Natural polyamines are found in all three domains of life and long-chain polyamines (LCPAs) play a special role in silicifying organisms such as diatoms and sponges where they are actively involved in the complex formation and nanopatterning of siliceous structures. With chain lengths extending up to 20 N-methylated propylamine repeat units, diatom LCPAs constitute the longest natural polyamines. Mixtures of natural LCPAs are typically purified in bulk using ion-exchange, size-exclusion and dialysis and then analyzed either by direct infusion mass spectrometry or by MALDI-TOF. Here, we describe a novel ion-pairing liquid chromatographic method that allows baseline separation, detection and structural elucidation of underivatized aliphatic methylated and non-methylated LCPAs with a wide range of chain lengths. Complete separation of synthetic mixtures of LCPA species differing by either a propylamine or an N-methylpropylamine unit is achievable using this method and chromatographic separation of natural, diatom frustule bound LCPAs extracted from sediment core samples is greatly improved. Using electrospray ionization mass spectrometry (ESI-MS), we detected singly [M+H]+ and multiply [M+nH]n+ charged protonated ions. The abundance of multiply charged LCPA species increased linearly as a function of LCPA chain length (N) and multiprotonated ions [M+nH]n+ were more abundant for longer chain polyamines. The abundance of multiply charged LCPAs along with the concomitant disappearance of the singly charged protonated molecular ion significantly increases the complexity of the MS spectra, justifying the need for good chromatographic separation of complex LCPA mixtures. This analytical procedure will likely constitute a powerful tool for the characterization, quantification, as well as the purification of individual LCPAs in natural and synthetic samples for studies of silica precipitation as well as nitrogen and carbon isotopic analysis used in paleoceanographic studies. Copyright © 2011 John Wiley & Sons, Ltd.