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Chinese Science Bulletin 2003 Vol. 48 No. 22 2438ü2443
Extraction of catechins
and caffeine from different
tealeaves and comparison
with micellar electrokinetic
chromatography
SONG Guanqun1, LIN Jinming1, Qu Feng1
& C. W. Huie2
1. Research Center for Eco-Environmental Sciences, Chinese Academy
of Sciences, Beijing 100085, China;
2. Department of Chemistry, Hong Kong Baptist University, Kowloon
Tong, Hong Kong, China
Correspondence should be addressed to Lin Jinming (e-mail: jmlin@
mail.rcees.ac.cn)
Abstract This work describes the simultaneous deter-
mination of catechins and caffeine in green, black tealeaves
and canned tea-drink using micellar electrokinetic chroma-
tography. The catechins analyzed include (+)-catechin, ()-
epicatechin, ()-epigallocatechin, ()-epicatechin gallate and
()-epigallocatechin gallate. Using UV absorption method at
280 nm, the limits of detections of catechins and caffeine are
106 mol/L, which is suitable for the real sample determina-
tion. Using this analytical method, the extraction of these
compounds from the tealeaves with hot water is compared
under different temperatures. The effects of temperature on
the amount of catechins and caffeine extracted are evident,
showing that ()-epigallocatechin gallate is the most easiest to
be extracted at 100ć. The stability of catechins and caffeine
in stocking solution of tea-drink at 4ć is also compared on
five consecutive days. The contents of catechins and caffeine
in green and black teas are discussed and the difference of
the content between different tealeaves can provide a refer-
ence for the assessment of tea quality.
Keywords: catechins, caffeine, tea, micellar electrokinetic chroma-
tography, pretreatment.
DOI: 10.1360/03wb0105
Tea is one of the most popular beverages in the
world because of its attractive taste, aroma and healthy
effects. Recently, several studies lead to acceptance of tea
as a useful cancer preventive agent[1]. Increased public
awareness of the health protective characteristics of tea,
which are generally considered to be associated with the
high flavonoid content of the leaves and extracts, has con-
tributed to the public’s general attitude toward the bever-
age[2]. The main components of tea are polyphenolic
compounds, commonly known as catechins, which repre-
sent a group of compounds belonging to the flavonoid
family. Catechins may be contained in up to 30% (w/w) of
the dried tealeaf[3], which has been found to possess
2438
anti-mutagenic, anti-oxidant and anti-tumorigenic proper-
ties[4ü7] and may exert prophylactic effects against hyper-
tension[8]. High levels of catechins also may render the tea
bitter and affect its astringency. The five major tea cate-
chins are (+)-catechin (C), ()-epicatechin (EC), ()-
epigallocatechin (EGC), ()-epicatechin gallate (ECG)
and ()-epigallocatechin gallate (EGCG). The structural
similarity of the various catechins makes the quantitation
and analysis difficult. Caffeine is another important bitter
component, which was contained in some popular bever-
ages like tea, coffee and cocoa and known for its stimula-
tory effect.
Many analytical methods have been developed to
measure these important tea components in order to esti-
mate the quality and taste of tea. Gas chromatography
(GC) methods[9,10] utilizing both glass or fused-silica cap-
illary column have been developed for the determination
of catechins. The methods give good separation of the
catechins but the derivatization step, along with the indi-
vidual isocratic runs, makes the methods time consuming
and labor intensive. High-performance liquid chromatog-
raphy (HPLC)[11ü14] is currently the most useful approach
for the routine analysis and research of non-volatile tea
constituents. However, these methods need large quantity
of elutant. Furthermore, with HPLC very little can be done
to manipulate the selectivity as the analytical columns are
used with simple buffers or dilute acid only.
Recently, capillary electrophoresis (CE) has been
complementary to chromatographic methods for it pro-
vides several promising features such as high separation
efficiency, fast analysis and low consumption of chemicals.
Capillary zone electrophoresis (CZE)[15,16] and micellar
electrokinetic capillary chromatography (MEKC)[17ü21] are
the CE methods of choice for the determination of cate-
chins. In general, the MEKC methods provide better
separation, resolution and quantitation for a larger number
of catechins than do the rudimentary CZE methods[22].
Although the previous methods had lower detection limit
and wider linear range, they were complex[16ü19] and the
analysis time was longer[15,18,21]. The present method was
simple, fast and without using organic solvent, applicable
for analysis of the real tea samples. Microemulsion elec-
trokinetic chromatography (MEEKC) is a relatively new
variant of CE, which also was used to analyze the cate-
chins[23,24], and Pomponio et al.[23] represented MEEKC was
superior to MECK in resolution, sensitivity and retention time,
but this method was not suitable for the real sample analysis.
The main purpose of this paper was to systematically
study the pretreatment of tea samples and analyze the tea
samples. The effects of several crucial parameters during
dealing samples, including temperature of sample prepa-
ration and time of deposition, on the contents of catechins
and caffeine extracted into the tea infusion were discussed.
Chinese Science Bulletin Vol. 48 No. 22 November 2003

