Display oriented mass spectrometer-computer system

reference 28 because of the large disparity (w factor of ten) in mass measurement accuracies achieved, indicating that these two approaches cannot be readily compared. Theoretical treatment of data such as described (14, 28) cannot permit meaningful conclusions about computer systems and tradeoffs in sensitivity, resolution, and so forth if standard deviations of mass measurement are 40-60 ppm. Only when a system has been studied and understood in detail and standard deviations of mass measurement in the 5-ppm range are routinely obtained, particularly above m/e 80 to 100 where high accuracy is required to distinguish among elemental compositions with very similar exact masses, can the worth of a computer system and mass spectrometer operating tradeoffsinvolved be assessed. ACKNOWLEDGMENT

The authors acknowledge the efforts of Mr. Rylee McPherron, Mr. Jan Hauser, and Mr. John McConnell for innovative

programming, and Mr. Robert E. Furey, Mr. Galen Collins, and Mr. Michael Issel for hardware interfacing. The authors also acknowledge the assistance of Dr. Donald A. Flory, NASA Manned Spacecraft Center, for technical and administrative assistance during this development program. RECEIVED for review April 12, 1971. Accepted July 23, 1971. This research was supported by the National Aeronautics and Space Administration, Manned Spacecraft Center, Contracts NAS9-9593 and NAS9-7889. The Sigma 2 computers were provided under NASA, MSC contract NAS9-7381; Principal Investigator A. L. Burlingame. The MS-902 mass spectrometer was provided by NASA NsG 243 Suppl. 5. The special version of the Hitachi Perkin-Elmer RMU6-D mass spectrometer was provided by Prof. K. Biemann, MIT, and coworkers R. Murphy, N. Mancuso, and R. Hebert, under separate NASA contract.

Display Oriented Mass Spectrometer-Computer System William F. Holmes Biomedical Computer Laboratory, Washington University School of Medicine, 700 South Euclid Avenue, S t . Louis, Mo. 63110

William H. Holland Department of Psychiatry, Washington Unioersity School of Medicine, 4940 Audubon Aoenue, St. Louis, Mo. 63110

John A. Parker Biomedical Computer Laboratory, Washington University School of Medicine, 700 South Euclid Avenue, St. Louis, Mo. 63110 A display oriented computer system for the LKB-9000 gas chromatograph-mass spectrometer has been de. veloped using the PDP-12 computer. The interface is very simple. Spectra may be obtained intermittently or with continuous scanning at a rate selected by the operator. Each spectrum is reduced and displayed on the computer oscilloscope as soon as the scan is done. Tape storage is optional so that the operator can monitor an entire GC run without storing unnecessary data. A description, clock time, and scan number are stored with the data. The spectrum display has expandable scales so that small regions can be highly magnified. Storage format is variable in length. The largest spectrum allowed contains 850 peaks in the range 0-1000 mass units. Over 220 spectra with 200 peaks in the range 20-500 mass units can be stored. Each spectrum contains calibration information which can be displayed as a deviation graph. This is used for calibration adjustments which are done about once a week. Calibration is routinely maintained to 850 mass units. Spectra can be plotted with a wide variety of options.

USEOF COMBINED gas chromatography-mass spectrometry requires automated data reduction to make full use of the information available. Several computer-mass spectrometer systems have been described that are capable of reducing data from continuously scanned gas chromatograph effluent, presenting the results on paper with a digital plotter or high speed line printer (2-5). A display oriented system is ( I ) R. A. Hites and K. Biemann, ANAL.CHEM., 40, 1217 (1968). (2) B. Hedfjall, P.-A. Jansson, Y. Marde, R. Ryhage, and S. Wikstrom, J . Sci. Imrrurn. Ser. 2, 1031 (1969). (3) W. E. Reynolds, V. A. Bacon, J. C. Bridges, T. C. Coburn, B. Halpern, J. Lederberg, E. C. Levinthal, E. Steed, and R. B. Tucker, ANAL.CHEM., 42, 1122 (1970). (4) C. C. Sweeley, B. D. Ray, W. I. Wood, J. F. Holland, and M. I. Krichevsky, ibid.,p 1505. (5) J. R. Plattner and S. P. Markey, Org. Mass Spectrom., 5, 463 (1971).

