Abstract
Various beliefs are common about the extraction of soluble compounds from leaf tea, suggesting that cold brewed tea or tea brewed for a shorter time than usual may contain a higher polyphenol-to-caffeine ratio, a selling point due to the potential health benefits of polyphenolic compounds. To test these beliefs, we investigated the effect of brewing time and temperature on the colour intensity and caffeine content of the extract of one type of black tea. Results showed that the extraction of the two components of interest had different half-lives, with an initial large variation of the ratio between them rapidly reaching a quite constant value. At different temperatures, a significantly different ratio between caffeine and colour compounds was observed. Although the difference in relative concentration was small, the range of brewing temperatures tested was only 20 Kelvin, so it seems possible to increase this difference. A difference in total extraction efficiency of both components together was noted too. To effectively change the ratio of the components using the extraction time would require using accurate times of less than 3 minutes and accurate temperatures unusual in a home environment.
1 Introduction
Caffeine is often called the world’s most consumed legal drug, with caffeinated beverages being some of the most popular drinks in the world. It is estimated that around 2.25 billion cups of coffee and 6 billion cups of tea are consumed per day [1,2].
Common beliefs about tea and brewing time include both that brewing the tea for a short time reduces caffeine extraction and that caffeine is released first. Any confusion is further enhanced by the common practice of discarding the first extract when brewing green tea and by strong beliefs as to the optimal temperature for tea extraction often without justification or measurement and local differences, suggesting culture is an important factor in this choice. The recommended infusion time of tea can also vary significantly depending on the type of tea used, traditions and the region it originates from.
For these reasons, we decided to investigate the effect of temperature and time on caffeine extraction from tea leaves and to compare that with a measure of the strength of the tea brewed using a colour index. The reason we chose a relative measurement is that it is simple for a user to dilute a brew as desired or to use more leaf, but the relative concentration of different components is more difficult to change. We chose to investigate black tea in this instance as it is the most common in many places and has a relatively high colour and caffeine content.
The time it takes for caffeine to be extracted into the hot water can depend on factors like the temperature of the water, hardness of water, or agitation of the water through mechanical processes such as stirring. As mentioned by Smit et al. [3] caffeine can also slow down surrounding water molecules, so the very release of caffeine might slow down its distribution in the water, although the concentrations seem too low for this to be significant except right next to the leaf [3]. As it has been shown [4] that calcium in water can reduce polyphenol extraction from tea leaves, distilled water was used in this study for reproducibility.
Black tea usually colours the water brown through the extraction of polyphenolic compounds, specifically known as thearubigins, which are formed during the enzymatic oxidation process in the black tea [5]. They are heterogenous polymers of tea catechins and account for up to 60% of the water-extractable material of black tea by dry weight [6]. Based on these numbers, thearubigins also seem to be the main source of bitterness of tea [7]. Theaflavins are closely related to the thearubigins and have similar reddish colour but are derived from flavanols instead of catechins. However, antioxidising properties of the mentioned flavonoid have suspected health benefits [8], and the high caffeine content of a drink seems to be often favoured over the bitter taste of a strong tea because of its stimulating properties. Black tea and oolong tea contain a higher concentration of caffeine compared to other types of tea [9].
Caffeine is an alkaloid, 1,3,7-trimethylxanthine that is sparingly soluble in water as the neutral form and very water soluble as the free base; however, as pK a is 10.4, it will be in the free base form during every normal tea brewing process for human consumption. Thearubigins and theaflavins are typically weak acids with a range of pK a values below 7 [10] and will therefore also be in their ionised form in a typical drinking water extraction and also in distilled water. Distilled water has a pH of around 5.8 at room temperature but moves towards 7 on heating as carbon dioxide is expelled; tap water can be more basic than this but not enough for a significant proportion of caffeine to be in the uncharged state. This difference in pK a allows caffeine and polyphenolics to be separated reasonably simply with an acid–base extraction into an organic phase. Although caffeine is known to bind to some of the polyphenolic compounds, forming particles that can be isolated, that bonding can be broken with a non-polar solvent, so a basic caffeine extraction into the organic solvent should not be affected [11]. More important are potential oxidation reactions that convert soluble species to insoluble ones and change the apparent colour of polyaromatic compounds.
