¹H NMR Analysis of Microalgal Long-Chain Alkenones and Development of a Sequential Extraction Protocol for Alkenone...

Polyunsaturated long-chain alkenones are unique lipids produced by certain species of microalgae with both well-established (i.e., as paleoclimatoligical indicators) and emerging applications (e.g., as a renewable hydrocarbon feedstock). Traditionally, alkenone-based research has relied on gas chromatography (GC) for alkenone detection and quantification, which can require long run times (ca. 1 h), multistep sample preparation, and suffer from coelution. ¹H NMR was therefore investigated as an alternative method to complement GC for the analysis of alkenones. Using a well-resolved singlet at 2.13 ppm corresponding to the methyl group adjacent to the carbonyl (CO) on methyl alkenones, alkenones were able to be detected in the various extracts of commercial Tisochrysis microalgae, including solutions prepared by soaking the microalgae in CDCl₃. This allowed for the differentiation between alkenone- and nonalkenone-producing algae by a streamlined CDCl₃ extraction/¹H analysis procedure. The ability of different solvents and conditions to extract alkenones from microalgae was also assessed by ¹H NMR, leading to the development of a sequential extraction protocol as a new method for alkenone isolation and purification.