A ComputerGompatible Digital Data Acquisition System for Fast-Scannirig, Single-Focusing Mass Spectrometers Ronald A. Hitesl and K. Biemann Department of Chemistry, Massachusetts Institute of Technology, Cambridge, Mass. With the advent of fast-scanning single-focusing mass spectrometers, it has become possible to produce a large volume of spectra in a short period of time, particularly when operated in direct conjunction with a gas chromatograph. In an effort to eliminate the time-consuming, manual conversion of oscillograph records into an interlpretable display of m / e values VS. intensities, a system was devised that produces the spectra in this form directly. The electron multiplier output of the mass spectrometer is digitized at a rate of 3000 data points per second and written on magnetic tape while the spectrometer scans from mass 20-500 in 1-3 seconds. The computer reads the tape, finds the peak centers, assigns them mass values and intensities, and prints the resulting spectrum in numerical form and plots it graphically. Thus the spectra are produced directly in compact, standardized form ready for interpretation. Beyond the timesaving aspects of this approach, the availability of the data in digital form now opens the way to easy and speedy manipulation of the spectra such as correction for background contributions, normalization, comparison with other spectra, or searching of libraries.
OVERTHE PAST FEW YEARS the instrumentation associated with organic mass spectrometry has steadily advanced. The most significant improvements, particularly the increase in resolving power, even of single-focusing instruments, and sensitivity, coupled with the availability of fast amplifiers, fast-scanning circuits, and fast-recording systems have led to mass spectrometers capable of recording a mass spectrum within a few seconds. The rate at which spectra are produced is thus only dependent on the time required to introduce the sample into the spectrometer and to remove it after the spectrum has been taken. Anyone familiar with the original form in which mass spectra are commonly -ecorded-i.e., an oscillogram (which permits one to register with sufficient speed the large number of sharp signals of a dynamic range of 1 :lo00 to 1 : 10,000)is aware of the relatively time-consuming processes necessary to convert such a record to a set of permanent data representing meaningful information. The mass-to-charge ratio of the up to a few hundred peaks of the spectrum has to be unambiguously identified, a process which for all practical purposes requires counting from an identifiable low mass up to the molecular weight of the compound. Sophisticated and relatively expensive so-called “mass markers”-i.e., electronic devices which produce 2. signal on the record every five or ten mass units-facilitate the manual process of mass identification but by no means eliminate it. Next, the peak heights have to be measured and converted to the values corresponding to equal attenuation, in order to restore the wide dynamic range of the signals recorded with a multi-trace oscillograph Finally, these data have to be tabulated and/or drawn in the form of a “bar graph,” to obtain them in a relatively standardized form suitable for interpretation ox comparison with other spectra. Obviously the methods of processing of the spectra between 1
NIH-predoctoral Fellow, 1966-68.
