Anal. Chem. 1983, 55, 1963-1970
1983
the ability of this combination to provide concentration data on biological soft tissue. The technique is equally applicable to a wide range of heterogeneous materials.
'g
ACKNOWLEDGMENT
....
The authors are grateful to Sarah Asher for her invaluable assistance throughout the work and to Adam Patkin for his help with the software. The implantation facilities of the National Research and Resource Facility for Submicron Structures a t Cornel1 were used. Registry No. Ca, 7440-70-2; B, 7440-42-8; BF2+,12355-90-7.
LITERATURE CITED
Flgure 6. r 2 correlation of successive images vs. time.
it does not become significantly greater than the system noise until 6 min have elapsed. This is longer than the analysis time. Thus, while sputtering does change the ion image, it does not appear to seriously affect these samples. At present, however, the system noise is a limiting factor. Since the implanted ion dose must be kept low to avoid gross damage to the tissue, and due to the need to detect small concentrations of elemenits, the imaging system must perform well a t high gains. Methods for noise reduction, either through hardware modifications such as high-performance micro-, channel plates or with the use of appropriate noise filtering software, will provide increased sensitivity. A second current limitation is the speed a t which images can be transferred to disk storage. At the sputtering rate of 1.2 nm/s used in these oxperiments, a minimum of 9.6 nm of material was eroded between images. The use of a videotape or videodisk unit would allow characterization of the implant with much improved depth resolution by providing real time image storage. In summary, the merging of quantitative ion implantation, with ita ability to provide a laterally homogeneous reference element in an otherwisle heterogeneous material, and the MIDAS image acquisition system's unique capability for characterizing this implanted reference element a t each dis. Crete location of the ion image provides a powerful tool for quantitative ion microscopy. This study has demonstrated
Morrison, G. H.; Slodzian, G. Anal. Chem. 1975, 47, 932A-943A. Castaing, R.; Slodzian, G. J . Microsc. 1962, 1 , 395-410. Galle, P.; Blaise, G.; Slodzian G. "IVth National Conference on Electron Microprobe Analysls"; California Institute of Technology: Pasada na, CA, 1969; Vol. 1, p 36. Galle, P. I n "secondary Ion Mass Spectrometry SIMS-11", Benninghoven, A., et al., Eds.; Springer: New York, 1979; Springer Ser. in Chem. Phys., Vol. 9, p 238. Spurr, A. K. Scannlng Electron Mlcrosc. 1960, 3 , 97-109. Truchet, M.; Trottier, S. C . R . Acad. Sci., Ser. D 1979, 288, 83 1-833. Lodding, A.; Gourgont, J. M.; Peterson, L. G.; Frostell, G. 2. Nafurforsch. A 1974, 29, 897-900. Burns-Bellhorn, M. S.; File, D. M. Anal. Blochem. 1979, 92,213-221. Zhu, D.; Harrls, W. C.; Morrison, G. H. Anal. Chem. 1982, 54, 4 19-422. Gries, W. H.; Rautenbach, W. L. 6th International Symposium on Microtechniques, Graz, Austria 1970. Leta, D. P.; Morrlson, G. H. Anal. Chem. 1980, 52,277-280. Leta, D. P.; Morrison, G. H. Anal. Chem. 1980, 52,514-519. Furman, B. K.; Morrlson, G. H. Anal. Chem. 1980, 52, 2305-2310. Chandra, S.;Chabot. J. F.; Morrison, G. H.; Leopold, A. C. Science 1982, 216, 1221-1223. Spurr, A. R. J . Ulffastfuct. Res. 1969, 26, 31-43. Salema, R.; Brando, I . J . Submicrosc. Cytol. 1973, 5 ,79-96. Ruberol, J. M.; Lepareur, M.; Autier, B.; Gourgout, J. M. 8th International Congress on X-Ray Optlcs and Microanalysis and the 12th Annual Conference of the Microbeam Analysis Soclety, Boston, MA, 1977. Patkin, A. J.; Chandra, S.; Morrlson, G. H. Anal. Chem. 1982, 54, 2507-2510. Fassett, J. D.; Roth, J. R.; Morrlson, G. H. Anal. Chem. 1977, 49, 2322-2329. Frank, J.; AI-AIL L. Nature (London) 1975, 256,376-379.
RECEIVED for review April 8, 1983. Accepted July 22, 1983. Funding for this project was provided by National Institutes of Health, the National Science Foundation, and the Office of Naval Research.
Relative Sensitivity Factors of Elements in Quantitative Secondary Ion Mass Spectrometric Analysis of Biological Reference Materials G . 0. Ramseyer and GI. H. Morrison* Department of Chemistry, Cornell University, Ithaca, New York 14853 Secondary Ion mass spectrometry is applied to the quantitative analysts of homogenized blological reference materials. The technique involves determlning relative sensllvlty factors ratloed to K for Na, Ca, Mg, B, Mn, Fe, N, P, Ai, and CI, and ratloed to P for Ci, F, CN, and SO in several types of bloioglcal matrlces.
