Detection Limits in Analysis of Metals in Biological Materials by Laser Microprobe Optical Emission Spectrometry William J. Treytl, James B. Orenberg, Kenneth W. Marich, Arthur J. Saffir,] and David Glick Division of Histochemistry, Department of Pathology, Stanford University Medical School, Stanford, Calif. 94305
RECENTSTUDIES from this laboratory on the operation of the laser microprobe, an instrument with considerable potential for high-sensitivity emission spectrometric analysis of elements in microscopic biological samples, dealt with effects of matrix material (1) and atmosphere ( 2 ) on the spectral emission, corrections for background and laser energy on spectral signal (3), and the use of photoelectric time differentiation (gating) to minimize signal noise (4). Detection limits in laser microprobe analysis as estimated by calculations from data of macroanalyses, and reported in (5), have been found to require revision when compared to data obtained by direct measurement in this investigation. The few values of analytical detectivity reported for elements in metallic and mineral matrices (6-8) cannot be validly applied to biological samples. The spectral emission of an element in a laser-generated plasma has been shown to be strongly dependent on the matrix in biological samples ( I ) . Therefore it is necessary that data of detection limits be derived from samples similar in composition to those to which the limits are applied. Dried samples of albumin and gelatin having approximately the same matrix effect as dried tissue were employed because it was not possible to prepare tissue samples conveniently with uniform known concentrations of added salts, as it was with the albumin and gelatin samples. Since many instrumental parameters affect analytical detectivity, the detection limits derived in this work apply only to the specific instrumentation employed. Detection limits of the elements, Li, Mg, Ca, Fe, Cu, Zn, Hg, and Pb, present in the form of their salts dissolved in a gelatin or albumin medium, were investigated using a gated photoelectric detection system ( 4 ) . EXPERIMENTAL
Materials and Methods. Stock solutions were prepared of the following reagent grade salts: Cu(NO&. 3H20, Zn(NOd2.6H20, Mg(NOd2.6H20, Hg(NO&, Pb(N03)?, Ca(N03)2.4H20, LiN03, and FeCl3.6Hz0. Stock solution I consisted of the final mixture (mM): 7.88 Cu, 15.3 Zn, 12.3 Mg, 17.5 Hg, 24.2 Pb, 43.7 Ca, and stock solutions I1 and I11 were 144mM Li, and 4.49mM Fe, respectively. Dilutions of 5 , 10, 50, 100, 500, and 1000 were made from each stock Present address, University of the Pacific, Department of Biochemistry, 2155 Webster Street, San Francisco, Calif. 94115. (1) K. W. Marich, P. W. Carr, W. J. Treytl, and D. Glick, ANAL.
CHEM., 42,1775 (1970). (2) W. J. Treytl, K. W. Marich, J. B. Orenberg, P. W. Carr, D. C. Miller, and D. Glick, ibid., 43, 1452(1971). (3) A. J. Saffir, K. W. Marich, J. B. Orenberg, and W. J. Treytl, Appl. Suectrosc., 26, in press. (4) W. J.Treyt1, J. B. Orenberg, K. W. Marich, and D. Glick, ibid.,
25,376(1971). ( 5 ) D. Glick, Ann. N . Y . Acad. Sci., 157,265 (1969). (6) M. V. Bobrova, Sci. Tech. Aerosp. Rep., 8 , No. 17(1970). (7) V. V. Penteleev, M. L. Petukh, 0. I. Putsenko, T. A. Iankovskaia, and A. A. Ionkovaskii, Zh. Prikl. Spektrosk., 12, 1106
(1970). (8) V. V. Panteleev, Sci. Tech. Aerosp. Rep.,9, No. 12(1971).
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I 2
I 4
I 6
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Delay t i m G ( p s e c )
Figure 1. Iso-contours depicting the effects of delay and integration times on values of S/U.Vfor a typical element (iron), in gelatin. Time parameters yielding optimal detectivity and given by the point (37)of maximum S/U,V solution. A 2-ml aliquot of each diluted solution was mixed with 18 ml of hot 5 % gelatin (USP) and gelled at 7 "C for 24 hr. Ten pthick frozen microtome sections were cut from pieces of the frozen (dry ice) gelatin solutions and mounted on 1 X 3 inch plastic slides. Since gelatin contains large amounts of Mg and Ca, solutions were prepared in 7 % human albumin at the p M concentrations of Mg: 53.5, 65.9, 83.4, 128, and Ca: 15, 190, 235, 292, 310. Droplets (12 nl) were deposited on plastic slides with a micro dispensing syringe ( I ) , air dried, and used as samples for detection limit determination. Apparatus. The Q-switched ruby laser microprobe used has been described (9). The laser was operated through a 2-mm transverse mode selector and delivered a beam of 12-16 mJ energy to the target. Laser light was focused by a 25-cm focal length, 5 or lox, microscope objective. Holes of approximately 50-1.1 diameter were produced in the gelatin sections from each laser shot, equivalent to 20 pl of sample. A gated detection system using EM1 9526B photomultiplier tubes (Whittaker Corp., Plainview, N.Y.) was employed for the measurements. This system was described earlier (4, IO). In most cases, the photomultiplier anode signals were recorded with a Biomation Model 802 Transient Recorder (Biomation, Palo Alto, Calif.). When this unit was employed, it replaced the gated integrators in the system. When necessary, the photomultiplier signals were preamplified with LRS Model 334 fast D C amplifiers (LeCroy Research Sys(9) N. A. Peppers, E. J. Scribner, L. E. Alterton, R. C. Honey, E. S. Beatrice, I. Harding-Barlow, R. C. Rosan, and D. Glick, ANAL.CHEM., 40,1178 (1968). (10) E. S. Beatrice and D. Glick, Appl. Specfrosc., 23,260 (1969).
