Anal. Chem. 1995,67,1159-1163
Trapping, Detection, and Mass Determination of Coliphage T4 DNA Ions of I O 8 Da by Electrospray Ionization Fourier Transform Ion Cyclotron Resonance Mass Spectrometry Ruidan Chen, Xueheng Cheng, Dale W. Mitchell, Steven A. Hofstadler, Qinyuan Wu, Alan L. Rockwood, Michael G. Sherman, and Richard D. Smith* Chemical Sciences Department and Environmental and Molecular Science Laboratory, Pacific Northwest Laboratory, P.0. Box 999, MS P8- 19, Richland, Washington 99352
The trapping, detection, and mass measurement of individual T4 DNA ions (nominal molecular weight, M,= 1.1 x lo8 Da) have been performed with a 7 T electrospray ionization Fourier transform ion cyclotron resonance (ESIFI'ICR) mass spectrometer. The ionic mass was obtained by direct measurement of the number of charges carried by individual T4 DNA ions. The ions detected are a factor of -20 larger than any molecule previously studied by mass spectrometry. These gas-phase macroions demonstrate a high degree of stability, which allows the acquisition of long transients (2476 s) from individual ions and the study of relatively slow reactions or dissociation processes. These results show the potential utility of ESIFI'ICR mass spectrometry for the analysis of very large biomolecules and microparticles and indicate that it is possible to transfer ions of 110 MDa size to the gas phase substantially intact. The potential applications of the FI'ICR analysis of such highly charged ions are briefly discussed. As an analytical tool, Fourier transform ion cyclotron resonance (FTICR) mass spectrometry has extraordinary advantages over conventional mass spectrometric methods, including unparalleled mass measurement resolution and accuracy, compatibility with a variety of external ionization techniques, and the ability to store, manipulate, and remeasure trapped ions (e.g., MY, where n 2 2) .1-7 However, only recently, with its couplings to electrospray ionization (ESI)8-14 and matrix-assisted laser desorption/ionization (MALDI) methods,15-17 have the high-performance capabili(1) Marshall, A G.; Grosshans, P. B. Anal. Chem. 1991,63,215A-229A. (2) Asamoto, B.; Dunbar, R C. In Analytical Applications of Fourier Transform Ion Cyclotron Resonance Mass Spectrometly; Asomoto, B., Ed.; VCH: New York, 1991; p 306. (3) Buchanan, M. V.; Hettich, R L. Anal. Chem. 1993,65, 245A-259A (4) Jacoby, C. B.; Holliman, C. L.; Gross, M. L. In Mass Spectrometly in the Biological Sciences: A Tutorial: Gross, M. L., Ed.; Wiley: New York, 1992; pp 93-116. (5) Koster, C.: Kahr, M. S.; Castoro, J. A ; Wilkins, C. L. Mass Spectrom. Reo. 1992,11, 495-512. (6) Marshall, A G.; Schweikhard, L. Int. J Mass Spectrom. Ion Processes 1992, 118/119, 37-70. (7) Marshall, A G.; Verdun, F. R Fourier Transforms in NMR, Optical, and Mass Spectromety: A User's Handbook; Elsevier: Amsterdam, 1990; p 460. (8) Whitehouse, C. M.: Dreyer, R N.; Yamashita, M.; Fenn, J. B. Anal. Chem. 1985,57,675-679. (9) Yamashita, M.; Fenn, J. B. J Phys. Chem. 1984,88, 2240-2249. (10) Wong, S. F.; Mann, M.; Fenn, J. B. J Phys. Chem. 1988,92,546-550. 0003-2700/95/0367-1159$9.00/0 0 1995 American Chemical Society
ties of FTICR been applied successfully to the analysis of biomolecules with molecular masses exceeding a few thousand daltons. A limiting feature of MALDI for larger molecules is that only low charge state ions with high m/z are generally formed, an experimental regime in which the FTICR performance is less favorable due to the inverse relationship between m/z and cyclotron frequency (and, hence, obtainable resolution) .7 In contrast, the multiple charging phenomenon inherent in the ESI process increases spectral resolution by shifting the molecular ions to the low m/z regime where ultra-high-resolution measurements are routine for FTICR mass spectrometry. Observations of very large charged particles, Le., aluminum particles in 1959,18have been reported previously using trapping methods. However, the mass and number of net charges for each particle could not be accurately determined. The trapping and detection of very large and highly charged synthetic polymer ions (poly(ethy1ene glycol) of up to 