Could it be that the later time points show lower absorbance simply because there's less left to release? This can be taken into account with some methodological variations to still perform a meaningful study.

The persistent decrease in absorption of released organic acids from your hydrogel system, despite regular media replacement, likely stems from multiple interrelated factors that require careful investigation. The hydrogel's polymer network structure may be too dense, physically trapping organic acids through excessive crosslinking or hydrophobic interactions, particularly if you're using synthetic polymers like PAAm or PVA. This issue can be addressed by modulating crosslinker concentration or incorporating more hydrophilic monomers to increase mesh size and enhance diffusion. The chemical nature of your organic acids also plays a crucial role - those with higher molecular weights or greater hydrophobicity will naturally diffuse more slowly, while charged species may interact electrostatically with oppositely charged groups in the hydrogel matrix. For accurate measurement, it's essential to validate your analytical method's sensitivity to concentration changes and rule out acid degradation during experiments through proper sample stabilization. The replacement media itself could be problematic if its pH or ionic strength differs significantly from your hydrogel environment, potentially causing swelling/deswelling that alters release kinetics. Consider characterizing your hydrogel's swelling behavior under experimental conditions using gravimetric analysis or FTIR-ATR spectroscopy. Mechanical factors like hydrogel thickness and surface area-to-volume ratio significantly impact release profiles, where thicker samples will exhibit slower apparent release rates. A systematic approach combining hydrogel formulation optimization with rigorous characterization of acid release kinetics under controlled conditions should help identify and resolve the underlying cause of your observed absorption decrease.

Thank you for your suggestions. One thing which I have noted is drug release is less compared to the dilution which is happening due to the addition of fresh media after every reading. From the absorbance I got the concentration of the drug release and I added the concentration of the previous to the current for a cumulative concentration.

A decreasing absorbance in your hydrogel organic acid release, despite media replacement, likely stems from limited acid diffusion, acid degradation, or assay inaccuracies. To fix this, optimize hydrogel properties for better release, verify acid stability, and consider more specific quantification methods like HPLC.