I want to separate our product that contains very complex components using HPLC.
First problem: it has seven active substances which polarities are ranged from most polar (vit C) to very non polar (coenzyme Q10).
Second problem: it has very different in concentration, which are ranged from 100 mg to 0.002 mg per tablet.
Based on that two problems, i divide the substance as 4 groups, that are: polar-high conc, polar-low conc, nonpolar-high conc, and nonpolar-low conc. High conc refers to the conc above 1 mg, conversely with low conc.
I decided to separate them based on their conc. This time i can analyse the 4 high conc comps. High conc comps are dominated by nonpolar comps with 1 polar comp (vit C). The nonpolar comps can be separated with gradient elution of THF-MeOH-1%TFA, but vit C can't be analyzed with this condition!
Any help?

You can either do vitamine C by HILIC (prefered) or try to retain it with a an ion-pair reagent. Have to tell you though, that I do not believe in ion-pair chromatography with gradients, since the reproducibility is not good.

You can use isocratic conditions (in terms of organic content) and verious acids as additive, check www.primesep.com. They have a column with embedded ion pairing reagent. I tried it for complex mixture (two acids, base, amide and amine and got good results)