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In-Furnace Standard Addition Method for the Determination Trace Metals in Biological Samples by Electrothermal Vaporization-inductively Coupled Plasma Atomic Emission Spectrometry
Hirofumi ISOYAMA, Syu]i OKUYAMA, TetSuo UCHIDA, MaSaaki TAKEUCHI, Chuzo IIDA and GenkiChi NAKAGAWA

of

Laboratory of Analytical Chemistry, Department of Applied Chemistry, Nagoya Institute of Technology, Gokiso-cho, Nagoya 466, Japan

Electrothermal vaporization-inductively coupled plasma atomic emission spectrometry was applied to the determination of trace metals in some biological samples on in-furnace standard addition method. Powdered samples were decomposed using a sealed PTFE vessel and a microwave oven; the sample solution served for measurements without any additional treatment. The sample solution was injected into a graphite furnace and a standard solution containing ammonium dihydrogenphosphate, as a matrix modifier, was added; the two solutions were mixed in the furnace. The present method was applied to the determination of trace metals in some reference materials; the analytical results agreed well with the certified values. Keywords Electrothermal vaporization-inductively coupled plasma atomic emission spectrometry, in-furnace standard addition method, ammonium dihydrogenphosphate as matrix modifier, certified reference materials, microwave sample decomposition

Since inductively coupled plasma (ICP) was independently suggested by Greenfield et al.', as well as Wendt and Fassel2, as being the excitation source of atomic emission spectrometry (AES), it has been well known to be an excellent source for a number of elements. Conventionally, samples have been introduced into ICP with pneumatic nebulization (PN) as aerosols of the sample solution. The sample introduction system with PN is simple, stable and rapid for sample changeover. The sample introduction efficiency of PN, however, is quite low, typically about 1- 3%3, compared with 5 10% in flame atomic absorption spectrometry. Though the sensitivity loss due to the low efficiency would be tolerable in routine work, it would sometimes be undesirable in ultra trace analyses encountered in environmental, clinical, forensic, biochemical, electronic and toxicological studies. To increase the introduction efficiency of a sample into ICP, many researchers have developed various heating devices to vaporize micro-volumes of a solution or solid sample (reviewed by Matusiewicz3). This electrothermal vaporization (ETV) technique provided fairly good introduction efficiency of ca. 20% or more4-6 and better detection limits. On the other hand, the matrix effects which are well-known in ETV-AAS might still remain in spite of the use of ICP as an excitation source. The matrix effect in ETV-ICP was so complex that a

different effect was reported for each sample. For example, though the Pb signal was suppressed by Mg, it was enhanced by a low concentration of Na, but suppressed by high Na;' Pb, however, was enhanced by Mg, but suppressed by Na, regardless of its concentration.8 To suppress or eliminate the matrix effects, either matrix matching or a standard addition method has been applied; a matrix modifier has also been used. Matrix matching could not be applied without any previous information concerning major elements in samples. With the standard addition method, the amounts of the standard added to the unknown samples could not be estimated if the concentration of the analyte was not known. Moreover, once an unsuitable amount of the standard was added, the sample solution could not be used for subsequent measurements. In the present study the in-furnace standard addition method (IF-SAM) was examined for the determination of trace metals in some biological samples prepared by National Institute for Environmental Study as Certified Reference Material (NIES-CRM) with ETV-ICP, after the decomposition of samples using a sealed PTFE vessel and a microwave oven. As a matrix modifier, ammonium dihydrogenphosphate was used.

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Experimental

Apparatus A Nippon Jarrel-Ash ICAP-575 ICP-AE spectrometer with a simultaneous background correction system9 was used in conjunction with a proto-type FAP-1 furnace atomizer and a non-pyrocoating graphite boat, as shown in Fig. 1. A FLA-100 power supply was used to heat the boat. At the wavelength setting of the monochrometer, a three-way valve was rotated toward a pneumatic nebulizer. Signals were sampled from the output terminal for a strip-chart recorder through a 12-bit A/ D converter interface made in our laboratory.9 The analog signal was recorded on the recorder and also sampled to a computer at 5 ms intervals and then stored onto a floppy disk. A NEC PC-9801 Vm2 personal computer was used for the sampling and storing of signals, and for a calculation of the standard addition method.' The operating conditions are given in Table 1. Since the flow rate of the carrier argon gas significantly affected the sensitivity, it was examined for every metal in order to obtain maximum sensitivity. The flow rate of the intermediate gas was twice that of the conventional in order to protect the inner tubes of the torch from melting due to plasma dropping during sample injection into the furnace. An injection volume of 40 mm3 was fixed for both sample and standard for adequate mixing of the solutions in the boat. The heating program of the boat was common for each

