Anal Bioanal Chem (2002) 374 : 561565

DOI 10.1007/s00216-002-1503-8

O R I G I N A L PA P E R

Janina Ohnsmann Guillermo Quints

Salvador Garrigues Miguel de la Guardia

Determination of caffeine in tea samples

by Fourier transform infrared spectrometry

Received: 30 April 2002 / Revised: 17 June 2002 / Accepted: 24 June 2002 / Published online: 10 September 2002
Springer-Verlag 2002

Abstract A sustainable and environmentally friendly procedure has been developed for the FTIR determination of
caffeine in tea leaf samples. The method is based on the
extraction with ammonia and CHCl3 and direct determination of caffeine on the chloroform extracts using peak
height absorbance measurements at 1658.5 cm1 and external calibration. The method provides a sensitivity of
0.2142 absorbance units mg1 mL and a limit of detection
of 1 mg L1, corresponding to 0.002% m/m caffeine in tea
leaves. As compared with a reference procedure, based on
UV absorbance measurement at 276 nm after low pressure column chromatography, the developed procedure reduces the consume of CHCl3 by a factor of 10, that of
NH4OH by a factor of 20 and avoids the use of diethyl
ether and Celite. The time required to do the analysis of a
sample is 15 minutes as compared with the 6 hours for the
reference one.
Keywords Caffeine Tea FTIR Solvent extraction

Introduction
Caffeine is the most important alkaloid present in tea and
thus, the determination of this stimulatory effect alkaloid
is an important factor in the quality control of tea.
Caffeine concentrations vary from one to another type
of tea with typical concentration levels of 3.2% (m/m) for
black tea and concentrations varying from 1 to 3% (m/m)
in green tea [1].
Caffeine has been determined in drinks by different analytical techniques such as UV-VIS spectrophotometry [2,

Janina Ohnsmann is on leave from the University of Mainz

(Germany)
J. Ohnsmann G. Quints S. Garrigues () M. de la Guardia
Department of Analytical Chemistry, University of Valencia,
Edifici Jeroni Muoz, 50th Dr. Moliner,
46100 Burjassot, Valencia, Spain
e-mail: salvador.garrigues@uv.es

3, 4] or amperometry [5] and most frequently by using

separation methods like high performance liquid chromatography (HPLC) [6, 7, 10], ion chromatography [11],
capillary electrophoresis [12, 13, 14], as well as high performance thin layer chromatography (HPTLC) [15, 16,
17] and micellar capillary electrophoresis [18, 19, 20].
The official method for caffeine determination in tea
leaves [21] is based on low-pressure column chromatography and spectrophotometric determination at 276 nm.
This method involves a sample preparation time of more
than 6 hours and the use of 200 mL diethyl ether and
50 mL CHCl3 for each sample. So it is a costly and tedious methodology, making the development of a fast,
simple and less contaminant procedure to determine caffeine in tea necessary.
Caffeine has been determined in other beverages like
coke [22] and coffee samples [23, 24] by the use of FTIR
spectrometry, because this technique offers an excellent
sensitivity and selectivity for this kind of analysis being
possible to do a practically direct determination after a
simple solid phase [22] or a solvent extraction [23, 24].
The main object of this work was the development of
an FTIR method for caffeine determination in tea samples
in order to reduce the time and the solvent volume required as much as possible for this determination.

Experimental
Apparatus and reagents
A Magna FTIR 750 system from Nicolet (Madison, USA)
equipped with a temperature-stabilised DGTS detector with a
Ge/KBr beamsplitter and precise digital signal processing (DSP)
was employed for all the FTIR measurements, with a nominal resolution of 4 cm1, accumulating 25 scans and using a micro flowthrough cell with ZnSe windows and a pathlength of 0.457 mm.
A stopped-flow system was employed for sample introduction
inside the IR cell with a Gilson Minipuls P2 (Villiers-le-Bel,
France) peristaltic pump. For sample transport, Viton (Isoversinic)
3 mm o.d. and 1 mm i.d. pump tubing and PTFE 0.8 mm i.d. connecting tubes were used. For processing the FTIR absorbance data,
the 2.1 version of the Omnic software of Nicolet Corp. was employed.

