Rigorous Optimization for Gradient Runs

The optimization by gradient operation starts with a linear gradient over the whole range of 100% water to 100% organic. If the solubility in different solvents is unknown, methanol or acetonitrile can be taken as starting solvents. Gradient time is adjusted to 10-15 fold the dead time of the column. If the target solutes elute at retention factors 1 the solutes are either polar or they ionize in aqueous solvents. Here, the interaction of polar solutes with the nonpolar adsorbent is very low and a polar solvent with low interaction (e.g., 100% water) is recommended. Ionization 

If the retention time is much higher than the time of the initial gradient (retention factor S 10) the substances seem to be too lipophilic for RP separation on the initial adsorbent. The polarity of the adsorbent should be increased by the use of amino or diol phases. If the adsorbent is already polar the phase system must be changed to NP-chromatography (Section 3.2.4). 

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