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Anal. Chem. 1990, 62, 1798-1804
On-Line Monitoring of Bioreactions of Bacillus polymyxa and Klebsiella oxytoca by Membrane Introduction Tandem Mass Spectrometry with Flow Injection Analysis Sampling M a r k J. Hayward,' Tapio Kotiaho,'s2 Anita a n d George T.T ~ a o * , ~
K.Lister,' R. Graham Cooks,**' Glen D. A ~ s t i nRamani ,~ Narayan:
Department of Chemistry and Laboratory of Renewable Resources Engineering, Purdue University, West Lafayette, Indiana 47907
Membrane Introduction mass Spectrometry with flow injectlon analysls sampllng has been utilized for on-llne monitoring of the major products and the volatile metabolites of fermentatlon by the Bacllus polymyxa and K l e M a oxjftoca organisms. A flow injection sampllng system was used to rapidly deliver fermentation broth or an external standard to the mass spectrometer. Analyte lntroductlon occurred vla a direct insertion membrane probe in which the aqueous solutions flowed past a membrane located within the ion source of the mass spectrometer. For both organisms, concentrations of the Uclukcphase products, acetic acid, acetoln, 2,3-butanedid, and ethanol, were monitored as a function of tlme after permeation through the membrane and ionization by chemical ionlzation. Tandem mass spectrometry conflnned that these measurements were made without interference. Off-line gas chromatography was utilized to test the accuracy of these measurements, and excellent agreement was found. Dissolved oxygen In the fermentation broth was also monitored, and carbon dioxide and oxygen were followed In the offgases. The O2 and C02 measurements were compared with other common measurement techniques (galvanic O2 electrode, infrared abgorptkn, and paramagnetic O2 analysis), and very good agreement was found. The use of tandem mass spectrometry has allowed the detection of addltionai compounds that were previously not known to be present In measurable amounts.
INTRODUCTION The use of analytical instrumentation for on-line measurement of the quality of process streams is a field of great fundamental and practical importance ( I ) . These measurements can yield information regarding process dynamics that can lead to model equations to describe the (time-variant) state of the system. Once realized, this knowledge can then be used to control the process so as to maintain near optimal conditions and maximize process economy. In many processes, especially bioprocesses, detailed structural specificity as well as high speed is essential to understand and control these processes. The urgent need to gain chemical information as a function of time on process systems has led to the development of a number of rapid sampling techniques, the most important of which is flow injection analysis (FIA). The advantages of FIA, high speed, the simplicity of the experimental setup, the economy of sample and reagent consumption, and the ability to utilize a wide variety of detection systems, have given the method broad acceptance (2). Because 'Department of Chemistry.
*On leave from Technical Research Center of Finland, Chemical
Laboratory, Biologinkuja 7 , 02150 Espoo, Finland. Laboratory of Renewable Resources Engineering.
0003-2700/90/0362-1798$02.50/0
it is usually desirable to obtain both quantitative and qualitative information in an on-line detector, optical methods predominate in FIA detection systems while electrochemical methods are becoming more common (2). While these detection methods can be manipulated for application to a wide variety of compounds, during any particular application they are often only capable of detecting a small class of compounds under what may be a very limited range of experimental conditions. Recent advances in flow-through membrane introduction methods in mass spectrometry (3) offer a more universal, molecularly specific method of detection when used with FIA. The incorporation of membranes into process sampling analytical instrumentation is gradually becoming more common. The use of membranes for sample introduction in mass spectrometry was first reported by Hoch and Kok in 1963 to study reaction kinetics during photosynthesis (4). A signifcant early use of membrane introduction was made by Llewellyn and Littlejohn, who built one of the earliest gas chromatography/mass spectrometry (GC/MS) interfaces (5). Much of the recent interest in membranes in chemical analysis can be traced to the work of Tou et al., who immersed sealed hollow fiber capillary membranes in liquid solutions and then evacuated the tubes via a mass spectrometer (6). Compounds that permeate the membrane are pumped into the mass spectrometer and are analyzed in the usual way. Further progress was made by Weaver and Abrams with the observation of the performance enhancements made possible by adjusting the pH of the sample matrix (7). Recently, membrane introduction mass spectrometry has begun to see more general acceptance as is evidenced by the construction of new flow-through membrane introduction devices (8, 9). Membrane introduction mass spectrometry has also been combined with FIA for rapid handling of environmental samples (10). Undoubtedly, the most common application of membrane introduction mass spectrometry is in fermentation monitoring. Reuss et al. were the first to report this application in 1975 (11). Since then, this area has fluorished; the subject has been reviewed (121,and symposia have been held (13). Typically, fermentation monitoring has been carried out by using a single-stage quadrupole mass spectrometer with electron impact ionization and a membrane inlet located in the reactor and connected to the mass spectrometer via long vacuum lines. These systems are often able to detect gases and volatile compounds within the reactor; however, such systems are commonly plagued with interference in monitoring volatile components due to the extensive fragmentation of their molecular ions in the ion source (14), with adverse chromatographic effects occurring in the long vacuum transfer lines (151, with clogging of the membrane by particulates and/or cell growth, and with inconsistenciesarising from the inability to standardize measurements. Nevertheless, previous work has sometimes yielded good results, especially for the measurement of dissolved gases and headspace or off-gases (15). 0 1990 American Chemical Society
ANALYTICAL CHEMISTRY, VOL. 62, NO. 17, SEPTEMBER 1, 1990
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Figure 1.
