I am currently using AEX-HPLC analysis with addition of 20% acetonitrile in the mobile phase (to reduce hydrophobic interactions during separation).
I cant find any information as to why the threshold is 20% organic and would like to know if I am able to use a higher percentage or alternative organic such as isopropanol.
I'd like to titrate higher amounts of organic into the mobile phase and analyse if the salt is precipitating out of solution at higher percentages of organic.
Has anyone got any advice on how to do this please?
Some times, you need to use 50% acetonitrile. It depends on the amount of buffer you added. MeOH and acetonitrile are the two common organic solvents. I didn't use isopropanol before. In summary, the best and easiest way is to prepare a small amount of final mobile phase and see any precipitation after 1-2 days.
There is a reason why MeOH and ACN are being mostly used; low UV cut-off. Unless you have a compound suggesting high hydrophobic interactions, do not spend time to optimize the % ACN.
OK, you are using anion exchange; this is a physical exchange process. You state that you are adding 20% ACN to reduce hydrophobic interactions, but your exchange process is anionic. You also don't tell us what your elution buffer is, so we have no clue as to what the "salt" that you are trying to measure is.
We also don't have a clue as to why you think you need additional organic or an alternative organic solvent in your eluant. IC typically runs fine without any solvent; there are good reasons for adding a little solvent to the IC buffer (20% is actually quite high) and really good reasons to not add too much solvent.
We don't know what your detector is. Andrei jumps to the conclusion that it is UV; I would assume that since it is AEC you are probably using some form of conductivity or electrochemical detector. We are both just guessing.
We really are hard pressed to help you if you don't post complete information. I can consult my Tarot cards if you wish, but I don't think that the answer will necessarily please you. To quote Rod's law, the more you don't tell us the more we can't help you.
Hongzhu
That's quite interesting to use 50% is that at quite low salt content in the mobile phase then?
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Mark, I am using AEX-LC for analysis of oligonucleotides, some are phosphorothioated and can display secondary hydrophobic interactions to the stationary phase. I am analysing at 260nm.
Its suggested to add up to 20% organic solvent to the mobile phase to reduce secondary hydrophobic interactions, which I have done, and it works, but not well enough for my work. So I thought about adding more to the mobile phase - but was worried about precipitation of the salt in my elution buffer (1.25 M NaCl or 0.4 M NaClO4).
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As it happens you can see visible phase separation at 30% organic, so I think I might have figured it out anyway.
Thanks for the responses everyone!
I shall also bear in mind to include more information in my future questions, sorry for the ambiguity!
I have used 2-propanol system (please see file; Lysozyme by RP-HPLC).
Solvent A is 0.1M sodium phosphate buffer (pH 2.0) and solvent B is a mixture of 2-propanol, ethylene glycol and solvent A (3 : 1 : 1 (v/v/v), pH is adjusted to pH 2.0 using usual pH meter by adding phosphoric acid). Solvent B contains 60% (v/v) of 2-propanol.
Please, use this reliable analytical system. After the work, HPLC pump should be washed by distilled water or HPLC-grade water every day.
By the way, I do not like AEX method, since it requires longer times in equilibration as compared to RP-HPLC. Gradient-elution-RP-HPLC requires usually 5 min using 5-cm length short column to return to the original condition, since gradient-elution-RP-HPLC method (like affinity method) does not require long column.
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