The addition of Na2CO3 in TPC estimation creates an alkaline medium for phenols to get unstable, however whenever I add 2 ml of 20% of Na2CO3 to 1 ml of gallic acid extract and 0.5 ml of FC reagent (1:1). it suddenly darkens the reaction mixture to a very dark blue colour beyond the range of the spectrophotometer. what can be the cause of such intense colour generation?
The intense dark blue color observed in the reaction mixture of gallic acid extract, Folin-Ciocalteu reagent, and Na2CO3 can be attributed to the formation of excessive amounts of colored complexes. The reaction between phenolic compounds and the Folin-Ciocalteu reagent, in the presence of alkaline conditions provided by Na2CO3, leads to the oxidation of phenols and the formation of intensely colored products.
In this case, the addition of 2 ml of 20% Na2CO3 to 1 ml of gallic acid extract and 0.5 ml of the Folin-Ciocalteu reagent might have resulted in an excessive amount of alkaline medium, causing an overly intense reaction. The excessive dark blue color observed may be beyond the detection range of the spectrophotometer, making it challenging to accurately measure the absorbance.
To address this issue, it is recommended to adjust the reaction conditions, such as reducing the amount of Na2CO3 added or diluting the reaction mixture to bring the color intensity within the range of the spectrophotometer. Additionally, it is essential to follow the recommended protocols and use appropriate standards and controls to ensure accurate and reliable measurements of total phenol content.
The intense dark blue color generated by adding 2 mL of 20% Na2CO3 to the reaction mixture with gallic acid extract and Folin-Ciocalteu (FC) reagent is due to the high Na2CO3 concentration, causing oversaturation and increased pH. This can lead to unstable phenolate ions and interference with the FC reagent, resulting in an exaggerated color response. To resolve this, use a lower Na2CO3 concentration, such as 2 mL of 7.5% Na2CO3, and adjust the reagent ratios to achieve stable color development within the spectrophotometer's linear range.
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