2590
Anal. Chem. 1984, 56, 2590-2592
Table I. Precision and Recovery Studies of Human IgG IgG added, pg mL-l
recovery
within-run precision, % RSD
0.4 0.6 1.0 1.5 2.0 2.5 3.0 3.5
98 98 97 96 94 97 97 96
k5.0 14.5 k3.6 k2.6 h2.0 k2.4 k1.5 k1.0
%
97% av
Chem. 1983, 55, 202R-214R. Wehmeyer, K. R.; Doyle, M. J.; Halsall, H. B.; Heineman, W. R. Methods Enzymol. 1983, 9 2 , 432-437. Alexander, P. W.; Maltra, C. Anal. Chem. 1982, 5 4 , 68-71. Gebauer, C. R.; Rechnitz, G. A. Anal. Blochem. 1982, 124, 338-348. Nakane, P. K.; Kawaoi, A. J . Hlstochem. Cytochem. 1974, 22, 1084-109 1. Meyerhoff, M. E.; Rechnltz, 0. A. Anal. Blochem. 1979, 9 5 , 483-493. Wisdom, 0. G. Ciin. Chem. (Winston-Salem, N . C . ) 1976, 2 2 , 1243-1 255. Shew, P. D.;Hager, L. P. J . 8/01. Chem. 1961, 236, 1626-1630. Hager, L. P.; Morrls, D. R.; Brown, R. S.; Eberweln, H. J. 8lol. Chem. 1986, 241, 1769-1777. Morrls, D. R.; Hager, L. P. J . 8/01.Chem. 1966, 1763-1768. Boorsma, D.M.; Kalsbeek, G. L. J . Hlstochem. Cytochem. 1975, 2 3 , 200-206.
patient samples are not available to us, we expect that this method could be useful for clinical purposes. LITERATURE CITED (1) Arnold, M. A.; Meyerhoff, M. E. Anal. Chem. 1984, 56, 20R-48R. (2) Davis, J. E.; Solsky, R. L.; Glerlng, Linda; Malhotra, Saroj. Anal.
RECEIVED for review June 12,1984. Accepted August 3,1984. The support Of the National Institutes of Health (Grant GM-25308) is greatly appreciated.
CORRESPONDENCE Thermospray Liquid Chromatographic Interface for Magnetic Mass Spectrometers Sir: The thermospray technique (1)is becoming recognized as a practical approach for on-line LC-MS. Up to now thermospray interfaces have been used on quadrupole mass spectrometers in which the ion source is a t or near ground potential. Recently it has been demonstrated that thermospray provides a "soft" ionization technique capable of producing intact molecular ions from large, nonvolatile molecules. Exploitation of this technique for large molecules has been limited by the mass range and resolution available from the quadrupoles presently used. Although the mass range available from commercial quadrupole instruments has increased substantially in recent years, it appears that magnetic deflection instruments will continue to enjoy a distinct advantage over quadrupoles in high-mass and high-resolution capabilities. In the thermospray system described earlier (1-4), the vaporizer and its associated control system must reside a t essentially the same potential ass the ion source, which in typical instruments is several kilovolts off ground. Also, the pressure in the source pump-out line is typically a few torr, a pressure range in which gas discharges are easily initiated at potential differences of a kilovolt or less. While it is possible to float the entire LC-MS interface a t ion source potential, it is desirable for operator safety and convenience to keep the LC and the mechanical vacuum pump at ground. The system developed to provide a practical solution to the problems associated with operating the thermospray interface on a magnetic mass spectrometer is shown schematically in Figure 1. The major portion of the interface is identical with that described earlier (I), but the necessary measures have been taken to deal effectively with the problems introduced by operating the ion source at high voltage. The LC effluent is coupled to the thermospray vaporizer by using a length of 150-pm-i.d. fused silica capillary (SGE,Austin, TX). The source is pumped by a glass dry iceacetone cooled trap backed by a 300 L/min mechanical vacuum pump. The line from the
