Anal. Chem. 1996,67,385-389
Durable Gold=CoatedFused Silica Capillaries for Use in Electrospray Mass Spectrometry M. Scott Kriger and Kelsey D. Cook* Department of Chemistry, University of Tennessee, Knoxville, Tennessee 37996-7600 Roswitha S. Ramsey
Chemical and Analytical Sciences Division, Oak Ridge National Laboratory, Oak Ridge, Tennessee 37831-6365
A simple procedure for preparing gold-coatedsilica capillaries for use in electrosprayionization mass spectrometry is described. The tip of the capillary is mechanically tapered to a fine point, and a thin film of gold is vapor deposited on the outer surface following treatment with an organofunctional silane. The performance characteristics of these durable capillaries as continuous infusion sources are examined, and their utility in on-linecapillary electrophoresis mass spectrometry is demonstrated. The use of small diameter fused silica capillaries has proven advantageous in several electrospray ionization (ES) mass spectrometric @IS) application^.'-^ Improved sensitivity, reduced background, and dramatically improved limits of detection have been reported. When these insulating capillaries are used, electrical contact must be established and maintained for successful ES. This has been accomplished by several distinct Probably the simplest and most widely used approach involves use of a sheath solvent flowing through a larger, metallic capillary concentric with the silica. This allows maintenance of total flow rates on the order of 1-5 ,uL/min, compatible with the requirements of "conventional" stainless steel needle ES emitters. Disadvantages which have been cited for this design include reduced stability and sensitivity due to dilution and/or the addition of ionizable species in the sheath solvent (such species compete with the analyte for available charge in the ES process).5 Other problems may result from changes in solubility or molecular conformation when sheath fluids with high organic solvent content are used.' VanBerkel et aL9and Smith et al.5have reported successful ES with electrical contact made at a metal (1) Smith, R D.; Loo, J. A; Loo, R R 0.; Busman, M.; Udseth, H. R Mass Spectrom. Rev. 1991, 10, 359-451. (2) Goodlett, D. R; Wahl, J. H.; Udseth, H. R; Smith, R D.J. Microcolumn Sep. 1993, 5, 57-62. (3) (a) Wahl, J. H.; Goodlett, D. R; Udseth, H. R; Smith, R D.Electrophoresis 1993,14,448-457. (b) Wahl, J. H.; Goodlett, D.R; Udseth, H. R; Smith R D. Anal. Chem. 1992, 64, 3194-3195. (4) Emmett, M. R; Caprioli, R M. J. Am. SOC.Mass Spectrom. 1994, 5, 605613. (5) Gale, D. C.; Smith, R D. Rapid Commun. M a s Spectrom. 1993,7(11), 10171021. (6) Olivares, J. A; Nguyen, N. T.; Yonker, C. R; Smith, R D. Anal. Chem. 1987, 59, 1230-1232. (7)Smith, R D.; Barinaga, C. J.; Udseth, H. R Anal. Chem. 1988,60, 19481952. (8) Lee, E. D.; Muck, W.; Henion, J. D.;Covey, T.R J. Chromatogr. 1988, 458, 313-321. (9) Van Berkel, G. J.; McLuckey, S. A; Glish, G. L. Anal. Chem. 1992, 64, 1586- 1593. 0003-2700/95/0367-0385$9.00/0 0 1995 American Chemical Society
section remote from the silica spray tip. This configurationworks well for conventional electrospray but is not compatible with the requirements of capillary electrophoresis (CE) ,wherein electrical contact must be made at the exit end of the silica capillary. Zare et allohave reported inserting a small wire into the silica capillary; while this risks clogging small capillaries and may distort the electric field at the tip, it has provided a useful CE/ES interface. Smith et aL1' and Jorgensen et al.12 have reported results with silica capillaries with metalized tips. Remarkable limits of detection (LODs), low-flow capabilities (compatible with CE), and tolerance of a wide range of solvents have been reported? Capillary tips may be etched with hydrofluoric acid or drawn before coating to provide sharper edges and enhanced field focusing. This facilitates stable ES with highly conductive solutions (including aqueous