Membrane Introduction Mass Spectrometrv A.
Tapio Kotiaho', Frants R. Lauritsen2, Tarun K. Choudhury, and R. Graham Cooks Department of Chemistry Purdue University West Lafayette, IN 47907
George T. Tsao Laboratory of Renewable Resources Engineering Purdue University West Lafayette, IN 47907
The introduction of samples into the mass spectrometer is different from sample introduction into most other types of analytical instruments. The differences lie less in the manipulations involved than in the danger that the vacuum will be breached. Not the least attractive feature of the membrane introduction systems dis cussed in this article is that they go a long way toward eliminating this concern while allowing either batch 'Permanent address: The Chemical Laboratory of the Technical Research Center of Finland. 2Permanent address: Odense University, Denmark.
0003-2700/91/0363-875A/$02.50/0 0 1991 American Chemical Society
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or continuous sample introduction. Another major advantage of membrane inlets is the high selectivity of membranes (particularly silicone membranes) toward organic compounds relative to water. Aqueous solutions can therefore be examined directly, and this feature has led to experiments in which chemical or biochemical reactors are examined continuously, on line, to follow their changing chemical environments. Because of these features, membrane introduction mass spectrometry (MIMS) is an increasingly popular sampling method both in analytical MS and in some areas less familiar to analytical chemists, such as fer-
through measurements of oxygen and carbon dioxide (1). It was later used extensively for analysis of photochemical systems (2) and for other physiological studies (3),including in vivo measurements of dissolved gases and organic compounds in blood (3-6). Another early application of membranes in MS was the use of a polymer as an interface in GC/MS (7),which depends on the low permeability of helium relative to organic compounds. The first application of MIMS in fermentation monitoring, was a n published by Reuss et al. (8), important development, and fermentation monitoring with MIMS is commonly used today (2, 9).
mentation monitoring and microbiology. In this REPORT we will review the basic theory and present some applications of MIMS in fermentation monitoring and environmental analysis. MIMS dates back t o t h e early 1960s, when it was introduced by Hoch and Kok for in situ analysis of the kinetics of photosynthesis
The use of hollow-fiber capillary membranes by Tou et al. in 1974 (IO) formed the basis for many subsequent advances. These researchers immersed sealed hollow -fiber capillary membranes in aqueous solutions and evacuated the tubes via a mass spectrometer to transfer dissolved or ganics into the instrument. A recent development of MIMS, the direct in-
ANALYTICAL CHEMISTRY, VOL. 63, NO. 18, SEPTEMBER 15, 1991 * 875 A
REPORT sertion membrane probe (11-13), resulted from the pioneering work of Tou et al. Direct insertion membrane probes, described below, are almost a s simple as the probes normally used to introduce solid samples into a mass spectrometer, but they allow continuous fluid introduction. MIMS has received less attention than alternative fluid introduction methods such as thermospray (14), electrospray (15), or continuous - flow fast atom bombardment (CF-FAB) (16). The operating principles of all these methods are compared in Figure 1. Thermospray, electrospray, and CF-FAB methods are of interest because they allow characterization of large biomolecules, especially peptides and proteins, by MS. MIMS does not yet offer this capability; however, membrane introduction is a simple, versatile, and extremely sensitive method for the introduction of small inorganic and organic compounds from aqueous solutions. Figure 1 also demonstrates that MIMS is the easiest fluid introduction method to interface with a mass spectrometer. Additional pumping, the use of high voltages, and heating or nebulization of the sample stream are not necessary, and conventional electron ionization (EI) and chemical ionization (CI) sources can be operated without modification. Membrane introduction devices can easily be interfaced with quadrupole, ion trap, or magnetic sector instruments. One enormous practical advantage is that they are very clean introduction systems-the ion source does not have to be removed for frequent cleaning, as is the case with other introduction methods. Devices The commonly used membrane introduction devices can be divided into two categories (Figure 2). The first includes devices t h a t are actually mounted in the fluid sample, reaction vessel, or fermentor (2, 9) (Figure 2a), whereas the second category includes devices in which the membrane is mounted within or near the ion source of the mass spectrometer (Figure 2b) (11-13).The first type of interface has been applied success fully in gas monitoring (2, 9),for example, in studying the kinetics of chemical (17) and biological reactions (18-20). The main advantage is that these interfaces are very easy to incorporate into any kind of reactor. Long transfer lines can be used, and one instrument can be used to analyze gases from several reactors (21). The long transfer lines, especially 876 A
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rFigure 1. Comparison of techniques for introducing and ionizing liquids in a mass spectrometer. (a) Thermospray, using heat (AH)and electrospray, using an electrical potential ( V ) . (b) CF-FAB, using energetic atoms or ions. (c) Membrane introduction, using El.
