Anal. Chem. 1995,67, 3515-3519
Capillary Isoelectric Focusing-Electrospray Mass Spectrometry for Protein Analysis Qing Tang, A. Kame1 Hamata, and Gheng 8. Lee* Department of Chemistry and Ames Laboratoty, Iowa State University,Ames, Iowa 5001 1
On-line combinationof capiUary isoelectric focusing(CIEF) with electrospray mass spectrometry (ESMS)as a twodimensional separation system is demonstrated. Mixtures of model proteins including cytochrome c (horse heart), myoglobin (horse heart), and carbonic anhydrase I1 (bovine erythrocyte) are focused and cathodically mobilized in a polyacrylamide-coatedcapihy. At the end of CIEF capihy, the mobilized protein zones are analyzed by mass spectrometry coupled on-line to an electrospray interface with a coaxial sheath flow codguration. The effects of carrier ampholyte concentration on the CIEF separation and the protein electrospray ionization mass spectra are presented and discussed. In this study, the focusing effect of CIEF permits analysis of very dilute protein samples. A typical concentration factor of 50100 times is observed. "he concentration detection limit of myoglobin for a full-scanCIEF-ESMSanalysis is in the range of lo-' M, 2 orders of magnitude over that possible with normal capillary zone electrophoresis ESMS. The traditional biochemical approach to protein characterization has been the use of two-dimensional gel electrophoresis. All the sample proteins are separated first by charge and then by size in a two-dimensional gel.' The separation by charge is canied out by isoelectric focusing in a column filled with a pH gradient medium. The medium does not contain sodium dodecyl sulfate (SDS) and separates the native proteins according to their overall charge. The gel containing the charge-separated sample is then applied to the top of a flat gel containing SDS, and the denatured proteins are electrophoretically separated by molecular weight in a second dimension. When the proteins are radiolabeled, their positions in the gel can be detected by autoradiography. As many as several thousand different protein chains-virtually the total protein content of Escherichia coli-can be detected and separated by a two-dimensional gel electrophoresis. Despite the selectivity and sensitivity of two-dimensional gel electrophoresis,this technique as practiced today is the collection of manually intensive procedures. Casting of gels, application of samples, running of gels, and staining of gels are time-consuming tasks prone to irreproducibility and poor quantitative accuracy. The objective of this study is, therefore, tc combine the strengths of both capillary isoelectric focusing (CIEF)2-5in the ease and speed of separation and electrospray mass spectrometry (ESMS)6-9 (1) Creighton, T. E. Protein Structure: A Practical Approach; IFU Press: New York, 1990; Chapter 3. (2) Hjerten, S.; Zhu, M. D. ]. Chromatogr. 1985,346, 265-270. (3) Hjerten, S.; Liao, J. L.; Yao, J. ]. Chromatogr. 1987,387, 127-138. (4) Kilar, F.; Hjerten, S. Electrophoresis 1989,10, 23-29. (5) Mazzeo, J. R; Krull, I. S . Anal. Chem. 1991,63, 2852-2857. 0003-2700/95/0367-3515$9.00/0 0 1995 American Chemical Society
in the accuracy of mass determination. In analogy to a twodimensional separation system, CIEF separates proteins on the basis of their differences in isoelectric point @I). The fused silica capillary contains not only ampholytes for the creation of a pH gradient but also proteins. The proteins are focused into discrete and narrow zones with local pHs corresponding to their isoelectric points. ESMS as the second dimension allows the formation of multiplycharged, high molecular weight ions and the precise mass determination of &0.01% for proteins up to 30 kDa. The integration of CIEF with ESMS exhibits superior resolving power, speed, and sensitivity for protein characterization in biological and biomedical studies. Various instrumentation arrangements for interfacing capillary zone electrophoresis (CZE) and MS, including the use of metalized capillary terminus, sheath (coaxial) interface, liquid junction, and gold wiring, have been introduced and dem~nstrated.'