I am using Agilent 1200 series HPLC to measure Xylose with the Aminex 87H column using 5mM H2SO4. When I am injecting Xylose standard,molecule is not retaining. No peak is there. In the next run when I am injecting the mobile solvent, I am getting peak for xylose. I cant understand the issue. Changing the flow rates it was not solved.
Pressure is 75bar, 0.6mL/min flow rate and 50'C temperature are my parameters.
Pradipta Patra It looks like isocratic elution. How did you decide the eluting condition? I recommend check what occur when you inject your sample and flow mobile phase longer 2 or 3 times.
Your column volume is about 14,3 mL?
After 25-40min ( wait atleast 1 hour for experiment) you should eluate all component after injection, i suppose
You should check solubiliy Xylose in mobile solvent and injection valve ( may be you shuold purge it)
I think you are using the wrong column type. It should be Aminex 87Ca which is the Calcium form. The column you are using was developed for organic acids. See the Biorad website!
Mr. Patra,
As "obvious"as it sounds like your peak is eluting too late to come out in the first run and finally showing up in the second run. Sounds crazy, but it's happened many, many times.-Paul
It does sound like your run time may be too short and the sample is eluting in the second run. Change mobile phase to reduce retention or extend run time to include the peak. Make sure your RID is set to the same temperature as your column. 50C seems too high, perhaps 35C or 40C would be more appropriate?
Bruce: The Aminex HPX-87H column IS suitable for carbohydrate analysis.
Can you include a chromatogram, injection volume and sample concentration ?
See the attached article below:
Article Quantification of glucose, xylose, arabinose, furfural, and ...
Something is not right: column type and length/method run time/gradient/column temperature - check these. It should not be very difficult to fix. Chromatography can always be manipulated according to the needs with the right tools.
If other factors are okay, I think you may run mobile phase for longer before next sample run. Again you may wash your column before the whole run process.
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