In addition, the content of catechins and caffeine in green
tea, black tea and canned tea drink were also compared
and the difference of the content between different tea-
leaves was primarily discussed.
1 Experiments
(ν) Reagents and tea samples
(1) Reagents. Five catechin standards, (+)-catechin
(C), ()-epicatechin (EC), ()-epigallocatechin (EGC),
()-epicatechin gallate (ECG) and ()-epigallocatechin
gallate (EGCG) (see Fig. 1) were purchased from Funa-
koshi (Tokyo, Japan). Caffeine was from Acros Organics
(USA). Sodium dodecylsulfate (SDS) was obtained
from Wako Pure Chemival Industries Ltd (Japan). All re-
agents were of analytical-reagent grade. All solutions were
prepared with distilled water that was deionized using a
Milli-Q purification system with 0.2 µm fiber filter (Barn-
stead, USA).
(2) Tea samples. The tea samples, including green tea,
Huang-Shan Maofeng, Dahong Pao, West-lake Longjing
and Xinyang Maojian were from Anhui, Fujian, Zhejiang
and Henan respectively, and black tea, Qi black tea and
Dian black tea were from Anhui and Yunnan respectively.
Canned tea drinks, Tongyi green tea and Tongyi black tea
(Tongyi Tea Inc. China) were obtained from the market.
(ξ) Apparatus. All experiments were carried out
with a lab-built CE system equipped with a high voltage
power supply (Beijing New Tech. Inc., China), a UV spec-
trometer (Jasco, Japan) and a plexiglass box in which the
buffer and sample reservoirs were. A fused-silica capillary
(Yongnian, China) with a total length of 60 cm and an I.D.
of 50 µm was used. A detection window was created at 45
cm from the capillary inlet by removing the polyimide
coating. HW chromatography software (Qianpu Inc.,
China) was used for both the operation of the detector and
data processing. THZ-82 constant-temperature shaker
(Changzhou Inc., China) was used to dispose the sample.
(ο) Analytical conditions. A 25 mmol/L SDS in a
20 mmol/L borate (pH 7.0) solution was used as a running
buffer. The potential applied for the separation was +25 kV
and the detection was performed by UV absorption at 280 nm.
The samples were injected by 1.5 kPa pressure for 10 s.
Stock solutions of catechins and caffeine were ob-
tained by dissolving these compounds in deionized water,
and the concentrations of all stock solution were 103 mol/L.
The stock solutions were stored at 4ć and used within 1
week.
The capillary was conditioned prior to use with 1.0
mol/L HCl for 10 min, followed by deionized water for 2
min, 1.0 mol/L NaOH for 10 min, deionized water for 2
min and finally buffer solution for 10 min. To achieve
reproducibility, between runs the capillary was rinsed
consecutively with 1.0 mol/L HCl (2 min), deionized water
(1 min) and finally for 2 min with buffer.
Chinese Science Bulletin Vol. 48 No. 22 November 2003
REPORTS
(π) Preparation of tea sample solution. 1.000 g of
tealeaves were weighted and placed into a 250 mL beaker,
adding 100 mL of deionized hot water at 90ć, stirring for
10 min by a constant temperature shaker at 90ćWhen
cooling at about 70ć,20 mL of tea infusion filtered with
common filter paper was diluted 10 times with 0.1%
metaphosphoric acid. After passing through the 0.45-µm
membrane filter, the filtrates were used as samples. Canned
tea drink was diluted five times with 0.1% metaphosphoric
acid and passed through the 0.45-Pm membrane filter and
the filtrate was used as a sample.
2 Results and discussion
(ν) Optimal conditions for the determination of
catechins and caffeine
(1) Effect of pH. In alkaline solutions catechins are
known to be unstable and the peak tailing significantly[25].
The pH of the electrophoretic run buffer must be carefully
controlled, as it influenced not only the EOF, but also the
resolution of the catechins. The influence of pH on elec-
trophoretic mobility of catechins and caffeine was tested.
The migration time decreased with increasing of pH. This
is because when the pH value of the solution is higher, the
density of the negative charges on the inner-wall of capil-
lary is increased, resulting in the value of the zeta potential
(]) higher and the velocity of the electroosmotic flow in-
creased. Therefore, the optimum pH was chosen at 7.0.
(2) Effect of SDS concentration. The SDS concen-
tration was particularly important for separating the com-
pounds with the similar structures. When its concentration
is above the critical micelle concentration (CMC), the
surfactant provided a pseudo-stationary phase, with which
the analytes were separated on their different hydrophobic
interactions. The SDS concentration was tested in the
range 0ü35 mmol/L at pH 7.0. An increase in the SDS
concentration helped to resolve the different analytes. But
higher SDS concentration did not increase the resolution
while caused peak broadening and unacceptable high
Joule heating. 25 mmol/L was chosen as the optimum con-
centration.
(3) Construction of calibration curves for catechins
and caffeine. As shown in Table 1, calibration curves for
each compound were established from six concentration
levels of mixed sample, which showed good linearity be-
tween peak area and sample concentration. Relative stan-
dard deviations (RSD) of peak area and migration time
were calculated based on four duplicate injections of a
standard sample. The limits of detection (LOD) were ob-
tained at a 3ĩ1 ratio of signal-to-noise (S/N). The deter-
mination ranges for caffeine and five catechins are from
their limit of detection to 5h105 mol/L The high repro-
ducibility and low LOD indicated that CE is reliable for
analysing catechins and caffeine in tea samples.
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Fig. 1. The chemical structures of the catechins and caffeine.

