The purification of chlorins from marine particles and sediments for nitrogen and carbon isotopic analysis

Methods are presented for the separation of chlorins from marine particles and sediments for nitrogen and carbon isotopic analysis. Carbon and nitrogen purities of 91 and 96%, respectively, were obtained for particulate chlorins, with recoveries of 88%. Sedimentary chlorins were obtained with carbon and nitrogen purities of 88 and 89%, respectively, and recoveries of 18%. The reproducibility of carbon and nitrogen isotopic analyses on particulate and sedimentary chlorins was 0.2‰. The low recovery of chlorins from sediments is primarily a result of their complex distribution, requiring that only the one or two most abundant chlorins from a sample are purified. The procedure for particulate samples includes ultrasonic extraction with solvents, two phase separations, and reversed- and normal-phase high-performance liquid chromatographic separations (HPLC). The procedure for sedimentary chlorins includes ultrasonic extraction by solvent, solid-phase extraction on silica gel, reversed-phase HPLC, size-exclusion chromatography, and normal-phase HPLC. The purification procedures require ca. 4 h for particulate samples, and ca. 8 h for sediment samples when two samples are processed simultaneously. The application of these methods to surficial Black Sea sediments demonstrated that different chlorins have distinct nitrogen and carbon isotopic ratios. The δ15N differences are interpreted in terms of changes in the seasonal flux of material out of the euphotic zone, while the δ13C differences are thought to derive from the presence or absence of the chlorin phytyl side-chain.