537

Bulgarian Journal of Agricultural Science, 12 (2006), 537-551

National Centre for Agrarian Sciences

AAS and ICP Determination of Heavy Metal Content

in Tobacco
P. S. ZAPRJANOVA1, V. R. ANGELOVA2, G. L. BEKJAROV3 and K. I. IVANOV2
1
Institute of Tobacco and Tobacco Products, BG - 4108 Plovdiv, Bulgaria
2
University of Agriculture, Dept. of Chemistry, BG - 4000 Plovdiv, Bulgaria
3
Gardering and Canning Institute, BG - 4000 Plovdiv, Bulgaria

Abstract

ZAPRJANOVA, P. S., V. R. ANGELOVA, G. L. BEKJAROV and K. I. IVANOV,

2006. AAS and ICP determination of heavy metal content in tobacco. Bulg. J. Agric.
Sci., 12: 537-551

There has been carried out a comparative study of the standard methods (dry ashing, acid
digestion and microwave digestion) and analysis (ICP - OES and AAS) of tobacco leaves of
Burley and Virginia varieties for the determination of Fe, Mn, Cu, Zn, Pb, Cd and Ni. It can be
concluded, that both measurement methods (ICP-OES and AAS) provide comparable results
with a good agreement and the results of this study are affected in greater extent by the decom-
position procedure than by measurement techniques. A careful choice of suitable digestion
procedures is critical step in ensuring correct results because even very similar material may
exhibit different behavior during the digestion. The use of HNO3 alone does not provide us with
good results even with the microwave digestion of tobacco samples. To achieve total decompo-
sition of sample matrices and dissolution of elements, combination of HNO3 and HCI, HF, HCIO4
or H2O2 have to be used. A careful approach should be applied when using dry ashing for sample
preparation. The lower temperatures (400 - 450 oC) and the longer heat treatment favor the
obtaining of correct results. Taking into consideration the good detection limits, large linear
dynamic range and simultaneous multi-element capability it seems that the combination of ICP
with microwave digestion procedure is a very rapid and accurate method for analyzing plant
materials.
Key words: tobacco leaves, sample preparation, heavy metals, AAS, ICP-OES

Introduction dustry, it is very important regarding hu-

man health, especially considering its ca-
Tobacco is a main agricultural crop for pacity to accumulate heavy metals
many countries, including Bulgaria. Be- (Adamu et al., 1989; Bell et al., 1992).
cause of its large use in the cigarette in- It is known that the content of metals
538 P. S. Zaprjanova, V. R. Angelova, G. L. Bekjarov and K. I. Ivanov
in tobacco varies a lot and it depends on
several factors such as soil type and pH
genotype, use of a metal-containing pesti-
cides, fertilizers, etc. Some of the metals
such as iron, manganese, zinc and copper
are important micronutrients and they are
also very important for plant growth and
yield. However, other metals such as lead,
cadmium and nickel are not important for
the plant growth, but they can cause seri-
ous health and ecological problems. In both
cases, we need to pay attention to their
right determination, which will allow us to
undertake due correcting operations with
serious economic consequences. It is
known that the latter depends on the right
selection of a method for test sampling and
suitable measuring equipment. Although,
a number of investigations have been car-
ried out on heavy metal concentration in
tobacco leaves (Golia et al., 2003;
Zaprjanova and Boshinova, 2004), only a
few studies have been done concerning
methods and techniques for samples
preparation and heavy metals determina-
tion. This made us consider performing a
comparative study of the basic methods
for samples mineralization (dry ashing, acid
digestion and microwave digestion) and
analysis (ICP - OES and AAS) of tobacco
samples, through which we could assess
the advantages and disadvantages for de-
termining the most important elements for
the mineral nutrition and the market quali-
ties of tobacco.
A lot of publications are dedicated to
the evaluation of various mineralization
methods and measurement techniques for
trace element analysis of different mate-
rials. Curdova et al. (1998) has compared
three decomposition methods (microwave
digestion, classical dry ashing and dry
ashing in a mixture of oxidizing gases) for
six plant materials and three measurement
techniques (ICP - MS, FAAS and
ETAAS) for the determination of Cd, Cu,
Pb and Zn. According to the statistical
evaluation, no significant differences be-
tween analytical methods are found. It's
concluded that the results obtained are
affected to a greater extent by the decom-
position procedure than by measurement
techniques. On the contrary, Pyle et al.
(1996) have registered a significant differ-
ence in cadmium determination of 50 soil
samples by four techniques (ICP - AES,
AAS, PSA and XRF). Analyzing the re-
sults of the laboratories participating in
Food Analysis Performance Assessment
Scheme (FAPAS) in the period 1991 - 2000
Rose et al. (2001) reported that percent-
age of satisfactory results are in the range
of 34 to 100 depending on the material,
sample digestion and detection techniques.
According to these authors, ICP-related
techniques produce better results than
AAS. Comparing five methods for sample
digestion they indicate that pressure ves-
sel is consistently associated with good
analytical performance. The same was
concluded by Poykio et al. (2000) on the
basis of a comparison of dissolution meth-
ods for multi-element analysis of some
reference plant materials. According to
these authors, microwave dissolution, com-
bined with ICP-AES or ICP-MS determi-
nation is a very rapid and accurate method
for analyzing botanical materials.
It can be summarized that a careful
choice of suitable digestion procedure and
detection techniques are of great impor-
tance for the ensuring of correct results.
This is especially important for the deter-
mination of plant materials because, as a
rule, plant material is not homogeneous and
usually contains variable matrices.
AAS and ICP Determination of Heavy Metal Content in Tobacco 539

