Instrumentation
R. D. Macfarlane Department of Chemistry Texas A&M University College Station, Tex. 77843
C alifornium-252 Plasma Desorption Mass Spectrometry
Large Molecules, Software, and the Essence of Time
Our first work with 252 Cf-plasma desorption mass spectrometry (252CfPDMS) began in 1973 as a spin-off from studies on the properties of /3-decay recoils desorbed from surfaces (/). In 1974, we published our first paper on the method, demonstrating that molecular ions of cystine and arginine could be desorbed from thin layers of these compounds excited by the highenergy nuclear fission fragments from 252 Cf-radioactive decay (2). The first indication that 252 Cf-PDMS might be useful in desorbing ions of "difficult" molecules came in 1976 when the molecular ion of tetrodotoxin was detected for the first time (3). This was a classic "difficult molecule" of the 1970s.
Since then, we have participated in a number of collaborations where our contribution was to determine molecular weights and correlate fragmentation patterns for molecules whose structures were being determined. 0003-2700/83/A351-1247$01.50/0 © 1983 American Chemical Society
Among the hundreds of molecules that we have studied, three in particular stand out in terms of the sense of achievement and contribution. First was the study of the Q*-nucleoside, where an unexpected naturally occurring modification was revealed that was so unusual that our data were, at first, treated with skepticism by our collaborators (4). Second, 262CfPDMS of bleomycin, an important an
titumor drug, gave a molecular weight that did not correlate with the published structure (5). This, along with additional data, led to a revision of its structure. Third, the 252 Cf-PDMS of palytoxin is included, because determination of its molecular weight (2880 u) accelerated the final solution of the structure of this legendary marine toxin, following a decade of extensive investigations (6). In some of our earliest studies, it became apparent that it was possible to
desorb molecular ions of high molecular weight species. A 252 Cf-PDMS spectrum of "platinum blue," a polymeric complex of platinum and thymine, showed molecular ions extend-
ing to m/z 3000 (3). We began to focus on these high molecular weight species beginning with the peptide /3-endorphin (3464 u) (7) and continuing with a series of synthetic protected oligonucleotides that brought us to m/z 12 500 (8). In 1982, a second group at Uppsala headed by Sundqvist became active in high molecular weight mass spectrometry using 252 Cf-PDMS. In a series of brilliant studies starting with insulin (9), and continuing with a set of small proteins of increasing molecular weight, they extended the mass range to ~m/z 14 000 (10). There are now 10 functioning 252 Cf-PDMS systems: four located in the U.S., five in Europe, and one in Sri
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Figure 1. Schematic of a 252 Cf-PDMS experiment
Fission Fragment Detector
The Big Event Excitation Area (100 nm 2 )
Desorbed Ions Molecular Ion
Sample Foil
Na + Acceleration Grid
Lanka. Seven of these are operated by groups whose roots lie in nuclear chemistry and physics. There is good reason for this. The 252 Cf-PDMS method is very much a classical arrangement for a nu clear spectroscopy experiment. Three of the 10 252 Cf-PDMS systems serve a dual purpose: as mass spectrometers for biomolecules and as nuclear spec troscopy systems when the group is scheduled for accelerator beam time. All of the variations used in data ac quisition and analysis in 252 Cf-PDMS that will be discussed in this paper de rive from methods developed for nu clear science studies. This methodolo gy is actually a general approach wide ly used in any measurement made at the event-by-event level and where many variables are being measured during that event. 252 Cf-PDMS is not only a unique ionization-desorption method for non-volatile molecules, but also a very different approach to obtaining mass spectra. The instrumental features of the method couple nicely with current developments in high-speed electron ics, minicomputers, and microproces sors and give an example of how the desires of the experimentalist can be met with computer software as well as hardware. We have a growing appreciation for what a dedicated computer can do for us as a partner in our experiments. This refers not only to the control of the hardware functions and data anal ysis, which are now commonplace in modern mass spectrometry. It also re fers to a unique feature of the dataacquisition phase where, through the medium of software, we come close to having a "Maxwell's Demon" that can interact, sort, and make decisions at the molecular level as information is being developed and transmitted by the mass spectrometer. It is this as pect of our work that is highlighted in these pages, especially as it relates to
Fragment Ions
obtaining mass spectra of high molec ular weight species.