5 x lo6 Da) by ESI-FTICR mass spectrometry in the 2000-3000 m/z range have been recently r e p ~ r t e d . ' ~ In - ~those ~ initial studies, the observation of individual (i.e., single) ions was confirmed and precise molecular weight measurements were facilitated by time-resolved ion correlation (TRIC), in which individual ions were monitored during transient acquisition and observed to undergo stepwise shifts in m/z due to charge exchange reactions.20 These measurements yielded the molecular weight determinations of individual ions with high precision. An independent and complimentary method for obtaining molecular weight information directly from a single m/z measurement is by direct and simultaneous measurement of the m/z value Henry, K D.; Williams, E. R; Wang, B.-H.; McLafferty, F. W.; Shabanowitz, J.; Hunt, D. F. Proc. Natl. Acad. Sci. U.S.A. 1989,86,9075-9078. Henry, K D.; Quinn, J. P.; Mclafferty, F. W.J Am. Chem. SOC.1991,113, 5447-5449. Winger, B. E.; Hofstadler, S. A ; Bruce, J. E.; Udseth, H. R; Smith, R D.]. Am. SOC.Mass Spectrom. 1993,4, 566-577. Smith, R D.; Loo, J. A.; Edmonds, C. G.: Barinaga, C. J.; Udseth, H. R Anal. Chem. 1990,62,882-899. Karas, M.;Hillenkamp, F. Anal. Chem. 1988,60,2299-2301. Castoro, J. A ; Wilkins, C. L. Anal. Chem. 1993,65, 2621-2627. Solouki, T.; Gillig, K J.; Russell, D. H. Anal. Chem. 1994,66, 1583-1587. Wuerker, R F.; Shelton, H.; Langmuir, R. V./. Appl. Phys. 1959,30, 342. Smith, R D.; Cheng, X.; Bruce, J. B.; Hofstadler, S. A: Anderson, G. A. Nature 1994,369,137-139. Bruce, J. E.; Cheng, X.; Bakhtiar, R; Wu, Q.;Van Orden, S.; Anderson, G. A.; Smith, R D. /. Am. Chem. SOC.1994,116,7839-7847. Cheng, X.; Bakhtiar, R; Van Orden, S.; Smith, R. D. Anal. Chem. 1994, 66, 2084-2087.
Analytical Chemistv, Vol. 67, No. 7, April 1, 1995 1159
and charge number, z. Initial results have been reported using this approach based upon the excitation and ejection behavior of individual where we also described the theoretical basis of such experiments. In theory, the number of charges carried by an individual ion, z, can be calculated as22-26
I
a) Experimental Pulse Sequence 1 Inject Hlnject H D e l a y H TOF HDelayH E x c i t e H Detect1
I’
in which C is the detection circuit capacitance (farad), Vp-pis the voltage drop in the detection circuit due to the orbiting individual ion (volt), q is the charge carried by an electron (coulomb), and R is the normalized ion radius (with respect to half of the cell edge dimension). The quantity of A (R)is detined by the equation A(R) = 0.81402R 0.03051R3 - 0.01369P 0.00200R7 for the elongated ICR cell used in this study (length-to-width ratio 1.5: 1). The value of A(R) is actually dependent on the z-oscillation amplitude as well as R. However, for ions with their z-oscillation amplitudes less than 60%of the maximum allowed value, A(R) is nearly independent of z-oscillation amplitude.27 Here, ions are assumed to have zero z-oscillation amplitude, which is a reasonable approximation when long initial ion relaxation times are employed, particularly with the use of collision gas cooling. Once the detection circuit capacitance (0, the induced voltage drop in the detection circuit (Vp-p),and the ion radius (R) are determined, the number of charges carried by a multiply-charged ion from eq 1 is readily calculated. The procedures for the determination of C and Vp-pwere demonstrated previously.22 As described below, a different approach to the measurement of R is used in the present study. In this paper, we report the trapping, detection, and molecular weight determination of the largest ions measured thus far by mass spectrometry, Coliphage T4 DNA (nominal M, = 1.1x lo8Da), based on the method described above. Although there are relatively large uncertainties (-10%) in our initial measurements, we believe that the uncertainties can potentially be reduced to (1% with the utilization of low noise detection circuitry having high sensitivity and stability coupled to a linearized trapped ion cella2* Nonetheless, the experimental results presented here strongly suggest that very large ions (Ilo8 Da) can be successfully transferred intact to the gas phase by ESI.