solutions without organic cosolvent).I3 In attempting to adapt this technology to studies with electrohydrodynamic (EH) MS and CE/FS MS in our laboratories, we tested a wide range of metalization methods, including silver paint, Tollen's reagent (for deposition of silver), vapor deposition of gold (with and without a vapor-deposited chromium underlayer), and e-beam coating with gold. None of these methods provided durable coatings suitable for long-term use. Silver coatings suffered from poor chemical stability, which has been attributed in part to the electrochemical oxidation of the silver." The various gold coatings, although electrochemically stable, suffered from poor physical stability (they did not adhere well to the silica surface). Goss et al have described the preparation of robust goldcoated glass plates for use as electrodes.'* We now report on the adaptation of this methodology for preparing gold-coated silica capillaries for ES. We describe as well a simple procedure for shaping the capillary tip to provide the strong fields needed to electrospray purely aqueous solutions without discharge. The capillaries have been evaluated for CE/MS and continuous infusion ES MS using an ion trap mass spectrometer (ITMS). (10) Fang, L.; Zhang, R; Zare, R N. Proceedings of the 41st ASMS Conference on Mass Spectrometry and Allied Topics; San Francisco, CA, May 1993; p 755A (11) Smith, R D.; Wahl, J. H.; Goodlett, D. R; Hofstadler, S. A Anal. Chem. 1993, 65, 574A-584A. (12) Dohmeier, D. M.; Austell, T. L.; Jorgenson, J. W. Fourth Intemational Conference on High Performance Capillary Electrophoresis, Amsterdam, The Netherlands, February 1992. (13) Chowdhury, S. K; Chait, B. T.Anal. Chem. 1991, 63, 1660-1664. (14) Goss, C. A; Charych, D. H.; Majda, M. Anal. Chem. 1 9 9 1 , 63, 85-88.
Analytical Chemistry, Vol. 67, No. 2, January 15, 1995 385
Figure 1. Electron micrograph (40x magnification) showing shaped gold-coated silica capillary tip. The capillary outer diameter is 365 pm, and the inner diameter is 25 pm. The diameter at the tip is estimated to be 50 pm.
EXPERIMENTAL SECTION
CapillaT Coating Procedure (developed at the University of Tennessee and under patent consideration by the University of Tennessee Research Corp.). A polyimidecoated fused silica capillary (10-50 pm i.d. x 365 pm 0.d. x 12-60 cm long; Polymicro Technologies, Inc., Phoenix, AZ) was shaped by gently applying pressure to the capillary tip with fine grit sandpaper (600 W grit; Sandpaper Inc., Rockland, MA) while the capillary was spun in a benchtop minilathe (Unimat-S1Model DB200, American Edestaal, NY). This treatment proceeded for about 10 min, until a profile like that shown in the electron micrograph of Figure 1 was achieved (obtained using an ETEC Autoscan (Los Angeles, CA) electron microscope). The capillary was removed from the lathe, and ca. 1cm of polyimide coating was burned off (starting at the shaped end). The capillary was then ultrasonically cleaned (Sonicor Model G1080/ET-l/PGl, Farmingdale, NY) in acetone for about 1 min. The capillary was next flushed with several capillary volumes of methanol using a syringe connected to the untreated end of the capillary with a 1/16 in. Swagelock (Solon, OH) union with Teflon ferrules. The shaped, uncoated end of the capillary was then cleaned by immersion for 20 min at 70 "C in "piranha solution" (1:4 mixture of 30%H202 and concentrated sulfuric acid). The capillary was then rinsed with several capillary volumes of deionized distilled water (Milli-Q Deionizer, Millipore Corp., Bedford, MA; minimum resistance, 15 MQ/cm), blown dry with argon gas, and then dried in an oven at 105 "C for 10 min. After being cleaned, ca. 1 cm of the capillary (shaped end) was immersed in a boiling coating solution (vessel equipped with a reflux condenser) for 10 min. The coating solution was comprised of 5 g of (3-mercaptopropy1)trimethoxysilane (95% Aldrich, Milwaukee, WI) dissolved in 200 g of 2-propanol and 5 mL of deionized distilled water. The reacted capillary (outer surface) was then rinsed with 2-propanol and cured in an oven at 105 "C for 10 min. The coating process (immersion, rinse, cure) was repeated two additional times. 386 Analytical Chemistry, Vol. 67,No. 2, January 15, 1995