source to a trap is a 1.2-cm-i.d.stainless steel tube which passes through the source housing by means of an electrically isolated flange. The LC connection and heater and thermocouple connections pass through a similar electrically isolated flange on the opposite side of the source housing. A discharge suppressor is installed inside the rubber hose connecting the trap to the mechanical vacuum pump. This device is similar to one described earlier by Wojcik and Futrell5 for coupling a chemical ionization source at high voltage to a grounded inlet system. The present discharge suppressor consists of 11disks of perforated stainless steel which are separated and supported by 2.5 cm long ceramic standoffs. Eleven 10-MO 2-W resistors are connected in series to this stack of discs, and the whole assembly is installed inside the 4-cm-i.d. rubber hose connecting the trap to the mechanical vacuum pump. The upper end of the resistor chain is connected to the ion source potential and the lower end to the grounded vacuum pump. The temperature controller for the thermospray system is similar to that used previously for the quadrupole interfaces, but it is mounted on high voltage standoffs inside a grounded outer case. The necessary controls are brought out on insulated shafts through a clear plastic panel which allows the temperature controllers and readouts to be monitored while protecting the operator from contact with the controller operating a t ion source potential. Power is supplied through an isolation transformer with insulation rated for 4 kV. This system has been successfully operated with a variety of solvents including 0.1 M ammonium acetate at ion source voltages to 3.5 kV, the maximum available with the power supply available with the present instrument. In initial trials, total ion currents in excess of A into the magnetic analyzer were obtained by direct thermospray ionization using the above buffer. The mass spectra obtained were essentially identical with those obtained under comparable operating conditions by using the quadrupole. Results of experiments on the current flow through the
0003-2700/84/0356-25~0$0 I .50/0 0 lg84 American Chemlcal Society
ANALYTICAL CHEMISTRY, VOL. 56, NO. 13, NOVEMBER 1984
2591
I
X I
-
t4KV
\
I
= M
THERMOSPRAY VAPORIZER -TRAP ION !&JRCE
LC
-
Y
Figure 1. Schematic diagram of modifications to the thermospray Interface to ailow operation at ion source potentials up to 4 kV.
Flgure 3. Mass spectra obtalned by direct thermospray ionization of 0.1 M ammonium acetate at 1 mL/mln and ion source temperature of 200 O C . An external source of ionization such as a hot filament is not required for direct thermospray ionization.
Table I. Observed Metastable Transitions in Thermospray Mass Spectrum of 0.1 M Ammonium Acetate
reaction NH4'sNHB 4"' NH,+*HZO "4' NH4+*(HzO)z NHd'*HzO
+ NHS + HzO
4
+
+ 2Hz0 + HzO NH4+.NH3*HZO NH4'*NH3 + HzO NH4'*(H20), NH4+*(HZO),+ HzO +
4"'
M* 9.26 9.0 6.0
+
capillary as a functlon of applied voltage for 0.01 and 0.1 M ammonium acetate flowing at 1 mL/min.