Desorbed Ion Detector
The Big Event
The fission of the 252 Cf nucleus is a big event at the atomic level. The nu clear fission of 252 Cf occurs sponta neously. The mean lifetime of a 252Cf nucleus exclusive of the competing α-decay process is ~ 8 5 y. It fissions because the nucleus of 252 Cf vibrates like a deformed liquid drop. Occasion ally at the extremes of vibrational de formation, it divides into two smaller droplets of nuclear matter—nuclear fission fragments. The process is high ly exothermic (~180 MeV/atom or 1.7 Χ 1010 kJ/mol) and the energy re lease is predominantly carried away by the two nuclear fission fragments as kinetic energy. Multiple atomic ion ization of the fission fragment ions is extensive, forming very high oxidation states not seen in chemical processes. The combination of energy release and ionization produces two heavy atomic ion pairs such as 110-MeV iooSr+20 a n d 75_MeV i50Ba+20. A wide range of fission fragment ion pairs contributes to the fission spec trum because of the statistical nature of the fission process. It is the interac tion of these fission fragments as they pass through matter that forms the basis of the 252 Cf-PDMS process. The theory of the interaction of high-energy charged heavy ions with matter was developed by Bohr fol lowing his monumental contributions to the quantum theory of atoms (11). This theory has been refined from its original formulation, but the basic principles remain unchanged. Accord ing to the theory, atoms and molecules of the matrix perceive the swiftly moving ion passing through the ma trix as equivalent to a short burst of
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photons. The frequency distribution of the photon spectrum depends on the ion velocity, and the intensity of the spectrum depends on the charge of the ion. The electromagnetic radiation induces electronic excitation, which quickly dissipates into a complex spectrum of atomic, molecular, and matrix excitation. The desorptionionization process in 252 Cf-PDMS de rives its energy from the relaxation of the primary energy deposition. A single fission fragment passing through a thin film can desorb many charged species (positive and negative ions, electrons) and up to 2000 neutral molecules (12), can release a burst of photons, and can excite a surface area ~10 nm in diameter (13). It is a big event. It is this big event that is studied on an event-by-event basis in 252CfPDMS. Each event constitutes a mea surement; a mass spectrum of the ions emitted is just one aspect of this event. The methodology of 252CfPDMS uses the versatility of the kinds of measurements and correlations that can be made on each event. Although one event can give a mass spectrum, there is a large statistical fluctuation from one event to another because of the small number of atoms excited in a fission track being probed. A large collection of fission tracks (~10 6 -10 7 ) formed at the rate of 2000 s _ 1 is used to obtain a statistical average of the desorption-ionization properties of the nuclear fission fragment track in the matrix of interest. Figure 1 portrays the general fea tures of the use of the nuclear fission
Figure 2 . H o w TOF data a r e c o n v e r t e d to digital i n f o r m a t i o n
fragments from 262 Cf decay in a 252 Cf-PDMS experiment. The 252 Cf source is mounted behind the sample and when a fission event occurs the two fission fragments are hurled in opposite directions, one toward the sample and the other toward a fission fragment detector. What follows is the big event, the emission of secondary ions, neutrals, and photons from the sample foil, and the subsequent detection and mass determination of the ionic products. Describing the Big Event in Electronic Language As shown in Figure 1, there are two kinds of detectors in the 252 Cf-PDMS system that are the sensors monitoring the big event. One of these, the fission fragment detector, determines precisely when a nuclear fission occurs by detecting one of the two fission fragments that are released in the 252 Cf fission process. This detector produces an electronic pulse that gives a precise time marker for the occurrence of a fission event. A desorbed ion detector located 50 cm away and in a line of sight with the surface of the fission track is sensitive to the ions and photons that are emitted. This sensitivity is enhanced by accelerating the ions toward the detector using an electric field (10-20 kV). Each ion that the desorbed ion
detector senses initiates the formation of an electronic pulse that is precisely synchronized with the arrival time of the ion at the detector. Every time a fission track is formed in the sample, there is a flurry of electronic activity at this detector as it senses each of the ions and announces each arrival by issuing an electronic pulse. This is followed by a quiescent period of ~50 jus before another fission track is formed in the sample. The experimental arrangement also includes the elements of a mass spectrometer. If we measure the time intervals between the initiation of the fission track and the arrival time of the ions at the ion detector, we have measured the time of flight (TOF) of each ion and hence its velocity and mass, since the kinetic energy of each ion is known from the electric field potential between sample foil and acceleration grid. We can use the electronic pulses from the fission fragment detector to measure the number of fission tracks forming per second (~2000), and the electronic pulses from the secondary ion detector to measure the number of ions that are desorbed per second (~8000) from the surface of the fission track. We also use the electronic pulse from the primary sensor to start a fast electronic clock. This clock "ticks" at the rate of 50 million times/s.