Schematic diagram giving a cross-sectional view and showing segmented flow through the direct-insertion sheet membrane probe.
In our laboratories at Purdue, a program aimed at developing and utilizing membrane inlet mass spectrometry is in progress. Initial results showed the utility of the method for monitoring organic compounds in an aqueous matrix (16) and for metabolite detection in vivo in blood (17). Later work resulted in the development of a hollow fiber membrane direct-insertion probe (18) that was unique in that it located the membrane in the ion souce of the mass spectrometer. The use of the flow-through principle, in which the solution flows across the inside surface of the membrane while the outside surface is exposed to the vacuum of the mass spectrometer, together with the location of the membrane, reduces problems due to analyte dilution, memory effects, and poor response times observed in interfaces where the membrane is located remote to the ion source. The flow-through design of the direct-insertion membrane probe facilitates analyte transfer to the mass spectrometer and allows FIA to be applied to the analysis process, thereby greatly facilitating the task of automated, continual monitoring. Early experiments of this type employed a hollow fiber membrane probe with several different types of mass spectrometers. For example, when used with an ion trap, a wide variety of organic compounds could be detected in well water at pbb levels (19). Recently, a second-generation membrane probe (Figure 1)that employs a sheet membrane and incorporates membrane temperature control has been developed (20). This probe has been well characterized (20), and initial results of its utilization for fermentation monitoring have been reported (21). The purpose of this paper is to present results of the application of FIA and membrane introduction mass spectrometry to the on-line monitoring of fermentation by the Bacillus polymyxa and Klebsiella oxytoca organisms. For both systems, the major product of interest is 2,3-butanediol, with acetoin, acetic acid, ethanol, and carbon dioxide also resulting from the biochemical pathways. The appearance of these products is dependent on oxygen availability (22) as shown in Figure 2. The primary interest in these organisms is the production of 2,3-butanediol a t very low cost. In the case of Bacillus polymyra, interest is focused on the ability to produce high optical purity (98%) (R$)-(-)-2,3-butanediol (22),which is a valuable specialty chemical that can be used in the synthesis of a wide variety of other optically active compounds. For Klebsiella oxytoca, interest is based on the ability to produce 2,3-butanediol almost exclusively in the meso form (23),which can easily be dehydrated to methyl ethyl ketone, a commodity chemical of special interest in the polymer industry. Other reasons for the selection of these systems is prior knowledge of their characteristics (22) and their similarity to many other systems to which this analytical method may be applied. The overall goal of this project is fully automated on-line monitoring and feedback control of bioreactors using FIA with membrane introduction tandem mass spectrometry (MS/MS). This paper presents the on-line monitoring aspects of that goal. The approach presented here has several advantages over previous uses of mass spectrometry for bioreactor monitoring: (i) the superior direct-insertion membrane
1 pyruvate
acetoin I
CH,-
CH
I
- C - CH,
I
OH
I
CH,
-CH,
Flgure 2. Major products of fermentation with the Bacillus po/ymyxa and Klebsiella oxyfoca organisms shown in the order of appearance with decreasing oxygen availability.
probe design; (ii) flow injection delivery of the sample and the standard allowing accurate near real-time concentration determinations to be made; (iii) chemical ionization of the major products, thus allowing greater sensitivity and selectivity; and (iv) availability of the powerful analytical capabilities of tandem mass spectrometry to provide structural information that is not otherwise available (24).