~-'~ The purpose of any CZE-MS interface is to establish the electrical connection at the CZE capillary terminus, which serves to define the electric field along the CZE capillary. The electrical connection at the capillary terminus also serves as the electrospray source by having a 3-6kV difference in applied voltage between the terminus and the sampling aperture of the mass spectrometer. The comparison between sheath interface and liquid junction has been made by Pleasance et a1.,I6 and the coaxial sheath flow appeared to have several advantages. The coaxial sheath flow interface is therefore employed in this study to increase the stability of the electrospray process in CIEF-ESMS. Mass detection of proteins in CZE-MS is typically in the high femtomole range.13J7 When the concentration of the sample injected is considered, the detection level in the range of 10-5 M is frequently insufficient for various biological and biomedical studies. An obvious approach to improving CZE-MS detection limits is to perform sample stacking during the injection step'* (6) Yamashita, M.; Fenn, J. B. J. Phys. Chem. 1984,88, 4671-4675. (7) Fenn, J. B.; Mann, M; Meng, C. W.; Wong, S. F. Mass Spectrom. Reo. 1990, 9,37-70. (8) Ikonomou, M. G.; Blades, A T.; Kebarle, P. Anal. Chem. 1991,63,19891998. (9) Kebarie, P.; Tang, L.Anal. Chem. 1993,65, 972A-986A (10) Olivares, J. A; Nguyen, N. T.; Yonker, C. R; Smith, R D. Anal. Chem. 1987, 59,1230-1232. (11) Smith, R D.; Barinaga, C. J.; Udseth, H. R. Anal. Chem. 1988,60,19481952. (12) Lee, E. D.; Muck, W.; Henion, J. D.; Covey, T. R]. Chromatogr. 1988, 458, 313-321. (13) Smith, R D.; Wahl, J. H.; Goodlett, D. R; Hofstadler, S. A Anal. Chem. 1993,65, 574A-584A. (14) Wahl, J. H.; Gale, D. C.; Smith, R D.]. Chromatogr. 1994,659,217-222. (15) Fang, L.; Zhang, R; Williams, E. R; Zare, R N. Anal. Chem. 1994,66, 3696-3701. (16) Pleasance, S.;Thibault, P.; Kelly, J.], Chromatogr. 1992,591,325-339. (17) Thibault, P.; Paris, C.; Pleasance, S. Rapid Commun. M a s Spectrom. 1991, 5, 484-490.
Analytical Chemistry, Vol. 67, No. 79, October 7, 7995 3515
by dissolving the sample in water or low-concentrationbuffer. A particularlyuseful approach invoiving oncolumn isotachophoretic sample preconcentration has recently been reported by Tinke et and by Karger and co-workers.2°s21With the proper selection of running buffers and the oncolumn combination of capillary isotachophoresis (CITP) and CZE, the concentration detection limits for a full-scan CZE-MS analysis are decreased by a factor of 100, to -low7 M.*I In this study, the focusing effect of CIEF permits analysis of very dilute samples with a typical concentration factor of 50-100 times. It will be shown that CIEF-ESMS provides a concentration detection level competitive with that obtainable with CI"-CZE-MS but 2 orders of magnitude over that possible with normal CZE-MS. EXPERIMENTAL SECTION
Capillary Isoelectric Focusing: UV Measurement. The capillary isoelectric focusing apparatus was constructed in-house using a CZE lOOOR high-voltage 0power supply (Spellman High-Voltage Electronics, Plainview, NY). Fused silica capillaries with 50 pm i.d. and 192pm 0.d. (Polymicro Technologies,Phoenix, AZ) were coated internally with linear polyacrylamide for the elimination of electroosmotic flow: The proteins, including cytochrome c (horse heart), myoglobin (horse heart), and carbonic anhydrase I1 (bovine erythrocyte), were obtained from Sigma (St. Louis, MO). A 2km-long capillary was filled with a solution containing proteins and carrier ampholyte, pharmalyte 3-10 (Pharmacia, Uppsla, Sweden). Focusing was performed at a 1GkV constant voltage for 15 min with the use of 20 mM phosphoric acid and 20 mM sodium hydroxide as the anolyte and the catholyte, respectively. Cathodic mobilization was initiated by replacing the sodium hydroxide catholyte with a solution containing methanol/water/acetic acid in a volume ratio of 50: 49:l at pH 2.6. A constant voltage of 10 kV was applied during the mobilization. The protein zones were monitored by UV detection (Linear Instruments, Reno, NV) at 280 nm. The distance between the injection point and the UV detector was 14 cm. All chemicals, including phosphoric acid, sodium hydroxide, acetic acid, and methanol, were purchased from Fisher (Fair Lawn, NJ). All solutions were filtered through a 1-pm filter (Whatman, Maidstone, England). Mass Spectrometerand ElectrosprayInterface. The mass spectrometer was a Finnigan MAT TSQ 700 (San Jose, CA) triple quadrupole equipped with an electrospray ionization source. The Finnigan MAT electrospray adapter kit, containing both gas and liquid sheath tubes, was used to perform the direct infusion experiment and also to couple CIEF with ESMS without any modifications. The electrospray needle was maintained at a 5 kV for all direct infusion and CIEF-ESMS measurements. The first quadrupole was used for the mass scanning of protein ions, while the second and third quadrupoles were operated in the radio frequency only mode. The electron multiplier was set at 1.3 kV, with the conversion dynode at -15 kV. Tuning and calibration of the mass spectrometer were established by using an acetic acid solution (methanol/water/acetic acid, 5049:l v/v/v) containing myoglobin and a small pepetide of MRFA (18) Chien, R L.; Burgi, D. S. Anal. Chem. 1992,64, 489A-496A. (19)Tinke, A P.; Reinhoud, N. J.; Niessen, W. M. A; 'Ijaden, U. R; van der Greef, J. Rapid Commun. Mass Spectrom. 