Gradient elution mode aqueous-organic mobile phase

Step 4. Then determine what the maximum pH of the aqueous portion of the buffer should be prepared at, taking into account the pH shift of the aqueous portion of the mobile phase upon addition of organic as shown in step 4 below. Therefore the optimal pH to analyze this compound would be at aqueous mobile-phase wpH of <2.7 using isocratic mode. This pH is also applicable for gradient mode separations given that the analyte of interest will elute at 50 v/v% of acetonitrile or less. 

Several column types (silica, cyano, amino, diol) function in an NP mode when used with organic solvents, such as acetonitrile, that contain a small percentage of water (usually <20%). In this mode of operation, gradient elution is performed by ramping the aqueous content of the mobile phase. Frequently, a small percentage of an ammonium buffer (<5 mM) is needed to disrupt secondary interactions that lead to peak tailing. 

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