sophistication. We believe that the relative intensity measurements, and consequently the rate constants, can be improved upon in certain cases. One factor of great importance is the intensity of the microwave absorption lines being used as the composition probes. In the present case, the spectral lines are all very weak because of the small electric dipole moments (-0.3 D in all cases) (14).Consequently, the signal-to-noise ( S I N )ratio is intrinsically rather poor, and thus the precision of the peak intensity ratios is not as high as it might be. For molecular systems having larger dipole moments, it should be possible to obtain a considerable improvement in this key factor.
(4) C. W. Gillies and R. L. Kuczkowski, J. Am. Chem. Soc., 94,6337 (1972). (5) H. W. Harrington, J. Chem. fhys., 46, 3698 (1967). (6) H. W. Harrington, J. Chem. fhys., 49, 3023 (1968). (7) A. S. Esbitt and E. B. Wilson, Rev. Sci. Instrum., 34, 901 (1963). (8) H. W. Harrington, J. Ch8m. fhys., 44, 3481 (1966). (9) See, for example, K. J. Laidler, "Chemical Kinetics", McGraw-Hill, New York, 1965, pp 19-20. (10) See, for example, Ref. ( I ) , Chapter 3. (11) J. P. Chesick, J. Am. Chem. SOC., 84, 3250 (1962). (12) S. N. Mathur, M. D.Harmony, and R. D.Suenram, J. Chem. fhys., 64,4340 (1976). (13) M. D.Harmony, C. S. Wang, K. B. Wiberg, and K. C. Bishop Ill, J. Chem. fhys., 63, 3312 (1975). (14) R. D. Suenram and M.D. Harmony, J. Chem. fhys., 56,3837 (1972).
LITERATURE CITED
RECEIVEDfor review April 16,1976. Accepted June 8,1976. Acknowledgment is made to the donors of the Petroleum Research Fund, administered by the American Chemical Society, and to the National Science Foundation (Grant MPS 74-22178) for support of this research.
(1) W. Gordy and R . L. Cook, "Microwave Molecular Spectra", Wiley-lnterscience, New York, 1970. (2) L. H. Scharpen and V. W. Laurie, Anal. Chem., 44, 378R (1972). (3) L. H. Scharpen, R. F. Rauskalb, and C. A. Tolman, Anal. Chem., 44,2010 (1972).
Direct Optical Encoding of Recorded Spectra with a Computer Interfaced Vidicon Television Camera J. A. de Haseth, W. S. Woodward, and 1.L. Isenhour" Department of Chemistry, Univeristy of North Carolina, Chapel Hill, N.C.
275 14
A vidicon tube television camera interfaced to a Raytheon 704 minicomputer enables the direct optical encoding of any spectral curve, provided the curve be a single-valued function of the abscissa value. The appllcation presented is the digitization of Infrared spectra. The algorithm is sufflciently flexible that it Is not iimlted by spectral range or physical size of the hardcopy medium being encoded. The encoder is accurate and able to digitize approximately 35 spectra per hour when operating in batch mode.
hours run-time is being recorded. The computer-time loss problem can be resolved to some degree if an intermediate device such as a paper tape punch with an analog-to-digital converter is used. This gives a computer readable format for the spectrum, but the digitization may still be time consuming. Of course Fourier transform infrared (FT/IR) spectrometers are available with their own processors but few give output in any format other than the absorption curve of the sample or its Fourier transform. A major drawback with these methods is that the spectrum must be obtained in its digitized form while being recorded. Thus, to obtain the digitized form of a spectrum that was recorded at some time in the past requires that the experiment be rerun and digitized. This approach may be difficult if the sample is unstable, in short supply, unavailable, or expensive. Another method that is commonly used is to digitize the spectrum manually. This involves reading the intensity a t each wavelength position and transcribing its value onto some form of input medium such as paper tape or computer cards. This is expensive and tedious to say the least. This paper presents an alternate method of digitizing spectra that is fast, efficient, and can be obtained from graphical representations of the spectra. A computer interfaced vidicon tube television camera is employed, a peripheral which permits the direct optical encoding and computer digitizing of curves. The apparatus has been described previously (28);however, some modification have been made to the computer such as the addition of 24K words of core. The only modification made to the equipment expressly for this project was the introduction of an inexpensive electronic bandpass filter to the TV camera-Raytheon 704 interface to enhance fine spectral lines. Vidicon tubes have recently been applied to several other chemical problems, mainly as spectrometric detectors (29-49) and as image encoders for bubble chambers (50-52). The camera is a relatively inexpensive piece of apparatus. The Panasonic WV-2007 vidicon tube television camera used for the interface cost only $220.
