Method to predict and compare the influence of the particle size on the isocratic peak capacity of high-performance liquid chromatography columns.

A kinetic plot based method has been used to experimentally predict the optimal particle size yielding the maximal isocratic peak capacity in a given analysis time. Applying the method to columns of three different manufacturers and characterizing them by separating a 4-component paraben mixture at 30 degrees C, it was consistently found that the classical 3 and 3.5 microm particles provide the highest peak capacity for typical isocratic separation run times between 30 and 60 min when operating the columns at a conventional pressure of 400 bar. Even at 1000 bar, the sub-2 microm particles only have a distinct advantage for runs lasting 30 min or less, while for runs lasting 45 min or longer the 3 and 3.5 microm again are to be preferred. This finding points at the advantage for high-resolution separations that could be obtained by producing 3 and 3.5 microm particle columns that can be operated at elevated pressures.