Secondary ion mass cipectrometry (SIMS) is a sensitive technique capable of determining most elements in a broad range of solid samples. Because the sensitivity of an element
is a function of the type of matrix in which it is located ( I ) , quantitative SIMS determinations are accomplished by either semitheoretical methods or empirical calibration methods using standards (2-4). When standards are available which closely approximate that of the sample, relative sensitivity factors (RSF's) have been demonstrated to consistently provide the most accurate results in inorganic matrices (2-5). RSF's have been applied to the elemental determinations in groups of similar inorganic matrices, including glasses (2, 5 , 6), metals (6, 7) and geologicals (8). Because of the wide diversity of biological materials and the lack of microstandards, the quantification of biological materials has been more
0003-2700/83/0355-1963$01.50/00 1983 Amerlcan Chemical Society
1964
ANALYTICAL CHEMISTRY, VOL. 55, NO. 12, OCTOBER 1983
Table I. Experimental Parameters instrument: CAMECA IMS-3f Ion Microanalyzer primary ions: 0,’ primary ion energy: 5.5 keV primary ion beam size: about 100 pm diameter primary ion raster size: 500 pm primary ion current density: 2 X A/cm2 mass resolution: 300 secondary ion energy range: 0-130 eV imaged field: 400 pm diameter circle field aperture: 1800 pm contrast diaphragm aperture: 60 pm entrance slit: 700 pm exit slit: 700 pm detection system: electron multiplier residual sample pressure: 4 x difficult. One approach has been the use of gelatin doped with elements of biological significance as biological matrix standards (9). Although the doped gelatins have given linear calibration curves (9, IO), there has been evidence that they are not universal biological standards (11). The purpose of this study is t o evaluate a variety of plant and animal homogenized bulk reference materials as SIMS microanalysis empirical calibration standards. RSF’s are determined to provide the most accurate results in biological matrices. The RSF’s from different types of biological matrices are compared t o determine the extent of any matrix effect. These RSF’s are also compared to those from gelatin matrices. The applicability of biological reference materials as empirical microanalysis standards for SIMS is established by the analyses of freeze substituted rat liver tissues.
EXPERIMENTAL SECTION Instrumentation. The study was accomplished with a CAMECA IMS-3f ion microanalyzer. The instrument was interfaced to a Hewlett-Packard 98451’ microcomputer. Instrumental parameters are listed in Table I. Computer Software. Analyses were performed under computer control. The cyclic nature of the CAMECA depth profile program provided sequential determination and storage of selected elemental isotopic secondary ion intensities as a function of time. The software was modified to automatically correct the measured secondary ion intensities for the dead time of the electron multiplier (70 ns). Reference Standards. The standards used in this study included the NBS biological standards Oyster Tissue SRM1566, Wheat Flour SRM1567, Rice Flour SRM1568, Trace Elements in Spinach SRM1570, Orchard Leaves SRM1571, Tomato Leaves SRM1573, Pine Needles SRM1575, Bovine Liver SRM1577, and Albacore Tuna RM50. National Biochemical Corporation standards for lung powder, heart powder, kidney powder, beef liver lyophilized, and duodenum pork (defatted) lyophilized were analyzed. Bowen’s Kale, one of the first biological reference materials, was also analyzed. Gelatin standards were prepared from Gelatin U.S.P. Powder (J.T. Baker) and Agar Flakes (Golden Harvest). The best values available, as listed in Table 11, were used for the elemental concentrations of the biological standards. The order of preference was NBS certified values, NBS for information only values, literature values (12, 131,and atomic absorption spectrometry values determined by this laboratory. Sample Preparation. A 1.5-g sample of each standard was placed in a Teflon container (4 cm length, 3 cm diameter), and two steel balls, which weighed 1.05 g and 7.2 g, were added. Because of the hardness of the gelatin samples, a steel container of the same dimensions as the Teflon container was used. After each standard was homogenized in a SPEX MIXER/MILL SO00 for 30 min, it was sieved through a series of 100,200 and 325 mesh monofilament nylon cloth screens (SPEX Industries). in. diameter 200 mesh copper screen was placed in the A bottom of a 3/8 in. diameter pellet press mold, and approximately 200 mg of standard of less than 325 mesh were added. A No. 4450 Briquetting Machine (Harry W. Dietert Co.) was used at 200000 Ib/in2 pressure for 2 min to press the standards into pellets. The
IXlE’
B o v i n e L i v e r SRM1577 t/t
a
lXlE@
IS
b
m
20
‘0
Ha:
”
B o v i n e L v e r SRM1577
$1-
Figure 1. Positive and negative secondary ion mass spectrum of
Bovine Liver SRM1577. copper screen became an integral part of the surface of the standard. Each pellet was silver-pastedto an aluminum disk, and degassed in a vacuum desiccator for several hours. The standards were individually spring loaded into a sample holder so that contact was made between the inner surface of the sample holder and the copper screen of the standard. Tissue Preparation. Approximately l-mm3pieces of rat liver were excised and quickly frozen in liquid nitrogen slush (-212 “C) in order to immobilize diffusible elements. Liver pieces were then freeze substituted with ether-acrolein (80:20) as substitution media for 5 days at -85 “C according to the method previously reported from our laboratory (14). Sections, 2 fim thick, were cut with a dry glass knife and LKB Ultrotome 111. Sections were pressed with silicon wafers. Procedure. Generally nine masses were sequentially integrakd for 2 s each, 45 cycles of data were acquired, and the mean intensities of the last 10 cycles were calculated. Thirty minutes was required for one complete analysis. Except for Wheat Flour SRM1567, each standard was analyzed at least four different times over a several month period. Because of the physical instability of the Wheat Flour SRM1567 pellet, it was only analyzed twice.
