Safe and Reliable Agricultural Production

Practical lessons

Quantitative method to determine

polyphenol content in tea

Ortega Nuñez Moises Alexander

2018 (30th year of Heisei era)

1. Objectives
a. To Determine the content of polyphenol in commercial bottled tea through interpolation
in a graph made with standard solutions of catechin.
b. To compare the quantities obtained with the ones presented in the labels so it can be
figured out which brand is sample A, B or C.

2. Materials and Equipment

- 3 samples of the three commercial bottled tea, diluted 10 times and labelled as A, B and C
- Folin-Ciocalteu reagent
- Solution of Sodium carbonate 10%
- Catechin diluted in ethanol (20% Standard solution)
- Spectrophotometer
- Glass pipets
- Bikers
- Test tube mixer
- 16.5mm Test tubes and caps

3. Methodology
a. Using the catechin standard solution (200mg/L), standard solutions of 200, 150, 100, 50,
and 0 mg/L were prepared in order to make up a graph.
b. In 5 different test tubes, 0.2 ml of every standard solution was poured.
c. 0.5 ml of phenol reagent was also poured in every test tube
d. Closing the test tubes with rubber caps, they were shaken in a test tube mixer for 10
seconds.
e. Then, 0.5ml of 10% Sodium carbonate and 2ml of distilled water were poured.
f. Once again, closing the test tubes with rubber caps, they were shaken for 10 seconds.
g. Covering the test tubes with a layer of aluminum paper, they were left to rest for 20
minutes at room temperature.
h. Then, the solutions were taken into different cuvettes, and their absorbance were
measured at 760nm in a spectrophotometer.
i. The results were used to make up a graph (Concentration in mg/L vs. Absorbance) in order
to interpolate the absorbance of next Tea samples and determine its polyphenol content.
j. Procedures from step “b” to step “g” were used with the three commercial tea samples.

4. Results
Using the results after measurement in the spectrophotometer, the next graph was
obtained.
Concentration Absorbance at
(mg/L) 760nm
0 0.077
50 0.236
100 0.333
150 0.516
200 0.582

Absorbance at 760nm
0.8 y = 0.0026x + 0.0908
R² = 0.9836
Absorbance

0.6
Absorbance at
0.4 760nm

0.2
Linear
0 (Absorbance
0 100 200 300 at 760nm)
Concentration (mg/L)

The absorbance and interpolated concentrations of the three samples of tea were as
follows:

Sample Absorbance Interpolated concentration

A 0.505 159.308
B 0.271 69.307
C 0.207 44.692
As the samples were diluted 10 times its original concentrations, the real values for the
three commercial bottle tea brands were 1593.08, 693.07 and 446.92 mg of polyphenol per
Liter. Knowing that, we can now determine the brands for each sample.

Catechin (mg/L) おーいお茶 おーいお茶 (濃い味) へルシア緑茶

Content indicated in label 400 800 1543
Measured Value 446.92 693.07 1593.08
Sample (A, B or C) C B A

5. Impressions
The quantities obtained are nearly close to the ones indicates in the labels. However, these
ones are higher, which is expected as the standard graph gives a positive value of
absorbance for a concentration of 0 mg/L. Even though the results were no as close as it
could have been expected, with more repetitions and statistical tests it could have been
proven that, statically, the differences between the commercial concentration and the real
one are not significant.
This a very simple but efficient method to measure the content of polyphenols in a liquid
product as bottled tea. Such methodology can be used then for quality control procedures
in factories that manufactures similar products.