Material and Methods lengths were as follows: Fe - 248.3 nm;

Mn - 279.5 nm; Cu - 324.8 nm; Zn - 213.9
Instrumentation nm; Pb - 217.0 nm; Cd - 328.8 nm and Ni
Two detection methods most frequently - 224.8 nm.
used for heavy metals determination in
plant materials ware compared: Reagents and standards
• ICP - OES. An ICP spectrometer The acids, including nitric acid, hydro-
Spectroflame-D (Spectro Analytical In- chloric acid, perchloric acid and hydrof-
struments, Kleve, Germany), equipped luoric acid, as well as H2O2 were obtained
with two monochromators: (i) spectral from E. Merck Company (Germany).
range 160 - 460 nm with nitrogen purged The calibration was performed using
optics and (ii) spectral range 240 - 790 nm five or six aqueous standard solutions in
with air purged optics, was used. The ana- 2 % v/v HNO3. A commercial multi-ele-
lytical operational parameters were opti- ment standard solution with concentration
mized with the aim to achieve the lowest 100 mg/l for ICP and individual standard
possible limit of detection for the determi- solution with concentration 1000 mg/l for
nation of led. For all elements, with the AAS were used as a stock solution. The
exception of Cu and Zn, a background calibration standard solutions had the fold-
correction was performed. The wave- ing concentrations: 0. 0.2; 0.5; 2.0 and 5.0
lengths of the analytical lines and wave- mg/l for ICP calibration and 0; 0.2; 0.4;
lengths used for background correction 0.6; 0.8 and 1.0 mg/l (Zn, Cu, Mn and Cd)
are listed in Table 1. and 0; 1.0; 2.0; 3.0; 4.0 and 5.0 mg/l (Fe,
• AAS. Specter AA-220 (Varian, Aus- Ni and Pb) for AAS calibration.
tralia) atomic absorption spectrometer was
used in flame mode. The working wave Materials
Two samples of tobacco leaves from
Table 1 the tobacco varieties, which are of great-
Analytical lines and wavelengths used for est importance for the Bulgarian econom-
background ics - Burley and Virginia, were used. Sam-
Background pling procedures followed Bulgarian stan-
Analytical line, dard 17728-91, including representative
Element correction line,
nm samples collection, washing, drying and
nm
grounding.
Fe 259.94 259.54
Mn 257.61 257.21 Digestion procedures
Three various sample digestion proce-
Cu 327.754 * dures were applied as follows:
Zn 206.19 * • Procedure for dry ashing at 525 o C in
muffle furnace, following Bulgarian stan-
Pb 220.353 220.313
dard 17365-94 was used. Samples (0.5 g)
Cd 226.502 226.543 were weighed in 50 ml glass beakers.
Ni 231.604 231.655 charged on a hot plate with stepwise in-
creasing temperature up to 350o C for 4
*Background correction not performed hours and finally ashed in a muffle fur-
540 P. S. Zaprjanova, V. R. Angelova, G. L. Bekjarov and K. I. Ivanov
nace at 525o C for 1 hour. After cooling,
ashes were dissolved in 20 ml of 1.5 %
HNO 3.
• Procedure for acid digestion, follow-
ing Bulgarian standard 11708 - 93, includ-
ing mineralization of the tobacco sample
by a mixture consisting of nitric and per-
chloric acid, was used.
• Procedure for microwave digestion
with different acid mixtures, following EPA
METHOD 3051, suitable for flame AAS
and ICP - OES determination of heavy
metals, was used. A microwave digestion
system (Milestone 1200 MEGA, Italy)
with 10 MRD 300 rotor with 10 positions
maxes. pressure of 30 bar and max. power
1000 W was used. A homogenized sample
of 0.5 g dry substance was weighed on
assay balance into a teflon bomb and 10
ml of concentrated nitric acid were added.
The microwave mineralization program
comprised of tree stages: (i) 5 min non
pulsed 250 W microwave irradiation; (ii)
5 min 400 W pulsed microwave irradia-
tion and (iii) 5 min 600 W pulsed micro-
wave irradiation. After a ventilation of one
minute the sample was cooled and diluted
to 50 ml.
Statistical evaluation of results
Statistical evaluation of the results was
performed by using of SPSS program
(Analysis of Variance, Independent
Samples T Test). The mean difference is
significant at the 0.05 level.
Results and Discussion
The most important elements for to-
bacco growth, yield and quality - Cu, Zn,
Fe, Mn, Pb, Cd and Ni, were object of our
investigation.
• Copper and zinc
Copper and zinc are essential metals
for tobacco growth and yield and their con-
centrations in tobacco leaves according to
Golia et al. (2003) are as follows: Burley -
1.1 - 34.5 mg/kg Cu; 6.8 - 165.5 mg/kg Zn
and Virginia -1.1 - 68.8 mg/kg Cu; 4.8 -
108.8 mg/kg Zn. The addition of Cu was
suggested to be related to the increasing
of nicotine content and decreasing of cit-
ric acid in tobacco leaves (Voss and Nicol,
1960). Zinc stimulates germination and
probably activates the enzymatic processes
of mobilizing the food reserves of seeds.
The results of Cu and Zn in tobacco
varieties Virginia and Burley, obtained by
various digestion methods and measure-
ment techniques together with standard de-
viation (SD) and relative standard devia-
tion (RSD) are presented in Table 2.
As it can be seen from the table, the
values obtained for copper and zinc con-
tent by means of the different digestion
methods vary in narrow limits. With the
Virginia variety they are 13.7-16.9 mg/kg
for the copper and 34.2-37.9 mg/kg for
the zinc. Apparently, the Burley variety
accumulates greater amounts of copper
content - 19.3-23.9 mg/kg. This difference
is well specified with the zinc, which val-
ues reach 63.0-69.4 mg/kg.
The results obtained show that all meth-
ods used for preparation of the samples
are appropriate for determining the cop-
per and zinc content in tobacco samples.
This conclusion is confirmed by the results
of the statistic data processing, presented
in Tables 3 and 4. In any case, the ob-
served significance level s exceeds the
significance level (0.05).
The comparison of the detection meth-
ods does not show significant differences
AAS and ICP Determination of Heavy Metal Content in Tobacco 541

