(13) F. Bruner, P. Ciccioli, and G. Bertoni, J . Chromatogr., 90, 239 (1974). (14) D. H. Desty, J. N. Haresnape, and B. H. F. Whyman, Anal. Chem., 32, 302 (1960). (15) E. L. Ilkova and E. A. Mistryukov, J . Chromafogr. Sci., 9, 569 (1971). (16) C. VidaCMadjar and G. Guiochon, J . Chromatogr. Sci., 9, 664 (1971). (17) A. V. Kiselev, N. V. Kovaleva, and V. S. Nikitin, J . Chromatogr., 58, 19 (1971). (18) A. V. Kiselev, J . Chromatogr., 49, 84 (1970). (19) R . G. Mathews, R . D. Schwartz, C. D. Pfaffenberger, S. N. Lin, and E. C. Horning, J . Chromatogr., 99, 103 (1974).
(20) S. N. Lin and E. C. Horning, J . Chromatogr., 112, 465 (1975). (21) S.Bekassy, P. Arpino, C. VidaCMadjar, and G. Guiochon, J. Chromafogr., in press. (22) A. L. German and E. C. Horning, J . Chromafogr. Sci., 11, 76 (1973). (23) R . S. Deelder, J. J. M. Ramaekers, J. H. M. Van Den Berg, and M. L. Wetzels, J . Chromafogr., 119, 99 (1976).
RECEIVED for review January 3,1977. Accepted January 21, 1977.
Identification of Components of Mixtures by Retrospective Computer Subtraction of Gas-Phase Infrared Spectra Bruce W. Tattershall Department of Inorganic Chemistry, The University, Newcastle upon Tyne, NE7 7RU, England
Retrospective dlgitization of plots of gas-phase infrared spectra of mixtures allows computer subtractlon, to provide fingerprint spectra of components whlch cannot be recognized in the orlginal plots. A spectrum sufficient for recognltion of one component of a binary mixture can be obtained if the other component can be recognized as corresponding to an archival plot obtained normally on a previous occaslon. Even If neither component Is recognlzed inltlally in the spectrum of a mlxture, relative enrichment of one component In an attempted separation, followed by computer subtractlon of the spectra, leads to spectra of each component separately. No spectrum of either pure component needs to be avallable lnltlally. The spectrum of a component of a mixture whlch was obscured by the spectra of two other components was successfully revealed.
Gaseous molecules of complexity in the range 2-15 atoms are frequently identified by IR spectrometry, since even when absorptions cannot be assigned to particular vibrations, the relative heights and shapes of band envelopes are useful as fingerprints to distinguish even between very similar substances. Although the spectra can be classified according to positions and heights of bands, much information about band shape is then lost, so the final identification, and in many preparative research groups dealing with a restricted range of compounds, the whole identification, is done by visual comparison of spectra with archives consisting of X-Y plots. We are concerned with preparative research involving gases which are so reactive as to make GLC separations difficult, and much time is spent in attempting to separate mixtures by the inefficient process of fractionation in a vacuum system, until the constituents can be identified by gas-phase IR as described above. Very frequently the fingerprint regions of constituents of a mixture overlap, and the resulting spectrum closely resembles that of one pure component. The presence of another major component is only revealed if another technique is applied, such as molecular weight or vapor pressure determination. This kind of research could be speeded if the spectrum of a recognized component could be subtracted from that of the mixture, to reveal obscured fingerprint information about other components. The measurement of difference spectra, by use of matched cells in a double-beam spectrometer, is very 772
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common, but suffers the overwhelming disadvantage that one needs archives not only of spectra, but of the compounds responsible for them, so that the matching cell may be loaded for the determination. This is inconvenient in the case of gases, and frequently impossible because of the instability of the samples on storage. To overcome the associated laborious process of matching pressures or concentrations of the reference substance by trial and error in difference spectroscopy, spectra have recently been recorded in digital form, from samples at arbitrary concentration, and the data scaled and subtracted by computer (1). Mixtures and reference substances have still been measured close together in time on a particular committed instrument fitted with digitization equipment and sometimes an on-line computer, to avoid instrumental variations. We have investigated whether spectra obtained without special precautions as X-Y plots from a communal common spectrometer, and stored over a period of time in that form, can be retrospectively digitized when they are needed, and when subtracted using a central university offline computer, can yield spectra which can be visually recognized as fingerprints. Retrospective digitization avoids both the installation of digitization equipment in a particular spectrometer, and the difficult storage of probably unwanted digitized spectra, e.g., as paper tape. One of th_e several commercially-available digitizers, now commonplace as service instruments in universities, is used to transfer spectra from X-Y plots to paper tape. A computer program (IRSPEC) has been written to perform subtractions interactively in an experimental manner, and allow plotting of the results on the same scale as the original spectra.
