Methods:

 

Alkaline hydrolysis/Methylation – determined chain lengths of the derived esters and amides using crude secretion and allowed for gas chromatography and mass spectrometry of 3 homologous alkanoic acids.

 

High Pressure Liquid Chromatography – revealed that secretion obtained from E. borealis contained a diverse mixture of macrocyclic polyamines.  Repeated preparative HPLC fractionation isolated the abundant trimeric, tetrameric and pentameric components of the eluting compound series. 

 

Column Chromatography – isolated the major component of the secretion as 10-(2-hydroxyethylamino)undecanoic acid, later distinguished and characterized by NMR spectra. 

 

NMR (Nuclear Magnetic Resonance) Spectroscopy – pure secreted sample from E. borealis revealed major components of secretion, which were previously unknown compounds of esters and amides derived from various alkanoic acids.