EXPERIMENTAL SECTION A schematic diagram of the FIA instrumentation is given in Figure 3. The mass spectrometer is a Finnigan TSQ 45 triple quadrupole instrument with the standard electron multiplier detector and is operated via an INCOS data system. The membrane probe utilizes a sheet membrane held at a constant temperature of 72 "C for these experiments. The membrane is a dimethylvinylsilicone polymer (Dow Corning Silastic) with a thickness of 0.005 or 0.010 in. The performance characteristics of this membrane introduction device have been described previously (20)and me very similar to the original direct-introduction membrane probe (18). A stream of H20 flowing at 1 mL/min is pumped through the probe at all times by an Ismatec multi-
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ANALYTICAL CHEMISTRY, VOL. 62, NO. 17, SEPTEMBER 1, 1990
It
-
/"i
/wl.ta
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Flgure 3. Schematic diagram of the flow injection sample delivery
system.
channel peristaltic pump. Plugs (250 pL) of fermentation broth or standard solution are injected into the stream of H 2 0 by a Waters filter acquisition module (FAM). The FAM consists of a tangential flow filter (25)equipped with a Millipore 0.2-pm pore diameter Durapore filter membrane, a TTL controllable sample/standard switching valve, and a TTL controllable Reodyne six-port high-performance liquid chromatography sampling valve. All other valves were obtained from Biochem Valve Corp. Fermentation broth is pumped through the tangential flow filter and is returned to the reactor at a rate of 500-1000 mL/min by a Masterflex pump. By regulating the pressure on the high-flow side of the tangential flow filter, the flow rate of filtered fermentation broth is held at 1mL/min. Excess fitered fermentation broth is recycled back to the fermentor. A switchingvalve prevents standard solutions from being admitted to the fermentor. Sample and standard plugs can be derivatized or otherwise reacted on-line by adding the reagent of choice to the stream and by mixing in a minicolumn packed with 75-pm glass beads (Supelco) just prior to the membrane probe. The operation of the FIA sampling system proceeds by injecting a plug of filtered fermentation broth into the H20 stream followed approximately 2 min later by a plug of standard solution. The timing is based on a 1 mL/min flow rate throughout the flow injection system, which allows sufficient time for the responses of each of the compounds of interest to return to base-lime values. The FIA system allows one to perform reactions on-line with the fermentation broth and standard solutions just prior to delivery to the membrane probe, and the operation of this part of the system is completely independent of the rest of the measurement system. In the present state of development, all valves are controlled via a custom-built FIA control unit. This unit supplies power to the FAM and adjustable pulse sequences to the sampling and switching valves. The major liquid-phase products, 2,3-butanediol, acetoin, acetic acid, and ethanol, were monitored in the selected ion monitoring mode by using isobutane or methane chemical ionization at a source pressure of 0.40 Torr. Quantification was achieved by comparison with signals generated by an aqueous external standard solution that contanied only these four major product compounds (obtained commercially) and the same buffer (phosphate, pH = 6.8) used in the fermentation. Quantification assumes linear response as a function of concentration; this has previously been demonstrated (19,20) and was reestablished for the conditions used in this study. The observation of acetic acid required that the solutions be acidified (7) prior to encountering the membrane probe because only the neutral molecule (not the anionic form) can permeate the membrane. Acidification was
performed on-line by mixing a 1:l ratio of 0.1 M HC1 with a plug of fermentation broth or standard solution. Off-line gas chromatography with flame ionization detection (GC FID) analysis with a Varian 3700 packed column gas chromatography (2 mm X 6 f t packed with Supelco Chromosorb 101) was carried out to confirm the concentrations determined by mass spectrometry for these major products. Samples for GC FID analysis were taken directly from the fermentor, centrifuged, and injected directly into the gas chromatography without further treatment. Dissolved O2 was monitored by using 70-eV electron impact ionization. A degassed water stream flowing through the membrane probe provided a zero reference, and the standard was saturated with air to provide a 100% signal reference. The degassed water stream is maintained gas-free by sparging with He, and the air-saturated standard is produced by sparging with air prior to the experiment and then maintained by storing the solution in a closed container during the experiment. The confirmation of dissolved O2measurements was obtained by utilizing an O2 galvanic electrode (26). COz and O2 concentrations in the off-gases were also monitored by using electron impact ionization by introducing the off-gases through the calibration gas inlet of the mass spectrometer. Air was used as a standard in these measurements. The confirmation of off-gas analysis was achieved spectroscopically by infrared absorption (Infrared Industries Model 702) for COz and by paramagnetic O2 analysis (Beckman