Finally, the capillary was coiled into a ca. 6 cm 0.d. circle, and all but the reacted tip was covered with aluminum foil. The coil was mounted on a rotator within a Denton (Cheny Hill, NJ) Model DV-515 bell jar evaporator. Followingevacuationto 6 x Torr, the capillary was gold coated by vapor deposition using a flash power of 70 W while rotating at approximately 30 rpm to ensure uniform coating of the exposed surface. The coated capillary was inspected with a 3x microscope to confirm complete coverage. Instrumentation. For ES applications,the metal-tipped silica capillary was placed within a platinum capillary (394 pm i.d. x 711 pm o.d.), with the coated tip projecting roughly 1cm beyond the end of the R A small quantity of silver paint (Micro-Circuits Co., New Buffalo, MI) was used to ensure good electrical contact between the two capillaries. High voltage (+1.5-4.5 kv) from a Universal Voltronics (Mount Kisco, W, Model BAP 161.5) power supply was applied to the platinum tube using an alligator clip. In some comparison experiments, 27 gauge (150 pm i.d. x 520 pm 0.d.) or 32 gauge (100 pm i.d. x 228 pm 0.d.) dometipped stainless steel needles (Hamilton, Reno, NV) were used in place of the coated silica/Pt combination. A nebulizer system was also used for comparisons. It was comprised of a 32 gauge needle concentric within a 22 gauge needle (394 pm i.d. x 711 pm o.d.), with nitrogen passing through the annulus. In direct infusion experiments, solution was supplied to the ES emitter using a Haxvard Apparatus (South Natick, MA) Model 11or 22 syringe pump with a gas-tight Hamilton syringe connected to the capillary using a 1/16 in. zero dead volume union (VICI, Houston, Tx). Flow rates ranged from 0.05 pL/min for the goldtipped capillaries to 1pL/min for the stainless steel needles. The CE apparatus consisted of a Spellman (Plainview, NY) CZE lOOOR high-voltage power supply, safety interlock box, and an ISCO (Lincoln, NE) S500 W absorbance detector. Separations were performed using a 60 cm long, 50 pm i.d. x 365 pm 0.d. gold-tipped fused silica capillary. The coated terminus was maintained at a potential of +2-3 kV to produce the electrospray and provide the bias necessary to drive the electrophoresis. The buffer reservoir was maintained at -22 kV. The inside of the column was coated with (3aminopropyl)triethoxysilane (Pierce, Rockford, IL) according to the method of Moseley et aI.l5 The CE mobile phase was 10 mM acetic acid buffer at pH 3.4. All injections were made electrokinetically from samples in aqueous solution. To characterize capillary durability and spray versatility, a benchtop apparatus was assembled. The emitter was positioned 1.5-2.0 cm from and perpendicular to a 3.5 cm x 3.5 cm x 1mm thick stainless steel collector plate, which served as the counter electrode. This plate was grounded through a Keithley (Cleve land, OH) Model 600A electrometer, thus providing a measure of the electrospray arrent. TNO measures of spray quality were assessed. With the room light dimmed, a good quality back-lit spray took on the appearance of a steady coneshaped mist that disappeared due to evaporation prior to striking the collector plate. A steady collector current (ca. 1pA) observed with the electrometer provided a second indication of stable electrospray. Mass spectrometric experiments were performed using a Finnigan-MAT ITMS (San Jose, CA) modfied for ES.16 Comparisons between various sources were made using identical data (15) Moseley, M. k, Jorgenson, J. W.; Shabanowitz,J.; Hunt, D. F.; Tomer, K B. J. Am. SOC.Mass Spectrom. 1992.3.289-300. (16) Van Berkel, G. J.; Glish. G. L; Mchckey. S. A Anal. Chem. 1990. 62, 1284- 1295.
