INDUSTRIAL
and
ENGINEERING
CHEMISTRY +
ANALYTICAL EDITION
Harrison E. Howe, Editor
Spectrographic Analysis of Biological Material Lead, Tin, Aluminum, Copper, and Silver JACOB CHOLAK AND ROBERT V. STORY Kettering Laboratory of Applied Physiology, University of Cincinnati, Cincinnati, Ohio
T
centrations of metals. However, a modification by means of which determinations of opacity could be substituted for determinations of density was found to be advantageous in the evaluation of very weak lines. This modification inyolves adherence to the authors’ earlier method (2, 9) of dealing directly with faint lines instead of attempting to isolate and concentrate the test metal ( 2 ) . In applying the modification, the quantity of internal standard used must be such as to produce a standard line the density of which is below 0.30 (opacity of 2) in a t least two steps of the spectrogram produced tiy means of the step sector. The measured opacities (galvanometer reading of emulsion/ galvanometer reading of line) for the two weak steps of the standard line are plotted as a straight line against the relative exposures of the steps. The opacities for the test line are also plotted and the distance between the two lines at a base opacity (1.30) can then be correlated n i t h the concentrations of the test metal (Figure 4). Figure 1 is a photograph of duplicate spectia with faint test lines and Figure 2 illustrates the niethod of ohtaining the separatiorii
ECHXICAL improvements developed during the past
few years have greatly reduced the uncertainties attending quantitative spectrum analysis and have resulted in a n increased application of emission spectrography to studies of trace metals in biological material (1-4, 6, I S , 14, 16, 19-21, 23,26, 27, 29, 35, 34). The purpose of this paper is to present a method for the simultaneous determination of lead, tin, aluminum, copper, and silver in biological material and to call attention to certain improvements in procedure which have been introduced since the authors’ earlier publications (2-4).
Photometry The earlier method of photometry (2, S), which gave sufficiently accurate results ( 5 ) but was applicable only to relatively low concentrations of metals, has been replaced after a study of other technics (IO,11, IS, 15, 28, SO) by the niethod of Preuss (25). This method. which employs the Hansen gage (17) to incorporate the blackening mark in the analytical spectrum, extends the analytical range, thereby reducing the dilutions otherv-iqe required to handle relatively high con-
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FIGURE I 619
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INDUSTRIAL AND ENGINEERING CHEMISTRY
620
T h e foregoing procedure is employed in the case of spectrograms in which the test lines appear in not more than three exposure steps. When the line is present only in the maximum exposure step, the plot is made by assuming a n opacity of 1.0 for the next lower step. When the test lines appear in three or more steps, the method is identical with that described by Strock (SO) in which the intervals of separation
1.5
,BLi
Pbi -Bil 15 4&-Biz = I3 6 AVE. = 14.5=0.035ma.Pb
1
RELATIVE EXPOSURES FIGURE 2
between the density curves for the test and standard lines a t a base density of 0.30 are correlated with the logarithms of the concentrations (Figure 3). I n borderline cases the choice of method depends upon whether or not the density curve for the test line can be accurately extrapolated to the base density of 0.30; when the density of the test line in the highest exposure step is less than 0.20, more accurate results can be obtained by resorting to the d a t a on opacities.
crater 3 mm. wide by 10 mm. deep into which the sample is introduced, while the negative, 70 mm. in length, is sharpened with a pencil sharpener to a fine point. This pointed electrode aids in centering and confining the arc, thereby reducing its tendency to wander. Among other impurities, graphite electrodes, as purchased, contain aluminum and copper in such quantities as to necessitate their removal. Preliminary arcing for 1.5 minutes accomplishes this but' also impairs the subse uent effectivenessof the electrodes because of increased arc wanlering. A satisfactory method of purification consists in steeping the properly cut rods in a mixture of equal parts of distilled hydrochloric and nitric acids maintained at 70" C. over a period of 48 hours during which the bath is changed 4 or 5 times, This treatment is followed by a corresponding period of immersion in 4 or 5 changes of tripledistilled wat'er also at 70" C., after which the electrodes are heated for 1 hour at from 900" to 1000" C. in an electric muffle furnace. Only boron, silicon, and traces of magnesium and vanadium then remain as impurities, and these may be reduced by other methods of purification (15, 18, 14,35). A sector for use in this work was designed with a relative exposure factor of 2 (log 0.30),in seven steps, each exposing a 2.5mm. length of the slit of the spectrograph. The sector is provided with a slotted plate that can be adjusted to eliminate any number of steps. A five-step exposure was found to be the most practical and was used in this study. The sector exposes the slit over but one-third of its circumference. (Sectors with higher total exposures are useful in analyzing less persistent lines than those referred to herein. A suitable sector for such lines is one which is cut out on both sides so as to double the total exposure given the slit of the spectrograph.) Density and opacity measurements were obtained with the Bauxh & Lomb nonrecording densitometer. I n order to measure 2.5-mm. sections of the spectrograms, it was necessary to increase the magnification by providing a longer arm for the projection mirror than that supplied with the instrument.
