Membrane Introduction Mass Spectrometry - American Chemical Society

Membrane Introduction Mass Spectrometry Tapio Kotiaho1, 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 t h a n in the danger that the vacuum will be breached. Not the least attractive feature of the membrane introduction systems discussed 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. 2

Permanent address: Odense University, Denmark.

0003-2700/91 /0363-875A/$02.50/0 © 1991 American Chemical Society

or continuous sample introduction. Another major advantage of membrane inlets is the high selectivity of m e m b r a n e s ( p a r t i c u l a r l y silicone m e m b r a n e s ) 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-

t h r o u g h m e a s u r e m e n t s 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, published by Reuss et al. (8), was an important development, and fermentation monitoring with MIMS is commonly used today (2, 9).

REPORT 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 d a t e s back to 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 (10) 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 organics 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 as 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 t h a n 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 t h a t 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 i n s t r u m e n t s . 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 successfully 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

(a)

(b)

(c)

Figure 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, 22), memory effects (22), 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 w i t h s h o r t connection tubes (centimeters in length) a slow transient signal resulting from interactions between the analyte and surfaces in 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 u s ing 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-

876 A · 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 probe— in which the membrane takes the form of a flat sheet (13)—are presented in the box. In general, interfacing a membrane introduction device 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

(a)

IJXjf) = -AD[dCm{x,t)ldx\

(1)

2

dCm(x,t)/dt = D^CJxfi/dx ] (2) where Im(x,t) is the analyte flow in side the membrane (mol/s), A is the membrane surface area (cm 2 ), D is the diffusion constant (cm 2 /s), Cm(x,t) is the concentration inside the mem brane (mol/cm 3 ), χ is the depth in the membrane (cm), and t is time (s). Equation 1 describes the rate of mo lecular flow inside the membrane; E q u a t i o n 2 describes the r a t e at which concentrations change with time. In many MIMS systems the mem brane is exposed to a continuous feed of analyte. For a step change in con centration, the concentrations inside the membrane can be calculated as a function of time from Equation 2 by the method of separation of vari ables. The flow through the mem brane is then found by inserting the calculated concentration into Equa tion 1 and solving it at the vacuum side of the membrane (x = /, where / is the m e m b r a n e thickness). In this way the 10-90% rise time is found as

(b)

Figure 2. Comparison of (a) flow-by and (b) flow-through configurations of 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 Membrane Membrane thickness/area Detection limits Response time3 Operation temperature Sampling frequency" Quantitative accuracy

Silicone membrane (Dow Corning silastic sheeting, medical grade) 0.25 mm or 0.13 mm/30 mm2 Benzene: 10 ppb; PFBOA derivative of formaldehyde: 1 ppb 8s 70 °C 15 sample and standard injections per hour 5%

a

Response time is the time it takes the signal to reach 50% of its maximum intensity. The response is for propan-2-one, 250-μί injections, flow rate of 0.9 mL/min.

0

External standard quantitation mode.

m e m b r a n e surface into v a c u u m . Each step depends primarily on the molecular properties of the analyte and the membrane material. In addi tion, the temperature of the mem brane 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 t h a t passes the membrane. Aqueous solutions are best analyzed

with hydrophobic polymers, which d i s c r i m i n a t e a g a i n s t w a t e r . The membranes typically used are organ ic 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 t h a t the constants for solvation and diffu sion are independent of partial pres

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(3) (3:

Using Henry's law (S = Cm/Ps) to ex press concentrations in partial pres sures, one can calculate the steadystate flow rate as

/ „ = ADS(PJl)

(4)

where 7SS is the steady-state flow through the membrane (mol/s), S is the solubility constant (mol/torr cm 3 ), Ps is the vapor pressure of the analyte on the sample side of the mem brane (torr), and / is the membrane thickness (cm). It is interesting t h a t the steady state flow depends on the product of t h e solubility a n d diffusion con stants, whereas the rate at which measured analyte concentrations change depends only on the diffusion constant. For most nonpolar com pounds the solubility increases and the diffusivity decreases in a homolo gous series of samples as the carbon chain length increases. This is ob served experimentally as longer re sponse times (diffusion constant de creases) and lower detection limits (solubility constant increases more than the diffusion constant decreas es). A vital parameter is the mem brane thickness. Equations 3 and 4 show t h a t doubling the membrane thickness results in a fourfold in crease in response time and a reduc tion in sensitivity by a factor of 2.


