Chromatography Forum

Dear all members

I am going to use hplc instrumentation (RPLC to be more specific) and its first time that I am going to setup a method from the beginning .I dont have experience on that as I am graduated analytical chemist without professional experience. I plan to run a trial sample before my real one and try get familiar with my instrumentation which is a HPLC ultimate 3000 dionex. My goal in general is check the purity of my sample ( protein ) and I have to use a blank and reference standard (10μg/ml). My question is about reference std. Should I make 3 different concentrations (ex.3,5,10μg/ml) so to have 3 injections of that making a calibration curve ? my steps are not clear and I am not sure how I have to think on that. what i have until now is:
blank (1 injection)
reference std solution (10μg/ml) ,3 injections
sample

You will first need to develop a proper HPLC method which is selective for the protein, retains the sample on the column and elutes it all off the column using good guidelines. Have you determined if SEC, RP-18, IC or other modes of chromatography are most applicable to your sample? For purity, you will need to know what other compounds may be present so you can select and develop a method that would resolve them, if present, along with your sample. This is an advanced method and we have not even scratched the basics yet. I hope you have some help with this!