+
1:
,
p
-5
L
b) Experimental Pulse Sequence 2
+
EXPERIMENTAL SECTION
The ESI-FI’ICR mass spectrometer used in this study has been described in detail e1se~here.I~Cell control and data manipulation are provided by an Extrel (Madison, wr) Odyssey data station. The detection circuit capacitance has been measured to be 96.71 p F and Vp-p @V) is calculated based on the calibration curve of y = 191,5Vp-p 92.93 under the experimental conditions used, where y is the measured magnitudemode FTTCR peak height (scaled abundance). In our studies, a population of ions is trapped,
+
(22) Chen, R; Wu, Q.; Mitchell, D. W.; Hofstadler, S. A; Smith, R D. Anal. Chem. 1994,66, 3964-3969. (23) Mitchell, D. W.; Rockwood, A L.; Chen, R.; Smith, R. D. J. Chem. Phys., in press. (24) Grosshans, P. B.; Shields, P. J.; Marshall, A G.J Chem. Phys. 1991,94, 5341-5352. (25) Limbach, P. A; Grosshans, P. B.; Marshall, A G. Anal. Chem. 1993,65, 135-140. (26) Rempel, D. L.; Huang, S. K; Gross, M. L. Int.J Mass Spectrom. Ion Processes 1986,70,163-184. (27) Chen, R Theory and Practice of Ion Trap Design in FITCRMS. Ph.D. dissertation, The Ohio State University, Columbus, OH, 1993. (28) Grosshans, P. B.; Chen, R; Limbach, P. A; Marshall, A. G. Int. J Mass Spectrom. Ion Processes 1994,139, 169-189. 1160 Analytical Chemistty, Vol. 67, No. 7, April 1, 7995
t’ P 2
’
0
t
Figure I. Experimental pulse sequences 1, for ion trapping and preselection, and 2, for ion remeasurements. No ion and neutral gases are introduced after sequence 1 is completed: Valve/lnject, gas pulse valve open and ion injection simultaneously; Inject, ion injection only; Delay, ion damping or relaxation; TOF, suspended trapping for ions’ free axial flight; Excite or Detect, dipole excitation or detection.
detected, and manipulated until an individual ion (or a few “individual” ions at different m/z values) is selected (isolated) prior to mass analysis. The experimental event sequences used in this study are outlined in Figure 1. Two somewhat different pulse sequences are used. First ions are injected from the ESI source, slowed by a collisional gas (Nz), and constrained in the ion trap by applied trapping potentials. After a short delay (less than 1 ms), the two trapping electrodes are grounded for a period of between 1 ms and 1 sZ9(marked as TOF in Figure 1because ions are undergoing axial free flight during this period). This TOF pulse can be adjusted to yield an appropriate ion population which, due to the relatively higher velocities of smaller ions, favors retention of higher molecular weight ions.20,22After ion injection and selection, the ions are excited and detected to ensure an appropriate ion population. After the first pulse sequence, we then employ a second pulse sequence for remeasurements (Figure lb). Note that there is no ion injection step in this second sequence. The ion population, prepared from the first pulse sequence, is now repeatedly excited and detected by standard dipolar methods until a maximum peak height is acquired for an individual ion. Note that the prepared ion population typically consists of several single ions at different m/z. If the individual ion being monitored is not distinguishable in two consecutive remeasurements due to possible m/z overlapping with other ions, one or more TOF pulses may be used to further decrease the ion population (and selectively eject lighter ions; see later sections for details) before remeasurements. The delay events, after the TOF events, are used to enhance the relaxation (Le., cooling) of ions to the center of the trapping well before excitation. Broad-band excitations are used exclusively in these studies. (29) Laude, D. A, Jr.; Beu, S. C. Anal. Chem. 1989,61, 2422-2427.
