Anal. Chem. lBSg, 61, 216-220
216
Synthetic Reference Material for Direct Analysis of Solid Biological Samples by Electrothermal Atomic Absorption Spectrometry Kunihiko Akatsuka* a n d I k u o Atsuya
Kitami Institute of Technology, 165 Koen-cho, Kitami 090, Japan
A reference material for the direct analysis of solid samples by electrothermal atomic absorption spectrometry ( E M S ) was prepared by copreclpitatlon of eight metal lons with magnesium( I I ) Oqulndlnate. The metal ions were quantitatively recovered and the distrlbutlon of the metal Ions In the reference materlal was conflrmed to be homogeneous. The reference material was more than adequate for mdtlelementai analysis of bidoglcai samples by EAAS. For the analysis, an Inner minlature cup, whlch was placed into a cupped-type graphHe furnace, was also used. Analyses were conducted wlthout any pretreatment of samples. The accuracy, preclslon, and limits of detecfh of the method are described. The accuracies obtained and the rapidlty of the method pennl! the anaiyds of solid samples on a routlne bask. The ihlts of detection for Ai, Cd, Co, Cu, Mn, Ni, Pb, and Zn in sollds are 0.087, 0.008,0.012, 0.05,0.003, 0.043, 0.14, and 0.013 pglg, respectlveiy
.
INTRO DUCT10N Electrothermal atomic absorption spectrometry (EAAS) with use of solid sampling technique is one of the most attractive methods for determining trace elements in a solid biological sample. There can be advantages in introduction of the sample into the furnace as a powder rather than in the form of a solution. I t eliminates many steps for sample pretreatment such as dissolution and dilution or concentration which increases the risks of contamination and loss of analyte. The authors (1-8) have reported the use of a newly devised inner miniature cup for the direct analysis of some powder samples. This solid sampling technique is very excellent in handling powder samples. They are easily and reproducibly placed into the graphite furnace and their residues after atomization are also easily removed. As differences in analytical sensitivity and accuracy between the miniature caps have not been found, it is possible to take the necessary numbers of sample aliquots a t the same time and then they can be measured continuously. Therefore the analytical time is considerably shortened. Although many works have been carried out to analyze various solid samples directly with EAAS (9-18), one of the most serious problems to be solved is to prepare a standard for calibration. Headridge and Riddington (18)employed the use of a metal powder and an aqueous solution as standard for preanalysis of alloys. They concluded that the aqueous standard is unsuitable for the direct analysis of the alloys. The authors (1,2,4,5) also investigated the suitability of the direct calibration procedure using simple reference solutions for the determination of trace elements in biological samples. In such a procedure, analytical results for copper, chromium, and manganese were apt to give lower values than the certified values ( 5 ) . The method of standard addition has been employed for analyses of biological samples (15). However, it is often difficult to verify that a substance for standard ad0003-2700/89/0361-0216$01.50/0
dition behaves in the same way as an analyte in an unknown sample (19). Many standard reference materials (SRMs) are available from the USA and other several countries. They are prepared from various natural occurring materials. However, for concentrations less than several micrograms per gram there is a scarcity of SRMs that could be applied for the calibration. Trace element standards must have small sampling error at the milligram or submilligram size and thus must be homogeneous at these levels. Recently, synthetic reference materials (SyRM) have been reported such as phenolformaldehyde resin (20,21), silica (22,23), gelatin (24-26), ion exchange beads (27), and copolymerization of acrylamide (281, but there are few synthetic reference materials (SyRMs) for multielement analysis of biological materials by EAAS with the solid sampling technique. In this paper, we have proposed a powder SyRM prepared by coprecipitation with magnesium(I1) 8-quinolinate for solid sampling EAA of biological samples. The procedure is the coprecipitation method that was previously investigated for preconcentration of trace elements in natural waters (7,8), in this work involving the quantitative introduction of eight elements in the same SyRM. The powder SyRM is stable and suitable for use at submilligram and milligram weights for the eight elements. Additionally, this SyRM is highly accurate for the determination of trace elements in different kinds of solid biological samples. EXPERIMENTAL SECTION Apparatus. A Hitachi Model Z-SOOO spectrometer equipped with Zeeman effect background correction was employed. The graphite furnace and miniature cup used in this study have been described in earlier publications (1-4) from this laboratory. A Hitachi data processor was used for the measurements of absorption signal as peak area and absorption-time profile. Differential thermal microbalance (Sinku Riko, Model TGD-5000) was used for thermogravimetric and differential thermal analysis. A Hitachi ICP atomic emission spectrometer (Super scan Model 306) was used for recovery testa. During the course of establishing favorable operating conditions for the furnace, the inductively coupled plasma (ICP) with electrothermalvaporization (ETV-ICP) system was also employed. The system consists of a electrothermal graphite furnace (Hitachi, controlled-temperature furnace atomizer) coupled to the Hitachi ICP system. The miniature cup and cupped-type furnace used were originally employed for the graphite furnace AAS. Emission signals observed in the plasma at the wavelength of interest were recorded with a chart recorder. Reagents. Acids and ammonia solution were of the purest available grade (ELSSand ELS, respectively, Kanto Chemical Co.). Ultraclean water from a Millipore Milli-Q purification system was used. 8-Quinolinol (8-Q;8-hydroxyquinoline, reagent grade, Wako Pure Chemical Co.) was preliminarily purified (8), and then dissolved in hydrochloric acid, and a 2% (w/v) solution was prepared. The solution of magnesium (10 mg/mL) as a carrier metal ion, which was prepared by dissolving the pure metal (99.995%) in hydrochloric acid, was purified by the addition of 8-Q solution and a subsequent extraction with chloroform at around pH 5 to remove any extractable metal chelates. Stock standard solutions (1.00g/L) for each element were prepared by 0 1989 American Chemical Society
ANALYTICAL CHEMISTRY, VOL. 61, NO. 3, FEBRUARY 1,
1989 217
Table I. Parameter Used for Analysis
matrix' A1/1,4,6 Cd/ 1-6 Cd/7 co/1,3,5,7 CU/l-6
CUI7 Mn/l-4,7
NiIl-6 Pb/1,3-7 Zn/ 1-5,7
nm
ash
atomize
weight, mg
256.8b
700/30 400130 500/90 1200/30 600/30 600130 700/30 1200/30 500160 500/30
2500110 250015 250015 2500115 2500/10 2500110 2500110 2500115 250015 200015
3 0.7 0.7 8 1.5 1.5 3 8 0.7 0.7
228.8 228.8 240.7 324.7 327.4 403.1b 232.0 283.3 307.6*
'NBS Standard Reference Materials: (1) SRM 1566, (2) SRM 1568, (3) SRM 1571, (4) SRM 1572, (5) SRM 1573, (6) SRM 1575, and (7) SRM 1577. *Toestimate the limita of detection, the most sensitive lines were also employed, i.e. 309.2, 279.5, and 213.8 nm
\
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O 4:
I''
\
600 I
800
I
I
1000
Ashing temperature ( " C )
Relation between zinc absorbance of peak area and ashing temperature (ashing time, 30 s): (1) NBS bovine liver; (2) NBS tomato leaves; (+) SyRM. Flgute 2.
for Al, Mn, and Zn, respectively.
a
h'
v
C
50
.
c c
I
0
n
c
400
200
L I
I
200
1
I
1
400 Ashing temperature
600
1
800
("c)
Relatlon between cadmium absorbance of peak area and ashlng temperature (ashlng tlme: (1) 90 s;(2,3)SyRM, 30 s): (1) NBS bovlne liver; (2)NBS oyster tissue; (3) NBS &chard leaves; (e)SyRM.
