490
Anal. Chem. 1986, 58, 490-493
sensitive to laser fluctuations; this is seen clearly as apparent noise in Figures 2a and 3a. This may be compared to the results obtained using an expansion of 1atm of argon (Figures 2b and 3b) where the cooling is complete; the background is negligible compared to the major peaks, so the base line shown corresponds to true zero signal. The mass spectra obtained for the laser photoionization of various PNAH's at 266 nm in a TOFMS are shown in Figure 5. In Figure 5a is a spectrum of acenaphthene, carbazole, and triphenylene obtained in a supercritical expansion of 160 atm of COZ at 40 'C. The TOF has not been maximized for resolution, which is only -150 here, but this is sufficient for the purpose of this experiment. The acenaphthene signal is strong under these conditions, while the other compounds are first beginning to dissolve in the fluid. In Figure 5b is a mass spectrum of acenaphthene, carbazole, phenanthrene, pyrene, and tetracene obtained from a supercritical expansion of 190 atm C 0 2 at T = 70 "C using 266 nm as the ionization source. These compounds all dissolve strongly in the supercritical fluid even a t 40 'C; however higher temperature was necessary to observe tetracene, which has a melting point >300 'C. Strong cluster formation is not observed in the mass spectra although this might be expected since cluster formation is proportional to Po2D. Although clusters of PNAH with COz are observed, the signal of any observed cluster peak is more than an order of magnitude smaller than any PNAH peak and the size of the clusters remains small. The small number of clusters formed may be due to minor shock waves in the orifice that may be sufficient to destroy weakly bound van der Waals complexes but may not be severe enough to destroy the supersonic flow. No clusters of the solute-solute type were observed in our experiments as expected at the fairly low seed concentration used. Thus, supercritical fluid injection of C02molecular beams can provide sufficient cooling to allow reasonably sharp spectral features for identification in chemical analysis. The attractive feature of this method is that low temperatures can be used to dissolve nonvolatile or thermally labile molecules into a supersonic jet. Although the spectra obtained in expansions of high-pressure C 0 2 are not as sharp as with Ar carrier gas, the cooling is sufficient to cool out the broad structureless rotational contours observed at room temperature. The use of the mass spectrometer allows identification of each compound by mass analysis in conjunction with the spectroscopic identification achieved by laser spectroscopy.
ACKNOWLEDGMENT We thank Werner Gerber of Ciba-Geigy, Basel, Switzerland, for helpful suggestions during the course of this work. Registry No. COz, 124-38-9;acenaphthene, 83-32-9; phenanthrene, 85-01-8; carbazole, 86-74-8; pyrene, 129-00-0; triphenylene, 217-59-4; tetracene, 92-24-0. LITERATURE CITED Lubman, D. M.; Kronick, M. N. Anal. Chem. 1982, 5 4 , 660-665. Sin, C. H.; Tembreull, R.; Lubman, D. M. Anal. Chem. 1984, 5 6 , 2776-2781. Zandee, L.; Bernstein, R. 8.J . Chem. Phys. 1979, 77, 1359-1371. Dletz, T. G.; Duncan, M. A.; Liverman, M. G.; Smalley, R. E. J . Chem. Phys. 1980, 73, 4816-4821. Sack, T. M.; McCrery, D. A.; Gross, M. L. Anal. Chem. 1985, 5 7 , 1290-1295. Johnston, M. V. Trends Anal. Chem. 1984, 3,58. Rhodes, G.;Opsal, R. 8.;Meek, J. T.; Reilly, J. P. Anal. Chem. 1983, 55, 280-286. Glddlngs, J. C.; Myers, M. N.; McLaren, L.; Keller, R. A. Science 1988, 162, 67-73. Jentoft, R. E.; Gouw, T. H. Anal. Chem. 1972, 4 4 , 681-686. Fjelsted, J. C.; Lee, M. L. Anal. Chem. 1984, 56, 619A-626A. Smith, R. D.; Udseth, H. R. Anal. Chem. 1984, 55, 2266-2272. Smith, R. D.; Udseth, H. R.; Kaiinoski, H. T. Anal. Chem. 1984, 5 6 , 2973-2974. Randall, L. G.; Wahrhaftig, A. L. Anal. Chem. 1978, 5 0 , 1703-1705. Otis, C. E.; Johnson, P. M. Rev. Sci. Instrum. 1980, 51, 1128-1129. McClelland, G. M.;Saenger, K. L.; Valentini, J. J.; Herschbach, D. R. J . Fhys. Chern. 1979, 83, 947-959. Lubman, D. M.; Rettner, C. T.; Zare, R . N. J . Phys. Chem. 1982, 86, 1129-1135. Smalley, R. E.; Wharton, L.; Levy, D. H. Acc. Chem. Res. 1977, IO, 139-145.