The coliphage T4 DNA is obtained from Sigma (St. Louis, MO). One unit (50 pg) of the T4 DNA is prepared in 500 pL of deionized water and then purified to remove buffer components (Tris-HC1, NaCl and EDTA) by microfiltration twice using Centricon-100 filters (Amicon, Danvers, MA). The prepared solution (0.1 mg/mL) is injected into the ESI source at a flow rate of 0.5 pL/min. RESULTS AND DISCUSSIONS
Estimation of Ion Radius. An interesting observation is that, contrary to the case of 5 x lo6Da PEG ions,22collisional relaxation of ion cyclotron motion for these larger T4 DNA molecules is relatively ineffective compared to lighter ions, possibly due to the low center of mass collision energy between the large ions and the N2 collision gas. As a result of this effect, the procedure used in our previous study, where ions were remeasured by excitation/ damping cycles,22 is not applicable here. Consequently, an alternative procedure (see Figure 1) is employed to estimate the ion's cyclotron radius. The concept is based upon the fact that the maximum peak height value occurs for an ion only when it is at its maximum possible cyclotron radius (Le., at R = 1). This procedure consists of two experimental pulse sequences, as described in the Experimental Section. The selected ion is first excited to a radius of R = 0.75 (Rma= 1.00) and subsequently subjected to remeasurement events at relatively low excitation power. After detection and Fourier transformation, the resulting peak height is measured. This low excitation remeasurement event is repeated numerous times for the same ion until a maximum peak height is determined. During these remeasurements, no collision gas is introduced into the mass spectrometer, and the ion undergoes no signscant collisional cooling (the pressure is -1 x Torr). We chose to use this approach since software control limitations and cyclotron frequency shifts during detection (see below) make it difficult to precisely control the delay intervals between any two remeasurement events. Thus, the phase of the excitation waveform during a remeasurement event does not generally exactly match that of the orbiting ion. When the phase difference is between 0" and 90" (i.e., approximately in phase), the ion is accelerated to a larger radius, and the resulting peak height is greater than in the previous measurement. If the phase difference is between 90" and 180" (Le., approximately out of phase), then the ion is decelerated to a smaller radius, and the resulting peak height is lower (this is not strictly true for cases in which a very long excitation duration is employed30). Statistically, a maximum peak height can typically be determined with an estimated uncertainty of -5% in 10 such remeasurements. Occasionally the ion of interest is ejected before 10 remeasurements can be made. In these instances the data are discarded. Such situations are minimized by using low excitation amplitudes (corresponding changes in cyclotron radii of 5-10% of Rm& for remeasurement events. To summarize, a particular ion is selected and monitored by a series of (typically 10- 15) excitation/detection cycles at relatively low excitation amplitude. The ion is excited to (or very close to) its maximum radius, which results in the maximum peak height being observed during these cycles. We have made the assump tion that the observed maximum peak height corresponds to an ion radius of 0.95. Substituting the Vp-p corresponding to this (30) Mitchell, D. W.; Hearn, B. A; Delong, S. E. Intl. /. Mass Spectrom. ion Processes 1993,125, 95-126.
100.0-
8
80.0
v A -
8 C
la
After Partial Ion Selection
J
60.0:
(II
2
40.01
2
20.0;
a
L
1
0.0-
2000.0
4000.0
2500.0
m/.
4500.0
100.0
-Ib $
After Complete Ion Selection
80.0
v
0.0
2000.0
2500.0
3000.0
3m.O
4000.0
4500.0
m/z
Figure 2. FTlCR magnitude-mode spectra of Coliphage T4 DNA molecules: (a) Typical spectrum obtained after initial ion trapping, selection (a suspended trapping period of 10 ms). Ions are excited to a small cyclotron radius. (b) Spectrum of an ion at m/z = 2883 obtained at its maximum radius after a number of remeasurements (including ion selection with suspended trapping of 1 ms).