Table 2
Comparison of results for Cu and Zn content of tobacco leaves obtained by different
digestion methods and measurements techniques.* average value (mg/kg);
**SD; **RSD
№ Type of Samples Content of Cu, mg/kg Content of Zn, mg/kg
tobacco preparation AAS ICP AAS ICP
X* + ∆ ** X+ ∆ X+ ∆ X+ ∆
1 2 3 4 5 6 7
1 Virginia Dry digestion 15.7 + 0.1 16.4 + 0.2 36.4 + 0.1 34.9 + 0.4
525o C, 1 h 0.6*** 1.2 0.3 1.1
15.4 + 0.1 16.9 + 0.1 35.6 + 0.3 35.0 + 0.6
0.6 0.6 0.8 1.7
13.7 + 0.1 14.7 + 0.2 35.2 + 0.1 34.6 + 0.3
0.7 1.4 0.3 0.9
13.7 + 0.1 14.7 + 0.1 35.3 + 0.2 37.4 + 0.3
0.7 0.7 0.6 0.8
Burley Dry digestion 22.9 + 0.1 21.3 + 0.2 65.2 + 0.3 65.1 + 0.4
525o C, 1 h 0.4 0.9 0.5 0.6
21.8 + 0.1 21.1 + 0.3 66.1 + 0.2 64.7 + 0.3
0.5 1.4 0.3 0.5
21.5 + 0.1 20.8 + 0.2 66.2 + 0.3 64.4 + 0.4
0.5 1.0 0.5 0.6
19.6 + 0.1 20.8 + 0.2 64.4 + 0.3 65.7 + 0.6
0.5 1.0 0.5 0.9
2 Virginia Microwave 15.1 + 0.1 12.9 + 0.1 35.2 + 0.2 35.4 + 0.7
Digestion 0.7 0.8 0.6 2.0
10 ml HNO3 15.2 + 0.1 13.3 + 0.2 35.1 + 0.1 35.2 + 0.2
0.7 1.5 0.6 0.6
15.1 + 0.1 13.2 + 0.3 35.8 + 0.2 36.2 + 0.2
0.7 2.3 0.6 0.6
15.2 + 0.1 13.3 + 0.5 35.3 + 0.1 35.0 + 0.9
0.7 3.8 0.3 2.6
Burley Microwave 23.1 + 0.2 20.3 + 0.3 65.2 + 0.1 65.5 + 0.8
Digestion 0.9 1.5 0.2 1.2
10 ml HNO3 23.4 + 0.2 20.3 + 0.3 66.1 + 0.1 64.7 + 0.4
0.9 1.5 0.2 0.6
21.1 + 0.1 22.3 + 0.1 66.2 + 0.1 64.4 + 0.7
0.5 0.4 0.2 1.1
21.1 + 0.1 22.3 + 0.2 64.4 + 0.2 65.7 + 0.4
0.5 0.9 0.3 0.6
542 P. S. Zaprjanova, V. R. Angelova, G. L. Bekjarov and K. I. Ivanov