if unheated, are also the greatest drawback of these interfaces in the analysis of condensable organic compounds. Poor response times (9, ZZ), memory effects (ZZ), and analyte dilution may occur because of condensation along the lines (23).Recently a theoretical model was used to describe the influence of adsorption and desorption processes at vacuum surfaces on the signals observed (12). The modeled data, compared with experimental measurements, showed t h a t even with short connection tubes (centimeters in length) a slow transient signal resulting from inter actions between the analyte and surfaces i n vacuum can be observed whenever polar compounds at low concentrations are analyzed. Such problems can be reduced or eliminated by heating transfer tubes or, as recently demonstrated, by using the flow-through mode of operation. In the latter case a direct insertion probe containing the membrane is introduced into the ion source of a mass spectrometer (Figure 2b) (11-13). In the flow-through mode, solution flows across the inside surface of the membrane while the out-
ANALYTICAL CHEMISTRY, VOL. 63, NO. 18, SEPTEMBER 15,1991
side surface is exposed to the vacuum of the mass spectrometer. This operating principle, together with the closeness of the membrane of the ion source, reduces problems such as analyte dilution and memory effects. Two additional advantages are the very low detection limits achieved and compatibility with continuous monitoring, especially when one uses flow injection analysis (FIA) (24) methods of sample handling. Typical characteristics of one type of direct insertion membrane probein which the membrane takes the form of a flat sheet (13)-are presented in the box. In general, interfacing a membrane introduction de vice to any kind of mass spectrometer is simple; in the worst case, only minor modifications need to be made to the existing ion source. Theory In MIMS, the introduction of analyte into the mass spectrometer is a result of transport through a polymer membrane. This permeation process involves absorption into the membrane, diffusion through the membrane, and evaporation from the
sure, the equations can be expressed as
c I
I
Figure 2. Comparison of (a) flow-by and (b) flow-through conllyurations
UI
membrane introduction devices. (a) The membrane is mounted inside the reactor and evacuated. (b) The sample is continuously withdrawn from the reactor and transported to a membrane inlet mounted inside or near the ion source.
Typical characteristics of a sheet direct insertion membrane probe Membranc Me rnicKness/area Detection limits Response timea Operation temperature
Silicone membrane (Dow Corning silastic sheeting, medical grade) 0.25 mm or 0.13 mm/30 mm2 -$ Benzene: 10 ppb; PFBOA de-:": formaldehyde: 1 ppb 8s 70 "C
.
Response time is the time it takes the signal to reach 50% of its maximum intensity. The respons is for propan-2-one, 250-pL injections, flow rate of 0.9 mumin.
a
* External standard quantitation modc
membrane surface into vacuum. Each step depends primarily on the molecular properties of the analyte and the membrane material. In addition, the temperature of the membrane can have a significant effect on permeation rates. Use of the right membrane thus can result in a large increase in mass fraction of analyte, relative to the mass fraction of the solvent that passes the membrane. Aqueous solutions are best analyzed
with hydrophobic polymers, which discriminate against water. The membranes typically used are organic polymers, such as polyethylene and Teflon for gas monitoring, and silicone - based polymers for organics in aqueous solution and in air. Mathematically, the permeation process can be described by Fick's diffusion equations. Assuming that the constants for solvation and diffusion are independent of partial pres-
Im(x,t) = -AD[aC,(x,t)/ax] (1) ac,(x,t)/at = D[a2Cm(x,t)/ax21 (2) where I,(x,t) is the analyte flow inside the membrane (molls), A is the membrane surface area (cm2), D is the diffusion constant (cm2/s), Cm(x,t) is the concentration inside the membrane (mol/cm3),x is the depth in the membrane (cm), and t is time (s). Equation 1 describes the rate of molecular flow inside the membrane; Equation 2 describes the r a t e at which concentrations change with time. In many MIMS systems the membrane is exposed to a continuous feed of analyte. For a step change in concentration, the concentrations inside the membrane can be calculated as a function of time from Equation 2 by the method of separation of variables. The flow through the membrane is then found by inserting the calculated concentration into Equation l and solving it a t the vacuum side of the membrane (x = 1, where I is the membrane thickness). I n this way the 10-90% rise time is found as
t,,-,,,
= 0.237(Z2/D)
(3)
Using Henry's law (S = Cm/PS)to express concentrations in partial pressures, one can calculate the steadystate flow rate as
I,, = ADS(P$Z)
(4)
where I,, is the steady-state flow through the membrane (molls), S is the solubility constant (molltorr cm3), P, is the vapor pressure of the analyte on the sample side of the membrane (torr), and I is the membrane thickness (cm). It is interesting that the steadystate flow depends on the product of t h e solubility and diffusion constants, whereas the rate a t which measured analyte concentrations change depends only on the diffusion constant. For most nonpolar compounds the solubility increases and the diffusivity decreases in a homologous series of samples as the carbon chain length increases. This is observed experimentally as longer response times (diffusion constant decreases) and lower detection limits (solubility constant increases more than the diffusion constant decreas es). A vital parameter is the membrane thickness. Equations 3 and 4 show that doubling the membrane thickness results in a fourfold increase in response time and a reduction in sensitivity by a factor of 2.
