I am trying to separate glucosamine and N-acetyl glucosamine from a filter-sterilized biological sample containing salt, amino acids, and other impurities. I used a standard solution containing an equimolar (2.5mM) solution of glucosamine+ N-acetyl glucosamine in milli-Q water. However, I am only getting one single peak of N-acetyl glucosamine (retention time 2.5 minutes). Can anyone help me with the separation process? I tried the following conditions:
Mobile phase A: 10mM sodium acetate+10 mM sodium octanesulfonate in water at pH 5.1
Mobile phase B: MeOH
Injection volume: 5 µl
Flow rate: 0.7ml/min
Column temperature: ambient
Column: Nucleodur C18 Gravity, 250x4.6 mm, 3 µm
You can't. Carbohydrates are simply too hydrophilic and don't interact with C18 columns. Either use another column which is more suitable for carbohydrates or look for a derivatization method that yields more hydrophobic analytes.
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