Previous studies have investigated the effect of salts in the water and milk content on reflected colour [12].
Here, we prepare tea brewed in distilled water at three different constant temperatures at a concentration fairly typical for home use and take samples after different times and measure caffeine content and colour index of polyphenolic compounds to determine whether the ratio of caffeine to phenolic compounds can be varied by the extraction time or temperature. We use solvent extraction to separate the components and measure spectroscopically.
2 Materials and methods
Water was distilled from tap water in a glass still, and tea was purchased as consumer grade cup portion teabags (Teekanne, NRW, Germany “Ostfriesen” black tea blend in individual bags). Caffeine was purchased from Sigma-Aldrich, Darmstadt, Germany, as >98.5% anhydrous powder. Saturated sodium carbonate was prepared from >99.5% anhydrous sodium carbonate obtained from Sigma-Aldrich and distilled water and allowed to equilibrate at 20°C for 2 days before use. Dichloromethane 99.5% (ACS reagent Sigma Aldrich with stabiliser) was used as supplied.
Ultraviolet (UV) spectroscopy was carried out using an Ocean Optics USB2000 spectrometer and a combination deuterium/tungsten light source with both bulbs on. Caffeine was measured in a 1 cm path length quartz cuvette, colour in disposable 1 cm path length polystyrene cuvettes.
2.1 Tea preparation
1 L of distilled water in a clean 2 L Pyrex beaker was stirred gently using a magnetic stirrer on a slow speed at a chosen temperature. Temperature was measured using a thermometer that was checked beforehand in ice water and boiling water, and results were adjusted accordingly. Distilled water was used to allow standardization and for others to repeat the measurements.
Ten teabags with a sum mass of 18.7 g including the paper and attachments were dipped in all at the same time at time 0 using a ring-shaped holder to place them all at a consistent depth and distance from the central stirring vortex. Stirring was gentle enough that they could hang almost vertically.
Dip samples (1.6 mL) of the brewing tea liquor were taken after certain brewing times and allowed to cool in capped tubes. Care was taken to attempt to get a representative sample, but that proved impossible under these circumstances at short brewing time (less than 30 s), where visible darker patches were present due to the slow rate of stirring.
Details can be seen in a video at https://www.youtube.com/watch?v=CadBqw-Q0tc and in the supplemental information.
2.2 Colour measurement
Samples were allowed to cool before being measured using UV-visible spectroscopy, but measurement was carried out as soon as possible to minimise the effect of oxidation. Cooling was rapid due to the small sample volumes, and the glass containers used. Samples stored overnight darkened considerably but spectra measured a few minutes apart in the first half hour were identical suggesting that they are representative of the state at the times each sample was extracted.
UV-visible measurements showed very high absorption at short wavelengths reducing the light to the detector below the levels where it could be measured accurately. At longer wavelengths, the development of colour could be followed. We chose 500–550 nm as a position to measure and normalised each measured wavelength against the value at the same wavelength measured at the highest temperature and time; all 100 wavelength normalised values in the range were then averaged to form a single value for each sample. As there are multiple chemicals contributing to this signal and probably also scattering, it is likely not to be linearly related to the concentration of any single compound, but it will be dependent on the concentration and extraction time. The wavelength has no chemical significance, but was chosen as it is a balance between being long wavelength to reduce the contribution of scattering from particles and short wavelength to detect the beginning of the extraction sensitively. As shown in Figure 1, the absorbance declines to longer wavelength like the tail of a large absorbance band. At shorter wavelengths, the value becomes too high for the spectrometer used when the tea is strongest, at longer wavelengths, the difference at low tea concentration is very small. The 100 values measured between 500 and 550 nm were averaged to reduce the statistical noise from the detector and scattering from any larger particles.
Development of UV-Vis absorbance of tea extracted at 77°C. Peak at 275 nm due to caffeine. Signal saturates between absorbance values of 2 and 4 on this spectrometer depending on settings.