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described below which allows the user close control and considerable flexibility in acquiring and analyzing mass spectra. The system uses an LKB-9000 gas chromatograph-mass spectrometer interfaced with a PDP-12 computer. This computer was selected because it contains a large built-in oscilloscope, and two small LINC tape units which serve as a convenient and inexpensive medium for data storage. Each LINC tape can store over 225,000 twelve-bit words. During data acquisition, spectra are reduced and displayed as soon as each scan is finished, with optional storage on tape; thus, the operator can monitor an entire gas chromatograph (GC) run by continuous scanning without storing any more data than are necessary. A noise display provides optimal threshold adjustment for measuring peaks with low abundance. The scan repetition rate and scan speed can be changed at any time during a GC run, or scanning may be stopped and restarted intermittently. Whenever a scan is not in progress, the computer displays the spectrum of the last scan. A programmable clock keeps track of the time, which is stored as part of each spectrum for comparison with GC retention times. Calibration is routinely maintained to 850 mass units by use of a calibration deviation display. Since calibration information is included with the reduced data, this display is available for every spectrum. The mass spectrum display is also used to examine data after it has been stored on tape. The mass abundance and mass unit scales can be greatly expanded for detailed observation of small peaks. Any spectrum on display can be plotted as a bar graph, whenever a record on paper is needed. Computer and Interface. The Digital Equipment Corporation PDP-12 is specifically designed as a laboratory instrument computer, The PDP-I:! is a typical small computer (in fact, a PDP-8 with an extra LINC instruction set), but contains in addition a number of input/output devices as

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L K B - 9000-PDP 12A INTERFACE Figure 1. Mass spectrometer-computer interface The PDP-12 input/output section is on the right. From top to bottom, two analog channels (10 and l l ) , four on/off level sensing lines (XLO, 1, 2, 3) and one relay (0) are used. No internal modifications were made to the computer. Two signal buffer amplifiers (top middle) are used to transfer the isolated mass abundance signal from the mass spectrometer galvanometer preamplifier to analog channels 10 and 11, splitting the signal into two parts with relative gains of 8 : 1. The other two interface amplifiers with their transistor drivers (left center) transfer the unit and tens mass marker voltage levels to the computer sense lines XL2 and XL3. Sense lines XLO and XL1 and relay 0 are connected to the scan control system. Their operation is described in the text

part of the basic computer. Besides the oscilloscope and LINC tape units, there are twelve on/off sense lines, six DPDT relays, and a sixteen-channel, ten-bit, 50-kHz analog to digital converter. The last three items greatly simplified the design of the mass spectrometer-computer interface, since no electrical modifications to the computer were necessary. Eight channels of the analog to digital converter are connected for external input; the other eight are attached to built-in potentiometers that are used whenever a program needs a continuously variable input. The potentiometers are especially useful with display programs to control such parameters as scale size and noise threshold. The system used here has 8K of memory, and two standard accessories, a programmable clock, and a plotter interface. The latter is connected to a Houston Instrument Company Complot digital plotter. The computer costs $36,500, plus $3,500 for the plotter. The LKB-9000 mass spectrometer is a standard instrument with the mass marker attachment. The mass marker senses the magnetic field with a Hall effect generator, and converts this signal into a signal proportional to the equivalent mass number, using an adjustable analog squaring circuit. This signal is then converted into digital levels and pulses, which are normally used for a numerical display and to provide mass marks on recorder tracings. The mass marker can be adjusted to 1 0 . 3 mass unit out to 1000 mass units, although only isolated peaks have been calibrated beyond 850 mass

