Most reproducible results are obtained by weighing the mobile phase mixtures. For water / ethanol 30/70 (v/v) 300.0 g (300 ml x 1.0 g/ml) water are mixed with 546.0 g (700 ml x 0.78 g/ml) ethanol.
Especially when working with alcohols as polar modifier, online mixing by the HPLC pumps should be avoided, as the viscosity of the components varies significantly and hinders proper blending.
Mobile phases should always be prepared fresh, as evaporation will alter the composition of a two component mobile phase.
A difference of 5°C can render +/- 10% shift in retention times. It is therefore recommended to work with a column oven even when working under ambient temperature.
The retention time of acidic and basic analytes depends upon the pH value. 0.1 pH unit can render a shift in retention time by 10%. It is therefore recommended to work at a pH value at least 2 pH units from the pKA.
It is recommended to equilibrate reversed phase columns with at least 10 column volumes, for buffered mobile phases with at least 20 column volumes before an analysis is initiated. Make sure all mobile phase channels are purged with the adequate mobile phase. Normal phase columns can require up to 10x longer equilibration times than reversed phase columns. Bare silica columns might take hours to equilibrate, special attention should be given to the water content in the normal phase systems. Most reproducible results are often obtained when deliberately adding 0.05% water the mobile phase.
Highly polar mobile phases can be expelled from the hydrophobic porous system, leading to less and less accessible surface area, and therewith causing shorter and shorter retention times. See also: "Can the column be operated with 100% aqueous mobile phase".
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