Effect of brewing time on mass percentage of gallic acid in green tea and black tea extract

How does the mass percentage of gallic acid, measured in g/L in tea extracts depends on the brewing time and the type of the tea extract – green tea and black tea used, determined using acid base titration with NaOH?

The ability to think about how I think, my meta-cognition has always inspired me to be an inquirer and explore scientific facts and principles that I come across. Having the habit of being awake late in the night to complete my assignments, tea and coffee has been an integral part of my life. To save time, I usually use hot water from the water heater and the tea bags to make my cup of tea. Often, I am so engrossed in my work, that the tea bag is dipped into the water for longer than usual. This has often made me realize that the taste of tea changes when the tea bags are soaked into the water for a longer time. This has made me inquire that what can be the possible reason behind this. After some research, I came to know that tea leaves contain a phytochemical – gallic acid which contributes towards the flavor and aroma of the tea we drink. The amount of gallic acid extracted depends on the brewing time; the time for which the tea leaves are soaked into the water. Recent researches about preparation of tea shows that the brewing time plays a great role in extracting the right chemicals from the tea leaves in the right amount to obtain the best flavor and aroma of that particular tea leaves and this depends on the type of tea leaves as well. Thus, can we surely claim that more the brewing time, more the amount of gallic acid extracted from tea leaves into the water? Is the extraction of gallic acid from tea leaves into the tea extracts happening in all cases in the same rate? To answer these questions, I decided to focus my Chemistry Internal Assessment on the research question stated above.

Gallic acid is a major constituent of tea leaves. On an average, gallic acid is a major nutritional ingredient of tea leaves and constitutes of around 1% of the dry mass of tea leaves. It is 3,4,5- tri hydroxy benzoic acid. It belongs to the class of hydroxy acids or phenolic acids. It has three phenolic OH groups and an aromatic carboxylic acid group. The compound is highly soluble in water due to the presence of three OH groups which can make inter molecular H bond with water. At room temperature, this is a white solid with the molecular formula – (OH)₃-C₆H₂-COOH. Two of the OH groups are at meta positions with respect to the COOH group while the other one is at para position.

The transport of gallic acid from tea leaves into the tea extract is an example of passive transport or diffusion. The molecules of gallic acid travels from the leaves to the water along the concentration gradient. This is a spontaneous process and does not require the consumption of energy to continue it. The kinetics of diffusion depends on two major factors; the concentration gradient and the temperature at which it occurs. As the diffusion is carried out at a higher temperature, there are more gallic acid molecules with higher kinetic energy and thus higher velocity to travel from the tea leaves to water. And more the difference of concentration of gallic acid in the tea leaves and in water, higher the concentration gradient and thus more molecules of gallic acid travels from tea leaves to water.

However, one more chemical factor plays a pivotal role here. As already informed, gallic acid can make inter molecular H bond with water and this imparts stability to gallic acid and increases the affinity of gallic acid for water. This allows more gallic acid to travel from the tea leaves to the aqueous extract. This process is an entropically favored process as it has a positive sign for the entropy change. The disorderness of the system increases as the gallic acid molecules moves from tea leaves to water.

The reaction between gallic acid and NaOH is an acid base reaction where the acid and NaOH reacts in the ratio 1:1. Gallic acid is a weak acid and it forms a salt 3,4,5-trihydroxy sodium benzoate on this reaction.

(OH)₃-C₆H₂-COOH (aq) + NaOH (aq) ------🡪 (OH)₃-C₆H₂-COONa (aq) + H₂O

This reaction can be used for the quantitative estimation of the amount of gallic acid in a given sample of tea leaves. As this titration is in between a weak acid and a strong base, the salt produced is a basic salt. Thus, the equivalence point lies around a pH of value greater than 7. This makes phenolphthalein as a suitable indicator for this titration.

According to the research paper – “Effect of Brewing Duration on the Antioxidant and Hepatoprotective Abilities of Tea Phenolic and Alkaloid Compounds in a t-BHP Oxidative Stress-Induced Rat Hepatocyte Model” by Derek J. Mc Phee reports that as the brewing time increases, the mass of gallic acid extracted consequently increases. This has also reported that anti-oxidant activity of tea leaves increases with the increase of brewing time.

Null hypotheses: There is no correlation between the mass of gallic acid extracted and the brewing time of the tea leaves.

Alternate hypotheses: There is no correlation between the mass of gallic acid extracted and the brewing time of the tea leaves.

Brewing time: Brewing time is the time for which the tea leaves are soaked in water. The brewing time of tea leaves is usually 2 minutes. In this investigation, the brewing time is 1.00 mins, 2.00 mins, 3.00 mins, 4.00 mins and 5.00 mins. A digital stop-watch was used to measure this time.

Type of tea leaves: To investigate that if the effect is same on all varieties of tea leaves or not, two different brands of tea leaves were used – green tea and black tea.​​​​​​​​​​​​​​

Mass percentage of gallic acid:

The tea extracts were allowed to react with a freshly prepared standard solution of NaOH. The titre value obtained was used to calculate the mass percentage of gallic acid in tea leaves.