EXPERIMENTAL IR plots with linear transmittance scales were produced by a Perkin-Elmer 457 spectrometer running at medium speed (3.33 cm-' s-'). The gas cell pathlength was 85 mm, and the baseline was arbitrarily set at about 80% transmittance by means of a double-comb attenuator in the reference beam. The plots were read, digitized, and punched on paper tape by means of a Ferranti Freescan Digitizer, which automatically measured points at preset intervals of frequency as a cursor was moved across the plots. Typically a 2-cm-' interval (0.4 mm) was used, which over a range of 750 cm-' yielded 16 m of tape. The tapes were processed by an IBM370/168 computer operating under the University of Michigan Terminal System (MTS). Intermediate storage was on magnetic disk. The program IRSPEC was written in IBM-extended FORTRAN IV, with calls to MTS subroutines;it contains about 800
FORTRAN statements, and uses about 44 kbytes of array storage in core. It specifically processes PE457 spectra. For use at other institutions or with other IR or UV/VIS spectrometers, the plotting section could be rewritten, and MTS subroutines replaced by local equivalents, by anyone suitably skilled in FORTRAN programming. Requests for source listings etc. should be addressed to the author.
THE PROGRAM If a small peak is to be isolated by subtraction of a large background, the spectrum to be subtracted can be scaled automatically until it is a least-squares fit to the mixture (2). The difference is then the desired peak. In our case, however, we are separating spectra of similar intensity, and the least-squares method would be unlikely to give a recognizable result. Our method depends instead upon testing a postulate by the user that he can recognize a peak in the spectrum of the mixture, which belongs only to the component to be subtracted. Using the program, he applies scaling so that upon subtraction, this scaling peak disappears. He then estimates visually the quality of the result, and tries to recognize it as the spectrum of a compound known to him. Consequently, IRSPEC was designed to be run conversationally from a graphics VDU terminal, so that results of a subtraction can be assessed visually and appropriate actions taken, before a final version is sent to the graph plotter. It can be used at greater speed but with less interaction at types of terminal restricted to character output, or with considerably less convenience in batch mode. Data are read, with suitable interpolation, into a storage array; one column of this contains wavenumber values at fixed intervals, and the remaining eight columns can be selected at will by the user to contain ordinate values of spectra or the results of transformation, subtraction, etc. Commands are by means of keywords, and include options to read data from magnetic disk; to write and reread it in internal format, so as to facilitate storage of intermediate results; to convert transmittance values to absorbance and vice versa; to subtract level or sloping, single or segmented baselines; to scale ordinate values; to move spectra to higher or lower frequency by fixed intervals of frequency; to subtract spectra from each other; to smooth spectra by convolution with a square wave, a simple quadratic function, or with the quadratic smoothing function tabulated by Savitzky and Golay (3);to tabulate maxima and minima at the terminal or on the line-printer; and to plot spectra on a grid calibrated in wavenumbers. Further options cause preset sequencies of these actions to be invoked, to allow more objective processing and to speed routine processing. The most extensive of these automatic options is invoked by the command AUTOxyabcd where x through d are numeric digits representing columns in the storage array. Column x contains the transmittance spectrum of a mixture; column y, the spectrum to be subtracted; and columns a through d are optionally specified columns which are to be preserved from overwriting. AUTO keeps a queue of columns available as workspace, and modifies the number of useful intermediate results that are preserved according to how much workspace it is given. First the spectra in x and y are converted to absorbance, then level baselines are optionally subtracted from each. Baselines are specified either by input of an ordinate value, or by input of a frequency at which a minimum in the spectrum is to be taken as the baseline value. The approximate frequency of the scaling peak is then input, and the highest ordinate value, within two intervals of frequency to either side of this value, is found for each spectrum. This was found to be a more accurate method of finding the maxima of peaks at the frequency intervals of 2 or 5 cm-' used, than the method of fitting a quadratic cap (3). Spectrum y is scaled so that the maxima found are