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Continuous feed of analyte to the membrane demands relatively large sample volumes, which may not be possible in some reaction and fermentation monitoring experiments when the sample cannot be returned to the reactor. For such purposes, FIA sampling h a s been combined with a membrane inlet (25),and the membrane is exposed to the analyte for only a limited period of time. A mathematical analysis of such sys tems was recently developed (26). Figure 3 shows a computer simulation of the analyte concentration on the sample side of the membrane together with its rate of flow through the membrane as a function of time. The simulation shows excellent
Figure 3. Typical simulated dynamil, responses of the membrane introduction/FlA apparatus for a dispersed input. Curve A is the analyte concentration; curve B is the analyte flux through the membrane. (See Reference 26 for the experimental details.)
agreement with experimental r e sults. Typical of this kind of sample delivery is an asymmetric response curve with fast rise times and slower fall times.
Fermentation monitoring Because MIMS can be used for online monitoring, fermentation moni toring is a primary application of this technique (2, 9). Fermentation monitoring experiments were first report ed by Reuss et al. in 1975 (8).Since then, in most fermentation experiments E1 has been used in a singlestage quadrupole mass spectrometer with a membrane inlet located in the bioreactor and connected to the mass spectrometer through a long vacuum line. Although these systems can detect 878 A
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gases and volatile compounds within the reactor, they have some drawbacks: the chromatographic effect th,at occurs in the long vacuum lines, poor response time, and inability to measure standards between samples. Moreover, the extensive fragrnentation of molecular ions attributable to E1 interferes with the monitoring of volatile compounds. Because of these and other factors, we suggest the following guidelines regarding the construction of membrane inlets for fermentation monitoring. First, the membrane should be located as close as possible to the ion source of the mass spectrometer to minimize response times and memory effects. This can be achieved by using direct insertion membrane probes. Second, FIA methods for sampling t h e fermentation broth should be used for on-line quantitation of the fermentation products. Third, the fermentation broth should be filtered prior to analysis with the membrane probe. Fourth, because acids and bases can pass through the membrane in the nonionic form only, it may be necessary to change the pH of the fermentation liquid on line to detect these compounds. Fifth, the extensive fragmentation caused by the use of E1 can be avoided by using CI. Finally, because gases such as oxygen and carbon dioxide a r e best measured using EI, it is important to use a mass spectrometer with both E1 and CI capabilities. To fulfill these demands a fully automated FIA sampling system, which allows on-line quantitation of the liquidphase products and the off-gases, was developed and adapted to a membrane inlet (25). As a n example of fermentation monitoring, consider data obtained during the fermentation of the microorganisms Bacillus polymyxa and Klebsiella oxytoca (25, 27-30). Bacillus polymyxa produces high optical purity (R,R)-2,3-butanediol (32), which is a valuable specialty chemical that can be used in the synthesis of other optically active compounds. Interest in KZebsiella oxytoca is based on its ability to produce 2,3- butanediol almost exclusively in the meso form (32), which can easily be dehydrated to methyl ethyl ketone-a commodity chemical of interest in the polymer industry. The biochemical pathways for the production of 2,3 -butanediol from these organisms are well known (33). Figure 4 shows the overall pathways with carbon dioxide, acetic acid, acetoin, 2,3 -butanediol, and ethanol as the major end products. The rela-
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nlz 91
Figure 4. Biochemical pathways for the formation of the main fermentation products of Bacillus polymyxa and Klebsiella oxytoca. Note that oxygen availability determines which products are favored. The major ions observed for these products during methane CI are also shown.
