Dear Sahasaha Ts,

column temperature in HPLC has the following effects:

low temperatures produce sharper peaks

correspondingly, higher temperatures lead to broader peaks (acceleration of diffusion processes in the column)

The eluent viscosity changes, this leads to changes in retention time. At higher temperatures the analytes should elute somewhat faster, the interactions with the stationary phase become more modest at higher temperatures, this also accelerates the elution.

You should work at the lowest possible temperature to obtain the best possible peak shape.

The best way is testing different temperatures, i think between 20 and 60 °C in 10° steps. Then it is your choyce. The retentiontime for HMF should be about some minutes, because degredation products are mostly smaller and eluates before HMF.

Do you have the possibility to test other stationary phase? Ion exclusion is suitable for separating ionic compounds, but electrooxidation can also produce neutral organic compounds that will not separate well with such a column. In our laboratory we are dealing with the degradation of pharmaceuticals from aqueous samples by UV oxidation, enzymatic degradation and electrooxidation, this produces many different neutral degradation products which we separate on RP phases and identify by mass spectrometry.

I hope my answer helped you, if you have any further questions, just contact me

Best regards

Joachim Horst

Joachim Horst

Dear Joachim Horst

Thankyou for your answer.

I hear that HPLC for HMF should be over 60℃ because HMF may precipitate, but I don't know that is true.

I experimented at40,50,60,65℃ , and, at 40℃and 65℃,I'm finding compounds that I can't identify.

Perhaps you mean that the peaks came out separately for ionized and non-ionized?

And,I want to know a lot about your answer " The retentiontime for HMF should be about some minutes, because degredation products are mostly smaller and eluates before HMF."

I don't think that changing the fixed phase is probably the way to go. Are ion-exclusion columns unsuitable for electrolytic oxidation? I honestly don't know why ion-exclusion columns were introduced.

In simple terms, changes to the column temperature will result in changes to the mobile phase's viscosity and the sample's SOLUBILITY IN the mobile phase (and the chemical and physical interactions it will have with the support).

As it takes a minimum of five years to attain a basic level of experience in HPLC analysis (and I do mean "basic"), please contact someone local who is experienced in practical HPLC method development to assist you with your project. Doing so will allow you to learn something about the technique and obtain useful and accurate data to move forward with.