Mass Spectrometry of Nonvolatile Compounds - ACS Publications

Dec 1, 1980 - Robert J. Cotter. Anal. Chem. , 1980, 52 (14), ... Kuangnan Qian, William E. Killinger, and Melissa Casey , Gordon R. Nicol. Analytical ...
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Robert J. Cotter Department of Pharmacology and Experimental Therapeutics The Johns Hopkins School of Medicine Baltimore, Md. 21205

Mass Spectrometry of Nonvolatile Compounds Desorption from Extended Probes The technique has been called "direct CI," "in-beam," "direct exposure," "desorption CI," and "surface ionization," and mass spectroscopists have been using variations of the technique ever since McLafferty and Baldwin (1) introduced it in 1973. The idea is very simple: Nonvolatile samples are coated on a surface and inserted directly inside an ion source by means of some extended direct insertion probe—an idea that can actually be traced back to R. I. Reed (2) and others who reported improvements in their spectra when the sample was placed inside of the source. This replaces the usual practice of vaporizing the sample outside of the source, which generally results in pyrolysis, and it gives a much better chance of observing molecular ions in the mass spectra. (See Figure 1.) Since its introduction, investigators have examined

many compounds on a number of different surfaces, examined heating rates (fast and slow), coated samples onto filaments or activated emitters (borrowed from field desorption sources), and argued about whether field desorption itself really needs the field. It has contributed to our understanding of other desorption methods, such as field desorption (FD), plasma desorption, and laser desorption, and demanded some revision of our idea of volatility, under conditions in which sample-surface interactions now become more important than the sample-sample interactions that normally govern the behavior of the vaporization process. The main advantage of the technique is its simplicity. Any mass spectrometer can be equipped with a direct exposure probe; and for many compounds, molecular ions will be ob-

tained where conventional solids probes would not do the job. It is not a substitute for field desorption, but it is less expensive and generally produces more fragmentation information than FD. It has also become popular enough at this point that mass spectrometer manufacturers are offering such probes for sale, along with programmable heaters, for the rapid vaporization of the nonvolatile! Direct Chemical Ionization Baldwin and McLafferty {1) reported the use of an extended glass probe tip for the sequencing of peptides without prior derivatization, and published a methane "direct CI" spectrum of ValAlaAlaPhe with a large molecular ion (MH + ) peak, and other peaks capable of yielding sequence information (Figure 2). The main advantages to the technique that they

(b) Direct Exposure Probe

(a) Conventional Solids Probe

Electron Impact or Chemical Ionization Source

Electron Impact or Chemical Ionization Source Vaporized Neutral Molecules and Decomposition Products

;/ /

Sample Molecules in Solid Phase

Figure 1. Comparison of conventional solids probe (a) and direct exposure probe (b). The conventional solids probe requires volatilization of the sample prior to entering the ion 0003-2700/80/A351 -1589$01.00/0 © 1980 American Chemical Society

source, while the direct exposure methods introduce the solid directly into the ion source, close to the electron beam

ANALYTICAL CHEMISTRY, VOL. 52, NO. 14, DECEMBER 1980 · 1589 A

suggests the opposite, i.e., that neutral molecules are desorbed and that a softer ionization then reduces the fragmentation in the same manner that is expected in conventional methods of sample vaporization. Surface Ionization

Figure 2. The direct CI spectrum of underivatized ValAlaAlaPhe; source temperature 160 °C, methane as ionizing reagent. Reprinted (with permission) from Ref. 1

cited were the use of lower temperatures to produce a molecular ion and small sample size (10~7 g). In-Beam El

In 1975, Williams et al. employed an analogous technique for an electron impact source in their determination of the structure of the antibiotic Echinomycin (3). On the basis of their NMR data, they questioned a previously reported structure (4), and sought confirmation of their revised structure through determination of the exact mass of the molecular ion using field desorption mass spectrometry. Because field desorption failed to give peaks other than the molecular ion, they then obtained a complete EI spectrum of Echinomycin using an extended probe tip that placed the sample up close to the electron beam. The sample was underivatized, and gave a clearly recognizable molecular ion at 1100 AMU. The use of "in-beam" electron impact for the production of molecular ions of nonvolatile and/or thermally labile organic compounds has been pursued vigorously by Ohashi and his co-workers (5-8), who have examined a number of compounds, including sugars (D-xylose, L-fucose, D-glucose, and saccharose), nucleosides (adenosine, guanosine), and long chain aliphatic alcohols. Ohashi used a quartz tip, and an ionizing voltage of 20 eV to reduce the fragmentation. In addition, he varied the distance between the probe tip and the electron beam, and determined that 2-3 mm gave optimal results (6). This was later noted by Hansen and Munson (9) for the CI case, and seems to have become a fairly accepted standard for extended probe tips. Ohashi made some important observations about the spectra produced by the "in-beam" EI method. First, the molecular ions were MH + ions, while the fragment ions generally corresponded to those observed in conventional EI spectra (6). The suspicion is,

