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- Posts: 16
- Joined: Mon Aug 02, 2010 2:05 pm
Hi
Im new to Chromatography technique. I need to use HPLC for enzyme assay in our lab specialised in Glycobiology. We got new Agilent 1200 Series HPLC instrument with Fluorescence and Multiple Wavelength detectors. Im in the process of setting up sugar standard calibration curve. Since our sample is a sugar, we have to use fluorescence detection in all our experiments with derivatized sugar molecules. In setting up the sugar standard curve, we dont have ready to use fluorescent labeled sugar standards. So I labeled Maltose oligomers such as Maltobiose, Maltotetraose and Maltohexaose with 2-Aminopyridine. After labeling, to remove the excess labeling reagent, I used ion-exchange column chromatography with Dowex 50X8 resin. Fractions were collected from the Column. The fractions detected with the labeled Maltose were concentrated by using freeze drier. The problem I'm facing here is that I am not able to calculate the concentration of labeled Maltose to be injected into HPLC. Since HPLC calibration curve requires known concentration of sugar, please help me to solve this problem to make the standard curve.
Im new to Chromatography technique. I need to use HPLC for enzyme assay in our lab specialised in Glycobiology. We got new Agilent 1200 Series HPLC instrument with Fluorescence and Multiple Wavelength detectors. Im in the process of setting up sugar standard calibration curve. Since our sample is a sugar, we have to use fluorescence detection in all our experiments with derivatized sugar molecules. In setting up the sugar standard curve, we dont have ready to use fluorescent labeled sugar standards. So I labeled Maltose oligomers such as Maltobiose, Maltotetraose and Maltohexaose with 2-Aminopyridine. After labeling, to remove the excess labeling reagent, I used ion-exchange column chromatography with Dowex 50X8 resin. Fractions were collected from the Column. The fractions detected with the labeled Maltose were concentrated by using freeze drier. The problem I'm facing here is that I am not able to calculate the concentration of labeled Maltose to be injected into HPLC. Since HPLC calibration curve requires known concentration of sugar, please help me to solve this problem to make the standard curve.