tive proportions of these products depend on the availability of oxygen, as illustrated. Monitoring the production of these products while control ling oxygen availability in response to the analytical data may therefore lead to higher yields of 2,3-butanediol. The major fermentation products were quantified on line using external standards with the automated FIA method of sampling (25, 27-30). These methods provide a sampling rate sufficient to give smooth curves displaying the concentration of the products during a complete fermentation. The concentration profiles of the products acetoin, ethanol, and acetic acid were derived from the abundances of protonated molecules, (M + H)+, appearing at m/z 89, 47, and 61, respectively, generated by methane CI. For 2,3-butanediol the (M + H-H,O)+ ion (m/z 73) was mon-
itored because it was far more abundant than the protonated molecule. As is typical, the mass spectrometer was used in the full scan mode so that all compounds were monitored during fermentation. Examples of the data extracted from these scans for a number of compounds of special interest, including acetic acid and 2,3-butanediol, are shown in Figure 5. The concentrations measured by on-line MIMS were confirmed by offline GC using flame ionization detection. During such on-line monitoring experiments, ferment a tion off - gases and dissolved gases can also be measured using a mass spectrometer. In this study the off-gases were measured by introducing the off-gas and a standard compressed air sample directly to the calibration gas inlet of the triple quadrupole mass spectrometer (25). Off-gas analysis pro vides useful information about the state of fermentation, as emphasized by Heinzle a n d co-workers ( 3 4 ) . Comparison of oxygen concentration in the feed stream with that of the gas stream produced by the aerobic fermentation and the concentration of carbon dioxide in the exit stream gives information about the respiratory state of the bioreactor. Concentrations of carbon dioxide and oxygen measured as a function of time by MS agree well with IR absorption and paramagnetic analyses, respec-
tively (25).The measurement of dissolved gases can also give valuable information on the enzyme kinetics, as demonstrated in novel stoppedflow MIMS experiments (35). MIMS is also useful for the identification of volatile metabolites produced in microbiological processes. Here, additional molecular structural information is obtained by using MS/ MS (25, 36). Differences i n membrane response times also provide some clues about molecular structure (36).MS/MS has been used on line in screening fermentation processes, as described in the example below, and in identifying volatile metabolites produced by microbial c u l t u r e s grown in small screw-capped tubes (36).In the latter case 2-3 mL of the growing culture is simply transferred to a small measuring cell fitted with a membrane inlet and analyzed. The MS/MS capability of a triple quadrupole mass spectrometer, although not essential to this type of analysis, does make it possible to confirm product identity by compar ing it with MS/MS spectra of authentic compounds. This capability is also the basis for analysis of optical isomers (27, 30);for example, the (R,R)and mem forms of 2,3-butanediol were distinguished by recording MS/ MS product spectra of phenylboric ester derivatives of these compounds. These derivatives were synthesized by mixing the fermentation liquid
with phenylboric acid in a small reaction chamber (Figure 6a) before the liquid was introduced to the membrane probe. Figure 6b compares the product spectra of the two isomers. A large difference in the ratio of the fragments at m/z 133 and m/z 73 for the two derivatives makes isomer distinction possible. MS/MS also facilitates screening the fermentation liquid for unknown metabolites. A simple example is the occurrence of
1
Mass spectromete
133
1
/ MH+ 177
Figure 6. Distinction of different optically active forms of 2,3-butanediol. Figure 5. Concentrations of products and feed oxygen as a function of time for a Klebsiella oxytoca fermentation. Data were obtained using a fully automated membrane introduction/FlA apparatus. A: ethanol, B: acetic acid; C: 2,3-butanediol; D: acetoin, E: oxygen.
(a) Formation of phenylboric ester derivatives of 2,3-butanediol. (b) MWMS product spectra for the derivatives of meso-2,3-butanedioland (R,R)2,3-butanediol. The large difference in the ratio of the fragments at m/z 133 and m/z 73 allows distinction of these isomers. (Adapted with permission from Reference 27.)
