I am working in oxidation reaction. I am facing a problem regarding product quantification. When i used water as a solvent then converted area of my reactant is not able to regenerate in product samples. Peak area stuck in first hour at very low value and kept stuck till completion of reaction. It look like reaction arrested in water. Although in solvents other than water area regenerates nicely. What could be the reason of this wiered pattern?
Hello, Bhanu. Can you describe your HPLC procedure and what kind of samples you're analyzing. That might help us troubleshoot your problem.
Thanks.
Question needs further clarification as Daniel King stated. Did you use water as MP too?
Hi Daniel King Thanks for replying. I am working on 5-Hydroxymethyl furfural (5-HMF)oxidation which give 2,5-Furandicarboxyllicacid (diacid). In my HPLC procedure mobile phase is 85% H2O(0.1%AcOH): 15% ACN. I am using 0.6mL/min flow at column temperature of 45 degree C and at lambda= 280 nm with Babd width 4. Column used by me is HI-plexH USP L17, 100x7.7 mm (agilent) whereas we are using agilent UHPLC 1260 quaternary pump. I had tried C-18 column also but the resolution of peaks was very poor and too early on time scale. We had also tried different mobile phase like 5mM H2SO4 and 70:30(water:MeOH) but we obtained best resolution and results for current mobile phase. Although peak area problem related to water as a solvent was persistent in all the cases.
Also i want to mention that when i make up the reaction crude i add NaOH in water along with my reactant and catalyst. This NaOH is required for the reaction to progress. The problem i faced arise only when base is used with water otherwise with other solvents its perfectly fine.
Hi, Bhanu. Sorry I lost track of this discussion. I do not know exactly what is going on but here are my thoughts. You are running into problems when using the NaOH. As I see on their website, the Hi-Plex 17 column is recommended with water for carbs and dilute acid with organic acids (like you have). I know you have acid in your buffer, but the NaOH in the sample may still alter the column and sample chemistry. Both the sulphonate (on the column) and the carboxylate on your compound will likely change protonation states depending on if they are in base or acid and should dramatically affect their binding affinities.
My suggestion is if you are committed to using this column, then see if you can do a cleanup step prior to injection to remove the NaOH, or at least bring the pH down. A quick solid phase extraction to switch solvents before analysis might help, or just add acetic acid to neutralize your sample. Hope you are able to figure things out.
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