Validating an hplc method

Consideration must be given to the following: Types of chromatography.Reverse phase is the choice for the majority of samples, but if acidic or basic analytes are present then reverse phase ion suppression (for weak acids or bases) or reverse phase ion pairing (for strong acids or bases) should be used. For low/medium polarity analytes, normal phase HPLC is a potential candidate, particularly if the separation of isomers is required.Reverse phase gradient HPLC is commonly used in peptide and small protein analysis using an acetonitrile–water mobile phase containing 1% trifluoroethanoic acid.


p H will only significantly affect the retention of weak acids and bases.

Cyano-bonded phases are easier to work with than plain silica for normal phase separations.


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