of course, that the use of the closed source, combined with the rapid rise in source pressure from the sudden burst of desorbed species from the probe tip, results in ion-molecular collisions that produce a CI-like spectrum. This is probably the case. In a later publication, Ohashi and Nakayama (8) reported the appearance of (2M + 1) + ions in the spectra of aliphatic alcohols, using the "in-beam" technique. Deuterium labeling studies showed that the extra hydrogen originated from the hydroxyl group, and the spectrum of a 1:1 mixture of 1-tetradecanol (Mt) and 1-pentacecanol (M p ) produced a mass spectrum with a 1:2:1 relative abundance of (2Mt + 1)+, (M t + M p + 1)+, and (2M P + 1)+. The second observation was that the spectra containing molecular ions appear immediately after insertion of the "in-beam" probe, and that later spectra begin to look like conventional EI spectra (5). There are really two explanations for this behavior. First, if the observed MH + ions are really dependent upon ion molecular collisions in the closed source, then the later spectra are occurring when the burst of molecules desorbed from the surface is over, and the source pressure has been reduced. Secondly, as has been borne out in later studies (9, 10), the heating rate upon insertion of a cold probe into a hot source initially favors the desorption of intact neutral molecules over the competitive process of pyrolysis, which becomes predominant as the sample temperature is further increased. The general question of mechanisms is an interesting one, and will be dealt with in more detail later. However, based upon Ohaehi's observations, the importance of the position of the probe tip with respect to the electron beam might seem to suggest some kind of direct ionization on the surface itself, followed by desorption of the ions. On the other hand, the fact that 10-20 eV electrons produce better molecular ion abundances

Hansen and Munson further explored the possibility that ionization was taking place on the surface of the probe tip (9). As a test sample they used creatine, a compound that undergoes dehydration to the cyclic lactone, creatinine, at temperatures lower than those required for volatilization. They noted that the relative abundance of the molecular ion MH + , using "in-beam" chemical ionization, is strongly dependent upon the distance between the probe tip and the electron beam, while the total ion current is not. The behavior led them to the conclusion that the process involved chemical ionization on the surface. Hansen and Munson used the temperature of the source block to determine the heating rate of their sample and probe tip. This practice has been used by subsequent investigators when nonmetallic probes have been used, since they cannot be heated directly. Higher source temperatures gave better relative abundances of the molecular ions of creatine, arginine, and choline chloride. But perhaps more interesting is their investigation of the time dependence of molecular ion production (after probe insertion) at different source temperatures, for this really corresponds to a heating rate, rather than a temperature effect. They investigated the MH + ion of creatine at source temperatures of 200, 220, and 240 °C. Not only did the total ion current peak earlier and sharper for the higher temperature (heating rate), but the relative abundance of the molecular ion was much greater. This same line of thinking has also prompted a number of other investigators to examine the role of heating rate in molecular ion production. Volatility Enhancement

Beuhler and co-workers {11, 12) had previously published results on the mass spectra of underivatized peptides containing arginine, using an "in-beam" chemical ionization technique, and had made the opposite assumption from Hansen and Munson (9), namely, that it was intact neutrals that were desorbed and then ionized, and not surface ionization. In their view, the problem was one of "volatility enhancement." Energy added to a sample to break the loose bonds between the sample and the surface (evaporation) is, under normal conditions, being distributed among the in-