Chung Hang Sin Ho Ming Pang David M. Lubman* Department of Chemistry The University of Michigan Ann Arbor, Michigan 48109
Jens Zorn Department of Physics The University of Michigan Ann Arbor, Michigan 48109 RECEIVED for review July 29,1985. Accepted October 7,1985. We gratefully acknowledge financial support from a Cottrell Research Grant and the donors of the Petroleum Research Fund, administered by the American Chemical Society. We acknowledge support of this work under NSF Grant CHE 83-19383 and partial support from the Army Research Office under Grant DAAG 29-85-K-1005.
Photoacoustic Spectroscopy in Supercritical Fluids
'
Sir: Laser-induced calorimetric techniques of spectroscopic detection are based on the direct measurement of the optical energy absorbed rather than that transmitted by the sample. In absence of photochemistry, optical excitation of sample molecules leads to either fluorescence or nonradiative decay of excited states that deposit, respectively, some or all of the excitation energy into the surrounding solvent as heat. Methods of detecting this thermalized energy fall into two classes where the evolved heat is measured either as a pressure change in the case of photoacoustic spectroscopy (1-3) or as a refractive index change in the case of thermooptical absorption techniques (4-6). The sensitivity of these calorimetric methods depends not only on the incident optical power used for excitation but also on the thermophysical properties of the solvent in which the measurement is made. For most solvents, heating the volume of sample liquid results in a 0003-2700/86/0358-0490$0 1.50/0
decrease in density due to the positive volume thermal coefficient of expansion, 0 (7, 8). Supercritical fluids or dense gases are a class of solvents that are generating considerable interest and applications in analytical chemistry, as both chromatographic (9-12) and spectroscopic solvents (13-15). Recent studies using supercritical fluids as solvents for thermooptical measurements have shown a more than 100-fold sensitivity improvement, relative to thermal lens and photothermal deflection measurements in carbon tetrachloride (14,15). These techniques share with photoacoustic spectroscopy a dependence of signal on the thermal expansion coefficient, that is, the thermally induced change in volume or density of the solvent. The large volume expansion coefficient of supercritical fluids responsible for the outstanding improvements in sensitivity for thermal lens spectroscopy,therefore, holds the promise for similar increases 0 1986 American Chemical Society
ANALYTICAL CHEMISTRY, VOL. 58, NO. 2, FEBRUARY 1986 TO
491
TUBING FOR CIRCULATING THERMOSTAT FLUID
FLOW . _ _ ..
SYSTEM
PA TRANSDUCER
PREAMP TEFLON GASKETS
CONNECTOR BNC
SIGNAL AVERAGER
Figure 1. Block diagram of the instrumentation for photoacoustlc spectroscopy (PAS) In a supercrltlcal fluid: BS, beam splitter; PD, photodiode detector; FC, flow cell: PZT, piezoelectrictransducer: EN and EX, effluent entry and exit tubes, respectively;PM, power meter.
in sensitivity for photoacoustic spectroscopy. Photoacoustic detection employing a piezoelectric transducer for efficient coupling to condensed phase samples has been demonstrated by a number of workers (16, 17); the method was refiied by Patel and Tam (I,18)for the sensitive measurement of small absorptions in liquids. The theory of pulsed photoacoustic spectroscopy for measuring weak absorptions in condensed matter has been considered by Patel and Tam (7) and is reviewed briefly below. When light is absorbed by a liquid sample, heat is produced which causes the liquid to locally expand, assuming a positive thermal coefficient of expansion. A resulting pressure differential forms which reaches the pressure transducer and is registered as a voltage signal. At low sample absorbances, the signal normalized to incident laser pulse energy S is proportional to the optical absorptivity a, the thermal expansion coefficient /3, and the square root of the speed of sound in the solvent c and inversely proportional to the solvent specific heat C,
(8)
The proportionality constant K depends only on geometrical parameters of the sample cell and illuminating beam and the sensitivity of the transducer. The above relationship indicates that the sensitivity of the photoacoustic measurement depends on the choice of sample matrix or solvent; Le., liquids with larger 6, larger c, and lower C, are predicted to increase the sensitivity of the photoacoustic measurement (3). The analytical sensitivity of photoacoustic absorbance measurements might be expected to increase without bound when the solvent in which the sample is dissolved is near its critical point following the divergent behavior of the thermal expansion coefficient, p. In this work, the sensitivity of the photoacoustic effect is measured as a function of solvent. Data are presented comparing the relative sensitivities of measurements made in carbon tetrachloride, water, methanol, acetone, and carbon dioxide as a supercritical fluid. The concept of the sensitivity enhancement in supercritical fluids as recently developed for thermooptical methods (14,15) is further tested in this study for photoacoustic spectroscopy.
