Effect of large volume injection of hydrophobic solvents on the retention of less hydrophobic pharmaceutical solutes in RP-LC

Injection of large volumes of samples in solvents other than mobile phase composition has been proved for some less hydrophobic compounds. Thus, the retention behavior of several compounds of pharmaceutical interest (isosorbide-2-nitrate, isosorbide-5-nitrate, tropicamide, pentoxifylline, and methyl p-hydroxybenzoate) was studied by using different hydrophobic solvents (n-hexane, n-heptane, or i-octane) as sample solvents. Two types of stationary phases were used: octyl and octadecyl modified silica (both of Zorbax Eclipse type). The experiments showed a linear dependence between capacity factor of each solute and sample injection volume, up to maximum volume values of about 680 μL for C8 stationary phase and 580 μL for C18 stationary phase, when the solutes are no longer retained in stationary phase. Injection of large volumes of these hydrophobic solvents is thus possible in RP-LC with a gradual reduction of retention and peak efficiency. Two major conditions are however necessary in order to apply such an injection approach: the solutes must have a proper solubility in hydrophobic solvents and meanwhile they have to be less hydrophobic than the sample solvent in order to avoid competition with solvent molecules in partitioning between mobile and stationary phases.