maximum peak height, the measured C value, and R = 0.95 into eq 1, the ion's charge number, z, is calculated, which in turn gives the ionic mass by multiplication of m/z by z. Determination of Charge Numbers and Ionic Masses. Figure 2a shows a mass spectrum of ions from T4 DNA. This spectrum was obtained after ion injection, ion trapping, and application of one suspended trapping event to reduce the likelihood of ion cloud expansion due to space charge effects which results from the high charge states (z -lo4) of the these macroions. After repeated application of TOF pulses, it is feasible to retain only one or two ions in the ion trap, which are then monitored throughout the remeasurement process described above. One example is shown in Figure 2b. The maximum peak height for this ion at m/z 2883 produces a Vp-p of 42.28 pV in the detection circuit. For an estimated ion radius of 0.95, the number of charges is calculated to be 31 530 (f3150), corresponding to an ion mass of 90.9 (f9.1) x lo6 Da. There are several reasons for the smaller signal-to-noise ratio in Figure 2a than that in Figure 2b. First, the ions in Figure 2a are excited to only a small radius, while the ion in Figure 2b is excited to near its maximum radius at the trap radial boundary. Second, due to the Coulombic interactions, the trapped ions repel each other, especially for highly charged ions.31 This results in a dephasing effect which decreases the signal-to-noise ratio of Figure la. Additionally, we have observed that lighter ions appear to be excited more readily than heavier ions, even though they are at nearly the same m/z value.20 On the basis of this observation, some portion of the peaks in Figure 2a are likely due to low-mass ions. The masses (31) Beebe-Wang, J.: Elander, N.: Schuch, R Phys. Scr. 1993,46, 560-568.
Analytical Chemistry, Vol. 67,No. 7, April 1, 1995
1161
Table 1. Molecular Mass of Individual Coliphage T4 DNA Ions
-5
-i
10022.6 5011.3 0.0
m/z
induced signal (ICV)
charge no.= (z)
Mru (x lo6 Da)
3619 4285 3724 3874 4175 2883
46.50 37.18 42.01 37.54 31.96 41.28
35 520 28 390 32 090 28 670 24 410 31 530
128.6 121.7 119.5
B
111.1
'17
42.31
2
13.312
5
6.65i 0.00
102.0 90.9
Gi -.
-10022.6 .I"VLL.Y
- -. ,
,
,
,
,
100000.0
,
,
,
,
,
,
,
200000.0
,
300000.0
Time, msec
400000.0
- 56.4
An uncertainty of f10%applies to charge number determination and, hence, to Mr. (I
determined for six of the largest ions are given in Table 1. Due to the relative errors of ion radius determination and the detection circuit calibration, the uncertainties of the results listed in Table 1are estimated to be -10%. Possible sources of error have been discussed in detail previously.22 Observation of Long Transients of Large Individual Ions. The unique features of Fourier transform ion cyclotron resonance mass spectrometry (FTICR-MS) include its ability to store and manipulate ions and to detect ions nonde~tructively.~~~.~ Once formed, ions can be trapped for as long as 5 x lo4 s prior to excitation and detection.32 The coherence of an ion cloud for a group of ions having a specific m/z value could be gradually lost due to the relaxation of ion motion, space charge effects, inhomogeneities in the trapping and magnetic fields, collisional damping with neutrals, and so on. On the other hand, if there is only one ion in an ICR trap and no signiticant dissipative processes are present, such as radiative damping, resistive damping, and collisional damping,33 then the individual ion will provide a measurable signal indefinitely. Usually radiative damping is insignificant for ICR motion, having typical relaxation times for ion cyclotron motion on the order of years. Resistive damping has typical relaxation times on the order of hours.33 In general, collisional damping is the most significant factor in FTICR for the signal decay of an ion cloud. However, the relaxation time of collisional damping increases as ionic masses increase, a trend that is strikingly obvious when ions of the present size are studied. Thus, it is possible for a large individual ion to display greatly extended relaxation times, perhaps extending to the regime of resistive damping. Figure 3 shows a 476 s transient (Figure 3a) from a T4 DNA ion and its corresponding frequencydomain spectrum (Figure 3b). The length of the transient was constrained by limitations in our computer memory and not by signal decay. A longer time-domain signal could be acquired with more memory (data points) and/ or a narrower mass range. We have recorded 20-35 megaword long transients using self-designed hardware, but appropriate software is not currently available to effectively handle such large data sets. A striking feature of Figure 3b is that, an first glance, it appears to be a spectrum resulting from an ensemble of ions as opposed to an individual one. However, by truncating the data at different time segments and then Fourier transforming these segments, we find that the spectrum is actually caused by complex series of cyclotron frequency shifts during detection (see Figure (32) Allemann, M.; Kellerrhals, H.; Wanczek, K P. Chem. Phys. Lett. 1980,75, 328. (33) Comisarow, M. B. In Ion Cyclotron Resonance Sfiectrometty II; Hartmann, H.,Wanczek, K-P., Eds.; Springer-Verlag: Berlin, 1982; pp 484-513. 1162
Analytical Chemistry, Vol. 67, No. 7, April 1, 7995
4 n
3260.0
a
3280.0
3320.0
3300.0
m/z
3340.0
B 112.279.204
c
6.13;
iI
.