1992,6,560-563. (20) Foret, F.; Szoko, E.; Karger, B. L.]. Chromutogr. 1992,608,3-12. (21) Thompson, T. J.; Foret, F.; Vouros, P.; Karger, B. L. Anal. Chem. 1993, 65.900-906.
3516 Analytical Chemistry, Vol. 67, No. 19, October 1, 1995
How Injection and Capillary Isoelectric Focusing: ElectrosprayMass Spectrometry. The solution mixtures of protein and pharmalyte were infused at 5 pL/min in 50% methanol, 49% water, and 1%acetic acid (v/v/v) by using a Havard Apparatus 22 syringe pump (South Natick, MA). The nitrogen sheath gas was operated at a flow rate of 2 Wmin. The first quadrupole was scanned from m/z 200 to 2000 at a scan rate of 3 s/scan. For the combination of CIEF with ESMS, a 2km-long CIEF capillary was mounted within the electrospray probe. The outlet reservoir containing 20 mM sodium hydroxide as the catholyte was located inside the electrospray housing during the focusing step. The inlet reservoir containing 20 mM phosphoric acid as the anolyte was kept at the same height as the outlet reservoir. The capillary dimensions and applied focusing voltage were the same as in CIEF-UV measurements. Once the focusing was completed, the electric potential was turned off and the outlet reservoir removed. The capillary tip was fixed about 0.5 mm outside the electrospray needle. The sheath liquid consisted of 50% methanol, 49% water, and 1%acetic acid (v/v/v) and was delivered at a flow rate of 3 pL/min with use of a Havard Apparatus 22 syringe pump. During the mobilization step, two HV power supplies (Spellman) were used for delivering the electric potentials at the inlet electrode and at the electrospray needle. Because most HV power supplies are not designed to operate as current sinks, a resistor ladder in parallel with the HV electrode connecting with the electrospray needle was incorporated. A constant electric field of 500 V/cm was applied for the mobilization of focused proteins in the CIEF capillary. The first quadrupole was scanned from m/z 700 to 1800 at a scan rate of 3 s/scan. No sheath gas was employed during the CIEF-ESMSmeasurements. RESULTS AND DISCUSSION
In a focusing experiment, the capillary tubing contains not only carrier ampholytes but also proteins. The carrier ampholytes are small amphoteric molecules with different pZs. When an electric potential is applied, the negatively charged acidic ampholytes migrate toward the anode and decrease the pH at the anodic section, while the positively charged basic ampholytes migrate toward the cathode and increase the pH at the cathodic section. These pH changes will continue until each ampholyte species has come to its isoelectric point, where it will then become concentrated. Because each ampholyte has its own buffering capacity, a virtually continuous pH gradient will be formed in the capillary. Because the proteins are amphoteric macromolecules, protein analytes will also focus at their pZ values in narrow zones in the same way as the individual carrier ampholytes. To prevent the ampholytes from migrating into the inlet and outlet reservoirs by either diffusion or gradient drift? the 20 mM phosphoric acid and 20 mM sodium hydroxide are generally used as the anolyte and the catholyte, respectively. To investigate the effect of carrier ampholyte concentration on protein separation in CIEF, the polyacrylamidecoated capillary was filled with a solution containing pharmalyte 3-10, myoglobin, and carbonic anhydrase 11. The concentration of pharmalyte 3-10 was varied between 5%and 0.1%. The final protein concentration in the solution was 0.2 mg/mL for each protein analyte before the focusing. The focused protein zones were mobilized by replacing the sodium hydroxide catholyte with a solution containing methanol/water/acetic acid (5049:l v/v/v) at pH 2.6. The catholyte used in cathodic mobilization was the same as the sheath liquid later employed in the electrospray interface.
