ANALYTICAL SCIENCES MARCH 2001, VOL.

17
2001 The Japan Society for Analytical Chemistry

391

Multielement Determination and Speciation of Major-to-Trace

Elements in Black Tea Leaves by ICP-AES and ICP-MS
with the Aid of Size Exclusion Chromatography
Hirotaka MATSUURA, Akiko HOKURA, Fumie KATSUKI, Akihide ITOH, and Hiroki HARAGUCHI
Department of Applied Chemistry, Graduate School of Engineering, Nagoya University,
Furo-cho, Chikusa, Nagoya 4648603, Japan

A multielement determination of major-to-trace elements in black tea leaves and their tea infusions was carried out by
ICP-AES (inductively coupled plasma atomic emission spectrometry) and ICP-MS (inductively coupled plasma mass
spectrometry). Tea infusions were prepared as usual tea beverage by brewing black tea leaves in boiling water for 5 min.
About 40 elements in tea leaves and tea infusions could be determined over the wide concentration range in 8 orders of
magnitude. The extraction efficiency of each element was estimated as the ratio of its concentration in tea infusions to
that in tea leaves. From the experimental results for the extraction efficiencies, the elements in black tea leaves were
classified into three characteristic groups: (i) highly-extractable elements (>55%): Na, K, Co, Ni, Rb, Cs and Tl, (ii)
moderately-extractable elements (20 55%): Mg, Al, P, Mn and Zn, and (iii) poorly-extractable elements (<20%): Ca, Fe,
Cu, Sr, Y, Zr, Mo, Sn, Ba and lanthanoid elements. Furthermore, speciation of major-to-trace elements in tea infusions
was performed by using a combined system of size exclusion chromatography (SEC) and ICP-MS (or ICP-AES). As a
result, many diverse elements were found to be present as complexes associated with large organic molecules in tea
infusions.
(Received October 6, 2000; Accepted December 4, 2000)

Introduction
Tea is one of the worlds most popular beverages. Thus, the
chemical components in tea leaves and their tea infusions have
received great interest because they are related to health and
The recent development of analytical atomic
disease.1,2
spectroscopy, such as ICP-AES (inductively coupled plasma
atomic emission spectrometry) and ICP-MS (inductively
coupled plasma mass spectrometry), allows the determination of
a variety of elements, even at trace and ultratrace levels.3,4
Consequently, ICP-AES and ICP-MS have been extensively
applied to the determination of trace and ultratrace elements in
biological and environmental samples.47 The multielement
determination of diverse elements in green tea leaves of tea
plant (Camellia sinensis) was also performed by ICP-AES and
ICP-MS.8,9
The concentrations and extraction behaviors of diverse
elements in black tea samples have been investigated by several
Natesan and Ranganathan determined the
workers.1012
concentrations of 20 elements in black tea leaves and tea
infusions.10 According to the extraction efficiencies from black
tea leaves, they classified analyte elements into the following
three groups; Ba, Ca, Fe, and Sr were less extractable (<10%),
Al, B, Cd, Co, Cr, Cu, Mg, Mn, P, Pb, Si, and Zn moderately
extractable (10 50%), and K, Na, and Ni highly extractable

To whom correspondence should be addressed.

A. H. present address: National Institute for Research in
Inorganic Materials, Science and Technology Agency, Namiki
1-1, Tsukuba 3050044, Japan.

(>50%).10 degrd and Lund also found that the extraction

efficiencies of the elements into tea infusion were 3 6% for
Ca, V, Fe, and Sr, and 29 85% for Mg, Al, Cr, Mn, Co, Cu,
Zn, Ni, Rb, and Tl.11 In addition, Lamble and Hill reported the
extraction efficiencies of 7 elements (Al, Ba, Ca, Cu, Mg, Mn,
and Zn) for 12 different origins of tea samples.12 The extraction
efficiencies were below 10% for Ba and Ca, and 10 50% for
Al, Mg, Mn, Zn, and Cu. In all of the samples, except for
Chamomile tea, Zn was most soluble among 7 elements.
In the case of tea infusions, the biological effects of Al have
received great attention in relation to neurological and
behavioral disorders,1315 for example, Alzheimers disease.
Thus, speciation analyses of Al by using HPLC coupled with
atomic spectrometric techniques have been carried out to obtain
some information about Al species in tea infusions.11,16,17 From
these results, it was suggested that Al in tea infusions was fully
or partly bound with relatively large organic molecules, which
might be polyphenolic compounds, antioxidants. In spite of
many studies on diverse elements in the black tea leaves and tea
infusion samples, their biological effects on human bodies have
not been well understood on a molecular basis. Therefore, the
multielement information about chemical species in tea leaves
and tea infusions is required, rather than the total concentrations
of some specific elements.
In a previous paper,9 we developed an analytical method for
the multielement determination of about 40 elements (major-toultratrace elements) in green tea leaves and their tea infusions
by ICP-AES and ICP-MS, and estimated their extraction
efficiencies. Therefore, in the present study, a similar method
was applied to the multielement determination of major-toultratrace elements in black tea leaves and tea infusions. Then,

