Chromatography Forum

Looking for expert !!!
I don't have much experience in HPLC analysis. Currently, I am exploring method to analyze a hydrophilic compound, which has PH=5, I tried use TFA BUFFER(ph=3.0), : methanol: acetonitrile (40:38:12), C18 column, which successfully separate the compound, RT=2.8min, however, since like methanol has the absorption at 4.5min. I never use RP-HPLC to develop method for hydrophilic compound, do I need to change to NP-HPLC, or how to optimize method to get better separation?

Thanks!!!

what is you column dimensions and flow rate? If you column is 4.6x250 mm and flow rate is 1 ml/min your compound is not retained, since void volume is 2.6 minutes. If you compound is ionic you can use ion-exchange column or mixed-mode ion-exchange column. If you can provide more details on your compound I will advice you on what to do.

regards,

Vlad

A HILIC column would be my first choice.

Questions:
1) Does your compound have a basic group (i.e., one with a (+) charge)? If not, then TFA won't be able to form a hydrophobic ion pair with it and so it will not increase retention.
2) You say that your compound has "PH=5". How can an individual compound have a pH? Do you mean that its pKa=5? That means that at pH=5, half of the compound will be in the ionized form and the other half will be unionized. I suppose that the implication is that your compound has a carboxylic acid and so would be unionized at your pH of 3.0.
3) TFA (= trifluoroacetic acid) has no buffering capacity at pH 3.0. What concentration of TFA are you using?
4) The concentrations of methanol and acetonitrile add up to 50% of the total mobile phase. That is much too high a concentration to use here. Try concentrations more like 5% if you're using reversed-phase for a hydrophilic compound.

If you want anyone to be able to provide detailed advice, then please tell us exactly what this compound is and answer the above questions regarding the composition of the mobile phase.