(45) H. L. Patdue, A. E. McDowell, D. M. Fast, and M. J. Milano, Clin. Chem., ( Winston-Salem,N.C.), 21, 1192 (1975). (46) F. L. Fricke, 0. Rose, Jr., and J. A. Ceruso, Anal. Chem., 47, 2018 (1975). (47) W. H. Woodruff and G. H. Atkinson, Anal. Chem., 48, 186 (1976). (48) N. G. Howell, J. D. Ganjei, and G. H. Morrison, Anal. Chem., 48, 319 (1976). (49) T. E. Cook, H. L. Pardue, and R. E. Santini, Anal. Chem., 48, 451 (1976). (50) R. N. Diamond, A. R. Erwin, and M. A. Thompson, Nucl. Instrum. Methods, 89, 45 (1970). (51) E. S.Belyakov, L. I. Bernstein, V. A. Klevalin, B. A. Lebedev, A. V. Petrakov, and V. M. Kharitonov, Prib. Tekh. Eksp., 5, 249 (1972). (52) R. G. Friday, D. W. G. S.Leith, and D. G. McShurley, U.S. Atomic Energy Comm., SLAC-Pub-792, (1970).
(53) J. R. DBnksand J. Gunn, "ISIS-Infrared Spectral Information SystemUser's Manual", Triangle Universities Computation Center Document No. LSR-98, Research Triangle Park, N.C., 1970. (54) Sadtler Standard Grating Spectra, Sadtler Research Laboratories, Inc., Phlladelphia, Pa., 1966.
RECEIVEDfor review April 12,1976. Accepted June 1,1976. The financial support of the National Science Foundation is gratefully acknowledged. Presented in part a t the 27th Pittsburgh Conference on Analytical Chemistry and Applied Spectroscopy, Cleveland, Ohio, March 4, 1976.
Standards for Iron, Cobalt, Nickel, Copper, and Zinc in Laser Microprobe Emission Spectrometry of Biological Material Elvio Giovannini" and Giovenni B. Princlpato lstituto di Siologia Generale, Faculty of Medicine and Surgery, Perugia University, 06 100 Perugia, Italy
Francesco Rondelli lstituto di Mineralogia, Faculty of Science, Perugia University, 06 100 Perugia, Italy
A laser microprobe was used to study and test a method for the determlnatlon of iron, colbalt, nickel, copper, and zlnc in animal organs and tissues. The relative standards were realized on a matrix prepared from a homogenate of rabbit muscle tissue by adding saline solutions of these elements In scaled concentrations; later they were ashed, ground and compressed into tablets. In this way, working stralght-line relationships for wide concentration Intervals referredto ashed material were prepared. Within the Indicated limits, these straight-lines are Ideal for carrying out quantitative evaluations of the above mentioned elements in animal organs and tissues that have previously been ashed, ground, and compressed Into tablets. As an Identical matrix Is obtained for standards and analysis samples, it is easy to arrive at the concentrations of the elements in the Initially damp material by determining the reduction In the weight after ashing for each case.
The laser microprobe has recently found a use in the spectroscopic determination of trace elements in solid state. In the biological field, the technique in question has been applied for studies in histochemistry (1-7). Considerable difficulties arise when it is required to use the laser microprobe in quantitative analyses, since the removal of a sample by the microprobe depends on the physical properties of the sample, which differ from case to case. Moreover, this use presupposes the preparation of standards with a matrix similar to that of the samples to be analyzed and, in view of the diversity of the latter, the problem appears hard to solve. Since there was no widely applicable method that could be used for quantitative analysis on biological preparations, we set ourselves to solve the problems connected with the use of this line of analysis in the determination of certain elements (Fe, Co, Ni, Cu, Zn)in a matrix that was easily reproducible for animal tissues and organs.
EXPERIMENTAL Apparatus, The analysis instruments used comprised the fol-
lowing.
