I am working on quantification of indole 3acetic ACID produced by endophytic bacteria, buy HPLC, it's resolved on reverse phase C18, with a solvent gradient program:
solution A: 10% methanol , 0.3 % acetic acid
B: 90% methanol ; 0.3 % acetic acid
time 0min ; 40%A 60%B
5min: 40%A 60%B
20min 0% A 100%B
I detect my peak before the gradient eluants, with a retention time of 3.5min? What is the explanation? I need help.
In acidic medium probably indole acetic acid is positively charged and usually positively charged species elute very fast from analytical columns in RPLC, usually faster than their non charged parents. Probably due to the negative potencial of the surface.
Try to avoid acetic acid in your mobile phase or diminishing their concentration on the mobile phase. Increasing the initial percentage of water (about 50%) and using low temperatures (about 15 or 20ºC) can also increasing the retention time of such an analyte.
It could mean that you analyte is simply too hydrophilic and goes a bit earlier then you expected. If you are sure, that it is your compound at 3.5 min and not just impurities or so, then simply adjust flow rate, reduce methanol content in eluent, i.e. reduce amount of solvent B. If it is still not enough and it is coming too early for you, try HILIC methods.
In the same time, your compound have a hydrophobic part, it means that it can come later than 20 min which you stated. Just wash column for about 10 min with 100% B and look if something is coming.
In acidic medium probably indole acetic acid is positively charged and usually positively charged species elute very fast from analytical columns in RPLC, usually faster than their non charged parents. Probably due to the negative potencial of the surface.
Try to avoid acetic acid in your mobile phase or diminishing their concentration on the mobile phase. Increasing the initial percentage of water (about 50%) and using low temperatures (about 15 or 20ºC) can also increasing the retention time of such an analyte.
I agree with Aurea and Marc, you are simply not polar enough with your gradient starting conditions. Therefore simply start your gradient more polar, then the retention time will increase.
Additionally check the attached file for an UHPLC method with an additional solvent system based on acetonitrile.
Good luck!
I can suggest you to Start the gradient at 10% you try this option and it'll work I think.
Good luck.
I agree with Oleg and Aurea, anyway, try starting at 0%B and decrease the amount of acetic acid from 0.3 to 0.1%. That could work. Good luck!!
Dear Affaf,
You can see our paper for quantification of indole 3acetic ACID produced by endophytic bacteria, by HPLC.
Cold-adapted and rhizosphere-competent strain of Rahnella sp. with broad-spectrum plant growth-promotion potential
Above paper will really work and solve your problem.
Regards,
Robin
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