392
Table 1 The operating conditions for ICP-MS and ICP-AES
instruments
ICP-MS SPQ 8000A (Seiko Instruments)
Plasma conditions
Rf frequency
27.12 MHz
Rf power
1.0 kW
Gas flow rate
Carrier gas
Ar 1.0 l min1
Auxiliary gas
Ar 1.0 l min1
Coolant gas
Ar 16 l min1
Sampling conditions
Sampling depth
12 mm from work coil
Sampling cone
copper, 1.1 mm orifice diameter
Skimmer cone
copper, 0.35 mm orifice diameter
Nebulizer
glass concentric type
Sample uptake rate
0.7 ml min1
Data acquisition
multielement mode by peak hopping
Data point
3 points/peak
Dwell time
10 ms/point
Integration
100 times
ICP-AES Plasma AtomComp Mk II (Jarrell-Ash)
Plasma conditions
Rf frequency
27.12 MHz
Rf power
1.0 kW
Gas flow rate
Carrier gas
Ar 0.5 l min1
Auxiliary gas
Ar 1.0 l min1
Coolant gas
Ar 20 l min1
Sampling conditions
Observation height
18 mm above work coil
Nebulizer
cross-flow type
Sample uptake rate
1.2 ml min1
Spectrometer conditions
Polychromator
PaschenRunge mounting
Focal length
75 cm
Grating
2400 grooves mm1
Entrance slit width
25 m
Exit slit width
50 m
Data acquisition
Integration time
10 s
Repetition
3 times

the extraction efficiencies of diverse elements in black tea

leaves were estimated from the concentrations of diverse
elements in tea infusions. Furthermore, speciation analyses of
diverse elements binding to organic molecules in tea infusions
were performed using a combined system of size exclusion
chromatography (SEC)/UV absorption/ICP-MS (or ICPAES).18,19

Experimental
Instrumentation
An ICP-MS instrument (Model SPQ 8000A from Seiko
Instruments, Chiba), which was equipped with a quadrupoletype mass spectrometer, was used for the determination of trace
and ultratrace elements in black tea leaves and tea infusion
samples. The major and minor elements in them were also
determined by an ICP-AES instrument (Model Plasma
AtomComp Mk II from Jarrell-Ash, Franklin, MA, USA) with
39 channels (photomultipliers) for simultaneous detection. The
operating conditions for the ICP-MS and ICP-AES instruments
are listed in Table 1. These conditions were established after
optimization of the instrumental parameters.

ANALYTICAL SCIENCES MARCH 2001, VOL. 17

A chromatographic system was composed of a pump (LC-9A,
Shimadzu, Kyoto), a sample injector (Rheodyne, Cotati, CA,
USA) with a loop volume of 200 l, an SEC column
(Superdex75 HR10/30, Pharmacia Biotech, Upsala, Sweden),
and a UV-visible absorption detector (870-UV, Jasco, Tokyo).
The permeation of the SEC column used was in the range from
1000 Da to 300000 Da. A molecular-weight calibration of the
SEC column was made by using standard molecules with
known molecular weights (MWs).9 The total exclusion and
total permeation volumes of the SEC column were 8.0 ml for
blue dextran and 24.4 ml for 0.5% HNO3, respectively, in
retention volume. This chromatographic system was coupled
with the ICP instrument via a Teflon tubing between the UV
detector and the nebulizer of ICP-MS or ICP-AES. In the
combined system, the ICP-MS and ICP-AES instruments were
used as element-selective detectors for simultaneous
measurements of multielement chromatograms.
Chemicals and samples
Nitric acid and hydrofluoric acid used for sample digestion
were of electronics industry grade, purchased from Kanto
Chemicals (Tokyo). As a mobile phase, 1 M ammonium acetate
buffer solution (pH 5.8) was prepared by mixing ammonia
solution and acetic acid solution. The following compounds
were used for the molecular-weight calibration of the SEC
column: vitamin B12 (1355), aprotinin (6500), cytochrome c
(12400), carbonic anhydrase (29000), albumin (66000), alcohol
dehydrogenase (150000), -amylase (200000), and blue dextran
(2000000), where the numbers in the parentheses indicate the
molecular weights in Da. All of these chemicals were obtained
from Sigma Chemicals (St. Louis, MO, USA). Deionizeddistilled water used throughout the present experiment was
prepared by a Milli-Q system (Model Milli-Q SP TOC from
Nihon Millipore Kogyo, Tokyo).
Commercially-available black tea leave samples (Darjeeling)
packed in tea bags were used for analysis, which were
purchased in a supermarket. They were ground into a powder
with an agate pestle and mortar for homogenization before use.
Digestion procedure of black tea leaves
The black tea leave samples were digested to prepare their
analysis solutions in a manner similar to that in previous
papers.9,20,21 First, 0.5 g of black tea leaves was taken in a PTFE
Teflon beaker. After adding 5 ml of conc. HNO3, the sample
was kept standing overnight at room temperature for predigestion. Then, 5 ml of conc. HNO3 was further added to the
beaker, and the sample was heated on a hot plate in sequence at
75C for 0.5 h, 130C for 0.5 h and 200C for 2 h. In order to
decompose siliceous materials in black tea leaves,21 2 ml of
conc. HF was added into the residual solution, which was then
further heated almost to dryness at 180C for 2 h and 230C for
3 h. The residue was dissolved with 7.5 ml of conc. HNO3 and
diluted to 100 ml with pure water. The solution was then
filtered with a membrane filter (pore size 0.45 m), which was
subjected as the analysis solution to determine the total
concentrations of diverse elements in black tea leaves by ICPAES and ICP-MS. In the ICP-MS measurement, the internal
standard elements (Ge, In, and Re) were added to be 10 ng ml1
each in the analysis solution for correcting matrix effects.22
Preparation of tea infusion samples
Tea infusion samples were prepared while taking into account
drinking habits. About 2 g of a powdered black tea leaves
sample was taken in a Teflon beaker and brewed in 100 ml of
boiling water for 5 min to extract the components of tea leaves.

