D i m t Emission Spectrographic Method for Trace Elements in Biological Materials A. J. Bedrosian, R. K. Skogerboe, and G . H. Morrison Department of Chemistry, Cornell University, Ithaca N . Y. 14850
A sensitive, rapid, and comprehensive spectrographic procedure has been developed for the direct determination of a large number of elements in biological materials without appreciable alteration of the samples. The high current density dc arc method utilizes 25-50 mg of dried material per analysis and provides detection limits of l ppm or less for 26 of the more than 60 elements studied. The precision of the method is consistently better than &15% and comparative analyses indicate comparable accuracies. The employment of universal analytical curves for such diversified samples as animal tissue, blood serum, stool, bone, and plant leaves is a particularly attractive feature of the method.
type of sample material and the sensitivities listed were usually unimpressive. The objective of this paper is to report the development of a comprehensive, rapid, sensitive, and direct quantitative spectrographic procedure for the analysis of a wide variety of biological materials. It is shown that representative 25mg biological samples can provide reliable analytical information for a large number of elements. Of the more than 60 elements studied, most provided detection limits ranging from 5 to 0.01 ppm. The employment of generally universal calibration curves is a particularly attractive feature of this method.
THE PRESENCE, absence, patterns of occurrence, and concentration levels of trace elements in a variety of biological materials are frequently of importance to life scientists (1-6). The multielement capabilities of emission spectroscopy coupled with its selectivity and sensitivity as an analytical tool for trace element research, has been recognized by many investigators (7-10). Most biological sample preparation techniques for emission spectrographic analysis involve either wet digestion, dry ashing, or “cold combustion” (11) to destroy the organic portion of the sample. Concurrent with or following this treatment, a preconcentration step is frequently employed prior to the addition of an internal standard-buffer mixture. Obviously these sample preparation steps provide the opportunity for sample contamination and loss of sample constituents. A reduction of sample handling between actual sample procurement and electrode preparation would be advantageous from the standpoint of reduced analytical errors and speed of analysis. Only a few spectroscopists have been successful in analyzing unaltered biological materials (12-15). In general, these reports have presented results for only one
EXPERIMENTAL
(1) C. L. Grant, The Spex Speaker (Spex Industries, Inc.), 6, No. 3 (1961). (2) H. A. Schroeder, Ibid., No. 2 (1961). (3) A. A. Kurnick, B. L. Reid, and J. R. Couch, Soil Sci., 85,106 (1958). (4) W. H. Allaway in “Trace Analysis Physical Methods,” G. H. Morrison, Ed., Interscience, New York, 1965, Chapter 3. (5) A. B. Ferguson, Jr., A. Yoshihiko, P. G. Laing, and E. S . Hodge, J. Bone and Joint Surg., &A, 317 (1962). (6) Ibid., p 323 (1962). (7) C. L. Grant, Science, 134,1207 (1961). (8) R. L. Mitchell, “Spectrographic Analysis of Soils, Plants and Related Materials,” Commonwealth Bureau of Soil Science (Great Britain), Technical Communication No. 44 (1948). (9) I. H. Tipton, M. J. Cook, R. S. Steiner, W. D. Fold, D. K. Bowman, and A. K. McDaniel, ORNL Rept. 56:3-60 (1960). (10) L. M. Paixasand J. H. Yoe, Clin. Chim. Acta, 4, 507 (1959). (11) C. E. Gleit and W. D. Holland, ANAL.CHEM., 34, 1454 (1962). (12) A. Paolini, Jr., and R. M. Kennedy, Appl. Spectr., 16, 15 (1 962). (13) J. H. Muntz and S . W. Melstead, ANAL.CHEM.,27, 751 (1955). (14) P. B. Zeeman and F. J. Coetzner, Appl. Spectry., 15 161 (1961). (15) Y. M. Priev, Sb. Rabot Molodnykh Unchenykh Nauch.-lssled. Inst. i Vuzor;. Min. Sel. Khoz. USSR, 1, 128 (1962). 854
0
ANALYTICAL CHEMISTRY
Apparatus. The instrumentation employed and the operating parameters are listed in Table I. Since the high dispersion grating (30,000 lines/inch) employed covers only 1250 A, one exposure must be made in each of two wavelength ranges to determine all elements studied. A single analysis would thus require two samples. By using a lower dispersion grating most of the elements could be examined with one sample but the decreased resolution would have deleterious effects on the line-to-background ratio and the spectral interference situation for some elements. Sample Preparation. Twenty-five mg each, of dry biological sample (preferably freeze-dried in the case of animal tissue), internal standard (60 ppm, each of lutetium and yttrium as oxides in National SP-IC, pelletizing graphite), and graphite (National SP-2X, nonpelletizing graphite), were mixed and pelletized in a 3/16-in~h diameter mold with a small hand press. The pellet was inserted into the cup of a inch diameter graphite electrode and a hole, approximately 1 mm in diameter, was made through the center of the pellet using a stainless steel pin and pin-guide. Standards. Two methods of standardization were examined. One was a “standard additions” technique while the other employed the well known “synthetic matrix” approach . STANDARD ADDITIONS.With one exception-Le., the addition of Spex Industries “G” standards and suitable dilutions thereof (with nonpelletizing graphite) in place of graphitethe standards preparation was identical to sample preparation. SYNTHETIC MATRIX. Two deviations from the sample preparation procedure were involved in the synthetic matrix approach. An organic chemical, p-nitrobenzene-azo-resorcinol (PNBAR) was added in place of biological sample and Spex Industries “G” standards and suitable dilutions thereof were added instead of the graphite. Selection of Analysis Conditions. Important analytical variables involved were determined by experimental observation of their effects upon line to background ratios, burning characteristics of the arc, and by previous experience. The variables examined are listed in Table 11. The appropriate ranges of examination are also listed in this table. Those variables having the most influential effects upon line to background ratios were examined further by factorial experimentation to develop the optimized conditions which have been incorporated into Table I and the sample preparation procedure.
