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Elemental analysis of bovine liver by inductively coupled plasma atomic emission spectrometry by using a simple dissolution procedure
T&ma, Vol. 39, No. 5, pp.563466, 1992 printed in Great Britain. All rights resewed
0039-9140/92 SS.00+ 0.00 CopyrightQ 1992F%rgamon F?w plc
TJKHNICAL NOT&
ELEMENTAL ANALYSIS OF BOVINE LIVER BY INDUCTIVELY COUPLED PLASMA ATOMIC EMISSION SPECTROMETRY BY USING A SIMPLE DISSOLUTION PROCEDURE T. N. ASP and W. LUND Department of Chemistry, University of Oslo, Box 1033, N-0315 Oslo, Norway (Received 31 May 1991. Revised 5 September 1991. Accepted 23 October 1991) Summary-A simple digestion with nitric acid followed by liltration of the undigested lipids was found to be suitable for the decomposition of bovine liver, prior to multielement determination by inductively coupled plasma atomic emission spectrometry. Using reference materials, accurate results were obtained for cadmium, copper, iron, manganese, molybdenum and zinc.
The determination of metals in bovine liver by inductively coupled plasma atomic emission spectrometry (ICP-AES) requires dissolution of the sample. Many different decomposition methods can be used for this purpose.’ Decomposition with a single acid or acid mixtures is performed either on a hot plate, in a microwave oven or under pressure in Teflon-lined steel bombs. Dry ashing is done either in a temperature-programmed mufile furnace, or by low temperature ashing in a low pressure oxygen plasma. Also solubilization with tetramethyl ammonium hydroxide may be used. De Boer and Maessen2 compared all of these methods for the decomposition of bovine liver prior to ICP-AES analysis, and showed that in skilled hands, most of the methods gave accurate results for at least some elements. Dahlquist and Knoll’ have compared wet and dry ashing of bovine liver for ICP-AES analysis, and found that wet ashihg gave poorer detection limits because of the dilution, whereas dry ashing gave rise to element losses. Other workers have also used dry ashing4 or low temperature ashing5 for multielement analysis of bovine liver and other biological tissues by ICP-AES. Apparently, there is not one decomposition method that stands out as the method of choice. However, the methods differ much with respect to the reagents used and the equipment and time needed for the digestion. An acid mixture
which includes perchloric acid will normally ensure a complete mineralization of the biological material, but the use of perchloric acid should be avoided whenever possible, for safety reasons. Dry ashing in a muffle furnace usually requires heating for 10-30 hr, and the choice of a suitable crucible material and temperature programming is often essential. In addition, the use of an ashing aid such as magnesium nitrate is not to be recommended for dry ashing in combination with ICP-AES, because a high concentration of magnesium gives rise to an enhanced background and a depression of the emission signals.68 The use of a microwave oven, high pressure bombs and low temperature ashing all require special equipment. For routine analysis, an attractive decomposition procedure would be one that utilizes standard equipment, has a short digestion time, low reagent consumption and a low dilution factor. The last point is important because of the moderate detection limits of ICP-AES. The suitability of a simple procedure based on heating with nitric acid was investigated in this work. A low dilution factor was considered essential, in order to dtiermine trace metals like cadmium. Bovine liver is not a difficult matrix to dissolve, but some fat usually remains after treatment with nit&z acid. Low values have been reported for copper and iron in bovine liver, using extraction with cold nitric acid.2 563