units because of a lack of satisfactory calibration compounds. Reproducibility from scan to scan is about 10.1 mass unit. Thus, the mass marker squaring and digitizing circuits correspond in resolution (but not linearity) to a fourteen-bit analog to digital converter. The mass spectrometer-computer interface is shown in Figure 1. It costs about $100 to construct. The mass abundance signal is split into two channels, with a relative gain of 8:l. This was necessary because the ten-bit analog to digital converter only has a dynamic range of 1000:1, while the mass abundance signal to noise ratio can be as great as 10,OOO:l. By use of two channels, the range is extended to thirteen bits or 8OOO:l. During data acquisition, the more sensitive channel is sampled first. If it is saturated, the other channel is used. The error at the transition point is il bit out of 7 bits or 11/128. A third channel could easily be added, giving a dynamic range of 64,000:1, equivalent to a sixteen-bit analog to digital converter. Thus, the dynamic range can be extended as needed without resort to unusual and expensive computer hardware. Scanning is started by an on/off switch at the mass spectrometer, connected to external level line 1 (XL1) on the computer, which senses on/off voltage levels. If the on condition is sensed, the program closes relay 0, which is connected in parallel with the manual scan button. The scan starts, and the program tests external level line 0 (XLO) until the closure of the mass spectrometer relay K2, which


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Figure 2. Unreduced mass abundance signal A highly expanded portion of a mass spectrum signal is shown, with a set of mass fragment peaks plus a metastable peak. The peak at the right appears flat because it goes off scale. A peak detection algorithm is simultaneously in use. The vertical lines designate the positions and abundances of those peaks that are identified by the algorithm

Figure 4. Threshold adjustment display Visual display of the base-line noise allows optimal adjustment of the threshold, using a computer potentiometer as input to vary the threshold level

Figure 5. Scan controls display This questionnaire controls the clock starting

Descriptive material is typed in with the Teletype, using the oscilloscope as a questionnaire. The description may be modified at any time by deleting or typing over the current entry. It is stored with each spectrum for subsequent viewing and plotting. The program can be easily changed to display and store a much more extensive description

time, storage of spectra, and the scan rate. The starting mass unit must also be supplied for correct data reduction. The answers to the questions are initially set by the program to those shown in the figure (N, N, 20, 10, 4); they can be quickly changed at any time during the operation of the mass spectrometer. The small dot above the upper left corner of the second ‘N”indicates the current typing position

actually turns on the magnet current drive. The program then tests line 2 (XL2) for a level change in the decades unit of the mass marker, meaning that mass 10 or 20 or 30 etc. has been reached. At this point data acquisition starts. Scans are started only at mass units divisible by ten so that the magnet can settle down from initial transients. In practice, the scan is started several mass units before the tens mass unit selected. During data acquisition, mass units are sensed by level changes on line 3 (XL3), while the mass abundance signal is sampled on channels 10 and 11. The current drive relay K2 is also tested via line 0 (XLO) during the scan. When this relay opens, the scan stops and the magnet field starts to collapse. The interface system has worked quite reliably. In particular, the starting decade signal and the subsequent mass unit signals have given no problems at all, to within the calibration specifications discussed above. Peak Detection. During the initial phase of program development, the unchanged mass abundance signal was sampled and stored on tape, with facilities for displaying and plotting any portion of the signal. Figure 2 shows a group of mass fragment peaks together with a metastable peak. A peak detection algorithm is in use simultaneously with the display. The vertical lines indicate the positions and abundances of the fragment peaks, as detected by the algorithm. Evaluation of peak detection algorithms is a straightforward procedure using this display. Errors arising from unusual peak shapes and noise conditions are readily apparent. The algorithm currently in use requires two conditions to be met before a fragment peak is recognized. First, the signal must rise above the last minimum by a threshold, which

can be changed by the mass spectrometer operator using a computer display. The threshold is measured from the last minimum rather than the base line so that fragments with overlapping peaks or combined with metastable ions will not be rejected by the threshold requirement (see Figure 2). The threshold is used as a simple filter for rejecting noise peaks. Peaks with high abundance have more noise than very small peaks near the base line. Thus, when the threshold was set high enough to prevent multiple peaks at abundant fragments, small peaks clearly above the base-line noise were not recognized. This problem was solved by adding to the constant threshold a variable threshold equal to one-fourth the momentary signal value. After the combined threshold is exceeded, and a tentative peak maximum is found, the signal must drop by an amount exceeding the combined threshold before the peak is identified as genuine. Using this algorithm, data can be acquired and reduced on-line at a sampling rate of about 10 kHz. All data acquisition programs have been written in a modular fashion, so that new algorithms and hardware for peak detection can be tested and installed without extensive program modifications. The current programs do not look for metastable peaks, although they are easily identified visually with the expanded computer display. On-Line Data Reduction. The system is normally operated using on-line data reduction to a bar graph form, retaining calibration information. During each scan, reduced data accumulate in the computer core. While the magnet field collapses, the scan is (optionally) stored on one of the tapes, the scan tape. Storage takes less than one second in all cases. The spectrum is then displayed until another