02139 REPEATING SCAN CONTROLLER
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, Figure 1. Block diagram of data acquisition system and interface with mass spectrometer
the primary recording step arid the final interpretation have not advanced as much as have the recording techniques. This situation leads to the present problem existing in many laboratories, namely that mass spectra are produced at a much faster rate than they can be interpreted. This dilemma is particularly aggravated when one uses a mass spectrometer directly coupled with a gas chromatograph, a very useful technique which was, in fact, one of the driving forces for the commercial development of fast scanning mass spectrometers. Because of the large number of fractions eluted in the course of a chromatogram and the desire to scan various portions of a gas chromatographic peak for the purpose of testing its homogeneity or identification of unresolved components, the number of mass spectra produced from a simple gas chromatogram of 0.5-1 hour of duration may easily exceed one hundred [see for example a recently published chromatogram of tobacco smoke ( I ) ] . It is obvious that such experiments call for a radically different approach to the data-acquisition and processing problems if the potential of the available data-producing instrumentation (the mass spectrometer) is to be fully exploited. Even the recently suggested recording of the signal in analog form onto magnetic tape which is then played slowly into a conventional recorder, while permitting even faster scan speeds, does not alleviate the problem (2). In the course of the development of techniques that permitted the digital recording of complete high resolution mass spectra (3-6), which represented a much more formidable (1) J. A. Vollmin, I. Omura, J. Seibl, K. Grob, and W. Simon, Helu. Chemica Acta, 49, 1768 (1966). (2) P. Issenberg, P. Bazinet, and C . Merritt, ANAL. CHEM., 37, 1074 (1965). (3) D. Desiderio and K. Biemann, 12th Annual Symposium of Mass Spectrometry,Montreal, June 1964. (4) D. Desiderio, Ph.D. Thesis, Massachusetts Institute of Technology, 1965. ( 5 ) K. Biemann, P. Bommer, D. M. Desiderio, and W. J. McMurray, “Advances in Mass Spectrometry,” Vol. 3, Pergamon Press, London, 1966. (6) K. Biemann, P. V. Fennessey, and J. M. Hayes, Proc. SOC. Photo-Optical Instr. Eng., 1966,p. 11-1. VOL. 39, NO. 8, JULY 1967
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Figure 2. Schematic representation of cycling magnetic field
SMOOTH AND FIND PEAKS
data-acquisition problem than do conventional mass spectra, we were forced at the outset to use automatic, digital recording systems and to process the data with a digital computer. During this work it was realized that similar approaches would be applicable in the case of conventional mass spectrometry, in spite of the fact that the basic objectives and the numerical problems are quite different in these two areas. It seemed, therefore, desirable to develop a digital, computer-compatible recording system that should fulfill two requirements: (1) to record a mass spectrum in the minimum time available for the (with respect to recording time) most demanding application, namely gas chromatography-i,e., 1-3 seconds per spectrum-and (2) to present the mass US. abundance data-i.e., the mass spectrum-in tabular and/or graphical form directly.
FIND REFERENCE MASS.( ~ 9 321 .
NORMALIZE INTENSITIES
1 DIFF. AT REF. MASS
EXPERIMENTAL
The system currently used in our laboratory in conjunction with a Hitachi-Perkin Elmer RMU6-D mass spectrometer consists of three parts (see Figure 1): (a) a 12-bit analog-todigital converter (supplied by Radiation Inc.) which samples the output of the electron multiplier on the mass spectrometer (via the high speed amplifier of the spectrometer) 3000 times per second; (b) a digital tape recorder (Ampex Model TM-2) which records these data at a packing density of 200 bpi (the system is also capable of a 12 Kc sampling rate at 800 bpi writing speed, which however cannot be conveniently handled with our present computer facilities); and (c) the triggering circuitry necessary to start the recording system at the start of the scan, terminate recording at the end, write an “end-of-file” after each scan, etc. For the purpose of monitoring the spectra being recorded or for preliminary checks, an oscilloscope is connected parallel to the recording system so that the mass spectrum is repetitively displayed on the screen. This feature enables the operator to actuate the tape unit whenever a spectrum or set of spectra is to be recorded, as an alternative to the continuous recording mode. The result of each triggering cycle is a complete mass spectrum of the compound, represented by 6-9000 intensity values taken at equal time intervals (every 1 / 3 ~of~ a~ second) recorded in binary form on magnetic tape. The scan characteristics of the spectrometer are such that at that sampling rate, scan speed and a resolving power of 1 :500 (lox valley) there are, for example, about 10 points per peak at mass 100 (but 25 points from center of m/e 99 to center of m / e 100) and 20 points per peak at mass 400 (but also only 30 points from center of m/e 399 to center of m/e 400). At the beginning of each scan a series of parameters are recorded which are, in part, set by the operator--e.g., compound identification, etc.-and in part set by the system itself (scan number, which is automatically incremented for each scan) to facilitate identification and addressing of the individual files. An end-of-file gap separates one scan from the next. Rather than relying on the reliability of an electronic “mass marker” consisting of a device which accurately keeps track of a reasonably fast sweep of the magnetic field (at constant accelerating potential), we use a continuous repetitive scan of the magnetic field. Slight variations in the latter can best be taken care of later in the treatment of a data because any irreproducibility in the magnet scan leads to a 966
ANALYTICAL CHEMISTRY
I
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CoHpps(E ALL PEAKS TO NEW CALI8 TABLE
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Figure 3. Flow diagram of program
relatively smooth contraction or expansion of the mass us. time relationship. The standard mass us. time relationship is established at the outset, using a compound of known spectrum under identical scan conditions, which holds as long as these conditions are not changed. It was found that after the first few scans, they become very reproducible (less than =k1 m.u. at mass 200, a variation easily corrected for in the mass identification program, which constantly keeps track of the deviation of the mass scale from the standard time us. mass relationship, computes the correction factor, and constantly corrects for the deviation). Thus, one is, in effect, using the mass spectrometer itself as a gauss meter which is continuously checked and recalibrated by the computer.
