for molecules with the nitrogen mustard group, N-C-C-Cl, might be very useful. This technique, known as substructure searching, is being developed in this laboratory both for the Wiswesser Line Notation (WLN) (16) and the Chemical Abstracts Service connection tables (17). In the case of the latter type of data base, a computer search system is under study which will allow for interactive file searching. While the search system is small and efficient, the files are quite large and require a large on-line disk storage capacity, available at few computer installations. The largest file is the full spectrum file, containing all the peaks and their intensities. One possible alternative to storing such a large file in the computer is to put the full spectrum file in a microfiche retrieval unit in the chemist’s laboratory driven remotely by the search system program. In such a device, the ID number would be a pointer to a given microfiche card and page number, in a (16) R. J. Feldmann and D. A. Koniver, J . Chem. Doc., 11, 151 ( 1971 ) . (17) R. J. Feldmann and S. R. Heller, ibid., 12, 48 (1972).
manner identical to the pointer system used for the peak, molecular weight, and spectrum lookup disk files described in the previous section. As the file grows in size, the microfiche becomes economically very attractive compared to the cost of on-line computer disk storage. Also, the microfiche reader can be operated manually and used for other storage purposes. ACKNOWLEDGMENTS
The author wishes to express his appreciation to Richard J. Feldmann for the extremely efficient intersecting list algorithm. The author also wishes to thank Henry M. Fales, G . W. A. Milne, Robert J. Highet, D. J. Pedder, and J. W. Wheeler for their generous use and criticism of the search system, and K. Biemann for the data base. RECEIVED for review March 23, 1972. Accepted June 13, 1972. Presented in part at the 163rd National Meeting of the American Chemical Society, Boston, Mass., April 9-14, 1972.
Computer Acquisition and Analysis of Gas Chromatographic Data R. A. Landowne,’ R. W. Morosani,* R. A. Herrmann, R. M. King, Jr.,3 and H. G. Schmus4 American Cyanamid Company, Central Research Division, Stamford, Conn. 06904 A computerized system for a multiple instrument gas chromatographic laboratory is described. Simultaneous operation of all chromatographs i s possible in real time even while the computer performs other functions. A set of resident programs controls the entire process which requires a minimal amount of operator interaction regardless of the complexity of the chromatographic analysis. In either method, development or routine analysis, only a few input parameters are required to choose several modes of data handling, with each instrument capable of operating in its own independent fashion. A teletypewriter is used almost exclusively for outputs of results, while most sample information and mode selection is entered through simple data switch boxes. Peak resolution and baseline determination is accomplished for almost all situations encountered without resorting to special routines.
THEUSE OF DIGITAL COMPUTERS at least in some, if not in all, stages of the handling of gas chromatographic data has been demonstrated and practiced in many laboratories for several years. The possible approaches are numerous depending upon the type of laboratory (e.g., research or analytical control), the volume of the work, the kind of computer and its peripheral equipment, the capital investment to be made, and the caliber and training of manpower available to operate the system. Examples of present day systems that fall into three major categories are: Off-line electronic integration followed by computerized data processing from the data collected on To whom inquiries should be addressed. 2 Present address, Laurel Ridge, Litchfield, Conn.
Present address, Xerox Data Systems, 1701 Research Blvd., Rockville, Md. Present address, Avon Products, Inc., Suffern, N.Y.
tape ( I ) ; on-line system totally dedicated to a large number of gas chromatographs (2); on-line system using a timeshared computer (3). Many of these systems are commercially available from either computer manufacturers or gas chromatograph vendors. This last type of configuration was determined to be most suitable for our laboratory which performs the complete spectrum of gas chromatographic functions from routine quality control analyses to complex method development work, in the midst of other analytical functions that could also be computerized (e.g., mass spectrometry, NMR, etc.). The computer on hand was, in fact, already functioning as an on-line instrument for mass spectrometry data acquisition. Subsequent reduction of data and other batch processes were time shared with the real time acquisition. This software was transferred from an existing XDS 930 to XDS Sigma 2 when the system was expanded to handle gas chromatographs. The chromatography system was designed to operate under the Real Time Batch Monitor of the Sigma 2. This consists of a Random Access Disc of 3 megabytes. Total memory available is 36 K words. New software was developed as a set of programs to cover various analytical situations encountered in the gas chromatography laboratory. These were capable of operating simultaneously with the acquisition of mass spectrometry data and other batch processing. For the most part, the software is written in X-Symbol assembly language. There are six controller tasks which handle (1) C. Merritt, J. T. Walsh, R. E. Kramer, and D. H. Robertson, in “Gas Chromatography 1968,” C. L. A. Harbourn, Ed., Institute of Petroleum, London, 1969, pp 338-40. (2) H. R. Felton, H. A. Hancock, and J. K. Knupp, Jr., I17strum. Contr. Syst., 40, 8 3 (1967). (3) R. D. McCullough, J . Gas Chromatogr., 5,635 (1967).