RESULTS AND DISCUSSION The positive and negative secondary ion mass spectra of Bovine Liver SRM1577 are shown in Figure 1. The elemental isotopic ions of potassium, sodium, magnesium, calcium, and copper are the predominant peaks in the positive secondary ion spectrum. The copper is mostly from the grid. The predominant negative secondary ion peaks are molecular ions and the elemental isotopic ions of carbon, hydrogen, oxygen, copper, chlorine, and fluorine. The absolute sensitivity factor (ASF) is defined as ASF, = (iJCJ,) (1) and the relative sensitivity factor is defined as
ANALYTICAL CHEMISTRY, VOL. 55, NO. 12, OCTOBER 1983
1965
_____________-_____I______.-__________
Table 11. Elemental Co'ncentrations of Biological Reference Materials (Values Are in Ppm Unless Otherwise Labeled) Trace Elements Orchard Tomato Oyster Tissue Wheat Flour Rice Flour in Spinach Leaves Leaves element SRM1566 SRM1567 SRM1568 SRM1570 SRMl571 SRM1573 B N F Na Mg A1 P S
c1
K Ca Mn
Fe element B N F Na
Mg A1
P
(30) (5.9%) (5.2)b 0.51 t 0.03%a 0.128t 0.009%
8.0 i: 1.5 466i 5e 17e
6.0 f 1.5 527 i 6e
0.136 i 0.004% 0.019 i 0.001% 8.5 + 0.5 18.3 i: 1.0
0.112 i: 0.002% 0.014 i 0.002% 20.1 i 0.4 8.7 f 0.6
11tje
(0.81%)
(0.76%) (1.0%)
0.969 t 0.005% 0.15 f 0.02% 17.5 i 1.2 1 9 5 f 34 Pine Needles SRM1575
Bovine Liver SRM1577
element
Albacore Tuna RM50
0.65 i 0.03% 3.56 i 0.03% 1.35 + 0.03% 165i 6 5 5 0 + 20
Bowen's Kale
gelatin
(5.0) .
I
522i 13c (0.7%) (0.12%) 0.34 i 0.02% 1.10 i 0.07%' 4.46 t 0.03 3.00 i. 0.03% 238 i 7 690i: 25
agar
48.8d (1.2%)
10.6
26t 4 0.15 i 0.02%' 5 4 5 i 30 0 . 1 2 t 0.02%
0.243 t 0.013% 604 i 9 45.6d
2 4 3 t 20' 0.37 i 0.02% 0 . 4 1 i 0.02% 6 7 5 i 15 2 0 0 t 10
(0.27%) 0.97 f 0.06% 124k 6 10.3 f 1.0 268 i 8
i
0.6% 0.11% 0.11 i O.O1%d
3 2 i 2d
(1~1%)
S
C1 K Ca Mn Fe
1.31 i 0.07%c 0.90 i: 0.06%c 870 i 50 0.55 i 0.02%
33i 3 2.76 i 0.05% (4) 82 t 6 0.62 * 0.02% 410d 0.21 i 0.01% (1900) (690) 1.47 i 0.03% 2.09 t 0.03% 91 i: 4 300 f 20
duodenum pork
1.60%d 0.15 i O . O 1 % d 1.22% 0.045 t 0.006%d 1.3 57f 2
kidney powder
4.92%d 0.25%d 0.16%d 38.2d 610d
0.989 i 0.009%e 0.072%e
0.055 i 0.002%e 0.047%e
0.34%d 2.46%d 4.09%d 14
0.0137 i 0.0003%e 0.082 i O.OO1%e
0.00295 i 0.00008%e 0.370 t 0.005%e
l l d
110 i l e
10.7 i 2.1e
lung powder
beef liver
heart powder
B N
F Na Mg
AI P
0.698 i 0.008%e 0.105%e 95"
0.753
1.129 * 0.018%e 0.0417 -i. 0.0013%e
17e
0.073%e 19
0.231 f 0.003%e 0.078%e 60e
0.528 i 0.004%e 0.13 0%e 42e
0.988 i 0.011%" 0.0553 i 0.0008%e
0.907 0.087
0.822 i 0.014%e 0.0118i O . O O 1 l % e
1.032 i 0.007%e 0.0210 i 0.0008%e
i
0.006%"
O.llO%e
0.701 t 0.009%e
S
c1 K Ca
i i
0.017%e 0.002%e
Mn
Fe 510f 3e 629 i 4e 786 + 5e 173 i 2e 2662 2e a NBS certified values. NBS values for information only. Reference 12. Reference 13. e Determined by atomic absorption spectrometry, this laboratory. where i is the measured secondary ion intensity, C is the elemental concentration, f is the isotopic abundance, and the subscripts x and ref refer to the analyte and reference elements, respectively. If the matrices of the reference standard and the sample are identical, accurate quantitative results have been obtained by using ASF's in inorganic matrices. If the matrices are similar, RSF's have been shown to give accurate quantitative results. The reference element included in the RSF compensates for experimental fluctuations such as differential sputtering. The local thermal equilibrium (LTE) model (1) is based upon the premise that ,a thermodynamic equilibrium exists in a plasma near the sample's surface. The relationship of the number of an element's ions ni to atoms no is then determined from this form of the Saha-Eggert equation In (ni/no) = 15.38 1.5 In T In (2Qi/Q,) 5040(1, - AE)/ T - In Ne- (3)
+
+
where 2 is the partition function of a free electron, Q, and Q, are the partition functions of the element's ions and atoms, AE is the Coulomb interaction potential of the charged particles, Ipis the element's first ionization potential, T is thie