REPORT Continuous feed of analyte to the membrane demands relatively large sample volumes, which may not be possible in some reaction and fer mentation 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 simula tion of the analyte concentration on the sample side of the membrane to gether with its rate of flow through the membrane as a function of time. The s i m u l a t i o n s h o w s e x c e l l e n t

Figure 3. Typical simulated dynamic responses of the membrane introduction/FIA apparatus for a dispersed input. Curve A is the analyte concentration; curve Β 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 on line monitoring, fermentation moni toring is a primary application of this technique (2, 9). Fermentation moni toring experiments were first report ed by Reuss et al. in 1975 (8). Since then, in most fermentation experi ments EI 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

gases and volatile compounds within the reactor, they have some draw backs: the chromatographic effect that occurs in the long vacuum lines, poor response time, and inability to measure standards between samples. Moreover, the extensive fragmenta tion of molecular ions attributable to EI interferes with the monitoring of volatile compounds. Because of these and other factors, we suggest the fol lowing guidelines regarding the con struction of membrane inlets for fer mentation monitoring. First, the membrane should be lo cated as close as possible to the ion source of the mass spectrometer to minimize response times and memo ry effects. This can be achieved by u s i n g direct i n s e r t i o n m e m b r a n e probes. Second, FIA m e t h o d s for s a m p l i n g the f e r m e n t a t i o n b r o t h should be used for on-line quantita tion of the fermentation products. Third, the fermentation broth should be filtered prior to analysis with the m e m b r a n e 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 EI can be avoided by using CI. Finally, because gases such as ox ygen and carbon dioxide are best measured using EI, it is important to use a mass spectrometer with both EI and CI capabilities. To fulfill these demands a fully a u t o m a t e d FIA sampling system, which allows on-line quantitation of the liquidphase products and the off-gases, was developed and a d a p t e d to a membrane inlet (25). As a n example of fermentation monitoring, consider data obtained during the fermentation of the mi croorganisms Bacillus polymyxa and Klebsiella oxytoca (25, 27-30). Bacillus polymyxa produces high optical purity (i?,i?)-2,3-butanediol (31), which is a valuable specialty chemical that can be used in the synthesis of other op tically active compounds. Interest in Klebsiella oxytoca is based on its abili ty 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 path ways with carbon dioxide, acetic acid, acetoin, 2,3-butanediol, and ethanol as the major end products. The rela-


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 de pend on the availability of oxygen, as illustrated. Monitoring the produc tion of these products while control ling oxygen availability in response to the analytical data may therefore lead to higher yields of 2,3-butane diol. The major fermentation products were quantified on line using exter nal 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 fermen tation. 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 2 0 ) + 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, fermentation 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 m a s s spectrometer (25). Off-gas analysis provides useful information about the state of fermentation, as emphasized by Heinzle a n d c o - w o r k e r s (34). 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 in 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 p r o d u c e d by m i c r o b i a l 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 comparing 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 meso 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

(a)

(b)

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/FIA apparatus. A: ethanoi, B: acetic acid; C: 2,3-butanediol; D: acetoin, E: oxygen.

(a) Formation of phenylboric ester derivatives of 2,3-butanediol. (b) MS/MS product spectra for the derivatives of meso-2,3-butanediol and (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.)


REPORT acetone in the Bacillus polymyxa fer mentation recognized t h r o u g h the presence of an unassigned m/z 59 ion in the CI mass spectrum and con firmed by collision-activated dissoci ation {25). The FIA/MIMS system should be capable of on-line feedback control of bioreactors. This has been demon s t r a t e d u s i n g t h e oxygen p a r t i a l pressure as the control parameter (28-30). From fermentations without feedback control it is known that the production rate of acetic acid is relat ed 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 an increase in the formation rates of acetoin, 2,3-butanediol, and ethanol. Furthermore, ethanol is known to be produced at high rates under anaero bic conditions. The amounts of the products acetoin and 2,3-butanediol might therefore increase if the oxy gen feed were adjusted during the fermentation, because this control is based on the measured rate of acetic acid formation. Figure 7 shows results of monitor ing by such an experiment (28-30). After approximately 2 h, the produc tion rate of acetic acid passed a pre set upper limit and the oxygen par tial p r e s s u r e 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 , 3 butanediol had started to decline. To m a i n t a i n an a d e q u a t e 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 sig nificant; it is the first demonstration of feedback control in conjunction with the powerful continuous analyt ical capabilities of FIA/MIMS.

