The analyte diffuses out of the solvent plug into the mobile phase and is distributed between the mobile and stationary phases according to its hydrophobicity. In such conditions, the hydrophobic solvent is readily adsorbed in the column head immediately after injection due to its strong stationary phase affinity and low mobile phase solubility. Injection of samples dissolved in these organic solvents is related to their hydrophobicity, which is higher than that of the analytes. Water-immiscible hydrophobic solvents like alkanes ( n-hexane, n-heptane and n-octane), aliphatic alcohols (1-octanol) ( 7, 9) or more common LLE solvents (ethyl acetate, isopropyl acetate, methyl isobutyl ketone and methyl tert-butyl ether) ( 5, 10) have successfully been employed as sample diluents for direct high or moderate volume injection in RP-LC. This possibility generates both increased sensitivity of the LC quantitation and increased throughput and accuracy of LLE procedures, due to elimination of the steps for solvent evaporation and dry residue re-dissolution ( 8). Several recent studies showed the possibility of injecting large volumes of hydrophobic solvents in RP-LC that are not compatible with the mobile phase without significant negative effects on retention, efficiency, width or symmetry of analyte peaks ( 6, 7).
These steps increase the duration of the sample preparation procedure and are prone to inducing errors in the analytical quantitation. LLE generally requires evaporation of the extracting solvent, followed by re-dissolution of the dry residue in a solvent compatible with the RP-LC mobile phase. Common solvents like benzene, toluene or hexane, highly non-polar in nature, are routinely used in LLE sample preparation. Sensitivity requirements for trace analysis with common spectrometric detection in LC are often addressed by increasing the injection volume of samples from liquid–liquid extraction (LLE). Additionally, poor chemical stability in the aqueous media of certain molecules requires the use of aprotic and non-polar solvents ( 5) as sample diluents in RP-LC. Often, poor solubility of various non-polar analytes in aqueous or polar organic solvents requires the use of hydrophobic solvents for dissolution. Otherwise, phenomena might occur like band broadening, fronting, tailing or other peak distortions ( 3, 4). Ideally, sample solvents should be water or the mobile phase itself. Generally, when injecting moderate to large sample volumes in reversed-phase liquid chromatography (RP-LC), the diluents should be compatible with and weaker than the mobile phase ( 1, 2). The critical conditions for a successful large volume injection of analytes dissolved in studied hydrophobic solvents are for the analyte to have lower hydrophobicity and for the specified solutes to have proper solubility. A very small retention decrease was observed when changing the sample diluent in the homologous series: benzene, toluene, ethylbenzene and propylbenzene. A moderate peak efficiency decrease was also observed, but peaks of model analytes remained undistorted with minimum band broadening up to 100 µL injection volume. A linear decrease in analyte retention factor was observed with an increase of injection volume in the range of 1–100 µL. Both hydrophobic interaction and π–π stacking account for the competitive interaction of both the diluent and model analytes with the phenyl-hexyl phase. The same possibility is investigated for a phenyl-hexyl stationary phase using aromatic solvents (benzene, toluene, ethylbenzene and propylbenzene) as diluents for several model analytes also containing aromatic rings. The use of a large volume injection of hydrophobic solvents as diluents for less hydrophobic solutes has already been proven for C18 and C8 stationary phases in reversed-phase liquid chromatography.