Figure 3. Description display

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Figure 6. Mass spectrum display Every spectrum is reduced and displayed after each scan. If the spectrum is stored, the description, the tape scan number, and the clock time when the spectrum was acquired are stored with it, and displayed at the top. The tiny bend on the top of the fragment at m/e 143 indicates that its abundance goes beyond the display scale. The letters at the bottom designate those control keys that are operationalwith this display

scan starts. Scans are stored sequentially on tape, so that several sets of scans from different experiments may be stored together. The description (Figure 3), the scan number, and a clock time permit later identification. The operation of the system is controlled by a sequence of four displays, the description, the threshold, the controls, and the mass spectrum displays (Figures 3-7). Instructions are given to the computer through questionnaires displayed on the oscilloscope. Figure 3, the description display, shows the initial step in data acquisition-typing in a description of the data. This description is stored on tape with each scan. It can be changed at any time by pausing between scans and returning to the description display. After the description is entered, a threshold adjustment display appears (Figure 4). Assuming the mass spectrometer is not focused on a peak, the base-line noise signal will be displayed as the difference between the momentary values and a time average. If any portion of the signal rises above the threshold, it is brightened, warning the operator that a higher threshold is needed. The threshold is changed using one of the computer potentiometers. The program stores the new threshold value for use with the peak detection algorithm, and displays the threshold as a horizontal line above the noise signal. The third display (Figure 5 ) controls the operation of the system. The programmable clock can be started when appropriate by typing in Y (yes) to the question SET TIMER? Thereafter, the time is available in seconds on the spectrum display, and is included with each scan just before the scan starts. Storing scans on tape is optional and quickly changeable by typing Y or N in answer to the STORE SCAN? question. The starting mass unit at which the computer will begin to accept data (10, 20, 30, etc.) must be entered. This datum is included with each scan. The mass spectrometer must be manually set several mass units before the starting tens unit, as described above. The scan repetition rate for automatic scanning may be selected according to the scan range and speed desired. The fastest scan regularly used goes from mass 20-500 in 4.2 seconds, mass 20-800 in 7 seconds, and mass 20-1000 in 12 seconds. The scan speed control on the mass spectrometer can be changed at any time without losing calibration, since the mass marker measures the magnetic

Figure 7. Expanded spectrum display

A small region of the spectrum in Figure 6 is shown, with the abundance and mass scales greatly expanded. The computer potentiometers are used for nearly continuous programmed gain control of the scale sizes. The initial mass value can be changed by increments of 5, up to 1000

field. The magnet collapse time varies with the scan range from less than one second to as high as four seconds; therefore, it seemed desirable to specify this time separately, because an inexperienced operator might misjudge the total scan plus collapse time and select a scan repetition rate that would start the mass spectrometer before the magnet had recovered, causing data loss. After the scan stops, another scan cannot start until the specified collapse time has passed, even if this slows down the scan repetition rate from the value selected. The correct clock time is stored with the new scan. The fourth display is the mass spectrum from the last scan taken (Figure 6). The current description, scan number, and time are displayed above the spectrum. The mass scale is initially displayed the way it was acquired, linear with time, but somewhat nonlinear with mass number, as the figure shows. The scale can be linearized by striking the “E” or “R” keys. The former retains the peak positions relative to the mass calibration marks, the latter produces a standard bar graph display. Both transformations appear nearly instantaneous. Any form can be plotted. The mass scale, the abundance scale, and the initial mass unit appearing on the display can all be changed by computer knobs. Figure 7 is an expansion of the spectrum in Figure 6 to show the smaller peaks between mass units 140-165 in greater detail. The dynamic range is sufficiently large that some of the peaks are not really visible on the other graph (Figure 6). So long as the knob settings remain unchanged, each new scan will display the same region of the spectrum, so that fragment peaks of interest can be monitored during the course of a GC run. Thus continuous scanning can be used for monitoring, without the need to store unnecessary data. Any spectrum can be stored by striking the “S” key, which also sets the computer to store subsequent scans. The four displays, description, threshold, controls, and mass spectrum, can be retrieved at any time by striking a single key which causes each to appear in turn. The program stores scans in sequence on tape as unnormalized spectra, packing each spectrum into one or more 256-word LINC tape blocks. The maximum size spectrum is dictated by the core size as 850 peaks in the range 0-1000 mass units. If more peaks are found during data acquisition,


