While using HPLC- C18 column, UV detector and derivatizing reaction with p-ABA in presence of reductive amination. The sugar standards (D-Glucose, D-xylose and D-Glactose) shows 2 different peak at different retention times. I have tried several times, but resulting same.
Will you please advise if this could be a normal phenomenon? or how can I overcome this to make standard curve of it? Thank you for your advises.
Such as a picture attached. The peak at time 35 is a labeling p-ABA and the other two perhaps resembles the Glucose substrate detection.
Hi Parveen, If you see the chromatogram the p-ABA peak got saturated due to high sample concentration. The %Area results at higher concentrations are not true. Reduce the sample concentration and then check the purity (%Area).
Thank you for the reply Sir. However, based on the reference I am the labeling are coming at right time and base area (Moreira 2010), Please see the attached figure. Also, I had tried different concentrations, but still multi peaks are occurring. I appreciate your suggestions.
The derivatizing agent must be at high concentration, so the peak before p-ABA could be an impurities. You have to check the p-ABA solution without the analytes (sugars). If the same peak occurs again it is from p-ABA.
On this way you can find which peak belongs to sugar.
Thank you dear Soleya, I had run one as a control, without any sample, it was perfectly showing only p-ABA peak, with no other band. I appreciate your further suggestions. what should be the standard concentration of the samples I run? I have used 0.5 g/L concentration.
try to reduce the sample concentration and then check the purity (mandatory]
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