Apparatus The spectral region employed (2600 A. to 3500 A.) is photographed with the large Bausch & Lomb quartz Littroiv spectrograph, a spherical quartz lens being used between the slit and the light source. Persistent lines of magnesium, manganese, iron, nickel, chromium, and zinc which also occur in this region provide means for the inclusion of these metals within the scope of the method. As in previous work (2-4) the source of excitation is a direct current arc betffeen graphite electrodes. This source and its modification involving the cathode layer effect (22) are generally the most satisfactory means for volatilizing the minute quantities of metals usually encountered in biological material. Other sources, such as various sparking procedures (7,8, 15, 16,52), the alternating current arc (7, 9), the "Abreissbogen" (15), or the flame (21), result in a lowered sensitivity of detection which offsets the other advantages of their use. The graphite electrodes (0.78 cm., 0.3 inch, in diameter) are prepared so that t,he positive rod, 40 mm. in length, contains a
VOL. 10, NO. 11
TABLEI. SPECTRAL LINES Metal Lead Lead Tin iiluminum Copper Silver
h Line 2833.07 2873.4 2840.0 3082.16 3273.96 3280.67
Internal Standard Bismuth Bismuth Bismuth Cobalt Cobalt Cobalt
X Line 2898.1
2898.1 2898.1 3082.6 3283.45 3283.45
Working Curves The working curves may be obtained from solutions prepared by adding the test metals and the internal standards to a salt solution of such composition as to be readily adaptable to that of the materials handled, with respect to inorganic salts. The material chosen as the base was the synthetically prepared ash of normal urine as described elsewhere ( 2 ) . A double internal standard, consisting of 5 mg. of bismuth and 100 mg. of cobalt per 100 ml. of solution, was employed. Table I lists the spectral lines of the metals and the corresponding lines of the internal standard, as used t o derive the calibration curves. Figures 3 and 4 illustrate the family of calibration curves used in this study. The extent of the analytical range for each line when used in either the density or opacity technique can be observed from the graphs.
Preparation of Samples T h e contaminations attending the chemical treatment of samples have been reduced to quantitative insignificance by employing purified acids and triple-distilled water ( 2 ) and by working in a laboratory equipped with a dust-removal system. Chemical treatments of the samples are preferred because they permit the use of solutions which in addition to guaranteeing the homogeneous nature of the samples greatly facilitates the introduction, into the craters of the electrodes, of the small amounts of material employed in the tests. FIGCRE3
Urine samples are prepared for analysis by the method described previously ( 4 ) ,except that the mixed internal standard (1 ml. = 0.5 mg. of bismuth and 10.0 mg. of cobalt) is added.
,4XALYTICAIJ EDITION
XOVEJIBER 13. 1938
621
of each of two purified electrodes and arced for 2 minutes, the TABLE11. CONCESTRATIONS OF METALSIS H u x m TISSUES data for the two spectrograms being averaged to determine the quantities of the metals present. (Case A. P . 1937. Age 7 5 years) In order to avoid contaminations or losses in handling small Metal Found samples of spinal fluid, the manipulations are kept at, a minimum. Tissue Sample Pb Sn hl cu The procedure is to collect or place the sample in a graduated 15Grams Mg. p e r 100 grams f r e s h tissue ml. quartz centrifuge tube, note the volume, add 0.1 ml. of distilled nitric acid for each 1 ml. of spinal fluid, and concentrate in a Heart Kidney g: : ::::5 0.45 0,005 n-ater bath to one-tenth of the original volume. Following the Brain 51.8 0.015 0.00 0.002 Stomach 50.0 0.03 0.02 0.14 0.09 0.00 addition of an equal volume of the salt diluent, 0.4-ml. portions are placed in the electrodes and their arc spectra are pliotoLiver Spleen :; 0.09 0.00 graphed. Such a procedure permit's duplicat'e analyses of SamSmallintestine 32.0 0,025 0.025 0.10 Skin 13.0 0.025 0.015 0.075 0.04 0.00 plesassmallas j m l . inwhichaslittleas0.05gammaofeachmetal can be determined quantitatively. Colon Lung :E: ::& 7
5":
13 0 24.0 5.4 10.0 5.2 9.2
Blood Crinarybladder Gall bladder Muscle Rib Femur
:::; :::: !;!: :::: :;"0 ::At5 :::; ::::! :::: :::! :;:is
0.025 0.01 0.015 0.005 0 39 3.29
0.45
0.00 0.01 0.00 0.00 0.00 0.00
0.11 0.00 0.17 0.10 0.21 2.50
0.065
0.02 0.04 0.01 1.09
0.00 0.005 0,005 0.00 0.00 0.00
Results I n Tables I1 and I11 are recorded thc findings for a COIIIplete series of necropsy specimens and for a number of other materials handled in the laboratory. The accuracy of the technique can be observed from the results listed in Table IV, in
Other biological materials, excepting spinal fluid, are prepared which are given the recoveries on duplicate samples prepared for not exceedby ashing in silica dishes at a temperature by adding known amounts of the nietak to the base salt stock. ing 500' C. The dried material is ashed directly or after digestion n-ith distilled nitric acid. The latter procedure reduces the time required for ashing and was employed for all materials TABLE 111. COSCEXTRATIONS OF METALS ISBIOLOGICAL MATEKIAL excepting feces, which are ashed c 3Ietal Foundafter drying t o constant weight Material Pb Sn AI cu .is ( 2 ) . Complete destruction of Ma. MU. Mu. Mu. xu. organic matter may the be hastened fluid
