Dr. Van Bramer
Testing the Caffeine Concentration in Tea
The determination of caffeine in coffee is a very important analysis in the
pharmaceutical and food industries due to legal restrictions imposed by the FDA. In this
experiment, the concentration of caffeine was determined in three Bigelow brand tea samples
(earl grey, green, and oolong) and a coffee bean sample using HPLC and GC methods. The
HPLC results showed that tea concentrations are less than 15 mg and that green tea has the
lowest concentration (6mg). In the case of coffee beans, the mass of caffeine could not be
determined due to noise problems in the chromatogram.
Caffeine is an alkaloid without smell and bitter taste found in many vegetable sources,
such as cocoa beans, coffee, tea, among others. Caffeine is widely used in the following
industries 1: i) pharmaceutical, for the elaboration of analgesics, anti-flu and diet remedies, ii)
food, in the elaboration of energizing and carbonated beverages. It is for this reason, that
caffeine has a high global demand (120,000 tons/year) 2.
Caffeine can be consumed daily through different sources, such as coffee, tea, chocolate
and energy drinks, as well as in anti-migraine drugs 1. The high concentrations of caffeine in
living beings can cause a series of disorders related to heart disease, urinary and asthma, and
even death (doses >200mg/day) 3.
The concentrations of caffeine in beverages as such as coffee and tea may vary
depending on the brand; however, the maximum concentrations are well regulated by the FDA
and may not exceed 10 g1. Because of legal restrictions, caffeine analysis is of great importance
in order to ensure adequate levels in beverages and to comply with regulatory standards.
For the analysis of caffeine in beverages, different analytical methods have been used, which
may vary depending on the matrix. In the literature, the most recommended techniques are
chromatographic and spectroscopic. Chromatographic techniques include high liquid
efficiency (HPLC) and gas GC 4. While spectroscopic techniques include UV-visible 5 and
This study focuses on the analysis of caffeine in tea samples using HPLC and GC
techniques to determine if the concentrations of the products used are within the limits
established by the FDA.
Materials and Method:
In this experiment three types of tea (earl grey, green, and oolong) Bigelow brand and a coffee
tree leaf from Widener greenhouse were used. The concentration of each sample was
determined by two chromatographic methods: LC and GC.
The teas (earl grey, green, and oolong) and the coffee were put in a 300 mL flask and 250 mL
of hot water (100 °C) was added. The ingredients were extracted from the tea for 5 minutes.
For chromatographic analysis, a column (Discovery RP - AmideC1615 cm x 4.6 mm I.D, 5 um
particles) was used, and the mobile phase was prepared with a composition of 75%DI water
and 25% methanol (25 mM K2HPO4, pH 7.0). The wavelength used in the UV detector was
254 nm. For the analysis of the standards (10, 20, 50, 70 and 100 ppm of caffeine) and the
samples 10µL were injected. The running time of the standards and samples was 8 minutes and
30 minutes for samples.
For sample preparation, each tea bag was taken and dissolved in 250 mL of water. Then 20 mL
of the dissolved tea was added to a separating funnel. Next, 20 mL of 2-methyl THF was added
to the separation funnel, the phases were shaken, so that the extraction of caffeine to the organic
phase occurred. We put the injection tubes that contain the standards and samples in the auto
samplers and start the GC test.
Results and Discussion:
The relationship of the areas of the standards with their concentration showed a linear trend
with an R2 equal to 0.9992 (Fig. 1). The adjustment obtained was:
𝐴𝑟𝑒𝑎 = 140.95 ∗ 𝐶𝑜𝑛𝑐𝑒𝑛𝑡𝑟𝑎𝑡𝑖𝑜𝑛 + 90.943
Concentration (ppm )
Figure 1. Calibration curve of caffeine
The analysis of liquid chromatography allowed determining the mg of caffeine present in the
different types of tea. Table 1 shows the caffeine mg for Earl grey, Green, and Oolong teas,
which were 13, 6, and 14 mg, respectively. These results indicate that the composition of
caffeine may vary with the type of tea. In the case of coffee beans, the concentration or content
of caffeine could not be determined because the chromatogram obtained was very noisy and
the peak corresponding to caffeine could not be integrated.
Table 1. Composition of teas and coffee
mg of caffeine
As initially mentioned, the caffeine content of tea samples may vary from brand to brand. With
respect to that fact, Bigelow brand teas have a low concentration of caffeine as they are below
The analysis of caffeine in the different tea samples using HPLC revealed that the amount
present depends on the type of tea: earl grey (13 mg), green (6 mg), and oolong (14 mg). These
levels of caffeine in teas are below of the legal restrictions.
1. Belay, A., Ture, K., Redi, M., & Asfaw, A. (2008). Measurement of caffeine in coffee beans
with UV/vis spectrometer. Food Chemistry, 108, 310–315.
2. Paradkar, M. M., & Irudayaraj, J. (2002). Rapid determination of caffeine content in soft
drinks using FTIR–ATR spectroscopy. Food Chemistry, 78, 261–266.
3. Pura Naik, J. (2001). Improved high-performance liquid chromatography method to
determine theobromine and caffeine in cocoa and cocoa products. Journal of Agricultural and
Food Chemistry, 49, 3579–3583.
4. Tzanavaras, P. D., & Themelis, D. G. (2007). Development and validation of a
highthroughput high-performance liquid chromatographic assay for the determination of
caffeine in food samples using a monolithic column. Analytica Chimica Acta, 581, 89–94.
Columnchromatographic extraction and separation of polyphenols, caffeine and theanine from
green tea. Food Chemistry, 131, 1539–1545.
6. Zou, J., & Li, N. (2006). Simple and environmental friendly procedure for the gas
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