If Analyte Structure Is Known

Determine if analytes are acidic, basic, or neutral. This will allow the chromatographer to choose a pH such that the analyte is being analyzed 

Once the probe gradient is run, check the diode array purity and if LC-MS is available, run as well to check for peak homogeneity. If you have any known precursors or impurities, run them as well to ensure resolution from the main component and to make sure they are adequately retained. The main analyte should elute between k 2-5. If the main component elutes at A = 2-5 and is spectrally pure and the impurities all elute k 1, the method is complete. If the retention factor of the impurities is below 1, then an isocratic hold at the initial organic composition should be implemented until the minor component (impurity) elutes k 1 and then a linear gradient can be implemented. The method could be further optimized by increasing the flow rate as long as the backpressure limitation of the system has not been reached. A general rule of thumb is that the backpressure should not exceed 85% of the maximum backpressure for a particular HPLC system. 

If resolution is not achieved between a critical pair, the use of a shallower gradient can be investigated. If that does not increase the resolution, then a longer column (15-cm column, packed with 3-pm particles of the same stationary phase type) should be used with a reduced flow rate of 0.7 mL/min (due to backpressure limitations). 

Note that multiple pH values and columns can be screened in gradient mode at the same time as well. This will increase the efficiency/probabifity of obtaining the best column/conditions and the best demonstrated chromatographic selectivity. Note that the aqueous phase pH values that would be chosen for these pH/column screening studies should be based on knowledge of the physicochemical properties of the molecule, taking into consideration the mobile-phase pH and analyte pKa shifts in the hydro-organic media. 

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