temperature of the plasma, and Ne- is the electron density of the plasma. Parameters other than Ne-and T a r e available from the literature. The CARISMA (15) software routine is normally used to determine T and Ne- for the best fit of ni to no for two internal standards. Carbon, potassium, and phosphorus were evaluated as reference elements for the RSF method. Carbon has been used as the reference element in gelatin (IO),but the carbon secondary ion yields in these types of biological materials did not change proportionally with the secondary ion yields of the analyte elements. Potassium and phosphorus are certified in all of the NBS standard reference materials, and the elemental isotopes of 39K+and 31P-do not have significant mass interferences. These elemental isotopes were used as the reference isotopes for positive and negative secondaries, respectively. Negative primary ions are sometimes used for the analyses of biological materials. The secondary ion intensities of sodium and potassium in Bovine Liver SRM1577 had not reached equilibrium after the sample had been presputtered for 1 h with negative primary oxygen ions. Less than 5 min was required to reach equilibrium by using positive primary
1968
ANALYTICAL CHEMISTRY, VOL. 55, NO. 12, OCTOBER 1983
Table 111. Mean Relative Sensitivity Factors standard
I95%
Confidence Limits Ratioed to Potassium for Positive Secondary Ions
Na/K
Ca/K
Mg/K Botanical Leaves
spinach orchard leaves tomato leaves pine needles Bowen’s Kale leaves mean
1.04 f 0.04 (12)a 0.79 f 0.11 (12) 0.87 i 0.15 (8) 1.88 i 0.50 (13) 0.82 f 0.09 (10) 1.08
wheat flour rice flour flour mean
1.99 0.34 (2) 2.42 f 1.07 (4) 2.21
bovine liver duodenum pork lung powder heart powder beef liver kidney powder animal mean
1.20 0.75 0.89 0.87 0.87 0.99 0.93
oyster tissue albacore tuna marine mean
1.08f 0.07 (8) 1.20 1.14
gelatin agar gelatin mean biological mean
0.184 0.165 0.157 0.108 0.205 0.164
f 0.032 I0.030 f 0.022 I0.019 f 0.066
(12) (12) (8) (13) (10)
0.193 f 0.047 0.176 f 0.032 0.259 I0.072 0.130 f 0.026 0.297 c 0.056 0.21 1
(12) (12) (8) (13) (10)
Botanical Flours
*
0.24 f 0.43 (2) 0.163 f 0.064 (4) 0.20
0.216 i 0.097 (2) 0.166 f 0.053 (4) 0.191
Animal Tissue 0.09 I0.12 f 0.13 f 0.05 f 0.01 f 0.04
(11) (4) (4) (4) (4) (4)
f f f f f f
0.017 0.017 0.031 0.042 0.007 0.017
f
0.043 (8) (8)
0.152 i 0.033 (8) 0.153 I0.026 (8) 0.153
0.50 f 0.17 (5) 0.56 c 0.05 ( 8 ) 0.53
0.124 I0.028 (5) 0.101 f 0.010 ( 8 ) 0.112
0.327 i: 0.098 (5) 0.262 c 0.039 (8) 0.295
1.10
0.156
0.248
f
0.142 0.136 0.164 0.143 0.117 0.188 0.148
(11) (4) (4) (4) (4) (4)
0.312 f 0.029 0.328 f 0.049 0.284 f 0.064 0.324 f 0.140 0.321 c 0.019 0.312 i. 0.039 0.314
(11) (4) (4) (4) (4) (4)
Marine Tissue 0.145 0.173 0.159
0.03 ( 8 )
f
I0.015
Gelatin
standard
Al/K
Mn/K
Fe/K
Botanical Leaves spinach orchard leaves tomato leaves pine needles Bowen’s Kale leaves mean
0.229 -i. 0.280 f 0.224 f 0.161 f
0.036 0.077 0.042 0.038
(12) (8) (8) (9)
0.062 I0.010 0.046 I0.024 0.072 c 0.010 0.027 i 0.007
0.224
(8) (8) (8) (13)
0.162 I0.032 0.291 I0.054 0.370 I0.088 0.100 f 0.016
0.052
(8) (9)
(7) (13)
0.230
Botanical Flours wheat flour rice flour flour mean
0.056 0.039 0.048
f
0.020 (2) 0.013 (4)
0.168 0.357 0.262
f
0.022 (11)
0.031 i 0.006 0.032 f 0.008 0.036 f 0.006 0.034 f 0.024 0.028 ~t 0.003 0.031 f 0.005 0.03 2
f
f f
0.076 ( 2 ) 0.121 (4)
Animal Tissues bovine liver duodenum pork lung powder heart powder beef liver kidney powder animal mean
0.078 f 0.026 (11) 0.159 f 0.024 (4) 0.247 c 0.173 ( 4 ) 0.166 f 0.381 (4) 0.097 f 0.026 (4) 0.161 f 0.051 (4) 0.151
0.106
(11) (4) (4) (4) (4) (4)
Marine Tissues oyster tissue albacore tuna marine mean
0.096 0.067
f
I0.015
(8)
0.078 0.165 0.122
0.025 (8)
f
f
0.017 (8) 0.020 (8)
Gelatins gelatin agar gelatin mean Biological mean standard
0.170
B/K (X10-2)
0.063
N/K ( ~ 1 0 - 4 )
0.135
P/K ( ~ 1 0 - 3 )
Cl/K (X10-3)
Botanical Leaves spinach orchard leaves tomato leaves pine needles Bowen’s Kale leaves mean
2.9 f 0.7 (4) 2.8 i. 0.7 (8) 6.5 4.1
f
0.79 1.70 1.85 2.36
1.3 (4) 1.68
f f f f
0.41 1.05 0.63 0.71
(4) (4) (4) (4)
0.81 -f: 0.67 f 0.81 f 0.30 f 1.44 ?: 0.81
0.13 0.23 0.28 0.26 0.05
(4) (8) (4) (4) (4)
0.71 i 0.13 (4) 0.32 f 0.06 (8) 0.97 c 0.24 ( 4 ) 0.78 0.70
f
0.08 ( 4 )
ANALYTICAL CHEMISTRY, VOL. 55, NO. 12, OCTOBER 1983
Table I11 (Continued) standard
B/K ( ~ 1 0 - 3 )
N/K.( ~ 1 0 - 4 )
P/K ( ~ 1 0 - 3 )
1967
Cl/K (X10-3)
Botanical Flours wheat flour rice flour flour mean Animal Tissues 0.46 f 0.09 (4)
bovine liver duodenum pork lung powder heart powder beef liver kidney powder animal mean
0.18
* 0.04 (4)
0.15
i
Marine Tissues oyster tissue albacore tuna marine mean
0.03 (4)
0.34
i
0.06 (4)
0.22 * 0.03 (4) 0.30 f 0.04 (4) 0.27
Gelatins gelatin agar gelatin mean biological mean -
a
4.1
1.43
Number of analyses.
