Abstract Previous paleoenvironmental studies reported the δD values of a mixture of coeluting alkenones. Here, we present a semi-preparative normal-phase high-performance liquid chromatography–mass spectrometry (NP-HPLC–MS) method for purifying long chain (C37 and C38) unsaturated methyl and ethyl ketones (alkenones) on the basis of chain length and degree of unsaturation. The method was applied to purify alkenones in suspended particles and surface sediments from a site in Chesapeake Bay, eastern USA. The hydrogen isotopic composition of di- and triunsaturated C37 and C38 alkenones differed significantly on the basis of chain length and the degree of unsaturation, demonstrating the importance of gas chromatography–isotope ratio-mass spectrometry (GC–irMS) analysis of individual alkenones for accurate paleoenvironmental reconstruction. Constant fractionation factors between alkenones with different chain length but the same degree of unsaturation ( α C 37 : 2 – C 38 : 2 and α C 37 : 3 – C 38 : 3 = 1.01 ) and those with the same chain length but different degree of unsaturation ( α C 37 : 2 – C 37 : 3 and α C 38 : 2 – C 38 : 3 = 0.97 ) in all samples suggest that the values may represent hydrogen isotope fractionation associated with elongation and desaturation during alkenone biosynthesis.