This method can help improve characterization of organic matter and environmental solid matrices, such as soils and sediments. The main advantage of this technique is that it can reduce mineral interferences in soil spectra that limit interpretation of the organic matter component of soils. The implications of this technique extend to other environmental samples dominated by minerals, beyond soils, such as sediments and fossil minerals.
Generally, individuals new to this method will struggle with determining an optimal subtraction factor. It is important to keep in mind that this is operationally determined by your experimental goals. Keep this mind when exploring subtraction factor values.
My laboratory has been doing subtraction work for about 10 years now, with the main objective of identifying absorbance bands associated with soil organic carbon quality. Originally, we wanted to study the role of wind erosion on the removal of different forms of soil carbon from agricultural soils. Visual demonstration of this method is helpful because the labor-intensive oxidation steps and adjustment of the subtraction factor requires some practice by doing, which can be informed by watching how others perform these procedures.
First, sieve soil to less than two millimeters, using a stainless steel mesh. Following this, adjust the pH of 6%sodium hypochlorite to 9.5, by adding one molar hydrochloric acid drop-wise to the solution, while mixing and measuring with a pH meter. Add 25 mL of pH 9.5 sodium hypochlorite to 4 grams of the sieved soil in a 50 mL conical tube and mix by sonication.
After sonication, incubate the mixture in an 80 degree celsius water bath for 15 minutes, to increase the oxidation rate. Centrifuge the mixture to obtain a clear supernatant. Then manually decant the supernatant into a waste container.
After the last oxidation step, add 20 mL of deionized water to the soil, and mix for five minutes using a horizontal shaker at 120 RPM. Centrifuge the sample at room temperature for 15 minutes at 4000 times g. Using a spatula and deionized water from a squirt bottle as needed, transfer the soil pellet from the bottom of the centrifuge tube to a plastic weigh boat.
Then, oven dry the sample at 60 degrees celsius for 48 hours. Once the soil sample is dry, quantify total organic carbon content by combustion gas chromatography, using a CN analyzer. For SOM removal by high temperature combustion, measure one to two grams of sieved soil into a porcelain crucible, using a spatula.
Then, heat the sample at 550 degrees celsius for three hours, using a muffle furnace. Grind untreated and treated soil samples to a similar consistency by hand grinding. Following this, load a previously ground KBr sample into the sample cup of an FTIR spectrometer.
To collect a background spectrum, open the dropdown menu for experiment, and select the desired experimental collection method in the software. Click the experimental setup icon to select spectral acquisition parameters. Under the collect tab, check that the number of scans and resolution are appropriate for experimental objectives.
Click okay to save changes. Then, click the collect background icon to collect a background spectrum. Next, pour the soil sample into the sample cup to the point of slightly overfilling, to ensure consistent loading and to minimize surface roughness.
Then, surface smooth the soil in the cup, using a flat edge, such that the height of soil sample is flush with the lip of the cup. To collect spectra of the untreated and treated soil samples, click experimental setup. Under the collect tab, select use specified background file, and load the background spectrum file.
Then, click okay to save changes. To commence spectral collection on the soil, click collect sample. To perform spectral subtractions, zero out peaks by using the subtraction option of the software program;to change the subtraction factor, to minimize or reduce target mineral peaks, and/or to maximize the linear baseline.
Simultaneously select the untreated and treated soil spectra, and click the subtract icon. The first untreated soil spectrum selected, will be the spectrum from which the second treated soil spectrum will be subtracted. Use the vertical toggle bar and arrows to increase or decrease the subtraction factor.
Observe the changes in the previewed subtraction spectrum. Finally, click add to load the calculated subtraction spectrum into a window. Common changes of mineral bands following high temperature ashing, include loss of OH peaks and peak losses and shifts in lattice silicon oxygen and aluminum oxygen peaks.
Soil A lost 89%of soil organic carbon by sodium hypochlorite oxidation, compared to 97%by ashing, while preserving mineral absorbance features altered by ashing. As the subtraction factor increases, the absorbance of peaks corresponding to minerals decrease, most notably, OH and silicon oxygen. Concurrently, absorbance increases for organic functional groups, such as aliphatic CH, amide CN and NH, and/or aromatic CC.Though zeroing out quartz-like silicon oxygen at 2100 to 1780 Wavenumber is achieved with a subtraction factor of 0.76, a prominent W-shaped inversion suggests that interpretation of the subtraction spectrum should be limited to greater than 1200 Wavenumber.
Enhancing the aliphatic CH stretch by over-subtracting the mineral reference spectrum, renders the remaining spectra uninterpretable, including the region corresponding to the majority of organic functional groups, relevant to SOM characterization. For a given method of SOM removal, differences are visually evident between the subtraction spectra of high and low organic matter soils that are less visible or absent in the untreated soil spectra. While attempting this procedure, it's important to remember to perform quality controls, such as checking the amount of organic matter removed by a particular method and for a specific sample.
It's also important to evaluate the subtraction factor for suitability for the experimental objectives. Following this procedure, other methods, like nuclear magnetic resonance or mass spectrometry, can be performed in order to provide complementary information on soil organic matter structure. With its ongoing development, this technique is helping researchers in the fields of soil science, geochemistry and sedimentology, to explore functional group composition of organic matter in mineral dominated samples.
After watching this video, you should have a good understanding on how to remove organic matter from soil samples, to produce a mineral-enriched reference spectrum for performing subtraction of mineral absorbances. Don't forget that working with oxidants, such as sodium hypochlorite, can be hazardous. Precautions such as personal protective equipment, should always be taken while performing this procedure.