EXPERIMENTAL SECTION A block diagram of the instrument for photoacoustic spectroscopy in a supercritical fluid is shown in Figure 1. A flashlamp-pumped dye laser (Chromatix, Model CMX-4) was used as the excitation source for the photoacoustic measurements, pro-
u
u
c (
1cm
Figure 2. Front (left) and side cross-sectional (right) views of highpressure flow cell.
viding pulses at 20 Hz of 1-ps duration and pulse energy 1.3 mJ. Rhodamine 6G dye covered the spectral region from 576 to 630 nm and was operated at 613 nm for these experiments. The laser beam was focused with a collecting lens (f = 29.4 cm). The piezoelectric transducer (PZT)is of lead titanate-zirconate composition with 4 mm diameter and 4 mm thickness (LTZ-2, Transducer Products, Goshen, CT). The stainless steel casing and interior design are similar to that described by Tam and Patel (7) with outer threads so that efficient acoustic coupling to the flow cell body was possible. The amplitude of the transient ultrasonic signal generated by the PZT disk for each laser pulse was amplified by a wide-band preamplifier (LM310, LF357) with a gain of about 200 and band-pass of 400 Hz to 3.2 MHz. (Nine-volt batteries were used to power the preamp in order to avoid electrical pickup.) A beam splitter was used to direct a small fraction of the beam to a reference photodiode detector, which provided a trigger signal to the boxcar signal averager. The transmitted dye-laser pulse was detected by a power meter (Coherent, Model 210). A conventional cylinder of COz (U.S.Welding, 99% grade) was connected to a Varian 8500 syringe pump. Transfer of GOz to the pump through distillation from the cylinder was facilitated by cooling of the syringe below ambient temperature by a jacket of circulating tap water (TN 16 "C). The compressed gas was then converted into a supercritical fluid in a preheating coil at constant pressure. The fluid proceeded through a stainless steel sampling valve (Rheodyne, Model 7125) equipped with a 2O-w.L sample loop where the sample is introduced directly into a 10 cm X 4.6 cm i.d. column packed with 10 pm diameter ODS/silica (Brownlee Labs) and the analyte separated from the solvent in which it was originally dissolved. The effluent from the column was then passed through the flow cell for spectroscopic analysis. All measurements not utilizing COz as solvent were carried out under static conditions at room temperature and pressure, while those involving C02were carried out under flow conditions, employing the above apparatus, at 33 "C and 82 atm. Sample handling was carried out in the flow system in a manner that has been described elsewhere (11). Photoacoustic measurements were made in a specially designed high-pressure flow cell shown schematically in Figure 2. The flow cell was made of stainless steel and has an effective sample volume of about 30 p L and 1-cm path length with cell windows of 3.2 mm thick by 12.7 mm diameter cylindrical sapphire. Gaskets made of Teflon were fitted to both sides of each optical window and each window was secured within the cell with an appropriate nut. The PA transducer was interfaced securely to the body of the cell and separated from the sample volume by 3 mm of stainless steel. A BNC connector attaches a 12-cm coaxial cable from the transducer to the preamp. A constant-temperature bath of ethylene glycol was circulated steadily through predrilled passageways in the walls of the flow cell to maintain isothermal conditions. A closed-pore foam rubber material was used to thermally insulate the flow cell and to aid in attenuating any external acoustic interference of mechanical vibrations. Mass flow rate measurements were obtained with a soap-bubble flowmeter, using the known density of COz at ambient pressure and temperatures. N
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ANALYTICAL CHEMISTRY, VOL. 58, NO. 2, FEBRUARY 1986
Table 1. Solvent Sensitivity Comparison for Photoacoustic Spectroscopy solvent
1038, K-'
c, km s-I
C,, J g-' K-l
relative sensitivity predicted" measured
