Isolation of Individual Saturated Fatty Acid Methyl Esters Derived From Groundwater Phospholipids by Preparative High-Pressure Liquid Chromatography for Compound-Specific Radiocarbon Analyses

Determining the biogeochemical pathways utilized by microbes living in groundwater is essential for understanding the subsurface C cycle and the fate of organic compounds, including pollutants. The radiocarbon signature (Delta C-14) of fatty acid methyl esters derived from microbial phospholipids (PLFA) provides useful information for differentiating microbial C sources and infering microbial metabolism. However, in subsurface environments, those analyses remain challenging. Here we present a method combining large volume groundwater filtration (up to 10,000L) and PLFA purification for subsequent compound-specific radiocarbon analyses. The analytical method involves conventional chemical extraction of PLFA followed by purification of individual compounds by semipreparative high-performance liquid chromatography. Different saturated PLFA in amounts of up to 10g each can be simultaneously separated on a C-18 high-load column using a mixture of MeOH/water and acetonitrile as the mobile phase. Our procedure introduced dead-C-ext contaminations of 0.57 +/- 0.29 and 0.35 +/- 0.18 mu g for the high-performance liquid chromatography and combustion/graphitization steps of the sample preparation, respectively. However, tests on different high-performance liquid chromatography C-18 columns revealed a large difference in dead C-ext associated with column bleed. Modern C-ext in the amount of 0.40 +/- 0.20 mu g was introduced by the combustion/graphitization step of the sample preparation, but other steps did not add modern C-ext. The entire method recovered approximate to 50% of the purified compounds on average, but this did not affect their C-14 content. This method will allow routine analysis of the Delta C-14 of PLFA isolated from groundwaters or other sample types, revealing the relationships between microbial and soil-derived C, sedimentary or dissolved C sources.