Mass of NaOH to be added = moles of NaOH × molar mass

= molar concentration × volume × molar mass = 0.10 × \(\frac{100}{1000}\) × 40.01=0.40 g

• A watch glass was placed on a top pan digital mass balance.
• The reading of the balance was tared to 0.00 ± 0.01 g.
• Solid white pellets of NaOH were transferred from the reagent bottle to the watch glass using a spatula until the balance displays 0.40 ± 0.01 g as the reading.
• The exact mass of NaOH was transferred to a 100cm³ glass beaker.
• To transfer the weighed solid completely the watch glass was washed and the washings were collected in the same beaker.
• Distilled water was added in the beaker till the level of 100 cm³ using a graduated measuring cylinder.
• A glass rod was used to stir the solution and the solid was allowed to dissolve completely.

• A watch glass was kept on the top pan digital mass balance and the reading was tared to 0.00.
• The black tea leaves were added to the watch glass until the balance reads 2.00 ± 0.01g.
• A 100 cm³ glass beaker was taken and filled with distilled water till the mark of 100 cm³ using a graduated measuring cylinder.
• The beaker was placed on a wire gauge resting over a tripod stand with a Bunsen burner under it.
• The burner was turned on and the flame was adjusted to a blue color.
• The beaker was covered with a watch glass.
• As soon as the water begins to boil, the burner was removed and the temperature of the water was checked using a laboratory thermometer.
• The weighed sample of tea leaves were immediately added to the water in the beaker.
• The beaker was covered with watch glass and the stop-watch was started.
• As soon as the stop-watch reads 1.00 ± 0.01 mins, the beaker was removed from the wire gauge and kept on the table to allow it to cool down.
• After the beaker cooled down, the content of the beaker was filtered using a filter paper and a funnel. The filtrate was collected in a 100 cm³ conical flask and the residue was discarded.
• The filtrate was labelled as the “Black Tea extract – 1.00 mins”
• Steps 1-12 were repeated for other values of time – 2.00 ± 0.01 mins, 3.00 ± 0.01 mins, 4.00 ± 0.01 mins and 5.00 ± 0.01 mins.
• Steps 1-13 were repeated for the green tea leaves.

• The burette was washed with 0.10 moldm^-3 NaOH solution and dried.
• It was then filled with the same NaOH solution till the mark of 0.00 cm³.
• The tea extract in the conical flask was taken and two drops of the phenolphthalein solution was added to it.
• The tea extract was titrated against the NaOH solution running down the burette.
• The burette readings were recorded and noted down at the end point indicated by the appearance of permanent pink color.
• Steps 1-5 were repeated for two more times.

For brewing time = 1.00 ± 0.01 minutes

Difference in burette reading (DBR) = Final burette reading (FBR) – Initial burette reading (IBR)

= (4.60 ± 0.05) cm³ – (0.00 ± 0.05) cm³ = 4.60 ± (0.05 + 0.05) cm³ = 4.60 ± 0.10 cm³

Mean volume of NaOH consumed =\(\frac{4.60+4.50+4.50}{3}\) = 4.53 ± 0.10 cm³

Standard deviation (SD) = \(\frac{(4.60-4.53)^2+(4.50-4.53)^2+(4.50+4.53)^2}{3}\) = 0.06

For brewing time of 1.00 ± 0.01 min in case of green tea,

C₆H₂(OH)₃(COOH) (aq) + NaOH (aq) -----🡪 C₆H₂(OH)₃(COONa) (aq) + H₂O

Gallic acid                                                         Sodium salt of gallic acid

Moles of NaOH required (n) = molar concentration × Volume = 0.10 × \(\frac{V}{1000}\)

V = Mean volume of NaOH required in cm³ (mean burette reading)

Moles of gallic acid = moles of NaOH = 0.10 × \(\frac{V}{1000}\)

Mass of gallic acid in 100 cm³ of tea extract = moles × molar mass = 0.10 × \(\frac{V}{1000}\) × 170.12

Mass of gallic acid in 1 L of tea extract = 0.10 × \(\frac{V}{1000}\) × 170.12 × 10 = 0.17 × V

Mass percentage of gallic acid in tea extract = 0.17 × V \(\frac{g}{L}\) = 0.17 × 4.53 = 1.10 g/L

Absolute error in mean volume of NaOH consumed = ± 0.10 cm³

As clear from the data processing, the major source of error is the absolute error in the burette reading.

Percentage error in mass % of gallic acid in tea extract

\(\frac{absolute\ error\ in\ mean\ volume\ of\ NaOH\ consumed}{mean\ volume\ of\ NaOH\ consumed}\) × 100 = \(\frac{±0.10}{4.53}\) × 100 = ± 2.20

Figure - 8 is a scatter plot of the mass percentage of gallic acid in g/L along the y axes and the brewing time in ± 0.01 minutes along the x axes.