ANALYTICAL SCIENCES MARCH 2001, VOL. 17

The extracted solution (tea infusion) was then filtered with a
membrane filter (pore size 0.45 m). One milliliter of tea
infusions was subjected to a speciation analysis, and the rest
was used for a quantitative analysis. The acidity of the black
tea infusion solution was adjusted to 1 M HNO3 by adding conc.
HNO3, in which the internal standard elements (Ge, In, and Re)
were also added to be 10 ng ml1 each. This tea infusion sample
was prepared just before the ICP-MS or ICP-AES measurement,
because tea infusion added with acid produced some
precipitates, perhaps organic materials, when the acidic tea
infusion sample was preserved for one or two days.23
Speciation analyses of major-to-ultratrace elements in black tea
infusions by the SEC/ICP-MS system
In order to obtain information about the chemical species of
diverse elements extracted from black tea leaves, a speciation
analysis was carried out using the SEC/UV absorption/ICP
combined system. In chromatographic measurements, the
organic buffer solution was constantly nebulized into the
plasma, which resulted in an energy loss of the RF power for
sustaining the plasma. Then, in a following experiment, the RF
power of the ICP-MS instrument was set at 1.3 kW, while it was
1.0 kW under the conventional operating conditions. The 1.0 M
ammonium acetate buffer solution (pH 5.8) was employed as
the mobile phase, whose pH was adjusted to that of the tea
infusion sample. The flow rate of the mobile phase was 1.0 ml
min1, and the injection volume of the tea infusion sample was
200 l.
In chromatogram measurements, the eluent from the column
was first detected at 260 nm by a UV absorption detector and
then by ICP-AES or ICP-MS. In the case of ICP-AES
detection, the chromatograms for 12 elements with the sign *
listed in Table 2 were obtained simultaneously. On the other
hand, in the case of ICP-MS detection, the data acquisition was
performed in the peak-hopping mode over 15 m/z positions
Consequently, the
during a 5 s measurement time.18,19
chromatograms of 15 elements (or at 15 m/z values) could be
obtained in real-time data acquisition for 90 min by the present
SEC/ICP-MS system.

Results and Discussion

Concentrations of major-to-ultratrace elements in black tea
leaves
The major-to-ultratrace elements in black tea leave samples
were determined by ICP-AES and ICP-MS after acid digestion.
Since the analytical figures of merit for the ICP-AES and ICPMS measurements, as well as the analytical reliabilities of the
present methods for plant sample digestion, were evaluated by
analyzing various plant reference materials in previous
papers,20,24 only the analytical results for the total concentrations
of 40 elements in the black tea leave samples are given in Table
2, where the concentration of each element was estimated on the
dry-weight basis. It can be seen from Table 2 that black tea
leaves contain diverse elements over a wide concentration range
from major to ultratrace levels. Among them, the highest value
is 19200 g g1 of K and the lowest value is 2.7 ng g1 of W.
The relative standard deviations (RSDs) are also given in Table
2. The RSD values are below 10% for almost all elements,
except for Fe, Zr, and Th, which are known as the refractory
elements. The rather large RSDs of such refractory elements
may be ascribed to incomplete decomposition or inhomogeneity
of the black tea leave samples.
In previous papers, we reported similar analytical results for