liquids water 0.257 1.50 4.18 0.054 f 0.004 0.1 f 0.056* methanol 1.20 1.10 2.54 0.355 f 0.003 0.4 f 0.1 acetone 1.46 1.17 2.15 0.526 f 0.005 0.6 f 0.1 carbon tetrachloride 1.23 0.93 0.85 1.00 1.0 supercritical fluid carbon dioxide (33 "C,82 atm) 49 0.26 2.27 8f2 7 f l OUsing eq 1 and data from ref 22-25 and 28-31. *Value corrected for the significant fluorescence quantum yield of methylene blue. Signal averaging was accomplished by a Nicolet 1170 signal averaging system with a Model 174,lO-MHz plug-in, which was used to integrate the sample absorbance across the chromatographic peak. The second positive excursion of the signal was usually chosen for processing, the actual time delay of which depended on the solvent. The results were subsequently recorded on either a Hewlett-Packard 7010B X-Y recorder or a Fisher Recordall Series 5000 chart recorder. Real-time monitoring of eluted peaks was accomplished with a Tektronix Model 3S2/3T2 sampling oscilloscope set at a fixed delay after the trigger pulse. The solvents used in the static samples were water (distilled, deionized), methanol (Fisher HPLC grade), acetone (Fisher ACS reagent grade) and carbon tetrachloride (Fisher ACS reagent grade). Absorbing solutes used were azulene (Aldrich), methylene blue (Matheson),and solvent green 3 (Aldrich). For injection into flowing COz,samples of azulene dissolved in methanol were most suitable. Molar absorptivities of the solutes at the laser wavelength were determined with a Cary 17D spectrophotometer. Solution concentration ranged from 1 ppm to 10 ppm.
RESULTS AND DISCUSSION In order to examine the sensitivity of photoacoustic detection in a supercritical fluid as compared to normal liquids, photoacoustic signals were measured as a function of solvent. To allow sufficient time delay to avoid electrical interference from the laser, the signal was digitized at the peak of the second acoustic pulse. Measurement sensitivity was determined by dividing the digitized signal by the respective sample absorbance. The sensitivities thus obtained for each solvent were divided by that obtained for CC14 to yield measured relative sensitivities with respect to that of CC4. The theoretical values for the relative sensitivity were calculated by using eq 1 and dividing by the value calculated for CClk Solutes for these measurements were chosen according to their respective solubilities. In the five solvents, solvent green 3 was used as the solute in carbon tetrachloride, acetone, and methanol, and methylene blue was chosen as the solute in water. While we have assumed solvent green 3 to be a nonfluorescing dye, based on the efficient nonradiative relaxation of azo chromophores (19),methylene blue, unfortunately, exhibits a near-unity fluorescence quantum yield (20). A photoacoustic signal much lower than that predicted by eq 1 would therefore be expected for methylene blue in water as the potential thermal energy is partially expended as fluorescence. T o remedy this, the thermal yield, &, of methylene blue was calculated by subtracting the average fluorescence photon energy of methylene blue from the excitation photon energy of the laser and dividing the result by the laser photon energy (21). The measured photoacoustic signals from methylene blue were corrected for fluorescence losses by dividing by the thermal yield, dt = 0.16 for the particular laser wavelength used, to produce a sensitivity value that can be compared to the response of nonfluorescent samples. The theoretical values of the photoacoustic sensitivity for the liquid solvents, relative to CC4, are calculated from thermophysical data in the literature (22-25) and compared
in Table I with the measured sensitivities. The results indicated excellent agreement between the observed signal amplitudes and those predicted by the solvent-dependent factors in eq 1. The same trend in the sensitivity ordering with liquid solvents is observed in photoacoustic spectroscopy as is found in thermooptical measurements such as the pulsed thermal lens method (26). The larger coefficient of thermal expansion and smaller heat capacity of nonpolar liquids produce greater sensitivity for both methods relative to polar liquids. The lower speed of sound in nonpolar liquids, however, limits the sensitivity increase over polar liquids with photoacoustic spectroscopy resulting in a smaller sensitivity range over the solvents tested compared to thermooptical methods. This limitation to the increase in photoacoustic sensitivity due to a lower speed