b
Figure 5. Plot of same section as shown in Figure 4, after smoothing of data Figure 4. Plot of section of original data in the region from mje 93 to 100
The cycling magnetic field is schematically illustrated in Figure 2, along with the events taking place automatically. Once the field is cycling continuously and the recording system is activated, the tape starts, the parameters are entered, and the intensity values are continuously written onto the tape in 12-bit digits. At the end of the scan a relay lets the magnetic field decay and simultaneously causes an end-of-file gap to be written on 1:he tape. When the field has decayed to the starting point a trigger pulse activates the scan and the tape again, after incretrienting the index-Le., running number of spectrum on the scan. These processes continue as long as one wishes to record spectra (a full reel holds 300 spectra). The recording mode can be temporarily stopped as well as restarted at any time, except that the system always finishes the current spectrum and restarts at the beginning of the scan cycle following tht: activation signal. Thus one has the option of recording either single spectra, groups of spectra at various intervals, or continuously, whatever the problem under investigation demands. The program permits a choice of processing either all spectra which are on the tape or any number of selected spectra by merely stating their index number. It may be recalled that mass standards have been routinely employed first in high resolution mass spectrometry, where they can be added as internal standards because the choice of a suitable calibration compound-e.g., perfluorokeroseneeliminates interference of its spectrum with that of the unknown. Such admixture would be intolerable when using a single-focusing mass spectrometer and it is for this reason that an external standard (we are currently using a saturated hydrocarbon) combined with a reproducible, repetitive scanning system is employed. The principal steps of the program (FORTRAN 11, for an IBM 7094) from reading the tape to presenting the final spectrum (list of mje us. relative abundance, and/or bargraph plot of these) are outlined below and summarized in the flow diagram (Figure 3). To eliminate electrical noise in the original data (Figure 4), they are smoothed (Figure 5 ) using the equation
Y,* =
&
+2
For explanation of letters see text
where C=I=Z= 1, C+1 = 4, CO= 6, and Yj and Yj* are the original and smoothed data points, respectively. The more commonly used coefficients ( 7 ) were found to be less useful. Next the center and height of each peak is identified by searching for three consecutively larger numbers (found at ‘‘a’’ in Figure 5 ) eventually followed by a pair of values of which the second is smaller than the first (“b” in Figure 5 ) . which is also taken as the peak height after correcting for base line value, see below) and finally a pair of equal (or increasing) values, the end of the peak (found at “c” in Figure 5 . Rather than taking the index of the highest value (b) as the center of the peak, its width at half height is determined, the in Figure 5 ) in index values-Le., units center of which (‘‘8’ of 1/3000 second-is taken as the actual center of the peak, to avoid incorrect positions of peaks of very low intensity. The reproducibility of the intensity measurements was found to be f5 when repeatedly scanning the spectrum of tetracosane and comparing the ratio of the peaks at mje 71 and 85. The resulting array of time cs. intensity values must then be converted to mass-to-charge ratio us. relative intensity. This is accomplished by comparing the time values with an internally generated table (see below) of time 6s. mass. At the start, one mass has to be unambiguously identified and mje 32 was chosen for this purpose. It can easily be characterized as the peak four mass units higher than the most abundant peak (mje 28) located at a certain time from the start of the scan if enough air is admitted via a constant (intentional or unintentional) leak. After mass identification the intensities of mje 28 and 32 are set to zero and all other intensities are normalized with reference to the remaining most abundant one (= 999 arbitrary intensity units). This simple approach has been found convenient and satisfactory; if for some reason the intensities of mje 28 and 32 are important aspects of a spectrum, neon rather than air can be bled into the system and mje 20 used as a starting point. After identifying mje 32 its time value (index) is set equal to that of mje 32 in the calibration table, and the time values of all other peaks are corrected correspondingly. The timevalues of all peaks are then converted to m/e values using the table as long as they agree within 0.4 mass unit with a nominal mass in the calibration table. If the difference is 0.3 to 0.4 mass unit it is assumed to be due to a slight difference