K 3260.0
3280.0
1
I
3320.0
3300.0
miz
3340.0
B 1 16.67
3
4
8.331 4.17 '
5
0.00~
n
a
e) I
3260.0
3260.0
3300.0
miz
3320.0
3340.0
Figure 3. A 476 s transient acquired from an individual T4 DNA ion (a) and its frequency-domain spectrum (b). Major noise peaks are indicated by asterisks. (c-e) are frequency-domain spectra of 5 s truncated data from the transient shown in (a) (0-5, 230-235, and 471 -476 s for (c-e), respectively). The last three spectra confirmed that the spectrum in (b) arises from an individual ion undergoing a complex series of changes in m/z.
3c-e, which are the Fourier transforms of 5 s long transient segments from 0 to 5,230 to 235, and 471 to 476 s, respectively). These frequency shifts are the principal reason why a relatively broad m/z range is selected for detection. Otherwise, it is possible that the ion may "move" out of the m/z range chosen. The origin of the complex frequency (Le., m/z) shifts remains to be elucidated. Possible mechanisms include combinations of charge loss and neutral gain or loss. CONCLUSIONS
Building upon previous work in which mass measurements were obtained from individual (i.e., single) ions, we have demonstrated the feasibility of mass spectrometry in the lo8 Da regime; to our knowledge these measurements represent the largest mass determination yet realized by mass spectrometry. T4 DNA (-1.1 x lo8 Da) was ionized by electrospray ionization; the highly charged molecular anions were trapped and m/z measurements made by Fourier transform ion cyclotron resonance mass spectrometry. A direct charge measurement scheme was employed which does not rely on collisional relaxation (e.g., axialization) of ions between excitation/detection events. Collision relaxation was found to be relatively ineffective for ions in excess of -lo7 Da. With this scheme, normalized ion radius was measured with -5-10% accuracy which, at present, is the factor that limits mass accuracy. Significant improvements in ion radius
determination, and thus mass accuracy, are anticipated when linearized trapped ion cell geometries are employed. The analysis of individual macroions by FTICR affords unique opportunities to investigate charged particle dynamics in the gas phase and to evaluate homogeneous vs inhomogeneous relaxation phenomena. We have observed time domain signals from individual macro-ions which demonstrate half-lives well in excess of 10 min. In conjunction with previously demonstrated timeresolved ion correlation methodologies, these methods begin to bridge the large molecule/small particle gap (we estimate that a fully extended 1.1x 108 Da DNA molecule is on the order of 100 pm in length!) and may lay the foundation for an entirely new approach to the study of ion/molecule reactions, photochemical processes, and ion/ion Coulombic interactions. As these studies were carried out in a relatively low m/z regime compared to that accessible by FTICR measurements (which can potentially be used for m/z > 100 000), we are confident that the methods described here will find broad applicability to species several orders of magnitude heavier than the present study. While at this point we cannot determine whether these large DNA fragments are ionized completely intact, it is clear that some fraction of the ions has masses corresponding to intact species (within experimental error). It should be pointed out that the “length of these large ions, which must be substantially extended by Coulombic forces, is nearly 2 orders of magnitude larger than typical ESI droplet sizes.
Because of the extremely high charge carried by large ions, the behavior of these ions should be very interesting when more than one ion is present in an ICR cell. Physicists have observed the phase transitions of an ion cloud (ordering structure) by lasercooling ions to IC3l We predict that, under the appropriate conditions, one should be able to observe this phase transition at room temperature for highly charged ions by use of collisional cooling instead of laser cooling, which is more complicated and not applicable to large ions. ACKNOWLEDGMENT
We thank Drs. Jim E. Bruce, Ray Bakhtiar, and Steve Van Orden for helpful discussions, and the US.Department of Energy for support of this research through internal Exploratory Research of Pacific Northwest Laboratory under Contract DE-ACOG76RLO 1830. Pacific Northwest Laboratory is operated by Battelle Memorial Institute.
Received for review February 9, 1995.@
November
8,
1994.
Accepted
AC941089S
@Abstractpublished in Advance ACS Abstracts, March 1, 1995.
Analytical Chemistry, Vol. 67, No. 7, April 1, 1995
1163