0.62
0.52
--
Table IV. Mean Relative Sensitivity Factors standard botanical leaves spinach (3)' orchard leaves (5) tomato leaves (4) pine needles (4) Bowen's Kale (5) leaves mean
95% Confidence Limits Ratioed to Phosphorus for Negative Secondary Ions SO/P Cl/P (X102) CN/P F/P ( ~ 1 0 3 ) ?:
0.40 i 0.15
9.4
f
1.0
1 , 2 i 0.7 5.3
animal tissue bovine liver ( 5 ) marine tissue oyster tissue (5) biological mean
1.00 i 0.20 0.20 i 0.10 18.5 i 2.6
5.0
4.4
i
1.7
4.9
0.067 f 0.008
0.93 i 0.20 0.46
15.0 f 1.4 8.5
0.35 r 0.08 0.69 i 0.15 0.24 f 0.14 3.1 i 0.4 0.08 i 0.02 0.89
0.83
i
0.09
0.137
i
0.052
0.107
Number of analyses. oxygen ions. The lower primary ion current density (4 X 10" A/cm2) for negative priimary oxygen ions accounted for only part of this instability. A comparison of the equilibria results indicated that sodium and potassium also diffused relative to the polarity of the primary ions. Sodium and potassium diffusion toward the surface of the sample was greater for negative primary oxygen ions than the diffusion away from the surface for positive primary oxygen ions. For positive primary oxygen ions Pine Needles SRM1575 required the longest presputtering time (20 min) to obtain secondary ion intensity equilibria. The analysis conditions were standardized by presputtering each biological standard for 24 min. Figure 2 compares the ASFc, and the RSF,,,, for each of the biological standards. The respective sensitivity factors are expressed with 95% confidence limits. The variation in the ASFca is almost 2 orders of magnitude greater than the variation of the RSFc,/K. The variation in the ASF values is indicative of solids which do not have identical matrices. The improvement in precision for the RSF values indicated that the matrices of these biological materials are somewhat similar. The mean RSF's determined for positive and negative secondaries with 95% confidence limits are listed in Tables I11 and IV, respectively. In these tables the biological
Figure 2. The
mean absolute sensitivity factors with 95% confldence
llmits vs. the mean relative sensitivity factors with 95% Confidence limits for Ca and Ca/K, respectively, for the biological standards.
standards are grouped according to types of biological material. Because only seven of the standards have known elemental concentrations for elements determined by negative secondaries, and all except two of these standards are biological leaves, only the statistical analyses of the RSFs of the positive secondary ions will be reported. Similar but less conclusive
1968
ANALYTICAL CHEMISTRY, VOL. 55, NO. 12, OCTOBER 1983 B i o l a g i c a l Standards
R5f(x/,ef 1
Na/K
fiRT
Mg/K
WF KL KP SP RT OL LP RF TL OY HP
fiI/K Ca/K
OL LP SP TL HP KD PN DP
h/K Fe/K B/K N/K
a
1s
1 s t f o n i z a t i o n P o t e n t K l (ev)
the first ionization potential for the blological standards.
--
Table V. Precision and Accuracy of LTE vs. RSF 'Results for Orchard Leaves SRM1571 RSF
LTE
Na B A1 Ca Fe Mn
c1 P
no. of analyses
error factor
RSD,
I 5 8
-1.54 2.84 1.41 -3.32 1.11 1.23 18.7 -15.1
20 64 42 25
9 9
9 5 5
%
99 23 62 22
BE BL
E
BE
BE KL BL DP
OL DP RG GL
GL
BE
PN FIG
BL RT
KD KL LP RG TL WF SP OL RF RT O Y E OL RF PN
OL WF RT SP
ELP
HP
DP
KD BL
BE
KL
m K
P/K
KL E
CI/K
TL
T
L PN EL OY BL OL AT OY
Flgure 4. Results of the Duncan's multiple range test for individual
Flgure 3. Positive secondary ion mean relative sensitivity factors vs.
element
DP GL HP
BL OY SP KP LP HP TL
biological reference materials. Solid ilnes indlcate that there is no slgnlflcant difference between the relative sensitivity factors of the standards below the line. SP = spinach, OL = orchard leaves, TL = tomato leaves, PN = pine needles, KL = Bowen's Kale, WF = wheat flour, RF = rice flour, BL = bovine liver, DP = duodenum pork, LP = lung powder, HP = heart powder, BE = beef liver, KD = kidney powder, OY = oyster tissue, AT = albacore tuna, GL = gelatin, and AG = agar.
