Glucose and phosphate identification HPLC


I need to analyse phosphate and glucose in the same analysis on HPLC I have tried using different columns such as Aminex HPX-87H Column 300 x7.8 mm, Agilent Hi-PLex H, 7.7 x 300 mm, NUCLEOGEL SUGAR 810 H, 300x 7.8 mm... but none of these manage to separate the two peaks which remain superimposed... does anyone perhaps know of a different column to use or suggestions for parameters considering the analytes? Or any suggestions considering the properties of the compounds ? 



  • Analyzing both phosphate and glucose in the same HPLC run presents a challenge due to their differing chemical properties. Phosphate is an inorganic anion, while glucose is an organic molecule, which means they have different affinities for various stationary phases and will elute differently under various mobile phase conditions. The columns you've tried, such as Aminex HPX-87H, Agilent Hi-Plex H, and NUCLEOGEL SUGAR 810 H, are typically used for carbohydrate, organic acid, or sugar alcohol separations and might not offer the selectivity needed for simultaneous separation of phosphate and glucose.

    • pH Adjustment: Adjusting the pH of the mobile phase can significantly impact the ionization state of phosphate, potentially altering its retention time and improving separation.
    • Buffer Strength: Using a buffer can help control the pH and ionic strength of the mobile phase, which can be crucial for the separation of ions and polar molecules.
    • Gradient Elution: If using a reverse-phase or HILIC column, consider using a gradient elution to help achieve better separation. The gradient can help in adjusting the elution strength throughout the run to separate compounds with close elution times.
  • Thanks for replying :) I will try to see if I change the pH maybe the retention time is also change 

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