metal, set to be as simple as possible. Bumping of solutions was avoided during the drying stage. A short ashing time was used only for making the drying sure. Rapid vaporization was carried out in the flash mode in order to obtain good signal-to-background ratios and the boat life. A Chyo JL-200 electronic balance was used for the preparation of both sample and standard solutions. Micropipets from Gilson and Shibata were also used for the preparation and injection of solutions. A Pierce Chemical Tuf-Tainer Teflon PFA vials (7 cm3), San-ai Kagaku Teflon vessels P-25 and a Mitsubishi RO-2500 microwave oven for kitchen use were used to decompose the samples. Reagents Standard stock solutions of 0.2 or 2% (w/w) were prepared from pure metals (99.999%, Mitsuwa Chemicals Co. or Nakarai Chemicals Co.) or pure metal compounds (99.999%, Johnson Matthey Chemicals "Specpure" or Soegawa Chemicals Co .) by dissolving in appropriate acids. Working standard solutions were prepared by dilutuon of the stock solutions with 0.1 mol dm 3 nitric acid by massll to the appropriate concentrations. The acids used were super special grade from Wako Pure Chemicals Co. and special grade from Hirota Chemicals Co. Milli-Q deionized water was used throughout this experiment.

Table 1 Operating FLA-100

conditions

for

ICAP-575,

FAP-1

and

Fig. 1 Schematic diagram of the graphite and the ICP torch. a, furnace atomizer; C, three-way chamber. valve; d, torch; e, pneumatic

furnace vaporizer b, transport tube; nebulizer and

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Sample decomposition A powdered sample of 100 mg in dry weight was decomposed as described previously.12 After evaporation of a decomposed sample, the residue was dissolved in 10 g of 0.1 mol dm 3 nitric acid, and used for determinations.

Measurements of IF-SAM The present IF-SAM was carried out based on signal area measurements according to the following steps: 1) As a blank, 40 mm3 of 0.1 mol dm 3 nitric acid was injected into the boat, followed by the addition of 40 mm3 of a 30 mmol dm 3 ammonium dihydrogen phosphate solution. The signal was measured under the operating conditions. 2) After injecting 40 mm3 of the sample solution, 40 mm3 of the ammonium dihydrogenphosphate solution was added. The mixture was measured as an indication of the sample only, i.e., without the addition of standard solution. 3) A mixture of 40 mm3 of the sample solution and 40 mm3 of a standard solution containing ammonium dihydrogenphosphate was measured, as an indication of the sample and standard added. 4) After subtracting the blank signal obtained in step 1 from the signals obtained in steps 2 and 3, the concentration of the concerned metal was given based on a standard addition method.

tion on the inner wall of the chamber and/or transport tube. The introduction efficiency with ETV-ICP was better than that with PN-ICP, about one order of magnitude or more, resulting in a better detection limit of ETV-ICP than PN-ICP, partly due to the calm base line observed in ETV-ICP. The reproducibility obtained with the present ETVICP, was ca. 5% at concentrations near the detection limit, while it was ca. 2% or better around the sub-ppm level.
Preliminary determination By changing the sample dilution from 25- to 200-fold (i.e., the samples of 100 mg each were converted to the sample solutions of 2.5 - 20 g), NIES-CRMs No. 1 Pepperbush and No. 6 Mussel were analyzed with IF-SAM without using a matrix modifier. The analytical results for metals in Pepperbush agreed with the certified values for Cd, Co, Cu and Pb, irrespective of dilution factors. A slightly low value of 180 ppm was obtained for Fe at every dilution factor. At 25-fold dilution, considerably high values of 2370 and 544 ppm were obtained for Mn and Zn, respectively. With an increase of the sample dilution, these high values came close to the certified values, i.e., 2080 ppm for Mn and 400 ppm for Zn at 200-fold dilution. On the other hand, the results for the same 7 metals in Mussel almost agreed with the certified values, irrespective to the dilution factors. Thus, the observed matrix effect was so