562
A Hewlett-Packard (Palo Alto, USA) 8452A UV-VIS DAD
system was employed for the UV-Vis determination of caffeine.
All the reagents employed in this study were of analytical
grade. Chloroform, stabilised with 150 ppm of amylene, was obtained from Scharlau (Barcelona, Spain), ammonium hydroxide
30% from Panreac (Barcelona, Spain), and caffeine from Fluka
(Buchs, Switzerland). The tea samples were obtained from the
Spanish market.

determination of caffeine peak height values at 1658.5 cm1, corrected using a baseline at 1800 cm1, obtained for samples were
interpolated in a calibration line obtained from caffeine calibrant
solutions directly prepared in CHCl3 and measured under the same
conditions as the sample extracts and using a background of
CHCl3.

Results and Discussion

Reference procedure
A 1-g ground sample was accurately weighed inside a 100-mL
beaker, 50 mL NH4OH (4 mol/L) was added and the mixture
warmed on a boiling H2O bath for 2 min. After cooling, transferred
quantitatively to a 100-mL volumetric flask and diluted to the volume with H2O. Then, 5-mL aliquots of the turbid solution were
carefully mixed with 6 g Celite 545 and the mixture was placed inside a 25250 mm tube column to obtain a homogenous and compact layer above a glass wool plug and a mixture of 3 g Celite 545
and 2 mL NaOH 2 mol/L. This basic column was mounted above
the acid column prepared from well mixed 3 mL 2 M H2SO4 and
3 g Celite 545 placed between glass wool layers inside a 25
250 mm tube. A 150-mL aliquot of H2O-saturated ether was sequentially passed through the basic and acidic columns and discarded. A 50-mL aliquot of H2O-saturated ether was then passed
through the acidic column and discarded. A 48-mL aliquot of H2Osaturated CHCl3 was also passed through the acidic column. The
first portions of the eluent were then passed through the basic column and the caffeine solution recovered in a 50-mL volumetric
flask. The column eluate was diluted to the mark with H2O-saturated CHCl3 and the absorbance read at 276 nm against an H2Osaturated CHCl3 blank, using caffeine CHCl3 solutions as calibrants.

FTIR spectra of caffeine and tea extracts

Figure 1 shows the FTIR spectra of the caffeine calibrant
solution in chloroform and a tea extract. Both spectra are
very similar and have a characteristic absorption that can
be used for the determination of caffeine the main difference being the presence at 1730 cm1 of an overlapped
band probably due to the absorption of other extracted
components present in tea samples. The carbonyl bands at
1704.8 cm1 and 1658.5 cm1 are the most intense bands,
but additionally there are others present at 1554.4 cm1,
1415.5 cm1 and 1361 cm1. So that could be useful for
the analysis of CHCl3 extracts obtained from tea leaves.
In Figure 2, the spectra of three different kinds of tea
present the shoulder at 1730 cm1 but demonstrate that is
possible to identify the main characteristic bands of caffeine in every tea sample extracted with CHCl3. So different studies were carried out in order to find the appropriate conditions for the quantitative extraction of caffeine
and its determination in the simplest way possible .

Recommended Procedure
A 250-mg sample of a blended tea was accurately weighed in a
glass tube, 5 mL NH4OH 2 mol/L was added and then the mixture
was mechanically shaken for 2 min. Then 5 mL CHCl3 were added
and the mixture mechanically shaken for an additional 2 min. After that, the mixture was centrifuged for 10 min.
The chloroform solution was introduced into a microflowthrough IR cell using a peristaltic pump and the FTIR spectra obtained between 4000 and 600 cm1 at 4 cm1 resolution and accumulating 25 scans for spectra. The background spectrum was established from the cell filled with a blank extract solution. For the

Caffeine extraction from an aqueous solution to a chloroform phase is a well known quantitative process. For this
reason, in the determination of caffeine in tea samples, the
main problem is to guarantee the extraction of the caffeine
from the solid to an aqueous phase. Previous studies [23,
24] proved that caffeine can be easily extracted by using






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Fig. 1 FTIR spectra of a caffeine

standard of 1.5 mg mL1 (), a sample extract (--) and a blank solution
(--). Every spectrum was obtained
for 25 cumulated scans at a resolution
of 4 cm1. For the sample extract the
spectrum was obtained using a blank
solution as background, the caffeine
standard was measured in the same
conditions as the sample extract but
using a background of CHCl3

Study of the extraction conditions




















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563
Fig. 2 FTIR spectra of a Jasmine,
Green and Ceylon tea samples extracted in CHCl3