ANALYTICAL CHEMISTRY, VOL. 52, NO. 14, DECEMBER 1980 · 1591 A

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ternal degrees of freedom of the molecule and results in several competing decomposition reactions. Beuhler et al. then made two suggestions for improving the desorption of intact neutral molecules. T h e first requires paying attention to the nature of the surface itself (12) (and they suggest the use of Teflon), in an effort to reduce the binding energy of the sample to the surface, and making desorption more competitive with decomposition. This has certainly been the thinking of the investigators previously mentioned, who have used glass (1), quartz (5-8), Teflon (9), and Vespel (10, 13). T h e second approach is "rapid heating," which favors the desorption of intact neutral molecules on kinetic grounds. Figure 3, taken from the work of Beuhler et al. (12), illustrates the principle. For nonvolatile molecules one can assume t h a t the energy of activation for the decomposition process is lower t h a n t h a t for the desorption of the intact neutral molecule. In addition, the energy of activation for decomposition will be the rate-determining step for evaporation of the decomposed neutral. Therefore, Arrhenius plots of the relative abundances of the molecular ion and a decomposition ion vs. 1/T will have the appearance shown in Figure 3 for Thyrotropin Releasing Hormone, and will intersect at some value of 1/T. T h e principle behind rapid heating is to get into the temperature region above the intersection before appreciable decomposition has taken place. T h e heating rates being used in this case are approximately 10 °C/s. In addition, Beuhler et al. (12) note the necessity of a data system for this work since individual spectra must be acquired approximately every 2-3 s. This results in about 10 spectra before the sample is consumed. T h e rapid heating approach has been taken u p by a number of investigators. In our own laboratory (10), we have used heating rates similar to those used by Beuhler et al. (12) by inserting a Vespel probe into a hot ion source block, according to the method used by Hansen and Munson (9), and have produced spectra with good molecular ion abundance for guanosine, deoxyguanosine, sucrose, and the glucuronide conjugate of p-nitrophenol. Ultrafast heating rates (up to 1200 °C in 0.1 to 0.2 s) have been used by Daves et al. (14-16) for a n u m b e r of sugars and peptides, a method which he has subsequently termed "flash desorption." This very rapid heating approach also forms t h e principle behind our current work using a pulsed CO2 laser for the desorption of intact neutral molecules, which are then ionized (17) using isobutane. However, it should be noted t h a t laser desorption

ANALYTICAL CHEMISTRY, VOL . 52, NO. 14, DECEMBER

1980

Figure 3. Relative intensity of m/e 363 and m/e 235 ions from TRH plotted or a function of 1/T. Spectrum was obtained by evaporation of TRH from a copper probe surface in a Teflon collision chamber: ( O ) protonated parent molecule of PCA-His-Pro-NH 2 , m/e 363; ( · ) m/e 235 ion formed by loss of pyrrolidinone carboxyl amide from PCA-His-ProNH 2 . Reprinted (with permission) from Ref. 12

methods can also produce ions directly on the probe surface. Both Kistemaker and co-workers (18) and Stoll and Rôllgen (19) describe experiments in which ions are produced by a CO2 laser in an ion source in which the electron beam is t u r n e d off. T h e particular wavelength used (10.6 μτή), the fact t h a t the desorption process is in­ dependent of wavelength (Kistemaker used a CO2 laser at 10.6 μπι and a N d YAG laser at 1.06 Mm), and recent work by Kistemaker (20) on t h e de­ pendence of the molecular ion abun­ dance upon power density, point to this as a thermal process, rather t h a n a photoionization (multiphoton) pro­ cess. It is therefore closely related to the very rapid heating experiments. Finally, desorption from activated emitters also involves rapid heating, since very high temperatures are reached when t h e emitter current is turned u p (21-23).

Activated Emitter Experiments Activated emitters, of the type com­ monly used in field desorption, are a logical choice as a sample substrate for the " i n - b e a m " type of desorption we are considering. First of all, they allow the sample to be distributed over an enormous surface area, which greatly reduces the intramolecular forces be-

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ANALYTICAL CHEMISTRY, VOL. 52, NO. 14, DECEMBER

Using this methodology, Soltmann, Sweeley, and Holland produced an EI/D (electron impact/desorption) spectrum of cholesterol with greatly reduced fragmentation (21). Hunt and co-workers (22), using the CI version, produced spectra with good molecular ion abundance for guanosine, cyclic AMP, creatine, choline chloride, arginine (HC1 salt), and several peptides. Fast-heated Wires