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Figure 9. Initial questionnaire for plotting This display specifies the range, size, and regions of enlargement for the mass scale. If the answers are left blank, a full scale plot will be made without enlargements,with a size of fifty mass unitsjnch

Figure 8.

Calibration deviation display

Every spectrum contains calibration information which can be used to display a graph of deviations between peak positions and the nearest mass calibration mark. The figure shown comes from a calibration mixture, PFK, which is used to keep the mass spectrometer calibrated to beyond 850 mass units

the rest are discarded and a warning bell rings. This would almost certainly indicate a noise problem. By using a more common spectrum size, 220 spectra containing 200 peaks in the range 20-500 mass units can be stored. Calibration. Mass marker calibration data are stored along with the mass spectrum, using a single computer word for each mass marker position. Each fragment peak requires two words. The first word specifies the peak position relative to the nearest two mass markers; the second word specifies the abundance. The mass calibration data could be eliminated, and peak mass values coded as a single bit for each mass, designating the presence or absence of a peak. Judging from spectra we have taken, this format would more than double the number of scans that can be stored on one tape. So long as the calibration data are present, they can be used to construct a display showing the deviation of each peak position from the nearest mass calibration mark (Figure 8). These displays can be obtained from any spectrum by striking a key. The figure shows a deviation display of the calibrating mixture perfluorokerosene (PFK), which is used to keep the mass marker system in calibration to past 850 mass units. As the display shows, there are enough peaks to clearly outline the entire calibration curve. There are a few half masses in the region of 100-200 mass units, which are rejected by eye without difficulty. The bunching at zero deviation is an artifact due to the near simultaneous occurrence of a mass mark and a peak maximum. The mass marker calibration needs adjustment at irregular intervals, averaging once a week. The mass marker has a gain control which causes a linear change in calibration with mass number. This is useful to compensate for mass defects. Since PFK is used for calibration, the gain control is normally adjusted to produce an effective negative mass defect, so as to roughly compensate for the positive mass defects of substances encountered in normal experimental work. Scans above 500 mass units sometimes require careful monitoring so that the larger deviations do not exceed one half mass unit. In practice, however, a large portion of the mass spectra obtained at this institution extend above 600 mass units, in order to study trimethylsilyl derivatives of sugars and sugar phosphates. Since there is a steady tendency to study higher molecular 1810

weight compounds, it seems desirable to plan for improvements in calibration which would allow for scans to higher mass units, and require less attention to mass defects at lower ranges. Observation of calibration deviation displays such as Figure 8 reveals a scatter of 10.1 mass unit to beyond 850 mass units. Therefore provisions have been made for the use of a calibration table constructed from the deviation display of a calibrating compound, which will reduce deviations to *O.l mass unit. The mass marker calibration deviations can be approximated by a series of straight line segments (Figure 8). The deviation display and the computer knobs can be used to position pointers designating the extent of each line segment. The set of lines will be automatically assembled into a calibration table for subtraction from each spectrum. Minor adjustments can be made to the calibration table in the same manner to compensate for gradual drift over the course of days. Since the mass marker operates out to 1000 mass units, there seems to be no reason why it cannot be completely calibrated in the region 850-1000 mass units, providing a suitable compound with sufficient peaks is available. At present this region is only calibrated at isolated peaks for specialized scans. A calibration table, coupled with the deviation display, would provide a straightforward method for adapting the computer system to other mass spectrometers. The clock is already available for time based calibration methods. Mass spectrometers with Hall effect generators would require a fourteen-bit analog to digital converter to achieve resolution comparable to the mass marker (11/10,000 in mass or 11/20,000 in magnetic field strength). If the Hall effect signal is first squared in a sufficiently stable analog squaring device, a thirteen-bit converter would suffice. Linearity is not important since the computer can compensate. Indeed, the mass marker squaring circuit converts the field strength signal to mass units by approximating it with twenty-five slightly adjustable straight line sections. Data Processing. Once data have been stored on tape, they are available for further processing whenever needed. The storage cost for a 200-peak 20-500 mass unit spectrum is four cents with calibration data included, and will be two cents without calibration data. This is comparable to permanent storage on paper or film. The tapes take up little storage space, since they are less than four inches in diameter. The spectra on tape can be displayed in sequence starting with the first one, using the same display previously described (Figures 6 and 7 ) to view the overall spectrum or a small section. Provided the clock was started at the time of injection, the clock time can be compared with the GC retention time observed on the total ion current recording from the mass spectrometer, The spectra can be displayed in sequence either backwards or forwards, by striking the ap-