2
0
i
6
8 Time (minj
io
i2
14
Figure I. CIEF separation of myoglobin and carbonic anhydrase II in the presence of various pharmalyte concentration. Capillary, 20 cm total length, 50 pm i.d. and 192 pm 0.d.; length to detector, 14 cm; voltage, 10 kV for focusing and mobilization; UV detection at 280 nm. Concentrations: (A) 0.1% pharmalyte, (B) 0.5% pharmalyte, and (C) 5% pharmalyte. Table I.Effect of Pharmalyte Concentration on Protein Separation in Capillary isoelectric Focusing.
pharmalyte 310 concn (%)
separation resoln, myoglobin/carbonic anhydrase
0.1 0.5 1 2 5
3.8 9.8 10.0 10.4 12.5
peak heightb (%) carbonic myoglobin anhydrase 19.5 65.5 76.0 92.8 100
21.3 66.8 74.2 83.0 100
a The experimental error in measuring the values of separation resolution and peak height was about 4-8% for various am holyte concentrations for over five runs. * The peak heights of myoglo!in and carbonic anhydrase, measured by W absorbance at 280 nm, were set to be 100%in the presence of 5%ampholyte.
mass spectra of these electrosprayed ions are characterized by the distribution of peaks, where each component peak of the distribution corresponds to a different charge state of the intact protein. The shapes of these charge distributions are determined by different factors, including protein ~onformation,”~~5 solution pH,2‘jsolvent compo~ition?~ instrumentation conditions,28temperature,% solution ionic ~trength,3O,~l and the number of basic or acidic A strong correlation has been observed between the number of basic amino acid residues present in the protein and the distribution of charge states seen in the positive electrospray ionization spectrum. To combine CIEF with ESMS, the effect of carrier ampholyte concentration on the charge distribution of protein analytes in the electrospray ionization has to be determined. In the direct infusion experiments, three model proteins of cytochrome c, myoglobin, and carbonic anhydrase I1 were dissolved separately in the solution containing methanol/water/acetic acid (50:491 v/v/v) at pH 2.6. The positive electrospray ionization mass spectra of cytochrome c at various pharmalyte concentrations are shown in Figure 2. The average charge states and the ion counts of cytochrome c, myoglobin, and carbonic anhydrase I1 at various pharmalyte concentrations are summarized in Table 2 for comparison. The ions of pharmalyte 3-10 were observed in the low m/z range up to m/z 800. The presence of pharmalyte not only caused a marked reduction in the protein peak intensity but also resulted in a decrease of the net charge of protein ions in the mass spectra compared to the average charge state measured in the absence of carrier ampholyte. Protein peak intensities in the mass spectra decreased as the solution pharmalyte concentrationincreased. The ion intensity of cytochrome c in Figure 2C was amplified l@fold after the mass scan of pharmalyte ions. Thus, the pharmalyte ions similar to simple electrolyte ions led to higher solution conductivity and contributed to the establishment of the charge excess known to exist in droplets formed during the electrospray process. The suppression of protein ion intensity due to the addition of pharmalyte ions could be qualitatively accounted for by the theories of Tang and Kebarle33J4and Wang and Cole.30 Furthermore, the pharmalyte ions seemed to participate in the process of charge attachment to protein analytes. As demonstrated by Mirza and Chait,31certain anionic species in the electrospray solution could cause a marked decrease in the average charge state of peptide and protein ions in the mass spectra. This charge neutralization effect was found to depend solely on the nature of the anion species and was independent of the source of the anion. A charge reduction mechanism was proposed that involved as the first step anion pairing with a
