For the conjugated linoleic acids (CLAs), we present the effects upon retention of the progressive addition of acetonitrile (ACN) to hexane-based mobile phases that contain background concentrations of acetic acid (AA) of 1.5, 2.0, and 3.0% by volume. An analysis of the variation in resolution across the range of elution conditions leads to unambiguous conclusions about the optimum conditions for separation and quantitation. Lower AA concentrations (≤ 1.5%) combined with low to moderate amounts of ACN (0.005–0.03%) yield good and uniform resolution amongst the major group of cis,trans/trans,cis (c,t/t,c) isomers that are believed to contain the demonstrated, desirable biological effects. For this range of %ACN, these major isomers elute around 22 and 16 minutes, respectively, and in the latter case the isocratic analyses are completed in 24 minutes. Extrapolations of the two sections of the linear dependence of the log k ′ versus %ACN plots back to zero %ACN yield data points for each background %AA that are compared with the directly measured retention of the CLAs in the presence of only AA as the strong solvent. The upper linear sections clearly relate to total retention due to all adsorption mechanisms and the extrapolations agree with the direct measurements. The lower linear sections appear to relate to that component of retention due to interaction between the CLA π bonds and Ag+ and the extrapolation thereof to the ability of AA to overcome this adsorption. These intercepts appear to reflect the diminishing general solvent strength for elution of the CLAs due to the Ag+-π bond adsorption as %AA decreases.