Model 775). Off-gasespass through a condenser and a filter/dryer to prevent interference in their measurement from volatile organics. The calculation of all gas concentrations (both off-gases and dissolved gases) assumes linear response, which was confmed for standard samples. The typical fermentation monitoring sequence occurs by first injection a plug of fermentation broth followed by a plug of standard and then an acidified pair of broth/standard plugs into the water stream flowing through the membrane probe as described above. During this time, m / z 47, 61, 89, and 91 ions produced by isobutane chemical ionization are monitored in the selected ion monitoring mode in order to follow ethanol, acetic acid, acetoin, and 2,3-butanediol concentrations, respectively. These ions were chosen on the basis of extensive tandem mass spectrometry studies on the authentic compounds. Then, the mass spectrometric conditions are changed to facilitate the observation of gases. The chemical ionization reagent gas is switched off (i.e., electron impact ionization is used), and the ions chosen to be monitored are m / z 28, 32, 40, and 44 in order to follow Nz,Oz, Ar, and COz,respectively. Another pair of broth/standard plugs is then directed to the membrane probe in order to measure dissolved gases. Finally, off-gas samples followed by air, as a standard, are introduced into the mass spelctrometer via the calibration gas inlet. At this point, the chemical ionization reagent gas is switched on and the monitoring cycle is repeated. Occasionally, especially during the latter part of the fermentation, the cycle is interrupted to obtain full chemical ionization mass spectra of the fermentation broth in an effort to detect trace components. Collision activation spectra (MS/MS spectra) were recorded under multiple-collision conditions (with Ar target gas) and 20 eV of collision energy in order to confirm the identity of the products. Collision activation spectra of some of the unassigned ions in the chemical ionization mass spectra, also recorded under 20-eV multiple-collision conditions, were examined in order to identify additional products. GC/MS was used to confirm unknown structural assignments with a Finnigan 4000 GC/MS system equipped with a 30 m X 20 pm Carbowax capillary column. The fermentation experiments were carried out in 1.7 L of medium and utilized an air flow rate of 0.5 L/min. An agitation rate of 750 rpm and an initial glucose concentration of 30 g/L were used for Bacillus polymyxa fermentation. In the fermentation with Klebsiella oxytoca, an initial glucose concentration of 20 g/L and agitation rates of 400-600 rpm were used. AU other fermentation parameters were set to their previously determined optima (22, 27, 28). RESULTS Experiments were carried out to test the performance of the FIA membrane introduction mass spectrometry system. A significant part of these experiments involved testing the performance of the membrane probe for the compounds and
ANALYTICAL CHEMISTRY, VOL. 62, NO. 17, SEPTEMBER 1, 1990
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Ethanol (m/z 47)
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Flgure 5. Intensities of m l z 47 and 61 (for ethanol and acetic acid, respectively) as a function of time during a Klebsiella oxytoca fer-
--4 m/2
2o
Figure 4. Collision activation (MS/MS) spectrum of m / z 91 ions derived from fermentation broth (top) compared with the spectrum from a 2,3-butanediol solution (bottom).
the concentration ranges of interest. Response curves as a function of concentration have been previously examined (20, 21) and were obtained again for all of the liquid-phase products over the concentration ranges observed in these fermentations. To a first approximation,responses were linear over the entire range of conditions and concentrations encountered. Matrix effects on membrane permeation, if any, were found to be minimal in the concentration ranges observed. However, it is worthwhile to note that significant nonlinearity and matrix effects are observed at higher concentrations (several weight percent or greater). Should the need arise to quantify products in the nonlinear concentration range, the wide dynamic detection range available combined with the ability of the FIA system to provide on-line dilution to the linear range via the mixer/reactor should make such measurements straightforward. It is also important that a sufficient abundance of ions of unique mass-to-charge ratio can be produced for each of the products to be monitored. Interference due to extensive fragmentation upon electron impact ionization and thus the lack of ions of unique mass-to-charge ratio has plagued previous attempts to follow the liquid-phase products of fermentation. The system presented here has overcome this problem by utilizing chemical ionization instead of electron impact ionization. While the long-term precision of chemical ionization is lower than that for electron impact ionization, possible errors resulting from reagent gas pressure drift are eliminated by standardizing each measurement. For the liquid-phase products monitored, isobutane chemical ioniza-
mentation. Injections alternate between fermentation broth and standard solution. Brothlstandard injection pairs alternate between the unreacted injection pairs and the acidified injectin pairs in which acetic acid may be observed.