Table 1 Calibration curves expressed as regression lines (y = ax+b)a)

Compound a b r RSDt (%) RSDA (%) LOD/molgL1

Caffeine 5.446h108 6764.1 0.9988 0.52 3.3 2.1h106

(+)-C 1.037h109 3225.8 0.9985 0.78 3.5 4.3h106
()-EGC 6.112h10 7
2200.6 0.9983 0.96 4.1 5.9h106
()-EGCG 3.572h108 99728 0.9989 0.58 3.3 2.2h106
()-EC 6.746h10 8
7769 0.9993 0.43 2.8 2.3h106
()-ECG 1.786h109 41589 0.9992 0.48 3.2 4.2h106
a) y, Integrated peak area; x, concentration of catechins and caffeine (mol/L); a, slope; b, intercept; r, relative coefficient. RSDt is relative stan-
dard deviation of retention time. RSDA is relative standard deviation of peak area. LOD is limits of determination at 3ĩ1 signal-to-noise ratio.

2440 Chinese Science Bulletin Vol. 48 No. 22 November 2003


(ξ) Application to real samples
(1) Effect of temperature on catechins and caffeine
extraction. The extraction of catechins and caffeine from
the tealeaves with hot water at 100, 90, 70, 50, 30ć for
10 min was studied. From Fig. 2, the concentration of in-
dividual catechin and caffeine increased with the increase
of extraction temperature. EGCG was the easiest to be
extracted at 100ć. In the temperature range from 90ć
to 100ć, the content of the compounds increased gradu-
ally with the exception of ECG. Chen et al.[26] reported the
effects of extraction temperature and time on the poly-
phenol content of Oolong tea and found an increase in
catechin content with increasing extraction temperature
and suggested the total polyphenol levels were highest
when extracted within 80ü90ć. The present work also
shows that the temperature for the extraction of catechins
and caffeine from tealeaves is suitable at 80ü90ć.
Fig. 2. Effect of temperature on catechins and caffeine extraction, as an
example of Xinyang Maojian tea, at different temperatures (100, 90, 70,
50 and 30ć).
(2) Effect of daytime on the content of catechins and
caffeine. It has been proved that drinking tea could prevent
diseases because the catechins in tealeaves played an impor-
tant role. In epidemiological studies, the intake of catechins
has normally been determined by tea consumption, but not all
cups of tea were drunk immediately. A decrease of the con-
centrations with time was found because catechins were un-
stable. Fig. 3 shows the changes of the catechins content in 5
days. Based on the drinking custom of Chinese, we prepared
the green tea sample at 90ć in the above method. Then the
tea sample was stored at 4ć all the time. The content of
the catechins was determined on 5 consecutive days.
A significant decrease of the concentration of EGCG
Chinese Science Bulletin Vol. 48 No. 22 November 2003
REPORTS
and EGC was shown. According to the mechanism of
antioxidative cooperative action, the antioxidative effect
of EGCG and EGC was the most significant. So, the
health protecting activities of tea was reduced with the
increase of the storage time of tea water. There was no
evident change in the concentration of C, EC and ECG,
but the concentration of caffeine increased quickly with
the longer storage time. The reason for the increase of
caffeine may be the degradation of complexes of caffeine
with polyphenols, which slowly released caffeine[27].
These results told us that the home-made tea drink could
be stored at 4ć and drunk at least 5 days even thought
part of EGC and EGCG were decomposed in the water so-
lution. This is why the canned tea is so popular in the world.
(3) Analysis of green and black teas. Green and
black teas are popular in the world, especially in China. The
most difference between them is the manufacture process.
Green tea is manufactured by steaming or drying fresh tea
leaves to prevent oxidation of the green tea polyphenols.
While the manufacture of black tea is characterized by a