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alkaline solutions such as ammonia. So the shaking mode,

shaking time and the volume and concentration of
NH4OH were studied in order to obtain the best conditions
for a quantitative extraction of caffeine from tea leaves.
Jasmine tea samples and peak height measurements at
1658.5 cm1, corrected using a baseline at 1800 cm1,
were employed in order to search for the best extraction
conditions.
Two different shaking modes, mechanical and ultrasonic, were assayed. For both modes, shaking times from
1 to 5 minutes were evaluated. From the results obtained
it can be concluded that there is no significant (at p=0.05)
difference between sonication and mechanical shaking,
the simplest method being the latter procedure. Using the
mechanical mode, caffeine was quantitatively recovered
after a 2-min shaking time.
The increase in NH4OH concentration from 0.25 to
4 mol/L does not influence the caffeine extraction. So a
NH4OH 2 mol/L concentration was selected in order to
guarantee a precise and robust procedure.
The effect of the volume of the NH4OH was evaluated
from 5 to 10 mL, it was found that 5 mL was enough to
obtain a quantitative recovery of caffeine from tea. The
use of reduced volumes of NH4OH and CHCl3 permitted
us to carry out all the different steps of the extraction,
such as solid-liquid extraction with ammonia, liquid-liquid extraction with chloroform and phase separation
through centrifugation.
Selection of the appropriate bands
for FTIR determination of caffeine
Figure 1 shows that at least two bands, namely 1704.8 and
1658.5 cm1, could be used for the quantitative determination of caffeine in tea extracts. For these bands, peak
height measurements were considered. When applying
two alternative baseline criteria for the carbonyl band at
1704,8 cm1, it is possible to compensate for an eventual







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Table 1 Calibration lines obtained for FTIR determination of caffeine using different bands
RSDc
%

LODd
(mg L1)

y =0.0017+0.1333Ca 0.9984

0.3%

1.5

y =0.0005+0.1053C

0.9995

0.3%

2.2

y =0.0016+0.2142C

0.9993

0.1%

y =0.0001+0.0361C

0.961

1.3%

4.8

y =0.0009+0.0177C

0.8995

5.1%

2.1

Band
(Baseline)

Calibration line

1704.8 cm1
(1800 cm1)
1704.8 cm1
(1683 cm1)
1658.5 cm1
(1800 cm1)
1554.4 cm1
(1800 cm1)
1415.5 cm1
(1800 cm1)

R2 b

Notes: Calibration lines were established in the concentration

range from 0 to 4 mg mL1 caffeine using 5 standard solutions;
aC: Concentration of caffeine in mg mL;
bR2 : Regression coefficient;
cRSD: Relative standard deviation for five independent measurements of a sample containing 1.45 mg mL1 caffeine;
dLOD Limit of detection established for a probability level of
99.6% (k=3).

matrix interference during the extraction step (see Table 1).

In order to choose between the criteria, the height ratio
based on the most intense bands was calculated for the
calibration solution, sample extracts and spiked samples.
In fact, a constant proportion between bands considered in
all types of solutions indicates a good comparability between natural sample extracts, external standards and
standard addition solutions.
Table 2 shows the peak height ratio between the three
bands considered working with a fixed baseline criterion
(at 1800 cm1) and with corrected values at 1883 cm1 for
the carbonyl bands at 1704.8 and 1658.5 cm1.
As it can be seen, the bands at 1704 and 1658.5 cm1
are those which provided the best sensitivity and the best
comparability when considering pure CHCl3 solutions,
extracts obtained from natural samples and those found

564
Table 2 Peak height ratio through bands presented by caffeine at 1704.8 cm1, 1658.5 cm1 and 1554.4 cm1 in different types of sample extracts and standards

Standards
Ceylon Tea
Jasmine Tea
Green Tea
Spiked Ceylon Tea
Spiked Jasmine Tea

H1704/H1658 sa

H1704/H1554 sa

H1658/H1554 sa

H1704/H1658 sb

H1704/H1554 sc

H1658/H1554 sd

0.632 0.002
0.6340.002
0.6320.002
0.6300.002
0.6330.002
0.6340.001

3.80.3
3.610.05
3.730.09
3.410.05
3.90.1
3.770.03

6.10.4
5.690.07
5.90.1
5.420.08
6.10.1
5.940.06

0.5650.004
0.5680.005
0.5790.006
0.5580.003
0.5730.002
0.5740.002

3.00.1
2.730.07
3.00.1
2.530.05
3.00.1
2.930.03

5.40.2
4.810.09
5.20.2
4.540.07
5.30.2
5.100.06

aUsing

a common baseline established at 1800 cm1 for all the bands.

a baseline correction at 1883 cm1for the bands at 1704.8 and 1658.2 cm1, and corrected at 1800 cm1 for the band at
1554.4 cm1.
b,c,dConsidering