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REPORT acetone in the Bacillus polymyra fermentation recognized through the presence of a n unassigned m/z 59 ion in the CI mass spectrum and confirmed by collision-activated dissociation (25). The FIA/MIMS system should be capable of on-line feedback control of bioreactors. This has been demonstrated using t h e oxygen partial pressure as the control parameter (28-30).From ferment a tions without feedback control it is known that the production rate of acetic acid is related to the availability of oxygen in the fermentation broth (31).A decrease in acetic acid production rate reflects a decrease in oxygen availability (large oxygen consumption) and a n increase in the formation rates of acetoin, 2,3 - butanediol, and ethanol. Furthermore, ethanol is known to be produced at high rates under anaerobic conditions. The amounts of the products acetoin and 2,3 -butanediol might therefore increase if the oxygen feed were adjusted during the fermentation, because this control is based on the measured rate of acetic acid formation. Figure 7 shows results of monitoring by such a n experiment (28-30). After approximately 2 h, the production rate of acetic acid passed a preset upper limit and the oxygen partial pressure was slowly reduced until a lower limit in the production rate was reached (hour 5). At this point the production r a t e of 2,3butanediol had started to decline. To maintain a n adequate production rate of 2,3 - butanediol, the oxygen feed was increased again. As Figure 7 shows, the production rate of 2,3butanediol increased again until the oxygen availability became too high (hour 6). Although only crude control was achieved, this experiment is significant; it is the first demonstration of feedback control in conjunction with the powerful continuous analyt ical capabilities of FIA/MIMS.
Replacement of water by the corresponding amine solution occurs at the time marked with the star. Addition of calcium hypochlorite solution (small arrow) and HCI solution (heavy arrows) occurs at the times indicated. (Adapted from Reference 47.)
Environmental monitoring Direct determination of environmentally significant compounds from aqueous solutions is another major application of MIMS, as proven by the recent increase in relevant publications (37-43).These types of exper iments demand detection of specific compounds at levels far lower than those needed for bioreactor monitor ing. Most studies involve the analysis of aqueous solutions, but work has also been published on the determination of environmentally significant compounds directly from air (41).The simplicity of the membrane introduc-
tion device has led to experiments aimed at developing it as an on-site monitoring system (39).The membrane introduction devices used in environmental applications are primarily of the flow-through type (3842).The mass spectrometer of choice is a quadrupole instrument (38,39, 41,431, an ion trap (38,42,43),or, less frequently, a magnetic sector instrument (37,40). The most important advantage of MIMS for trace analysis of organic compounds from aqueous solutions is that the analytes can be measured
880 A
I
-
Y-
Acetic acid
\
Oxyien
,
/' *r ,Acetoin
I
I
Figure 7. Fractional yields (product formation rate/Z product formation rates) of the products and feed oxygen concentration during a feedback-controlled experiment.
Figure 8. On-line monitoring of individual products of chlorination reactions of 0.2 ppm aniline.
ANALYTICAL CHEMISTRY, VOL. 63, NO. 18, SEPTEMBER 15,1991
directly a t part - per - billion levels without any preconcentration or de rivatization steps, which allows continuous monitoring. The detection limits obtainable with most membrane introduction devices are some where between a few parts per billion and a few hundred parts per billion, depending on the analyte. The lowest detection limits achieved to date have been published by Slivon et al. (43).They used a silicone rubber hollow-fiber membrane in which aqueous sample solution flows continuously over the membrane while the
’’
The way you inject sample right at the start of an LC analysis has a major effect on the final results. In fact, using the right injection technique at the start is essential for high accuracy. You can learn all about proper techniques from Rheodyne literature. Three publications cover the subject at differing depths: 1.Most popular is “Tips on LC Injection,”a concise single-sheetthat clears up common misconceptions and guides you through several
critical steps. 2. If you want a deeper understanding, read “Technical Notes 5, Achieving Accuracy and Precision with Rheodyne Sample Injectors.” This 6-pageguide describes the subtle processes that occur during sample injection and explains why certain techniques are recommended. 3. Finally, Rheodyne offers little 5 cm x 10cm booklets you can hang around an injector handle for every user to read. These mini-manuals, CIRCLE 122 ON READER SERVICE CARD
one for each model, contain brief reminders of what to do -and what not to do- when using the injector. For copies use the reader service card, call your Rheodyne dealer, or contact Rheodyne,Inc., PO. Box 996, Cotati, California 94931, U.S.A. Phone (707) 664-9050. Fax 707-664-8739.