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tween molecules and makes the evaporation process dependent upon sample-surface forces, rather than the sample-sample forces predominant when bulk samples are vaporized. Secondly, the sample-surface attraction can be overcome with rapid heating by passing an electrical current through the emitters. It is, in fact, possible to achieve much faster heating rates with such emitters than with the "inert," nonconductive types of probes that must be heated by some indirect means. Two publications, which appeared simultaneously, are of great interest: one by Soltmann, Sweeley, and Holland (21), who used an activated emitter as a solids probe in an electron impact source, and another by Hunt, Shabanowitz, Botz, and Brent (22), who used a chemical ionization source. In both of these investigations, a best emitter temperature (BET) was determined for each compound by scanning the current passed through the emitter. The best emitter temperature is defined as "the emitter temperature (current) that affords the highest abundance of ions characteristic of the molecular weight" (22), and is analogous to the "best anode temperature" (BAT) used in field desorption. The best emitter temperature concept for this type of desorption had been previously described by Holland et al. (23). (This laboratory has also published an emitter current programmer (24) for scanning the emitter current to determine the best emitter temperature.) Upon determining the BET for a particular compound, the sample is recoated on the emitter, and the current is programmed up to a few milliamperes above the BET for taking the complete mass spectrum.

1980

Activated emitters, because of their physical size, are difficult to adapt as solids probes for some mass spectrometers. In addition, they require some type of emitter preparation unit for activation, which may not be available to some laboratories. Apart from their ability to disperse the sample over a large surface area, however, they are also attractive since a high current can be passed through the wire to give extremely high heating rates. Thus, there is a strong motivation to desorb samples from "unactivated" wires or filaments to achieve similarly high

Figure 4. Single ion current profiles for the [ M H ] + ion of sucrose at several source temperatures in ° C .

heating rates. Daves and co-workers have published a number of articles using this technique (14-16). As mentioned before, temperatures as high as 1200 °C are reached within 0.1 to 0.2 s. An interesting problem arises with this approach. Because the heating is so rapid, the time frame during wbich desorption occurs is so short t h a t scanning of the mass spectrum is not possible. For this reason, Daves used photoplate detection for recording all of the ions simultaneously. Ohashi pointed out (5) t h a t there is a timedependence for the molecular ion, and t h a t later spectra produce only fragm e n t ions. Therefore, the easily recognizable, b u t low relative abundance of the molecular ion obtained by Daves for sucrose (15) may result from integration of the desorption and pyrolysis spectra, which cannot be time-resolved using the photoplate. In our laboratory, we have demonstrated t h a t this is in fact the case, using much lower heating rates resulting from introduction of a Vespel probe at room temperature into a source block whose temperature is varied prior to insertion (10). Our results for sucrose are shown in Figure 4. At low heating rates, achieved by a source temperature of 180 °C, almost no molecular ion is observed, as is expected from the type of arguments presented by Beuhler et al. (12). At source temperatures of 230 and 280 °C, the total ion current shows t h a t there are clearly two processes involved, and the single ion currents for the molecular ion (MH+) and for MH+ - H 2 0 ion suggest t h a t desorption of intact neutral molecules (with some fragmentation) is followed by desorption of decomposition products formed on the surface. When the source is heated to

Single ion current profiles for the [MH — H 2 0 ] + ion

Figure 5. Mass spectrum of androsterone glucuronide using the direct exposure probe. Reprinted (with permission) from Ref. 17

310 °C prior to insertion of the probe (providing an even faster heating rate), the two processes begin to coalesce. At this point, the inability to distinguish the two processes may result from the inability to acquire spectra in a period of time short enough to resolve the processes and not the absence of two processes.

1598 A · ANALYTICAL CHEMISTRY, VOL. 52, NO. 14, DECEMBER 1980

One possible way to increase the heating rate and avoid the decomposition problem is through the use of short pulsed lasers for thermal desorption of neutral molecules. High temperatures are reached in a very short period of time, b u t are dissipated before the pyrolysis process can occur. For example, the conventional isobu-

desorption, and therefore they postulated a thermal desorption process. This drew immediate response from Beckey and Rôllgen (25) who challenged the suggestion t h a t field desorption could take place without the field. T h e debate continued in the Letters to the Editor column of Organic Mass Spectrometry (26, 27), and makes interesting reading. It is in fact known that, at very high temperatures, ions as well as neutrals can be desorbed, or evaporated, from a metallic surface, according to the Langm u i r - S a h a equation (28): n + / n ° = exp[e(W - I)/kT]

