These are two completely different assays based on different chemical reactions. Not all phenols are flavonoids.

Greetings!

Suggestions related to your issue:

1. Explore and test various TFC protocols. Depending on your raw sample, different reactions may occur that can affect the final results and potentially lead to false results. Ensure that the final reaction is neither turbid nor produces any precipitation.

2. Reperform the calibration curve for both TPC and TFC.

Good luck!

It looks like the standards you use for calibration are inappropriate, do not correspond with the compounds in your sample.

Hi Sheva.

Yes, it is possible, but unusual, for the TFC value to be higher than the TPC value. Typically, since flavonoids are a subclass of phenolic compounds, the total phenolic content (TPC) should be equal to or greater than the total flavonoid content (TFC). However, there are some valid reasons this might occur, mainly related to the limitations of the methods themselves. 1) Folin–Ciocalteu (TPC): this method measures the overall reducing capacity of compounds, which usually correlates with total phenolics. However, not all phenolic compounds react equally well with the FC reagent. Some may react weakly or not at all—especially complex, glycosylated, or polymerized phenolics. In addition, the method may be affected by interfering substances like ascorbic acid, sugars, or amino acids. 2) AlCl₃ method (TFC): this method specifically targets flavonoids that can form complexes with aluminum chloride, such as flavonols and flavones. However, it can also overestimate flavonoids if other compounds in the extract can form similar complexes.

Be sure to compare the units and standards used. For example: TPC is often expressed in mg gallic acid equivalent (GAE)/g, TFC in mg quercetin or rutin equivalent (QE or RE)/g. Different standards can give different absorbance responses, so direct numerical comparison may be misleading. In conclusion: while the TFC > TPC result seems counterintuitive, it can occur due to: underestimation of phenolics in the FC assay, overestimation of flavonoids in the AlCl₃ assay, or both.