Notes from my research:

To determine the total anthocyanin content of aqueous extract C.ternatea flower, you can use the pH differential method, which is a simple and widely used technique for anthocyanin analysis (REF 1/2). This method is based on the fact that anthocyanins change their color depending on the pH of the solution. At acidic pH (around 1), they are red, while at neutral or slightly basic pH (around 4.5), they are purple or blue (REF 3). By measuring the absorbance of the extract at two different wavelengths (520 nm and 700 nm) and two different pH values (1 and 4.5), you can calculate the total anthocyanin content using this formula:

Total anthocyanin content (mg/L)=(A520​−A700​)pH1​−(A520​−A700​)pH4.5​×MW×DF×1000​ / ϵ×L

where A520​ and A700​ are the absorbance values at 520 nm and 700 nm, respectively; MW is the molecular weight of cyanidin-3-glucoside (449.2 g/mol), which is used as a reference compound; DF is the dilution factor; ϵ is the molar absorptivity of cyanidin-3-glucoside (26,900 L/mol/cm); and L is the path length of the cuvette (usually 1 cm) (REF 3).

To perform this method, you need to prepare two buffer solutions: one with pH 1 using potassium chloride (KCl) and one with pH 4.5 using sodium acetate (CH$_3$COONa). The exact concentrations of these buffers may vary depending on the source, but one possible recipe is:

• For pH 1 buffer, dissolve 0.2 g of KCl in 100 mL of distilled water and add 0.1 N hydrochloric acid (HCl) until the pH reaches 1.
• For pH 4.5 buffer, dissolve 0.4 g of CH$_3$COONa in 100 mL of distilled water and add 0.2 N acetic acid (CH$_3$COOH) until the pH reaches 4.5.

Then, you need to dilute your aqueous extract with these buffers in a certain ratio. Again, this may vary depending on the source, but one possible ratio is:

• For pH 1 dilution, mix 0.5 mL of your extract with 24.5 mL of pH 1 buffer.
• For pH 4.5 dilution, mix 0.5 mL of your extract with 24.5 mL of pH 4.5 buffer.

This means that your dilution factor (DF) is equal to 25/0.5​=50. After diluting your extract, you need to measure the absorbance of both solutions at 520 nm and 700 nm using a spectrophotometer. Then, you can plug in the values into the formula above and get the total anthocyanin content in mg/L.

Here is an example with some hypothetical values:

• For pH 1 dilution, A520​=0.8 and A700​=0.1.
• For pH 4.5 dilution, A520​=0.6 and A700​=0.05.

Using the formula above, we get:

Total anthocyanin content (mg/L)=26900×1(0.8−0.1)−(0.6−0.05)×449.2×50×1000​

Total anthocyanin content (mg/L)=2690014.46−7.35​

Total anthocyanin content (mg/L)=269007.11​

Total anthocyanin content (mg/L)=0.26

According to some sources, the total anthocyanin content of aqueous extract C.ternatea flower ranges from 181.826 mg/L to 212.585 mg/L (REF 4/5). This indicates that C.ternatea flower is a rich source of anthocyanins, which are natural pigments with antioxidant and other health benefits 12.

I hope this answer helps you understand how to determine the total anthocyanin content of aqueous extract C.ternatea flower. If you want to learn more about the extraction methods, stability, antioxidant activity, and applications of anthocyanins from C.ternatea flower, you can check out these sources (1/2/3/4/5).

Gina Lauffer Thank you for the answer. The thing is my aqueous extract is in freeze-dried extract. Do i need to dissolve it first in distilled water or directly put it in the buffer? Do i have to weight it first ?

Thank you so much for the explanation! I get the picture

Good day Ashraf Hadi,

To determine the total anthocyanin content of aqueous extract C.ternatea flower utilizing the freeze-dried extract, try using these steps:

• First, you need to dissolve your freeze-dried extract in distilled water to obtain a liquid solution. The amount of water you need depends on the concentration of anthocyanins you want to measure. A common ratio is 1 g of extract per 10 mL of water (REF. 3).
• Second, you need to prepare a buffer solution that has a pH of 1.0. This is because anthocyanins are most stable and have the highest absorption at this pH (REF. 4). You can use hydrochloric acid (HCl) and sodium chloride (NaCl) to make the buffer solution.
• Third, you need to mix your aqueous extract solution with the buffer solution in a volumetric flask. The volume ratio of extract to buffer should be 1:4 3. For example, if you have 10 mL of extract solution, you need to add 40 mL of buffer solution and then fill up the flask to 100 mL with distilled water.
• Fourth, you need to measure the absorbance of your mixed solution at 510 nm using a spectrophotometer. This is the wavelength where anthocyanins have the maximum absorbance (REF. 4).
• Fifth, you need to calculate the total anthocyanin content using the following formula:

Total anthocyanin content (mg/L)=ϵ×lA×MW×DF×1000​

where A is the absorbance at 510 nm, MW is the molecular weight of cyanidin-3-glucoside (449.2 g/mol), DF is the dilution factor, ϵ is the molar absorptivity of cyanidin-3-glucoside (26,900 L/mol$\cdot$cm), and l is the path length of the cuvette (1 cm) (REF. 3).

I hope this helps you with your experiment.

ϵ×lA×MW×DF×1000​ is the formula?

Referencing the Beer-Lambert Law:

The formula for calculating the total anthocyanin content is based on the Beer-Lambert law, which states that the absorbance of a solution is directly proportional to the concentration of the absorbing substance, the path length of the light, and the molar absorptivity of the substance (REF. 11).

The formula can be derived as follows:

Let C be the concentration of anthocyanins in the solution (mol/L), then according to the Beer-Lambert law, we have:

A=ϵ×l×C

where A is the absorbance at 510 nm, l is the path length of the cuvette (1 cm), and ϵ is the molar absorptivity of cyanidin-3-glucoside (26,900 L/mol$\cdot$cm).

To convert C from mol/L to mg/L, we need to multiply it by the molecular weight of cyanidin-3-glucoside (MW), which is 449.2 g/mol. Therefore, we would have:

C×MW=ϵ×lA​×MW

To account for the dilution factor (DF), which is the ratio of the final volume of the mixed solution to the initial volume of the extract solution, we need to divide both sides by DF. Therefore, we have:

DFC×MW​=ϵ×l×DFA​×MW

Finally, to convert from L to mL, we need to multiply both sides by 1000. Therefore, we have:

DFC×MW​×1000=ϵ×l×DFA​×MW×1000

This then is equivalent to:

Total anthocyanin content (mg/L)=ϵ×l×A×MW×DF×1000

(Devil's always in the details and Appologies: I think I missed keying the X sign (between the l and A) in the original note) 😊