24.0 23.1 40.5 46.15 NHdt*(CH3COOH) NH4'*(CH&O) + HzO NH4'.NHS(CHSCOOH) NH4'.NH3(CHZCO) + HzO 62.41 NHd+*(CH&OOH)+ HzO 63.38 NH,*.H,O.(CH&OOH)
liquid column passing through the fused silica capillary are summarized in Figure 2. These results show the expected ohmic behavior, and the conductances are consistent with the conductivity expected for ammonium acetate when corrected for ionic strength by using the Debye-Huckel-Onsager theory (6). These experiments used a 1.2-m length of the 150-pm capillary which gives a pressure drop for the flowing liquid of about 120 psi/mL/min of flow. Since this pressure drop is small compared to that in the vaporizer and in the LC column, a longer length could be used if it is necessary to reduce the current flow. The conductivity of the capillary can also be reduced by decreasing the inside diameter, but, since the resistance goes as the inverse square of the diameter and the pressure drops as the inverse fourth power of the diameter, this approach can lead to unacceptably high pressure drops a t flow rates in the milliliter per minute range. The performance of the thermospray LC-MS interface on the magnetic instrument is similar in most respects to that observed earlier for a similar interface on a quadrupole ( I ) . The mass spectrum obtained by direct thermospray ionization of 0.1 M ammonium acetate at 1mL/min is shown in Figure 3. This spectrum which was obtained at an ion source temperature of 200 OC is essentially identical with that obtained with the quadrupole. The only noticeable difference is the presence of a number of small peaks, some a t nonintegral masses, which are clearly due to metastable dissociations which are not observed when using the quadrupole. These are particularly apparent in the lower trace where the vertical scale has been expanded by a factor of 100. The transitions associated with the more intense metastable peaks (ca. 0.3-3% of the base peak) are identified in Table I. While the structure of some of the observed ions has not been established, the metastable transitions tend to support the iden-
tifications discussed earlier ( I ) . For example, mass 77 is produced by loss of water from the cluster of NH4+ with ammonia and acetic acid, and mass 60 is produced by loss of water from the cluster of NH4+ with acetic acid. The other major metastables are mostly due to loss of one or more waters from the hydrated ammonium ion. The pumping system on the mass spectrometer used in these studies consisted of a 1200 L/s diffusion pump backed by a 300 L/min mechanical pump on the source housing and a small (110 L/s) turbomolecular pump on the analyzer. With this system, in addition to the mechanical pump connected to the ion source as described above, the pressure in the analyzer increased by 8 X torr as the liquid flow into the thermospray vaporizer was brought from 0 to 1.5 mL/min while the pressure in the source housing increased to about 1X torr according to the Penning discharge gauge available. The former pressure was read on a Bayard-Alpert ionization gauge and should be reasonably reliable; the pressure in the source housing was probably somewhat higher than indicated by the discharge gauge which may not be accurate for water vapor. Stable operation was obtained at all flow rates from 0.5 to 2.0 mL/min with 0.10 and 0.01 M ammonium acetate, both neat and in mixtures with methanol. No problems with electrical discharges were encountered. The mass spectrometer used in these initial tests is not interfaced to a data system, so obtaining on-line LC-MS data is cumbersome. Nevertheless, the present results indicate that the approach described here provides a practical method for incorporation a thermospray LC-MS interface on a magnetic mass spectrometer. To date, we have not extended our tests to source potentials (ca. 8 kV) used in some high-performance instruments, but this should not present any serious difficulties.
0
I
2
3
V (kV)
--+
+
Flgure 2. Measured current flow through 1.2 m of 150-pm fused slllca
+
+
4
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Anal. Chem. 1984, 56,2592-2594
ACKNOWLEDGMENT I gratefully acknowledge the technical assistance of C. C. Wu and Leonard Wasicek in constructing the thermospray interface. Special thanks are extended to Curt Brunee and Finnigan/Mat for Lending the CH-5 mass spectrometer used in this work. LITERATURE CITED (1) Blakely, C. R.; Vestal, M. L. Anal. Chem. 1983, 57, 750-754. (2) Vestal, M. L. I n f . 3 . Mass Spectrom. Ions Phys. 1983, 46, 193-197. (3) Vestal, M. L. "Ion Emission From Liquids"; Miinster, A. B. Ed.; Sprlnger-Verlag: Berlin, tg83; pp 246-263. (4) Vestal, M. L. Mass Specfrom. Rev. 1983, 2 , 447-480.
(5) Futrell, J. H.;Wojcik, L. H. Rev. Sci. Instrum. 1971, 42, 244-251. (6) Atklns, P. W. "Physlcal Chemistry", 2nd ed.; Freeman: New York, 1982; pp 900-902.
Marvin L. Vestal Department of Chemistry University of Houston Houston, Texas 77004
RECEIVED for review March 30,1984. Accepted June 20,1984. This research was supported by NIGMS under Grant GM 29031 and by the Robert A. Welch Foundation.