Data Processing. A modified computer program (16) and an IBM 360140 computer system were utilized for the conversion of percent transmission values to background corrected intensity ratios. These ratios were employed to obtain calibration curves, limits of detection, and sample composition for a large number of elements. RESULTS AND DISCUSSION
Selection of Apalysis Conditions. The dc arc and the 103-0 emulsion were chosen on the basis of sensitivity. Gordon’s (17) static atmosphere excitation system was selected for initial study because this technique was felt to have considerable potential. After the examination of a number of variables, optimum analytical conditions were found which were in agreement with those recommended by Gordon. However, a subsequent comparison of static atmosphere excitation with dynamic atmosphere excitation indicated that improvtd sensitivity was obtained with the latter. This was attributed to the fact that the combustible biological samples produced large amounts of gases upon excitation as contrasted to the inorganic materials examined by Gordon. In addition, biological samples apparently display an effect similar to carrier distillation and the gases released during excitation produce cyanogen band spectra even in a static, inert gas atmosphere. Lutetium and yttrium were chosen as internal standards since they tend to occur in insignificant amounts in most biological samples. Lutetium has also been reported to possess desirable characteristics as an internal standard (18) and previous work in this laboratory has verified this. Volatilization behavior of these elements closely paralleled those of most elements studied. Analysis lines and internal standard lines were paired to minimize wavelength differences. Analysis lines (listed in Table 111) were generally selected for sensitivity and freedom from spectral interference. Elements normally present at higher concentrations-e.g., the nutrient elements-required the use of less sensitive lines. Initial screening experiments using dynamic atmosphere (Stallwood jet) excitation were conducted to assess the importance of a number of variables. The screening experiments were followed by sequential factorial experiments (19) to simultaneously examine the effects of arc current, exposure time, electrode geometry, analytical gap, inert gas, inert gasoxygen ratio, and total gas flow rate upon line to background ratios. The optimized analysis conditions resulting from this experimentation have been incorporated into Table I and the sample preparation section. Appropriate comments with regard to the effects of the principal variables are given below. ARCCURRENT.In general, results concerning the effect of high current density were similar to those of Gordon (17). Impurity lines not detectable or weak were markedly more intense under conditions of high current density. Maximum line to background ratios were obtained at 25 A (shorted electrodes). It is felt, also, that high current density tends to erase variations which might otherwise arise from moderate matrix differences. (16) W. A. Gordon and A. K. Gallagher, NASA Technical Memorandum NASA TM X-1220, Cleveland, Ohio, April 1966. (17) W. A. Gordon, NASA Technical Note NASA TN D-2598. Cleveland, Ohio, Jan. 1965. (18) P. Tymchuk, D. S. Russell, and S . S . Berman, Appl. Spectry., 17, 163 (1963). (19) 0. L. Davies, Ed., “The Design and Analysis of Industrial
Experiments,” 2nd ed., Hafner Publishing Co., New York, N. Y., 1956, p 247.