Sample Removal Unit. This consists of a neodymium rod laser, Jarrell-Ash Laser Microprobe Mark 111,which emits 0.4 MW pulses having a beam convergence of 20 f t of arc. With the reflected light microscope, associated with the generator, it is possible to explore the surface of the sample under examination and focus the laser beam on the area to be analyzed; this area should be not less than 50 p in diameter. Electronic excitation is provided by a 2000-V spark made to shoot between AGKS graphite electrodes. Spectrograph. This is a Jarrell-Ash 75-150 model having a focal length of 1 m, with a diaphragm of F = 1:6.7 and a completely illuminated grating of 150 cm2with 1180 lines/mm. Resolution is 0.12 8, for a line exposed to a 20% transmission. The spectrograph is thermostatically controlled at 19 "C. The spectrum is recorded on a Kodak 130-0 spectrographic plate (4 X 10 inc) and development is carried out in an automatic developing tank using Kodak D-19 at 19 O C for 4 min, with a continuous agitation (8). Reader. The reading of the plate is carried out by means of a comparative microdensitometer, Jarrell-Ash 21-001-M, allowing percentage measurements of transmittance or absorbance to be made either with a mirror galvanometer or with a paper recorder. Procedure. Prelzrninary Tests. It is well known that any spectroscopic method is influenced by the matrix effect (9);in particular, the quantity of sample removed by a laser microprobe is strongly dependent upon the color, opacity, thermal conductivity, melting point, and physical structure of the sample. In order to use this microprobe in quantitative analyses on animal organs and tissues having characteristics that vary from case to case, it was therefore necessary to employ treatments such as would achieve a uniform matrix, as regards both the standards and the samples to be analyzed. For this purpose, various rabbit organs and tissues (kidney, liver, spleen, and muscle) were dehydrated in a oven and ashed at 450 "C in closed porcelain dishes which were placed in a thermostatic muffle furnace until the weight of the ashed material remained constant. The time required for this process was about 24 h. The ashed material relative to the various samples was finely ground in a mortar and then reduced to tablets by means of a micropress; the grinding made it possible to obtain a degree of homogeneity suited to the quantity of substance removed by the microprobe. Samplings were carried out with this microprobe on tablets of ashed organic material prepared from various organs, employing a laser intensity of 0.4 J, since, in these conditions, the laser emission of the instrument is not subjected to the action of filters, as occurs with the lower intensities, or to emission overloading in the case of higher intensities. The average size of the craters produced in the various materials studied were about 300 p in diameter and 180 p in depth; the variation coefficients for the removal of the sample were below
ANALYTICAL CHEMISTRY, VOL. 48, NO. 11, SEPTEMBER 1976
1517
Table I. Response/Lo Concentration Relations Observed b y Means of t h e Laser Microprobe in Standard Preparations of Fe (A = 3719.935 ), Co ( h = 3453,505 A), Ni (X = 3414.765 A), Cu (A = 3273.962 8)and Z n ( h = 3345.020 A), Prepared o n a Matrix of Homogenate of Ashed Rabbit Muscle Tissues
1
Log ppm
Z (mean
f
SE)
Analysis of variance
RSD, %
F values
Source of Fe
co
3.21748 3.52061
2.090 3.030
0.0554 0.1710
6.5 5.8
3.89371 4.14740
4.680 i 0.0998 5.862 i 0.2158
5.2 9.0
3.24797
2.292
f f
f
0.0416
4.5
3.52737
3.102
f
0.0458
3.6
3.91719
4.722 5.667
f
0.1880 0.3230
9.8
4.17088
f
ss
variation
Linear regr. Curvature Error Total Source of variation
Linear regr. Curvature Error Total
13.9
cu
3.24576
1.675
3.52582
2.608
3.91519 4.16941 3.28058
?
0.0524
7.7
i
0.0526
4.9
4.257
f
5.530
?:
0.1166 0.3350
6.7 15.7
1.755
i
0.0487
6.8
3.56074
2.577
0.0842
8.0
3.95056 4.20423
3.923 k 0.1299 5.020 ?; 0.2295
8.1 11.2
2
PO.05
ss DF MS F P 0.05 41.915 1 41.915 194.0506 4.35 0.158 2 0.079 0.366 3.49 4.312 20 0.216 46.385 23 Regression line: I = 3.730 log ( p p m ) - 9.915
P 0.01
8.10 5.85
PO.01
8.10 5.85
F values
ss
DF
MS
F
PO.05
52.554 1 52.554 241.628b 4.35 0-3 7 7 0.814 3.49 0.354 2 4.351 20 0.217 57.259 23 Regression line: I = 4.173 log ( p p m ) - 11.981
PO.01
8.10 5.85
F values
Source of variation
Linear regr. Curvature Error Total
F
F values
variation
Linear regr. Curvature Error Total
MS
50.579 1 50.579 752.664b 4.35 0.383 2 0.191 2.842 3.49 1.345 20 0.067 52.307 23 Regression line: I = 4.094 log ( p p m ) - 11.21
Source of Ni
DF
ss
DF
MS
F
P 0.05
37.375 1 37.375 316.737b 4.35 0.160 2 0.080 0.675 3.49 2.369 20 0.116 39.904 23 Regression line: I = 3.523 log ( p p m ) - 9.889
PO.01
8.10 5.85
F values
Source of variation