Figure 1.
dissolution of the pure metals; working standards were obtained by serial dilution with water. Preparation of SyRM. The powder SyRMs were prepared by coprecipitation of Al(III), Cd(II), Co(II),Cu(II), Mn(II), Ni(II), Pb(II), and Zn(I1) at levels from 1pglmL to 0.05 ng/mL. In the dtandard procedure, 100 mL of the standard solution was placed in a Tefron beaker, into which 20 mg of magnesium ions and a definite amount (5-10 mL) of 2% (w/v) 8-Q solution were added. This solution was adjusted to pH 9 by adding an aqueous ammonia solution and then allowed to age for 1h at 70 "C on a hot plate. It was then filtered through a glasa filter (No. 4G).The precipitate (referred to as SyRM) was weighed accurately and then stored at room temperature in a desiccator after being dried at 110 "C for 1 h in a drying oven. A blank standard sample was also prepared by the use of 100 mL of deionized water. Coprecipitation Recovery Yields of Metal Ions and Homogeneity of SyRM. The recoveries were determined by analyzing the respective elements in the filtrate with ICP-AES. The homogeneity of the SyRM was tested by AAS analysis of nine aliquots of the samples for each sampling weight at random locations on the filter. Determination of Trace Metals in SyRM and Biological Samples by EAAS. The miniature cup was first weighed with a microbalance (Mettler, Model M-3). An appropriate amount of the solid sample was placed in the miniature cup with a small tantalum spoon, and the cup was again weighed (difference = net weight of the sample). The miniature cup was inserted into the graphite furnace, and the sample was dried (at 200 "C for 30 s), ashed, and atomized successively according to the instrumental conditions in Table I. In order to examine the ashing condition or other thermal conditions, at least five aliquots with different weights were measured, and the absorbance value corresponding
600
800
Ashing temperature ( " C )
between lead absorbance of peak area and ashing temperature (ashing time, 60 s): (1) NBS orchard leaves; (2) NBS oyster tissue; (3) NBS bovine liver; (+) SyRM. Flgure 3. Relation
Ll
li l
'
500
l
"
"
'
"
'
'
t
1000
1500
Ashing temperature ( " C )
Relation between manganese absorbance of peak area and ashing temperature (ashing time, 30 s): (1) NBS cltrus leaves; (2) NBS orchard leaves; (e)SyRM. Figure 4.
to 1mg of the sample was calculated from each absorbance, and in Figures 1-4 the mean absorbance values for each sample were plotted in arbitrary units, respectively.
RESULTS AND DISCUSSION Previous works (7,8)showed that some elements (Cd, Cu, Mn, Pb, and Zn) have been quantitatively coprecipitated with more than 40 mg of 8-Q from natural water samples containing 20 mg of magnesium ions as a carrier. The conditions for the preparation of SyRM are essentially the same as those reported previously. In the present procedure, amounts of 8-Q in the range from 100 to 200 mg were added into the standard solution containing 20 mg of magnesium, because the complete
218
ANALYTICAL CHEMISTRY, VOL. 61, NO. 3, FEBRUARY 1, 1989
Table 11. Coprecipitation Recovery Yields of Metal Ions with Magnesium(I1) 8-Quinolinate at pH 9
(i
element
recovery, %
element
recovery, %
Cd(I1) Co(I1) Cu(I1) Mn(I1) Ni(1I) Pb(I1)
100 f 1
Zn(I1) AUIII) Cr(II1) Mo(V1) v (IV)
100 f 1 97 f 1 83 f 2" 64 f 3 70 f 2
100 f 1 100 f 2 100 f 1 100 f 2 100 f 1
Recovery of 98% was obtained at pH 10.5.