393
green tea leaves9 and tea leaves standard reference material.20,24
Therefore, the total concentrations for 40 elements in green tea
leaves and tea leaves standard reference material (NIES No. 7)
are also summarized in Table 3 for a comparison. In order to
compare the concentrations of the elements in three tea leave
samples comprehensively, the concentration ratios of the
elements in black tea leaves or tea leaves standard reference
material to those in green tea leaves are given in Table 3. In
addition, the elemental concentration profiles for three tea
leaves are also illustrated in Fig. 1. Since raw materials and the
preparation methods, as well as the production places, for these
three kinds of tea leave samples are different from each other, it
is difficult to draw any general or common conclusion from
Table 3 or Fig. 1. Even so, the following comments can be
made here. It can be seen from Table 3 that the elemental
concentrations of black tea leaves are generally higher than
those of green tea leaves. In the production process, black tea
leaves are air-dried before fermentation, while green tea leaves
are steamed with water vapor. Thus, there is a possibility of
some losses of the elements in tea leaves during the steaming
process in the case of green tea leaves. Such losses of the
elements may be the reason why the elemental concentrations in
green tea leaves are lower than those in black tea leaves. On the
other hand, the elemental concentrations of tea leaves standard
reference material are lower than those in green tea leaves. In
preparing standard reference material, tea leaves are first ground
into powder and filtered to collect only fine particles for sample
homogeneity. In these preparation processes of tea leaves
reference material, only soft tissues of tea leaves may be
collected and hard tissues are discarded. Consequently, it is
considered that the elements enriched in hard tissues of tea
leaves may be lost in the case of tea leaves reference material.
Extraction efficiencies of diverse elements in tea infusions from
black tea leaves
In general, the concentrations of the elements in tea infusions
are related to the dietary intake amounts of mineral nutrients
from tea leaves. Such intake amounts depend on the extraction
efficiencies of the constituents under the brewing conditions, as
well as on the total concentrations of the elements in tea leaves.
Therefore, in the studies on elemental analysis for tea samples,
the extraction efficiency is generally examined to characterize
the dissolved components of analyte elements in tea
infusions.1012
The tea infusion samples extracted from black tea leaves were
also analyzed in the present experiment in order to evaluate the
extraction efficiencies; 35 elements could be determined by
ICP-AES and ICP-MS. The results are summarized in Table 2.
It can be seen from Table 2 that various elements over a wide
concentration range are extracted in black tea infusions,
although some elements, such as Cd, W, Pb, Th and U, found in
black tea leaves sample could not be determined in tea
infusions. The RSDs for analytical values are also listed in
Table 2. These RSD values were around 10% for most
elements, expect for Mo. The poor precision for Mo may be
ascribed to its extremely low concentration, close to the
analytical detection limit obtained by the ICP-MS instrument.
In the present study, the extraction efficiency for each element
was estimated as the ratio (%) of its concentration in tea
infusions to its total concentration in black tea leaves, which
could be calculated from the analytical results in Table 2. The
extraction efficiencies of 35 analyte elements are shown in Fig.
2. The confidence limits for the extraction efficiencies were
calculated from the law of propagation of errors, which are also
shown as the error bars in Fig. 2.

394

ANALYTICAL SCIENCES MARCH 2001, VOL. 17

Table 2 Analytical results for the concentrations of diverse elements in black tea leaves and tea infusions determined by ICP-AES
and ICP-MS

Elementa
K*
Ca*
P*
Mg*
Al*
Mn*
Fe*
Rb
Na*
Zn*
Sr*
Ba*
Cu*
Ni
Sn
Pb
Co
Y
Ce
La
Nd
Cs
Zr
Pr
Tl
Dy
Gd
Sm
Yb
Mo
Er
Cd
Th
Eu
Ho
Tb
Lu
Tm
U
W

Black tea leaves

Wavelengthb
or m/z
766.4 nm
317.9 nm
213.6 nm
279.0 nm
308.2 nm
257.6 nm
259.9 nm
85
589.0 nm
213.8 nm
407.7 nm
493.4 nm
329.7 nm
60
118
208
59
89
140
139
143
133
90
141
205
163
157
147
174
98
167
111
232
151
165
159
175
169
238
184

Concentration/g g
I
II
I
II
I
II
II
I
I
II
II
I

19200 1000
4550 66
2470 30
2070 30
807 59
503 7
134 48
44.2 0.8
37.0 4.1
36.6 0.7
30.2 0.8
29.5 1.0
27.7 0.7
8.06 0.19
0.786 0.065
0.709 0.020
0.506 0.042
0.360 0.010
0.336 0.040
0.335 0.018
0.257 0.015
0.200 0.026
0.143 0.034
0.069 0.004
0.063 0.006
0.057 0.002
0.055 0.0003
0.052 0.003
0.043 0.001
0.041 0.001
0.037 0.001
0.018 0.0003
0.017 0.008
0.013 0.0001
0.012 0.001
0.0086 0.0001
0.0070 0.0003
0.0061 0.0002
0.0057 0.0007
0.0027 0.0003

Tea infusions
c

Concentration/g ml1

RSDc, %

5
1
1
1
7
1
36
2
11
2
3
3
3
2
8
3
8
3
12
5
6
13
24
6
9
3
0.5
5
3
3
3
2
48
1
4
1
4
3
13
11

305 21
4.03 0.02
20.0 0.6
19.6 0.6
5.54 0.24
5.16 0.18
0.18 0.03
0.906 0.014
0.717 0.070
0.288 0.002
0.0329 0.0005
0.0447 0.0013
0.102 0.004
0.125 0.005
0.00034 0.00003
< 0.00038
0.0057 0.00001
0.00086 0.00002
0.00021 0.00001
0.00028 0.00001
0.00032 0.00002
0.0041 0.00001
0.00012 0.00002
0.000070 0.000001
0.0010 0.00002
0.00013 0.00001
0.00010 0.000003
0.000068 0.000008
0.000092 0.000009
0.00012 0.00005
0.000096 0.000009
n.d.d
n.d.d
0.000021 0.000001
0.000028 0.000003
0.000019 0.000001
0.000013 0.000001
0.000015 0.000002
< 0.000022
< 0.000039

7
0.4
3
3
4
3
16
2
10
0.8
1
3
4
4
10

0.1
2
4
2
6
0.1
20
2
2
7
3
11
10
38
9

4
9
4
10
10

RSD , %

a. The elements with * were determined by ICP-AES, and other elements by ICP-MS. b. I and II indicate atomic and ionic lines,
respectively. c. RSD: relative standard deviation. d. n.d.: not detected.

