Can I operate my column with a 100% aqueous mobile phase?
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Reverse phase columns should always be operated with at least 5% organic modifier, unless otherwise stated. Under 95 to 100 % aqueous conditions, the columns are likely to undergo dewetting/phase collapse which means the mobile phase will be expelled from the porous system due to surface tension. The problem usually occurs when the flow is stopped. If a column has been dewetted, it can usually be restored by pumping 10 to 20 column volumes of pure Methanol/Acetonitrile or a mobile phase containing a minimum of 50% organic modifier. This phenomenon is extremely frequent with C18 and C8 columns and should also be avoided with every other reverse phase columns in order to assure robust chromatography.
In fact, the C8 and C18 chains form a very hydrophobic layer so when the organic content of your mobile phase is under 5% its surface tension increases to a point where the mobile phase can’t no longer wet the interior of the pores and that obviously shows up as a reversible loss of analyte retention. Reverse phase HPLC columns with low carbon loading (C6, C4, Phenyl, PFP etc.) columns have, on the other hand, always been able to tolerate lower concentrations of organic without any shift of retention.
Because conventional C18 HPLC columns show poor performances in these high aqueous conditions, a new class of RP-AQ phases, that show unaltered performances in these harsh conditions, are now available on the market. Their versatility proves them an ideal choice for routine HPLC analysis as well as for polar samples analysis in 100% aqueous conditions.