Table 111. Results of Calibration Curves for Cadmium and Cobalt
Table IV. Homogeneity of Trace Elements in SyRM mean sampling wt," mg (n = 9)
A1
0.14 0.53
4.5 4.1 3.1
1.07
relative standard deviations: W Cd Co Cu Mn Ni Pb 4.7 2.2
ND'
4.9 1.8 1.8
4.5 3.0 2.4
2.5 1.2
1.0
4.4 3.9 2.1
Zn
4.8 3.8
4.3 4.0
ND
ND
"Sampling weight in each range is mean i 0.03 mg. *Eachvalue is precision of nine measurements in which the measured absorbance is divided by respective sample weight submitted (not mean weight). The amounts of doped elements are 0.2-0.5 ng of Cd and Zn and 3-8 ng of the other six elements in mg of SyRM, respectivelv. ND, not determined.
cadmium in SyRM" cobalt in SyRM" sampling absorbsampling absorbweight, ance, weight, ance, mg peak area A-s ng-' mg peak area A.9 ng-' 0.057 0.107 0.148 0.174 0.210 0.290 0.395
0.0268 0.0503 0.0683 0.0836 0.0967 0.1203 0.1817
0.902 0.903 0.886 0.923 0.884 0.796 0.902 0.885b 4.33'
0.180 0.282 0.304 0.345 0.408 0.427 0.525
0.1131 0.1694 0.1903 0.2214 0.2506 0.2525 0.3428
0.08028 0.07675 0.08001 0.08194 0.07845 0.07822 0.08337 0.07986b 2.64'
"The amounts of doped elements are 0.521 ng of Cd and 7.826 ng of Co in mg of SyRM. bMean (n = 7). 'Percent relative standard deviation.
; 100
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recovery of the doped elements is required. Furthermore, metals not previously reported, such as Al(III), Co(II), Ni(II), Cr(III), Mo(VI), and V(V), form Bquinolinate complexes. The effect of pH on the recoveries of these elements was investigated to find the conditions necessary for simultaneous coprecipitation of multielements with magnesium(I1) 8quinolinate. A p H value in the range from 8.5 to 9.5 was appropriate for simultaneous coprecipitation and the recovery yields at pH 9 are given in Table 11, in which the elements reported previously are also included. The recovery of the eight elements is 100% within the analytical error, except that aluminum was 97% recovered. As a result, the contents of these elements in the SyRM were simply calculated from the sample weight taken and the quantities of spiked elements, with the exception of aluminum being 97% of the added amounts. In this study Cr, Mo, and V were not studied fwther because their recoveries were less than 85%. Stability and Homogeneity of SyRM. By the thermogravimetric and differential thermal analysis, no appreciable change in weight of powdered SyRM was observed up to 480 "C under the heating rate at 30 "C/min in N2 carrier gas, except the dehydration occurring around 150-190 "C (about 9.6% weight loss). The SyRM can be preserved for one or more years in a silica gel desiccator without any loss of the analytes in SyRM. As described in detail below, a good linear relationship between the peak area (absorbance seconds) versus the mass of each element was obtained, when the ashing step was carried out prior to the atomization. This experiment was made by submitting increasing masses of the SyRM to EMS. For cadmium and cobalt, Table I11 shows a part of data where the precision of working curve slopes (relative standard deviation) is less than 5% even when SyRM in submilligram sample sizes ranging from 0.1 to 0.5 mg was submitted. To assess homogeneity, relative standard deviations of the measured absorbance (n = 9) were calculated for each sampling weight. From the values in Table IV, it can be seen that
I
I
70 Time ( s )
Figure 5. Emission signal for zinc vs heating time with electrothermal vaporization sample introduction into the I C P sample, (a, b) aqueous standard solution containing 150 ng of Zn, (c, d) SyRM containing 140 ng of Zn; heating conditions, (stage I ) 200 OC for 30 s, (stage 11) (a) 400, (b, c) 500, and (d) 600 "C for 30 s, respectively, (stage H I ) 2500 OC for 10 s.
