Glucose and phosphate identification HPLC

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 

For glucose a 87H type is ok as it uses Ion Exclusion Chromatography for the separation, the phases contain benzenesulfonic acid group (and will interact with anions in ion exchange mode). Phosphoric acid does not interact with sulfonic acid groups as it is a cation needing something like quaternary amines to interact with.

You could do a solid phase extraction first either remove the glucse by Bond Elut PBA or the phorphate with Bond Elut SAX, otherwise I see little chance with LC alone unless you use a strong ion exchange column to retain the phosphate very long and have glucose elute early.