T. N.
564
ASP
EXPERIMENTAL
Apparatus
A Perk&Elmer ICP 5500B sequential inductively coupled argon plasma atomic emission spectrometer was used. The plasma generator, operating at 27.12 MHz, and the torch box was identical with those of the Perkin-Elmer ICP 6500, but the data system was a Model 3600 computer with a PR-100 printer. An all-quartz torch was used instead of the demountable torch. A peristaltic pump and a cross-flow nebulizer were used for sample introduction. Materials
All reagents were of analytical-reagent grade. Multielement standard solutions were prepared from 1000 f 0.5 ppm single-element standards (Spectrascan, Oslo). The bovine liver materials analysed were SRM 1577a from the National Institute of Standards and Technology (NIST; Gaithersburg, USA) and NSC-2 from Czechoslovakia.g Digestion procedures Procedure A (recommended). To a 0.5-g sample, add 10 ml of 65% nitric acid in a beaker and boil on a hot plate until ca. 1 ml of acid remains (l-2 hr). Transfer to a lo-ml standard flask and dilute to volume with water. Filter through a filter paper (Black Ribbon) before the ICP analysis. Procedure B. Same as procedure A, except that the solution, including the water used for washing the beaker is filtered, before dilution to 10 ml in a standard flask. Procedure C. To a 0.8-l .0-g sample, add 7 ml of nitric acid and 7 ml of water in a beaker and let it stand for 24 hr at room temperature. Filter the solution (and the washings) through a filter paper into a 25-ml standard flask and dilute to volume. Procedure D. Same as procedure A, except that the remaining solution (ca. 1.5 ml) is finally
and W. LUND
diluted to 10 ml with methanol instead of water, and the filtration is omitted. Procedure E. To a l-g sample, add 16 ml of an acid mixture consisting of 9 ml of nitric acid, 6 ml of perchloric acid (70%) and 1 ml of sulphuric acid (95.5%). Heat on a water bath by slowly increasing the bath temperature to loo”, then continue the digestion on a hot plate until ca. 1 ml of acid remains. Cool, transfer to a 25-ml standard flask and dilute to volume with water. Measurements The plasma conditions used in this work were: plasma power 1.0 kW, plasma gas flowrate 15 l./min, nebuliser gas flow-rate 1 l./min and viewing height 18 mm. The wavelengths used for the emission measurements were (in nm): Cd: 228.80, Cu: 324.75, Fe: 261.19, Mn: 257.61, MO: 281.62, Zn: 213.85. Background correction was used for cadmium, manganese and molybdenum. The instrument was standardized with two multielement standard solutions. For each digestion procedure, the standards were matrix-matched with the samples, with respect to the concentration of acids. Also, three blank digestions were carried out for each procedure. Of the resulting solutions, one was run as a blank, while the other two were run as unknowns (control). The blank values were usually less than 10% of the sample concentrations. RESULTS AND DISCUSSION
The two bovine liver materials SRM 1577a and NSC-2 were analysed. NSC-2 was obtained from Czechoslovakia and had a certified value only for cadmium.g The results obtained, using the digestion procedures A, B, C and E (see Experimental) are given in Tables l-3. The two sets of results reported for procedure A in Tables 1 and 3 refer to digestions carried out at different dates. Each value given represents
Table 1. Results @g/g; n = 3) for cadmium, copper and iron in SRM 1577a (NUT) bovine liver Cadmium Procedure Certified value A A B C
Mean
S.D.
0.44 0.48 0.40 0.30 0.21
0.06* 0.06 0.02 0.03 0.03
*95% confidence limit.
Copper Mean SD. 158 160 167 125 125
7+ 5 3 6 2
Iron Mean
SD.
194 188 206 154 115
20’ 9 12 5 5
Elemental analysis of bovine liver
56.5
Table 2. Results @g/g; n = 3) for manganese, molybdenum and zinc in SRM 1577a (NIST) bovine liver Manganese Mean Certifkxi value Procedure A
S.D.
9.9 10.7
zinc
Molybdemun Mean
S.D.
Mean
S.D.
