urban landscapes and lawns, which can pollute water supplies. Here, scientists set up test plots to simulate urban landscapes and better understand this process. A variety of chemical tests were used to analyze specific nutrients such as nitrates and ammonia in the collected runoff, and combustion-based elemental analysis was used to measure the levels of dissolved organic carbon and nitrogen.

Finally, analyzing the C:N ratio in herbivore carcasses revealed an interesting link between predation risk and the decomposition rate in soil. In this study, grasshoppers were reared with or without the risk of predation by spiders. Carcasses of these grasshoppers were then allowed to decompose in plots of soil, and plant detritus were later added to the soil for decomposition.

Elemental analysis showed slightly increased C:N ratio in grasshoppers reared with predation risk, but this in turn led to significantly decreased rate of decomposition in soil in which the stressed grasshopper was decomposed, pointing to unexpected complex dynamics in ecosystem nutrient cycling.

You've just watched JoVE's video on carbon and nitrogen analysis of environmental samples. You should now understand the principles behind this method of analysis; how to perform it using a flash combustion elemental analyzer; and some of its applications in environmental science. As always, thanks for watching!

Results

A chromatogram for each sample is produced showing the amount of nitrogen and carbon in the sample (Figure 7).

The areas under the curve at each of the peaks in the sample chromatogram are compared to the standard curves (Figures 8 and 9), and the amount of nitrogen and carbon in the sample is calculated. Based on the weight of the original sample, the %N and %C is calculated (Figure 10).

Figure 7
Please click here to view a larger version of this figure.
Figure 7. Chromatogram showing nitrogen and carbon peaks.

Figure 8
Figure 8. Assay standard curve for nitrogen.

Figure 9
Figure 9. Assay standard curve for carbon.

Figure 10
Figure 10. Calculation of %N and %C, based on the weight of the original sample.

Applications and Summary

The Carbon to Nitrogen (C:N) ratio in soil is a ratio of the mass of carbon to the mass of nitrogen in the soil sample. The C:N ratio of soil and anything put on the soil (like crop residue cover) can affect crop residue decomposition and nutrient cycling. Soil microorganisms have a C:N ratio of approximately 8:1. To maintain this ratio, they must acquire their carbon and nitrogen from the environment. However, since some of the carbon that the microorganisms acquire must be used as a source of energy in addition to what it needs for body maintenance, the microorganisms require a C:N ratio of approximately 24:1. If leaf litter or soil cover with a C:N ratio of higher than 24:1 is placed on the soil (e.g., corn stover with a C:N ratio of 57:1), the microorganisms will be required to use nitrogen from the soil in order to decompose the litter material. This results in a nitrogen deficit in the soil. If leaf litter or soil cover with a C:N ratio of lower than 24:1 is placed on the soil (e.g., alfalfa hay with a C:N ratio of 13:1), there will be some nitrogen remaining after the decomposition of the litter material, which will be released into the soil as nutrients.

Elemental analysis not only can be used to determine the C:N ratio of the soil samples, but can also be used to determine the C:N ratio in plant materials, such as tree leaves and crop residue. This information is important for farmers in order to help them decide what type of crop cover to use. The C:N ratio of the crop residue added to cover the soil influences how quickly the residue will decompose. This has implications for whether or not the soil is protected for the desired length of time.

1. Preparation of Soil Samples

  1. Dry soil samples at 60 °C for 48 h.
  2. Pass the soil through a 2 mm x 2 mm sieve.
  3. Put approximately 5 g of the soil into the ball mill grinder and grind for 2 min. It is important to get a homogeneous sample since your sample size will be very small.
  4. Put milled soil into a small container and store in a desiccator until ready to use.

2. Setting up the Instrument Parameters

  1. Turn on the Flash EA 1112 instrument in the back by flipping the switch up.
  2. Turn on the computer.
  3. Double click on the “Eager 300” icon to start the software program that runs the instrument.
  4. Double click on the “NC Soils” icon to open the method that runs the instrument setup for soils.
  5. Heat up the instrument by opening up the “Edit Elemental Analyzer Parameters” and clicking on the “Send” button. The parameters should be as follows (See Figures 1–3):
    a. Temperatures:  Left = 900 °C, Right = 680 °C, Oven = 50 °C
    b. Gas flow:  Carrier = 130 mL/min, Oxygen = 250 mL/min, Reference = 100 mL/min
    c. Cycle Runtime = 360 s
    d. Sampling Delay = 12 s
    e. Oxygen Injection End = 5 s
    f. Detector = Filament On
  6. Create a sample table by clicking on “Edit Sample Table” and then “Fill Sample Table”. Change the filename to today’s date. Input the number of samples you plan to run, including the standards and blanks. Then click “Replace” to replace the last sample table that was created with your new sample table.