+
tion yields primarily protonated molecules, (M H)+. Collision activation spectra (MS/MS spectra) have been obtained for each of the protonated molecules derived from the fermentation broth and from aqueous standard solutions in order to confirm that no interference exists in following the protonated molecule abundances. Figure 4 illustrates the collision activation spectra for the ions, m / z 91, formed from protonated 2,3-butanediol present in fermentation broth and in an aqueous standard solution. For all of the products monitored, identical collision activation spectra were recorded when this comparison (fermentation broth vs standard) was made. Another vital part of performance testing of the system is ensuring the rapid and reproducible delivery of sample. Repeatability was tested by measuring and comparing the sensitivity of a wide variety of compounds injected as both sample and standard plugs. Testing also included, but was not limited to, on-line acidification of the plugs. In all cases, excellent agreement was found. Typically, concentration determinations can be made with better than 5 % relative precision. This precision is available at a sampling rate at the present maximum of 15 times/h. This rate is limited only by the desire to maintain a 1 mL/min flow rate through the membrane probe. Higher flow rates and/or smaller injection plugs would allow considerably faster sampling rates, if needed, but experience suggests that the present capabilities are quite sufficient for fermentation monitoring. The sampling capabilities are reasonably illustrated in a case where it is desirable to alternate between delivery of acidified and unacidified analyte to the membrane probe. For example, Figure 5 shows mlz 47 and 61, the protonated molecular ions of ethanol and acetic acid, respectively, as a function of time
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ANALYTICAL CHEMISTRY, VOL. 62, NO. 17, SEPTEMBER 1, 1990
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Figure 8. Comparison of 2,3-butanedbl concentration measurements made by on-line membrane introduction mass spectrometry and by off-line gas chromatography for a Klebsiella oxytoca fermentation.
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Figure 6. Dissolved O2and major liquid-phase product concentrauons as a function of time for a Bacillus po/ymyxa fermentation.
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during a fermentation where every other sample/standard injection pair is acidified just prior to reaching the membrane probe in order to allow acetic acid to be monitored (7). Note that acidification has no effect on ethanol response other than decreasing signal intensity by the dilution factor introduced by adding reagent to the flow injection stream. Acetic acid, acetoin, 2,3-butanediol, and ethanol production during fermentation has been monitored as a function of time by using chemical ionization mass spectrometry. Concentration profiles for fermentation with Bacillus polymyxa are shown in Figure 6. The calculation of the concentrations assumes linear response and is based on the response given by standard solutions measured just after sampling of the fermentation broth. For Bacillus polymyxa fermentation, acetic acid, acetoin, and ethanol were monitored as the protonated molecules ( m / z 61,89, and 47, respectively) generated by methane chemical ionization. For 2,3-butanediol, the (M + H - H20)+ ion, m / z 73, produced by methane chemical ionization was followed since it was far more abundant than the protonated molecular ion. Representative data are presented in Figure 6. Dissolved oxygen was also monitored in order to gauge the progress of the batch reaction process. The measurement of dissolved oxygen was accomplished by following the molecular ion of O,, m / z 32, produced by electron impact ionization. Again, the calculation of concentration assumes linear response and is based on the response given by a standard solution (saturated with air) measured just after analysis of the fermentation broth. The data for Bacillus polymyxa fermentation (Figure 6) were obtained under manual control of all valves and with standards injected by hand. Similar experiments have been carried out during Klebsiella oxytoca fermentation. Figure 7 presents the concentration profiles of the fermentation broth constituents followed in these experiments. The experimental conditions utilized for monitoring Klebsiella oxytoca fermentation differ somewhat from those used for Bacillus polymyxa. Isobutane chemical
0
2
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Time
6
8
10
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Figure 9. Comparison of acetate concentration measurements made by on-line membrane introduction mass spectrometry and by off-line gas chromatography for a Klebsiella oxytoca fermentation.