ANALYTICAL CHEMISTRY, VOL. 44, NO. 12, OCTOBER 1972
1961
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Figure 1. Block diagram of system hardware routine input-output and the sequence of jobs. Two major tasks handle the data reduction and data analysis. The latter, which contains some novel approaches to this problem, is written in Fortran IV. Detailed descriptions of the software are available from the authors or Xerox Data Systems. As designed and put into operation, the system contains many features from earlier reported systems with a variety of modes of data analysis (4-13). The most unique aspects of this new second generation system are of more concern to the chromatographer, however, particularly with regard to curve analysis and overall simplicity of operation for those not computer oriented. The result is a set of resident programs, using only 7000 16-bit words of core memory, simultaneously and completely accessible by several operators through eight instruments, and capable of performing any type of gas chromatographic task, from the simplest method development scan to the most complex analysis. Furthermore, these programs are utilized with a minimum of operator interaction through a specially devised communicator box which serves as the major link between operator and computer. Admittedly, operation of the system was not designed for the disinterested button pusher, but neither is it overly complex. Training in its operation is no more difficult than the training required for a relative newcomer to a gas chromatography laboratory. In fact, the training process is much simpler with the com(4) H. A. Hancock, Jr., L. A. Dahm, and J. F. Muldoon, J . Chromatogr. Sci., 8,57 (1970). (5) F. Tivin, ibid., p 13. (6) F. Baumann, A. C . Brown, and M. B. Mitchell, ibid., p 20. (7) W. 0. Wilson and J. G. W. Price, ibid., p 31. (8) A. J. Raymond, D. M. G. Lawrey, and T. J. Mayer, ibid., p 1. (9) B. Goldberg, ibid., 9, 287 (1971). (10) A. W. Westerberg, ANAL.CHEM.,40, 1595 (1968); 41, 1770 (1969). (11) E. Ziegler, D. Henneberg, and G. Schomburg, ibid., 42 (9), 51A (1970). (12) A. H. Anderson, T. C. Gibb, and A. B. Littlewood, ibid., p 434. (13) L. L. Hegedus and E. Petersen, J . Chromatogr. Sci., 9, 551 (1971).
1962
puterized system because much of the art formerly required in area allocation and measurement procedure is eliminated by the automated system. And yet an operator can exert his own influence and use any extensive experience and knowledge he has in order to obtain the best results. On the other hand, someone with no knowledge of the system at all can be shown how to make two or three simple entries with every sample injection and obtain routine reports without difficulty. In effect, the system was designed for use by all types of chromatographers, doing all kinds of work on most standard instruments in any typical laboratory. Although the extremes can be handled in most cases, many features of the system are compromises that were reached in order to prevent the overall program and its manipulation from becoming too complex. However, there are ways of altering some features in the basic system for use in special applications that would not affect operations as normally carried out. Physical Description of the System. Initially eight gas chromatographs with flame or thermal conductivity detectors were put on-line with the XDS Sigma 2 computer through the appropriate interface hardware also supplied by XDS. The system is diagrammed in Figure 1. It should be noted also, that the chromatographs are an additional 100 ft away from the remote part of the interface. Included in the remote devices are two Model KSR 7012 teletypewriters, although only one is required for the chromatography laboratory, a Model CD5l/DM 40 low level analog-to-digital multiplexer, a Model AD20 analog-to-digital converter, two shaft encoders (A and B), the Enter or Communicator Boxes, and the "Run-End'' Boxes for each chromatograph. Encoder A handles the gas chromatographs, routing data through the 15-bit variable gain A/D converter which can handle peaks in the 1O-pV to 10-V range. Encoder B is reserved for real-time data acquisition from the mass spectrometer photoplate in an adjoining laboratory (14). (14) D. M. Desiderio, Jr., and T. E. Mead, ANAL.CHEM.,40, 2090 (1968).
ANALYTICAL CHEMISTRY, VOL. 44, NO. 12, OCTOBER 1972
Figure 2a. Run-end box
Figure 2b. Communicator box
Transmission to the local site is via a 5 W f t cable into a Model 7930 digital 110 adapter, which then presents data to the computer through a DIO processor. Other data which may bypass the special interface are sent via a low-level A p multiplexer to a Model 7915 analog controller. Finally, the input data rate is controlled by a Model 7969 frequency controller according to programmed instructions. Complete details can be found in the appropriate XDS system manual (Is). The Run-End and Enter Boxes were designed and built in this laboratory and will be described in somewhat more detail. Additional information on them is also available, however. Run-End Button Boxes. The Run-End Box mounted on each instrument is shown in Figure 2a. The box consists of two buttons marked “Run” and “End,” and a yellow indicator light marked “Active.” Model KB encoding switches provide and generate interrupt and control signals to start and end data transmission from the chromatographs. The indicator light, under program control, is lit while its chromatograph is being monitored. Communicator Boxes. For the gas chromatographer, the Communicator Box is his major connection to and means of controlling the system. Only one is required for all the chromatographs, but a second box was provided for more convenience and easier accessibility to the system. They are placed a t separate locations and can be operated simultaneously. The device is shown in Figure 26. It is an input device similar in function to the Run-End Box. However, in addition to its ability to transmit test and control signals through similar closure switches and lights, it also is used to input data parameters through the multiplexer. These parameters control the operation of the system as described later. Eight Digitran Series 9000 DCI Coded Output thumbwheel switches are used for this transmission. These include one hexadecimal switch (0-15) for channel (instrument) identification, two hexadecimal switches (0-9, a-f) for parameter or “function code” identification and five switches (0-9) for data transmission. The information is not transmitted until the (I>)
.Xerox uata systems, “lechmcal Manual No. Y80199,”
Rockville, Md., 1970.