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Figure 10. Typical plot This unretouched plot shows an overall expansion in the abundance scale, as well as a regional expansion ( X 10) from 350 onwards. The label above the graph was typed in just before plotting; the information underneath the graph was stored with the spectrum

propriate control key. It takes about one second/spectrum to move forwards, slightly more backwards. Any spectrum on display can be plotted. After plotting is complete, the spectrum just plotted reappears on the display. There is considerable flexibility in the plots. Figure 9 shows the initial questionnaire display, one of five. If a standard plot is desired, these displays may be skipped, since if nothing is entered into a set of blanks, a sensible conclusion is assumed. For example, if no mass unit range is specified, the entire spectrum is plotted. The display shown in Figure 9 specifies the mass unit range and scale. With a 0.01-inch incremental plotter, plots can be as compact as one hundred mass units/inch, but the bars will merge. A 0.1-millimeter version of the plotter is available, which will work without changing the program. All plots will be reduced to 4 0 z of the size specified without loss of detail. Since it may be desirable to have a compact complete spectrum and still expand the mass unit scale around certain groups of fragments for detailed analysis, the mass scale may be expanded in as many as four regions of the spectrum. The height of the abundance scale is also adjustable, In addition, the entire spectrum or as many as four regions can be expanded in the standard manner to see peaks with low abundance. Figure 10 shows a typical plot with expansions of the abundance scale. The description, clock time, and scan number stored with the data are automatically plotted at the bottom. In addition, a one-hundred character detailed description may be typed in, which is plotted above the bar graph. The computer system has been set up to provide for further developments in data analysis. In particular, visual and automatic procedures are under development for identification of mass spectra using files of known spectra (6, 7). The present system uses one LINC tape for data storage, and the other tape for programs. Major program segments are (6) R. A. Hites and K. Biemann, Adcun. Muss Spectrom., 4, 37 (1968). (7) B. A. Knock, I . C. Smith, D. E. Wright, R. G. Ridley, and W. Kelly, ANAL.CHEM , 42, 13 (1970).

loaded from tape as a series of overlays. The computer memory is divided into two thousand words of permanently resident subroutines, two thousand words for program overlays, and four thousand words for data. The programs are written in LINC assembly language. Since most of the program tape is empty, it is available for files. A study of a computer file of known spectra (8) shows that well more than five hundred complete scans may be stored on one tape. A much larger number of major peak spectra can be stored. Exchange of computer files is a significant problem with small computers, since the standard exchange media are punch cards and IBM tape, which require hardware that adds considerably to the cost of the computer. The Washington University Computer Center has a remote input/output system over telephone lines between its IBM 360-50 and small computers. In this way, standard computer files can be sorted, formatted, and sent to the PDP-12 for storage on LINC tape, and spectra collected locally can be converted to standard computer media for distribution. The system has been in routine daily use for a number of months at this institution, and is now in operation at several others. Copies of the program tape, program documentation, and circuit notes for constructing the interface are available from the authors. ACKNOWLEDGMENT

The authors thank Dr. George Drysdale and Dr. William Sherman for numerous discussions on the experimental and instrumental aspects of mass spectrometery.

RECEIVED for review June 1, 1971. Accepted August 16, 1971. This work was supported by Grants Number 5R01-CA-10926 and RR00396-04, and a Health Science Advancement Award Number 5-S04-FR-06115 from the National Institutes of Health. (8) Mass Spectrometry Data Center, Aldermaston, England, 3000 MSDC Spectra.


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