Milli q biocel system

Standards of 3′-SL (purity 98.55%) and 6′-SL (purity 98.75%) were provided by GeneChem Inc. (Daejeon, Korea). Methanol and acetonitrile (HPLC grade) were purchased from Burdick & Jackson (Muskegon, MI, USA). Acetic acid (HPLC grade) was purchased from Sigma-Aldrich (St. Louis, MO, USA). Ammonium acetate (HPLC grade) was purchased from Fluka (Muskegon, MI, USA). Deionized water was obtained using a Millipore Elix and Milli-Q Biocel system (Millipore, Milford, MA, USA). An internal standard, [1,2,3-¹³C³]3′-sialyl[3 -¹³C^glc] lactose sodium salt, was purchased from Omicron Biomedicals, Inc. (South Bend, IN, USA).

Anaerobic Rifle Artificial Groundwater (RAGW) was prepared based on the aqueous geochemical composition of site groundwater [which has been described elsewhere (Williams et al., 2011 (link); Fox et al., 2012 (link))]: 7.7 mM NaHCO₃, 0.4 mM KCl, 4 mM MgSO⁴.7H₂O, 4.8 mM CaSO₄, and 2.6 mM NaCl. As the RAGW did not include a source of N or P, these would have to be provided by the aquifer sediment, as is presumably the case under in situ conditions. The basal solution (excluding NaHCO₃) was made sterile and anaerobic by autoclaving, immediately followed by purging under filtered, anaerobic 90% N₂—10% CO₂, using methods described previously (Beller et al., 2012 (link)). Anaerobic and sterile sodium bicarbonate (1 M stock solution) was prepared separately in a serum bottle, as described elsewhere (Beller et al., 2012 (link)). The bicarbonate stock was added to the artificial groundwater basal solution in an anaerobic chamber (Type B, Coy Laboratory Products, Inc., Grass Lake, Mich.) when both solutions had cooled. The final pH was 7.03. Highly purified water (18Ω resistance) obtained from a Milli-Q Biocel system (Millipore, Bedford, MA) was used to prepare all aqueous solutions described in this article.

The solvents used were acetonitrile ≥99.9% v/v (Fisher Scientific, Glee, Belgium), methanol ≥99.9% v/v, hexane ≥95% v/v, ethanol ≥99.9% v/v, and chloroform ≥99.9% v/v (Sigma-Aldrich, Bellefonte, PA, USA). Deionized water (DI) was purified through a Milli-Q Biocel system (Millipore, Burlington, MA, USA).
Various combinations of mixed solvents were prepared and used, such as acetonitrile and ethanol (1:1 v/v), acetonitrile and water (1:1 v/v), ethanol and water (1:1 v/v), and acetonitrile and ethanol and water (2:2:1 v/v/v).
Individual external standards were purchased from Merck-Sigma Aldrich Co. and included palmitic acid ≥99% w/w, linolenic acid ≥99% w/w, linoleic acid ≥99% w/w, oleic acid ≥99% w/w, arachidic acid ≥99% w/w, cholesterol ≥99% w/w, p-benzoquinone ≥98% w/w, 2-methyl-p-benzoquinone ≥98% w/w and 1-pentadecene ≥98% v/v. In addition, n-alkane standard C7–C40 (1000 mg/mL in hexane) was purchased from (Supelco, Bellefonte, PA, USA).
The 50/30 µm divinylbenzene/carboxen/polydimethylsiloxane (DVB/CAR/PDMS) SPME fiber with a 2 cm coating was supplied by Sigma-Aldrich, Bellefonte, PA, USA. Prior to use, the fiber was activated according to the manufacturer’s recommendations by exposing the fiber’s coating to 270 °C for half an hour.