of sound was also observed for measurements made in supercritical COz. Azulene was selected as the absorber in these measurements due its solubility in COZ and its negligible fluorescence quantum yield (27). As a result of the sparse thermophysical data available for COz under the particular conditions of this experiment (28-30), cubic spline interpolative techniques (31) were employed to estimate the speed of sound, thermal expansion coefficient, and heat capacity a t 33 OC and 82 atm to determine the predicted relative sensitivity with eq 1. Note that the conditions of the experiment are close to the critical point of COz, T,= 31.0 "C and P, = 72.8 atm. Performance of sample manipulation for supercritical fluids is illustrated by a chromatogram of a sample injection of 6.26 X 10" M azulene in methanol shown in Figure 3. The average retention time of azulene was 12.7 min, well separated from the methanol peak caused by refractive index gradients deflecting the beam into the detector cell walls. The flow rate of the syringe pump was set at 30 mL/h producing a COPmass flow rate of 2.7 g/s. The azulene peak was entirely eluted within approximately 2 min. A laser pulse rate of 20 Hz indicated 20 data points were to be collected per second. The signal averager was therefore set to collect 4096 photoacoustic transients over the azulene peak, corresponding to an integration time of 3.4 min. The average absorbance over the integration time was determined by multiplying absorbance of the injected sample by the ratio of the injection volume to the total volume of material flowing through the cell during the time of data collection. The photoacoustic sensitivity results for supercritical fluid conditions, listed in Table I, are in good agreement with the value relative to CC14 predicted by eq 1. The higher uncertainty for the predicted value for supercritical COz reflects the rapidly changing thermophysical parameters of the dense gas, which magnify any error in the measurement of temperature and pressure. The actual sensitivity gain of performing photoacoustic detection in a fluid near its critical point, compared with normal liquids, does not correspond to the nearly 2 orders of magnitude increase in the coefficient of thermal expansion which predominates the response of thermooptical absorption measurements (14,15).As observed
Anal. Chem. 1906, 58, 493-496
493
using entirely dielectric materials. These modifications could reduce the background noise level by more than an order of magnitude. Registry No. COz, 124-3&9; azulene, 275-51-4; methylene blue, 61-73-4; solvent green 3, 128-80-3.
LITERATURE CITED Patel, C. K. N.; Tam, A. C. Appl. Phys. Lett. 1979, 3 4 , 467-470. Oda, S.;Sawada, T. Anal. Chem. 1981, 5 3 , 471-474. Volgtman, E.; Jurgensen, A.; Wlnefordner, J. Anal. Chem. 1981, 5 3 , 1442- 1446. Hu, C.; Whinnery, J. R. Appl. Opt. 1973, 12, 72-79. Hordvik, A. Appl. Opt. 1977, 16, 2827-2833. Dovlchl, N. J.; Harrls, J. M. Anal. Chem. 1979, 5 1 , 728-731. Tam, A. C.; Patel, C. K. N. Appl. Opt. 1979, 18, 3348-3357. Patel, C. K. N.; Tam, A. C. Rev. M o d . Phys. 1981, 5 3 , 517-550. Kiesper, E.; Corwln, A. H.; Turner, D. A. J. Org. Chem. 1982, 2 7 , 700-701. Giddings, J. C.; Manwaring, W. A.; Myers, M. N. Science 1966, 154, 146-1 50. Novotny, M.; Sprlngston, S.R.; Peaden, P. A.; Fjeldsted, J. C.; Lee, M. L. Anal. Chem. 1981, 5 3 , 407A-414A. Gere, D. R. Science 1983, 222, 253-259. Shafer, K. H.; Griffiths, P. R. Anal. Chem. 1983, 5 5 , 1939-1942. Leach, R. A.; Harrls, J. M. Anal. Chem. 1984, 5 6 , 1481-1467. Leach, R. A.; Harris, J. M. Anal. Chem. 1984, 56, 2801-2805. Hordvik, A.; Schiossberg, H. Appl. Opt. 1977, 16, 101-107. Farrow, M. M.; Burnham, R. K.; Auzanneau, M.; Olsen, S.L.; Purdle, N.; Eyring, E. M. Appl. Opt. 1978, 17, 1093-1098. Tam, A. C.; Patel, C. K. N.; Kerl. R. J. Opt. Lett. 1979, 4 , 81-83. Parker, C. A. ”Photoluminescence of Solutlons”; Academic: New York, 1968. Melhulsh, W. H. NBS Spec. Publ. ( U S . ) 1973, No. 378, 137-150. Brannon, J. H.; Magde, D. J. Phys. Chem. 1078, 8 2 , 705-709. Harrison, D.; Moeiwyn-Hughes, E. A. Proc. R , SOC.London, A 1957,
Figure 3. Photoacoustic detection of azulene in supercritical C02. elution of 20-pL injected sample of azulene 6.26 X M (second peak) in methanol (first peak).