Figure 7. Fractional yields (product formation rate/Σ 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. 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 environmen tally significant compounds from aqueous solutions is another major application of MIMS, as proven by the recent increase in relevant publi cations (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 determi nation 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 mem brane introduction devices used in environmental applications are pri marily of the flow-through type (3842). The mass spectrometer of choice is a quadrupole instrument (38, 39, 41, 43), an ion trap (38, 42, 43), or, less frequently, a magnetic sector in strument (37, 40). The most important advantage of MIMS for trace analysis of organic compounds from aqueous solutions is that the analytes can be measured


directly a t p a r t - p e r - b i l l i o n levels without any preconcentration or derivatization steps, which allows con tinuous monitoring. The detection limits obtainable with most mem brane 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 hol low-fiber membrane in which aque ous sample solution flows continu ously over the membrane while the

REPORT inside of the membrane is continu ously purged with helium directed into the ion source of a mass spec trometer. 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 r a n g e s are highly compound d e p e n d e n t ; t h e y s t a r t at part-per-billion levels and extend to several orders of magni tude higher concentrations. The dy namic ranges obtained are sufficient for most MIMS applications. For ex ample, in drinking water samples it h a s been demonstrated t h a t chloro form h a s a l i n e a r dynamic r a n g e from 10 ppb to 100 ppm (10), and in swimming pool samples it h a s been measured at concentrations as low as 6 ppb (44). The direct analysis capability of MIMS also allows reliable determi nation of compounds t h a t are both toxic and labile. Examples include in organic (45, 46) and organic chloramines (47). Both types of compounds can be present in drinking water, and inorganic c h l o r a m i n e s t h e m s e l v e s a r e u s e d as d i s i n f e c t i n g a g e n t s . MIMS allows on-line monitoring of the formation and interconversion of these toxic compounds (46, 47). Recently in our laboratory we dem onstrated the direct determination of acrolein, acrylonitrile, and pentafluorobenzyl hydroxylamine (PFBOA) derivatives of aldehydes from aque ous s a m p l e s (48). EI a n d MS/MS were used in the determination of ac rolein and acrylonitrile. Aldehydes were derivatized in aqueous solu tions and subsequently analyzed u s ing negative CI together with mem brane introduction. This combination adds to the advantages of MIMS the very low detection limits obtainable in electron capture negative CI. As an example of the determina t i o n of labile compounds directly from aqueous samples, Figure 8 il lustrates an experiment in which the formation of mono-, di-, and trichlorinated derivatives of aniline are moni tored on line (47). The results were recorded using isobutane CI by mea s u r i n g t h e protonated aniline a n d two different isotopic forms of proto nated mono-, di-, and trichlorinated reaction products. In Figure 8, only the signal of the molecular ions con taining the 35 C1 isotope is presented. The m e a s u r e m e n t s were per formed as follows. First, water was pumped through the membrane probe and then replaced by an aque ous solution of 0.2 ppm amine (indi-