Digital Electrochemical Transient Analysis: Diagnosis of an Elementary Reaction Mechanism as an Illustration of Data Acquisition Strategy Sir: With the advent of the digital and the approach of the cybernetic (1) ages of electroanalytical chemistry, a need has arisen for computational algorithms which might prove to be useful in making decisions concerning the information content of current transients. In this communication, the digitally computed function A(1n i)/A(ln t ) vs. t is offered as a most useful component of the artificial intelligence system of a cybernetic instrument because of its ability to discriminate on line and under high-level software control those processes which may rate limit the current in electroanalysis. We first became aware of the utility of this function during the development of what we have identified as Riemann-Liouville transform polarography (2). Even though it is now our contention that each rate-limiting process produces a unique current response that may be readily distinguished through the computation of this function, we present herein an illustration of its utility only in the treatment of chronoamperometric transient theory for the ECE mechanism. This treatment suggests that this function might find wide use as the digital equivalent of the "working curves" employed so successfully during bygone analogue days in studies of the mechanism of electrode processes. In the development of this illustration we have concurrently been forced to develop the data acquisition strategy that is necessary for the implementation of this method in the analysis of experimental current transients by a cybernetic instrument. This aquisition strategy is also presented herein in prelude to its utilization in subsequent efforts.
THEORY For the ECE mechanism the current subsequent to the application of a potential step to diffusion-limiting conditions has shown ( 3 ) to be
i ( t ) = (nFACD1/2/(~t)1/2)(2 - e-kt)
(1)
where all symbols have conventional electroanalytical meaning, or upon rearrangement This equation may be expressed in logarithmic form and differentiated with respect to In t to obtain an exact expression for the function A(ln i)/A(ln t ) to be digitally computed from the synthetic transient obtained from eq 2 d(ln i)/d(ln t ) = Izt/(2ekt - 1) - 7 2
(3)
Synthetic results for i ( t )were generated within a BASIC program by assigning values to kt in eq 2 in order to obtain a traditional dimensionless working curve (4-8) by plotting the dimensionless current as a function of log k t ; several of these previous treatments suggest ways to perform the appropriate mechanistic diagnosis and to obtain the first-order rate constant k. For comparison, an exact representation of the dimensionless d(ln i)/d(ln t ) working curve was obtained by assigning values to kt in eq 3. This is shown in Figure la. As would be anticipated, a 2 orders of magnitude time domain of kinetic activity separates those periods when the electrode process appears to be diffusion controlled (when, by definition, d(ln i)/d(ln t ) = 0.5). Because this function is completely independent of bulk concentration (C), electrode area (A), and mass transport parameters (D), no further treatment of experimental data would be necessary in the elucidation of the mechanism. This could be accomplished in real time by direct comparison with eq 3; it may be noted that
k = 0.768/tm
(4)
where t , represents the real time associated with the achievement of the maximum value of d(ln i)/d(ln t ) .
RESULTS AND DISCUSSION The theoretical merit of this approach having been demonstrated, a study was undertaken to determine the feasibility of using this method in the interpretation of digitally acquired current transients. This feasibility rests upon the ability to obtain an accurate representation of eq 3 through the computation of A(ln i)/A(ln t ) . The accuracy of the digital computation of the function A(ln i)/A(ln t ) depends upon two factors: (1) the resolution of i ( t )so as to obtain a significant logarithmic difference in the determination of A(1n i) from any two successive data points in the current transient; this resolution depends, to an extent, upon the combined quantization noise (9) and random noise of the measurement of i ( t ) ;and (2) the selection of the proper acquisition rate so as to obtain a significant logarithmic difference in A(ln t ) over all time domains. For example, it is clear that A(ln t ) is not equally spaced for equally spaced time increments used in conventional data acquisition systems. Instead, the value of A(1n t ) tends to become smaller as the value o f t gets larger. To overcome the problem of the variability of A ( h t ) ,we have developed an acquisition strategy that allows A ( h t ) to exhibit a similar variation over all time domains. This strategy calls
0003-2700/84/0356-2592$01.50/00 1984 American Chemical Society