In our previous study on green tea leaves,9 the extraction

efficiencies of analyte elements were classified into 3 groups:
highly-extractable (>55%), moderately-extractable (20 55%),
and poorly-extractable (<20%).
Thus, the extraction
efficiencies of analyte elements in black tea leaves shown in
Fig. 2 were also classified into 3 groups, followed by the above
classification guideline.
(i) highly-extractable elements (>55%):
Cs, Rb, Na, Tl, K, Ni, Co
(ii) moderately-extractable elements (20 55%):
Mn, Mg, P, Zn, Al
(iii) poorly-extractable elements (<20%):
Cu, Mo, Y, Ba, Fe, Sr, Ca, Zr, Sn, lanthanoid
elements,
(Pb, Cd, Th, U, W)

In Fig. 2, the extraction efficiencies of analyte elements from

green tea leaves are also shown as the black bars. It is seen in
Fig. 2 that the extraction efficiencies of the elements in black tea
leaves are significantly larger, except for Co and Sn, than those
in green tea leaves. This may be explained by the fact that the
cell tissues of black tea leaves are broken during fermentation in
the tea production process, which makes it easy to extract the
elements inside the cells. Even so, the classification of the
elements according to the extraction efficiencies is almost the
same, except for Tl, for both black tea leaves and green tea
leaves.
All alkali elements (Na, K, Rb, Cs) together with Co, Ni and
Tl are highly-extractable from tea leaves. The common
chemical properties of these elements are highly or quite ionic.
It is generally known for plants that Na is more abundant inside
the cells, while K is more abundant outside the cells. However,
both Na and K were highly extractable, as can be seen from Fig.

ANALYTICAL SCIENCES MARCH 2001, VOL. 17

395

Table 3 Concentrations (g g1) of diverse elements in black tea leaves, green tea leaves, and tea leaves standard reference material
(NIES No. 7) determined by ICP-AES and ICP-MS
Elementa
K*
Ca*
P*
Mg*
Al*
Mn*
Fe*
Rb
Na*
Zn*
Sr*
Ba*
Cu*
Ni
Sn
Pb
Co
Y
Ce
La
Nd
Cs
Zr
Pr
Tl
Dy
Gd
Sm
Yb
Mo
Er
Cd
Th
Eu
Ho
Tb
Lu
Tm
U
W

Black tea leavesb

19200 1000
4550 66
2470 30
2070 30
80 59
503 7
134 48
44.2 0.8
37.0 4.1
36.6 0.7
30.2 0.8
29.5 1.0
27.7 0.7
8.06 0.19
0.786 0.065
0.709 0.020
0.506 0.042
0.360 0.010
0.336 0.040
0.335 0.018
0.257 + 0.015
0.200 + 0.026
0.143 + 0.034
0.069 0.004
0.063 0.006
0.057 0.002
0.055 0.0003
0.052 0.003
0.043 0.001
0.041 0.001
0.037 0.001
0.018 0.0003
0.017 0.008
0.013 0.0001
0.012 0.001
0.0086 0.0001
0.0070 0.0003
0.0061 0.0002
0.0057 0.0007
0.0027 0.0003

[0.93]
[1.17]
[0.85]
[0.94]
[1.04]
[0.69]
[1.20]
[1.27]
[0.51]
[1.30]
[2.03]
[2.42]
[2.64]
[1.73]
[11.4]
[0.97]
[2.24]
[2.09]
[1.58]
[1.75]
[2.02]
[0.86]
[1.13]
[1.97]
[1.20]
[2.44]
[2.02]
[2.16]
[3.52]
[1.32]
[2.59]
[0.44]
[1.63]
[2.29]
[2.53]
[2.21]
[3.50]
[3.21]
[1.22]
[0.38]

Green tea leavesc

20600
3900
2890
2200
776
730
112
34.9
72.9
28.1
14.9
12.2
10.5
4.65
0.069
0.734
0.226
0.172
0.213
0.191
0.127
0.233
0.127
0.035
0.053
0.023
0.027
0.024
0.012
0.031
0.014
0.041
0.010
0.0057
0.0048
0.0039
0.0020
0.0019
0.0047
0.0071

Tea leaves standard

reference material (NIES No. 7)d
18000
3090
3000
1400
670
611
88
6.4
15.6
31.7
3.77
5.76
7.6
6.29
0.21
0.784
0.11
0.078
0.077
0.068
0.052
0.021
n.d.e
0.014
0.0059
0.0084
0.012
0.0097
0.0042
0.012
0.0053
0.025
0.0039
0.0023
0.0019
0.0018
0.00062
0.00062
0.0018
0.006

