I apply mobile phase stepwise elution in HPLC to separate glucose isomerization to desired fructose. However, I got unreasonable result showing reaction selectivity of fructose is larger than 1. I wonder if RID is NOT suitbale for stepwise elution? Thank you in advance!
RID systems are not appropriate for gradient elutions even it is linear, step, convex, concave, or flow gradient due to the refractivity that can dramatically change during unstable mobile phase composition flows. Elutions must be done under isocratic flow. For perfect resolution, temperature optimization and column selection is crucial. Use HILIC chromatography (ACN-Water mixture) and amide linked column applying high column temperature. Longer column lengths and Triethylamine additive to mobile phase can improve resolution. Temperature optimization can help for identifying anomer mutorotations of isomers and amide bond can prevent Schiff base formation disadvantage that could be occurred as RT shifting.
I hope this may be helpful for your research.
Good Luck,
Emir.
HPLC Refractive Index Detectors (HPLC RI Detectors) are used with high-pressure liquid https://www.biocompare.com/Lab-Equipment/13033-HPLC-Refractive-Index-Detector-HPLC-RI-Detector/
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