ionization was employed, and this allowed the use of the protonated molecule, (M + H)+, m / z 91, for monitoring 2,3butanediol. In addition, all sampling was handled by the FIA control unit, which permitted it to be accomplished in a much more rapid and reproducible manner. An area of considerable importance to the development of this FIA mass spectrometric process monitoring system has been the accuracy with which concentration measurements can be made by using the system. Therefore, other common methods of measuring these values have been utilized for comparison purposes for the Klebsiella oxytoca fermentation presented above. For the liquid-phase products, off-line GC FID was used to verify concentration measurements, and in all cases excellent agreement was found. For example, Figure 8 shows the agreement found when 2,3-butanediol (the product of primary interest) in Klebsiella oxytoca fermentation was monitored by using off-line GC FID and on-line FIA membrane introduction mass spectrometry. Figure 9 illustrates the same comparison for acetic acid in the same Klebsiella oxytoca fermentation, thereby demonstrating that good quantitative information can be obtained by utilizing the mixer/reactor capabilities of the FIA system. A comparison of dissolved O2measurements was made between the FIA mass spectrometric method and galvanic O2electrodes constructed on-site. Excellent steady-state agreement was found, but in dynamic systems, the slow response times of the O2electrodes often resulted in a delay of several minutes in electrode signal response as a function of time. It is often important to measure the consumption of O2and the evolution of C 0 2 in order to estimate the state of a fermentation process, and as a consequence, CO, and O2 have been measured in the headspace or off-gases. Since it is desirable to perform this measurement in the same mass spectrometric system, the off-gases and a standard compressed air sample were alternately admitted to the calibration gas
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C 0 2 concentration measurements for the off-gases of a Klebsiella oxytoca fermentation made by mass spectrometry (points)
and by infrared absorption (iine).
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Figure 11. 0,concentration measurements for the off-gases of a Klebsiella oxytoca fermentation made by mass spectrometry (points) and by paramagnetic 0, analysis (line). inlet of the same mass spectrometer. The gases were admitted under the manual control of three valves used to introduce the two gases (off-gas and standard) and to pump out the tubing connecting the gases to the mass spectrometer. Electron ionization was used to follow m / z 28,32,40, and 44 for N2,02,Ar, and COP,respectively. Since the concentration of each of these gases can vary significantly, the concentration-dependent response for each component was considered to be the ratio of the intensity for the ion yielded by that component to the sum of ion intensities for all four components. The calculation of concentration assumes a linear response and is based on the response given by the standard (air) measured just after analysis of the off-gases. Good agreement was observed between the mass spectrometric and the other common measurement methods. Figure 10 shows C02concentrationsas a function of time for Klebsiella oxytoca fermentation measured by mass spectrometry (points) and by infrared absorption (solid line). The sharp drop in COz a t 7.5 h coincided with the addition of an antifoam agent. Figure 11 illustrates O2 concentration for the same fermentation measured by mass spectrometry (points) and by paramagnetic oxygen analysis (solid line). The differences present between the mass spectrometric and other methods of gas analysis are probably due to inconsistencies in the manual sampling used in the mass spectrometric system. The use of flow injection sampling with membrane introduction tandem mass spectrometry (MS/MS) has allowed the discovery of minor products that were not previously known to be present in measurable amounts. In the chemical ionization mass spectra of Bacillus polymyxa fermentation broth,
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Flgure 12. Collision activation (MS/MS) spectrum of mlz 59 ions derived from fermentation broth (top) compared with the spectrum recorded for an acetone solution (bottom). an unwigned ion at m / z 59 was observed. Therefore, collision activated dissociation (MS/MS) was used to fragment the m / z 59 ion (Figure 12, top), and it indicated that the source of the m / z 59 ion was acetone. Confirmation of this identification was made when the collision activation spectrum of the m / z 59 ion from an authentic sample (Figure 12, bottom) was recorded and found to be in g o d agreement with the one from the fermentation broth. Further confirmation was obtained by GC/MS. In Klebsiella oxytoca fermentation, it has been observed that the m / z 47 ion abundance produced by chemical ionization increases very slightly upon acidification of the broth plug. This observation (7) combined with some knowledge of the biochemical pathways likely to be utilized by this organism (29) suggests that formic acid was present. Unfortunately, small amounts of formic acid cannot be directly isolated by isobutane chemical ionization tandem mass spectrometry in the presence of relatively large amounts of ethanol because protonated ethanol (also m / z 47) yields collision activation fragments at the same mass-to-charge ratios. Ethanol does, however, have a fragment in its collision activation spectrum at m / z 27 that is not possible for formic acid. When acidified, the collision activation spectra of m / z 47 from the fermentation broth shows small increases in the abundance of all fragment ions except m / z 27, which is consistent with the presence of formic acid in the broth containing ethanol. GC/MS was used to confirm the presence of formic acid. There are probably many more trace compounds present in these fermentation broths, but only a preliminary study has been carried out so far because most effort thus far has
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been directed toward the development of the quantitative methodology.