[0.87]
[0.79]
[1.04]
[0.64]
[0.86]
[0.84]
[0.79]
[0.18]
[0.21]
[1.13]
[0.25]
[0.47]
[0.72]
[1.35]
[3.04]
[1.07]
[0.49]
[0.45]
[0.36]
[0.36]
[0.41]
[0.09]

[0.40]
[0.11]
[0.36]
[0.44]
[0.40]
[0.34]
[0.39]
[0.37]
[0.62]
[0.38]
[0.40]
[0.40]
[0.46]
[0.31]
[0.33]
[0.38]
[0.85]

a. The elements with * were determined by ICP-AES, and other elements by ICP-MS. b. The values in [ ] are the ratios of the
concentrations of the elements in black tea leaves to those in green tea leaves. c. Cited from Ref. 9. d. Cited from Ref. 24. The values in [ ]
are the ratios of the concentrations of the elements in tea leaves standard reference material to those in green tea leaves. e. Not detected.

2. Thus, it may be stated here that the ionic elements are highly
extractable from black tea leaves. On the other hand, as for
alkaline earth elements, Mg (47%) was moderately-extractable,
while Ca (4.4%), Sr (5.4%), and Ba (7.4%) were poorlyextractable. These results suggest that Mg is a labile element in
black tea leaves, while Ca, Sr and Ba are immobilized there.
According to our unpublished data for the seasonal variations of
the concentrations of the elements in plant leaves,25 Ca, Sr and
Ba were significantly accumulated along with plant growth,
although Mg was not. Thus, the present results in Fig. 2 suggest
that most of Ca, Sr and Ba are accumulated inside the cells and
are hardly extracted during brewing, while Mg, which is a
component of chlorophylls in tea leaves, is quite easily
extracted in a manner similar to that of the ionic elements, such
as alkali elements.
As can be seen from Fig. 2, the extraction efficiencies of
lanthanoid elements were very small, rather similar to those of

Ca, Sr and Ba. In addition, it is noted here that the extraction

efficiencies of lanthanoid elements became larger for an
increase of their atomic numbers. These differences of the
extraction efficiencies for lanthanoid elements may be partly
explained by their stabilities in complex formations with
organic compounds extracted into tea infusions. In fact, the
formation constants of heavy lanthanoid elements with biogenic
large organic molecules are generally larger than those of light
lanthanoid elements.26 Among the elements in group (iii)
(poorly-extractable elements), Pb, Cd, Th, U and W are shown
in parentheses, because these elements in tea infusions could not
be detected or were below the detection limit.
It should also be pointed out that transition metals belong to
the following three groups: (i) Co, Ni, (ii) Mn, Zn, and (iii) Cu,
Fe. These differences in the extraction efficiencies for
transition metals seemingly reflect their ionic and covalent
characteristics, although further study is required to explain

396

Fig. 1 Comparison of the concentrations of analyte elements in

black tea leaves, green tea leaves, and tea leaves standard reference
material (NIES No. 7). , black tea leaves; , green tea leaves; ,
tea leaves standard reference material (NIES No. 7).

ANALYTICAL SCIENCES MARCH 2001, VOL. 17

Fig. 2 Extraction efficiencies of diverse elements in tea infusions.

The confidence limits for the extraction efficiencies are shown as
error bars. The black bar in each element indicates the extraction
efficiency in the case of green tea leaves.9 Extraction condition,
100C for 5 min.

their chemical and biological behaviors in black tea leaves, or

more generally in plant leaves.
In relation with Alzheimers disease, Al in black tea leaves
provided a relatively large extraction efficiency (ca. 34%). On
the other hand, most of the refractory elements, such as Fe, Mo,
Sn and lanthanoid elements, provided small extraction
efficiencies. In order to explain the behaviors of these
refractory elements, their extractabilities should be taken into
consideration together with their solubilities in tea infusions.
After the brewing procedure, the pH of the tea infusion was 5.8.
Thus, it is considered that the above refractory elements may
hardly be soluble at pH 5.8 in aqueous solution, because organic
substances for complex formation are reduced in tea infusions
as a result of the denaturalization of proteins in black tea leaves.
Speciation of diverse elements extracted in tea infusions
In order to inspect the soluble forms of analyte elements in the
extracted solution from black tea leaves, the tea infusion sample
was analyzed by the system of SEC combined with a UV
detector for molecular absorption detection and with the ICPMS or ICP-AES instrument for element-selective detection.
The chromatogram obtained by UV absorption detection at 260
nm is shown in Fig. 3, together with the molecular-weight
calibration curve for the present SEC column. The calibration
curve for the molecular weight was linear in the range from
1000 Da to 30000 Da, as shown in Fig. 3(a). In the
chromatogram for organic molecules detected by the UV
absorption, shown in Fig. 3(b), the largest peak is apparently
obtained near the total permeation limit at 24.3 ml. In addition,
the large peaks are found at retention volumes of 17.4, 19.5, and
20.5 ml, and the small peaks at 7.8 and 14.3 ml. Some small
peaks were further observed at the larger retention volumes
after the permeation limit, although they are not shown in Fig.
3(b). The chromatogram in Fig. 3(b) suggests that various
organic molecules are present in tea infusions. According to
nutritional studies on tea samples,1,27 it is known that tea leaves
contain 2 4% caffeine on the dry-weight basis, and that most
of the caffeine is extracted through the brewing process. In the
present system, thus, the largest peak at ca. 24 ml was assigned
to caffeine extracted in tea infusion.
Chromatograms for alkali and alkaline earth elements and for
other metallic elements in tea infusions are shown in Figs. 4 and
5, respectively. The molecular weights of the species, in which

