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!