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Analytica Chimico Acfa, 100 (1978) 139-144

0 Elsevier Scientific Publishing Company, Amsterdam - Printed in The Netherlands




Department of Chemistry, University of Oslo, Oslo 3 (Norway)

(Received 16th January 1978)


Various atomic absorption spectrometric methods - including the solid sampling

technique - are described for the determination of cadmium, manganese and lead in
hard tissue. The methods were used in the analysis of hydroxyapatite, ivory and animal

Interest in the biolcgical effects of trace metals has Ctiated a considerable

number of studies of their concentration and distribution in human and
animal soft and hard tissues. The trace metals in bone are normally deter-
mined by atomic absorption spectrometry (aas.) 11-41, emission spec-
troscopy [ 5,6], spectrophotometry or neutron activation analysis [4]. Interest
in the environmental effects of lead has generated surveys [7, S] on the use
of various analytical techniques for its determination in bone.
Most methods for trace analysis of bone involve ashing, separation and/or
concentration steps, but these operations are time consuming and may intro-
duce serious systematic errors.
The present paper describes direct a,a.s. methods for the determination of
cadmium, lead and manganese in bone, and of lead in ivory. In a previous
paper [9] the present technique was applied to the determination of silver
and zinc in animal bone.


Perkin-Elmer 303 and 400 S atomic absorption spectrometers were used;
both instruments were equipped with arc source deuterium lamps for back-
ground correction, and the 400 S spectrophotometer had also a tungsten
lamp for corrections in the range 300-800 run.
Solid and liquid samples were atomized in a graphite furnace, the con-
struction of which has been described elsewhere [lo] _ These analyses were
done with the 303 instrument and were based on measuring peak areas.

Manganese was also determined by atomization in an a.+acetylene flame

with the 400 S spectrometer, which ws?sfitted with the appropriate back-
ground corrector.
The samples of hydroxyapatite and bone were ground in agate mortars and
pestles. The ivory platelets were sampled by filing.
Liquid samples or sample-standard mistures were transferred to the graphite
furnace with plastic-tipped micro-pipettes.
Weighings were made with micro or semi-micro balances.
Instrument settings
The measurements were made at the following wavelengths: cadmium
228.8 nm and lead 283.3 nm; manganese was determined with a slit width of
0.7 nm, corresponding to the use of the triplet at 279.5 nm.
The folIowing settings were used in the determination of cadmium and
lead by solid sampling: drying at 100C for 60 s, ashing at 400C for 60 s,
atomization at 1200C for 30 s, and cleaning of the tube at about 1950C
for 15 s. In the determination of manganese in the Weider sample by the same
technique, the same drying step was used, ashing was made at 500C for
60 s, atomization at 1400C for 30 s, and cleaning as for the other two
Solid samples and standards were analyzed in the following order: sample-
standard-sample-standard etc. For each element five samples and five standards
were atomized.

Reagents and standard solutions

Hydroxyapatite for column chromatography (BioGel HTP, Bio-Rad Labs.)
was employed as the solid standard; the reagent was not dried. (Earlier
results [ll] showed the content of hygroscopic water in this batch to be
2.3%) Before use, the reagent was finely pulverized in an agate mortar and
intimately mixed with equal amounts of graphite powder.
Cadmium, lead and manganese standard solutions were prepared by dissolv-
ing the appropriate amount of high-purity metal in a small excess of nitric
acid. In the diiuted solutions pH was maintained at or below 2.0 by adding,
where required, nitric acid (Suprapur quality, IMerck).
The furnace was purged with argon (purity 99.970, by volume).
Graphite powder of the quality used in the home-made furnaces was em-
ployed as a dispersing agent. The powder was produced from rods by filing,
and was heated in the furnace until the signals from the elements to be deter-
mined could no longer be detected.

The major components of bone are organic collagen and inorganic apatite;
water is bound to both substances_
Thermogravimetric analysis [12] of a human femur bone showed a content
of about 28% protein and about 8% water, the total weight loss up to about
1000C being approximately 36%.
The following materials were analyzed:
(1) a reference sample of calcined animal bone (code name A-3/1 (1974))
issued by the International Atomic Energy Agency (IAEA), Vienna, Austria;
(2) a sample of purified animal bone for medical purposes. (The bone was
from Argentine cattle and had been processed by Weiders Farmas&tiske A/S,
Oslo, Norway. A typical analysis of the product showed the following con-
tents (given in %): calcium, 34; phosphorus, i5; magnesium, 0.17; fluorine,
0.05; and silicon, 0.01.);
(3) eight samples of an ivory tusk which were issued [ 13 ] in connection
with a cooperative investigation of the content and distribution of lead in
hard tissue; they were received as small plates measuring about 20 X 8 X 1 mm
and weighing approximately 0.25 g.
The two samples of animal bone and the hydroxyapatite mere ground to
pass a 270-mesh (53~pm) sieve.
Before analysis by the solid sampling technique, the IAEA and Weider
samples were mixed with equal amounts of graphite powder; the samples
of ivory were not mixed with graphite.
The samples were not dried before analysis.