error factor
RSD, %
R5f (x/rat)
-1.15 -1.67 1.37 -1.23 1.38 1.13 -2.6 -1.26
22 28 34 29 25 32 20 40
Na/K Ng/K RIIK
Flours
Marlnes
Leaves
Animals
Gelatins
Flours
Leaves
Marlnes
Rnlmals
Gelatins
Leaves
Rnlmals
Marlnes
CalK
Rnimals
Gelatlns
Leaves
Flours
Marines
nn/K Fe/K N/K P/K CI/K
Rnlmals
Marlnes
Leaves
Flours
Marlnes
Animals
results were determined for the negative secondary ions. Andersen and Hinthorne (1)have demonstrated the relationship of the positive secondary ion yield of an element to the first ionization potential of that element. Figure 3 indicates the correlation of the mean RSF,p values for all of the biological materials analyzed (Table 111) and the first ionization potential of that element. The correlation coefficient is -0.97. This relationship is in part the basis of the qemitheoretical LTE method of quantification. Orchard Leaves SRM1571 have previously been analyzed by the LTE method (16). Our results indicated that the internal standards magnesium and potassium gave the best accuracy for the LTE quantification. Our L T E results are compared to our RSF results in Table V for the analysis of Orchard Leaves SRM1571, and the table shows that the RSF method is the most accurate quantification method for the elemental analyses of biological materials. The Duncan multiple range test (17)was used to determine if a matrix effect existed for individual biological standards and groups of similar biological standards. Alpha was 0.05. The Duncan multiple range test results for RSF's of positive secondary ions are shown in Figure 4. Lines indicate that the elements's R S F s are not significantly different. Although the RSF's for magnesium and aluminum are not significantly different, the other elements have one or more RSF which are significantly different. Therefore, a SIMS matrix effect must exist for different biological materials. The Duncan multiple range test results for groups of standards (flours, leaves, animals, marines, and gelatins) are shown in Figure 5. The results indicated that there are significant differences for all of the R S F s determined for these groups of biological materials. With the exception of iron, the RSF's for animal and marine standards are not significantly different. Except for the RSFca K, the RSFs in gelatins were significantly different from the R$Fs of the other groups. There is a SIMS matrix effect associated with different types of biological materials.
Bi o 1 og ical Groups
Flours
Leaves
Leaves
Rnlmals
Leaves
Rnimals
Marlnes
Leaves
Rnimals
Marlnes
Flgure 5. Results of the Duncan's multiple range test for groups of bloiogicai reference materials. Solid lines indicate that there is no slgniflcant differences between the relative sensitivity factors of the groups of standards below the line.
There is a large variation in the RSFFeIKin Table 111, and the Duncan multiple range test determined that each group's RSF,,,, was significantly different. High mass resolution has been used to determine the extent of the mass interference of the molecular ion CaO' on 56Fe+for several standards. The molecular interferent was negligible for animal materials, but significant for most of the botanical materials. Atomic absorption spectrometry of the SRM's indicated that the botanical materials were also contaminated by iron during the homogenization procedure. If the determination of iron in biological botanical material is important, it is recommended that the steel balls not be used in the homogenization procedure, and that high mass resolution be used (18). High mass resolution has also determined that there is a significant mass interference of the molecular ion KO+ on 55Mnf for Bovine Liver SRM1577 and Oyster Tissue SRM1566. However, this molecular interferent was negligible for the botanical leaves standards. From Table I1 the concentration of manganese in the animal biological reference materials is significantly lower than in the botanical standards. Iron has been excluded from further statistical analyses. Manganese for Bovine Liver SRM1577 and Oyster Tissue SRM1566 has also been excluded. Other significant molecular interferences were not found. Error factors have previously been used to compare RSF quantification of glass (2, 5 , 6) and metallurgical (6, 7) matrices. Error factors are defined as the determined concentration divided by the true concentration. If the error factor is less than one, then by convention the negative recipical of the error factor is used. The elemental concentrations of the biological standards were determined by the RSF method by solving eq 2 for C,.
ANALYTICAL CHEMISTRY, VOL. 55, NO. 12, OCTOBER 1983
1969
0
_.
Table VI. Error Factor Distributions for RSF Quantifications of Elements in Reference Materials for Experimentally Determined RSFxlref RSF, ref used for each element
percentage of error factors values _- between these_____ r l . 2 t1.4 i1.6 r2.0 t3.0
biological mean group mean
29 63
group meana
62
57 84 88
71 91 96
88
97
97
100 100
100
This is for certified elements with estimated uncertainties of less than 1G% of the certified values. a
Table VII. Error Factor Distributions for RSF Quantifications of Elements in Reference Materials with Gelatins and Respective Group RSF,/,,f percentage of error factors RSF,lref used between these values for each elementa k1.2 r1.4 t1.6 i 2 . 0 r3.0 gelatinmean groupmean a
44 67
52
91
63 98
88
94
100
100
Results are for sodium, magnesium, and calcium.