“Enter” button is pressed. This permits the operator to check visually everything he has dialed in and to make corrections before committing the information to the computer. Acceptance of the data is indicated by the green “Correct” light. Only in a few cases does the software check the validity of the data just input. If invalid data are noted, the red “Error” light will be lit instead. It will stay on until the operator corrects his input, re-enters and obtains the correct light, or the “Reset” button is pressed. Should the operator realize a mistake was made even after obtaining the correct light, he may simply override the previous input by entering the new, correct data. Teletypewriter. One KSR-35 teletypewriter is dedicated for use in the gas chromatography laboratory. For the most part, it is used as an output device, furnishing completed reports to the operator from all of the gas chromatographs. Reports are generated in the same order that analytical runs are terminated by pressing the end button on the instruments. Since the TIY is the slowest part of the system, and could be quite active during peak operation periods, its use as an input device is kept to a minimum. Only in the preparation or editing of standards for computer filing, where compound names and weights are transmitted, is the TTY output function interrupted by the operator. All other necessary data are input through the communicator box. OPERATION OF THE S Y m M
Selection of Modes. The choice of three modes, via the communicator box, and subselections within each, tells the computer how to handle the data it will acquire and/or reduce. Table I summarizes these inputs. However, most of them are optional. In addition, a key feature of the system is that there is no need t o repeat an entry for every analysis unless the data are to be changed or a new mode is entered. DBVELOPMENT MODB. The development mode takes all peaks that were found and saved after reduction of the raw data, and reports them after doing a simple area per cent -ml-..lnd--
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Table I. Communicator Box Inputs Development mode Sample mode All zeros means remove solvent peak Reference standard number
Update mode
Standard number for update Sample identification Sample identification Sample identification Teletype request (no data) N.A. Teletype request (no data) Relative retention time peak number Time window N.A. Internal standard peak number Internal standard amount N.A. N.A. Sample amount N.A. Density Density factor Data N.A. Dilution volume Volume factor codes N.A. Injection volume N.A. N.A. N.A. N.A. Save command (no data) Abort command (no data) Abort command (no data) Abort command (no data) Base-line tolerance Base-line tolerance Base-line tolerance Peak threshold Peak threshold Peak threshold - Indicates computer ignores this left hand function digit. e Indicates that any digit (0-9) used in this left hand function switch is a negative exponent for the data according to floating point notation, e.g., 1234 X lo-'. 0
number and resolution of peaks are significant. However, where a sample is to be quantitatively analyzed, without resort to component response factors, this mode is also used, rather than the sample mode described below. Also, the final chromatographic step in most method development problems, the running of a known standard, is performed in the development mode. SAMPLE MODE. Use of the sample mode is obvious. The computer compares the retention times and areas of the components in the sample that has just been analyzed to the weight-area ratio or response of the components with matching retention times contained in a standard previously run and stored for reference in the computer. The result is given as weight and weight per cent for each component. Both external and internal standard methods of analysis can be used in this mode. In the latter case, the additional computation of the response factor, the relative response of the component to that of the internal standard, is used in arriving at the weight per cent for each component. UPDATEMODE. In the update mode, the computer performs a combination of parts of the other two modes. The main purpose in using this mode is to permit the checking of a previously stored standard with respect to reproducibility of retention times and areas of components that could have changed. If required, subsequent editing of the first standard may be done, thus updating it with a new standard. The first standard is saved in its original form, however, and the new standard is identified with a separate number. In effect, the update mode is a way of preparing a new standard, by running the standard somewhat like a sample is run-i.e., comparing it to an old standard already on file. This procedure allows for simpler monitoring of any chromatographic or standard sample changes and is useful in routine or control analyses where the same standard may be run once or twice a day. Should changes be required in the standard file, less teletype input is required than in the usual standard development mode. Selection of Software Controlling Parameters. The collected time US. voltage data eventually reduced by the software into a chromatogram for analysis can be treated in a variety of ways during this process of raw data reduction and reduced data analysis. Such treatment is affected by the use of' certain parameters, a few of which can be varied by communicator box input. In no case is this input mandatory, however. 1964
PEAKTHRESHOLD.One choice the operator has is peak threshold, defined as the minimum value, in millivolts, that the height of any peak must exceed before being saved by the computer as part of the chromatogram. The entire peak is saved, not just that portion above the threshold value. In other words, this is not a means of setting the base-line level, but it is an additional method for discarding small peaks, noise, or minor components, when they are not of interest. If no threshold is chosen, the software will reduce the raw data using a value of 0.501 mV. For most flame detector instruments and their associated electronics, 0.501 mV is suitable. For thermal conductivity detectors, 0.050 mV or less is required, since overall signal size is low. Even a value of 0.000 mV can be selected, but the hardware was optimized for peak heights of 0,010 mV to 10 V. Another parameter that can be BASE-LINETOLERANCE. altered using the communicator box is the base-line tolerance. This value is an aid in setting the base line for the peaks found by the reduction program, so that their areas can be calculated properly. A tolerance of zero draws a base line that is virtually straight from beginning to end. This is also the default value. The maximum value of 0.9999 selects an ever changing base line that will be drawn through almost every valley point. Between these two extremes, the base line can be made to follow a particular chromatogram according to the needs and accuracy of the analysis. In practice, only small numbers, no higher than 