For the studies relating to the use of large chemical data bases, it has become clear that the acquisition of these data files has not always been easy. One such series of compilations is infrared spectra. Various research ideas have depended on large infrared data files such as studies involving pattern recognition (1-7), and search systems (8-27). Originally collections of these data bases were kept on coded index cards, but this form of data processing proved unwieldy. Emphasis then shifted to converting the files into a computer manageable format, but the conversion of a spectrum from an analog representation to a computer readable format has thus far been a tedious and expensive process. Spectra are available in digitized formats such as the binary file of 135 000 spectra assembled by the American Society for Testing and Materials. However, this file is available only in a peak-no-peak format and may therefore be unsuitable for some applications. Other files may be expensive or difficult to obtain. Therefore, many workers compile their own files. There are several methods of obtaining digitized spectra. One commonly employed method is to directly interface the spectrometer to a computer. Such interfacing can now be done routinely and is neither difficult nor expensive. Often a disadvantage to this approach is the extent to which the computer is devoted to the spectrometer and thus unavailable during the recording of a spectrum. This can lead to serious computer-time losses if a high-resolution spectrum of several
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Figure 1. A bit-no-bit representation of the infrared spectrum of methyl acetate from 600 cm-” to 2000 cm-’ The four large spots, one in each corner, are the field of view corner markers. This photograph represents an integratedrecording of the hardcopy spectrum; hence the grid lines appear more intense than on a raster-to-raster image
The direct optical encoding of spectra with a vidicon tube television camera needed to meet five criteria. 1)The spectra were to be digitized directly from hardcopy graphical output of the spectrometer. The spectra should be of any spectral scale or any range and not restricted in physical size. No intermediaries such as tracings, photographic, or Xerox copies of the original spectra were to be required. 2) The algorithm was to be fast to minimize computer and operator time. In addition to speed, high accuracy in the digitized output was required. 3) Operator interaction was to be kept to a minimum. Once spectra were presented to the computer, the operator was only to supply two values so that the spectra could be calibrated. This criterion was a necessary minimum for an accurate digitization. 4) The encoder and digitizer were to be flexible enough to accept a variety of graphical representations. Although infrared spectra digitization was the original application, the algorithm was to be general enough to digitize any curve with only slight modifications being made to calibrate and output the data. The only stipulation was that the curve be a singlevalued function of the abscissa variable. 5 ) As in the previously cited article (28), the scanning hardware had been designed to minimize cost but the encoder was t o in no way limit the operation of the Raytheon 704 minicomputer. It is noted that there are other “direct” optical encoders available such as the “flying spot” and photodiode arrays. These instruments are commercially available but are unable to meet the above mentioned criteria. Frequently a photographic reproduction of the spectrum is required, or operator interaction is high. The system presented here is not a simple image encoder; that is, the entire image is not encoded, only the curve is recorded. In an image encoder, the entire viewing field must be stored. On a system of the same size as the current system, the storage would entail a minimum of 76.8 k-bits, Le., an image of 240 X 320 bits in dimensions. The graph encoder can represent the same data in a more useful format in only 10.3 k-bits. This figure accounts for 320 x,y-coordinates of a single spectral curve. Input Medium Format. To encode and digitize spectra, the system must accept any hardcopy graphical representation. This can be done as long as the spectral line and the grid 1514