2.3 Caffeine extraction
Caffeine extraction was carried out using a variation of the protocols used in other studies [10,11]. Because it was not necessary to extract the full amount, but rather to determine the relative concentration and because the absorption peak for caffeine at 275 nm is very strong, a single partial extraction was carried out. Typically 100 μL of tea sample was pipetted into a separation funnel along with 200 μL of saturated sodium hydrogen carbonate solution to render the polyphenolic compounds more soluble and the caffeine less soluble in water. 2 mL of dichloromethane was added and shaken to extract the caffeine. 1 mL of the dichloromethane was then added into a 1 cm path length quartz cuvette and stoppered; the UV-visible spectrum was measured. As the beam was close to the bottom of the cuvette, any water droplets entering the cuvette did not interfere with the measurement.
Caffeine concentration was estimated using solutions prepared in the same manner from caffeine purchased from Sigma Aldrich (98.5%). No attempt was made to add polyphenolic compounds to the reference sample.
2.4 Extraction models
The rate of extraction was compared to two simple models from the literature, the Peleg model, which was developed for the ingress of water into plant matter and an exponential decay model, as is common in diffusion phenomena. Both treatments are empirical but have been shown to fit similar systems.
3 Results and discussion
The UV-Vis absorbance of tea extracts showed no obvious features apart from the caffeine peak and what appears to be noise around 450 and 590 nm, probably arising from the light source, external lighting, detector combination superimposed with what looks like the long wavelength tail of a peak absorbance quickly reaching peak absorbance values greater than 3 in stronger brews and being cut off by the physical limitations of the detector.
Figure 1 shows the absorbance of tea extract after different times with the broad absorbance of the polyphenolics quickly swamping the much sharper caffeine peak at around 275 nm. The reference curve for caffeine concentration remained linear until the absorbance value of slightly above 2 before becoming nonlinear. This means that the highest two points of the highest temperature caffeine concentration curve are probably inaccurate as they exceed this value. The curve being linear to such a high absorbance value suggests that the spectrometer is working very well for such a small device and that the extraction is proportional to the original concentration.
Extraction time profiles were compared with the established models [13] using a simple least squares consideration with a simple generalised reduced gradient nonlinear iteration. Extraction of both components (caffeine and brown component) followed the type of exponential profile expected with a rapid increase initially tending towards a maximum value at infinity time; however, measurements at short times did not extrapolate to zero at zero time, and fitting using equation (1) (exponential) required time zero as a fit parameter and removal of the first few points, suggesting that extraction did not begin immediately. As shown in Figure 2a, these points represent an initial delay and cannot be modelled with either equation (1) or equation (2):
where [X] is the concentration of the extracted species (caffeine or polyphenol); A, B, and C are variables to be fitted, t is the time, and t 0 is the delay time.
(a) Fitted curves of “brownness” (top curves) and caffeine concentration (lower curves) against time in tea extracts brewed at 77°C. Both fits appear reasonable within the accuracy of the data, but the Peleg fits are closer to the data points and these are used for later analysis. (b) Fits of curves for normalised brownness (lower curves left axis) and caffeine (upper curves right axis) showing the different curve shapes and maxima.
The offset in time is not entirely unexpected, the hot water must first penetrate the teabags and enter the tea itself, warm it and the extract make its way back out. It is entirely plausible that this introduces an offset in the time profile. In the case of the measurements described here, it was not possible to accurately measure the values of caffeine concentration or “brownness” at these early times, and they represent unrealistic times for home tea brewing, so no attempt was made to model them.
Previous papers have noted effects of tea being enclosed, most notable was the study by Astil et al. [14], who did not show a time shift due to filter material, but rather a reduction in the extraction rate. Looking at their figures, however, many of their tea extraction curves including some for loose leaf do not extrapolate to zero at time zero. These authors used high-performance liquid chromatography to separate and measure components, suggesting that the results are also not due to the methodology.
In a different style of experiments, Spiro and Jago [15] used a rotating disc to show that the extraction of tea components is independent of transport in the water phase under their conditions. The differences we observe may be due to gas in the teabags or the very low agitation used here.
Alternatively, the results could also be fitted to Peleg’s equation (originally devised for water absorption, but also sometimes applied to extraction) [16], equation (2):
where [X] is the concentration of the extracted species (caffeine or polyphenol); A, B and C are variables to be fitted; and t is the time.
