Anal. Chem. 1995,67, 1084-1091
Multivariate Approach to Fingerprint identification of Organic Compounds Using an Oscillating Glow Discharge Detector for Gas Chromatography Diane L. Smith* and Edward H. Piepmeier* Department of Chemistry, Gilbert Hall 153, Oregon State University, Corvallis, Oregon 97331-4003
Fingerprint identification information obtained from an oscillating plasma glow discharge detector for gas chromatography is improved by changing the cell pressure, applied voltage, and electrode spacing. Changes in the discharge operating 'conditions produce changes in the analyte peak responses. The relative magnitudes of the analyte current and frequency peak responses also change with respect to each other under different discharge conditions. Unique fingerprints or patterns of responses are created for each analyte by changing the discharge operating conditions. The detector responses toward nine organic compounds, representing six different functional groups, were recorded under 56 different combinations of discharge conditions. The ratios of the frequency-tocurrent peak responses (heights and areas) for three of the 56 sets of conditions investigated provide enough information to distinguishbetween the nine compounds. Principal component analysis and hierarchical cluster analpis, multivariate exploratory techniques, are used to observe natural clustering in the data. A low-pressureglow discharge detector for gas chromatography was first reported in 1956.' A voltage change was observed across an argon discharge when organic impurities, on the order of 10-l2 mol, were present in the gas. More recently, a lowpressure oscillating plasma glow discharge detector for gas chromatography was describedS2A linear relationship between the oscillation frequency and impurity concentration in the support gas of the discharge was demonstrated. The frequency-tocurrent peak height ratios were observed to differ from one compound to the next, suggesting that the detector could help to identify analytes and perhaps even provide analyte structural information. The variations in the detector responses of 25 organic compounds have been studied under constant discharge condit i o n ~ . This ~ study showed that fingerprint identification of compounds is possible by using the ratios of the frequency-tocurrent peak responses (areas and heights) of the oscillating plasma glow discharge detector. The compounds clearly respond differently. However, limited identification information can be obtained due to the overlapping responses of some compounds caused by peak measurement uncertainties. ' Present address: Wyeth-Ayerst Laboratories, P.O. Box 8299, Philadelphia, PA 19101-1245. (1) Harley, J.; Pretorius, V. Nature 1956, 178, 1244. (2) Kuzuya, M.; Repmeier, E. H. Anal. Chem. 1991, 63, 1763-1766. (3) Smith, D. L.; Piepmeier, E. H. Anal. Chem. 1994, 66, 1323-1329. 1084 Analytical Chemistry, Vol. 67,No. 6,March 15, 1995
The operating conditions of the plasma are known to influence the the oscillations! and the analyte detector responses. The geometry and type of electrode materials used also influence the di~charge.~ In the present work, the effects of electrode spacing on the baseline signals and detector responses under constant applied voltage and cell pressure using a stainless steel anode and a brass cathode are reported. In addition, the useful applied voltage and cell pressure ranges are studied at two electrode spacings using a brass anode and cathode. The relationships between the discharge conditions and the baseline signals and detector responses are discussed. The changing detector responses with discharge conditions are used to improve analyte identification. Fifty-six different combinations of applied voltage, cell pressure, and electrode spacing were investigated. The frequency and current chromatographic peak responses of nine organic compounds, representing six functional groups, are monitored and recorded under each combination of discharge conditions. The discharge conditions clearly influence the detector response. More important,however, is the fact that the analyte responses shift relative to one another with changing discharge conditions. The ratios of the frequencyto-current peak responses (heights and areas) for three of the 56 sets of conditions provide enough information to distinguish between the nine compounds. The multivariate exploratory techniques of principal component analysis'O and hierarchical cluster analysis11J2show separate groups or clusters of responses representing replicate injections of the nine compounds studied. EXPERIMENTAL SECTION A detailed description of the detector cell and experimental setup is presented in a previous publication? The following moditications have been used in the present work. A regulated, adjustable 1600-V dc power supply (Model 65154 Harrison/ Hewlett-Packard) operated between 300 and 600 V was used (4) Von Engel, A Ionized Gases; Oxford University Press: Oxford, U.K, 1955. (5) Tong, S. L.; Harrison, W. W. Anal. Chem. 1984, 56, 2028-2033. (6) Mchckey, S. A,; Glish, G. L.; Asano, IC J.; Grant, B. C. Anal. Chem. 1988, 60, 2220-2227.