Table 2 continue
1 2 3 4 5 6 7
3 Virginia Wet digestion 16.2 + 0.1 16.4 + 0.3 36.0 + 0.5 35.6 + 0.3
HNO3+HCIO4 0.6 1.8 1.4 0.8
16.1 + 0.1 16.4 + 0.2 36.2 + 0.6 37.9 + 0.5
0.6 1.8 1.7 1.3
15.4 + 0.1 14.8 + 0.2 36.8 + 0.1 37.6 + 0.7
0.6 1.4 2.7 1.9
15.6 + 0.1 14.9 + 0.2 34.7 + 0.4 34.2 + 0.5
0.6 1.4 1.2 1.5
Burley Wet digestion 23.9 + 0.1 22.8 + 0.2 67.9 + 0.4 63.0 + 0.5
HNO3+HCIO4 0.4 0.9 0.5 0.8
23.9 + 0.1 23.4 + 0.3 69.4 + 0.2 67.0 + 0.2
0.4 1.3 0.3 0.3
19.9 + 0.1 19.5 + 0.2 66.2 + 0.1 65.1 + 1.0
0.5 1.0 0.2 1.5
19.3 + 0.1 19.4 + 0.3 65.4 + 0.3 64.8 + 0.9
0.5 1.5 0.5 1.4

in the results, obtained by ICP and AAS
(Table 4). In all cases the observed level
of significance s (0.05). The relative stan-
dard deviations are low and do not ex-
ceed 2.7 % for AAS and 3.8 % for ICP.
• Iron and manganese
Iron and manganese are essential met-
als for tobacco growth and yield too, and
their concentrations in tobacco leaves are
as follows (Golia et al., 2003; Zaprjanova
and Boshinova, 2004): Burley - 90.0 - 232.0
mg/kg Fe; 16.7 - 662.5 mg/kg Mn and Vir-
ginia - 1.1-68.8 mg/kg Fe; 4.1-897.5
mg/kg Mn. Manganese is important for
plant metabolism and tobacco plants re-
quire Mn for growth, but are sensitive to
toxicity.
The results of Fe and Mn in tobacco
varieties Virginia and Burley, obtained by
various digestion methods and measure-
ments techniques together with standard
deviation (SD) and relative standard de-
viation (RSD) are presented in Table 5.
As it can be seen from the table, the
values obtained for iron and manganese
content at the different digestion methods
vary in large limits. With Virginia variety,
they are 44.8-68.1 mg/kg for iron and 22.2-
35.3 mg/kg for manganese. In this case
the Burley variety accumulates significantly
larger quantity from the two elements as
well - the iron is from 72.2 to 94.8 mg/kg.
and the manganese - from 60.2 to 85.9
mg/kg.
The results of the statistic data process-
ing show that in any case, the observed
significance level αs does not exceed the
significance level (0.05), which means that
the differences of the values, obtained
through the different methods are statisti-
cally significant. The only exception oc-
curs with the comparison of microwave
and wet digestion for manganese content
AAS and ICP Determination of Heavy Metal Content in Tobacco 543

Table 3
Statistical evaluation of the relationship between digestion methods at α (0.05)
DD – dry digestion, 5250 C; MD – micro wave digestion; WD – wet digestion; * α s < α
Ele me n t T y p e to b a cc o M eth o d s M ea n d iffere n ce Std . erro r Sig . α s
Fe Virg in ia DD-M D * -12.563 0.816 0.000
DD-W D* -18.300 0.816 0.000
M D-W D * -5.736 0.816 0.000
Bu rle y DD-M D * -7.675 0.697 0.000
DD-W D* -18.725 0.697 0.000
M D-W D * -11.150 0.697 0.000
Mn Virg in ia DD-M D * -12.636 2.469 0.000
DD-W D* -12.388 2.469 0.000
M D-W D 0.250 2.469 0.920
Bu rle y DD-M D * -8.300 0.985 0.000
DD-W D* -16.725 0.985 0.000
M D-W D * -8.425 0.985 0.000
Cu Virg in ia DD-M D 0.238 0.414 0.572
DD-W D -0.575 0.414 0.180
M D-W D -0.813 0.414 0.063
Bu rle y DD-M D -0.388 0.856 0.655
DD-W D -1.163 0.856 0.189
M D-W D -0.775 0.856 0.375
Zn Virg in ia DD-M D -0.350 0.364 0.347
DD-W D -0.575 0.364 0.129
M D-W D -0.225 0.364 0.543
Bu rle y DD-M D -4.763 7.227 0.517
DD-W D -9.313 7.227 0.212
M D-W D -4.550 7.227 0.536
Pb Virg in ia DD-M D * -0.375 0.174 0.043
DD-W D* -0.675 0.174 0.001
M D-W D * 0.300 0.174 0.100
Bu rle y DD-M D 0.275 0.268 0.316
DD-W D* -0.875 0.268 0.004
M D-W D * -1.150 0.268 0.000
Cd Virg in ia DD-M D * -0.825 0.046 0.000
DD-W D* -0.763 0.046 0.000
M D-W D -4.62E-02 0.046 0.322
Bu rle y DD-M D * -1.931 0.103 0.000
DD-W D* -2.254 0.103 0.000
M D-W D * -0.323 0.103 0.005
Ni Virg in ia DD-M D * 0.875 0.306 0.009
DD-W D* -0.838 0.306 0.012
M D-W D * -1.713 0.306 0.000
Bu rle y DD-M D * 2.025 0.703 0.009
DD-W D -0.913 0.703 0.208
M D-W D * -1.725 0.703 0.000
544 P. S. Zaprjanova, V. R. Angelova, G. L. Bekjarov and K. I. Ivanov