REPORT inside of the membrane is continuously purged with helium directed into the ion source of a mass spectrometer. A detection limit of 0.1 ppb was measured for benzene and for vinyl chloride. For quantitation purposes, a wide linear dynamic range is desirable for any analytical method. In the case of MIMS, linear dynamic ranges are highly compound dependent; they start at part-per-billion levels and extend t o several orders of magnitude higher concentrations. The dynamic ranges obtained are sufficient for most MIMS applications. For example, in drinking water samples it has been demonstrated that chloroform has a linear dynamic range from 10 ppb to 100 ppm (IO),and in swimming pool samples it has been measured at concentrations as low as 6 ppb (44). The direct analysis capability of MIMS also allows reliable determination of compounds that are both toxic and labile. Examples include inorganic (45,46)and organic chloramines (47). Both types of compounds can be present in drinking water, and inorganic chloramines themselves a r e used a s disinfecting agents. MIMS allows on-line monitoring of the formation and interconversion of these toxic compounds (46, 47). Recently in our laboratory we demonstrated the direct determination of acrolein, acrylonitrile, and pentafluorobenzyl hydroxylamine (PFBOA) derivatives of aldehydes from aqueous samples ( 4 8 ) . E1 and MSiMS were used in the determination of acrolein and acrylonitrile. Aldehydes were derivatized in aqueous solutions and subsequently analyzed using negative CI together with membrane introduction. This combination adds to the advantages of MIMS the very low detection limits obtainable in electron capture negative CI. As a n example of the determination of labile compounds directly from aqueous samples, Figure 8 illustrates a n experiment in which the formation of mono-, di-, and trichlorinated derivatives of aniline are monitored on line (47). The results were recorded using isobutane CI by measuring the protonated aniline and two different isotopic forms of protonated mono-, di-, and trichlorinated reaction products. In Figure 8, only the signal of the molecular ions containing the 35Cl isotope is presented. T h e m e a s u r e m e n t s were p e r formed as follows. First, water was pumped t h r o u g h t h e m e m b r a n e probe and then replaced by an aqueous solution of 0.2 ppm amine (indi882 A
cated by the star), raising the pH to 10-11. Calcium hypochlorite solution was then added (indicated by the small arrow), and the pH was lowered to < 1 by adding HC1 (indicated by the heavy arrows). Figure 8 demonstrates that the efficient chlorination of aniline i n aqueous solutions requires acidification and that the end product of the reaction is trichloroaniline. MSiMS was used to confirm that the chlorination occurred on the benzene ring, whereas i n t h e case of aliphatic amines, chlorination occurs on the nitrogen atom (47). Note also that, because formation of all the possible reaction products can be determined, the starting amine concentration of 0.2 ppm represents the concentration limit at which the reaction sequence can be followed when continuous monitoring MIMS is used.
Future perspectives MIMS displays several outstanding characteristics: rapid response times, ease of automation, very high chemical specificity, simplicity, adaptability to any type of instrument, and low impact on instrument cleanliness. The detection limits achieved are exceptionally low for small (molecular weight < 3001, less polar organic compounds (e.g., less polar than 2,3butanediol), and on-line derivatization can be used to convert analytes into suitable forms. Unfortunately, the types of compounds t h a t can be examined are limited. To examine polar or even ionic compounds, alternative membranes with different selectivities must be developed. This requires the synthesis of new polymers, evaluation of their physical properties, and their incorporation into devices that allow on-line analysis. The technique used for on-line detection of gases and small, less polar organic compounds, however, can be regarded as having reached maturity; its usefulness has already been demonstrated in analytical chemistry as well as disciplines such as process monitoring and control, microbiology, and medicine. Because of the simplicity of the technique, in the near future we can expect a large increase in the use of MIMS i n t h e a r e a s already d i s cussed, especially in fermentation and environmental monitoring.