:::
0 5+ 0:2
123 123
s* 3
*95% confidence limit.
the mean of the results from three parallel digestions. The corresponding standard deviations are also given, The standard deviations for repeated ICP-analyses of each sample solution are not shown, but these were generally lower than the standard deviations for parallel digestions. From the Tables it can be seen that only procedure A gave results in agreement with the certified values for all metals and both samples studied. For copper and iron in NSC-2, the results should be compared with those obtained by procedure E, due to the absence of certified values. The concentration of cadmium (Tables 1 and 3) was close to the detection limit. When the detection limit was determined experimentally,‘0 a value of 4 pg/l. was obtained, based on three times the standard deviation of the back~ound signal. This corresponds to 0.08 pg/g dry liver, when procedure A is used. For the other elements, the detection limits were of little interest, because these were at least a factor of 0.002 lower than the actual concentrations in the liver material, except for moly~enum, where the factor was ea. 0.05. All samples were apparently dissolved completely, after being boiled with nitric acid for l-2 hr (procedure A). However, when the digest was cooled, some fat appeared on the surface, and some was also formed when the digest was diluted with demineralized water. For this reason, the solution was filtered before the ICP analysis. From Tables 1
Table 3. Results &g/g;
n = 3) for cadmium, copper (Czechoslovakia) bovine liver
Cadmium Procedure
and 3 it can be seen that the filtration must be done after the sample has been diluted to volume (procedure A). If the digest is filtered before the final dilution to 10 ml (procedure B), low results are obtained, because of adsorption losses during the filtration. It was found that the adsorption losses could be minimized by a more thorough washing of the filter paper, but the resulting larger dilution factor prevented the quantitative determination of cadmium in this case. Low results were also obtained with procedure C, indicating that treatment with diluted nitric acid at room temperature for 24 hr does not release all metal. The method is similar to the extraction procedure used by De Boer and Maessen,’ who also obtained low results for copper and iron. Obviously a high temperature is more effieient than a long time for releasing these metals. Somewhat better results were obtained when concentrated nitric acid was used instead of the 1 + 1 diluted acid in procedure C, but the values were still low. To avoid the formation of fat, attempts were made to dilute the solution to volume with methanol instead of water (procedure D). It was found that the resulting solution (85% methanol) could be handled by the ICPinstrument, provided a more narrow injection tube (0.85 mm), a higher power (1.3 kW), and an auxiliary gas flow (1 l./min) were used. However, the methanol depressed the emission signals, and caused a high and structured background around the three most useful
Copper Mean S.D.
Mean
S.D.
Certified value A A
0.43 0.49 0.43
0.03* 0.03 0.07
52 49
: E
0.29 0.30 0.48
0.06 0.04 0.05
40 41 49
‘95% confidence limit.
and iron in NC-2 Iron Mean
S.D.
1 2
301 303
8 16
41 2
;: 297
26 4 13
T. N. ASP and W. LUND
566
cadmium lines (214.44, 226.50 and 228.80 nm). Therefore, cadmium could not be determined in the samples diluted with methanol. In addition, the plasma was unstable and carbon was deposited in the injection tube. Therefore, the use of a methanol-water mixture as solvent appears to be unattractive for the determination of trace metals in biological material by ICP-AES.
2. J. L. M. De Boer and F. J. M. J. Maessen, Spectrochim. Acta, 1983, 38B, 739. 3. R. L. Dahlquist and J. W. Knoll, Appl. Spectrosc., 1978, 32, 1.
REFERENCES
9. P. Mader, J. Kucera, J. Cibulka and D. Miholova, Chem. L.isty, 1989, 83, 765. 10. G. F. Wallace and P. Barrett, Analytical Methoak Development for Inductively Coupled Plasma Spectrometry, p. 3-3. Perkin-Elmer, Norwalk, 1981.
K. A. Wolmk, J. I. Rader, C. M. Gaston and F. L. Fricke, Spectrochim. Acta, 1985, 4OB, 245. K. Shiraishi, G. Tanaka and H. Kawamura, Talanta, 1986, 33, 861.
T. N. Asp and W. Lund, unpublished results. G. F. Larson and V. A. Fassel, Appl. Spectrosc., 1979, 33, 592. 8. M. Thompson and M. H. Ramsey, Analyst, 1985, 110,
1413.
