I am purifying cell samples for HPLC by SPE to analyze nucleotides and want to improve the SPE procedure to get rid of an impurity causing a broad peak in the HPLC analysis. I can get rid of most of this interfering peak by reducing the elution volume in SPE but then I also loose a significant portion of the nucleotides (especially GTP with 30% losses). I now wonder if I can improve the separation between GTP and the impurity in SPE. The SPE cartridge is 60 mg OASIS WAX (similar results has also been obtained by Varian DEA). Elution is made with 1.5ml 80/15/5 methanol/water/ammonium hydroxide. I have tried a lot of changes of the composition of the elution solution and the trend is always similar With this impurity starting before GTP is completely eluted. I therefore wonder if there is anyway to improve the resolution to get GTP out in a more narrow frame compared to the impurity (regarding flow rate etc). It is now going through by gravity but the flow rate can be reduced by adding smaller volumes at a time.
Thanks, I will try more with that then. So you do not think the flow rate can be a limiting factor for the resolution?
If you are using a flowrate stongly differing from the optimal value it will affect the resolution.
Regards
G
Ok, then I think I cannot improve the resolution by optimizing the flow rate (I use gravity flow).
When it comes to the elution strength, I do not understand it completely since it is a combination of water content and ammonium hydroxide concentration. If using pure water solutions, it is enough with 2 ml 0.5% ammonium hydroxide. If mixing 0.5% ammonium hydroxide in methanol, I only elute impurities (this is my regular washing step). It should be way higher than the pKa value of 6.5. Is it the pKa that changes so much depending on the organic content or is it the solubility of my compounds that is so much lower in the methanol solutions? I am wondering if I should keep the water content of the eluting solution and only vary ammonia or if I should vary both. I have tried to use pure water solutions with different ammonia contents and that was nearly impossible since it requires almost no ammonium hydroxide to elute my compounds.
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