Results

and

Discussion

Detection limit and introduction efficiencyof ETV ICP The detection limits and the introduction efficiencies are summarized in Table 2 for the concerned metals in the present work along with peak area measurements. The former was defined as the concentration of metal giving a signal corresponding to 3-times the standard deviation of the net signal near the detection limit. The latter was calculated from the ratio of the slope of the calibration curve with the present ETV system to that with PN in discrete nebulization using the same excitation source of ICP. For this calculation, it was assumed that the introduction efficiency with PN-ICP was 2.5% for each metal, because it was obtained for Mn with continuous nebulization in our laboratory. The introduction efficiency considerably varied from IA + - 1nnm_fr aonh Al im of rrn~~~ly Aa fr Aannei_

Fig.

Signal

profiles 1 ppm

of Fe in IF-SAM a matrix

for

Pepperbush a, sample ppm

at only;

200-fold dard.

dilution

without

modifier. c, sample+2

b, sample+

Fe standard;

Fe stan-

Table

Introduction

efficiencies

and detection

limits

with

ETV-I C P

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complex that it could not be avoided with a simple standard addition method. A typical signal profile for Fe is shown in Fig. 2 as evidence of a complex matrix effect due to the main components of Pepperbush. Even at a sample dilution of 200-fold, the profile for only the sample had double peaks. Though the first peak was smaller than the second, it became bigger than the second upon the addition of Fe to the sample. A similar behavior of the two peaks was also observed for the sample solutions at different dilution factors. No two-peak was observed for Fe in Mussel. The peak splitting of Fe for Pepperbush was mainly caused by the coexistence of the Ca and perchlorate used for sample decomposition and by the low content of P, as described later. The reason for the peak splitting and its mechanism are not yet known. Choice of matrix modifier The addition of a matrix modifier was attempted in order to avoid any matrix effect. To combine the observed split Fe signals for Pepperbush to one peak, some compounds were examined as a matrix modifier. With increased additions of Na, K, Ca, P, Mg, Mn and Zn to the sample, though the profile of Fe tended to give a single peak, Al accelerated the split. Taking into account the single peak of Fe observed for Mussel and higher contents of Na and P in Mussel than those in Pepperbush, disodium hydrogenphosphate was used at concentrations of 10 mmol dm 3 for a sample solution diluted to 100-fold. Though it gave a single peak, the analytical result for Fe was still slightly lower than the certified value. In order to prevent the denitrification of a quartz torch, disodium hydrogenphosphate was replaced by ammonium dihydrogenphosphate. Moreover, the sample solution was further diluted in order to minimize the matrix effect due to the main components, considering the detection limits of the analytes. In Table 3 the effect of the concentration of the matrix modifier is shown for the metals in Pepperbush at dilutions of 1000-fold for Fe, Mn and Zn and of 100fold for others. As can be seen in Table 3, the analytical results for Cd, Co, Cu and Pb agree with the certified values, even with no modifier. Moreover, the result of Mn is also good at a dilution factor of 1000-

fold, without a modifier. For accurate analyses of Fe and Zn, a modifier is required. Ammonium dihydrogenphospahte was sufficient in the range from 10 to 100 mmol dm 3. At 300 mmol dm 3 of ammonium dihydrogenphosphate, the plasma sometimes turned off. Peak-height measurements in the present IF-SAM offered fairly accurate values for 7 metals in the presence of 30 mmol dm 3 ammonium dihydrogenphosphate. Even in calibration-curve methods with a peak-area or peak-height measurement, the analytical results were close to the certified values at the same concentration of the modifier. Therefore, 30 mmol dm-3 ammonium dihydrogenphosphate was recommended as the matrix modifier. The profile of the Fe signal in the presence of the modifier is illustrated in Fig. 3. The signals of other metals, including Zn, showed single profiles without any interference. Application of the present method NIES-CRMs No. 1 Pepperbush, No. 5 Human Hair, No. 6 Mussel and No. 7 Tea Leaves were analyzed with the present method; the results are summarized in Table 4. The dilution factor was 100-fold, except for elements marked with a mark. The obtained results agree well with the certified values for 7 metals in NIES-CRMs analyzed with peak-area measurements.