1. F. J. M. J. Maessen, in P. W. J. M. Boumans (ed.), Inductively Coupled Plasma Emission Spectroscopy, Part 2, p. 100. John Wiley, New York, 1987.
0039-9140/92 SS.00+ 0.00 CopyrightQ 1992F%rgamon F?w plc
TJKHNICAL NOT&
ELEMENTAL ANALYSIS OF BOVINE LIVER BY INDUCTIVELY COUPLED PLASMA ATOMIC EMISSION SPECTROMETRY BY USING A SIMPLE DISSOLUTION PROCEDURE T. N. ASP and W. LUND Department of Chemistry, University of Oslo, Box 1033, N-0315 Oslo, Norway (Received 31 May 1991. Revised 5 September 1991. Accepted 23 October 1991) Summary-A simple digestion with nitric acid followed by liltration of the undigested lipids was found to be suitable for the decomposition of bovine liver, prior to multielement determination by inductively coupled plasma atomic emission spectrometry. Using reference materials, accurate results were obtained for cadmium, copper, iron, manganese, molybdenum and zinc.
The determination of metals in bovine liver by inductively coupled plasma atomic emission spectrometry (ICP-AES) requires dissolution of the sample. Many different decomposition methods can be used for this purpose.’ Decomposition with a single acid or acid mixtures is performed either on a hot plate, in a microwave oven or under pressure in Teflon-lined steel bombs. Dry ashing is done either in a temperature-programmed mufile furnace, or by low temperature ashing in a low pressure oxygen plasma. Also solubilization with tetramethyl ammonium hydroxide may be used. De Boer and Maessen2 compared all of these methods for the decomposition of bovine liver prior to ICP-AES analysis, and showed that in skilled hands, most of the methods gave accurate results for at least some elements. Dahlquist and Knoll’ have compared wet and dry ashing of bovine liver for ICP-AES analysis, and found that wet ashihg gave poorer detection limits because of the dilution, whereas dry ashing gave rise to element losses. Other workers have also used dry ashing4 or low temperature ashing5 for multielement analysis of bovine liver and other biological tissues by ICP-AES. Apparently, there is not one decomposition method that stands out as the method of choice. However, the methods differ much with respect to the reagents used and the equipment and time needed for the digestion. An acid mixture
which includes perchloric acid will normally ensure a complete mineralization of the biological material, but the use of perchloric acid should be avoided whenever possible, for safety reasons. Dry ashing in a muffle furnace usually requires heating for 10-30 hr, and the choice of a suitable crucible material and temperature programming is often essential. In addition, the use of an ashing aid such as magnesium nitrate is not to be recommended for dry ashing in combination with ICP-AES, because a high concentration of magnesium gives rise to an enhanced background and a depression of the emission signals.68 The use of a microwave oven, high pressure bombs and low temperature ashing all require special equipment. For routine analysis, an attractive decomposition procedure would be one that utilizes standard equipment, has a short digestion time, low reagent consumption and a low dilution factor. The last point is important because of the moderate detection limits of ICP-AES. The suitability of a simple procedure based on heating with nitric acid was investigated in this work. A low dilution factor was considered essential, in order to dtiermine trace metals like cadmium. Bovine liver is not a difficult matrix to dissolve, but some fat usually remains after treatment with nit&z acid. Low values have been reported for copper and iron in bovine liver, using extraction with cold nitric acid.2 563