Figure 6. Laser desorption/chemical ionization spectrum of the molecular ion region of androsterone glucuronide. Reprinted (with permission) from Ref. 17

tane CI spectrum of the glucuronide conjugate of the steroid androsterone shows mass spectral peaks characteristic of the steroid and glucuronic acid moieties only, resulting from decomposition in the direct insertion probe. A direct exposure CI spectrum shows better results (Figure 5) since the (MH — H 2 0 ) + ion characteristic of the conjugate is now present. T h e laser desorption/chemical ionization spect r u m (Figure 6), however, does produce a molecular ion (17). T h e 40-ns pulses used in this experiment are spaced 1 s apart, and produce t h e same relative intensity of molecular ion for as long a time as the sample is kept in the source. Desorption M e c h a n i s m s

It may be difficult, and perhaps erroneous, to propose a single mechanism for the variety of desorption techniques t h a t have been discussed. T h e wide range of conditions of fast and slow heating, inert and activated surfaces, and the types of samples themselves may result in different

processes from the many competing possibilities. However, the questions about mechanisms, as they have been asked by the investigators involved, have generally centered around two different areas: 1) the nature of the desorption process where FD emitters have been used, and 2) the question of whether neutral desorption or surface ionization occurs first in the case of inert, indirectly heated probes. T h e principal reason for looking at these two cases separately is t h a t there is the possibility with directly heated emitters (or untreated wires and filaments for t h a t matter) of "thermal ionization." For indirectly heated inert probes, this is not a possibility; ionization results from the electron beam (EI) or the reagent gas ions (CI), and the question is only whether this takes place on the surface or not. An article by Holland, Soltmann, and Sweeley (23) about the nature of the field desorption process noted t h a t the "best emitter t e m p e r a t u r e " (with the field off) corresponded closely to the "best anode t e m p e r a t u r e " in field

1600 A · ANALYTICAL CHEMISTRY, VOL. 52, NO. 14, DECEMBER 1980

where n + / n ° is the ratio of positive ions to neutral molecules evaporated as a function of the work function (W), first ionization potential (I), and the temperature (T). While it is not t h e intention of this author to comm e n t upon whether or not the electron tunneling mechanism is operative for field desorption, it does appear possible t h a t a portion of the ions originating from an activated emitter operated at high temperature is the result of some thermal process, and t h a t this is in competition with a much larger n u m b e r of desorbed neutrals. Generally, however, thermal ionization sources are employed for elemental analysis (28), and the desorption of N a + and K + is well known, having been reported as early as 1918 by Dempster (29). It is possible, therefore, t h a t some thermal process plays a role in the formation of M + N a + ions t h a t are observed in field desorption. There are, in fact, two other observations t h a t are relevant to this discussion. Daves, using fast-heated wires, has not been able to observe molecular ions when the electron beam has been turned off (30). This suggests t h a t even at very high temperatures, the desorption of neutrals greatly exceeds t h a t of ions. On t h e other hand, we have noted in experiments using a pulsed CO2 laser for thermal desorption in a chemical ionization source t h a t for compounds which we have studied u p to this point, M + N a + can be produced with the electron beam turned off, while M H + ions are observable only when the electron beam is restored (31). This appears to point to a thermal process in the ionization of intact molecules by N a + attachment, while desorption of neutrals is predominant in the formation of M H + ions. Stoll and Rôllgen have reported similar results (19). Using a quadrupole mass spectrometer and a continuous wave (CW) CO2 laser, they observed for polar compounds such as sucrose and stachyose t h a t only cationized molecular ions were produced whose relative