Fig.

3.

Signal

profiles with

of Fe in IF-SAM 30 mmol

for Pepperbush dihydroFe standard;

at

1000-fold

dilution

dm-3 ammonium ppm

genphosphate. a, sample; b, sample+0.4 C, 0.4 ppm Fe standard only.

Table

Effect

of the concentration

of matrix

modifier

for metals

in Pepperbush

at 100-fold

sample

dilution

a.

Sample

dilution:

1000-fold.

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Table

Analytical

results (ppm) for trace metals in NIES-CRMs

(n=3)

ND.,

not

determined;

), reference value;

>>, estimated

value.

a. Measured

with

1000-fold

dilution.

In addition, peak-height measurements also offered results in good agreement with the certified values, except for the slightly lower results of Cd and Pb in Mussel and Mn in Tea Leaves. Calibration-curve methods with peak-area and peakheight measurements were also carried out in order to evaluate the effect of 30 mmol dm 3 ammonium dihydrogen phosphate as a matrix modifier. As shown in Table 4, some results are not so close to the certified values, even with the modifier. Without the modifier, however, most of the results deviated from the certified using the calibration curve method.

The present method would determinations of many trace various minute samples.
The Matano authors and wish T. Ooe to thank

be useful for accurate or ultra trace elements in

Messrs. Jarrell-Ash

S. Co. and

Hamada,

Y.

of Nippon

for the kind many

permission to use the furnace atomizer useful suggestions throughout this work.

for their

References

Even if the ETV-ICP technique could reveal high sensitivity, severe matrix effects would exist and could not be avoided just by using IF-SAM for certain elements. The matrix effect could be either minimized or eliminated by the addition of ammonium dihydrogen phosphate as a matrix modifier in the present IF-SAM. Sample solutions could be kept intact using the present method, since the modifier was added not to the sample but only to the standard solutions; the standards were added directly to the sample within the furnace. This method was quite convenient for unknown samples, since many preliminary measurements were allowed with a minimum consumption of the sample solution.

1. S. Greenfield, I. L. Jones and C. T. Berry, Analyst [London], 89, 713 (1964). 2. R. H. Wendt and V. A. Fassel, Anal. Chem., 37, 920 (1965). 3. H. Matsusiewicz, J. Anal. At. Spectrom.,1, 171 (1986). 4. D. L. Millard, H. C. Shan and G. F. Kirkbright, Analyst [London], 105, 502 (1980). 5. S. E. Long, R. D. Snook and R. F. Browner, Spectrochim. Acta, 40B, 553 (1985). 6. S. M. Schmertmann, S. E. Long and R. F. Browner, J. Anal. At. Spectrom., 2, 687 (1987). 7. H. Matusiewicz, F. L. Fricke and R. M. Barnes, J. Anal. At. Spectrom., 1, 203 (1986). 8. J. Alvarado, P. Cavalli, N. Omenetto, G. Roddei and J. M. Ottaway, J. Anal. At. Spectrom., 2, 357 (1987). 9. H. Isoyama, T. Uchida, C. Iida, G. Nakagawa, Y.

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Matano, T. Ooe, S. Hamada, H. Iwata and K. Goto, Anal. Sci., 5, 49 (1989). H. Isoyama, T. Uchida, T. Niwa, C. Iida and G. Nakagawa, J. Anal. At. Spectrom., 4, 351 (1989). T. Uchida and Y. Hirao, Bunseki,1987, 203.

12. H. Isoyama, T. Uchida, K. Oguchi, C. Iida and G. Nakagawa, Anal. Sci., 6, 385 (1990). (Received February 21, 1990) (Accepted May 25, 1990)