Table I. Solutions Used To Test the Chemical Stablllty of Capillary Coatings’
solution*
stable
water methanol ethanol propanol acetic acid buffer(l0 m M pH = 3.5) concentrated HCl(l2.1 M) aqua regia piranha solution ammonium acetate buffer (10 mM;pH = 7) NaHzPOd(10 mM; pH = 4.5) boric acid (10 m M pH = 4) potassium hydroxide (1 M)
xxx xxx xxx xxx xxx xxx xxx xxx xxx xxx
unstable
xxx xxx
“Stabilitybased on visual inspection with a microscope. The coated capillary terminus was immersed in the solution and sonicated for 1 min. bAll solutions were aqueous, except for the alcohols.
acquisition parameters. Ions were injected for periods ranging from 10 to 500 ms and then subjected to a “heating” ramp to promote des01vation.l~Resonance ejection was used in all cases to extend the m/z range by factors up to 5 beyond the normal limit (Le., m / z 650) for the trap. The ES emitter was positioned 0.5-2.5 cm away from the front aperture plate to maximize signals. Materials. Except as noted, all reagents were analytical reagent grade from Mallinckrodt (St. Louis, MO) and used as received. Also used as received were 95%ethanol (Midwestern Grain, Weston, MO), cytochrome c m e V, bovine heart), monobasic sodium phosphate, and leucine enkephalin (Sigma, St. Louis, MO). Cytochrome C and leucine enkephalin solutions were prepared using HPLC grade solvents 0. T. Baker, Phillipsburg, NJ) and were filtered through 0.22 pm poly(vinylidene fluoride) filters prior to use (Alltech Associates, Inc., Deerfield, IL). Safety Considerations. Aqua regia, piranha solution, hydrochloric acid, and the potassium hydroxide solution are all corrosive and should be handled with care. Piranha solution is a strong oxidant and should be treated by addition to dilute sodium iodide, neutralization with base, and treatment with bisulfite (to reduce 12) prior to disposal. Preparation of piranha solution is highly exothermic; it was normally prepared in volumes of 1 mL. The aqua regia, hydrochloric acid, and potassium hydroxide solutions may be neutralized prior to normal acid/base disposal. (3Mercaptopropy1)trimethoxysilaneis a combustible liquid irritant with a strong stench. It should be handled within a fume hood and may be disposed of with other combustible organic materials. Normal care should be taken when using the high voltages necessary for capillary electrophoresis and electrospray. RESULTS AND DISCUSSION
CapillaryDurability. In a preliminary test of coating stability, treated capillaries were sonicated for 1min in a variety of solvent systems (see Table 1). Microscopic inspection (3x amplification) after sonication revealed perceptible coating deterioration only by aqua regia and piranha solution. In a second test, physical adhesion was assessed by application and removal of a piece of adhesive cellophane tape. As reported by Goss et al.,l4 gold deposited on untreated silica was easily removed by adhesive tape, whereas there was no perceptible deterioration or removal of the (17) McLuckey, S. A; Glish, G. L.;Van Berkel, G. J. Anal. Chem. 1991, 63, 1971-1978.
gold layer from a capillary reacted with (3mercaptopropyl)trimethoxysilane prior to gold coating, as outlined above. The coating longevity under normal electrospray conditions was also evaluated. A 10 mM solution of acetic acid in water (PH = 3.50) was sprayed at 0.2 pL/min from a single 25 pm i.d. coated capillary for more than 100 h. This was accomplished in 10 sessions of approximately 10 h each. The ES onset voltage at the beginning of a session varied (randomly) from 2.7 to 3.3 kV. The voltage was maintained just above the onset in each session, resulting in an average of 3.1 f 0.1 kV (uncertainties here and subsequently reflect the standard deviation of the mean). The collector current was recorded at 5 h intervals, averaging 61 f 17 nA The rather large uncertainty in current (28% relative standard deviation) reflects slow, random drift (between 40 and 100 nA), rather than short-terminstability withii a single session. We estimate that over a period of 10 min, the collector current was stable to within f 2 nk However, at the conclusion of 100 h of testing, currents became erratic, and the setup was disassembled. In removing the capillary from its platinum support, the gold coating was inadvertently scraped off by abrasion, tewinating further evaluation of this capillary. It was subsequently determined that a faulty connection at the grounded counter electrode had caused the instability. The 100 h benchmark has been surpassed in a second longevity experiment currently underway. During this