ANALYTICAL CHEMISTRY, VOL. 44, NO. 11, SEPTEMBER 1972
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Table I. Optimal Time Parameters and Detection Limits of Metals in Organic Matrices Delay IntegraWavetime, tion time, Detection limit, Element length, A psec psec gram Li 6104 4 5 2 x 10-13 4 7 2 x 10-15 2796 Mg Ca 3934 5 15 1 x 10-14 Fe 10 6 3 x 10-13 3020 cu 3248 15 3 2 x 10-16 Zn 2139 5 2 5 x 10-14 3 x 10-13 2537 7 3 Hg Pb 4058 16 5 1 x 10-13 Dried droplets of salts dissolved in 7 x albumin used as samples for Mg and Ca, all others in 5% gelatin matrix. Laser energy 12-16 mJ; 25 nsec pulse duration. Table 11. Comparison of Capabilities of Highly Sensitive Techniques for Elemental Analysis Detection limits Referppm gram ence Technique Sample size Ion 10-1' g 100-10-2 10-17-10-'Q (11) microprobe 1-5 p diam. Electron 10-12 g 103-101 10-15-10-17 (12,13) microprobe 0.2-1 p diam. Laser 10-8 g io*-io-1 10-1*-10-15 microprobe" 10-200 p diam. Neutron mg-g 10-3-10-6 10-lO-lO-la (14) activation Atomic mg 10-1-10-5 10-9-10-1* (15) absorption a Present data.
tems, West Nyack, N.J.) before being fed into the transient recorder. RESULTS AND DISCUSSION
The detection limit is commonly defined as the amount of material yielding a signal detectable within the 95% confidence level, Le., a signal whose magnitude is twice that of the standard deviation of the total noise of the system, S1.N = 2, where S is the noise corrected signal and uN is the standard deviation of the noise. In a gated pulse-integration system, the portion of the signal pulse measured is selected by a combination of delay time (the time interval after the laser burst at which measurement commences) and integration period (the duration of the measurement). Since any combination of delay and measurement times may be selected, it is necessary to determine optimum times (that yield maximum s / ~ to ~ obtain ) maximal sensitivity. If the uncertainty of the background (11) M. Bayard, h e r . Lab., 3 (4),15(1971). (12) J. A. Chandler, ibid., p 50. (13) R. H. Heidel, ANAL.CHEM., 43,1908 (1971). (14) W. W. Meinke, Proc. Jap. Conf. Radioisotop., 9th, Tokyo, 1969, 535 (1969). (15) J. D. Winefordner and R. C. Elser, ANAL. CHEM.,43 (4), 254A (1971).
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were a well-defined function of its magnitude, then a reasonable number of measurements of equal duration and successive delays could be made, the data could be appropriately combined to yield other delay and integration times, and the S / c N values could be calculated. Unfortunately, no well-defined functional relationship between N and uN was found. If each signal or noise pulse, P, can be divided into n equal time intervals, rn successive measurements can be made such that
. . '&I 2P = [*Sl,2S2, %a,. . . 2S,] "P = ["Sl,"S2, "Sg,. . . . "S,] 'P = ['Sl, 5 2 ,
where each S is the signal within a particular time interval. Then, for a measurement of duration, W ,and delay, D,the average signal value, Snw and its standard deviation can be calculated:
with no assumptions concerning the relationship of the standard deviation to its variable. Tabulations or graphs of S/uN US. delay and integration periods can then be assembled. The Biomation Model 802 Transient Recorder is suitable for such measurements. Each interval, i, was 0.5 psec, and 10 spectra of each signal and background pulse were taken. An IBM 360/50 computer was used to perform the calculations. The result of a typical measurement is shown in Figure 1 in which contours of equal S/uN values are plotted as functions of delay time and integration time. Similar measurements were made for the other elements studied. The optimum timing values are read from the contour plots. Samples containing varying dilutions of each element were analyzed, and the s / g N values obtained. The best linear regression curve was fitted to a plot of SicN US. amount of the particular element, and the detection limit in each case was determined. Optimal time parameters and detection limits are given in Table I. The detection limit values reported here, compared with those obtained for other sensitive methods, are shown in Table 11. Although it is beyond the scope of this communication to deal with the relative and particular advantages and limitations of the different techniques indicated in Table IT, each of which has its unique applicability, the values are presented to give perspective with regard to the detection limits derived in this study. It may be noted that the laser microprobe can be particularly useful when biological samples of -10-8 gram are to be measured and manipulations of sample preparation must be minimized.
RECEIVED for review February 2, 1972. Accepted May 1, 1972. Supported by research grant G M 16181 and research career award 5K6AM18, 513 (to D.G.) from the National Institutes of Health, U S . Public Health Service.
ANALYTICAL CHEMISTRY, VOL. 44, NO. 11, SEPTEMBER 1972