coordinate lines are of a sufficiently different color to or of a different intensity from one another. The vidicon tube is blue sensitive and “peaks”in this region; therefore blue lines are detected as white. Red is seen as black. Hence, a red spectral curve on blue-lined paper results in having only the red line perceived because of the inherent color discrimination of the vidicon tube. Should the original spectral recording have been made with a less opportune color combination, the spectral and grid lines can be distinguished by color filtration. For example, should a spectrum have a green grid and a red spectral line, only the spectral line will be transmitted to the camera if a green filter is placed in front of the camera lens. Again, if the grid is red and the spectral line is blue, only the grid will be seen. To reverse the situation, a red filter need simply be placed in front of the camera lens. The grid lines will disappear and the spectral line becomes clearly visible. In elementary terms, to enhance the spectral line over the grid, a filter of the same color as the grid lines is placed directly in front of the camera lens. Unfortunately, not all spectra are recorded on chart paper with pure color grid lines and in some cases no satisfactory filter may be available. If the situation arises where the grid and spectral lines are of the same color, the spectrum may be encoded if the grid lines are fainter than the spectral line. The camera-computer interface has a contrast threshold adjustment so that areas above a certain intensity may be ignored whereas all those below that intensity may be recognized. The output from the camera interface is in a bit-no-bit format so that a point is either present or not. The contrast threshold adjustment is useful when encoding printed spectra from tabulations or texts as the publishers usually make the grid lines fainter than the spectral line. This technique makes the spectra visually more readable. The only other aid the operator need provide is to mark the four corners of the spectrum so that the encoder has a frame of reference. The authors have found that a simple method is to mark a small solid circle on each of four separate small pieces of clear, colorless acetate. The solid circles should be of the same color as the grid lines. This is easily accomplished if the circles are drawn with a felt-tipped marking pen of the appropriate color. The resulting corner markers are transparent and can be removed along with the grid lines at the time of encoding by use of the correct color filter. It has been found that flat, even illumination over the spectrum during encoding is a desirable situation. The more uniform the illumination, the greater signal-to-noise response since there is less interference from shadows. The algorithm was designed to differentiate between signal and noise. Therefore, some care was taken to regulate the illumination; however, special baffles and lights proved unnecessary. A spectral curve as interpreted as bit-no-bit input by the vidicon tube television camera is illustrated in Figure 1. Encoding Algorithm. Encoding begins as the four corners of a spectrum are marked and the spectrum is placed within the viewing area of the television camera. The total viewing area is expressed as an array of 240 X 320 bits. At the beginning of the routine, the computer awaits a signal from the operator to search for corners. After the signal is given, the computer commences the scan using a discrete 16 X 16 bit window. The window spirals from one of the corners of the viewing area until it encounters an image, that image being one of the corner markers. The computer then centers its sampling window over the marker and calculates its center of mass with respect to the coordinates of the total viewing area. Three corner markers are found in this manner and then the position of the fourth is calculated. The fourth marker is searched for and, after it is found, its position is compared to its calculated value. If the two positions do not coincide within small tolerances, the process is repeated continuously until
ANALYTICAL CHEMISTRY, VOL. 48, NO. 11, SEPTEMBER 1976
Figure 2. The graph encoder interpretation of Figure 1 The white regions of the curve are recorded points: the black regions indicate where linear interpolations have been made
Figure 3. The same as Figure 2, except the curve has been calibrated In this case the linear scale is in reciprocal centimeters and the output is appropriately marked \