In this case, it was possible to fit the curve simply by allowing the first term, A to be negative. This first term represents the initial concentration, so setting it to a negative value is the same as shifting the time before anything happens. This is misusing the variable slightly as a negative initial concentration is not physically meaningful, but it does allow the fit of most of the data without adding an extra variable.
Of course neither equation fit the measured data near time zero due to the time shift required to fit the data. Both fits extrapolated to non-physical negative values at low time.
Either type of equation could be used to fit the data, which in itself is not overly surprising as they are both semi-empirical models of diffusion. That the tea extraction measurements follow a curve shape mathematically expected from other extraction processes is encouraging and suggests our measurements are representative of the true process. As shown in Figure 2a, equation (2) is a better fit for both curves and hence used to smooth the data.
These results are similar to those measured by Saklar et al. [17] with green teas, also indicating most of the extraction being complete within 5 minutes at 85°C with mostly side reactions of phenolic compounds continuing afterwards. The results presented by Musilová and Kubíčková [18] showed similar extraction kinetics of caffeine from teas although they did not vary temperature and extraction time together.
Of interest to our research question is whether the concentrations of the two components are multiples of each other at all times and temperatures or whether they follow different courses from one another. Any differences in the relative compositions could theoretically be used by the consumer to modify their caffeine intake while maintaining a similar polyphenol profile.
3.1 Extraction vs time behaviour
Figure 3a shows the fitted ratio of caffeine to polyphenols and how it changes over time. As can be seen, the slight mismatch in the curvature (modelled by constant C) of the two components mean that caffeine is extracted faster than polyphenols at higher temperature and at a similar rate at lower temperatures. There is a swing in caffeine ratio at the highest temperature as initially caffeine is predominantly extracted followed later by polyphenols.
(a) How the ratio between caffeine and “brownness” varies with time. (b) Extrapolated data for very short and very long times, suggesting changing the temperature further is likely to have a greater effect.
Astil et al. [14] also measured the effect of brew time on the caffeine ratio of their teas. They only brewed using “boiling” water and generated a curve much like our one at the highest temperature. The drop to low values at short times was not observed in their data, but their shortest time point was 50 s and their temperature was higher and so this is not surprising. Data measured by Spiro and Siddique [19] also showed the same behaviour and also only measured at a higher temperature, although they separated the colour component measured here into theaflavins and thearubugins using chromatography and found different kinetics for these components.
3.2 Extraction vs temperature behaviour
Extracting the tea at different temperatures changed the ratio between the tea components. As we were trying to remain in a range that people might realistically use for hot brewing at home, we did not vary the temperature very much. However, the values used revealed that the relative efficiency of tannin and caffeine extraction could be altered (somewhat) by controlling the temperature, which also affected the total extraction efficiency of both components. Figure 3b shows the effect of extrapolating the data, suggesting that increasing the temperature further could extract more polyphenolics, particularly at longer times. The point at low temperature and time is significant, but the extract would be extremely weak in both components and the extraction efficiency is extremely low. The highest ratios of caffeine to polyphenolics were observed at low temperature, long times.
Hajiaghaalipour et al. [20] measured the antioxidant power of tea extracts and found considerably different results from ours. This is possibly due to their measured radical reactivity being mostly from smaller phenolic extracts; however, the darkness of the tea arises more from larger molecules. Lantano et al. [21] also observed a similar effect with various tea types. This does not mean that the same effect would be observed in the body; indeed, various reports show that changes during digestion of polyphenolic compounds increase their bioavailability and antioxidant power [22,23,24], suggesting that the antioxidant power of the tea extract itself is not a good measure of the effect on the body after drinking it.
4 Conclusion
The experiments carried out here reveal that if only the relative caffeine and polyphenol concentration are considered after a swing in ratio at short times, there is a long plateau where the ratio of the components remains constant but both are extracted together.
On the other hand, there was a difference, although limited, in relative concentration when the extraction temperature was changed with the efficiency of caffeine extraction being less affected by reduced temperature than that of polyphenol extraction.