(7) Marcus, R IC, Ed. Glow Discharge Spectroscopies;Plenum Press: New York, 1993; pp 41-43. (8) Pekhrek, L. Ion Waves and IonizationWaves. loth Intemational Conference on Phenomena in Ionized Gases, 1971; Donald Parsons & Co. Ltd.: Oxford, England, 1971; pp 365-403. (9) Llewelyn-Jones, F. The Glow Discharge; Methuen: London, 1966. (10) Massart, D. L.;Vandeginste,B. G. M.; Deming, S.N.; Michotte,Y.; Kaufman, L. Chemometrics: a textbook; Elsevier: New York, 1988. (11) Shard, M. A; Illman,D. L;Kowalski, B. R Chemometn'cs;Wiley: New York, 1986. (12) Everitt, B. Cluster Analysis; Wiley: New York, 1974. 0003-2700/95/0367-1084$9.00/0 0 1995 American Chemical Society
instead of the Heath Model EUW-15 for improved stability. A pointed brass anode was used with a 60” occluded angle and an exposed surface area of 0.18 cmz. A l/sin.-i.d. glass tube, 1 cm long, is slipped over the pointed end of the anode. Three to four turns of Teflon tape are wrapped around the outside of the anode to provide a press fit to help secure the glass tube. The distance from the tip of the anode to the open end of the glass tube is 4 mm. The highly visible anode glow of the glow discharge appears in the glass tube. The purpose of the glass tube is to confine the discharge to this region to improve the oscillations. The 1 . k m diameter brass cathode is concave with a l.&m radius of curvature. The electrode spacing is measured in a coaxial direction from the edge of the cathode to the end point of the anode. A computer-interfaced Fluke Model 45 multimetes monitors the voltage across a 1-kB cell-current-sampling resistor. When the signal is below 10 V, the meter is set to obtain a resolution of 100pV. When the signal exceeds 10 V, the meter is set to obtain a resolution of 1 mV. The oscillation frequency was measured by a Philips Model PM 6654C high-resolution timer/countes set to measure to within 2 ns the exact time (0.4 s) it takes to accumulate an integral number of oscillations of the signal. Both instruments were controlled by a computer with a GPIB interface. Mixtures for injection were prepared by combining equal volumes of five compounds. No additional solvent was added to the mixtures. For example, one mixture contained equal volumes of 1-hexene, 1-heptyne, 2-pentanone, 1-decene and 1-decyne. Another contained equal volumes of n-hexane, di-n-propyl ether, n-decane, 1-pentanol,and heptanal. All chemicals used were of the highest purity available (9899+%) and were purchased from one of the following companies: Aldrich Chemical Co., Alfa Products, EM Scientific, JTBaker, or Sigma Chemical Co. A l/&-o.d. x 2-mm4.d. x 3-m-long glass column (Alltech Associates) of 3%OV-225 on Chromosorb W-HP, 100/120 mesh, was used. All 0.01-pL injections (Dynatech precision syringe,Alltech Associates) of each mixture were made in triplicate under each combination of discharge conditions. The frequency and current peak heights and areas from the chromatograms were determined for each compound using a QuickBasic program. Heights and areas were determined relative to the baseline signals. In addition, the frequency-tocurrent peak ratios (heights and areas) are calculated. The principal component analysis and hierarchical cluster analysis were done using Pirouette (Infometrix, Seattle, WA). Data were preprocessed using autoscaling. RESULTS AND DISCUSSION First, the results of a study of how the oscillating glow discharge detector responds under different operating conditions will be presented to provide a background for understanding why fingerprintidentiiication can be improved by including results from different operating conditions in the data set. Electrode Spacing. Figure 1 shows how the electrode spacing alters the baseline frequency and current signals under constant applied voltage and cell pressure. The baseline frequency goes through a minimum near an electrode spacing of 1.6 an. Cook and Piepmeier13suggest that the oscillation frequency shifts with electrode spacing as the discharge adjusts to maintain an integral number of effective wavelengths between the electrodes. (13) Cook,B.;Piepmeier, E. H.Anal. Chem. 1994,66,1249-1253.