the operator changes the position of the marker that is out of place. Only a rectangle will be accepted as being correct. At this point, the computer pauses so that the operator can place a filter in front of the camera lens and/or adjust the contrast threshold so that the grid lines are invisible. The encoding scan begins a t the upper left hand corner of the frame of reference and proceeds downward. A column, one bit in width (1/& of the total viewing area width), is scanned. If nothing is present, the column one bit to the right is scanned. This procedure is repeated until the spectral line i s encountered. The center of mass of this line is then determined. The computer examines the position one bit to the right of the first recorded position. If the bit is “on”, Le., the line is present, the process is continued. If the line is not present, it looks a t the position one bit to the right and one bit above the last recorded position, Failing to relocate the line, it checks the bit position downward to the right. A fourth sample is taken one bit to the right and two bits upward. This one-dimensional spiral process is repeated within this single column until either the line is found or the computer has looked at five bits above and five below the calculated center of mass. If the line is found, as is generally the case, an upward or downward trend for the curve has been established and the encoder follows that trend. Should the trend reverse, as will happen a t extrema, the algorithm verifies that change and then follows that path. Should the line be “lost” due to a discontinuity, the computer checks the general area of the last point in order that it might find the line again before it has to resort to a column search in order to relocate the line. Resolution may be a problem in the hardcopy representation of the spectrum. This is most prevalent in tabulated spectra, where peaks are often represented as spikes when the spectrum is laterally compressed to fit onto the printed page. This is a potential problem for the algorithm as although it may detect the peak, it may not correctly record the intensity. As the algorithm emphasizes encoding in a horizontal direction, a change in upward or downward trend is established rather than the peak being followed to its final intensity. Consequently, once a peak has been found, the scanner returns to the peak position and searches for a spike. Should one be found, the spike is followed to its termination and the termination value is recorded for the peak intensity. The algorithm is not totally dependent upon the camera since it does not have to return to the camera every time a new bit is sampled. As mentioned above, a 16 X 16 bit window is
sampled a t one time; thus the computer can manipulate these data mathematically. The computer need return to the camera only when the bit position requested did not fall within the last 16 X 16 bit area. The computer can return to the television camera only once every fiG0 of a second because of the continuous cycling of the television raster. This is adequate time for any computations that need to be done, and thus the encoding speed is limited simply by the raster. When 16 consecutive points are recorded from a single sampling window position, the optimum position for the next window is calculated using the last three recorded data points taken. The method of quadratic least squares is used to calculate the expected position of the next data point. This way, the sampling window is placed in the most opportune position for the next point and subsequent points. As can he seen, this algorithm is considerably more efficient than a column-by-column search. Once the spectrum has been encoded, it is plotted onto a television monitor which functions as a display scope and printer. An example of the display is shown in Figure 2. The vertical axis is not labeled hut is marked in increments of‘10% T . The horizontal axis is unmarked. The encoded spectrum is displayed in one of‘ two ways: with simply the recorded points shown or with the points connected to form a line spectrum. Consecutivc poin1.s arc shown in white and, where discontinuities occur, a linear interl)olation is made and shown in black. Rel‘ore complet.e digitiznt,ion is iicc:omplish(4.t ht. spectrum must he calibrated. This need he don(> only o t i c c x for batch processing, i.e., a series of spectra, assuming ;dl t.hc1 spectra are of the same physical size and the s a m e range is encoded. T h e first step involves the computer selecting the largest,peak and indicating the peak o n t,he display. ‘l’h(> operator decides whether the peak is sat wtory lor c:ilihtion. lf the peak is unsatisfactory, tho computer will choose the next largest.peak. This continues until a suitable peak has been h i n d . The operator is thus given the option 01 picking the m o s t easily read peaks for calibration purposes. The operator must. supply some information, the linear scale ol‘the spectrum (micrometers or reciprocal cc?nt,irncters)i i n d t.hr viiluc. of’ thr peak position. The compui or Ih r n procwds i o f i n d :j .;cvond peak stitisfactory to t . 1 operator ~ who t h ~ providw n II s posithn value. As soon as the spectrirm is (~;dil)r:>i~(ld t lip horizontal axis is marked and 1aI)eled in the app-opriak s d e . T h i s i s illustrated in Figure 3.