T. N.
564
ASP
EXPERIMENTAL
Apparatus
A Perk&Elmer ICP 5500B sequential inductively coupled argon plasma atomic emission spectrometer was used. The plasma generator, operating at 27.12 MHz, and the torch box was identical with those of the Perkin-Elmer ICP 6500, but the data system was a Model 3600 computer with a PR-100 printer. An all-quartz torch was used instead of the demountable torch. A peristaltic pump and a cross-flow nebulizer were used for sample introduction. Materials
All reagents were of analytical-reagent grade. Multielement standard solutions were prepared from 1000 f 0.5 ppm single-element standards (Spectrascan, Oslo). The bovine liver materials analysed were SRM 1577a from the National Institute of Standards and Technology (NIST; Gaithersburg, USA) and NSC-2 from Czechoslovakia.g Digestion procedures Procedure A (recommended). To a 0.5-g sample, add 10 ml of 65% nitric acid in a beaker and boil on a hot plate until ca. 1 ml of acid remains (l-2 hr). Transfer to a lo-ml standard flask and dilute to volume with water. Filter through a filter paper (Black Ribbon) before the ICP analysis. Procedure B. Same as procedure A, except that the solution, including the water used for washing the beaker is filtered, before dilution to 10 ml in a standard flask. Procedure C. To a 0.8-l .0-g sample, add 7 ml of nitric acid and 7 ml of water in a beaker and let it stand for 24 hr at room temperature. Filter the solution (and the washings) through a filter paper into a 25-ml standard flask and dilute to volume. Procedure D. Same as procedure A, except that the remaining solution (ca. 1.5 ml) is finally
and W. LUND
diluted to 10 ml with methanol instead of water, and the filtration is omitted. Procedure E. To a l-g sample, add 16 ml of an acid mixture consisting of 9 ml of nitric acid, 6 ml of perchloric acid (70%) and 1 ml of sulphuric acid (95.5%). Heat on a water bath by slowly increasing the bath temperature to loo”, then continue the digestion on a hot plate until ca. 1 ml of acid remains. Cool, transfer to a 25-ml standard flask and dilute to volume with water. Measurements The plasma conditions used in this work were: plasma power 1.0 kW, plasma gas flowrate 15 l./min, nebuliser gas flow-rate 1 l./min and viewing height 18 mm. The wavelengths used for the emission measurements were (in nm): Cd: 228.80, Cu: 324.75, Fe: 261.19, Mn: 257.61, MO: 281.62, Zn: 213.85. Background correction was used for cadmium, manganese and molybdenum. The instrument was standardized with two multielement standard solutions. For each digestion procedure, the standards were matrix-matched with the samples, with respect to the concentration of acids. Also, three blank digestions were carried out for each procedure. Of the resulting solutions, one was run as a blank, while the other two were run as unknowns (control). The blank values were usually less than 10% of the sample concentrations. RESULTS AND DISCUSSION
The two bovine liver materials SRM 1577a and NSC-2 were analysed. NSC-2 was obtained from Czechoslovakia and had a certified value only for cadmium.g The results obtained, using the digestion procedures A, B, C and E (see Experimental) are given in Tables l-3. The two sets of results reported for procedure A in Tables 1 and 3 refer to digestions carried out at different dates. Each value given represents
Table 1. Results @g/g; n = 3) for cadmium, copper and iron in SRM 1577a (NUT) bovine liver Cadmium Procedure Certified value A A B C
Mean
S.D.
0.44 0.48 0.40 0.30 0.21
0.06* 0.06 0.02 0.03 0.03
*95% confidence limit.
Copper Mean SD. 158 160 167 125 125
7+ 5 3 6 2
Iron Mean
SD.
194 188 206 154 115
20’ 9 12 5 5
Elemental analysis of bovine liver
56.5
Table 2. Results @g/g; n = 3) for manganese, molybdenum and zinc in SRM 1577a (NIST) bovine liver Manganese Mean Certifkxi value Procedure A
S.D.
9.9 10.7
zinc
Molybdemun Mean
S.D.
Mean
S.D.