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The baseline current signal shows the opposite behavior, going through a maximum near 1.6 cm. These results show that the plasma oscillates over this entire region of electrode spacing, unlike the results of Cook and Piepmeier,13which showed breaks in electrode spacing where the oscillations stop. The main difference between these cells is that Cook and Piepmeier introduced the mobile phase through a hole in the anode, coaxial with the plasma, while our cell introduces the mobile phase from the side, perpendicular to the axis of the plasma. The influence of the electrode spacing on the frequency peak height for three of the compounds that show the greatest variations is shown in Figure 2a. Each point represents the average of triplicate runs. The analyte response tends to decrease as the electrode spacing increases. However, the slopes differ for each of the three compounds shown, and two of the lines intersect. Although not shown, the frequency peak area tends to be highly correlated with the frequency peak height. Figure 2b shows the dependence of the average current peak area response on electrode spacing for 1-pentanol,heptanal, and 1-hexene. As with the frequency peak response, the current peak response tends to decrease with electrode spacing for shorter spacings, and intersections are observed. However, the current peak response rises again at larger spacings for some analytes. Similarly, the relative differences between the analyte responses change with electrode spacing. As with the frequency heights and areas, the changes in the current heights and areas tend to be highly correlated. Although the electrode spacing intluences the detector response, the variation in the responses between analytes indicates that there is not a simple relationship between the electrode spacing and the detector response. Similar trends in the frequency and current peak responses are observed for each of the 10 analytes investigated. Although the trends are similar, the differences are sufficient to provide unique fingerprint information for each analyte. The relationships between the baseline signals and the analyte peak responses for the different discharge conditionswere studied to determine if the behavior of the analyte responses can be predicted from the magnitude of the baseline signals. Figures 1 and 2a show that the baseline frequency and the frequency peak Analytical Chemistry, Vol. 67, No. 6, March 15, 1995
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height tend to decrease with increasing electrode spacing for electrode spacings less than 1.5 cm. Figures 1 and 2b show an inverse relationship between the baseline current and the current peak area for electrode spacings less than 1.25 cm. The dependence of the analyte response on the baseline signals varies for each analyte, making it difficult to use the baseline signal to predict how the detector will respond. Voltage/Pressure Influences on Baseline Signals. Figure 3 shows the useful voltage and pressure operating ranges at the two electrode spacings investigated. Outside of the operating ranges shown, the discharge either went out or the current signal stopped oscillating. A relatively low voltage (315-400 V) and a relatively high pressure (0.95-1.45 Torr) are required to obtain useful oscillations at an electrode spacing of 1.016 cm compared to the high voltage (340-575 V) and low pressure (0.30-0.60 Torr) required to sustain the same oscillation at 2.184 cm. At a given electrode spacing, however, lower applied voltages tend to be useful only at lower cell pressures, and higher voltages tend to be useful only at higher pressures. The baseline current increases almost linearly with both the applied voltage and cell pressure for both electrode spacings investigated. However, parts a and b of Figure 3 show that at fixed pressures the increase in the baseline current with voltage is smaller at lower pressures, as indicated by the smaller slopes in the curves obtained at lower pressures. Similarly, parts c and d of Figure 3 show that at fixed voltages the increase in the baseline current with pressure is smaller at lower voltages. The curve obtained at 315 V has a much smaller slope than the curve obtained at 400 V, indicating that the baseline current increases with pressure at a faster rate at 400 V than at 315 V. The influence of the applied voltage and the cell pressure on the baseline frequency differs significantly from the influence on the baseline current. Parts a and b of Figure 4 show that the baseline frequency tends to increase with applied voltage at several different pressures, while the baseline frequency exhibits significant curvature with changes in voltage. The relationship between the baseline frequency and cell pressure is illustrated in parts c and d of Figure 4. Again, unlike the baseline current, the baseline frequency exhibits significant curvature with increasing pressure for each voltage investigated. 1086 Analytical Chemisfty, Vol. 67, No. 6, March 15, 1995