ANALYTICAL CHEMISTRY, VOL. 48, NO. 1 1 , SEPTEMBER 1976
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32 pm range can be examined; however, this is out of the scope of most infrared spectrometers. In conclusion, the system described permits rapid and precise encoding of hardcopy spectra into a digital format. These data, once collected, can be used for various purposes such as pattern recognition, structural analysis, or search and retrieval operations. As with the Chemical Structure Encoder previously cited (28),the total equipment cost is on the order of $33,000.
-
LITERATURE CITED
Figure 4. The methyl acetate infrared spectrum after a 60 % Tcut-off has been established The peaks below the cut-off have been detected and their wave lengths in micrometers (to an accuracy of 0. l,urn) are displayed in black. The intensities in YO T corresponding to these peaks are displayed in white below the respective peak positions
A t this point the spectrum may be considered digitized. It would be a simple matter to print out all the spectral positions and their intensities; however, an alternate result was chosen. It was decided that the operator should have the option to pick any transmittance cut-off value and then select all peak positions and their intensities below that value. This was done so that the digitizer would be compatible with retrieval systems such as the Infrared Spectral Information System (ISIS) developed at the Triangle Universities Computation Center (TUCC) in North Carolina (53).ISIS is an infrared search system that uses the ASTM file of IR spectra. The encoder is sufficiently flexible that it allows the operator to choose the output device. This can be the display screen, a magnetic tape, the line printer, the teletype, or paper tape. When running the system in batch mode it is necessary to define the cut-off and output device only once. An example of the final output displayed on the monitor is illustrated in Figure 4.
RESULTS AND DISCUSSION The above described algorithm was tested on a random sampling of 11 Sadtler Standard Grating Spectra ( 5 4 ) .The spectra were encoded in batch mode and each was encoded in the range 1600 cm-1 (6.25 pm) to 600 cm-l(l6.7 pm). The total time required to run the job was 20 min, 15 s. This included a run-time of 2:45 for the first spectrum which had to be calibrated and have a percent transmittance cut-off set. This left 17:30 for the remaining ten spectra, or 1:45 per spectrum. From these times, it can be seen that approximately 35 spectra per hour could be digitized or almost 280 per eight-hour working day. Most of the time requred required was spent registering the spectra for the encoding step. Had a properly constructed register board been available it would not be unreasonable to expect that the encoding rate could be doubled. The entire system is efficient and extremely flexible as the system can be used for individual or multiple encoding. Output can be stored in a number of forms. Spectra can be encoded in sections if the spectral scale changes with grating changes, or if a higher resolution is desired. At present, to maintain a resolution of 0.1 pm, a section of no greater than 1516
ANALYTICAL CHEMISTRY, VOL. 48, NO. 11, SEPTEMBER