Table 4
Statistical evaluation of the relationship between detection methods at α (0.05) * αs< α
AAS-ICP Mean Std.error
t df Sig. αs
Element Tobacco difference difference
Fe Virginia -0.414 22 0.683 -1.3667 3.3003
Burley 0.1 22 0.921 0.3333 3.3254
Mn Virginia 0.13 22 0.898 -0.2583 1.9905
Burley 0.202 22 0.842 0.6167 3.0550
Cu Virginia -1.076 22 0.293 -1.075 0.9988
Burley 0.979 22 0.338 0.6833 0.6979
Zn Virginia 1.737 22 0.096 0.508 0.2927
Burley 1.361 22 0.187 0.800 0.5879
Pb Virginia 2.714 22 0.013 0.4250* 0.1566
Burley 2.183 22 0.040 0.5917* 0.2710
Cd Virginia 0.66 22 0.516 0.1008 0.1527
Burley 0.135 22 0.894 6.08E-02 0.4494
Ni Virginia 1.599 22 0.124 0.5833 0.3648
Burley 0.195 22 0.848 0.125 0.6424

in Virginia variety, where αs = 0.920. The Their concentrations in tobacco leaves

most significant losses are observed with
the dry digestion method, and the fullest
extraction is with the wet digestion.
The comparison of the detection meth-
ods does not show significant differences
in the results, obtained by ICP and AAS
and αs >α (0.05). The relative standard de-
viations are also low and do not exceed
2.0 % for AAS and 2.9 % for ICP.
• Lead, cadmium and nickel
Lead, cadmium and nickel are not es-
sential metals for tobacco growth and yield
(Voss and Nicol, 1960). However, their
content in tobacco is of great importance
for reducing the risks for smokers' health,
especially, having in mind their capacity to
accumulate in tobacco leaves.
Nickel is important as a regulator of
the oxidation process occurring in plants.
usually are as follows (Golia et al., 2003;
Zaprjanova and Boshinova, 2004): Burley
- 0.04-9.1 mg/kg Pb; 0.04-3.6 mg/kg; 0.04-
.6 mg/kg Cd, 6.8-165.5 mg/kg Ni and Vir-
ginia - 0.01-10.2 mg/kg Pb; 0.01-3.4
mg/kg Cd, 0.7-4.7 mg/kg Ni.
The results of Pb, Cd and Ni in tobacco
varieties Virginia and Burley, obtained by
various digestion methods and measure-
ments techniques together with standard
deviation (SD) and relative standard de-
viation (RSD) are presented in Table 6.
As it can be seen from the table, the
values obtained for lead, cadmium and
manganese content at the different diges-
tion methods vary in large limits. With Vir-
ginia variety they are 1.1-2.6 mg/kg for
lead, 0.28-1.25 mg/kg for cadmium and
1.6-4.7 mg/kg for nickel. In this case as
well the Burley variety accumulates sig-
AAS and ICP Determination of Heavy Metal Content in Tobacco 545

Table 5
Comparison of results for Fe and Mn content of tobacco leaves obtained by different digestion
methods and measurements techniques. * average value (mg/kg); **SD; **RSD