Fig. 3 FTIR spectra from the residue

remaining after evaporation onto a
BaF2 plate of 100 L of caffeine standard solution and the chloroform extracts of tea samples. The spectra have
been shifted to avoid band overlapping



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for spiked samples, thus indicating that a method based on

the aforementioned bands could be a robust alternative for
caffeine determination in tea leaves. However, the band at
1704.8 cm1 could be affected by that at 1730 cm1 which
appears in some sample extracts and, because of that peak
height measurements at 1658.5 cm1 established with a
baseline at 1800 cm1, seems the best alternative for FTIR
determination of caffeine in tea.
On the other hand, Figure 3 shows the FTIR spectra of
the solid residue obtained after evaporation, onto a BaF2
plate, of a few microliters of the chloroform extracts of
different tea samples considered and a calibrant solution
of caffeine. On comparison with the caffeine standard,
one can see differences in the relative intensity of the
band at 1704.8 cm1 especially for Jasmine tea samples;
which confirms the selection of the band at 1658.5 cm1
for quantification of caffeine in the organic extracts of tea
samples.

Analysis of natural samples of tea

As can be seen in Table 3, caffeine concentrations found
by FTIR in three types of tea samples are comparable to
those found in the literature and data found are of the
same order to those obtained by the official method.

Table 3 Results obtained for the determination of caffeine in tea

samples by different procedures
Sample

Caffeine % (m/m)
FTIR determination

Ceylon Tea
Green Tea
Jasmine Tea

LLEa
preparation

AOACb
preparation

2.670.03
2.700.02
2.290.04

2.590.02
2.210.02
2.190.07

Reference
method

2.10.4
2.10.2
1.90.1

aLiquid-liquid

extraction;
determination after the official AOAC preparation method.
Data reported are the average of 3 independent determination the
corresponding standard deviation.

bFTIR

In fact, it seems that the results from the reference

method based on UV detection at 276 nm are a little bit
low and that the simple liquid-liquid extraction and FTIR
measurement gives errors a little too high compared with
FTIR determinations carried out after the AOAC preparation procedure.
In order to verify the accuracy of the developed procedure, recovery experiments were carried out by spiking
Ceylon and Jasmine tea samples with amounts of caffeine

565
Table 4 Recovery of caffeine from tea samples
Sample

Caffeine
added (mg)

Caffeine
found (mg)

Recovery
(%)a

Mean
recovery (%)

Ceylon Tea

2.50
5.00
7.35

2.62
4.75
6.95

104.70.2
94.90.2
94.60.2

986

Jasmine Tea 2.51

4.92
7.33

2.51
4.82
6.84

100.20.6
98.00.1
93.30.3

973

Acknowledgements. The authors acknowledge the financial support of the Generalitat Valenciana Project GV01249. G. Quints
acknowledge the grant provided by the Laboratorio de Higiene
Laboral y Ambiental of the Universitat de Valencia and J. Ohnsmann acknowledge the Erasmus grant of the European Union.

References

aValues correspond to the average recovery the standard deviation of 3 independent determinations

ranging from 2.5 to 7.35 mg. Data in Table 4 show that

average recovery percentages vary from 97 to 98% thus
indicating the absence of systematic errors of the FTIR
measurements carried out after solvent extraction of samples with ammonia and CHCl3.
Analytical performance of the developed procedure
The methodology developed for caffeine determination in
tea leaves by FTIR measurement of CHCl3 extracts obtained after extraction of the solid samples with ammonia
gives a sensitivity of 0.2142 absorbance units per mg
mL1 caffeine with a limit of detection of 1 mg mL1, corresponding to a 0.002% m/m concentration in the solid
sample and a limit of quantification of 3.4 mg L1 thus of
the order of 0.007% m/m caffeine in tea leaves (taking
into account the extraction of 250 mg sample with a final
volume of 5 mL CHCl3).
On comparing the solvent extraction FTIR procedure
with the reference one using low pressure chromatography and UV determination, the consumption of CHCl3
was reduced by a factor of 10 and that of NH4OH by a
factor of 20, thus providing a cheaper alternative. The use
of diethyl ether is also avoided and as well as that of
Celite, thus providing an economic sustainable procedure
which drastically reduces the environmental impact. In
addition, the time required for each determination is approximately 15 minutes, compared with 6 hours needed
for the reference procedure. So, the new procedure not
only increases the productivity of the laboratory but also
reduces the cost of each determination.

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