Each elemental concentration was determined by using the biological mean value of RSFXjrefthat was experimentally determined for all of the biological materials (Table 111). The elemental concentrations were also calculated by using each standards respective group mean RSF,/,,f. Each determined elemental concentration was compared to the known bulk value concentration (Table 11) by using error factors. The distributions of error factors (Table VI) for each of these RSFXjrefvalues clearly demonstrated the improvement in accuracy when group mean RSF’s were used. This improvement in accuracy iindicates that there is a matrix effect which is dependent upon types of biological material and that improved accuracy by the RSF method is achieved by using biological standards which closely approximate the matrix of the sample. The accuracy of the RSF comparisons ‘Is a function of the accuracy of the standards’ original elemental concentrations. Several of the elements in the NBS biological SRM’s have estimated uncertainties which are less than 10% of the certified values. The accuracy of the elemental concentrations for these elements deteirmined by the RSF method is compared with the accuracy for all of the elements in Table VI. The distribution of the error factors for these well characterized certified elemenhi is similar to the distribution of error factors for all of the elements. These results verify that it is reasonable to compare the determined concentrations for well characterized certified elements and uneertified elements. Gelatins and similar biological reference materials were compared to determine their relative usefulness as RSF standards. The bulk concentrations of potassium, sodium, magnesium, and calcium were determined in the gelatin standards. The distribution of error factors for RSF quantification of the biological reference standards using gelatin mean RSF,/,,f is compared in Table VI1 to the distribution of error factors using the group mean RSFXjreffor sodium, magnesium, and calcium. The biological reference materials are better standards than gelatins for empirical reference standards for the RSF method of quantification. The 95% confidence limits determined by SIMS were compared to the estimated uncertainties determined by NBS for certified elements using bulk analytical techniques. The SIMS results were from replicate analyses of picogram amounts of homogenized material determined over a several
Table VIII. Triplicate Analyses of Freeze Substituted Rat Tissues by RSF’s with Bovine Liver SRM1577 as the Standard SIMS (wt/wt % r std dev) Na
Mg Ca
0.179 r 0.012 0.085 t 0.006 0.0142 t 0.0005
AA re1 error, (wt/wt %) % 0.188
0.080
0.0160
-5
6 -11
month period, while the NBS results are from the replicate analyses of milligram sample sizes. The correlation coefficient of the linear regression is 0.24. The mean SIMS relative confidence limit is 4.8 times that of NBS, and the median is 2.7 times. Because a few elements have very small bulk estimated uncertainties, the median is lower than the mean. Although only picogram weights of materials were being determined in the SIMS analyses of the homogenized SRM’s, the confidence limits determined were reasonably similar to the estimated uncertainties determined by bulk analytical techniques for milligram weights. The usefulness of the biological reference materials as RSF standards for the quantification of biological tissue is established by using the RSF’s determined for Bovine Liver SRM1577 in the elemental analyses of freeze substituted rat liver tissue. The accuracy of the analyses is determined by comparison with the atomic absorption results for the original rat tissue. The accuracy of the analyses in Table VI11 demonstrates that these biological reference materials are important SIMS empirical standards for RSF quantification of biological tissues. In summary, it has been determined that there is a matrix effect associated with different types of biological materials. The RSF’s for different elements are generally similar but not identical within groups. The best accuracy can be expected when the matrix of the standard and the sample are as nearly identical as possible. The usefulness of this technique extends beyond the elemental bulk quantification of biological tissues. An important application of SIMS is the determination of the relative elemental distributions in heterogeneous solids. RSF’s are applicable to not only such ion probe determinations but also ion microscopy determinations. The homogeneity of the reference element determines in part the precision of the RSF method. The matrix element of inorganic materials is normally used as the reference element. However, we found that carbon was not a good reference element for biological materials. If the reference element is grossly heterogenous, then a homogeneous internal standard must be added to the sample. The implantation of boron into biological materials has been shown to provide a homogeneous internal standard for the RSF method ( 1 1 ) . The implantation of boron into a biological reference material which closely approximates the matrix of a sample should provide an ideal reference standard for ion microscopy. The actual RSF’s reported here were those from our CAMECA IMS-3f over a period of several months. When several elements were analyzed in glass matrices by different SIMS instruments in a round robin study, different RSF’s were determined for not only different types of SIMS instruments but also different SIMS instruments of the same type (19). The RSF’s reported here are not universally applicable for other instruments, However, the methodology developed here can be used to determine the RSF’s for a homogenized biological reference material, and a biological sample can then be analyzed under the same experimental conditions. If the matrix of the standard and the sample are similar, then the most accurate elemental quantification by SIMS can be expected.
1970
Anal, Chem. 1983, 55, 1970-1973
LITERATURE CITED (1) Andersen, C. A.; Hinthorne, J. R. Science 1972, 175, 853-860. (2) Newbury, D. E. Scanning 1980, 3 , 110-118. (3) Krauss, A. R.: Krohn, V. E. I n "Mass Spectrometry, Vol. 6"; The Royal Society of Chemistry: London, 1981; pp 118-152. (4) Turner, N. H.; Colton, R. J. Anal. Chem. 1982, 5 4 , 293R-322R. (5) Ganjei, J. D.; Morrison, G. H. Anal. Chem. 1978, 5 0 , 2034-2039. (6) Smith, D. H.; Christie, W. H. Int. J . Mass Spectrom. Ion Phys. 1978, 26,61-76. (7) Ganjei, J. D.: Leta, D. P.; Morrison, G. H. Anal. Chem. 1978, 50, 285-290. (8) Havette, A.: Slodzian, G. J . Phys. Left. (Orsay, Fr.) 1980, 41, L247L250. (9) Burns-Bellhorn, M. S.;File, B. M. Anal. Biochem. 1979, 92,213-223. (10) Zhu, D.; Harris, W. C., Jr.; Morrison, G. H. Anal. Chem. 1982, 5 4 , 4 19-422. (11) Harris, W.C., Jr.; Chandra, S.; Morrison, G. H. Anal. Chem. 1983, 5 5 , 1959-1963.