high degree of enzymatically catalysed aerobic oxidation of
the leaf polyphenols followed by a series of chemical con-
densations. The compositions of tea varied with species,
season, age of the leaf, climate and horticultural practices.
Four brand green teas, two brand black teas and canned tea
drink were analyzed and the content of catechins and caf-
feine was compared. A typical chromatogram for Xinyang
Maojian tea is shown in Fig. 4. Spiking with standards
identified the peaks.
Fig. 3. Dependence of the content of catechins and caffeine in the tea
extraction solution on the stocking time during 5 days at the same time.
The stocking temperature of the tea solution: 4ć.
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Fig. 4. Electropherogram of the Xinyang Maojian tea sample. The conditions are the same as in Fig. 2, except the buffer pH 7.0. Peak
identification: 1, caffeine; 2, (+)-C; 3, (-)-EGC; 4, (-)-EGCG; 5, (-)-EC; 6, (-)-ECG.

Fig. 5 shows a comparison of the concentration of

catechins and caffeine for different green and black teas.
EGCG was the major constituent of the green tea and the
trends of their individual catechin contents (EGCG>EGC
>EC>ECG>C) were similar. The caffeine content of green
tea was found to be higher than that of catechins except
that in Xinyang Maojian tea. That was probably because
of the difference in resources, agronomic conditions, stor-
age, etc.
The two kinds of black tea reported in this study are
classified as fully fermented tea and showed an overall
similar trend among individual catechins (EGCG>ECG>
EC>EGC>C). The caffeine content of black tea was still
high. Generally, fermentation has little effect on caffeine
levels in tealeaves; therefore, the variation could be due to
the variety of tea and the structure of the leaf that can in-
fluence the kinetics of the infusion process. Fig. 5. Analytical results for the individual five catechins and caffeine
in green tea, black tea and commercial canned green and black tea sam-
At the process of manufacturing canned drink, ples (n=3). Green Tea: 1, Huang-shan Maofeng tea; 2, Dahong Pao; 3,
ascorbic acid was added to maintain the quality, and the West-lake Longjing tea; 4, Xinyang Maojian tea; Black Tea: 5, Qi black
composition of catechins was changed by the high-tem- tea; 6, Dian black tea; Canned tea drink: 7, Tongyi Green tea; 8, Tongyi
black tea.
perature treatment to kill the microorganisms. In our ex-
periment, the contents of the catechins and caffeine in 3 Conclusion
canned drink were a little lower.
An easy and fast analytical method to determine
It is known that the amount and the proportion of
catechins and caffeine in tea was presented and could be
various catechins depend on the agronomic conditions, successfully applied to analysis the real tea samples. In
leaf age, and degree of fermentation, which are directly this research, the relationship between the tea health effect
correlated with the final quality of the beverage[28]. It is and dealing conditions was elucidated and the mechanism
considered that the finest tea is made from the young tea of health protective characteristics will be studied further.
shoots containing the highest levels of catechins. Green
tea prior to black tea manufacturing generally contains a Acknowledgements This work was supported by the National Science
Fund for Distinguished Young Scholars of China (Grant No. 20125514)
high catechin content, which may also account for some and the National Natural Science Foundation of China (Grant Nos.
of the variation in catechin contents. 50273046 and 20275043).

2442 Chinese Science Bulletin Vol. 48 No. 22 November 2003


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(Received June 16, 2003; accepted September 4, 2003)
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