Portions of hydroxyapatite (about 1 g) were dissolved in 5.0 ml of nitric
acid, and the clear solution was diluted to 25 ml.
The IAEA and Weider samples were decomposed, where specified, in
polytetrafluoroethylene-lined bombs. To portions of about 1 g, concentrated
nitric acid (5.0 ml) was added, and the mixture was heated for about 1.5 h at
ca. 150C. The solutions were diluted to 25 ml. Clear solutions were obtained.

Analysis of hydroxyapatite
The contents of cadmium, lead and manganese in the solid standard were
redetermined by two a.a.s. methods, both based on the use of thestandard
addition technique. In one of the methods, known volumes of metal standard
solution were added to weighed amounts of solid hydroxyapatite. In the
other method, metal standard solutions were added to portions of hydroxy-
apatite solution.
The mixtures were atomized in the graphite furnace, and gave the following
averages (previous data [9,11] for the same batch of material are given in
parentheses): cadmium, 0.34 ppm (0.35); lead, 2.5 ppm (2.4); and manganese
0.89 ppm (0.90). The data established in the present analyses were employed
as the standard values.

Before the start of the measurements with the graphite furnace, the hollow-
cathode and deuterium lamps were heated for about 15 min. The flow of
argon was adjusted to 6 ml s-l, and l-15 mg of the sample, sample-graphite
or standard-graphite mixture was weighed in a small tantalum scoop and
placed in the middle of the preheated graphite tube by means of a specially

constructed adjustable inserting device. The scoop was reweighed, and the
furnace was moved into its preadjusted position.
Before the atomization of lead and manganese, any chloride in the sample
had to be removed (see below). This was done by adding 20 ~1 of nitric
acid (l+Z) to the solid sample or standard in the graphite furnace, and heat-
ing the mixture for 1 min at about 100C. To ensure the complete removal of
chloride, a new portion of acid was added, and the operation was repeated.
Lead was not detected in the nitric acid blanks. In the present work, chloride
was removed only from the IAEA sample.
In an alternative procedure for the removal of chloride prior to the deter-
mination of manganese, 5 ml of the sample solution wzs evaporated to a small
volume with 5 ml of concentrated nitric acid in a platinum dish at about
120C; the mixture was transferred to a lo-ml volumetric flask and diIuted to
the mark with water.


Interference of chloride
During the preliminary analyses, it, was observed that the values for lead and
manganese obtained by solid sampling of the calcined animal bone from IAEA
were persistently lower than the recommended values, and than the data
obtained by analyzing solutions in the graphite furnace. The results for the
Weider sample and hydroxyapatite did not exhibit this discrepancy. A pos-
sible explanation for this disagreement was the presence of an interfering
element, e.g. chlorine.
Solutions of the two samples of bone and the hydroxyapatite standard were
tested for chloride; the only solution giving a precipitate with silver nitrate
solution was that of the IAEA sample. Similarly, a piece of one of the ivory
samples was decomposed, and the solution was tested for chloride with
negative results.
The interfering effect of chloride on lead in analysis by solid sampling was
confirmed by the following experiments. To portions of the solid Weider
sample (in which chloride could not be detected) varying amounts of potas-
sium chloride solution were added. Samples with and without the addition
of potassium chloride solution, and a potassium chloride blank were then
atomized as described above. The integrals obtained per mg of sample decreased
distinctly as the amount of chloride added was increased. The interfering
effect of chloride on lead.was removed by adding nitric acid to a weighed
portion of the solid sample deposited in the graphite furnace, and
evaporating the mixture to dryness; complete removal of chloride was
secured by repeating the operation.
The interfering effect of chloride on manganese may be removed by
evaporations either in the graphite furnace as described for lead, or in a
platinum dish heated on a hot plate.,Interfering effects of chloride on cadmium
were not found during application of the present techniques.

Analysis for manganese

The high content of manganese in the IAEA sample (about 30 ppm) com-
plicated the analysis by solid sampling. Measurements at the less sensitive
line 403.1 nm could not be made, because of the low intensity of the back-
ground corrector at this wavelength. Reduction of the sample size to below
1 mg and/or further dilution of the sample with graphite were found to
introduce inconveniences and errors; consequently, manganese in the IAEA
sample was determined by atomizing solutions prepared by decomposition
in the PTFE-lined bomb. When this method was used, chloride was removed
from sample solutions by evaporation with nitric acid in a platiium dish.
Samples containing chloride, and in which the manganese content is
relatively low (less than about 5 ppm), may be analyzed by removing the
chloride (as described above for lead) by evaporation with nitric acid in the
graphite furnace.
Samples, such as the present Weider sample, may be analyzed by solid
sampling if hydrovyapatite is used as the solid standard.