Figure 7. Total ion current profiles for p-nitrophenyl/3-D-glucuronide at several ion source temperatures in °C. abundance could be increased by the addition of N a l to the sample. If the laser techniques (17, 19, 20) are viewed as a form of ultrafast heating, then it appears t h a t there is a need for a definitive experiment to demonstrate the production of molecular ions of large nonvolatile compounds from purely thermal processes. Unfortunately, the experiments carried out by Holland et al. (23), while making a good case for some thermal process because of the close correspondence between the B E T and BAT, are not unambiguous. Since the authors found t h a t the electric field is still necessary, supposedly to extract the ions, the possibility of competing processes is still there, and the ionization and evaporation events are difficult to separate. Cationized species have been extracted from surfaces by L i + a t t a c h m e n t by Rôllgen et al. (32, 33) using untreated wires, high temperatures, and an electric field strength below t h a t expected for field ionization. However, the compounds studied were generally volatile. T h e most impressive result using this method is a spectrum of t h e thermally labile sugar, sucrose, with an M + L i + abundance of 0.2%. However, for purposes of this discussion, the sucrose was first volatilized from an oven and interacted with L i + ions in the gas phase, (33) so t h a t the process still clearly relied upon the desorption of intact neutral molecules. Ligon (34) used a similar technique, which he called "cation extraction," in which polyphosphoric acid was coated on a bare tungsten wire in a field desorption source. T h e source was operated a t low field strength, and volatile organic compounds were introduced to

— Direct exposure probe temperature vs. time. Reprinted (with permission of Heyden & Son, Ltd.) from Ref. 10

the source in the gas phase. T h e field then extracted MH+ and (2M + H)+ ions. T h e implication of both of these experiments is, of course, t h a t the field may be used for extraction of either cationized or protonated molecular ions, and t h a t this may be its role in the field desorption process, as Holland et al. (23) have suggested. However, in both of these experiments, the organic components are volatile and are already in the gas phase. What, then, is the mechanism when activated emitters are used strictly as solids probes with no extracting fields? It is clear t h a t the E I / D method of Soltmann, Sweeley, and Holland (21 ), the CI/D method of H u n t and co-workers (22), and the untreated, fast heated wires of Daves et al. (1416) all required a secondary means of ionization provided by the electron beam or CI reagent ions. Although high temperatures are reached very quickly, thermal ionization does not seem to have a major role. Nor does thermally induced cationization. Rather the major process appears to be t h a t suggested by the Rrookhaven group (12), namely, t h a t the rate of the evaporation process is enhanced over the competing decomposition processes. However, results from the laser experiments by Kistemaker et al. (20), Stoll and Rôllgen (19), and our own laboratory (31) indicate t h a t thermal ionization can occur, mainly through a cationization process, when high enough temperatures are reached in a short period of time. T h e work of Rôllgen et al. (32) and Ligon (34) shows t h a t cationized species can be extracted when the organic neutral is in the gas phase, or very close to the surface. A model consistent with all of

1602 A · ANALYTICAL CHEMISTRY, VOL. 52, NO. 14, DECEMBER 1980

these results might picture fast heating as resulting in codesorption of cations and organic neutrals, which are then associated closely above the surface. As a purely thermal phenomenon, this result has at the moment occurred only through the use of laser heating. Mechanisms of the Inert Probe Desorption I t is clear t h a t the lower temperatures achieved in indirectly heated inert probes cannot result in thermal ionization. However, the question of whether the reagent gas performs its role of ionization on the surface or after the neutral molecules have been desorbed needs to be considered. One way to answer this question is to examine the total ion current as a function of time after insertion of the sample probe into the ion source, with the reagent gas present (CI), and with the reagent gas absent (EI). Figure 7 shows the behavior of p-nitrophenyl/3-D-glucuronide at several source temperatures (10). T h e first and last traces, taken at the same source temperature, with and without the reagent gas present, are nearly identical. This suggests t h a t the desorption rate is independent of the presence of the reagent gas. This could hardly be expected to be the case, if in the first instance, ions were being desorbed, while in the EI case, neutrals were being desorbed. T h e two maxima in the total ion current traces can be interpreted: T h e first involves desorption of largely intact neutral molecules, which are then ionized to produce both molecular ions and fragm e n t ions. T h e second maximum is primarily the desorption of neutral pyrolysis products. This can be illus-

Figure 8. Mass spectrum of p-nitrophenyl-/3-D-glucuronide obtained 15 s after insertion of the direct exposure probe.

Source temperature 220 °C

Figure 9. Mass spectrum of p-nitrophenyl-/3-D-glucuronide obtained 90 s after insertion of the direct exposure probe.