This suggests that brewing tea for longer times at lower temperatures would increase the amount of caffeine that a tea drinker consumed compared with shorter times or less leaf at higher temperatures. Equivalently, it would reduce the dose of polyphenolic compounds the consumer would consume for the same caffeine intake.
The tactic of brewing this tea at nearly boiling point for a short time would result in a higher relative caffeine concentration than brewing for a longer time and diluting the resulting extract. This would suggest that should a consumer wish to brew tea with a lower caffeine content but the same flavour, using a high temperature extraction and diluting to the desired colour would be advisable. Particularly a practical tea extraction carried out in a home kitchen may begin at over 90°C, but in reality, it takes place over a falling temperature profile particularly if small quantities are prepared.
4.1 Implications for sun tea
Extrapolating these results to very long times at considerably lower temperature would disagree with the typical claims made for sun tea (brewed at room temperature for a long time) that it is “healthy,” implying it contains more polyphenolics (and possibly less caffeine) than hot brewed tea. It would appear that the polyphenolic extraction efficiency at low temperature would be reduced and the consumer faced with a choice of having more caffeine or a lower polyphenolic concentration.
One advantage of cold extraction not considered here has been noted before [25] that volatile components are better preserved by cold extraction although they also noted a lower polyphenolic extraction under colder conditions (room temp 120 min).
In addition, the extraction curves did not extrapolate to zero at zero time, but exhibited an offset that could be accounted for in equations using a time offset. This suggested a short time delay of 5–10 s from first contact with water to extraction starting properly, but our experiment was not designed to measure this accurately (Table 1).
Data summary of both measured caffeine (mg/100 mL) and normalized absorbance
| Time | Caffeine | “Colour intensity” | ||||
|---|---|---|---|---|---|---|
| 62°C | 77°C | 85°C | 62°C | 77°C | 85°C | |
| 15 | 0.406072 | 4.097639 | 0.959807 | 0.042835 | 0.065837 | 0.055805 |
| 30 | 0.332241 | 6.460241 | 4.503711 | 0.054179 | 0.055785 | 0.077269 |
| 45 | 9.745736 | 11.70227 | 0.114467 | 0.132776 | ||
| 60 | 5.574265 | 12.47749 | 21.41109 | 0.118225 | 0.18774 | 0.234572 |
| 90 | 22.1494 | 36.02969 | 0.288277 | 0.362441 | ||
| 120 | 24.77041 | 36.2881 | 52.12492 | 0.281547 | 0.386378 | 0.470246 |
| 150 | 44.88945 | 0.470957 | ||||
| 180 | 39.27827 | 59.69263 | 0.387627 | 0.627258 | ||
| 210 | 52.3095 | 0.553913 | ||||
| 240 | 44.74179 | 64.75008 | 0.454299 | 0.71239 | ||
| 300 | 49.98381 | 64.41784 | 68.29398 | 0.508078 | 0.66355 | 0.778571 |
| 420 | 71.1734 | 84.31538 | 0.772959 | 0.905968 | ||
| 480 | 61.16926 | 0.625175 | ||||
| 600 | 79.99625 | 88.11769 | 0.87971 | 1 | ||
| 660 | 72.46545 | 0.694199 | ||||
| 900 | 76.63692 | 0.742907 | ||||
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Funding information: This work was funded by the Faculty of Technology and Bionics, Rhein Waal University (Appl. Sci.) for teaching purposes
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Author contributions: Conceptualization: NS; data curation: NS, KA, JN, and JS; formal analysis: NS; funding acquisition: NS, WS, and AS; investigation: NS, KA, JN, and JS; methodology: NS, AS, and WM; project administration: NS; resources: NS; software: N/A; supervision: NS; validation: WS and AS; visualization: NS; writing –original draft: KA, JN, and JS; writing – review and editing: NS, AS, and WM.
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Conflict of interest: This study was carried out as part of a course on science for communicators. There is no association with any part of the tea industry or the wellness industry, woo or legitimate. Students taking part received credit for the course, so participation was not entirely voluntary. The text was compiled using the reports students provided for the course.
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Ethical approval: The conducted research is not related to either human or animal use.
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Data availability statement: The data presented in this study are available on request from the corresponding author.
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