I have synthesized a sensor, which has sulphonic acid grp (-SO3H) and Boronic acid as well in it. And has a Molar mass of around 899 and also has an amide bond in it.. I am using Reverse phase HPLC to purify using ACN and Water(0.1%) as mobile phase. As I separate the compounds of one peak, the next very day it is splitting into two peaks in the chromatogram. Is TFA creating a problem? I could not figure it out. Should I need to use a buffer as an additive instead of TFA? Which buffer and how much is good for use? Could you please suggest?
Hello, better with anion suppression Ion chromatography with NaCO3 and NaHCO3 mix 0.25M in water.
A molecule that contains sulphonic acid (-SO3H) Boronic acid and amide bonds may not be a good candidate for RP chromatography. Weak cation exchanger resins and IEX application, or diol phases (maybe bare silica) coupled with HILIC application would also be efficient. Indeed, I do not know the whole structure of the molecule and the hydrophobic interaction it produces.
Sharing a chromatogram could be beneficial.
In RP, TFA acts as a weak ion pairing agent and it improves the resolution. Remain the TFA at both phases at 0.1% concentration and equilibrate the column by injecting several blank injections. Then test your sample. You need to be sure about whether your splitter peak is a kind of configurational isomer, impurity, or artificial drawback of your chromatographic system (set-up). Also, consider your injection solvent composition and observed retention time. Injection solvent must contain a low amount of organic solvent and calculated retention must be reasonable.
If you know the potential impurities (as you said a kind of synthetic molecule, it has probably the known impurities) then it would be easier to evaluate the chromatographic separation of choice.
Good luck,
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