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 (Δ14C) 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 10,000 L) 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 10 μg each can be simultaneously separated on a C18 high‐load column using a mixture of MeOH/water and acetonitrile as the mobile phase. Our procedure introduced dead‐Cext contaminations of 0.57 ± 0.29 and 0.35 ± 0.18 μg for the high‐performance liquid chromatography and combustion/graphitization steps of the sample preparation, respectively. However, tests on different high‐performance liquid chromatography C18 columns revealed a large difference in dead Cext associated with column bleed. Modern Cext in the amount of 0.40 ± 0.20 μg was introduced by the combustion/graphitization step of the sample preparation, but other steps did not add modern Cext. The entire method recovered ∼50% of the purified compounds on average, but this did not affect their 14C content. This method will allow routine analysis of the Δ14C of PLFA isolated from groundwaters or other sample types, revealing the relationships between microbial and soil‐derived C, sedimentary or dissolved C sources. Key Points Fatty acid methyl esters derived from microbial phospholipids were purified and concentrated for compound‐specific radiocarbon analyses The method involves chemical extraction followed by semipreparative high‐performance liquid chromatography of diverse saturated FAME Large differences in the degree of extraneous C contamination were observed depending on the HPLC columns used for separations