Direct analysis of gramicidin double helices using packed column supercritical fluid chromatography

Direct analysis of the monomeric and four double helical dimeric conformations of gramicidin has been achieved using packed column supercritical fluid chromatography (pSFC). Using a PRP-1 polymeric column and typical conditions of 40 °C column temperature, 25 MPa column pressure, and 35% n-pentanol modifier addition, all of the gramicidin conformers were readily separated. To evaluate the method, the dynamic characteristics of the monomer and dimer species were monitored as a function of solvent type, incubation time, solvent temperature, and initial concentration. The findings agree with those previously obtained by other methods but also yield new information about the relative amounts of two closely related dimers (species 1 and 2) as well as the simultaneous changes in the full dimer/monomer distribution. Results indicate that the developed pSFC method can be an informative complimentary tool for readily monitoring changes in the full profile of gramicidin species present in different environments.