Fig. 3 Molecular weight calibration curve (a) and SEC

chromatogram (b) for organic molecules in tea infusions obtained by
UV absorption detection at 260 nm.

analyte elements were detected, are summarized in Table 4.

Those molecular weights were estimated from the calibration
curve, shown in Fig. 3(a). As can be seen from Fig. 4, K
provided an extremely large peak with a shoulder before the
peak top. Other alkali elements, such as Rb and Cs, also
provided similar peak shapes to K, while no peak of Na could
be obtained by ICP-AES. All alkaline earth elements were
observed in the chromatograms detected by ICP-AES and/or
ICP-MS. It is noted that the retention volumes of Mg, Ca, and
Sr were slightly different from each other, and that their peak
widths became wider in the order from Mg to Sr. These results
suggest that Mg, Ca, and Sr are bound with some organic
molecules with molecular weights of 1400 1200, 1200 1000,
and <1000 Da, respectively. In the case of Ba, the peak
appearances were completely different from those for other
alkaline earth elements. Barium provided two peaks at retention
volumes of 12.5 ml and 7.8 ml, indicating that Ba may be

ANALYTICAL SCIENCES MARCH 2001, VOL. 17

Fig. 4 SEC chromatograms for alkali and alkaline earth elements in

tea infusions obtained by ICP-AES or ICP-MS detection. K, Mg, and
Ca: ICP-AES detection; Sr and Ba: ICP-MS detection.

associated with large organic molecules of the molecular

weights corresponding to 18000 20000 Da and those larger
than 70000 Da.
The SEC chromatograms for other elements (Al, Mn, Fe, Cu
and Zn) detected by ICP-AES are shown in Fig. 5. All of these
elements provided relatively simple peaks near to 18 ml (MW
1200 2300 Da), although the small peaks were overlapped
with their main peaks in the cases of Al and Fe. In the present
SEC system, Al, Zn, Sr and Ba were detected by both ICP-AES
and ICP-MS. In the case of Ba, the peak at 12.5 ml obtained by
ICP-MS could not be detected by ICP-AES because of its low
concentration in tea infusions.
The molecular weights of the chemical species binding with
diverse elements, obtained in the present study, can be
summarized as follows. The molecular weights of the species
for Fe, Ni, Cu, Y, Zr, Sn and W were in the range of 1600
2300 Da, while those for Mg, Al, P, Mn and Zn were in 1200
1600 Da. Furthermore, the molecular weights of the species for
Ca, Rb and Cs were in the range of 1000 1200 Da, which were
close to the permeation limit. In addition, Fe, Zr, Sn, and Ba
provided some extra peaks at the retention volume beyond the
permeation limit, maybe because of the possibility of adsorption
of these elements on the column.
A speciation analysis for Al was investigated by several
groups in relation with Alzheimers disease.11,16,17 degrd and
Lund found Al-binding compounds with MWs of 4000 6000
and 6500 8500 Da in black tea infusions.11 Other workers
reported the Al species with MW smaller than 4000 Da in black
tea infusions17 and with MW of 6100 Da in tea infusions at pH
5.5.16 In the present study, the molecular weight of Al-binding
compound was estimated to be about 1200 1500 Da. Although
the molecular weights of Al-binding compounds were different
among these studies, most of the organic molecules bound with
Al in tea infusions may be polyphenolic compounds,28 which
are potential chelating compounds for Al.

Conclusion
The major-to-ultratrace elements in the black tea leaves and tea

397

Fig. 5 SEC chromatograms for Al, Mn, Fe, Cu, and Zn in tea
infusions obtained by ICP-AES detection.

Table 4 Retention volumes of analyte elements in black tea

infusions obtained in SEC separation detected by ICP-AES or
ICP-MS
Element
Mg
Al
P
K
Ca
Mn
Fe
Ni
Cu
Zn
Rb
Sr
Y
Zr
Sn
Cs
Ba
W
Tl

Retention volumea/ml
17.75*
17.92***
17.92*
18.83*
18.33*
17.83*
17.17*
17.42**
17.00*
18.00***
18.42*
19.67***
17.25**
17.33**
17.08**
18.33**
7.83***
12.50***
17.17**
22.75**