with the photoacoustic sensitivity comparison of nonpolar and polar liquids above, the decrease in the speed of sound in supercritical fluids moderates the effect of the expansion coefficient and limits the overall increase in sensitivity. Since the speed of sound and expansion coefficient both depend on the degree of intermolecular interaction in the fluid, they tend to change inversely to one another as the fluid is taken from liquid to dense gas conditions (28-30). As a result, the sensitivity of photoacoustic detection is not as strongly affected as thermooptical detection methods by the approach to the critical point of the solvent. The observed photoacoustic sensitivity in supercritical COz near its critical point is nearly an order of magnitude greater than CC14, the best liquid solvent tested. While this increase in sensitivity is not as large as that encountered with thermal lens measurements, the result could, nevertheless, provide a significant benefit for photoacoustic detection in supercritical fluid chromatography, for example. Flowing the sample for photoacoustic measurements does not reduce the sensitivity of detection as observed with the thermal lens method (32). Furthermore, the high frequency, band-pass filter on the preamplifier eliminates the low-frequency acoustic noise associated with the flowing sample. Although the experiments in this study were designed primarily to test the relative sensitivity of photoacoustic detection in supercritical fluids, they also provide an opportunity to evaluate noise contributions. The predominant source of background noise was found to be from scattered laser radiation striking the walls of the stainless steel flow cell. The small internal diameter of the flow cell precluded time resolving this background from sound generated within the solvent. Absorption detection limits in COz, estimated from the base line noise as in Figure 1, were not outstanding, Amin = 1.4 X lo4. Since the major source of stray light within the cell was reflections and scatter from the sapphire windows, one might improve on these results by using antireflectioncoated windows with a higher quality surface polish. Absorption of the stray light by the cell walls might also be reduced by coating the surfaces with a material that is more reflective than stainless steel or by construction of the cell
---. Bok, R. E., Tuve, G. L., Eds. “Handbook of Tables for Applied Engl239. 230. ---
neerlng Science”; CRC Press: Cleveland, OH, 1973; pp 88, 92. Weast, R. C.. Ed. “Handbook of Chemistry and Physics“, 57th ed.; 1977, CRC Press: Cleveland, OH, 1977; p E-47. Washburn, E. W., Ed. “International Critical Tables”; McGraw-Hili: New York, 1930; Vol. 3; pp 25-28. Morl, K.; Imasaka, T.; Ishlbashi, N. Anal. Chem. 1982, 5 4 , 2034-2038. Birks, J. B. “Photophysics of Aromatic Molecules”; Wlley: London, 1970, Chapter 5. Mlchaeis, A.; Hamers, J. Physlca (Amsterdam) 1937, 10, 995-1006. Mlchels, A.; Blaisse, B.;Michels, C. Proc. R . SOC.London, A 1937, 160, 358-375. Herget, C. M. J. Chem. Phys. 1940, 8 , 537-542. Burden, R. L.; Faires, J. D.; Reynolds, A. C. “Numerical Analysis”; 2nd ed.; Prlndle, Weber and Schmidt: Boston, MA, 1981; pp 107-123. Dovlchi, N. J.; Harris, J. M. Anal. Chem. 1081, 5 3 , 689-692.
‘
Present address: Corporate Research Sciences Laboratories, Exxon Research and Englneerlng Company, Clinton Township, Route 22 East, Annandale, NJ 08801. *Present address: E 3021126, Du Pont Experlmentai Station, Wilmington, DE 19898.
F. D. Hardcastle’ R. A. Leach2 J. M. Harris* Department of Chemistry University of Utah Salt Lake City, Utah 84112
RECEIVED for review August 16, 1985. Accepted October 7, 1985. This research was sponsored in part by the National Science Foundation under Grants CHE 82-06898 and CHE 85-06667 and by fellowship support to J.M.H. from the Alfred P. Sloan Foundation.
Intracellular Localization of Diffusible Elements in Frozen-Hydrated Biological Specimens with Ion Microscopy ,
Sir: Diffusible elements such as K, Na, and Ca play vital roles in biological processes, and their cytochemical localization 0003-2700/86/0358-0493$0 1.50/0
has become a major area of research. Ion microscopy, based on secondary ion mass spectrometry (SIMS), provides a 0 1986 American Chemical Society
