What was the treatment given to the sample before injection? Injection volume? What was the catalyst used for glucose dehydration? 

You should increase the temperature of analysis to ensure that the HMF is liquid, start at 60°C. At low temperature even the column will promote HMF precipitation. Since HMF is nonionic you can have only water as mobile phase. 

I only dilute the reaction mixture to the final concentration of less than 0.001M and centrifuge to remove the catalysts. In the publications people use 5 mM sulfuric acid as mobile phase, that is why i choose it. The catalyst is carbon material.

If it precipitates, than it could also precipitate in the connections leading to the column or in the syringe of the autosampler....These parts we can not heat up....

Hi Katarzyna,

Yes its possible for HMF to polymerize under specific conditions. But I doubt in your situation you have in-situ polymerization during the analysis. Have you considered changing the flowrate of the mobile phase? You can try 0.6ml /min at 60 degs.

I hope this helps.

Regards

No. I haven´ tried higher flow rate as we are having problems with increasing pressure on the column during the analysis. At the moment, with 0.5 mL/min, the pressure of the column surpasses the limit. We do not have the heating equipment to heat up the column...The HMF can be seen in our analysis on the UV detector, but for some reason the system gets blocked with it. What is the reason for heating up the column apart from decreasing the retenstion time?

Thanks in advance

Omar suggested to ensure HMF is liquid instead of become solid, I don t now the fusion point of HMF...but I think that heating up can promote polymerization.

Increasing temperature will favor the hydrophobic interaction between the stationary phase and hmf. Is the pressure high if you bypass the column?

Dear Kasia

try that the quantification with NMR......the resonances of HMF are fairly outside the region of glucose resonances!

If you want so, you can send me a sample to Empa.

Best regards and good luck

Dani

Thanks for the suggestion Dani, I have other products in the mixture apart from glucose and fructose that I can not identify. Some of them can overlap with HMF, which could be a problem. Anyway, I would be interested to send you a sample for products identification, but it is in ultrapure water..

Kasia

 The pressure without column drops from 90 bar to 14 bar, without pre-column it drops to 1 bar. The problem is that it looks like the HMF is staying on the column and I would like it to come out from the column. So if the heating up improves the interaction with stationary phase than it would accomodate on the column even more, wouldn't it?

The problem is the temperature, if HMF is in solution in your sample but close to its fusion point when it enters the column the precipitation process will be favored due to the hydrophobic nature of the stationary phase. 

Thanks for your help Omar. Is it that with higher pressure on the column, the melting point of HMF will increase so in room temperature it will have a tendency to precipitate?Correct? So heating up the column should solve our problems...

Yes and no, the high pressure is most likely being caused by the precipitation of HMF. I recommend that you wash (also regenerate)  the column according to the manufacturer and then run standards at high temperature, if column is fine you should not have any problems

Thanks Omar. We have tried to wash the column by heating it up in a water bath and running the eluent overnight, also in reverse flow with no luck. Now we have changed for a new column of the same kind and we are scared to ruin this one as well . That  is why I am asking for the maximum concentration of the HMF that could be analysed... Maybe the concentration that we used was too high, becouse we also had problems with autosampler (needle blockage) and with the connecting tubing.

Start with very diluted solutions and work your way up. 

Thanks again

Hi Kasia

ultrapure water is not a problem for NMR......the question is, how concentrated is your stuff!? Perhaps you send me 2 or 3 samples of interest (1 mL each) and I try what can be figured out, you know my address! Perhaps you should inform me by e-mai as soon as you sent of the samples.

All the best

Dani

HMF is often measured using reversed-phase HPLC, there are many publications on the subject, maybe you want to try that instead of the organic acids column ??  Here are some publications:-

doi:10.1016/j.talanta.2005.08.003

http://dx.doi.org/10.1016/j.foodchem.2013.07.135

http://dx.doi.org/10.1016/j.talanta.2013.11.081

I have a GC FID, can I quantify HMF with it in juice concentrate? Which protocol recomend and the concentration of standard solution to use?