0.00100 V/min X 100, are used for best accuracy on a drifting base line. Otherwise the default value is quite adequate. TIMEWINDOW. A third parameter that can be used to alter the software action in analyzing the data is the time window. This value is significant only in the sample mode when it is necessary to match and identify sample peaks with stored standard peaks based on their absolute or relative retention times, The time window is the allowable error for this peak matching process; i.e., if it is set at is%, a sample peak whose time is within 5 of the time for a standard peak will be identified with the standard peak. If the difference is greater than &5%, the identification or match will not be made for that sample peak. The default value is in fact 5 %, which has proved satisfactory in most cases. It is obvious that when retention times can fluctuate during a series of repeated analyses based on the same standard, a larger window would be beneficial as long as it is not made too large so that another peak can then fall within the window. Conversely, where
ANALYTICAL CHEMISTRY, VOL. 44, NO. 12, OCTOBER 1972
peaks are very close together, so that more than one could conceivably fall within a five per cent window, then a smaller window must be used. In this instance, retention times must be very reproducible for successful peak identification. The use of relative retention times, using a peak in the middle of the chromatogram as the relative time base, an option easily chosen, helps greatly in avoiding peak matching problems. Running a Sample. After placing the instrument in one of the three possible modes, and choosing any of the data reduction and analysis parameters, the sample to be injected is readied in the syringe or other sampler and the run button pressed. For five seconds, the computer records data on the base line, then the active light comes on and the injection is,made as quickly as possible. Should the operator fail to inject within a second or two, the active light can be turned off and the process repeated. COMPUTER DATA HANDLING PROCEDURE
Reduction of Raw Data. The computer performs two basic data handling functions-preliminary data reduction during acquisition, and analysis of the reduced data afterwards. The primary task consists of taking the data points of voltage cs. time and reducing them to segmented areas each with its own retention time. These areas do not necessarily correspond to final peak areas at this point. However, the number of peaks is usually known since during data acquisition curve smoothing is performed to eliminate noise. The smoothing process is actually a combination of hardware and software filtering. In addition to the customary isolation amplifiers, removal of all ground loops, and RCL filtering of high frequency spikes, a unique data acquisition routine is used to eliminate the 60-Hz noise. Each of the “data points” used in computations is the arithmetic average of eight analog-to-digital conversions equally spaced over second. In actual practice this was found to provide 1000 to 1 rejection of 60-Hz noise, making it possible to measure G C signals at low microvolt levels. As an additional guard against single spikes, each “data point” is compared with its neighbors and if not between them is assigned their average value. The first derivative is estimated using a centered 15-point least squares fit of the voltage. A change in sign is a key point, but to guard against slight perturbations in following the first derivative sign change it must last for at least 8 consecutive data points to be properly noted. Similarly the second derivative is calculated from the first derivative using the same 15-point least squares smoothing function. However, first derivative values are rounded off before they are included in the calculation in order to avoid numerical overflow in regions of large derivative. Again, eight successive second derivatives of opposite sign must occur before a sign change is noted. These successive derivatives must also be greater than a fixed constant in absolute value in order to warrant the sign change. Thus the data reduction process saves, in only 18 words for each peak, all the key points from the chromatographic curve-the points at which first and second derivative sign changes occur. Whether the change is from positive to negative, or the reverse, and the order in which these changes occur obviously determines peak start and peak end points as well as the top of the peak or its retention time. Peaks with heights below the peak threshold value are discarded during this process also. Area Allocation. Following the completion of a chromatographic run and the concurrent data reduction process,
Figure 3. Model trace of unresolved pair of peaks showing key points on curve used by resolution routine
the reduced data are further refined by an analysis program so that final results can be computed and reported according to the prior specifications of the operator. This is a non-resident program that replaces any background program in progress at the time in order to carry out the analysis. The size of this non-resident program, 18,000 words, is a function of the number of peaks including noise that is allowed. At present, up to 300 peaks can be handled from a single chromatograph. BASE-LINE SETTING. The initial task in this procedure is the setting of the base line under the two or more peaks found. The general goal of this part of the program is to arrive at the tightest base line under the curve, with all valleys or trough points connected, but within the limitations of the base-line tolerance. First, a base line is drawn between all trough points. These points would include all peak valleys plus an artificial trough point which occurs at the start of the chromatogram. Once all the trough points have been found and connected by the initial base line, this line is pulled taut from both ends as if it were a string under the points. Conceivably this could leave some trough points above the base line. Now the program attempts to re-include these trough points, one by one, by redrawing a segment of the base line through each point. If a redrawn segment forms an angle with the previous base-line segment whose tangent exceeds the operator pre-set base-line tolerance, then this correction is not made and that trough point is kept above the base line. Use of a low tolerance permits only those trough points slightly above the base line to be included. Higher tolerances are reserved for following a drastic base-line shift due to temperature programming or where a small peak falls on the front of a very large one. A zero tolerance is also practical because the taut string mechanism will properly include most trough points. PEAKRESOLUTION.Where an integrated area segment is adjusted by subtraction of excess area below the base line, leaving a segment containing a normal peak with a single maximum whose beginning and end are both on the base line, no further refinement is necessary. However, if more than one peak maximum is found in the base-line-corrected area segment, a resolution routine comes into play in order to allocate the area to each of the unresolved components. When the beginning or the end of a peak, or both, is not on the base line, one of several methods for dropping a perpendicular from the curve to the base line is resorted to in order to allocate the area to the unresolved peaks. First, if the trough point (the point between a pair of peaks where the sign of the first derivative first changed) is one-tenth or less in height (above base line) than the height of the smaller of the two unresolved peaks, the division is made at this trough point or drop point, D (see Figure 3). When this height ratio of 0.1 is exceeded, a different point in the valley between the peaks is searched for on either side of the trough point but between the neighboring inflection points. This point, P, lies somewhere between D (where