Source temperature 220 °C. Reprinted (with permission of Heyden & Son, Ltd.) from Ref. 10

trated by examining the spectrum of p-nitrophenyl glucuronide a t each of the maxima (Figures 8, 9). Recently, Hansen and Munson (35) have also observed this "double maxima" phenomenon for arginine and confirmed this interpretation by single ion monitoring of the MH+ and (M - OH)+ ions for this compound. Conclusions In most of the experiments described above, it has been neutrals t h a t have been desorbed from the extended probes inserted into the ion

source. Therefore, rather than being a radically new form of ionization, the " i n - b e a m " methods appear to be ways to enhance the volatility of what are normally thought to be nonvolatile compounds. Samples are placed inside of the source, as close to the ionization area as possible. T h e sample is dispersed to minimize the intramolecular attractions. T h e sample is then rapidly heated to make evaporation of intact neutrals competitive with decomposition. Finally, a data system, capable of scanning and acquiring data rapidly is essential, since the rapid

1604 A · ANALYTICAL CHEMISTRY, VOL. 52, NO. 14, DECEMBER 1980

temperature " b u n c h e s " the desorbed neutral molecules into a short time period, after which pyrolysis becomes predominant. T h e possibility of desorbing ions should not be abandoned. While highly polar compounds (sucrose, stachyose, guanosine, peptides, etc.) may desorb more easily as intact neutrals, it is clear t h a t ionic compounds, such as the quaternary ammonium salts, may desorb far better as ions. Laser desorption experiments may provide some of the answers, b u t experiments using fast heating with N a + and L i +

ion a t t a c h m e n t in sources not de­ signed for field desorption also need to be carried out. Another important area t h a t needs to be investigated is t h e desorption of high mass compounds. T h e " i n - b e a m " methods need to be applied to com­ pounds in t h e molecular weight ranges t h a t have been examined by plasma desorption mass spectrometry (36). At this point, however, for mass spectroscopists t h e use of an "inb e a m " probe, whether of t h e " i n e r t " type or rapidly heated type, provides the opportunity for obtaining spectra of compounds generally thought not amenable to mass spectral analysis, and greatly increases t h e potential a p ­ plications of mass spectrometry.

NewLC clean-up technique is simpler and faster. Rheodynes Technical Note 2 tells how to prepare samples with greater speed and lower cost than purification steps using liquid/liquid partition, column chromatography or commer­ cial separation cartridges. The rapid clean-up technique uses Rheodyne's Model 7125 Syringe Loading Sample Injector with a short column in the sample loop. It allows complex samples to be injected directly into the chromatographic system. This technique can be used to: • Eliminate interferences from complex matrices • Concentrate samples to improve detection limits • Shorten analysis time by trapping late-eluting peaks

Send for Tech Note # 2 The simplified procedures are fully described in this well-illustrated 4-page technical note. Contact Rheodyne, Inc., P C Box 996, Cotati. Calif. 94928. U.S.A. Phone(707) 664-9050.

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Acknowledgment T h e author acknowledges t h e many helpful and stimulating discussions with Burnaby Munson and Gordon Hansen, Al Yergey, Mamoru Ohashi, Jack Holland, and Doyle Daves on t h e subject of desorption of nonvolatile solids. In addition, he is grateful to Catherine Fenselau a n d Al Yergey for their comments and suggestions on this manuscript. This work was supported by grants from the U.S. National Institutes of Health, GM-21248, and the National Science Foundation, CHE-7818396, and was carried out at the Middle Atlantic Mass Spectrometry Facility, supported by NSF, at the Johns Hopkins University. References (1) M. A. Baldwin and F. W. McLafferty, Org. Mass. Spectrom., 7,1353 (1973). (2) R. I. Reed, "Some Problems in Mass Spectrometry," in "Mass Spectrometry," R. I. Reed, Ed., Academic Press, London, 1965; ρ 401 and references contained therein. (3) A. Dell, D. H. Williams, H. R. Morris, G. A. Smith, J. Feeney, and G. C. K. Roberts, J. Am. Chem. Soc, 97, 2497 (1975). (4) W. Keller-Schierlein, M. Lj. Mihailovic, and V. Prelog, Helv. Chim. Acta., 42, 305 (1959). (5) M. Ohashi, N. Nakayama, H. Kudo, and S. Yamada, Mass Spectrosc. (Japan), 24, 265 (1976). (6) M. Ohashi, K. Tsujimoto, and A. Yasuda, Chem. Lett.(Japan), 439 (1976). (7) M. Ohashi, S. Yamada, H. Kudo, and N. Nakayama, Biomed. Mass Spectrom., 5, 579 (1978). (8) M. Ohashi and N. Nakayama, Org. Mass Spectrom., 13, 642 (1978). (9) G. Hansen and B. Munson, Anal. Chem., 50,1130 (1978). (10) R. J. Cotter and C. Fenselau, Biomed. Mass. Spectrom., 6, 287 (1979). (11) R. J. Beuhler, E. Flanagan, L. J. Greene, and L. Friedman, Biochem. Biophys. Res. Commun., 46,1082 (1972). (12) R. J. Beuhler, Ε. Flanagan, L. J. Greene, and L. Friedman, J. Am. Chem. Soc, 96, 3990 (1974). (13) R. J. Cotter, Anal. Chem., 51, 317 (1979). (14) W. R. Anderson, Jr., W. Frick, G. D. Daves, Jr., D. R. Barofsky, I. Yamaguchi, D. Chang, K. Folkers, and S. Rosell, Bio­ chem. Biophys. Res. Commun., 78, 372 (1977). (15) W. R. Anderson, Jr., W. Frick, G. D. Daves, Jr., J. Am. Chem., 100,1974 , NO. 14, DECEMBER 1980

