Characterization, Quantification and Compound-specific Isotopic Analysis of Pyrogenic Carbon Using Benzene Polycarboxylic Acids (BPCA)

The overall goal of this molecular marker method is to characterize, quantify and isotopically analyze the Pyrogenic Carbon fraction in environmental samples. This method can help answer key questions in various fields, ranging from archaeology and environmental forensics, to biochar and Carbon cycling research. The main advantages of this technique are detection of pyrogenic Carbon over a broad range of the combustion continuum.

Simultaneous characterization, quantification, and isotopic analysis of pyrogenic Carbon. And applicability to a very wide range of environmental sample materials. Demonstrating the sample preparation and chromatography will be Ulrich Hanke, a grad student from our laboratory.

The subsequent isotopic analysis of the purified target compounds will be shown by Negar Haghipour, lab coordinator at ETH Zurich. To begin this procedure weigh previously freeze-dried and homogenized samples into quartz digestion tubes, and cover against dust with aluminum foil. Use only clean, decalcified, and combastic glassware, thoroughly cleaned tools, and ultra-pure chromatography grade water and solvents for the entire procedure.

Add two milliliters of 65%nitric acid into the digestion tubes. Use a vortex mixer to assist thorough wetting of each sample. Following this, insert the digestion tubes into a pressure chamber.

Close the pressure chamber according to the manual and place it into a preheated oven at 170 degrees Celsius for eight hours. After removing the digestion tubes from the cooled pressure chamber, filter the samples with water into Volumetric flasks, using disposable glass fiber filters. Then adjust the volume to 25 milliliters with water.

For each sample, prepare two glass columns with 11 grams of cation exchange resin per column. Condition the resin inside the columns by consecutively rinsing them with water, two molar sodium hydroxide, and two molar hydrochloric acid. Check the conductivity of the water.

Which is rinsed through the resin after its conditioning. Next transfer one half of the sample onto each column, rinse sequentially five times with 10 milliliters of water. Then freeze-dry the aqueous solutions afterwards.

Condition C18 solid phase extraction cartridges according to the manufacturers instruction manual. Following this, re-dissolve each freeze-dried residue in three milliliters of methanol and water. Elute each half of the sample over a separate C18 solid phase extraction cartridge into 2.5 milliliter test tubes.

Then, rinse the cartridges with one milliliter of methanol and water. Dry the test tubes with the sample solutions using a vacuum concentrator heated to 45 degrees Celsius, and with a vacuum of approximately 50 millibar. After drying, re-dissolve each residue with one milliliter of water and mix with a vortex mixer.

Then, transfer the solutions to 1.5 milliliter auto-sampler vials. For the chromatographic analysis, prepare solvent A by mixing 20 milliliters of 85%orthophosphoric acid with 980 milliliters of water and filter the solution through a disposable glass fiber filter using vacuum. Prepare standard solutions of commercially available benzene polycarboxylic acids of BPCAs to produce an external standard concentration series.

Conduct the chromatography, and quantify the BPCA contents, by comparing the respective BPCA peak areas to the measurements of external standard series. Purity requirement for chemical and laboratory equipment are especially high for compound specific Carbon-14 analysis of BPCAs. Blank assessment and swipe tests are important to monitor potential source of sample contamination.

If compound specific Carbon-13 and Carbon-14 analyses of the pyrogenic Carbon fraction are intended, repeat the chromatography several times for each sample, and collect the individual BPCAs in sufficient quantity using a fraction collector connected to the HPLC. Remove the solvent, by blowing down the fractions with a gentle nitrogen stream, while heating them to 70 degrees Celsius. Next, prepare the oxidizing reagent by dissolving two grams of sodium persulfate in 15 milliliters of water.

Re-dissolve each residue with four milliliters of water, and transfer the samples to 12 milliliter gas-tight, borosilicate vials. Then, add one milliliter of oxidizing reagent, and close each vial with a standard cap containing a butyl rubber septum. Purge the gas-tight vials containing the aqueous solutions with helium for eight minutes to remove Carbon dioxide from the vial and the solution.

Following this, oxide the samples in the gas-tight vials by heating them at 100 degrees Celsius for 60 minutes. After allowing the samples to cool to room temperature, directly analyze the Carbon dioxide from the oxidation on an isotope ratio mass spectrometer for Carbon-13 content and on an accelerated mass spectrometer for Carbon-14 content. The described procedure allows baseline separation of all BPCA target compounds by HPLC and chromatograms of the referenced materials tramazem and grass-char are shown here.

By adjusting the chromatography parameters the separation can be further modified for specific needs. Quantitative analysis of the referenced materials chromatograms with external standards should yield the pyrogenic carbon values depicted here. The Carbon-13 and Carbon-14 values that are obtained when purified BPCAs of referenced materials are analyzed for their carbon isotopic content are listed here.

Once mastered this technique can be done in three working days. Therefore, sample throughput is a matter of how many samples are processed simultaneously. Measuring a dozen samples per week in duplicates and with isotopic information is easily achievable.

After watching this video, you should have a good understanding of how to perform the indubitable steps for the successful analysis of pyrogenic Carbon on the compound specific level.