ANALYTICAL CHEMISTRY, VOL. 44, NO. 12, OCTOBER 1972
1965
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where Il and Iz are the inflection points on either side of I,. The perpendicular again is drawn to the curve at t,, but the area is now allocated by assuming the curve is a parabola passing through M and the two neighboring inflection points. The ratio into which the perpendicular cuts the area under this parabola is then applied to the actual area between inflection points, and the partial areas are added to the neighboring peak areas as before. The peak center (retention time) of the “bump” peak, the smaller of the two, is defined as the point at which the second derivative is a minimum since the first derivative will not pass 1966
through zero here either. The distance between the adjacent inflection points on either side of the bump peak center must be at least 100 data points, and the distance between the adjacent inflection points on the bump peak and on the main peak, Le., the width of the trough between the unresolved pair of peaks of this type, must be at least 16 points. These minimum widths have been arbitrarily set and, if not met, the bump peak is ignored and the entire area is considered a single peak. Less rigid restrictions would lead to more unreal bump peaks as a result of noisy second derivatives. Poor chromatograph signals could even necessitate more restrictions on bump peak characterization. The other special situation for peak resolution arises when one of the unresolved peaks is a solvent or other exceptionally large tailing peak. Such a peak is presently defined as one whose height exceeds a voltage of 2.5 V. The operator must choose to use this “tangent” routine through the communicator box, however. When so chosen, and the 2.5-V value is exceeded, smaller peaks on the tailing side of the large peak have their areas allocated by drawing a tangent between their trough points. The tangent is only an extra base line for such peaks; the original base line remains below the entire larger unresolved peak areas. The area below the tangent but above the base line is allocated to the large peak. If two small peaks on the side of the large peak are themselves unresolved, a single tangent is drawn below them and a simple perpendicular (to the base line, not the tangent) is drawn to resolve them from each other. If more than one small peak falls on the tail of the larger peak, but they are resolved from each other, individual tangent lines will be drawn below them. It should be noted that bump peaks are ignored on solvent type peaks. Also, the tangent routine is used only on the rapidly receding backside of large, asymmetric peaks, not on the rising front of large overloaded peaks. In the latter case, the normal base-line following routine is adequate for handling this situation with at least equivalent accuracy. Finally, in all cases an empirical rule has been set to void any peak resolution that results in two adjacent peaks with an absolute retention time difference of less than 2 x . Should this occur, the peaks are merged into one, using their average time as a retention time. The 2x value can be changed if high resolution capillary columns are in use that are capable of yielding such separation. This minor merging restriction was necessary particularly on low, long retention time peaks. Even the best flame detector had difficulty in producing a smooth signal at low microvolt levels just above the noise threshold. Calculation and Reporting of Results. With the final peak areas and their respective retention times at hand, the system is now in a position to compute and report the results. These steps depend upon the mode called for by the gas chromatographer via the communicator box, and other steps he may have taken prior to the analysis. DEVELOPMENT MODE, In the development mode, the total of the peak areas is found and the area per cent of each peak given along with its time, in minutes, and area in volts X minutes x 100. A typical report is shown in Figure 4. Table I1 defines the peak codes that appear in the last column of these reports, Where the solvent or other large peak is to be eliminated, the area per cent calculation is performed without that peak even though its time andarea aregiven (Figure 5). The development mode was so named because it is used for the development of a standard data file for storage for future use. Up to 30 standards can be accommodated at once for
ANALYTICAL CHEMISTRY, VOL. 44, NO. 12, OCTOBER 1972
Table 11. Peak Type Codes Peak type Explanation Solvent Height above 2.5 V ; exclusion from calculations requested. Tangent Resolved from solvent peak by tangent routine. Usual Totally resolved. Trough points on base line. Usual Resolved by perpendicular drop routine before and/or after peak; trough points not on base line indicated by absence of asterisk. Bump Resolved by bump peak subroutine. Merged Area is sum of two or more peaks. Time is average of these peaks.
Code
S T
*U*
*U,U*,U
B M
OEVEOPMENT MODE 1MT.NO. 1 SAMPLE 1.0. 00200 BSLN TK PEAK THRESHULO: ,501 MY. PK. W.
12/02/70
ARM
AREA PCNT
.01 .14 .39
0.1598E-02 0.2Q4E-02 0.2610E-03 0.6714E-04 0.717b.E-04 0.67M)E-04 0.3229E-03 0.1936EMl 0.2396E-01 0.2023E-03 0.2723E-03 0.2593E-03 0.1692E-03 0.1881E-02 0.3234E-02 0.3615E-02 0.7786E-03 0.1209E-01 0.1884E-02 0.2418E-02 0.1764E-02 0.6717E-03 0.5903E-03 0.1064E-02 0.3396E-02 0.6707E-03 0.3079E-32
2.385 3.930 e389 -100 .lo7 .lo1
NO.
AREA TINE 0.1443EWO 2.123 0 . ~ 2 9 1 ~ ~ 0 5.057 0.5678EM0 5.530
MUE ACRYACIO ACRYAMIO HYORACRN
1
2 3
TllTAL AREA
2 3 4
5 6 7 8 10 7 11
12 13 14 1 56 17 18 19 20 21 22
23 24 25 26 27 TUTAL A a E A
-43 .50 .59 .65 .88 1.91 2.18 2.36 2.93 2.68 2.87 3.22 3.35
3.L5 3.67 4.00 4.20
2-41 4.59 1.76 5.17 6.15 1.39 8.98
.482 35.750
-302 A06 .387 252 2.806 1.825 5.394 1.162 18.043 2.811
,4928
RESPONSE 1.1971 . ~ 7 4 .E679
R.F.
yT/VL
.002
.005 .005
Figure 7. Report of saved standard after preparation INST NE. 7 SAMPLE 1.0.
SAMPLE
00000 T I M E WINDBY ,0500 SAMPLE VI. lCO.0000
NO. NAME 1 ACRYACID
2 3
STANDARO USED
AREA
ACRYAMIO
0.1096EM1 0.3618EM0
HYORACRN
MOOE
ESLN i a INT.STD.YT.