(1978). (16) G. D. Daves, Jr., Acc'ts. Chem. Res., 12 359 (1979). (17) *R J. Cotter, Anal. Chem., 52,1767 (1980). (18) M. A. Posthumus, P. G. Kistemaker, H. L. C. Meuzelaar, and M. C. Ten Noever de Brauw, Anal. Chem., 50, 985 (1978). (19) R. Stoll and F. W. Rollgen, Org. Mass Spectrom., 14, 642 (1979). (20) P. G. Kistemaker, presentation at the "Middle Molecules Symposium," the Johns Hopkins University, Baltimore, Md. (May 1980). (21) B. Soltmann, C. C. Sweeley, and J. F. Holland, Anal. Chem., 49,1164 (1977). (22) D. R Hunt, J. Shabanowitz, F. K. Botz, and D. A. Brent, Anal. Chem., 49, 1160(1977). (23) J. F. Holland, B. Soltmann, and C. C. Sweeley, Biomed. Mass Spectrom., 3, 340 (1976). (24) J. W. Maine, B. Soltmann, J. F. Hol­ land, N. D. Young, J. N. Gerber, and C. C. Sweeley, Anal. Chem., 48,427 (1976). (25) H. D. Beckey and F. W. Rollgen, Org. Mass Spectrom., 14,188 (1979). (26) J. F. Holland, Org. Mass Spectrom., 14, 291 (1979). (27) H. D. Beckey, Org. Mass Spectrom., 14, 292 (1979). (28) J. Roboz, "Introduction to Mass Spec­ trometry," Interscience, New York, 1968, pp 127-32. (29) A. J. Dempster, Phys. Rev., 11,316 (1918). (30) D. Daves (private communication). (31) R. J. Cotter and C. Fenselau, Paper presented at the 28th Annual Conference on Mass Spectrometry and Allied Top­ ics, New York, 1980. (32) F. Borchers, U. Giessmann, and F. W. Rollgen, Org. Mass Spectrom., 12, 539 (1977). (33) U. Giessman and F. W. Rollgen, Org. Mass Spectrom., 11,1094 (1976). (34) W. V. Ligon, Jr., Science, 204, 198 (1979). (35) G. Hansen and B. Munson, Anal. Chem., 52,245(1980). (36) R. D. MacFarlane, D. Uemura, K. Ueda, and Y. Hirata, J. Am. Chem. Soc, 102,875(1980).

Robert J. Cotter received his BS de­ gree at Holy Cross College and his PhD degree in physical chemistry at The Johns Hopkins University. Cur­ rently he is on the research faculty of The Johns Hopkins School of Medi­ cine and is the facility manager for the Middle Atlantic Mass Spectrome­ try Laboratory, an NSF instrumenta­ tion facility. His current research in­ terests include developments in in­ strumentation electronics, mass spec­ trometry of nonvolatile compounds, and new ionization techniques, in­ cluding laser desorption methods.