Table I. Experimental Apparatus and Conditions Excitation system Source unit Jarrell-Ash Custom Varisource (Model 40-750) 25 A dc arc (shorted electrodes) Discharge Sample electrode National L-3900 (anode) Counter electrode National L-3951 Analytical gap 4-mm Exposure time 20 sec-no preburn Excitation chamber Spex Industries enclosed Stallwood jet Gas mixture 7.5 liter/min of 20 O d O He Electrode-chamber base 6.5mm distance Optical system Jarrell-Ash 3.4 meter Ebert (Model Spectrograph 71-0oo) 30,000 lines/inch blazed for 2700 ,& 1st Grating order (one Wavelength coverage 2300-3500 A and 3350-4550 sample required for each wavelength region) 50 microns Slit width 3-lens condensing system with vignette External optics mask passing central 2 mm of discharge. 8-step rotating sector (step factor = Step sector 1.585)-5-darkest steps utilized 4 X 10-inch Kodak 103-0 plates Detector Developing equipment Jarrell-Ash Photoprocessor (Model 34100) Developing conditions Development Eastman D-19 (full strength) for 3 min Running water for 30 sec Stop bath Eastman Rapid Fixer with hardener for Fixing 3 min. Running water for 10 min, distilled water Washing for about 1 min Drying Warm air for 5 min Jarrell-Ash console microphotometer Densitometer (Model 2 3 - 1 0 ) Table 11. Analytical Variables Examined Variable Range of study Arc current 10-35 A Electrode polarity lis to l/z-inch crater depth, boilerElectrode geometry caps, undercut electrode cups Exposure time 5-60 sec Analytical gap 3-8 mm Sample size 5-100 mg Spectroscopicbuffer, carrier, Graphite, LizCOa,GeOz, InnOa,AgCl or diluent Sample-to-buffer ratio 1:0 to 1:4 Sample form Packed powder, pellet, pellet with hole Excitation chamber Static and dynamic atmosphere chambers Gas composition Air, A, He, mixtures of 0 2 with A and He Gas flow rates 0-10 l/min
ELECTRODE POLARITY.To assess the merit of slower sample volatilization coupled with the cathode layer effect, both cathode and anode sample excitation were examined. Results clearly favored the use of anode excitation. ELECTRODEGEOMETRY. Shallow-cratered sample electrodes without boiler caps were found to produce better line to background ratios. No appreciable difference was observed when these electrodes were undercut. Since sample preparation was easier when sample electrodes were not undercut-Le., less possibility of breakage during sample loadingthese were selected. VOL 40, NO. 6, MAY 1968
855
Element Ag 3280.7 A1 3082.2 As 2349.8 Au 2428.0 B 2497.7 Ba 2335.5 Be 2348.6 Bi 3067.7 Ca 3006.9 Cd 3466.2 Ce 4460.2 c o 3453.5 Cr 2835.6 Cu 2484.4 Dy 4ooo.5 Er 3692.6 Eu 3972.0 Fe 3024.0 Ga 2943.6 Gd 3422.5 Ge 2651.2 Hf 3134.7 Hg 2536.5 Ho 3456.0 In 3256.1 Ir 3220.8 La 3949.1 Li 3232.6 Mg 2779.8 Mn 2576.1 Mo 3132.6 Na 3303.0 Nb 3094.2 Nd 4303.6 Ni 3414.8 P 2535.7 Pb 2833.1 Pd 3404.6 Pr 4255.5 Pt 3064.7 Re 3460.5 Rh 3434.9 Ru 3436.7 Sb 2877.9 Sc 3613.8 Si 2516.1 Sm 4424.3 Sn 2840.0 Sr 3464.5 Ta 3311.2 Tb 3676.4 Te 2385.6 Th 3392.0 Ti 3361.2 T1 2767.9 Tm 3462.2 U 2941.9 V 3184.0 W 2724.4 Yb 3694.2 Zn 3345.0 Zr 3392.0 (
) =
Table 111. Comparison of Detection Limits Gg/Electrode) Method AshDigestion-AshPreconcentration ( 2 4 Preconcentration (22) Sample ash (20) 0.001 0.002 0.015 0.02 0.008
0.15 0.08 0.002 0.008
0.0008 0.02 0.24
0.008 (0.5)
0.008 0.008 2.5) 0.4 0.8 (>2.5) 0.005 0.03 0.1
0.036
0.008
0.03
0.8
0.24 0.02
(0.8) 0.03
3.0
(>2.5) 0.02 (>2.5) . 0.02 (0.005) 0.03
Most sensitive line not used.
EXPOSURE TIME. Moving plate studies indicated that sample constituents were completely volatilized after 30 seconds. Maximum sensitivity as indicated by maximum line to background ratios was Obtained for a 20-second exposure with no preburn. ANALYTICAL GAP. Factorial experimentation indicated 856
0.1
Present-direct 0.002 0.004 1 .o 0.05 0.05 (0.4) 0.0002 0.005 (0.1) (0.1) 0.5 0.01
ANALYTICAL CHEMISTRY
(20) S. R. Koirtyohann and C . Feldman, presented in part at the Fourth Conference on Analytical Chemistry and Nuclear Reactor Technology, Gatlinburg, Tenn., Oct. 1960. (21) C. Feldman, Oak Ridge National Laboratory, personal cornmunication, 1966. (22) R. L. Mitchell and R. 0. Scott, AppI. Spectry., 11, 6 (1957).