Results for cadmium, manganese and lead in hydroxyapatite and two samples of animal
[Method A (standard addition). Standard solution is added to the sample solution, with
atomization in the graphite furnace.
Method B (standard addition). Standard solution is added to the solid sample, with
atomization in the graphite furnace.
Method C. Standard solution is added to the sample solution, with atomization in the
Method D. Solid sample is atomized in the graphite furnace with solid hydroxyapatite as

Sample Method Cd Mn Pb

X s T
(ppm) (lo) (ppm) &) ippm) k)
Hydroxyapatiteb A 0.33 18 0.94 3 2.5 12
B 0.34 18 0.84 5 2.5 12
Animal bone Values
IAEA recommended
by IAEA not given 32 16c 6.8 15
C - - 33 12- -
A - - 32 11 6.9 15
D 0.072 12 - - 6.8 18
Animal bone C - - 5.4 20- -
Weider A - - - - 9.8 14
D 0.036 22 6.0 22 9.7 10

aAverage result with relative standard deviation (So). bThe sample employed as the solid
standard. Relative standard deviation of the mean value.


Results for lead in 8 samples of an ivory tusk

Sample Content of Sample Content of

designation lead (ppm) designation lead (ppm)
A 4 0.45 2 o-14= B 4 0.15 ? 0.02
A 14 0.26 -c 0.03 B 14 0.17 L 0.03
A 24 0.15 5 0.02 B 24 0.18 * 0.01
A34 0.35 f. 0.08 B 34 0.15 f 0.01

=Average 2 one standard deviation.

Analysis of animal bone and ivory

Solutions of the IAEA and Weider samples were analyzed by atomization in
the graphite furnace; these determinations were made with the standard
addition technique_ Portions (10 ,ul) of the sample or sample-standard mix-
tures were transferred to the furnace, dried by heating for 30 s at about
lOOC, and atomized as described above. Two standard addition curves were
plotted for each element.
Manganese was also determined by atomization in the acetylene-air
flame, the analysis being based on plotting one standard addition curve.
Table 1 lists the results for cadmium, manganese and lead in the two
samples of animal bone, and in the hydroxyapatite employed as the solid
standard. The recommended values for manganese and lead in the IAEA sample
(Table 1) compare favourably with the results of the present investigation.
No data are given by IAEA on the content of cadmium, and nothing seems
to have been published on the concentration of cadmium in this sample.
Table 1 shows that the data from the various techniques employed are in
satisfactory agreement.
The present data on lead in ivory obtained by the solid sampling technique
are listed in Table 2; the values given are the averages of five determinations
with the standard deviation. Hydroxyapatite was used as the solid standard.
The content of lead is practically constant in the four samples designated
B, while the values of the four samples of the A-series vary considerably.
The results obtained by other workers who participated in this survey appear
to have varied in the range < l-20 ppm, with polarographic data being
in closest agreement with the present results [ 131. Whether the variations
are due to procedural faults or sample inhomogeneity is not known.

1 C. D. Strehlow, AEC Report _NYG-3086-7. Part V. 1967.
2 C. D. Strehlow and T. J. Kneip. Am. Ind. Hyg. Assoc. J., July-August (1969)372.
3 H. Kawamura, G. Tanaka and Y. Ohyagi, Spectrochim. Acta Part B, 28 (1973) 309.
4 P. Bdttcr,D. Gamlik, J. Lausch and U. Rijsick. J. RadioanaLChem., 37 (1977) 393.
5 R. 0. Becker, J. A. Spadaro and E. W. Berg, J. Bone Joint Surg., 50-A (1968) 326.
6 N. Avram, G. Draganescu and I. Marki, Stud. Biophys., 51 (1975) 69.
7 J. S. Hislop and A. Parker, AERER-6987 (1971).
8 3. S. Hislop, A_ Parker, G. S. Spicer and M. S. W. Webb, AERER-7321 (1973).
9 F. J. Langmyhr, T. Lind and J. Jensen, Anal. Chim. Acta, 80 (1975) 297.
10 F. J. Langmyhr and Y. Thomassen, Fresenius Z. Anal. Chem, 264 (1973) 122.
11 F. J. Langmyhr, A Sundli and J. Jonsen, Anal Chim Acts, 73 (1974) 81.
12 J. J. Lim, J. Biol. Phys., 3 (1975) 111.
13 W. Tanti-Wipawin, Chulalongkorn University, Bangkok, unpublished report.