In summary the separation is performed on a UHPLC using:
Long (250 mm) but narrow (2.1 mm) column packed with either sub-2 μm fully porous particles (FPP) or modern sub-3 μm superficially porous particles (SPP) bound with C18 alkyl chain ligands as the stationary phase.
Starts with very high aqueous, sometimes as high as 100%, care should be exercised here as not all columns are able to operate under such polar mobile phase conditions. The organic is then ramped up to mid concentrations, typically around 50%, over approximately 30 minutes.
Standard mobile phases are 0.05 – 0.1% TFA (trifluoroacetic acid) in aqueous as the polar (A) solvent and 0.05 – 0.1% TFA in acetonitrile (MeCN) as the organic (B) solvent.
This very much depends on the ID of the column, but volumetric flow rates of 200 – 300 μL/min for 2.1 mm ID columns are the norm. Wider ID columns will require higher volumetric flow rates to maintain a suitable linear velocity.
Elevated temperatures are common with 60 °C often favored.
In routine production and QC environments dual wavelengths are typically monitored – 214 and 280 nm
Each of these parameters will be reviewed in the following section so their impact and optimization is fully understood.