c a t e d by t h e s t a r ) , r a i s i n g t h e p H to 1 0 - 1 1 . Calcium hypochlorite solu tion w a s t h e n added (indicated by t h e s m a l l a r r o w ) , a n d t h e p H w a s low e r e d t o < 1 by a d d i n g HC1 ( i n d i c a t e d by t h e heavy arrows). F i g u r e 8 d e m o n s t r a t e s t h a t t h e ef f i c i e n t c h l o r i n a t i o n of a n i l i n e i n a q u e o u s s o l u t i o n s r e q u i r e s acidifica t i o n a n d t h a t t h e e n d p r o d u c t of t h e r e a c t i o n is t r i c h l o r o a n i l i n e . M S / M S w a s u s e d t o confirm t h a t t h e chlori n a t i o n o c c u r r e d on t h e b e n z e n e r i n g , w h e r e a s i n t h e c a s e of a l i p h a t i c a m i n e s , c h l o r i n a t i o n o c c u r s on t h e n i t r o g e n a t o m (47). N o t e a l s o t h a t , b e c a u s e f o r m a t i o n of all t h e possible reaction products can be determined, t h e s t a r t i n g a m i n e c o n c e n t r a t i o n of 0.2 p p m r e p r e s e n t s t h e c o n c e n t r a t i o n limit a t which t h e reaction sequence c a n b e followed w h e n c o n t i n u o u s m o n i t o r i n g M I M S is u s e d . Future perspectives MIMS displays several outstanding characteristics: rapid response times, e a s e of a u t o m a t i o n , v e r y h i g h c h e m i cal specificity, simplicity, a d a p t a b i l i t y t o a n y t y p e of i n s t r u m e n t , a n d low impact on i n s t r u m e n t cleanliness. The detection limits achieved a r e ex c e p t i o n a l l y low for s m a l l ( m o l e c u l a r weight < 300), less polar organic c o m p o u n d s (e.g., less p o l a r t h a n 2 , 3 butanediol), a n d on-line derivatizat i o n c a n b e u s e d to c o n v e r t a n a l y t e s i n t o s u i t a b l e forms. U n f o r t u n a t e l y , t h e t y p e s of c o m pounds t h a t can be examined are limited. To e x a m i n e polar or even ionic c o m p o u n d s , a l t e r n a t i v e m e m b r a n e s w i t h different selectivities m u s t b e developed. T h i s r e q u i r e s t h e s y n t h e s i s of n e w p o l y m e r s , e v a l u a t i o n of t h e i r p h y s i c a l p r o p e r t i e s , a n d t h e i r i n c o r p o r a t i o n i n t o devices t h a t allow o n - l i n e a n a l y s i s . T h e t e c h n i q u e u s e d for o n - l i n e d e t e c t i o n of g a s e s a n d small, less polar organic com pounds, however, can be regarded as h a v i n g r e a c h e d m a t u r i t y ; i t s useful ness h a s already been demonstrated i n a n a l y t i c a l c h e m i s t r y a s well a s disciplines s u c h a s p r o c e s s m o n i t o r ing a n d control, microbiology, a n d medicine. B e c a u s e of t h e s i m p l i c i t y of t h e technique, in t h e near future we can expect a l a r g e i n c r e a s e i n t h e u s e of M I M S in t h e a r e a s a l r e a d y dis cussed, especially in fermentation and environmental monitoring.

References (1) Hoch, G.; Kok, B. Arch. Biochem. Biophys. 1963, 101, 160-70. (2) Degn, H.; Cox, R. P.; Lloyd, D. Methods Biochem. Anal. 1985, 31, 165-94. (3) Woldring, S. /. Assoc. Adv. Med. Instrum. 1970, 4, 4 3 - 5 6 . (4) Brantigan, J. W.; Dunn, K. L.; Albo, B.J. Appl. Physiol. 1976, 40, 443-46. (5) Lundsgaard, J. S.; Jensen, B.; Groenlund, J.J. Appl. Physiol. 1980, 48, 3 7 6 - 8 1 . (6) Brodbelt, J. S.; Cooks, R. G.; Tou, J. C ; Kallos, G. J.; Dryzga, M. D. Anal. Chem. 1987, 59, 454-58. (7) Llewellyn, P. M.; Littlejohn, D. P. U.S. Patent, 3 429 105, 1969. (8) Reuss, M.; Piehl, H.; Wagner, F. Eur. J. Appl. Microbiol. 1975, 1, 323-25. (9) Heinzle, E.; Reuss, M. Mass Spectrome try 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, 5 6 8 - 7 1 . (11) Bier, M. E.; Cooks, R. G.; Brodbelt, J. S.; Tou, J. C.; Westover, L. B. U.S. Patent 4 791 292, 1989. (12) Lauritsen, F. R. Int. J. Mass Spectrom. Ion Proc. 1990, 95, 259-68. (13) Bier, M. E.; Kotiaho, T.; Cooks, R. G. Anal. Chim. Acta 1990, 231, 175-90. (14) Arpino, P. Mass Spectrom. Rev. 1990, 9, 631-69. (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 - 4 8 5 A. (17) Degn, H.; Lauritsen, F. R. /. Phys. Chem. 1989, 93, 2781-83. (18) Calvo, K. C ; Weisenberger, C. R.; Anderson, L. B.; Klapper, M. H. /. Am. Chem. Soc. 1983, 105, 6935-41. (19) Jensen, B. B.; Cox, R. P. In Methods in Enzymology, Volume 167, Cyanobacteria; Packer, L.; Glazer, A. N., Eds.; Academ ic Press: New York, 1988; pp. 467-74. (20) D a s , J.; Timm, H.; Busse, H. G.; Degn, H. Yeast 1990, 6, 2 5 5 - 6 1 . (21) Bohatka, S.; Berecz, L; 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, Ε. Η. Flow Injec tion Analysis, 2nd éd.; John Wiley and Sons: New York, 1988; Vol. 62. (25) 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. (26) Tsai, G-J.; Austin, G. D.; Syu, M. J.; Tsao, G. T.; Hayward, M. J.; Kotiaho, T.; Cooks, R. G. Anal. Chem., in press. (27) Lister, A. K. Ph.D. Thesis, Purdue University, 1990. (28) Hayward, M. J. Ph.D. Thesis, Purdue University, 1990. (29) Austin, G. D. Ph.D. Thesis, Purdue University, 1990. (30) Kotiaho, T.; Hayward, M. J.; Choudhury, T. K ; Lister, A. K.; Cooks, R. G.; Austin, G. D.; Syu, M. J.; Tsao, G. T. Presented at the 39th Annual Conference on Mass Spectrometry and Allied Topics, Nashville, TN, May 1991.

