Volatile and Semi Volatile Organic Compound Sampling and Analysis

their compositional distribution and identification of biomarkers. Higher molecular weight hydrocarbon data furthered the understanding of hydrocarbon formation

mechanisms at these two sites of continental serpentinization and helped to determine potential abiotic and/or biotic reactions.

2.4.1 Sampling of Volatile and Semi Volatile Organic Compounds

Sampling for volatile and semi volatile organic compounds. Fluids for

dissolved high molecular weight hydrocarbons were collected by filtering water through a pre-weighed, pre-combusted GF/F (0.7 μm, 25 mm ID) glass microfibre filter. The fluid was collected using a Cole-Parmer Masterflex E/S 07571-00 Portable Sampling Drive equipped with a Masterflex L/S easy-load pump head and L/S 16 (1/8 in ID) silicon tubing. Tubing was flushed for a few minutes with sample water before collection began. When the pump was unavailable collection and filtering was done manually by using a sterile 60 mL syringe and acid washed filter holder. Filtered fluid was collected into a pre-combusted 1 L glass bottle and sealed with a Teflon lined cap. The samples had minimal headspace and were kept cold and dark until analysis. Replicate bottles were collected when possible.

2.4.2 Analysis of Volatile and Semi Volatile Organic Compounds

The filtered water was divided in the laboratory for various analyses. For volatile organic compound concentrations 3 x 40 mL pre-combusted glass vials were filled and sealed with Teflon lined caps and no headspace. The remaining water was saved for semi volatile organic compound concentrations.

Dissolved volatile organic compounds. Samples were measured on an Agilent 6890A GC equipped with a DB-624 (30 m x 0.25 mm ID, 1.4 μm film thickness) column

and a FID with He as the carrier gas. Volatile organic compounds were extracted and concentrated using a direct headspace technique designed to optimize headspace analysis of trace level dissolved volatile organic compounds adapted from Slater et al. (1999). An aliquot (10 mL) of sample was transferred into a 20 mL glass vial with 6 g of NaCl and sealed with an open aluminum cap with a Teflon septa tightly fitted inside. The samples were saturated with NaCl to increase the ionic strength of the water which reduced the solubility of volatile organic compounds and drove them into the headspace. To further optimize the partitioning of dissolved volatile organic compounds into the headspace the vials were heated to 60°C and simultaneously shaken by an auto sampler for 30 minutes before 300 μL of the headspace was injected into the gas chromatograph. The elevated temperature reduced the solubility of gases and shaking caused an increase in NaCl dissolution which further striped the solution of any gas into the headspace. The standards were prepared and extracted using the same method as described for the samples. The samples were split 5:1 in an Agilent split/splitless injector and the carrier gas flow was constant at 1.3 mL/min. An oven temperature program of 40 °C hold for 5 minutes, ramp 10 °C/min to 260 °C, hold at 260 °C for 3 minutes was used to separate the volatile organic compounds. The auto sampler syringe was flushed with N2 between every

injection to eliminate carryover from one sample to the next. Volatile organic compounds were quantified using a 5 point calibration curve (1000, 500, 100, 50, 10 μg/L) of the following organic compounds: cyclohexane, methylcyclohexane, n-heptane, 1-heptene, benzene, toluene, ethylbenzene, xylene (p, m and o), isopropylbenzene and naphthalene. To identify unknown organic compounds, a selection of samples were also run on an Agilent 6890N GC with a 5975C mass spectrometer detector equipped with the DB-624

column using the same temperature program. To quantify these analytes the calibration curve generated for the standard with the most similar chemical structure were used. The reproducibility on replicate field samples for The Cedars ranged from 1.7 to 47.9 % RSD and for the Tablelands ranged from 8.3 to 12.7 % RSD.

Dissolved semi-volatile organic compounds. To extract semi- and non- volatile organic compounds from aqueous samples a separatory funnel liquid-liquid extraction following the Environmental Protection Agency (EPA) method 3510C was used. The pH of the sample was adjusted to ≥11 using a small amount of a concentrated sodium

hydroxide stock solution, unless the sample was already highly basic. The sample was than transferred to a pre-combusted 2 L separatory funnel and extracted three times with 60 mL of dichloromethane. The sample was then acidified to a pH of ≤2 using sulphuric acid and extracted another 3 times with 60 mL of dichloromethane, which gave a total extract volume of 360 mL. With each round of extractions 1 L of nano pure UV water was also extracted as a blank. Extracts were collected in a pre-combusted Erlenmeyer flask and then concentrated down to <1 mL using a ThermoElectron Savant SC250EXP SpeedVac Concentrator. If sample clean up was necessary, then the samples were

subjected to gravity-fed solid-liquid chromatography modelled after EPA method 3600C using activated (140 °C, minimum 8hrs) 100-200 mesh silica gel in order to separate organic compounds based on their polarity using different organic solvents. The different fractions were hexane (F1), 2:1 hexane/dichloromethane (F2), and methanol (F3). A consistent amount of internal standard (o-terphenyl and 5α-cholestane) was added to all gas chromatograph vials (standards and samples) before being analyzed. F1 and F2 extracts were analyzed for aliphatic and aromatics, respectively, using an Agilent 6890N

GC with a 5975C MSD equipped with a HP-5MS (30 m x 0.32 mm ID, 0.25 μm film thickness) column or an Agilent 6890N GC with a 5973 inert MSD equipped with a DB-5 (30 m x 0.32 mm ID, 0.25 μm film thickness) column. The oven temperature program was 50 °C hold for 1 min, ramp 8 °C/min to 310 °C, hold for 20 min with He as the carrier gas. Blank chromatograms were subtracted from sample chromatograms after the retention factor (compound area/internal standard area) was calculated for all integrated areas. Sample chromatogram peaks were compared to 4 point calibration curves (r2_>0.99) of aliphatic standard RESTEK 31459 and polycyclic aromatic hydrocarbons standard RESTEK 31011 by using the calibration curve of the standard with the most similar chemical structure. Compounds were identified using Wiley and NIST libraries for references and comparing sample retention times to standards. The reproducibility on replicate field samples for The Cedars ranged from 4.8 to 33.9 % RSD and for the Tablelands ranged from 1.5 to 12.5 % RSD.