PEAK THRESHBLO: .WO UV. D E H S I T Y FCTR 1.0000
.ioooo
YEIGHT .1727 .7094 .I149
.15
1505
.OOOO
V K U M E FCTR
TIME 2.12 5.08 5.49
12/03/70 75
1.0000 YT.PCNT .7094 .3149
*U
.0003 .OD00
.0003
*U*
.OOOO
*U*
.5493 .OOOO ~
3493
*U*
.OOOO . . ~ . .
*U*
-0242
*U*
U'
UYKYOWM: 0.3077E-03 0.9819E-05 0.6076EKlO 0.11105E45 . .-.- .. 0.2672E-01 0.6120E-06 0.2800E-03 0.2372E-05 0.1305E-04 0.7776E-Ob 0.2011E-04 0.1369E-04 0.6782E-04
1 .U*
3
*U U'
4
*Us
6 7 8 9 10 11 12 13
5
*U'
*U* *Us
.s
1 1 1 1 1
TOTAL AREA
U
.42 .69 2.02 -..2.28
-0242 .OOOD
4.06
,0000 'U' .0003 M'
.0003 .OOOO .OOOO
4.31 4.51 10.86 11.74 12.81 13.48 14.96
0.2093EM1
.
.OOOO
*M'
.OOOD .0001
*Uo
,0001
.OOOO .OOOO
.OOOO .OOOO
.0001
.0001
*M* *M' *M*
TOTAL KNUUN IYI.PCNT
*Me
0.1024E101
M M
Figure 8. Sample report
U M M
3.Q8
H
2.631 1.002 481 1.587 5.067 1.001 4.593
M U
1 6 1 vow 7 UPOAlE NODE 00500 S T A I I D A R O USED 82 SAMPLF 1.0. PEAK THRESH0.U: .Do0 MV.
BSLN
12/16/70 1344 .50500
IK
M* DEL-1
*I
llAME UNKYEHN UYKNOUY UYKYOWN 1 UYKNOUN 5 UIIKNYIYN 6 ACRACIC 7 UNKNONN 8 UVKNWY 9 ACRYAMID 10 HYOACRCH 11 UWUOYN 12 UUKUO#N 13 UVKNOWN 14 UYKNOA" 15 UVKNOjN 1 6 UNKYOXN 17 UVKkOIN
AREA 0.2107E-03 0.1375E-05 0.3755E-05 0.2002EW0 0.9 360E 6 0 O.l435E+00 0.1447E-03 0.9523E100 0.1510E+00 0.7033E-05 0.2168E-04 0.1486E-04 0.8817E-06 0.3393E-04 0.139EE-03 0.2106E-04 0.1607E-03
NO.
1 2
M
*M* *1*
0.6703E-01 -.095
Figure 5. Development mode report with area per cent computation excluding a solvent type peak Figure 6. Development mode teletype input used for a three-component standard file preparation
KIGHT .1727
r).1541E+01
2 1
12/03/70 1424 75 VOL. 0.0000EWO
1414
.01000
TIME
STANDARD MOOE IMT NO. 7 STANDARD NO. SAMPLE 1.0. 00000 0.0000EMO OIL. VOL. 0.1000EWl INJ.
DEMITY
-188 2.998
~ ~ : ~ ~ 2 ~ ~ ~ c & d [ $INST* c $ No* O 7 81.53699ACRYAMI0 9,. 4928,HYDRACRV
0,
TOTAL AREA
use as references when analyzing in the sample mode by either the internal or external standard procedure. With periodic, simple deletion of unused standard files, there is adequate space for this reasonable number of stored standards for this size laboratory. For the preparation of a standard, the standard sample is run in the development mode and the usual area per cent report is obtained. After any report is received, the data are saved by the computer until a new run is made or the mode is changed for that particular instrument. Thus, the operator can scan his report and, if it is satisfactory, he will input via the teletype the additional names and weights for those components in the standard he wishes to analyze for in future samples. This is shown in Figure 6. The initial "ready" statement is generated automatically when permission for teletype input is requested of the system via the proper communicator box signal. Then the operator will characterize only those peaks he wishes to be part of the standard file by typing the peak number from his previous report, its weight in the standard sample, and its designated name using up to eight characters in the manner shown. The ''g" sign erases the prevous character if a mistake in typing is made. A whole line is easily
TIME .ll .45 .49
YEIGHI
RESPOYbE
.193il
1.3449
.68 .e1 1.64 ?.65 3.43 3.87 7.15 7.44
U
.a00 3.P223 .192027298.4135
'U
8.00 8.52 8.86 11.52 14.48 17.37
0.1890Et01
Figure 9. Report after standard update run INS1 NO. 7 SAMPLE 1.0. NO.
1 2
NAME ACRYACID ACRYAMIO
EDIT REPORT
WOOO
TIME
AREA 0.1443Ec00 D.P293E+00
2.12 5.06
#EIGHT .1727 .5369
12/04/70 RESPONSE 1.1971 .6474
1356
R.F.
