That is very interesting, tried several times, but thanks god, developed good assay:Column: C18, 250X4.6 mm, 5 um.
Mobile Phase: 0.1 % H3PO4, use H2O as solvent.
Flow rate: 1.0 ml/min, 200 nm.
Today, I was asked :" Why not use 0.1 % H3PO4 as mobile phase or Why use buffer as mobile phase?
OOOPsss.. That is really good questions. Now I still try my best to answer this question in one words, however I failed all the time.
(1) Covalent attachment of the stationary phase yields a thermally and hydrolytically stable bonded phase.
(2) Hydrophobic Surface, Particle Size Distribution, Porosity, Pore Size and Surface Area.
(3) Less Polar ( more Hydrophobic) analytes are more attracted to the hydrophobic bonded phase. So more hydrophobic spends more time associated with the bonded phase... then are eluted last... That is the I always Love Methanol: it is a active solvent.
(4) Hydrophobic Theory...Partition Theory..Adsorption Theory [Chromatography of “cavities” in solvent created by hydrophobic portion of analyte molecule Surface Tension, Interaction of polar functions with solvent, Stationary phase is passive]
Cool, See that "Surface Tension" ... that is the reason why we use the Sodium Phosphate/Potasium Phosphate...
At the same time, always remember that "Water is “weak” solvent, Increased organic ---> decreased retention"