brought to the same value in the two absorbance spectra. Two separate routines are then followed consecutively. In the first, the scaled absorbance spectrum in y is simply subtracted from the spectrum in x. In the second, spectrum y is moved, one frequency interval at a time, until the total of negative absorbance values produced upon subtraction from x is minimized. So that this movement does not lead to a grossly false alignment of peaks, the movement is limited to four intervals of frequency in either direction. Then in both routines, the result of subtraction is converted to transmittance, plotted, smoothed by the Savitzky and Golay method over a convolution range equivalent to about 34 cm-l, plotted again, and the maxima and minima of the smoothed result are tabulated for the line printer. By use of this AUTO option, the user is thus provided with plots and listings of the results of subtraction, both with an attempt at aligning the spectra, and without one. In response to a suggestion by a referee, we have provided an option to compare two spectra, e.g., a result of subtraction vs. an archival reference spectrum, so as to provide a goodness of fit statistic 6.A command COMPxyabcd, of the same form as the AUTO command, causes absorbance versions of x and y to be produced in columns x' and y', elsewhere in the workspace. To account for differences of scale and baseline respectively, the parameters m and k are calculated by the linear least squares method, to minimize Cr=1e:, where m, k , and e; are related by y/ = mx/ + k + ei. The goodness of fit is then given by q5 = 100
(1
-
variance of errors variance of y' about its mean EeT c y ; * -(Zy;)z/n
To account for errors in alignment, 4 is reported for the initial alignment and then for successive frequency moves of y' until 6 reaches a maximum or a specified number of moves have been made. Here, in contrast to the main use of IRSPEC, the least squares criterion can be used, both for scaling and for optimizing frequency movement, since it is supposed that x and y do resemble each other, and the resemblance is to be maximized, over the whole frequency range to be considered. 6 varies between 4 cm-') signify that x and y represent different compounds. This is a test of similar value to judgments about intermediate values of 4, providing the same spectrometer has been used for all the spectra involved, and it is kept in a reasonable state of adjustment. However, the eye and judgment of the experienced chemist appraising the graphical results is superior to a single numerical statistic. Graphical evidence is presented here. In addition, Table I gives values of 4 for some comparisons of the figures presented.
RESULTS AND DISCUSSION For thorough testing of the method, compounds were sought which would provide spectra which were sufficiently characteristic to be useful for fingerprint identification, but which overlapped so as to obscure each other in mixtures of the compounds. We selected three materials which were rather easy to handle: CClF2CClzF(F113), CCIF&CIFz (F114), and (CClFz)2C0(DCTFA). Their spectra, digitized, and reproduced by IRSPEC, are shown as Figures la, 2a, and 3a, respectively. A ca. 1:l mixture of F113 and F114 gave a ANALYTICAL CHEMISTRY, VOL. 49, NO. 6, MAY 1977
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Table I. Statistical Comparison of Spectra Fit statistic @ No. of After frequency Before alignintervals Figures compared alignment moved ment Results vs. components they represent 57.8 ... la 30.3 32.8 Id la ... le la 76.3 62.0 *.. 4c la 88.8 ... 2b 2a 65.8 ... 2c 2a 84.0 ... 2d 2a
1000
1200
~~
800
cm-'
0 2 0 0 0 0 0
I C
3b 4a 4b
Mixtures vs. obvious component 87.0 87.9 3a 64.7 66.8 2a 78.6 79.7 2a
1 1 1
3b 4a 4b
Mixtures vs. obscured component 59.8 57.8 2a 65.4 61.6 la *.. la 38.1
2 2 >4
Single components vs. different single components la 2a 12.3 ... >4 la 3a 7.9 ... >4 2a 3a 25.9 ... >4 1200
1000
800
cm-'
Figure 2. Spectra of F114. (a) F114; (b) 3b - 3a; :) 3b - 3a, without attempted alignment; (d) 4b - 4a 1800
1600
1400
1200
1000
800 cm-'
Figure 1.