CtYI+i i = -2
(7) A. Savitzsky and M. J. E. Golay, ANAL.CHEM., 36, 1627 (1964). VOL. 39, NO.
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Figure 6. Mass spectrum of cholesterol derived from raw data and plotted by computer Scan time from m/e 28 to m/e 386 was 1.85 seconds
(slow drift) in the magnet scan between the time the calibration compound was run and the spectrum in question. A correction is then applied by multiplying all of the following time values by a factor representing the average ratio of the found time values of the last five points to their corresponding time values in the calibration table. This continuous self-correction approach also takes care of one other possible discrepancy between the calibration data and the compound spectrum: The former is derived from a saturated hydrocarbon and the fractional mass of the various peaks becomes increasingly larger with increasing hydrogen content and reaches 0.5 at about m/e 449 (C3*Hc5: 449.509) while a polyhalogenated compound has a fractional mass which may even be below the integral mass-e.g., CsBr5: 454.592. Considering all the fragment ions, this divergence becomes larger when proceeding from low mass to high mass and is thus continuously corrected by the process outlined in the previous paragraph. To avoid complications due to the relatively greater difficulty in determining the exact center of peaks of very low intensity, only peaks of an intensity >1% of the most abundant ion are used in establishing the mass scale by correlating the time values with those of the calibration spectrum in the manner outlined above. A second calibration table is now constructed from these more abundant peaks and used for identification of the less abundant ones (within 1 0 . 2 m.u.). This temporary calibration table holds only for that particular spectrum from which it is derived and is erased after use while the primary calibration table is retained throughout. After the mass assignment process there may remain a few time-values unassigned which represent one of the following: (a) Doubly charged ions of odd mass (recognizable by their mass falling exactly between two integral masses and lying below one half of the highest mass in the spectrum). (b) Maxima of “metastable peaks,” which are generally characterized by the discrepancy (with respect to normal peaks) of the peak height 6s. width and can be recognized in this manner. (c) Spurious maxima, usually being of low intensity and appearing much narrower than normal peaks. Obviously (a) and (b) contain useful information while maxima of type (c) should be discarded. 968
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The resulting list of mje cs. abundance is then either printed as a table of mje vs. intensity, plotted in graphical, standardized form (see Figures 6, 7, 9 and 10) or retained in the computer’s secondary memory (on tapes or discs) for further treatment, such as comparison with previously determined spectra on file in the secondary memory. The previously mentioned calibration table-i.e., time values us. mass-to-charge ratio-is assembled as follows, using the mass spectrum of an aliphatic hydrocarbon-e.g., dotriacontane (C3*H66,MW 450)-recorded under the same conditions (accelerating potential and cycling magnetic field). The mass 28 peak is located as being the largest peak near the beginning of the spectrum. Because the spectra of such hydrocarbons exhibit intense alkyl ion peaks, they are easily located by the computer by searching for the largest peak within a given range from the last found peak. Once all the ions in this short list (28, 43, 57, . . ., 421, 450 for dotriacontane, for example) have been found, the calibration table is completed by interpolation. RESULTS
