Hello, I have to develop a UPLC and a UHPLC method for a new active product soon- assay and impurities and although im very experienced with chromatography, its more from the end-user point of view ie using validated methods, running and processing samples etc. I don't know where to start with choosing the right column, isocratic or gradient, best solvent etc, are there any good guides out there to jump into so at least I have some kind of background to it and start creating new methods, test Resolution, sensitivity, peak shape etc?
Also, obvious question here but is it better to run a fixed concentration of standard and run a sample to confirm you are recovering the total amount? How do you know that your peak areas etc match up to what you injected? Thanks.