CONCLUSIONS The results here clearly illustrate the ability of flow injection sampling with membrane introduction mass spectrometry to be used to monitor and quantify the major products and the metabolites of Bacillus polymyxa and Klebsiella oxytoca fermentations. It has been shown that membrane introduction mass spectrometry can provide continual on-line quantification of the liquid-phase products of comparable quality to off-line gas chromatography. Furthermore, it has been demonstrated that mass spectrometry is an excellent method for monitoring the dissolved gases and off-gases in these fermentations. The results also show that membrane introduction tandem mass spectrometry can enable one to detect the presence of trace metabolites. ACKNOWLEDGMENT The help of J. Williams, K. Cox, and R. Jullian in preparing this document is acknowledged. Registry NO. 02,7782-44-7;COP,124-38-9;acetic acid, 64-19-7; acetoin, 513-86-0; 2,3-butanediol, 513-85-9;ethanol, 64-17-5. LITERATURE CITED (1) Riebe, M. T.; Eustace, E. H. Anal. Chem. 1990, 62, 65A. (2) Ruzicka, J.; Hansen, E. H. Anal. Chlm. Acta 1988, 779, 1. (3) Cooks, R. G.; Bier, M. E.; Brodbelt. J. S.; Tou, J. C.; Westover, L. B. U S . Patent 4,791,292,1989. (4) Hoch, G.; Kok, B. Arch. Blochem. Biophys. 1983, 707, 160. (5) Llewellyn, P. M.; Lmlejohn, D. P. U S . Patent 3,429,105,1969. (6) Westover, L. 6.; Tou, J. C.; Mark, J. H. Anal. Chem. 1974, 46, 568. (7) Weaver, J. C.; Abrams, J. H. Rev. Sci. Inshum. 1979, 50, 478. (8) Dheandhanoo, S.;Dulak, J. Rapid Common. Mass Spectrom. 1989, 3,175.
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RECEIVED for review February 20, 1990. Accepted May 14, 1990. Support from the National Science Foundation (EE 7-87 12867) is acknowledged. Support from the Emil Aaltonen Foundation and Suomen Kulttuurirahasto is acknowledged
(T. K.).
Variations in Detection Efficiency of Halobenzenes Studied by Using Gas ChromatographylLaser Ionization Mass Spectrometry: Correlation with Excited-State Lifetimes Charles W. Wilkerson, Jr.,l and James P. Reilly* Department of Chemistry, Indiana University, Bloomington, Indiana 47405
Fluoro-, chloro-, bromo-, and lodobenzene are studied wRh gas chromatographyAaser Ionization mass spectrometry. Picosecond llght pulses are found to be much more effective at lonirlng the heavier halogenated species (Cl, Br, and I ) than are nanosecond pulses. I n a separate experiment, the S, exclted-state IHetlmes of these species are measured by uslng pkosecond pump-probe laser Ionization mass spectrometry. The variation of the observed ilfetlmes expialns the GC/MS results.
INTRODUCTION The laser multiphoton ionization efficiency of a molecule is a function of a number of parameters, and it is necessary Current address: Los Alamos National Laboratory,Los Alamos, NM 87545.
to understam. them if laser ionization techniques are to become viable tools for quantitative analysis. Arguably, the two molecular characteristics that most influence laser-induced ion yield are the ground-state absorption cross section and the lifetime of the intermediate electronic state that is initially excited. Laser ionization is significantly more efficient if it proceeds through a resonant state, and currently available lasers can be tuned to absorption features of many molecules. Nevertheless, different types of transitions are characterized by different line strengths, and each exhibits an absorption contour over which the cross section can vary significantly. If a species has a radiative lifetime that is very short compared to the laser pulse duration, it may relax after excitation and not be ionized. Pulsed lasers used in most laser ionization experiments have multinanosecond durations. Therefore, molecules with subnanosecond lifetimes can be difficult, if not impossible, to detect (1-6). This problem can be solved by employing laser pulses that are at least as short as the excited-state lifetime. We have recently reported an improve-
0003-2700/90/0362-1804$02.50/00 1990 American Chemical Society
