HPLC+RI Detector for Sugar Separation

[vanbeld]

I'm in an undergrad quantitative analysis course and we just recently created standard curves of fructose, glucose, and sucrose through HPLC with an RI detector. Our lap does not say what our stationary phase is (although it mentions sugars will be separated by charge/polarity). Our mobile phase was acetonitrile:water 80:20, so I assume this is reverse phase LC.

We ran 3 different concentrations of each sugar and then ran an unknown. We're supposed to construct a standard curve, but my chromatogram has 2 peaks for each standard.

-Will our mobile phase generate a peak on the chromatogram? We made our mobile phase that we used to dilute our samples, if we made a slight concentration difference in the mobile phase, would a peak appear?

-I read in another post that anomers create the two peaks, but does sucrose have an anomer?

I noticed that the line between sucrose peaks hits the baseline, while fructose and glucose do not...

I just have no idea how to analyze this and generate my standard curve (if only one peak represents my sugar, which peak do I choose?)

I'm aware of over injection of sample, and column void space, but I doubt either of those are creating the problem...

[Rob Burgess]

Sounds like this method is being run on an amino NH2 column - if you search down the topics list you will come across further details much like your own.

The basic environemnt at the stationary/mobile phase interface should stop you seeing two anomer peaks for each sugar. So not sure why you are seeing two? it could be that you are running a C18 column, in which case it might not be appropriate for sugar analysis.

Your mobile phase per se will not produce a peak but could have an impact on the performance of your separation - an earlier post explains what concentration of AcN you could get away with (about 50% with a 10µL injection).