Molecular weight/Da
1400 1600
1200 1500
1200 1500
<1000
1000 1200
1300 1600
1800 2100
1600 1900
2000 2300
1200 1400
1000 1200
<1000
1700 2000
1700 2000
1900 2200
1000 1200
>70000
18000 20000
1800 2100
<1000

a. The values with the signs of *, **, and *** were obtained by ICPAES, ICP-MS, and both ICP-AES and ICP-MS, respectively.
Among them, Al, Zn, Sr and Ba were detected at 27, 66, 88 and 138
of m/z, respectively, in the ICP-MS measurement.

infusions were determined by ICP-AES and ICP-MS. Then, the

extraction efficiencies of diverse elements into tea infusions
could be estimated from the concentration data for the black tea
leaves and their tea infusions. Furthermore, speciation analysis
using the hyphenated system of SEC with ICP-MS or ICP-AES
provided some information about the molecular-weight
distributions of metal-binding compounds extracted in tea
infusions. In the present experiment, the chemical species of
trace elements in black tea leaves could not be identified only
from the chromatograms detected by UV absorption, ICP-AES

398
and ICP-MS. However, it has been confirmed that most of the
diverse elements in tea infusions bind with some organic
molecules when they are extracted from black tea leaves. Thus,
further research for the elucidation of the complexes of trace
elements associated with organic molecules is now in progress
in order to understand the bioavailabilities of trace elements in
tea leaves.

Acknowledgements
The present work was partly supported by a Grant-in-Aid for
Scientific Research (No. 90001934) from the Ministry of
Education, Science, Sports and Culture, Japan. The author
(Akiko Hokura) expresses sincere thanks to the Japan Society
for Promotion of Science for the Research Fellowship for
Young Scientists (1997 1999).

References
1. G. V. Stagg and D. J. Millin, J. Sci. Food Agric., 1975, 26,
1439.
2. A.-M. Coriat and R. D. Gillard, Nature, 1986, 321, 570.
3. C. Vandecasteele and C. B. Block, Modern Methods for
Trace Element Determination, 1993, John Wiley & Sons,
Chichester.
4. H. Haraguchi, Bull. Chem. Soc. Jpn., 1999, 72, 1163.
5. H. J. M. Bowen, Environmental Chemistry of the
Elements, 1979, Academic Press, London.
6. H. Haraguchi, E. Fujimori, and K. Inagaki, Free Radical
and Antioxidant Protocols, ed. D. Armstrong (Methods in
Molecular Biology, Vol. 108), 1998, Humana Press,
Totowa, 389 411.
7. A. Taylor, S. Branch, D. J. Halls, L. M. W. Oven, and M.
White, J. Anal. At. Spectrom., 1999, 14, 717.
8. T. Takeo, Jpn. Agric. Res. Quart., 1985, 19, 32.

ANALYTICAL SCIENCES MARCH 2001, VOL. 17

9. H. Matsuura, A. Hokura, and H. Haraguchi, Bunseki
Kagaku, 2000, 49, 397.
10. S. Natesan and V. Ranganathan, J. Sci. Food Agric., 1990,
51, 125.
11. K. E. degrd and W. Lund, J. Anal. At. Spectrom., 1997,
12, 403.
12. K. Lamble and S. J. Hill, Analyst, 1995, 120, 413.
13. S. S. Krishnan, J. E. Harrison, and D. R. C. McLachlan,
Biol. Trace Element Res., 1987, 13, 35.
14. D. R. C. McLachlan, W. J. Lukiw, and T. P. A. Kruck,
Can. J. Neurol. Sci., 1989, 16, 490.
15. C. Moon and M. Marlowe, Biol. Trace Element Res., 1986,
11, 5.
16. L. M. W. Owen, H. M. Crews, and R. C. Massey, Chem.
Speciat. Bioavail., 1992, 4, 89.
17. A.-K. Flaten and W. Lund, Sci. Total Environ., 1997, 207,
21.
18. A. Itoh, C. Kimata, H. Miwa, H. Sawatari, and H.
Haraguchi, Bull. Chem. Soc. Jpn., 1996, 69, 3469.
19. H. Haraguchi, A. Itoh, C. Kimata, and H. Miwa, Analyst,
1998, 123, 773.
20. F. Katsuki, A. Hokura, D. Iwahata, T. Ikuma, S. Oguri, and
H. Haraguchi, Bunseki Kagaku, 1998, 47, 835.
21. A. Hokura, S. Oguri, H. Matsuura, and H. Haraguchi,
Bunseki Kagaku, 2000, 49, 387.
22. H. Sawatari, E. Fujimori, and H. Haraguchi, Anal. Sci.,
1995, 11, 369.
23. K. R. Koch, M. A. B. Pougnet, and S. D. Villiers, Analyst,
1989, 114, 911.
24. A. Hokura, H. Matsuura, F. Katsuki, and H. Haraguchi,
Anal. Sci., in press.
25. F. Katsuki, Master Thesis of Nagoya University, 1998.
26. K. Inagaki and H. Haraguchi, Analyst, 2000, 125, 197.
27. T. Goto, Y. Yoshida, M. Kiso, and H. Nagashima, J.
Chromatogr. A, 1996, 749, 295.
28. N. D. Michie and E. J. Dixon, J. Sci. Food Agric., 1977, 28,
215.