References (1) Hoch, G.; Kok, B. Arch. Biochem. Biop h y ~ .1963, 101, 160-70. (2) Degn, H.; Cox, R. P.; Lloyd, D. Methods Biochem. Anal. 1985, 31, 165-94. (3) Woldrine, S . I. Assoc. Adv. Med. I n strum. 1 9 7 6 4, 4j-56. (4) Brantigan, J. W.; Dunn, K. L.; Albo, B. J. Appl. Physiol. 1976, 40, 443-46. 15) Lundsgaard, J. S.; Jensen, B.; Groenlund, J.J. Appl. Physiol. 1980, 48, 376-81. (6) Brodbelt, J. S.; Cooks, R. G.; Tou, J. C.; Kallos, G. J.; Dryzga, M. D. Anal. Chem. 1987, 59, 454-58. 17) Llewellvn. P. M.: Littleiohn. D. P. U.S. Pate&, 3 429 105, 1969*. ( 8 ) Reuss, M.; Piehl, H.; Wagner, F. Eur. J. Appl. Microbiol. 1975, I , 323-25. 19) Heinzle, E.; Reuss, M. Mass Spectrometry in Biotechnological Process Analysis and Control; Plenum Press: New York, 1987. (10) Westover, L. B.; Tou, J. C.; Mark, J. H. Anal. Chem. 1974,46, 568-71. (11) Bier, M. E.; Cooks, R. G.; Brodbelt, J. S.; Tou, J . C.; Westover, L. B. US. Patent 4 791 292, 1989. 112) Lauritsen, F. R. Int. J. Mass Spectrom. Ion Proc. 1990, 95, 259-68. 113) Bier, M. E.; Kotiaho, T.; Cooks, R. G. Anal. Chim. Acta 1990,231, 175-90. (14) Arpino, P. Mass Spectrom. Rev. 1990, 9 R.?1-69 --(15) Fenn, J . B.; Mann, M.; Meng, C. K.; Wong, S.F.; Whitehouse, C. M. Mass Spectrom. Rev. 1990, 9, 37-70. (16) Caprioli, R. M. Anal. Chem. 1990, 62, 477 A-485 A. ~~. 117) Degn, H.; Lauritsen, F. R. J. Phys. Chem. 1989, 93, 2781-83. (18) Calvo, K. C.; Weisenbereer, C. R.; Anderson, L. B.; Klapper, MTH. J A m . Chem. Sot. 1983, 105, 6935-41. (19) Jensen, B. B.; Cox, R. P. In Methods in Enzymology, Volume 167, Cyanobacteria; Packer, L.; Glazer, A. N., Eds.; Academic Press: New York, 1988; pp. 467-74. (20) Das, J.; Timm, H.; Busse, H . G . ; Degn, H. Yeast 1990, 6, 255-61. (21) Bohatka, S.; Berecz, I.; Langer, G.; Szilagyi J. Vakuum-Technik 1986, 35, 79-86. (22) Brodbelt, J. S.; Cooks, R. G. Anal. Chem. 1985, 57, 1153-55. (23) Schmidt, W. J.; Meyer, H-D.; Schugerl, K.; Kuhlman, W.; Bellgardt, K-H. Anal. Chim. Acta 1984, 163, 101-09. (24) Ruzicka, J.; Hansen, E. H. Flow Injection Analysis, 2nd ed.; John Wiley and Sons: New York, 1988; Vol. 62. 125) Hayward, M. J.;Kotiaho, T.; Lister, A. K.; Cooks, R. G . ; A u s t i n , G . D . ; Narayan, R.; Tsao, G. T. Anal. Chem. 1990, 62, 1798-1804. 126) Tsai, G-J.; Austin, G. D.; Syu, M. J.; Tsao, G. T.; Hayward, M. J.;Kotiaho, T.; Cooks, R. G. Anal. Chem., in press. 127) Lister, A. K. Ph.D. Thesis, Purdue University, 1990. 128) Hayward, M. J. Ph.D. Thesis, Purdue University, 1990. (29) Austin, G. D. Ph.D. Thesis, Purdue University, 1990. I301 Kotiaho, T.; Hayward, M. J.; Choudhury, T. K.; Lister, A. K.; Cooks. R. G.; Austin. G. D.: Svu. M. J . : Tsao. G. T. Presented a t t h e 39th Annual Conference on Mass Spectrometry and Allied Topics, Nashville, TN, May 1991. - 1
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Our work on environmental and bioreactor monitoring was supported by the Environmental Protection Agency (R.G.C., EPA CR-815749-01-0) and the Iiational Science Foundation (G.T., NSF E E - 7 87 128671, respectively. The contributions to these developments by the co-workers cited in the references are gratefully acknowledged. The support provided for T. K. by the Emil Aaltonen Foundation and for F.R.L. by the Danish Center for Process Biotechnology is much appreciated.