• The graph clearly suggests that as the brewing time increases from 1.00 ± 0.01 minutes to 5.00 ± 0.01 minutes, the mass percentage of gallic acid increases from 0.77 g/L to 1.92 g/L in green tea and from 0.17 g/L to 0.85 g/L in black tea. This claims as that the tea leaves are brewed for a longer time, more mass of gallic acid is extracted in the tea extract.
• However, the way the mass % of gallic acid increases with brewing time is not the same in case of black tea and green tea. The final value for black tea and green tea are 0.85 g/L and 1.92 g/L respectively. This shows that the mass of gallic acid extracted is more in case of black tea than that in green tea. At the same time, it also depends on the initial mass of gallic acid present within the tea leaves. Though the mass of tea leaves were kept constant yet it cannot be ensured that the initial mass of gallic acid present within the leaves was not the same in all cases.
• The graph also displays equation of trend line for both green tea and black tea. The equations are; y = 0.4169x and y = 0.17x for green tea and black tea respectively. The magnitude of the gradient is positive in both case which again claims that as the brewing time increases, the mass percentage of gallic acid increases. The value of the gradient is not the same in both cases. The magnitude of gradient is higher in case of green tea; 0.42 and lower in case of black tea; 0.17. This indicates that for the same increase in brewing time, the increase of mass percentage of gallic acid is more in green tea than that in black tea.
• Hence, it is again confirmed that the extraction of gallic acid from green tea is faster than that in black tea. Moreover, as clearly shown in the graph, for all values of x axes, the values in the y axes for green tea is higher than that in black tea. This again confirms that the mass percentage of gallic acid extracted increases with the increase in brewing time more significantly in green tea than that in black tea.
• For both the trend lines, the trend line is passing through the origin. The equation of trend line shows that the intercept is 0.00. This fact proves that as the brewing time is 0.00 ± 0.01 minutes, the mass percentage of gallic acid is 0.00 g/L. This is absolutely acceptable as if the tea leaves are not brewed, there will not be in any gallic acid extracted from the tea leaves.

The gallic acid is extracted from the tea leaves and by the process of diffusion through the leaves it goes into the solution. Thus, as brewing time increases, there is more movement of the gallic acid molecules from the cells in the leaves to the aqueous medium of the extract. Diffusion is a spontaneous process and also depends on the concentration gradient. Thus, as more gallic acid moves from the cellular matrix to the aqueous layer, the concentration difference between the cells and the aqueous extract decreases. This in turn slows down the rate of diffusion. This is evident from Figure 8. As the brewing time increases, the gap between consecutive data points are decreasing which indicates that with more gallic acid passing down from the cells in tea leaves to the aqueous extract, the difference of concentration is decreasing and thus rate of diffusion is decreasing too.

How does the mass percentage of gallic acid, measured in g/L in tea extracts depends on the brewing time and the type of the tea extract – green tea and black tea used, determined using acid base titration with NaOH?

• As the brewing time increases from 1.00 ± 0.01 minutes to 5.00 ± 0.01 minutes, the mass percentage of gallic acid increases from 0.77 g/L to 1.92 g/L in green tea and from 0.17 g/L to 0.85 g/L in black tea. This claims as that the tea leaves are brewed for a longer time, more mass of gallic acid is extracted in the tea extract.
• For the same increase in brewing time, the increase of mass percentage of gallic acid is more in green tea than that in black tea.
• However, the way the mass % of gallic acid increases with brewing time is not the same in case of black tea and green tea. The final value for black tea and green tea are 0.85 g/L and 1.92 g/L respectively. This shows that the mass of gallic acid extracted is more in case of black tea than that in green tea.
• As the brewing time increases, the gap between consecutive data points in Figure 8 is decreasing which indicates that with more gallic acid passing down from the cells in tea leaves to the aqueous extract, the difference of concentration is decreasing and thus rate of diffusion is decreasing too.

• The values of the brewing time chosen are continuous and regular. Moreover, it covers a wide range from 1.00 minutes to 5.00 minutes. This allows us for a fair and more comprehensive comparison of the change in gallic acid content against brewing time.
• The conclusion has been deduced in a coherent manner. Both the line displayed in the graph as well as the equation of trend line obtained indicates a positive correlation between the mass percentage of gallic acid and brewing time.
• The values of independent variable have a realistic reference as the values chosen covers the usual range of brewing time chosen for making tea extracts.
• The values of standard deviation have been computed in the raw data table and the low value confirms that the data collected is really precise.

Apart from gallic acid, another major constituent of tea leaves is caffeine. The caffeine level of tea leaves depends on the time for which the tea leaves are brewed. I would like to carry out an investigation to understand how the amount of caffeine extracted from tea leaves depends on the brewing time. The caffeine can be extracted from the tea extracts by liquid-solid extraction method where first sodium bicarbonate is added and then the filtrate is crystallized using an organic solvent like DCM.

• PubChem. Gallic Acid. https://pubchem.ncbi.nlm.nih.gov/compound/370. Accessed 30 Nov. 2021.
• Zhou, Xiaochen, et al. “Metabolism of Gallic Acid and Its Distributions in Tea (Camellia Sinensis) Plants at the Tissue and Subcellular Levels.” International Journal of Molecular Sciences, vol. 21, no. 16, Aug. 2020, p. 5684. PubMed Central,https://doi.org/10.3390/ijms21165684.