Our work on environmental and bioreactor monitoring was supported by the Environmental Protection Agency (R.G.C., EPA CR-815749-01-0) and the National Science Foundation (G.T., NSF EE-787 12867), 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.


Discover new solutions (31) de Mas, C ; Jansen, Ν. Β.; Tsao, G. T. Biotechnol. Bioeng. 1988, 31, 366-77. (32) Qureshi, N.; Cheryan, M. Appl. Micro biol. Biotechnol. 1989, 30, 4 4 0 - 4 3 . (33) Gottschalk, G. Bacterial Metabolism, 2nd éd.; Springer-Verlag: New York, 1986. (34) Heinzle, Ë.; Oeggerli, Α.; 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.; N i e l s e n , 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, Κ. V.; Cooks, R. G.; Noon, Κ. R. Biomed. Environ. Mass Spectrom. 1989, 18, 1063-70. (39) Dheandhando, S.; Dulak, J. Rapid Commun. Mass Spectrom. 1989, 3, 1 7 5 77.

(40) Sturaro, Α.; Doretti, C ; Parvoli, G.; Lecchinato, F.; Frison, G.; Traldi, P. Biomed. Environ. Mass Spectrom. 1989, 18, 707-12. (41) LaPack, M. Α.; Tou, J. C ; E n k e , C. G. Anal. Chem. 1990, 62, 1265-71. (42) Bauer, S. J.; Hayward, M. J.; Riederer, D. E.; Kotiaho, T.; Cooks, R. G., un published results. (43) Slivon, L. E.; Bauer, M. R.; Ho, J. S.; Budde, W. L. Anal. Chem. 1991, 63, 1335-40. (44) 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. Α.; 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.; K o t i a h o , T.; Cooks, R. G., unpublished results.

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Tapio Kotiaho (far left) received his M.Sc. degree in organic chemistry from the Univer sity of Helsinki, Finland, in 1987 and is pursuing his Ph.D. at Purdue University. His re search interests include MIMS and the use of charge exchange and ion-molecule reac tions in analytical MS. George T. Tsao (second from left) is a professor of chemical engineering and serves as a director of the Laboratory ofRenewable Resources Engineering, an interdisciplinary or ganization that does research on bioreaction and bioseparation processes as well as utili zation of wastes. He has worked for many years on various types ofbioprocesses, including microbial fermentation, immobilized enzyme 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 spec trometers. His research interests include surface analysis, ion scattering, and energy dis posal in chemical reactions. Tarun K. Choudhury (fourth from left) received his M.Sc. degree from Rajshahi Uni versity, 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 as sociate 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 biochemistry from Odense University, Denmark, in 1986 and 1990, re spectively. 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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