ANALYTICAL CHEMISTRY, VOL. 63, NO. 18, SEPTEMBER 15, 1991
(31) de Mas, C.; Jansen, N. B.; Tsao, G. T. Biotechnol. Bioeng. 1988, 31, 366-77. (32) Qureshi, N.; Cheryan, M. Appl. Microbiol. Biotechnol. 1989, 30, 440-43. (33) Gottschalk, G. Bacterzal Metabolism, 2nd ed.; Springer-Verlag: New York, 1986. (34) Heinzle, E.; Oeggerli, A.; Dettwiler, B. Anal. Chim. Acta 1990,238, 101-15. (35) Carlsen, H. N.; Joergensen, L.; Degn, H. Appl. Microbiol. Biotechnol. 1991, 35, 124-27. (36) L a u r i t s e n , F. R.; Nielsen, L. T.; Degn, H.; Lloyd, D.; Bohatka, S. Biol. Mass Spectrom. 1991, 20, 253-58. (37) Harland, B. J.; Nicholson, P.J.D.; Gillings, E. Water Res. 1987, 21, 107-13. (38) Lister, A. K.; Wood, K. V.; Cooks, R. G.; Noon, K. R. Biomed. Enuiron. Mass Spectrom. 1989, 18, 1063-70. (39) Dheandhando, S.; Dulak, J. Rapid Commun. Mass Spectrom. 1989, 3, 17577.
(40) Sturaro, A.; Doretti, C.; Parvoli, G.; Lecchinato, F.; Frison, G.; Traldi, P. Biomed. Environ. Mass Spectrom. 1989, 18, 707- 12. (41) LaPack, M. A.; Tou, J. C.; Enke, C. G. Anal. Chem. 1990, 62, 1265-71. (42) Bauer, S. J.; Hayward, M. J.;Riederer, D. E.; Kotiaho, T.; Cooks, R. G., unpublished results. (43) Slivon, L. E.; Bauer, M. R.; Ho, J. S.; Budde, W. L. Anal. Chem. 1991, 63, 1335-40. ( 4 4 ) L a u r i t s e n , F. R. P h . D . T h e s i s , Odense University, Denmark, 1990. (45) Savickas, P. J.; LaPack, M. A.; Tou, J. C. Anal. Chem. 1989, 61, 2332-36. (46) Kotiaho, T.; Lister, A. K.; Hayward, M. J.; Cooks, R. G. Talanta 1991, 38, 195-200. (47) Kotiaho, T.; Hayward, M. J.; Cooks, R. G. Anal. Chem. 1991, 63, 1794-1801. (48) C h o u d h u r y , T. K.; Kotiaho, T.; Cooks, R. G., unpublished results.
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Tapio Kotiaho (far left) received his M.Sc. degree in organic chemistryfrom the University ofHelsinki, Finland, in 1987and is pursuinghis Ph.D. at Purdue University.His research interests include MIMS and the use of charge exchange and ion-molecule reactions in analytical MS. George T. Tsao (secondfrom left) is a profissor of chemical engineering and serves as a director of the Laboratory of Renewable Resources Engineering, an interdisciplinary organization that does research on bioreaction and bioseparationprocesses as well as utilization of wastes. He has worked for many years on various types of bioprocesses, including microbial fermentation, immobilized enzymb conversions, and chromatographic separa tion, and is considered a pioneer in the field of biochemical engineering, R. Graham Cooks (third from left) is Henry Bohn Hass Professor of Chemistry. He earned his Ph.D.s in organic chemistry from the University of Natal in South Africa in 1966 and Cambridge University in England in 1967. He pioneered the MS/MS method for determining trace organic compounds and has built several new types of mass spectrometers. His research interests include surface analysis, ion scattering, and energy disposal in chemical reactions. Tancn K. Choudhury flourth from left) received his M.Sc. degree from Rajshahi University, Bangladesh, in 1970 and his Ph.D. from Southern Illinois University at Carbondale in 1986. He joined R. Graham Cooks’s research group as a postdoctoral research associate in 1990. His research interests include applying MIMS to determining trace amounts of organic compounds in aqueous solutions and monitoring bioreactor reaction products. Frants R. Lauritsen (right) received his M.Sc. degree in experimental surface physics and his Ph.D. in biochemistryfrom Odense University, Denmark, in 1986 and 1990, respectively. He began work on MIMS development in 1986 and is primarily interested in on-line monitoring with membrane inlets and instrument design.
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