A few eyamples will illustrate various aspects of the technique described above. A sample of cholesterol was placed into the ion source via the vacuum lock of the direct introduction system. The sample temperature was slowly raised while the magnet scanner was continuously cycling, covering the region of m/e 10-600 in 3 sec with aresolving power of 1 :400 (10% valley). Each scan was displayed on the face of a memory oscilloscope. First is seen only the spectrum of the background (mainly air bleeding through the inlet system for reasons mentioned earlier). When the sample temperature reached the point where cholesterol vaporizes, its spectrum appeared, and the data acquisition system was activated to record the following scan cycles. The spectrum reached a maximum and then subsided because of depletion of the sample. The first several spectra were processed and plotted by the computer. Figure 6 represents one of the plots and agrees very closely with the mass spectrum recorded conventionally with an oscillograph, and measured and plotted manually.
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Figure 7. Mass spectrum of 1-phenyldodecane
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Figure 8. Gas chrom;atogram of fatty acid methyl esters (CB-C2,,, even carbon iiumbers only), recorded using flame ionization detector Numbered sections reprefient periods during which the spectra in Figure 9 were recorded (3% SE-30, 12 ft X inch, programmed
from 170"-270' at 20°/minute)
The mass spectrum of 1-phenyldodecanewas recorded in an effort to demonstrate the ability of the system to establish correctly the mass scale in the case of a compound which exhibits a spectrum with a large part devoid of peaks and is thus often difficult to "count" correctly in manual operation. Figure 7, produced by the computer, shows the molecular ion correctly at mass 246. The major use for this recording technique lies, however, in the area of gas chromatography, for reasons outlined in the introduction. Figure 5, represents spectra taken at the front of the last fraction of the gas chromatogram (Figure 8) of fatty acid methyl esters (C8-C2,). Comparison of scans 1, 2, and 3 (Figure 9) reveal's a relative increase in certain peakse.g., 74, 87, and 326-while others-e.g., 73, 207, and 281sinlultaneously decrease. The latter are typical of silicone compounds and are due to the liquid phase (SE-30) of the gas chromatographic colurnn which at that point has reached 270" C and bled someryhat. Because the emerging fraction contributes more and more to the spectra of consecutive scans of the rising portion of the peak, the relative ratio of the two components (methyl eicosanoate and silicone oil) changes continuously. The apparent decrease of the silicone peaks are, however, due to the fact that the computer automatically normalizes the intensities with respect to the most abundant one ( m / e207 in scan 1 and 2, but mje 74 in scan 3). The availability of the spectral data in digital form in the memory of the computx (or in any convenient storage form, such as punched cards, punched paper tape, magnetic tape or
*
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Figure 9. Spectra obtained are of sections labeled 1, 2, and 3 in Figure 8 Top: C 20 methyl ester. Scan 3 Middle: C 20 methyl ester. Scan 2 Bottom: C 20 methyl ester. Scan 1 magnetic discs) opens the way to many simple or more complex forms of information processing. For example the somewhat obliterating effect of the silicone peaks in the spectra shown in Figure 9 can be eliminated by subtracting the spectrum of the background (Le. column bleed alone) from the raw spectrum. The lower part of Figure 10 represents the result of subtracting the spectrum obtained when the column had reached 270" C during a blank run, and is now unmistakeably that of methyl eicosanoate. The upper specVOL. 39,
NO. 8, JULY 1967