№ Type of Samples Content of Cu, mg/kg Content of Zn, mg/kg

tobacco preparation AAS ICP AAS ICP
X* + ∆ ** X+ ∆ X+ ∆ X+ ∆
1 2 3 4 5 6 7
1 Virginia Dry digestion 47.9 + 0.8 51.7 + 0.7 24.2 + 0.6 26.7 + 0.3
o
525 C, 1 h 1.7*** 1.4 2.5 1.1
44.89 + 0.6 49.59 + 0.7 25.8 + 0.4 24.5 + 0.3
1.3 1.4 1.6 1.2
45.49 + 0.8 50.2 + 0.7 25.3 + 0.7 22.2 + 0.1
1.8 1.4 2.8 0.5
47.19 + 0.8 49.3 + 0.8 22.7 + 0.4 20.9 + 0.2
1.7 1.6 1.8 1
Burley Dry digestion. 74.09 + 0.7 72.2 + 0.7 69.2 + 0.5 66.8 + 0.6
o
525 C, 1 h 0.9 1.0 0.7 0.9
75.19 + 0.9 73.5 + 0.7 66.6 + 0.6 65.6 + 0.5
1.2 1.0 0.9 0.8
76.59 + 1.2 72.7 + 0.8 66.3 + 0.5 60.2 + 0.5
1.6 1.0 0.8 0.8
74.79 + 1.4 75.1 + 0.8 66.6 + 0.5 65.4 + 0.5
1.9 1.1 0.8 0.8
2 Virginia Microwave 60.09 + 0.7 62.0 + 0.6 34.1 + 0.5 34.6 + 0.3
Digestion 1.2 1.0 1.5 0.9
10 ml HNO3 59.69 + 1.2 59.0 + 0.8 33.7 + 0.4 34.7 + 0.3
2 1.4 1.2 0.9
60.39 + 0.9 61.3 + 0.7 33.5 + 0.3 32.6 + 0.2
1.5 1.1 0.9 0.6
62.49 + 0.8 61.4 + 0.8 32.1 + 0.3 33.1 + 0.2
1.3 1.3 0.9 0.6
79.39 + 1.4 81.3 + 0.9 71.7 + 0.3 74.7 + 0.5
Burley Microwave 1.8 1.1 0.4 0.7
Digestion 83.29 + 0.7 80.2 + 1.2 73.6 + 0.2 75.2 + 0.3
10 ml HNO3 0.8 1.5 0.3 0.4
81.89 + 0.7 82.8 + 0.8 74.3 + 0.2 73.2 + 0.3
0.9 1.0 0.3 0.4
83.49 + 0.13 82.4 + 1.1 75.3 + 0.3 75.1 + 0.2
1.6 1.3 0.4 0.3
546 P. S. Zaprjanova, V. R. Angelova, G. L. Bekjarov and K. I. Ivanov

Table 5 continue
1 2 3 4 5 6 7
3 Virginia Wet digestion 67.09 + 0.8 66.2 + 0.4 30.1 + 0.3 33.8 + 0.1
HNO3+HCIO4 1.2 0.6 1 0.3
65.79 + 1.1 66.1 + 1.2 33.5 + 0.4 35.3 + 0.1
1.7 1.8 1.2 0.3
66.49 + 0.7 65.3 + 0.8 31.9 + 0.6 32.9 + 0.1
1.1 1.2 1.9 0.3
67.19 + 1.2 68.1 + 0.8 35.1 + 0.3 33.8 + 0.1
1.8 1.2 0.9 0.3
Burley Wet digestion 92.79 + 0.8 93.4 + 0.5 85.9 + 0.4 84.4 + 1.0
HNO3+HCIO4 0.9 0.5 0.5 1.2
91.39 + 1.1 94.4 + 2.7 83.2 + 0.2 82.4 + 1.1
1.2 2.9 0.2 1.3
93.19 + 0.9 91.1 + 0.0 81.2 + 0.1 82.3 + 1.0
0.9 0.1 1.2
92.89 + 1.0 94.8 + 0.0 80.7 + 0.4 80.4 + 0.9
1.1 0.5 1.1

nificantly larger amounts of the three ele-
ments as lead is from 0.8 to 3.4 mg/kg,
cadmium - from 0.8 to 3.5 mg/kg and
nickel - from 2.8 to 7.7 mg/kg.
Here, just as it was with the copper
and manganese, the largest losses of Pb
and Cd are observed with the dry diges-
tion method, and the fullest extraction is
with the wet digestion. Only with the nickel,
the results of the dry digestion are com-
mensurable with these of the wet diges-
tion.
The comparison of the detection meth-
ods does not show significant differences
in the results, obtained by ICP and AAS
for most of the elements. Only with the
lead, the observed significance level is
αs<α (0.05). In this case the relative stan-
dard deviations are also high (up to 50%),
which reduces to a large extent the cor-
rectness of the obtained results.
The analysis of the results shown in
Tables 2 - 6 show that the results obtained
by the dry digestion method and micro-
wave mineralization do not provide us with
appropriate results for most of the tested
elements. Other authors have also ob-
served lowered results for the content of
heavy metals with dry digestion of the
samples on studying other materials (Soon
and Bates, 1982). However, in most of the
cases, the microwave mineralization is
considered the most appropriate method
for preparation of the samples, especially
with the easily volatile elements. Most
probably, the reason for the differences
observed is the need of specific working
conditions, in dependence with the com-
position and the properties of the exam-
ined material. To make this problem clear,
we changed some of the basic parameters
of the digestion process - we reduced the
temperature and increased the calcinating
time of the dry mineralization and we used
stronger oxidizing mixtures with the mi-
crowave mineralization.The results ob-
tained are shown in Table 7.
Table 6
Comparison of results for Pb, Cd and Ni content of tobacco leaves obtained by different digestion methods and measurements
techniques.* average value; **SD; **RSD

