7 |
No peaks (oligosaccharides) are observed in SAX- or RPIP-HPLC and UV detection |
Enzyme is inactive |
Check for enzyme activity on a GAG standard |
Insufficient enzymatic treatment |
Extend the digestion time |
Baseline chromatogram in SAX- or RPIP-HPLC and UV detection is noisy and contains spurious peaks |
Solvent inadequately degassed |
Use an on-line vacuum degasser to the HPLC system |
Purity of reagents has been compromised |
Reprepare fresh reagents and HPLC solvents |
GAG sample was impure |
Repurify GAG sample and treat again with lyase |
12 |
Overlapping of oligosaccharides species in RPIP-HPLC-ESI-MS |
pH values of HPLC solvents are different than 7.0 |
Carefully check for pH of HPLC solvents. Reprepare fresh HPLC solvents |
Sample contains salt |
Either repeat purification and depolymerization of GAG sample or desalt oligosaccharides |
Elution time periods change or become unstable from run to run in RPIP-HPLC-ESI-MS |
The column has not been well conditioned from run to run |
Reequilibrate the C18 column(s) in solvent C for 20 min at a flow rate of 0.5 ml min−1 (for the Discovery C18 column) or 0.3 ml min−1 (for the Gemini C18 column) |
No peaks are evident by mass spectrometry |
Solvent flow is inadequate |
Make sure the solvent is flowing from the needle |
Make sure that the divert valve setting is correct |
High voltages and spay chamber currents are off |
Make sure the electrospray high voltages are switched on |
Drying gas flow is insufficient |
Check the drying gas flow and temperature |
Pressure is too low |
Make sure the fore and high-vacuum pressure are within normal ranges |