Applicability of alkyl‐bonded ultra‐pure silica stationary phases for gradient reversed‐phase HPLC of folates with conventional and volatile buffers under highly aqueous conditions

Applicability of several alkyl-bonded silica stationary phases was tested for gradient RP-HPLC of folates under highly aqueous conditions. High retention of folates was achieved on alternative phases with enhanced polarity and classical phases with higher carbon content. Phases exhibiting polar secondary interactions were found to provide better selectivity for late-eluting folates, whereas selectivity for early-eluting folates was mostly dependent on hydrophobic interactions. Best selectivity in phosphate buffered mobile phase was achieved on polar-endcapped silica phases (Aquasil C18 and HyPurity Aquastar) followed by alternative Atlantis dC18. Classical phases exhibited poorer separation of 10-formyl-folic acid and 5-formyl-tetrahydrofolate, but it could be considerably improved by increasing the buffer pH. Strong secondary interactions of ion-exchange character on polar-embedded phases resulted in marked peak deterioration, loss of recovery and dramatic changes in retention behaviour for early- and late-eluting folates when changing the mobile phase composition and pH. Therefore, polar-embedded phases such as HyPurity Advance were found to be unsuitable for separating folates. Stationary phases exhibited peak deterioration when using volatile buffer of low ionic strength. Better results were obtained with classical phases, whereas alternative phases showed not only peak deterioration but also a decrease in recovery and poorer selectivity due to increased secondary interactions in volatile buffer.