Figure 10. Report received after editing standard in the update mode UPOATE OF SAWPLE WOOO STORED AS STANDARD
76
0 IS I N T . STD. PK. NO. YAYE
ACRYAMIO
TIME 2.123
MIGHT .5369
RESPONSE 3.721f
Figure 11. Report received after decision to save edited or updated standard
corrected by retyping new data for that peak number. The final zero and comma is the input sign-off code which returns the teletype to its output function. Upon completion of this input, it will be combined with the chromagraphic data being held and a standard mode report will be generated (Figure 7). The response listed for each
ANALYTICAL CHEMISTRY, VOL. 44, NO. 12, OCTOBER 1972
-
~~
1967
Toluene Temp, "C 90 90 110
110 125 125 130 140 140 155
155 170 170 170 Average Std dev
RRT 0.666 0.651 0.731 0.728 0.793 0.784 0.807 0.826 0.828 0.863 0.861 0.900 0.900 0.900
z
Table 111. Area Per Cent Computations rn, p-Xylene o-Xylene RRT RRT 40.574 1.120 11.468 1 .Ooo 1.125 1 .ooo 40.693 11.384 1 .Ooo 39.903 1.092 12.057 1.000 40.557 1.093 12.049 ... ... ... ... ... ... ... ...
z
47.958 47.923 48.040 47.394 47.959 47.314 47.933 48.213 47.957 47.718 47.700 47.022 47.154 47.124 47.672 0.392
...
z
...
... ... ...
... ... ... ...
...
...
...
...
...
... ... ...
...
... ...
...
...
...
...
... ... ...
...
...
...
Total xylene v
RRT
/o
(52.042) (52.077) (51.960) (52.606) 52.041 52.686 52,067 51.787 52.043 52.282 52.300 52.978 52.846 52,876 52.390 0.422
I . .
... 1.000 1.000 1.000 1,000 1.000
1.000 1.000 1.000 1.000 1.000
ii.739 0.363
40.431 0.357
1 I
a
I1
b
C
I
1
2
2
I
_i_ 4
4
2
2
MINUTES
MINUTES
MINUTES
Figure 12. Chromatogram examples testing accuracy of area allocation routines: 1, toluene; 2, mdecane solvent
+ p-xylene; 3, o-xylene; 4, n-
a. 90 "C b. 110 "C c . 170 "C
Table IV. Sample Analysis Results' Sample No. 1 Sample No. 2
Temp, "C 90
tA/tBb
Z B 0.095 0.137 0.128
1.261 110 1.217 1.226 120 130 not resolved not resolved 140 not resolved 140 Average found 1.234 0.120 Standard deviation 0.023 0.022 Theory 1.234 0.121 a Each entry is itself an average of at least three runs. * A = 2-rnethylcyclohexanol, B = n-heptanol.
1968
0.776 0.843 0.861 0.885 0,92O(U) 0.912(B)
z-4
z-4
0.705 0.692 0.683 0.672 0.659 0.653 0.677 0.019 0.686
ANALYTICAL CHEMISTRY, VOL. 44, NO. 12, OCTOBER 1972
Z B 0.588 0.600 0.609 0.620 0.632 0.640 0.614 0.019 0.607
Sample No. 3 % A
j7B
0.142 1.088 0.151 1.080 0.146 1.083 0.139 1.092 not resolved not resolved 0.144 1.085 0.005
0.005
0.137
1.094
0 MINUTES
MINUTES
I
I
C
A
J MINUTES
MINUTES
Figure 13. Chromatogram examples testing area allocation routines: A , 2-methylcyclohexanol ; B, n-heptanol Sample mixture No. 2, 90 "C b. Sample mixture No. 3, 90 "C c. Sample mixture No. 2, 120 "C d. Sample mixture No. 1, 120 "C e . Sample mixture No. 2, 140 "C a.
0
2 MINUTES
component is the weight divided by the area. The response factor (R.F.) appears only for internal type standards and is the response for each component divided by the response of the internal standard. The internal standard peak would be recognized by its response factor of 1.000. Most of the data from this report have been stored in memory and are available for future use with any instrument for the analysis of related samples.
SAMPLE MODE. In the sample mode, the task of identifying peaks in a sample by comparison with peaks in a stored standard is carried out before any computation can be performed. The peak matching routine is critical for proper identification of sample components, but with reproducible retention times easily obtained with modern chromatographs and the simple use of the time window, no problems arise. The time window is used to ascertain whether the closest
ANALYTICAL CHEMISTRY, VOL. 44, NO. 12, OCTOBER 1 9 7 2
1969
I
I
1
I
0
I
T
0
2
C
b
MINUTES
MINUTES
I 0
0
MINUTES
Figure 14. Chromatogram examples testing tangent peak measurement : S, methanol; T, n-pentanol a. 110 "C b. 140 "C c . 170 "C
peaks in absolute or relative time that are matched, one each from the sample and standard, do not differ by more than the time window value as given in per cent. The use of relative retention time leads to much better accuracy and avoids matching errors. Here it is important that the base peak for relative retention time calculations is properly chosen by the computer from the sample data. This is readily recognized by the relative retention time of 1.OOO for that peak when reported. The sample mode report (Figure 8) consists of the computed weight per cents (these could also be volume or mole per cents) for those components matched or found. There are then two types of unmatched components, those in the standard that are unfound and those in the sample that are unknown. The unfound components are listed in the usual increasing retention time order with the found components. The time and weight given is from the standard file even though this is a sample report. Of course, no area is listed and no weight per cent can be calculated and the lack of entries in these columns pinpoints these unfound components (e.g., Peak No. 1, Figure 8). In a sample containing an internal standard, its weight per cent is not calculated but there must be an area given, the internal standard component having been programidentified by virtue of its being closest in time to that of the internal standard peak in the original standard sample. At the bottom of the report, a listing of unknowns from the sample is given. Here a weight and weight per cent value is computed from each area using either the average response of all components from the external standard or a response factor of unity if the standard was the internal standard variety. Sometimes it is possible to observe in the report the presence of unknown and unfound components with comparable retention times. This may occur after the first of a series of samples is run and some time has elapsed since the standard itself was run, possibly even on another instrument. Alteration of chromatographic conditions or a slight change in the normal 1970