Figure 3. Spectra of DCTFA and mixtures. (a) DCTFA; (b) DCTFA F114; (c) DCTFA F113 4- F114
spectrum (Figure 4a) which closely resembled that of pure F114, and gave little indication of the identity of the second compound. Likewise, in a mixture of F114 and DCTFA
(Figure 3b), only the DCTFA was obvious. Revelation of an Obscured Component. Even when the spectrum of a 1:3 mixture of F113 and F114 (Figure 4b) was taken, subtraction of the obvious component F114 gave a plot
Spectra of F113. (a) F113; (b) IC, before smoothing; (c) 4b - 2a; (d) 4b - 2a, with attempted alignment; (e) 4a - 4b
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ANALYTICAL CHEMISTRY, VQL. 49, NO. 6, MAY 1977
+
+
1200
1QOO
800
cm-’
I
(a)
I: b)
(cl
Figure 4. (a) 1:l F113 (c) 3c - 3a - 2a
+ F114;
(b) 1:3 F113
+ F114;
(Figure IC)which was quite recognizable as F113. The peak of F114 at 843 cm-’ was used for scaling. The smoothed result is presented, since the crude result of subtraction, converted to transmittance (Figure l b ) , is typically much less recognizable to the human eye, although it contains more information. Except for Figure lb, all of the results of subtraction presented in the Figures are after smoothing by the Savitzky and Golay method. In all of the described work on 2-component mixtures, the AUTO command to IRSPEC was used; in this case, movement of one spectrum before subtraction (see description of IRSPEC)produced a less recognizable result (Figure Id). In a typical preparative experiment, neither component of a mixture is obtained in a pure form, but some degree of enrichment of one component is attained. Even in this case, the spectrum of one component may be obtained by sub-
,traction. Figure l e shows the result of subtracting the 1:3 mixture (Figure 4b) (i.e., enriched in F114) from the 1:l mixture of F113 and F114 (Figure 4a). This result can still be recognized as the spectrum of F113. As a further example, Figure 2b shows the result of subtracting the spectrum of DCTFA (Figure 3a) from that of the mixture of DCTFA and F114 (Figure 3b) with scaling on the peak of DCTFA at 1798 cm-l. This result is clearly recognizable as F114. In this case, in contrast to that of the mixture of F113 and F114, alignment of the spectra by AUTO produced the better result. The result of subtraction without movement (Figure 2c) was much poorer. Subtraction of a n Obscured Component. If at least one peak of an obscured component in the spectrum of a mixture is sufficiently attributable to that one component for use as a scaling peak, then subtraction of the spectra of two mixtures containing different concentrations of the constituents can produce an excellent spectrum of the more obvious component. Figure 2d shows the result of subtraction of the 1:l mixture from the 1:3 mixture of F113 and F114, and bears a much closer resemblance to the spectrum of pure F114 (Figure 2a) than does that of the 1:3 mixture itself (Figure 4b). Scaling was on the peak of F113 at 813 cm-’. If an archival spectrum of an obscured component were subtracted, the production of a good spectrum of a previously known obvious component would be a reasonable test of the hypothesized identity of the obscured component. Revelation of an Obscured Component in a 3-Compoaent Mixture. As a final experiment, we attempted to reveal a component obscured by two other components. From a spectrum of a mixture of F113, F114, and DCTFA (Figure 3c) was subtracted f i t the spectrum of DCTFA and then that of F114, with scaling on the peak of DCTFA at 1798 cm-’ and on that of F114 at 922 cm-’. The result, shown in Figure 4c, is recognizable as corresponding to F113, though extension of the method to the subtraction of more than two components would probably be unfruitful. Best results were obtained by allowing movement of the spectrum of DCTFA before subtraction, but not that of F114, just as has been found for the 2-component mixtures.
ACKNOWLEDGMENT The author thanks C. E. Small for carrying out some initial experiments with IRSPEC, the Department of Geography of the University of Newcastle for the use of their Digitizer, and the Computing Laboratory of the University for facilities.
LITERATURE CITED (1) “Computers for Spectroscopists”, R. A. G. Carrington, Ed., Adam Hilger, London, 1974. (2) H. A. Willis. Reference 1, p 209. (3) A. Savitzky and M. J. E. Golay, Anal. Chem., 36, 1627 (1964).
RECEIVED for review September 14,1976. Accepted February 23, 1977.
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