second experiment, onset voltages have varied from 3.1 to 3.5 kV (average 3.3 f 0.10 kV), and collector currents have ranged from 160 to 210 nA (average 180 f 20 nA). Stability is similar, but the values are higher than those with the first emitter, probably reflecting emitter-toemitter variation (current changes due to deliberate variations within the range of uncertainty in solution concentrations and capillary positioning using a single capillary were at least an order of magnitude less). We estimate that other capillaries used in untimed MS applications (see below) have approached the 100 h benchmark. We thus conclude that the gold coating is stable for periods in excess of 100 h of ES. In general, the capillaries have remained useful until they have been accidentally scratched through rough handling. Spray Versatility. Plots of collector current as a function of needle potential using a 25 pm i.d. capillary (Figure 2) illustrate the distinct conditions necessary to sustain stable electrospray (plateaus below 1jiA) and the voltages that give rise to a discharge (3-5 kV) in various solvent systems. As expected? the discharge onset voltage for the tapered silica capillary is lower than that observed with a blunt stainless steel needle (with correspondingly higher flow). Nevertheless, there is in all cases a wide range of voltages where a stable spray was observed without a discharge. A stable spray was obtained with a wide variety of solutions (Table 2), including purely aqueous (no cosolvent) solutions without a discharge suppressor. Finally, it is noteworthy that the data presented in Figure 2 were acquired using a single gold-coated capillary. The coating survived multiple discharges (evident from collector currents greater than 1PA), indicative of its durability. Mass Spectrometric Applications. The use of the goldtipped capillaries for ES MS was initially evaluated by continuously infusing cytochrome c in aqueous solution containing 1%(v/v) acetic acid or in 75%methano1/24%water/l% acetic acid (v/v/v). Spectra were averaged over a 9 s period (ca. 33 scans) to simulate the peak width that we obtain for this compound by CE (using Analytical Chemistry, Vol. 67, No. 2, January 15, 1995
387
100% = 18600
6
I
?
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508
Needle Voltage (kV) Figure 2. Current-voltage curves obtained for electrospraying through a 25 pm i.d. gold-coated capillary (0,0.2 pUmin) or a 150 pm i.d. stainless steel needle (A, 1 pUmin). Solutions sprayed were (a) pure methanol; (b) 50:49:1 methanoVwater/acetic acid (v/v/v); and (c) pure water. In each case, the current plateau is indicative of stable electrospray, and the subsequent sharp increase in current indicates the onset of a discharge. Data for the stainless steel needle are offset by +1 pA for clarity. Table 2. Solutions Used To Test EiectrorprayStability with 25 pm i.d. Coated Capillaries
solution" HPLC water methanol acetic acid methanol/water/ acetic acid ammonium acetate NaHzP04 NazHP04 Nd&P04 boric acid tris-phosphate
concentration neat neat 10 mM 5049:l (v/v/v) 100 mM 10 mM 10 mM 50 mM 100 mM 75mM
onset voltage (kV) 2.8 1.8 2.9 2.4
3.1 3.0 3.0 3.0 2.8 3.1
collector current (nA) 50
30 70 300 750 300 500 250 500 1000
4 1 solutions were aqueous, except that labeled methanol.
an acetic acid buffer and a 50 pm i.d. column treated with (3aminopropyltriethoxysilane). F w e 3 compares the data obtained from the analyte at 21 pM concentration in the organic solvent solution using a 32 gauge needle (typical for infusion experiments in our laboratory) and using a 10 pm gold-tipped capillary. The flow rates indicated in the iigure captions were those that provided the greatest electrospray stability (Le., the least amount of variation in total ion current from scan to scan). The spectra are similar, with multiply charged molecular ions ranging from the 11+to the 17+species, indicating that the protein is in a denatured state, as 388 Analytical Chemistty, Vol. 67, No. 2, January 15, 1995
,
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i~ee
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,
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Figure 3. Mass spectra of 21 pM cytochrome c in 75% methanol/ 24% water11YOacetic acid (v/v/v) using (a) a 32 gauge stainless steel needle and a flow rate of 1 pUmin (3.2 pmol of analyte were consumed during the 9 s data averaging period) and (b) a 10 j i m i.d. gold-tipped capillary and a flow rate of 0.1 pUmin (0.32 pmol of analyte consumed). Numbers at upper right compare absolute intensities.