(1) H. B. Woodruff, S. R. Lowry, and T. L. Isenhour, Appl. Spectrosc., 29, 226 (1975). (2) H. B. Woodruff, G. L. Ritter, S. R. Lowry, and T. L. Isenhour, Technometrics, 17, 455 (1975). (3) H. B. Woodruff, S. R. Lowry, G. L. Ritter, and T. L. Isenhour, Anal. Chem., 47, 2027 (1975). (4) B. R. Kowalski, P. C. Jurs, T. L. Isenhour, and C. N. Reilley, Anal. Chem., 41, 1945 (1969). (5) P. C. Jurs, B. R. Kowalski, T. L. Isenhour, and C. N. Reilley, Anal. Chem., 41, 1949 (1969). (6) R. W. Liddell 111 and P. C. Jurs, Appl. Spectrosc., 27, 371 (1973). (7) R. W. Liddeli 111 and P. C. Jurs, Anal. Cbem., 46, 2126 (1974). (8) L. E. Kuentzel, Anal. Chem., 23, 1413 (1951). (9) A. V. Baker, N. Wright, and A. Opler, Anal. Chem., 25, 1457 (1953). (10) R. A. Sparks, “Storage and Retrieval of Wyandotte-ASTM Infrared Spectral Data Using an IBM 1401 Computer”, ASTM, Philadelphia, Pa., 1964. (11) L. D. Smithson, L. B. Fall, F. D. Pitts, and F. W. Bauer, “Storage and Retrieval of Wyandotte-ASTM Infrared Spectral Data Using a 7090 Computer”, Tech. Doc. Rept. No. RTD-TDR-63-4265, Research and Technology Division, Wright Patterson AFB, Ohio, 1964. (12) T. A. Entzminger and E. A. Diephaus, “Storage and Retrieval of Wyandotte-ASTM Infrared Spectral Data Using a Honeywell 400 Computer”, U.S. Public Health Service, Robert A. Taft Sanitary Engineering Center, Cincinnati, Ohio, 1964. (13) D. H. Anderson and G. L. Covert, Anal. Cbem., 39, 1288 (1967). (14) L. H. Cross, J. Haw, and D. J. Shields, in “Molecular Spectroscopy, Proceedings of a Conference Held at Brighton, England, 17-19 April 1968”, P. Hepple, Ed., The Institute of Petroleum, London, 1968, p 189. (15) G. A. Massios, Am. Lab., 3 , p 0 5 , Sept. 1971. (16) D. S. Erley, Anal. Cbem., 40, 894 (1968). (17) F. E. Lytle and T. L. Brazie, Anal. Chem., 42, 1532 (1970). (18) D. S. Erley, Appl. Spectrosc., 25, 200 (1971) (19) F. E. Lytle, Anal. Cbem., 42, 355 (1970). (20) P. C. Jurs, Anal. Cbem., 43, 364 (1971). (21) E. C. Penski, D. A. Padowski, and J. B. Bouch, Anal. Chem., 46, 955 (1974). (22) Yu. P. Drobyshev, R. S. Nigmatullin, V. I. Lobanov, I. K. Korobeinicheva, V. S. Bocharev, and V. A. Koptyug, Vesfn. Akad. Nauk SSSR,40 (a), 75 (1970); Chem. Abstr., 74, 69g (1971). (23) Yu P. Drobyshev, R. S. Nigmatullin, V. i. Lobanov, I. K. Korobeinicheva, F. S. Bochkarev, and V. A. Koptyug, lzv. Sib. Ofd.Akad. Nauk SSSR,Ser. Kbim. Nauk, 1972 (l), 108; Chem. Abstr., 77, 96627 y (1972). (24) C. S. Rann, Anal. Chem., 44, 1669 (1972). (25) K. Schaarschmidt, R. Reimer, and E. Steger, 2.Chem., 14, 374 (1974). (26) K. Tanage and S. Saeki, Anal. Chem., 47, 118 (1975). (27) H. B. Woodruff, S. R. Lowry, and T. L. Isenhour, J. Chem. Inform. Comput. Sci., 15, 207 (1975). W. S. Woodward and T. L. Isenhour, Anal. Chem., 46, 422 (1974). R. E. Santini, M. J. Milano, H. L. Pardue, and D. W. Margerum, Anal. Chem., 44, 826 (1972). M. A. Kwok, E. F. Cross, and T. A. Jacobs, Rev. Sci. Instrum., 43, 1043 (1972). K. W. Jackson, K. M. Aldous, and D. G. Mitchell, Spectrosc. Lett., 6,315 (1973). D. G. Mitchell, K. W. Jackson, and K. M. Aldous, Anal. Chem., 45, 1215A (1973). W. H. Woodruff, D. W. Magerum. M. J. Milano, H. L. Pardue, and R. E. Santini, lnorg. Chem., 12, 1460 (1973). P. H. Lloyd and M. P. Esnouf, Anal. Blochem., 60, 25 (1974). K. W. Jackson, K. M. Aldous, and D. G. Mltchell, AppI. Spectrosc., 26,569 (1974). M. J. Milano, H. L. Pardue, T. E. Cook, R. E. Santini, D. W. Margerum, and J. M. T. Raycheba, Anal. Chem., 46, 374 (1974). K. W. Busch, N. G. Howell, and G. H. Morrison, Anal. Chem., 46, 575
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/107A\
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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 following.
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 a t 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 a t 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 t o 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
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