5 Z time window would bring these unknowns and unmatched peaks into a matched situation. UPDATE MODE. For the update mode, the peak matching procedure is again performed for the peaks obtained, matching them with peaks in the standard that are closest in time. No time window is used. Instead the output (Figure 9) shows an additional column at the left, "DEL-T," which is the actual time difference in minutes between the old standard and the newly run standard peaks that were matched. Otherwise the report is similar to the standard mode report except the weights are taken from the old standard. All other data including unknown peaks are new, taken and calculated from the chromatogram just compiled. If the update report is satisfactory, i.e., it duplicates the stored standard being checked, nothing more need be done and samples can be run immediately using the original standard. If the comparison is poor because of chromatographic conditions, these must be changed and a new update run is then performed. If only minor editing is required, such as the renaming or adding or changing names and weights, this is possible via the teletype. When this procedure, usually simpler than standard input, is complete, an edit report is generated as in Figure 10. This edit report data, or the original update data, can then be saved by transmitting a simple communicator box signal, A report of the filing of these data as a new standard is then received (Figure 11). This abbreviated report is all the computer stores in its standard files, but any other data such as areas and response factors could be computed from this information if required. PERFORMANCE OF THE SYSTEM
Area Per Cent Test. A solution of toluene and xylene about 1% each in normal decane was analyzed on a 10-ft X 'bin. 0.d. column of 15% SE-30on 60-80 mesh Chromosorb W. Various column temperatures from 90 to 170 "C were used
ANALYTICAL CHEMISTRY, VOL. 44, NO. 12, OCTOBER 1972
Table V. Tangent Peak Areas 140 "C
170 "C
Run No.
RRTa
1 2 3 4
1.63 1.55 1.62 1.58 1.59
Area units 0.2426 0.2393 0.2610 0.2311 0.2436 0.0126
110 "C
RRT
Area units
RRT
Area units
2.09 2.09 2.17
0.2554 0.2611 0.2537
3.38 3.44 3.52
0.2535 0.2593 0.2526
Average 2.11 0.2567 3.44 0.2551 Std dev 0.0038 0.0036 a RRT = retention time of n-pentanol relative to solvent peak whose absolute times were 0.53 min at 110 "C,0.42 min at 140 "C, and 0.38 min at 170 "C.
so that the initially resolved component peaks would merge as the temperature increased. At 110 "C or below, the o-xylene isomer was also resolved from the other xylene isomers. A complete summary of the data, given in Table 111, shows the excellent reproducibility of the area measurement process and the ability of the system to allocate these areas properly. The samples of chromatograms in Figure 12 show that various degrees of resolution still yielded these equal results. Sample 6s. Standard Analyses. Two solutions were prepared, one containing 1.832 grams of 2-methylcyc1ohexano1, the other 1.6234 grams of n-heptanol each in 100 ml of methylene chloride. Using class A pipets, three standard solutions were made to have various amounts of each component in a mixture of the two. One standard contained about equal amounts of the two alcohols, the others had one or the other alcohol predominate in the ratio of about 9 :1. Their gas chromatography was performed on a 10-ft X li4in. 0.d. column of 15% Carbowax 20M on 80-90 mesh Anakrom ABS at various temperatures from 90 to 140 "C. With a response factor (flame detector) for the n-heptanol of 1.085 (us. 1.OOO for the 2-methylcyclohexanol), the resulting data shown in Table IV were obtained. Again, equivalent results were achieved even with minimal resolution. Examples of some of the chromatograms from these runs are in Figure 13, Tangent Routine Test. A simple test was made of the ability of the system to accurately measure the area of a peak on the side of a sloping solvent via the tangent routine. A solution of about 1% of n-pentanol in methanol was
chromatographed several times on the same Carbowax column described above at temperatures of 110, 140, and 170 "C. The chromatograms in Figure 14 show the varying position of the n-pentanol peak on the solvent tail at the different temperatures. Yet the resulting areas are all in good agreement as shown in Table V. CONCLUSIONS
The basic advantages of this system rest on its versatility in handling all the common problems of gas chromatography data reduction without requiring the selection of special routines for different situations. Furthermore, teletype input is kept at a minimum while the communicator box permits the simple input of any required information which itself is minimal. The entire system is controlled by a set of resident programs still capable of base-line correction refinements, resolution of unresolved peaks that may even greatly overlap, or where one peak is overwhelmed by a larger peak such as in the tangent or bump peak resolutions. And all this is accomplished with satisfactory accuracy of area measurement and peak identification that can be improved even further by a few optional inputs via the communicator box if the gas chromatographer so desires. The system is expandable without alteration to 16 instruments. All that would be required is the additional dedication of about 300 words of core to handle the signals from each instrument added. An automatic injector has already been added to the laboratory instrumentation and the utility of this device has proven compatible with the overall system, thereby further enhancing its high value to the analyst. It mechanically closes the Run and End switches, however, and is not computer controlled. ACKNOWLEDGMENT
We thank A. S. Kucis, P. L. Fagan, and other members of the instrument group for their work in the construction and installation of the signal boxes and in the connection of the gas chromatographs to the interface hardware.
RECEIVED for review March 2,1972. Accepted June 19,1972.
ANALYTICAL CHEMISTRY, VOL. 44, NO. 12, OCTOBER 1972
e
1971