№ Type of Samples Content of Pb, mg/kg Content of Cd, mg/kg Content of Ni, mg/kg
tobacco preparation AAS ICP AAS ICP AAS ICP
X* + ∆ ** X+ ∆ X+ ∆ X+∆ X+ ∆ X+∆
1 2 3 4 5 6 7 8 9
1 Virginia Dry diges tion 2.0 + 0.3 1.5 + 0.5 0.35 + 0.04 0.31 + 0.04 3.4 + 0.4 2.9 + 1.0
o
525 C, 1 h 15.0*** 30 11.4 12.9 11.8 34.5
1.7 + 0.4 1.1 + 0.5 0.35 + 0.05 0.30 + 0.11 4.2 + 0.6 3.4 + 1.0
23.5 45.5 14.3 36.7 14.3 29.4
1.5 + 0.2 1.6 + 0.4 0.35 + 0.05 0.32 + 0.14 3.7 + 0.3 3.0 + 1.1
13.3 25 14.3 43.8 8.1 36.6
Burley 1.6 + 0.2 1.9 + 0.3 0.30 + 0.06 0.28 + 0.11 4.1 + 1.1 3.6 + 1.1
12.5 15.8 20 39.3 26.8 30.6
Dry diges tion 3.0 + 0.3 1.5 + 0.2 0.80 + 0.06 0.82 + 0.11 7.0 + 0.3 6.5 + 0.4
o
AAS and ICP Determination of Heavy Metal Content in Tobacco

525 C, 1 h 10 13.3 7.5 13.4 4.3 6.2

2.9 + 0.3 1.3 + 0.2 1.10 + 0.07 1.10 + 0.03 7.6 + 0.4 7.0 + 0.3
10.3 15.3 6.3 2.7 5.3 4.3
2.5 + 0.4 2.0 + 0.2 1.20 + 0.06 1.2 + 0.09 7.2 + 0.7 6.4 + 0.4
16 10 5 7.5 9.7 6.2
3.1 + 0.3 1.6 + 0.2 1.05 + 0.05 1.00 + 0.08 6.8 + 0.8 6.3 + 0.4
9.7 12.5 4.8 8.0 11.8 6.3
2 Virginia M icrowave 2.5 + 0.5 1.4 + 0.1 1.15 + 0.04 0.92 + 0.06 3.5 + 0.7 1.6 + 1.2
digestion 20 7.1 3.5 6.5 20 75
10 ml HNO 3 2.1 + 0.6 1.6 + 0.3 1.10 + 0.05 0.91 + 0.08 2.9 + 1.2 1.6 + 1.0
28.6 18.8 4.6 8.8 41.3 62.5
2.5 + 0.4 2.3 + 0.2 1.15 + 0.04 0.95 + 0.06 3.5 + 0.6 2.0 + 1.0
16 8.7 3.5 6.3 17.1 50
547
 548
Table 6 continue
1 2 3 4 5 6 7 8 9
Burley 2.2 + 0.3 1.3 + 0.3 1.25 + 0.06 0.90 + 0.07 3.9 + 0.2 2.3 + 1.0
13.6 23.1 4.8 7.8 5.1 43.5
Microwave 2.5 + 0.4 1.0 + 0.5 3.30 + 0.05 2.72 + 0.09 5.2 + 0.4 3.8 + 1.1
Digestion 16.0 50.0 1.5 3.3 7.7 28.9
10 ml HNO3 2.1 + 0.3 1.4 + 0.2 3.35 + 0.6 2.73 + 0.08 4.5 + 0.2 3.9 + 1.1
14.0 14.3 1.8 2.9 4.4 28.2
2.0 + 0.3 0.8 + 0.4 3.10 + 0.04 2.68 + 0.07 4.1 + 0.7 2.8 + 0.3
15.0 50.0 1.3 2.6 17.1 10.7
2.6 + 0.4 2.3 + 0.2 3.10 + 0.06 2.74 + 0.09 4.2 + 0.3 3.1 + 0.4
15.4 8.9 1.9 3.3 7.1 12.9
3 Virginia Wet digestion 2.5 + 0.5* 2.0 + 0.5 1.15 + 0.06 1.22 + 0.06 4.2 + 0.6 4.7 + 0.7
HNO3 + HCIO4 20.0** 25.0** 5.2** 4.9** 14.3** 14.9**
2.3 + 0.6 2.2 + 0.7 1.15 + 0.08 1.11 + 0.05 4.3 + 0.5 4.7 + 0.7
26.1 31.8 7.0 4.5 11.6 14.9
2.5 + 1.2 2.2 + 1.2 1.05 + 0.07 1.03 + 0.05 3.9 + 0.4 4.5 + 0 .5
48.0 55.0 0.7 4.9 10.2 11.1
Burley 2.6 + 0.9 2.0 + 0.9 1.05 + 0.09 1.05 + 0.05 4.2 + 0.7 4.5 + 0 .5
Wet digestion 34.6 45.0 2.6 4.8 16.7 11.1
HNO3 + HCIO4 3.0 + 0.9 3.3 + 08 3.4 + 0.08 3.27 + 0.04 6.2 + 0.8 7.7 + 1.2
30.0 24.2 2.4 1.2 2.0 15.6
3.2 + 0.8 3.4 + 0.7 3.4 + 0.07 3.04 + 0.07 5.9 + 0.3 7.6 + 1.2
25 20.6 2.1 2.3 5.1 15.6
3.0 + 0.8 3.1 + 0.9 3.5 + 0.09 3.44 + 0.06 6.5 + 0.6 7.5 + 0.8
26.7 29 2.6 1.7 9.3 10.7
2.9 + 0.7 3.0 + 0.8 3.2 + 0.08 3.23 + 0.08 6.3 + 0.9 7.4 + 0.8
24.1 26.7 2.5 2.5 14.3 10.8
P. S. Zaprjanova, V. R. Angelova, G. L. Bekjarov and K. I. Ivanov
Table 7
Comparison of results for Cu, Zn, Fe, Mn, Pb, Cd and Ni content of tobacco leaves obtained by different digestion
*average value, mg/kg; **SD; ***RSD

