# Effect of Temperatures on Polyphenols during Extraction
## 7 Challenges in the Analysis of the Extractant
### Overestimation of Total Phenolic Content by Folin-Ciocalteu Assay  
We need to develop a further understanding of how the TPC, as measured by the Folin-Ciocalteu method, corresponds to the TPC measured by HPLC. It is difficult to measure TPC using HPLC, as a standard of each phenolic acid is needed to identify the peaks and quantify the area and, as a result, TPC calculated by HPLC is often much lower than that measured by the Folin-Ciocalteu method [22, 73].  
As the Folin-Ciocalteu method depends on the reducing power of phenolic hydroxyl groups to estimate the TPC, it accounts for all the phenols and their degraded products. This lack of specificity often results in an overestimation of TPC.  
According to [PERSON] et al. [47] the TPC calculated by Folin-Ciocalteu increased with increasing temperature while the measurements of individual polyphenols and total polyphenols as calculated by HPLC suggested the opposite. The various polyphenols tested were hydroxyphenyl propanoic acids (HPPA), hydroxycinnamic acids, flavones, and flavanols/flavanones. Except for HPPA, the yield was highest at 100 \({}^{\circ}\)C and lowest at 200 \({}^{\circ}\)C for all compounds. HPPA and TPC calculated by the Folin-Ciocalteu assay showed increased yield with increasing temperature. The total phenols calculated by the sum of areas of peaks in the HPLC chromatogram is lowest at 200 \({}^{\circ}\)C and highest at 100 \({}^{\circ}\)C, in contrast to the TPC measured by Folin-Ciocalteu assay. At temperatures above 160 \({}^{\circ}\)C, water is able to solubilise even lignin and hemicellulose [47]. With the increase in solubility and hydrolytic reactions at high temperatures, these compounds may break down into phenolic acids, and the breakdown of lignocellulosic may release not just phenolic materials but also reducing agents and sugars [54]. These additional compounds could also be detected by the Folin-Ciocalteu assay causing further errors in the estimation of TPCs.  
[PERSON] et al. [73] performed the quantification of phenolics (TPC) by HPLC and compared the result to the TPC values obtained by Folin-Ciocalteu assay. The study included parsley, buckthorn, mint, caraway and birch extracts and found no significant correlation between the results of HPLC and the Folin-Ciocalteu method. However, the difference between the results was not very high for parsley, mint and buckthorn. TPC in birch and parsley was overestimated by the Folin-Ciocalteu method, while it underestimated caraway. This suggests the effectiveness of measuring TPC using the Folin assay varies depending on the source material.  
[PERSON] et al. [87] found that the TPC and TFC calculated by the Folin-Ciocalteu and HPLC methods is highly correlated (r = 0.90). However, the Folin-Ciocalteu method resulted on average in a TPC 1.5-2.5 times higher than the HPLC results. [PERSON] et al. [88] found that TPC (HPLC) correlates to AOA with r\({}^{2}\) = 0.628 and TPC (Folin-Ciocalteu) correlates to AOA with r\({}^{2}\) = 0.649. Once again, the TPC (HPLC) values are on average 2.9 times lower than TPC (Folin-Ciocalteu) values, suggesting that other compounds present in the extracts that are accounted for in the TPC have little influence on the overall AOA.