The electropherogramsof CIEF separation under the influence of various pharmalyte concentrations are shown in Figure 1. The elution order was myoglobin (PI 7.2 and 6 4 , followed by carbonic anhydrase II (PI 5.9). The migration time of protein zones during the cathodic mobilization step increased with increasing pharmalyte concentration. A signjficant increase in band broadening of protein analytes was observed when the pharmalyte concentration was reduced to 0.1%. The quantitative dependence of pharmalyte concentration on the separation resolution and the peak height of focused proteins in CIEF is summarized in Table 1. In (23) Fenn, J. B. J. Am. SOC.Mass Spectrom. 1993,4, 524-535. comparison with protein separation at 5%pharmalyte, a 22%loss 214-217. (24) Katta, V.; Chait, B. T.Rapid Commun. Mass Spectrom. 1991,5, in the separation resolution and a 34% reduction in the W (25) Loo, J. A; Ogorzalek Loo, R R; Udseth, H. R; Edmonds, C. G.; Smith, R D.Rapid Commun. Mass Spectrom. 1991.5,101-105. absorbance intensity were measured in CIEF with 0.5%carrier (26) Chowdhury, S. IC; Katta, V.; Chait, B. T. /. Am. Chem. SOC. 1990,112, ampholyte. Significant deterioration in both the separation and 9012-9013. the peak intensity was observed when the pharmalyte concentra(27) Edmonds, C. G.; Loo, J. A; Barinaga, C. J.; Udseth, H. R; Smith, R D. J. Chromatogr. 1989,474, 21-39. tion was further reduced to 0.1%. Clearly, a 0.1% ampholyte (28) Ashton, D. S.; Beddel, C. R; Cooper, D. J.; Green, B. N.; Oliver, R W. A solution was insufficient to provide a smooth and continuous pH Ow. Mass Spectrom. 1993.28,579-583. gradient for CIEF protein separation. (29) Mirza, U.A; Cohen, S .;.I Chait, B. T. Anal. Chem. 1993,65, 1-6. (30) W a g , G.; Cole, R B. Anal. Chem. 1994,66,3702-3708. Electrospray ionization is a highly effective means for producing gas phase peptide and protein ions from the s o l ~ t i o n The . ~ ~ ~ ~ ~(31) Mirza, U.A; Chait, B. T. Anal. Chem. 1994,66,2898-2904. (22) Fenn, J. B.; Mann, M.; Meng, C. IC;Whitehouse, C. M. Science 1989,246, 64-71.
(32) Smith, R D.; Loo, J. A; Ogorzalek Loo, R R; Busman, M. Mass Spectrom. Rev. 1991,10, 359-451. (33) Tang, L.; Kebarle, P. Anal. Chem. 1991,63, 2709-2715. (34) Tang, L.; Kebarle, P. Anal. Chem. 1993,65, 3654-3668.
Analytical Chemistry, Vol. 67, No. 19, October 1, 1995
3517
I
I
Time (min)
I. 06 1.36
Figure 3. CIEF-ESMS reconstructed ion electropherogram of 0.1 mg/mL each of (1) cytochrome c, (2) myoglobin (p/7.2), (3) myoglobin (pl6.8),and (4) carbonic anhydrase II. Capillary, 20 cm total length, 50 pm i.d. and 192pm 0.d.; applied voltages, 10 kV for focusing and mobilization, 5 kV for electrospray; sheath liquid, methanol/water/ acetic acid (50:49:1 v/v/v) at pH 2.6, 3 pUmin.
0 b .i
1
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d
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2
0
, , , , . , , , . . j
oa
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.
.
,
,
,
.
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.
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.
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.
.
.
,
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,
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.
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,
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,
.
,
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Time (mm)
Figure 4. CIEF-ESMS reconstructedion electropherogram of 0.01 mg/mL each of (1) cytochrome c, (2) myoglobin (pl 7.2), and (3) carbonic anhydrase II. Other conditions are the same as in Figure 3.