would be expected for solutions with a high methanol content.' The total amount of sample consumed using the 10 pm capillary was an order of magnitude lower than for the 32 gauge needle, while the intensity of the base peak was ca. 2.9 times greater. Such sensitivity enhancement derived from use of small inside diameter capillaries has been observed elsewhere and has been attributed to increased ionization efficiency (resulting from the formation of smaller droplets) and a decrease in mass flow (with a corresponding reduction in background).3 In electrospray experiments using an aqueous protein solution and a 25 pm gold-tipped capillary (Figure 4a), only three charge states were apparent, with the 8+ ion forming the base peak. Differences in the spectrum from those shown in Figure 3 may be related to the conformation of the protein under different solvent conditions.' Stable electrospray for the aqueous solution using the 32 gauge needle could only be obtained using a sheath flow of nitrogen gas to assist the nebulization process. Signal levels were found to be ca. 6 times higher, and sample consump tion was an order of magnitude lower for the gold-tipped capillary (Le., an overall 6@fold enhancement in the signal/sample ratio; Figure 4). For continuous infusion of 0.17 pM cytochrome c in acidfied aqueous solution, a detectable signal (with a S/N = 2) could be obtained from single scans using a 50 ms ion injection time into the ion trap, corresponding to 7 amol of sample consumed (Figure 5). CE/ES/MS. Figure 6a shows the total ion electropherograms obtained for aqueous solutions of leucine enkephalin varying in concentration from 9 to 45 pM. Quantities injected ranged from 710 to 17 fmol; a 50 pm i.d. gold-tipped column was employed. The S/N in the mass spectrum for the lowest quantity analyzed (Figure 6b) indicates that the full scan detection limit is ca. 10
(M+s~s+
100% = 21950
lee!
I
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4 59
5 59
6 59
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4 59
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6 58
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2588
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mlz Figure 4. Mass spectra obtained from a 21 pM aqueous solution of cytochrome c using (a) a 25 pm i.d. gold-tipped capillary (infusion rate 0.1 pllmin) and (b) a 32 gauge stainless steel needle with nitrogen nebulization (infusion rate, 1.OpUmin).
'7
200
400
m/z
see
see
Figure 6. (a) Total ion current electropherograms for multiple injectionsof leucine enkephalin on a 50 pm i.d. gold-tipped capillary. (b) Mass spectrum from a 17 fmol injection of leucine enkephalin.
(M+8H)"
i
columns have been shown to be useful in ES MS and CE/ES MS, providing interfaces that are quite durable. They are capable of withstanding multiple discharges and have lifetimes that exceed 100 h of operation. ACKNOWLEDGMENT I/ I ill I I'"""''l """"'l""""""""'I" 110 1500 2888 2588 I
500
3880
mlz
Figure 5. Single scan (50 ms injection time) mass spectrum of cytochrome c obtained from a 0.17 pM aqueous solution. The spectrum was acquired under continuous infusion conditions at a flow rate of 0.05 pUmin using a 25 pm i.d. gold-tipped capillary.
fmol. Similar values have been .reported for CE/ITMS using a 50 pm i.d. column and a liquid sheath interface.'* Our column efficiency (theoretical plate number), however, was a.3-5 times lower, suggestingthat a better limit of detection could be obtained by optimizing chromatographic conditions. We also anticipate that use of smaller inside diameter columns will enhance the sensitivity in CE/ES, as it did in the continuous infusion experiments. CONCLUSIONS
Gold-tipped capillaries prepared using an organofunctional silane to improve the adhesion of the gold film to the silica
Capillary development was supported in part by the National Science Foundation (Grant CHE-8822787 to the University of Tennessee). ES/MS and CE/ES/MS studies were supported in part by the National Institutes of Health under Grant GM45372 (to Martin Marietta Energy Systems). Oak Ridge National Laboratory is managed for the US. Department of Energy by Martin Marietta Energy Systems, Inc., under Contract DEAC840R21400. The assistance of Mr. Danny Totten in capillary preparation is greatfully acknowledged. K.D.C. and M.S.K. wish to thank Mr. Richard Williams of the UTK Life Sciences Electron Microscopy Facility for use of the vacuum deposition apparatus and for the electron micrograph of the capillary. M.S.K. wishes to thank the UTK Scholarly Activity Research Incentive Fund (SARIF) for fellowship support. Received for review July 15, 1994. Accepted October 27, 1994.8 AC940709M
(18) Schwartz, J. C.; Jardine, I. Proceedings ofthe 40th ASMS Conference on Mass Spectrometry and Allied Topics; Washington, DC, May 1992; p 707.
@Abstractpublished in Advance ACS Abstracts, December 1, 1994.
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