all the elements were distributed homogeneously in the SyRM. As the absorbance decreased with decreasing the sample mass submitted (see Table 111), the increase in relative standard deviation owing to smaller sample size is primarily the result of the uncertainty in the measurement step. Ashing Prior to Atomization. The operating conditions for the furnace were investigated. The temperature of 2500 "C was the highest attainable in this apparatus when the cupped-type f&nace was used. An increase in the steady-state temperature of the graphite furnace led the peak profiles to sharpening. The absorbance of both peak area and peak height for the analyte increased with increase in temperature, with the exception of peak area for Cd and Zn. Peak areas for cadmium and zinc did not vary significantly over the temperature ranging from 1800 to 2300 "C and slightly decreased in the temperature higher than 2300 "C. However, no difficulties were encountered in examining peak areas for Cd a t an atomization temperature of 2500 "C. Under thermal conditions for the furnace, the ashing condition was very important. Accurate determinations are dependent on successful ashing of the sample without volatilization of the analyte. Therefore, the effects of the ashing temperature were investigated in detail for each matrix. Typical results are shown in Figures 1-4. In Figures 1-4, the highest ashing temperature in the plateau region can be used when the loss of the analyte did not occur in the ashing stage. This fact was confirmed by using the ETV-ICP system. As shown in Figure 5, for example, loss of Zn in SyRM did not occur during the ashing stage at 500 O C , which was the highest ashing temperature in the plateau region for Zn in SyRM (see
ANALYTICAL CHEMISTRY, VOL. 61, NO. 3, FEBRUARY 1, 1989
Table V. Sensitivity and Detection Limit wavelength, element nm A1 A1 Cd Go Cu Mn Mn Ni Pb Zn Zn
309.2 256.8 228.8 240.7 324.7c 279.6 4O3.lc 232.0' 283.3 213.8 307.6c
sensitivity (0.0044 AU), absolute dl," ng ng 0.045 0.068 (0.26) 0.38 0.55 (1.4) 0.005 0.002 (0.005) 0.055 0.023 (0.090) 0.051 0.023 (0.075) 0.009 0.007 (0.009) 0.13 0.057 (0.13) 0.053 0.16 (0.34) 0.065 0.036 (0.095) 0.003 0.0038 (0.0088) 4.8 1.0 (4.5)
219
preanalysis was carried out against an aqueous standard at an ashing temperature of 500 "C. However, analytical results for lead thematrix were apparently lower than-the certified values as follows (found): NBS tomato leaves, 4.9 f 0.3 pg/g; NBS citrus leaves, 12.1 0.7 pg/g (certified value is shown in Table VI). The reason for this is not clear but may be the result of inadequate ashing of the analyte in standard and/or different matrix compositions of low volatile elements (these samples contain around 0.6-0.7 wt % magnesium and 3 wt % calcium). Such difficulties have been overcome when the calibration was made with the SyRM. For lead in SyRM the ashing temperature of 500 "C for 60 s was recommended because it was the highest temperature in the plateau region for P b in SyRM (Figure 3) and a two-peak pattern of profiles for the analyte was occasionally observed for 30 s at the temperature. Under this ashing condition no problems were encountered for P b in any biological samples. Cadmium is more volatile than Pb, thus the ashing condition of 400 "C for 30 was employed without a drying step prior to atomization because the loss of Cd in orchard leaves was at temperatures higher 4o0 "c. For bWhe liver, however, the atomization profile of cadmium was two peaks under the ashing condition of 400 "C. This is attributable to the incomplete ashing of the sample. On the basis of the thermogravimetric analysis of bovine liver, its complete ashing is made at temperatures higher than 500 "C, but the loss of Cd was significant at temperatures higher than 550 "C. Consequently, the ashing condition of 500 "C for 90 s was recommended for bovine liver. Sensitivity and Detection Limits. With the provision of background correction, the maximum permissible sample weights in milligrams can be estimated by the restriction of background absorbance, which should be less than 1.0 (12). In this work, the maximum values were estimated by the linear range in the relation between the analyte absorbance and the sample weight. For the eight elements in the biological samples, the linear ranges mainly depend on the ashing temperature employed rather than the kind of analyte or matrix. The results are shown in Table I. The leaf materials (tomato leaves) can be ashed at relatively low temperatures (