:::
0 5+ 0:2
123 123
s* 3
*95% confidence limit.
the mean of the results from three parallel digestions. The corresponding standard deviations are also given, The standard deviations for repeated ICP-analyses of each sample solution are not shown, but these were generally lower than the standard deviations for parallel digestions. From the Tables it can be seen that only procedure A gave results in agreement with the certified values for all metals and both samples studied. For copper and iron in NSC-2, the results should be compared with those obtained by procedure E, due to the absence of certified values. The concentration of cadmium (Tables 1 and 3) was close to the detection limit. When the detection limit was determined experimentally,‘0 a value of 4 pg/l. was obtained, based on three times the standard deviation of the back~ound signal. This corresponds to 0.08 pg/g dry liver, when procedure A is used. For the other elements, the detection limits were of little interest, because these were at least a factor of 0.002 lower than the actual concentrations in the liver material, except for moly~enum, where the factor was ea. 0.05. All samples were apparently dissolved completely, after being boiled with nitric acid for l-2 hr (procedure A). However, when the digest was cooled, some fat appeared on the surface, and some was also formed when the digest was diluted with demineralized water. For this reason, the solution was filtered before the ICP analysis. From Tables 1
Table 3. Results &g/g;
n = 3) for cadmium, copper (Czechoslovakia) bovine liver
Cadmium Procedure
and 3 it can be seen that the filtration must be done after the sample has been diluted to volume (procedure A). If the digest is filtered before the final dilution to 10 ml (procedure B), low results are obtained, because of adsorption losses during the filtration. It was found that the adsorption losses could be minimized by a more thorough washing of the filter paper, but the resulting larger dilution factor prevented the quantitative determination of cadmium in this case. Low results were also obtained with procedure C, indicating that treatment with diluted nitric acid at room temperature for 24 hr does not release all metal. The method is similar to the extraction procedure used by De Boer and Maessen,’ who also obtained low results for copper and iron. Obviously a high temperature is more effieient than a long time for releasing these metals. Somewhat better results were obtained when concentrated nitric acid was used instead of the 1 + 1 diluted acid in procedure C, but the values were still low. To avoid the formation of fat, attempts were made to dilute the solution to volume with methanol instead of water (procedure D). It was found that the resulting solution (85% methanol) could be handled by the ICPinstrument, provided a more narrow injection tube (0.85 mm), a higher power (1.3 kW), and an auxiliary gas flow (1 l./min) were used. However, the methanol depressed the emission signals, and caused a high and structured background around the three most useful
Copper Mean S.D.
Mean
S.D.
Certified value A A
0.43 0.49 0.43
0.03* 0.03 0.07
52 49
: E
0.29 0.30 0.48
0.06 0.04 0.05
40 41 49
‘95% confidence limit.
and iron in NC-2 Iron Mean
S.D.
1 2
301 303
8 16
41 2
;: 297
26 4 13
T. N. ASP and W. LUND
566
cadmium lines (214.44, 226.50 and 228.80 nm). Therefore, cadmium could not be determined in the samples diluted with methanol. In addition, the plasma was unstable and carbon was deposited in the injection tube. Therefore, the use of a methanol-water mixture as solvent appears to be unattractive for the determination of trace metals in biological material by ICP-AES.
2. J. L. M. De Boer and F. J. M. J. Maessen, Spectrochim. Acta, 1983, 38B, 739. 3. R. L. Dahlquist and J. W. Knoll, Appl. Spectrosc., 1978, 32, 1.
REFERENCES
9. P. Mader, J. Kucera, J. Cibulka and D. Miholova, Chem. L.isty, 1989, 83, 765. 10. G. F. Wallace and P. Barrett, Analytical Methoak Development for Inductively Coupled Plasma Spectrometry, p. 3-3. Perkin-Elmer, Norwalk, 1981.
K. A. Wolmk, J. I. Rader, C. M. Gaston and F. L. Fricke, Spectrochim. Acta, 1985, 4OB, 245. K. Shiraishi, G. Tanaka and H. Kawamura, Talanta, 1986, 33, 861.
T. N. Asp and W. Lund, unpublished results. G. F. Larson and V. A. Fassel, Appl. Spectrosc., 1979, 33, 592. 8. M. Thompson and M. H. Ramsey, Analyst, 1985, 110,
1413.
1. F. J. M. J. Maessen, in P. W. J. M. Boumans (ed.), Inductively Coupled Plasma Emission Spectroscopy, Part 2, p. 100. John Wiley, New York, 1987.