*
‘O‘O
.oI f
I
4
I
I
IO 40 Sn,ppm by weight
I
I
100
400
1
1000
Figure 1. Typical analytical curves for tin (Apple leaf data not considered in curve) 0
Liver; A Apple Leaves;
Bone; + PNBAR; V Blood Serum; X Kidney Tumor;
that a 4 mm analytical gap provided best line to background ratios. In addition, a distance of 6.5 mm between the top of the sample electrode and the base of the excitation chamber was found sufficient to prevent an occasional tendency of the arc to strike between the counter electrode and the chamber base. GAS COMPOSITION AND FLOWRATE. Initial experiments comparing air, argon, and helium as excitation atmospheres indicated the superiority of the inert gases and comparison of the latter with and without oxygen indicated helium and oxygen to be the best choice. Although the presence of oxygen was found to significantly increase the sensitivity, the effect was essentially the same for either 10 or 20% oxygen with helium. An examination of gas flow rates between 0 and 10 liter/min indicated a flow rate of 7.5 liter/min. SAMPLESIZE. Although an examination of sample size indicated a general improvement in sensitivity with decreasing sample size, (even at the 5 mg level), a 25 mg sample size was selected to reduce difficulties originating from inhomogeneity, weighing, and handling. SPECTROSCOPIC BUFFERAND SAMPLE-BUFFER RATIO. An examination of the influence of several commonly used spectroscopic buffers indicated highly improved line-to-background ratios with graphite. The most desirable sample-tobuffer ratio was found to be one part sample to two parts graphite. Improvement of arc burning characteristics also resulted from the use of a 1 :1 mixture of pelletizing and nonpelletizing graphite rather than all pelletizing graphite. SAMPLE FORM.Comparison of arc burning characteristics of samples in various forms indicated the superiority of samples in pellet form with a hole in the center of the pellet
+ Lung
to allow for the release of gases formed very rapidly on initiation of the discharge. Standard Additions. With the technique of standard additions, analytical curves were obtained for samples of liver, lung, kidney tumor, blood serum, stool, bone, apple, cherry, and citrus leaves. Element concentrations in these samples were obtained by applying residual corrections (23). Upon examination of the analytical curves, it was observed that with the frequent exception of bone and the occasional exception of plant leaves they were identical within experimental error for all these matrices. Figures 1 and 2 are presented as typical examples. The majority of the analytical curves were comparable in terms of both slope and linearity and were generally useful over two orders of magnitude. The behavior of bone and plant leaf curves was not surprising since bone is primarily a calcium phosphate and the plant materials normally contain about 10% ash (primarily oxides of potassium, calcium and magnesium) in comparison to the other materials which contain about 1% or less of ash. The bone curves and the plant leaf exceptions were generally parallel to the curves for the other biological materials. Synthetic Matrix. The similarity of the standard additions analytical curves led to the conviction that matrix effects were not serious so a “clean” synthetic matrix could be employed in place of the biological material, consequently simplifying and speeding the analytical application of the method. Although the material employed, PNBAR, was found to contain some metallic impurities at low concentration (23) N. H. Nachtrieb, “Principles and Practice of Spectrochemical Analysis,” McGraw-Hill, New York, 1950, p 139. VOL. 40, NO. 6, MAY 1968
857
4
0
I .O A
I
r 0
0.4
0.I
.O4
I
I
$01’ .I
0.4
I
I 4 Cr, ppm by weight
I IO
I
40
100
Figure 2. Typical analytical curves for chromium (Apple leaf data not considered in curve) 0 Liver; A
Apple Leaves;
Bone;
0 Stool
+ PNBAR; V Blood Serum; X Kidney Tumor; + Lung;
m n by Emirion Smcttometn
Figure 3. Comparison of results obtained by atomic absorption and direct emission spectrographic procedure for diversifled biological samples Zinc; A Copper;
858
ANALYTICAL CHEMISTRY
Manganese; +Lead; V Iron; X Aluminum;
+ Boron
Comparison of Emission Spectrographic and Mass Spectrographic Analysis (ppm by weight) Lung A Lung B Standard additions analysis Synthetic matrix calibration Emission spectrographic Mass spectrographic Emission spectrographic Mass spectrographic Element