(12) Nadkarni, R. A. Radlochem. Radioanal. Left. 1977, 3 0 , 329-340. (13) Nadkarni, R. A,; Morrison, G. H. J . Radioanal. Chem. 1978, 4 3 , 347-369. ... (14) Ross, G.D.; Morrison, G.H.; Sacher, R. F.; Staples, R. C. J . Microsc. 1883. 129. 221-228. (15) Andersen, C. A.; Hinthorne, J. R. Anal. Chem. 1973, 4 5 , 1421-1438. (16) Tamura, H.; Ishitanl, T.; Izumi, E. Shltsuryo Bunseki 1981, 29, 81-87. (17) Carmer, S. G.;Swanson, M. R. J . A m . Stat. Assoc. 1973, 68. 66-74. (18) Burns, M. S. Anal. Chem. 1981, 5 3 , 2149-2152. (19) Newbury, D. E. I n "Secondary Ion Mass Spectrometry 11"; Benninghoven, A., Evans, C. A., Jr., Powell, R. A,, Shimizu, R., Storms, H. A,, Eds.; Springer-Verlag: New York, 1979; pp 51-57.
RECEIVED for review April 8, 1983. Accepted July 10, 1983. This work was funded by the National Institutes of Health under Grant No. R01 GM-24314.
Voltammetric Determination of Water in an Aluminum Chloride-N-n -Butylpyridinium Chloride I onie Liquid Saeed Sahami and Robert A. Osteryoung* Department of Chemistry, S t a t e University of New York a t Buffalo, Buffalo, New York 14214
The electrochemical behavior of water has been investigated in an ambient-temperature molten salt, aluminum chloride-Nn -butylpyridinium Chloride. I t was found that throughout the entire range of melt composition, water undergoes a chemical reactlon to generate HCI, which can be eiectrochemlcaliy reduced at a platinum electrode. By use of rotating platinum disk experiments, a caiibratlon curve was obtained In the basic melt. This calibration curve was found to be linear up to a water concentration of 50 mM.
The N-n-butylpyridinium chloride (BuPyC1)-aluminum chloride mixtures are molten at ambient temperatures (-30 O C ) over a wide compositional range varying from 0.75:l to 2:l (mole ratio of AlC1,:BuPyCl) (1,2). The Lewis acid-base properties of these ionic liquids change as the mole ratio of AlCl, to BuPyCl changes. In acidic melts there is excess A1C13 while for basic melts BuPyCl is in excess. It has been shown potentiometrically that equilibrium 1provides an adequate description of the system over the entire composition range (2). 2AlC1,- e A1,Clf 4- C1-
(1)
Due to the low melting point, aprotic, and relatively anhydrous nature of the AlC1,BuPyCl melts, they have been employed as solvents for electrochemical and spectroscopic investigations of both organic and inorganic species (1-13). Although various authors have assumed that AlC1,:BuPyCl melts are totally anhydrous (1, 6, 12, 13) and even explained the remarkable stability of some radical cations in these melts due to the absence of water ( l ) ,no unambiguous evidence exists t o support this assumption. The present work was intended to obtain information on the electrochemistry and analytical determination of water in these melts. No direct investigations on the electrochemistry of water in A1C13-BuPyC1 are reported in the literature. In a recent electrochemical study of oxide and water addition in the basic
AlCl,-BuPyCl melts containing titanium chloride, it was suggested that the addition of water resulted in removal of oxide from the melt (9). Tremillon et al. (14) have studied the electrochemistry of water and HCl at a platinum electrode in a basic melt of AlC1,-NaCl at 210 "C. They observed two waves on the voltammogram which were assumed to be due to the reduction of dissolved HCl and dissolved water.
EXPERIMENTAL SECTION Anhydrous aluminum chloride (Fluka A.G.) was purified by sublimation at 220 "C from a mixture containing a small amount of sodium chloride and aluminum wires in a Pyrex tube sealed under vacuum. N-n-Butylpyridinium chloride was prepared by refluxing n-butyl chloride and pyridine (Fisher ACS). These procedures as well as the preparation of melts have been described elsewhere (1). Water was repurified by the use of a Milli Q purification system (Millipore Corp.). Before water is taken into the drybox, it was deaerated by bubbling with prepurified argon. Water was introduced into the stirred melt with a gastight microsyringe. Upon addition of water to the melt a local white precipitate formed which redissolved on stirring. All measurements were made after complete dissolution of the precipitate. Hydrogen chloride gas was obtained from Linde. The aluminum wire (Alfa Products) was cleaned in a 30:30:40 volume mixture of H2SO4:HNO3:H3PO4, rinsed with water, and dried. Chemicals were stored and all experiments performed under argon atmosphere in a Vacuum Atmospheres Co. drybox. A Metrohm glass cell was employed. It was covered with a Teflon lid which had several holes for reference and counterelectrode compartments, working electrode, and thermometer. The entire cell assembly was placed in a simple furnace and the temperature was controlled at 40 f 1 OC by a Thermo Electric Selector 800 temperature controller. Reference and counterelectrode compartments were aluminum wires dipped into the 2:1 AlC1,:BuPyCl melt and both were separated from the working compartment by fine glass frits. Glassy carbon (GC), tungsten (W), and platinum (Pt) disks were used as working electrodes. The GC (0.071 cm2) and W (0.077 cm2) disk electrodes were polished with 0.3-pm alumina (Buehler) by using a polishing cloth and Milli Q water as lubricant. The Pt disk electrode (0.049 cm2), which was obtained from Pine Instrument Co., was polished with successively finer grades of 1.0,0.3, and 0.05 pm alumina, treated
O003-2700/83/0355-1970$01.50/00 1983 American Chemical Society
