Separation of PA-labeled glycans was carried out on a Shimadzu HPLC system equipped with a fluorescence detector. In case of RP-HPLC, a Hypersil ODS column (C18; Agilent) was used with 100 mM ammonium acetate, pH 4.0, and 30% (v/v) methanol; a gradient of the latter (1% per minute) was programmed. The column was calibrated daily in terms of glucose units (g.u.) with a pyridylaminated partial dextran hydrolysate. For 2D-HPLC, selected fractions were reapplied to a combined hydrophilic-interaction anionic-exchange HPLC (HIAX, Dionex IonPac AS11) with an inverse gradient of acetonitrile in 800 mM ammonium acetate, pH 3.85, as previously described.32 (link) For further details refer to the Supporting Information .
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Chromatographic Analysis of PA-Labeled Glycans
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Acetonitrile
Ammonium acetate
Dextran
Fluorescence
Glucose
Glycans
Hplc
Hydrophilic interaction
Methanol
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Other organizations : BOKU University
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Variable analysis
independent variables
- Gradient of 30% (v/v) methanol, 1% per minute
- Inverse gradient of acetonitrile in 800 mM ammonium acetate, pH 3.85
- Separation of PA-labeled glycans
- Shimadzu HPLC system equipped with a fluorescence detector
- Hypersil ODS column (C18; Agilent)
- 100 mM ammonium acetate, pH 4.0
- Pyridylaminated partial dextran hydrolysate for column calibration
- Combined hydrophilic-interaction anionic-exchange HPLC (HIAX, Dionex IonPac AS11)
- Pyridylaminated partial dextran hydrolysate as a positive control for column calibration
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