ss DF MS F P 0.05 P 0.01 98.252 1 98.252 347.180b 4.35 8.10 4.220 i 0.1189 3.57276 6.9 1.074 2 0.537 1.897 3.49 5.85 5.669 20 0.283 6.468 f 0.2231 3.96265 8.4 104.995 23 4.21635 10.2 8.280 i 0.3455 Regression line: I = 5.700 log ( p p m ) - 15.946 a T h e concentrations of t h e metals in ppm in the ashed material are expressed in logarithmic units. The intensity of the response ( I ) is expressed in conventional units proportional to t h e data provided b y t h e instrument, Mean values ?: standard error and relative standard deviations (RSD) relative t o 6 measurements; response/log concentration regression lines and statistical evaluation of t h e response of the instrument and of its linearity by means of variance analysis. SS = sum of the squares; D F = degrees of freedom; MS = mean squares; F = variance ratio. b Highly significant. Zn
3.29270
3.000
i
0.0759
6.2
Linear regr. Curvature Error Total
10%in all cases. Craters were almost circular, the edge was free from scoriae and fusion products. Preparation of the Standards. For the preparation of the standards matrix, use was made of rabbit muscle tissue, since, in carrying out some preliminary tests with semiquantitative measurements, it was found to contain lower amounts of Fe, Co, Ni, Cu, and Zn than the other organs and tissues. In particular, five rabbits were used, killed by blood-letting from the carotid artery, from which the skeletal muscles of the hind limbs were removed in a quantity of about 100 g per animal. This tissue, freed as far as possible from adipose, connective, and tendon elements, was homogenized with bidistilled water (100 ml per 100 g of tissue) in an E. Buhler mincing homogenizer until the tissue was completely disintegrated. Samples of equal volume, correspondingto 50 g of fresh tissue, were removed from the homogenate pool; to each of these samples were added 2 ml of a solution of NaaCo(NO&, (NH4)2Fe(SO4)2,NiC12, CuSO4, and ZnS0.I in concentrations of 0.1, 0.05, 0.025, and 0.0125 M. These concentrations were obtained by dilutions I'rom ii 0.2 M mother solution.Some samples of homogenate were used nh control without further manipulation. 'I'he above materials were dehydrated in a oven at 80 "C for 72 h ;itid then at 120 "C for 48 h; they were then ashed and reduced to tahlets by the methods already described in the preliminary tests. The reduction in weight resulting from these treatments, expressed as a
1518
percentage of the initial quantity of tissue, was found to average about 98%. Six analyses were carried out on each tablet.
RESULTS AND DISCUSSION T h e spectrographic d a t a recorded by t h e reader for each element, at t h e various concentrations, were processed according to t h e ASTM method (10). F r o m t h e overall values of t h e intensities of t h e single s t a n d a r d s were subtracted, for each element, those relative t o normal muscle, measured in t h e samples t o which n o saline solutions had been added. T a b l e I shows these values, with the statistical assessments made by analysis of variance. T h e responses of t h e apparatus relative t o the analysis of each element at t h e various concentrations show a good degree of reproducibility, since the relative standard deviations measured for each group of data exceed a value of 10%only with t h e higher concentrations of t h e elements studied; this higher variability in t h e responses is probably t o be a t t r i b u t e d t o t h e lower degree of homogeneity of t h e material analyzed, which m a y result, in the presence of high saline contents, from t h e technique that we followed in preparing t h e standards.
ANALYTICAL CHEMISTRY, VOL. 48, NO. 11, SEPTEMBER 1976
The effecthog dose regression lines relative to the intensity of response of the instrument for the concentrations of the elements studied show, in each case, a significantly straightline relationship. Having constructed a working-straight-line relationship for each of the elements under examination, for a given interval of concentration, it is possible to make quantitative measurements on any type of biological material, provided that this has previously undergone ashing, grinding, and compressing into tablets. The values of concentration determined by means of these working relationships are referred to the ashed material; knowing the reduction in weight resulting from ashing, i t is easy to calculate back to the concentration in the initial damp material. The method described is simple to apply for the determination of the elements that we studied and may be extended by similar methods to the determination of other elements, within the limits of the possibilities of the line of analysis and of the actual possibility of achieving consistent standards. At present, the analysis technique here described is usefully employed in studies that are being carried out on the distribution of trace elements in certain rabbit organs and on their physiological implications.
