I am using UPLC system (Water ACQUITY UPLC H-Class System) to determine carbohydrate in biomass. However, the maximum temperature of column’s heater is 65 c.
I ran the system today, in order to get glucose standard profile (concentration of glucose 0.2 mg/ml, 4 mg/ml, 10mg/ml) with water as a mobile phase, 0.33 ml/min flow rate (pressure of column in this flow rate was about 700psi), 10,20,25,30 µL injection sample, 70 min running time and 65 c, but there was no peak on the profile, it was just a straight line at different injections and concentrations.
By the way, since increasing of the flow rate lead to increasing the pressure of column (maximum pressure of column 800 psi according to the user guide), I also was not able to do that.
I would appreciate for any recommendation. Thank you.
Hi Majid,
I think that your problem is related to the mobile phase. You only use water? Ideally you should acidify your mobile phase in order to get retention of sugars in your column.
Best regards.
Good day dear Vitor. Yes the mobile phase is only water and I can not use acidity compounds, because the column is packed with calcium (Ca++) , therefore, the presence of cations such as Na+ or H+ in mobile phase will cause a performance loss in the column.
In my opinion this problem related to temperature and flow rate, but I dont know what to do!
anyway thanks for your answer.
In that case are you sure your column is suitable for sugar analysis?
yes, I am. it is specified for sugar analysis. My column is equivalent to the Bio-Rad Aminex HPX-87C HPLC column. and I am following the Technical Report NREL/TP-510-42618 Revised July 2011, which it attached as follow.
The conditions you are trying should produce a visible glucose peak. There is a small chance that glucose would elute after 70 minutes, but I doubt that. I would suggest double checking the injection system and the detector more than the column and eluant.
For the detection of monosaccharides you're most likely using the wrong column after all. Your column is equivalent to an Bio-Rad Aminex HPX-87C as you say, but an Bio-Rad Aminex HPX-87H column is more suitable in this case, since you can acidify your mobile phase (like 5mM of sulphuric acid for example).
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