№ Type of Samples Content of Fe, M n, Cu, Zn, Pb, Cd and Ni, mg/kg, X* +
tobacco preparation Fe Mn Cu Zn Pb Cd Ni
1 Virginia Dry diges tion. 66.0 + 0.9 33.4 + 0.5 13. + 0.2 32.2 + 0.5 2.2 + 0.8 0.73 + 0.04 3.6 + 0.3
o
450 C, 3 h 1.4*** 1.5 1.5 1.6 36.4 5.4 8.3
Burley 94.2 + 1.1 79.0 + 0.4 20.8 + 0.3 70.0 + 0.3 3.0 + 0.8 2.62 + 0.03 6.3 + 0.5
1.2 0.5 1.4 0.4 26.7 1.1 7.9
2 Virginia Dry diges tion. 62.1 + 1.2 32.2 + 0.2 13.9 + 0.3 32.0 + 0.3 2.3 + 0.8 0.80 + 0.02 3.3 + 0.5
o
400 C, 12 h 1.9 0.6 2.2 0.9 34.8 2.5 15.2
Burley 94.1 + 1.1 80.2 + 1.2 20.3 + 0.1 69.0 + 0.5 2.9 + 0.9 2.80 + 0.03 6.0 + 0.6
1.2 1.5 0.5 0.7 31 1.1 10
3 Virginia 60.8 + 0.4 34.6 + 0.2 15.1 + 0.3 36.8 + 0.2 2.5 + 1.0 0.92 + 0.04 4.8 + 0.5
AAS and ICP Determination of Heavy Metal Content in Tobacco

Microwave
digestion. 0.7 0.5 0.4 0.5 40 4.3 10.4
Burley 9 ml HNO3 + 2 ml HF 97.4 + 2.1 77.7 + 0.4 22.9 + 0.2 68.4 + 0.3 3.2 + 1.0 3.2 + 0.03 8.9 + 0.9
+ 2 ml H2 O2 2.2 0.5 0.9 0.4 31.3 0.9 10.1
4 Virginia Microwave 63.4 + 1.7 33.8 + 0.3 14.5 + 0.3 36.8 + 0.3 2.4 + 0.8 1.02 + 0.01 4.6 + 0.4
digestion. 2.7 0.9 2.1 0.8 33.3 1 8.7
Burley 9 ml HNO3 + 2 ml HCI 94.2 + 1.2 81.2 + 0.5 23.1 + 0.3 67.2 + 0.4 3.0 + 0.9 3.31 + 0.04 8.0 + 0.6
+ 2 ml H2 O2 1.3 0.6 1.4 0.6 30 1.2 7.5
549
550 P. S. Zaprjanova, V. R. Angelova, G. L. Bekjarov and K. I. Ivanov
The results presented in show that re-
ducing of the temperatures and increas-
ing the calcinating time of the dry diges-
tion and the use of H2O2 and HF with the
microwave mineralization are favorable for
the complete extraction of the elements
and make the results obtained completely
comparable.
Conclusions
• It can be concluded, that both mea-
surement methods (ICP-OES and AAS)
provide comparable results with a good
agreement and the results of this study are
affected in greater extent by the decom-
position procedure than by measurement
techniques. A careful choice of suitable
digestion procedures is critical step in en-
suring correct results because even very
similar material may exhibit different be-
havior during the digestion.
• The use of HNO3 alone does not pro-
vide us with good results even with the
microwave digestion of tobacco samples.
To achieve total decomposition of sample
matrices and dissolution of elements, com-
bination of HNO3 and HCI, HF, HCIO4
or H2O2 have to be used.
• A careful approach should be applied
when using dry ashing for sample prepa-
ration. The lower temperatures (400 -
450îÑ) and the longer heat treatment fa-
vor the obtaining of correct results.
• Taking into consideration the good
detection limits, large linear dynamic range
and simultaneous multi-element capability
it seems that the combination of ICP with
microwave digestion procedure is a very
rapid and accurate method for analyzing
plant materials.
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Received March, 23, 2006; accepted June, 14, 2006.