3. Creating a Standard Curve

  1. Using forceps, remove one tin disc from the pack and mold it into a cup shape using the special sealing device. Avoid touching the tin disc with your fingers to avoid transferring oils from your fingertips. (See Figures 4–5)
  2. Using forceps, place the tin disc on the microbalance and zero the balance.
  3. Using forceps, remove the tin disc from the microbalance and using a microspatula, place approximately 1 mg of aspartic acid standard into the tin disc.
  4. Weigh the tin disc with the aspartic acid standard on the microbalance. Enter this weight into the data table in the Eager 300 software on the computer.
  5. Seal up the tin disc with the forceps so that none of the aspartic acid standard will spill out of it. Place the tin package into the autosampler. (See Figure 6)
  6. Repeat steps 3.1 – 3.5, using approximately 5 mg of aspartic acid standard.
  7. Repeat steps 3.1 – 3.5, using approximately 7.5 mg of aspartic acid standard.
  8. Repeat steps 3.1 – 3.5, using approximately 10 mg of aspartic acid standard.

4. Loading the Autosampler with Soil Samples

  1. Using forceps, remove one tin disc from the pack and mold it into a cup shape using the sealing device. You should not touch the tin with your fingers to avoid transferring oils from your fingertips.
  2. Using forceps, place the tin disc on the microbalance and zero the balance.
  3. Remove the tin disc from the microbalance and place approximately 50 mg of the homogenized soil into the tin disc using a microspatula.
  4. Weigh the tin disc with soil sample on the microbalance. Enter this weight into the data table in the Eager 300 software on the computer.
  5. Seal up the tin disc using the forceps so that the soil is contained. Transfer the tin package to the autosampler tray.
  6. Repeat steps 4.1 – 4.5 for all of your samples. It is recommended to run triplicate trials of each sample. A triplicate experiment is considered a good rule of thumb to rule out experimental errors.

5. Running the Samples

  1. When the appropriate temperatures have been reached on the instrument, the green “Temperature Ready” light will turn on. At the bottom of the screen on the computer, it will also say “Ready for Analysis”.
  2. Before starting your sample run, click on “File” and “Save Method” to save the data you just input. It is recommended that you save the method with your last name and the date.
  3. To begin the run, click on the green arrow and push “Start Now”.
  4. It will take approximately 6 min per sample to run.
  5. After the run is complete, you can see the results by clicking on “Recalculation” then “Summarize Results”.

Figure 1
Figure 1. Flash EA 1112 parameters setup screen 1.

Figure 2
Figure 2. Flash EA 1112 parameters setup screen 2.

Figure 3
Figure 3. Flash EA 1112 parameters setup screen 3.

Figure 4
Figure 4. Removing a tin disc with forceps.

Figure 5
Figure 5. The tin disc molded into a cup shape using the sealing device.

Figure 6
Figure 6. The tin package being placed into the autosampler.

Analyzing the amounts of the carbon and nitrogen in environmental samples - a process known as "elemental analysis" - provides important insight into the ecological properties of the environment.

Carbon and nitrogen are two of the most important elements for life. Carbon is the foundation of organic compounds that form the basis of all living things, and is particularly useful as a measure for molecules such as carbohydrates, the primary energy source for organisms. On the other hand, nitrogen is found in molecules such as nucleic and amino acids. These serve, respectively, as genetic material and as the building blocks of the proteins used by organisms for structure and function.

Because these different classes of organic molecules have different biological roles, organisms require them at different amounts. For example, microorganisms in soil typically require food sources with a C:N ratio of 24:1. Because different plant residues have different C:N ratios that range from 13:1, such as alfalfa, to 57:1, as in corn, they will be decomposed by microbes at different rates and to different extents, in turn affecting how nutrients are returned to the soil.

This video will introduce the principles of analyzing carbon and nitrogen elemental composition; a protocol for performing elemental analysis on soil samples; and finally, some applications of this analysis method to environmental research.

Elemental analysis can be performed in a number of ways, such as the use of specific chemical reactions, often involving strong acids, resulting in characteristic products that can be detected. A major improvement in elemental analysis methodology was the development of the flash combustion technique, which removed the need for using dangerous chemicals, greatly simplified and sped up the process, and allowed for automation.

The basis of flash combustion-based elemental analysis is to oxidize the sample in an "oxidation chamber", by burning it in the presence of oxygen at high temperatures of around 1,000 °C in the presence of a catalyst, which speed up the reaction. This converts the carbon in the sample into carbon dioxide gas, and the nitrogen into nitrogen oxide and nitrogen gases. An inert "carrier gas" such as helium is then used to transport these combustion products to a "reduction chamber" with copper filling, where the nitrogen oxides are further converted into nitrogen gas. Excess water vapor is removed from the gas mixture by filtration with a desiccant such as magnesium perchlorate.