mlz Flgure 2. Positive electrospray ionization mass spectra of cytochrome c obtained at different pharmalyte concentrations: (A) 0% pharmalyte, (B)0.5% pharmalyte, and (C) 2% pharmalyte. Cytochrome c concentration, 0.2 mg/ml. The solution mixture of cytochrome c and pharmalytewas infused at 5 pUmin in 50% methanol, 49% water, and 1% acetic acid (v/v/v). Table 2. Effect of Phannalyte Concentration on Average Charge State and Ion Count of Proteln Mass Spectra. phmdyte concn (%) 0 0.5 1 2 (I
average charge stateo CYT MY0 CAB
ion count ( ~ 1 0 ~ ) ~ CkT My0 CAB
13.0 11.0 11.0 11.0
47.5 11.1
18.0 15.0 15.0
14.5
26.5 23.0 22.5 21.0
5.2
1.1
74.2 23.1 10.8 2.1
8.86 5.12 0.07
0.01
CYT, cytochrome c; MYO, myoglobin; CAB, carbonic anhydrase
11. The ion counts were measured for cytochrome c, myoglobin, and carbonic anhydrase I1 at m/z 1124, 1304, and 1320, respectively.
positively charged basic amino acid of protein in the solution.The second step occurred during the process of desolvation or in the gas phase, where the ion pair dissociated to yield the neutral acid and the protein with reduced charge state. The different propensities for charge neutralization of the different anionic species were 3518 Analytical Chemistry, Vol. 67, No. 79, October 7, 1995
assumed to reflect the avidity of the anion-protein interaction. These findings from Mirza and Chait3I could be used to support the possible ion pair formation between the anionic moiety of carrier ampholyte and a positively charged basic amino acid of protein. The ion pair formation, in combination with the subsequent desolvation and dissociation processes, produced the charge neutralization effect and the observed shift in the charge distribution of protein mass spectra. Considering the effect of ampholyte concentration on the C E F separation and the protein electrospray ionization mass spectra, a solution containing 0.5%pharmalyte and standard proteins was used in the CIEF-ESMS measurements. The reconstructed ion electropherograms of the protein mixture with final concentrations of 0.1 and 0.01 mg/mL for each protein analyte are shown in Figures 3 and 4, respectively. The reconstructed ion electropherograms were obtained from the mass scan between m/z 700 and 1800 at a scan rate of 3 s/scan. All protein peaks were directly identified on the basis of mass spectra of protein analytes taken from the average scans under the peaks. There was no measurable protein mass spectrum by averaging the scans between peak 3 (myoglobin, PI 6.8) and peak 4 (carbonic anhydrase IT) in Figure 3. An example of the mass spectra obtained from the average scans under the peaks of Figure 4 is shown in Figure 5 for carbonic anhydrase I1 with a concentration of 0.01 mg/mL, or 3.45 x M. Initial preconcentration during the focusing step
1266..5
1450.6
mlz
Figure 5. Positive electrospray ionization mass spectrum of carbonic anhydrase II taken from the average scans under the peak in Figure 4.
was responsible for improving detection limit by 2 orders of magnitude in comparison with that obtained with normal CZE MS. In comparison with CIEF-UV experiments (see Figure l),the longer migration distance from the focused regions to the end of the capillary in CIEF-ESMS accounted for the increase in the migration time. However, the average velocity of myoglobin (8 cm/11.2 min = 0.71 cm/min) during the cathodic mobilization (35) Goodlett, D. R; Wahl, J. H.; Udseth, H. R; Smith,R D.J. Microcolumn Sep. 1993,5,57-62.
in CIEF-EMS was faster than that of myoglobin in CIEF-UV (2 cm/4.05 min = 0.49 cm/min). This difference in the average migration velocity contributed to a smaller Af of 1.6 min between the myoglobin and carbonic anhydrase II peaks in CIEF-ESMS than that of 2.2 min in CIEF-W. In CIEF-ESMS, the scan rate of MS might be insufficient to truly reflect the high separation efficiency of CIEF. The reduced elution speed method, as demonstrated by Goodlett et al.,35could be applied to circumvent the speed limitations of scanning mass spectrometry. It is known that selected ion monitoring (SIM) of quadrupole mass spectrometry yields signiscantly enhanced detection limits compared with those obtained with scanning MS operation because of the greater dwell time for signal acquisition at each selected m/z value. For samples with prior known analyte molecular weights and m/z values, SIM can further reduce the detection limits of CIEF-ESMS. ACKNOWLEDGMENT
Support for this work by the Microanalytical Instrumentation Center of the Institute for Physical Research and Technology at Iowa State University is gratefully acknowledged. C.S.L. is a National Science Foundation Young Investigator (BCS9258652). Received for review April 7, 1995. Accepted July 1 1 ,
1995.B AC950348Q @Abstractpublished in Advance ACS Abstracts, August 15, 1995.
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