Gradient Elution

[mobunvar]

Right now i developed method for 4 drugs using Isocratic elution. But 2 drugs are eluting between 3-4 minutes. When I did plasma extraction, those two peaks are masked by plasma peaks (EDTA peak). So i am planning to shift to Gradient so that i can move those two drugs.
I already tried flow rate, pH to move those peaks but still plasma peaks are interfering.

So please any one can help me how to solve the problem or guide me for gradient elution.

my drugs are acidic with pKa (ranges from 3.5 - 7) and soluble in water. Right now my mobile phase is Hepatanesulfonic sod salt with 0.05% TEA pH = 2.3 and MeOH

how can start gradient ? any guidance ...?

[Alexandre]

Have you considered non ion pairing and column chemistries, eg phenyl retains aromatics more than C18. SPE can help a lot. If you don’t want to mess with extra step of SPE, try to slow down separation at the beginning. EDTA will shoot through unretained, then your peaks that are now eluting at 3 and 4 min. You can play with pH to slow down that analytes, this should have limited effect on EDTA, but keep it ionized. If they anyway masked, you need to do SPE, that is the reason you see it in many assays, without good sample prep, you may also have all sorts of validation problems.

[carls]

What is the purpose of the anionic ion pairing agent? Could you try a cationic ion pairing agent for your acidic compounds such as tetrabutylammonium phosphate at pH 7?

[mobunvar]

@ Carls... TEA is used to to reduce peak tailing. Any how ur question was thinkable, my drugs are acidic and i should go as per ur suggetions, but can u tell me how much conc should i use...any suggestions.

[carls]

5-10mM is usually sufficient. You can play around with the concentration to adjust retention.

[HPLCaddict]

Do you really need an anionic ion-pairing reagent (heptanesulfonic acid) for ACIDIC drugs?
Do you really need TEA to reduce peak-tailing of ACIDIC drugs (usually this was used for BASIC drugs on "old" columns)?

[carlos.teixeira]

Dear Mobunvar,

Do you have any update? I am curious about your next chapter, for sharing suggestion.

Hoping the best,

Carlos de Souza Teixeira
teixeiracs@yahoo.com

[mobunvar]

@Carlos
Thank You for the concerned

Right now I am workless (no bench work) because of few technical problems and also Holidays here (malaysia) due to Chinese new year. So it may take next week to people come to solve my problem and I can start work again. I am surely update about my work....My next step will be Gradient.

I already ran Scount Gradient. So one peak among four is clearly separated where as other three were inseparable. I need to separate them.

Tq