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Figure 10. Bottom: Spectrum shown on top of Figure 9, after subtraction of silicone oil background. Top: Same, after subtraction of spectrum shown on bottom of Figure 9. For further explanation see text trum in Figure 10 was obtained by subtraction of scan 1 from scan 3 (Figure 9), thus using a spectrum (scanned during the same chromatogram) in which column background dominates. Again the “pure” spectrum emerged. The latter approach can also be used to obtain the “pure” spectrum of a component of a gas-chromatographically unresolved doublet (for more complex fractions a more involved iteration procedure is required). CONCLUSIONS A method for the fast and facile digital recording and permanent graphical display of mass spectra produced by fast-scanning, single-focusing mass spectrometers has been developed, It reduces the tedious and time-consuming manipulation of conventionally obtained oscillograph records. Even more important is the availability of the data in a form which lends itself to enhancement of the information content using simple and obvious approaches too time-consuming or boring for manual execution. For example, when introducing a compound directly into the ion source it is convenient to scan fast (a few seconds) and record a number of consecutive spectra while the compound vaporizes, because this eliminates the need for careful control of the rate of vaporization. One then has the choice of having the computer select the most intense spectrum for processing and presentation or have it compare the consecutive scans for identity to uncover fractionation of an impure sample or thermal decomposition of a labile one. Furthermore, only the direct computer-generation of digital mass spectra makes it practically feasible to compile libraries
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of mass spectra (8-10) and to generate the spectra of unknowns for identification by computerized comparison. This technique is far superior to the tedious punching of data on cards, a process that itself may be more time-consuming than a manual search of a suitably-arranged card index. For similar reasons it will be more realistic and desirable to develop computer-based techniques to aid in the interpretation of the spectra of compounds not yet contained in such libraries along lines recently suggested (IO). The most important use of this technique will undoubtedly be the area of combined gas chromatography and mass spectrometry because, when used efficiently, it produces an enormous amount of spectra, the majority of which are suitable for, or even require, further processing along the lines indicated above. Many of the components turn out to be known compounds or have been encountered before. Their automatic identification by searching a collection of spectra in the secondary memory of a computer relieves the chemist of a large volume of routine work. Other fractions may be due to more than one component and need subtraction of consecutive scans. Conversely the test for homogeneity of a fraction by careful comparison of consecutive scans throughout the emergence of the peak is an important (but manually very tedious) task. The need for fast scans, requiring only a small part of the time of the emergence of the gas chromatographic peak is too well known (11) to require further emphasis. The method of mass identification is simple, reliable, and inexpensive. The elimination of the need for an electronic mass marker, which would also require frequent checking with a calibration compound and is quite expensive if it has to be reliable at such fast scan speeds, partially compensates for the cost of the A/D converter and digital tape. While computer time is still generally considered an expensive factor, the actual cost per spectrum is much less than that of manual processing of a conventional oscillograph record. Processing one complete spectrum takes only 20 seconds on an IBM 7094 computer, thus costing very little even at industrial peak time rates. ACKNOWLEDGMENT
The authors are indebted to Mrs. Vivian Beecher for her help with the programming, to the Perkin-Elmer Corporation for supplying us with the analog-to-digital converter and tape unit, as well as designing of the interface to the mass spectrometer to our specifications, and to the MIT Computation Center.
RECEIVED for review March 31, 1967. Accepted May 5, 1967. (8) S . Abrahamsson, S. Stenhagen-Stallberg, and E. Stenhagen, Biockem. J., 1964,92. (9) S . Abrahamsson, G. Haggstrom, and E. Stenhagen, 14th
Annual Conf. of Mass Spectrometry and Allied Topics, Dallas, Texas, May 1966. (10) B. Pettersson and R. Ryhage, Arkiv Kemi, 26, 293 (1966). ill) F. A. J. M. Leemans-and J. A. McCloskey, J. Am. Oil Chemists’ Soc., 44, 11 (1967).