ACKNOWLEDGMENT The authors thank G. Cocco and F. S. Liotti for their critical reading of the manuscript. LITERATURE CITED (1) R. C. Rosan, F. Brech, and D. Glick, Fed. Roc., 24, 5-126 (1965). (2) M. Kozik, J. Warchol, and B.Arcimowicz, Biochemie, 26, 212 (1971). (3)W. J. Treytl, J. B. Orenberg, K. W. Marich, A. J. Saffir, and D. Glick, Anal. Chem., 44, 1903 (1972). (4) M. Kozik, J. Warchol, and B. Arcimowicz, Biochemie, 30, 289 (1972). (5) K. Mietkiewski, J. B. Warchol, and B. Arcimowicz, Acta Histochem., 42, 95 11972). (6) I . Harding-Barlow, "Laser Application in Medicine and Biology", M. L. Wolbarsht, Ed., Vol. 2, Plenum Press, New York, 1974, p 133. (7) D. Glick, and K. W. Marich, Clifl. Chem. ( Winston-Salem,N.C.), 21, 1238. (1975). (8) Kodak S.p.A., Roma, Italia, "Lastre e Peilicole Kodak per usi Sclentlfici ed Industriali", Kodak S.p.A., Ed., 1968. (9) K. W. Marich, P. W. Carr, W. J. Treytl, and D. Glick, Anal. Chem., 42, 1775 (1970). (IO) American Society for Testing and Materials, Committee E-2, Philadelphia, Pa., "Methods for Emission Spectrochemical Analysis", 1971.
RECEIVEDfor review December 15, 1975. Accepted June 2, 1976.
Considerations for lmplementing Spatiall y-Resolved Spectrometry Using the Abel Inversion Alexander Scheeline and J. P. Walters* Depan'ment of Chemistry, University of Wisconsin, Madison, Wis. 53706
Obtaining spatially-resolved information from a plasma dlscharge is not necessarily a straightforward operation. Such phenomena as self-absorption, contlnuum background, discharge wander, and measurement noise may make meaningful spatially-resolved emission line profiles difficult or imposslble to obtain unless the numerous optical properties of the discharge are considered carefully and systematically. Herein, several measurement-distorting phenomena are discussed in terms of the Abel Inversion, a common calculational technique for elucidating radial emlsslon llne proflles.
The essential problem in spatially-resolved spectrometry is to determine the distribution in space of the species giving rise to emitted light and of the species perturbing that emitted light by absorption or scattering. For a plasma discharge (spark, arc, etc.), two factors determine the distribution of these species: (1)the geometry of the plasma and vapor clouds incident to that plasma and (2) the excitation conditions within the plasma as it intersects the vapor cloud. If the excitation conditions in the plasma are completely understood, the geometry of the discharge can be readily determined; similarly, if the geometry is known, excitation conditions may be ascertained. For the purposes of this paper, it will be assumed (1) one wishes to determine the excitation conditions within a discharge so as to understand its behavior and (2) the geometry is fixed so that all properties of the discharge are cylindrically symmetrical about the discharge axis. It will be evident shortly that the latter assumption is not overly restrictive.
A plasma discharge may be spatially resolved in several dimensions, as shown in Figure 1B and 1C. The discharge ( 5 in Figure I C ) takes place between two electrodes (4 in the figure); resolution along that axis, Le., parallel to arrow 6 is axial resolution, and a single resolution element in this direction is an axial slice through the discharge (for example, the disk-shaped region in Figure IC). The only other dimension viewable directly in the laboratory is a lateral dimension, shown by arrow 7 in the figure. The distribution of observed intensity across the inhomogeneous discharge in a lateral direction may be termed a lateral intensityprofile, and may in turn be viewed in a wavelength-resolved or wavelength-integrated fashion. As the discharge is cylindrically symmetric, the chemical processes occurring within the plasma are unique in a radial direction (arrow 3, Figure 1B) and radial information is not directly viewable in the laboratory. To obtain fundamental chemical and physical information, one must convert laterally and axially resolved data into radially resolved information (radial emission profiles). The mathematical tool for so processing the observable information is known as the Abel Inversion. Various computational methods for performing the Abel Inversion have been reviewed by Stanisavljevic and Konjevic ( I ) . A very thorough article by Bracewell (2) not only enumerates various inversion techniques, but also contains tables and figures showing pairs of lateral and radial profiles. A spatially-resolved study of an arc discharge has been performed by Olsen ( 3 ) ,and the method of Cremers and Birkebak ( 4 ) has been employed in studying inductively coupled plasmas ( 5 ) .Analog as well as digital inversion techniques have
ANALYTICAL CHEMISTRY, VOL. 48, NO. 11, SEPTEMBER 1976
1519