The flash combustion products can then be separated by gas chromatography, during which the gas molecules pass through tubing, called a column, containing a thin coating of liquid or polymer. The gases repeatedly dissolve and vaporize from this substrate as they pass through the column, at rates that are dependent on how strongly the molecules interact with the substrate and the carrier gas. A species that spends more time dissolved in the substrate will travel more slowly through the column, thus allowing the gases to be differentiated.

Once they exit the column, the gases can be identified by, for example, detecting how well they conduct heat, a property known as thermal conductivity. By plotting the time it takes each gas to travel through the coil, scientists obtain a "chromatogram" with peaks that represent each gas. By calculating the detected amounts of carbon dioxide and nitrogen gases using the area under the respective peaks, the C:N ratio in the original sample can then be deduced.

Now that you understand the principles of carbon and nitrogen elemental analysis using the flash combustion method, let's go through a protocol for performing this using an automated elemental analyzer.

To prepare the soil samples for analysis, first, dry the samples in a 60 °C oven for 48 h. Then, pass the dried soil through a 2 x 2-mm sieve, and discard any soil particle that doesn't pass through. Next, use a ball mill grinder to grind approximately 5 g of the soil for 2 min to make a homogeneous powder. Put the milled soil into a small container such as a polyethylene vial, and store it in a desiccator until ready to use.

Set the analysis parameters on the elemental analyzer according to manufacturer's instructions. These include the temperatures of the oxidation furnace, the reduction furnace, and the gas chromatography oven, the flow rate of the carrier gas, the oxygen injection rate, the flow rate of the reference gas, the cycle run time, the delay between sample drop and oxygen injection into the oxidation chamber, and the duration of oxygen injection.

In order to quantitatively determine the composition of the sample, a standard curve is first created using different amounts of a compound of known composition, such as aspartic acid.

To do this, first use forceps to remove a tin sample-holding disc from a pack and mold it into a cup shape using the specialized sealing device. Avoid touching the tin disc with your fingers, as that could lead to the transfer of oils onto the disc.

Now, place the tin cup on a microbalance, and set the tare mass. Remove the tin cup, then use a microspatula to place approximately 1 mg of the aspartic acid standard into the cup. Weigh the cup and record the mass. Then, seal the tin cup, and place it into the autosampler, which will automatically deliver each sample into the reaction chamber.

Repeat the above steps for several amounts of the standard. Then, place all standards into the autosampler.

Dispense and weigh the soil samples in tin cups similarly as the standards, using approximately 50 mg of each homogenized soil sample. Prepare each sample in triplicate.

Once all samples are placed into the autosampler, and the appropriate temperatures have been reached in the instrument, set the measurements to run. The instrument software will produce a chromatogram for each standard and sample.

Depending on the parameters used, the peak for nitrogen gas should be at about 110 s on the chromatogram, while the carbon dioxide peak is detected at around 190 s. Standard curves are generated with aspartic acid, which has a carbon to nitrogen ratio of 4 to 1. With this knowledge, along with the concentration of each standard, the area under each peak can be used to calculate the amount of nitrogen and carbon in each sample.

Based on the mass of the original sample, the percent-nitrogen and percent-carbon of each sample can be calculated. In this demonstration, the C:N ratio of this soil sample was found to be approximately 13:1, lower than the ratio of 14.25:1 usually found for soil under open woodlands and indicative of woods dominated by the invasive European buckthorn trees.

Carbon and nitrogen content analysis can be applied to a variety of environmental samples in addition to soil, and has wide applications in environmental research.

In this example, researchers collected water samples from various marine habitats, such as coral reefs. To understand the availability of organic nutrients to marine microbial communities, various chemical parameters were measured, including carbon and nitrogen elemental analysis. Levels of dissolved organic carbon were directly measured from the water sample, while particulate organic matter was filtered from the water and analyzed.

Elemental analysis can also be used to monitor nutrient loss in runoff from the irrigation of urban landscapes and lawns, which can pollute water supplies. Here, scientists set up test plots to simulate urban landscapes and better understand this process. A variety of chemical tests were used to analyze specific nutrients such as nitrates and ammonia in the collected runoff, and combustion-based elemental analysis was used to measure the levels of dissolved organic carbon and nitrogen.

Finally, analyzing the C:N ratio in herbivore carcasses revealed an interesting link between predation risk and the decomposition rate in soil. In this study, grasshoppers were reared with or without the risk of predation by spiders. Carcasses of these grasshoppers were then allowed to decompose in plots of soil, and plant detritus were later added to the soil for decomposition.

Elemental analysis showed slightly increased C:N ratio in grasshoppers reared with predation risk, but this in turn led to significantly decreased rate of decomposition in soil in which the stressed grasshopper was decomposed, pointing to unexpected complex dynamics in ecosystem nutrient cycling.

You've just watched JoVE's video on carbon and nitrogen analysis of environmental samples. You should now understand the principles behind this method of analysis; how to perform it using a flash combustion elemental analyzer; and some of its applications in environmental science. As always, thanks for watching!

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