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Figure 2 outlines how the TOF measurement is made. When an electronic pulse from the secondary ion detector enters the clock, the clock circuitry performs two tasks: It determines whether this was the first, second, third, or nth ion to be detected from the current fission track formed in the sample and also how long it took for that ion to travel from the site of the fission track to the secondary ion detector located 50 cm away. Figure 2 portrays a big event where five ions were detected whose TOF values ranged from 977.260 to 18 339.675 ns. Each time interval is measured to within 1/200 of a "tick," or 78 ps. The electronic marvel that performs these measurements is the time interval digitizer (TID) (14). The TID is at the heart of the 252 Cf-PDMS measurement. Its function parallels that of the magnet in a magnetic sector mass spectrometer. At 10 kV, an m/z 104 ion takes ~30 /is to make the 50-cm trip. If the ions were monoenergetic and there were no instrumental aberrations, the peak width would be 78 ps, the limit of precision of the TID. This corresponds to a mass resolution of 200 000. The upper limit in time of the TID is 320 ms, which means that the flight time of an m/z 10 12 ion could be measured using this TID, if it could be desorbed and detected. Data Acquisition The description of the big event in electronic language is in the form of a set of electronic words sent by the TID to a computer, as shown in Figure 2. For a particular fission track, as many as 36 ions have been detected (15), each producing an electronic word that contains the relative posi(continued on p. 1255 A)
tion of the ion in the 36-word list as well as the precise TOF with the full precision of the TID. For example, in the event portrayed in Figure 2, the third ion was one in a sequence of five ions desorbed by a fission fragment, and its TOF was 7.2445 μβ. Before being processed, these electronic words are stored in a holding circuit (a buffer) until all the electronic words from a single event have been collect ed (~40 yus). The words then pass through an interrogation module, where each word is subjected to a se ries of tests that have been previously established by the experimenter. As shown in Figure 3, there are nu merous options available depending on what is being measured. Some of these will be discussed below. After leaving the interrogation circuit, some of the words will be discarded because they don't meet criteria established by the experimenter, others will be stored in the memory of the computer with their full complement of significant figures, while most will have some sig nificant figures removed before stor age. Some might be subjected to mathematical manipulation prior to storage. The memory of the computer serves as a multichannel analyzer in these ex periments. The array size varies from 25 000 to 50 000 channels, depending on the mass range covered for a partic ular experiment. Each channel is re sponsible for recording the number of ions whose TOF lies within a time in terval of 1.25 ns. For example, if the TOF of a particular ion is 12 501 ns, it will be recorded by the channel that covers the time range from 12 500 to 12 501.25 ns. At the end of the experi ment, each of the 25 000 channels will have recorded the number of times an ion has been detected whose TOF value has fallen within the time inter val assigned to it. This information is then transferred to long-term storage (a disk storage unit) to await data analysis. One of the most interesting aspects of data acquisition in 252 Cf-PDMS is the ability to interact with the data through the interrogation module. The interrogation is handled and con trolled by software. This is readily modified depending on what new in formation we might want to obtain in studying the dynamic properties of fission tracks. For this concept in data acquisition to be used to the fullest, it is desirable to collect the full spectrum of ions emitted independent of kinetic energy spread or metastable lifetime and to allow selections from the full spectrum to be made by software. Data Analysis We now discuss some aspects of our data analysis, one of these being the
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ANALYTICAL CHEMISTRY, VOL. 55, NO. 12, OCTOBER 1983 ·
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How to switch HPLC columns using valves. Different analyses sometimes use different columns. But even when the same column can handle more than one kind of sample, many chemists dedicate a column to each analysis. This prolongs column life, reduces interferences, and eliminates equilibration delays. Rheodyne's Technical Notes 4 tells how to use switching valves to connect as many as five columns to a chromatograph. Any column can be selected, while the off-line columns remain sealed at each end. The effect on resolution is shown to be negligible in most cases.
Send for Tech Note # 4 For the well-illustrated tech note, contact Rheodyne, Inc., P.O. Box 996, Cotati, California 94928, U.S.A. Phone (707) 664-9050.
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Maxwell's Demon Becomes a Mass Spectroscopist Figure 3. How the digital output of the time interval digitizer is processed at the event-by-event level The interrogation module investigates each electronic word and makes decisions (established by the human factor) that determine the ultimate fate of an electronic word. Special words are given the V.I.I, (very important ion) treatment
conversion of a TOF spectrum to a mass spectrum. But because we have so many software options available at the data-acquisition level, the data analysis can provide much more insight about the mechanism of the desorption-ionization process. The mass calibration of any mass spectrometer is an important and sometimes troublesome operation, particularly at high masses. For TOF mass spectrometers, the mass (m/z) of an ion and its TOF are related by the equation: TOF = d yf^Tz + C 2 where C\ and C