The baseline signals may provide insight into how the operating conditions influence the discharge processes. However, the changes in the baseline frequency are not related by a simple relationship to the changes in the baseline current. Voltage/Pressure Influences on Analyte Responses. The chromatograms in Figure 5 clearly show that the system operating parameters have a strong influence on the detector response. For 340 V, 0.95 Torr, and 1.016 cm, the five compounds in parts c and d of Figure 5 are all responding negatively with respect to both the baseline frequency and baseline current signals. A change in the system operating parameters to 500 V, 0.50 Torr, and 2.184 cm dramatically alters the detector response, as illustrated in parts a and b of Figure 5. The same five compounds now respond positively with respect to the baseline frequency signal, yet most continue to respond negatively with respect to the baseline current signal. Although the current peaks tend to be negative under both sets of conditions, the relative sizes of the peaks differ significantly within and between the chromatograms. For example, l-hexene (first peak) has a larger response than l-heptyne (second peak) in Figure 5b but a smaller response in Figure 5d. Although l-decene (fourth peak) has a small negative response in Figure 5d, it has a smaller positive response in Figure 5b. l-Decyne (last peak) has a small response in Figure 5d, but it is virtually undetectable in Figure 5b. All possible combinations of positive and negative frequency and current peaks are observed (not shown) by changing the discharge operating conditions. The ratios of the frequency-to-current peak areas for six analytes under five different combinations of cell pressures and applied voltages are shown in Figure 6. The combinations of responses for each analyte under the five sets of discharge conditions are grouped together to show patterns in the responses. All six analytes have negative response ratios at 375 V and 1.25 Torr (left bar in each group) and positive response ratios at 325 V and 0.95 Torr (right bar). The response ratios are mixed for the remaining three sets of intermediate conditions. Thus, each analyte has a unique pattern or fingerprint of responses that provides a means to more clearly distinguish different compounds. The change in the frequency and current peak heights with cell pressure at different cell voltages for di-n-propyl ether are
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shown in Figure 7. The frequency peak heights tend to increase with pressure at constant voltage. However, the frequency peak heights tend to decrease with voltage at constant pressure. The fact that lower responses are observed under high-pressure and high-voltage conditions indicates that the applied voltage has a stronger influence on the frequency response than the cell pressure. The current responses behave in a similar manner but tend in the opposite directions compared to the frequency responses. With some exceptions, the current peak heights tend to decrease with pressure at constant voltage but increase with voltage at constant pressure. As observed with the frequency responses, the cell pressure and applied voltage have opposite influences on the analyte current responses. Under high-pressure and high-voltage conditions, large current responses are observed, indicating that the applied voltage also has a stronger influence on the current response than does the cell pressure. The same trends observed for the di-n-propyl ether peak responses shown in Figure 7 are observed for the other nine analyte species investigated. However, the absolute magnitudes
of the responses differ significantly between compounds. For example, the di-n-propyl ether frequency peak height has a 6@ kHz change over the range of applied voltages and cell pressures investigated, but the 1-decyne frequency response (not shown) changes by only 20 kHz. In addition, the slopes of the curves obtained in plots similar to those in Figure 7 vary for each of the other analytes. The discharge operating conditions influence both the baseline signals and the analyte peak responses. Although the baseline signals may provide insight into the discharge processes, they cannot be used to predict how an analyte will respond under changing voltage and pressure. A d y t e Identitication. The an& responses obtained under 56 different combinations of discharge operating conditions were carefully reviewed to identify three sets of conditions which provide the most reproducible responses yet show the greatest variation between the nine analytes. Figure 8 shows a plot of the frequency-tocurrent peak area ratios versus peak height ratios for one set of operating conditions. These ratios are used because they are independent of analyte c~ncentration.~ Each point of the Analytical Chemistv, Vol. 67, No. 6,March 15, 7995
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triplicate measurements is plotted to indicate the spread in the points. A similar plot for a second set of conditions in this study is shown in Figure 12 of ref 3. Separate groups or clusters of replicate injectionsrepresenting the individual analyte responses can be observed in these twodimensional plots. Figure 12 in ref 3 shows that several of the analyte species (1-heptyne, 1-decene, 1-decyne, n-decane, l-pentanol, and possibly 2-pentanone) are distinguishable from each other. Although many of the analyte responses in Figure 8 are very close and perhaps overlapping,the ratios have clearly shifted (from positive to negative values) with respect to each other when compared to Figure 12 in ref 3. Now, 1-heptyne (b) and n-hexane (0 may be the only distinct groups under these conditions. The third set of responses chosen for this study is for a plasma operating at 425 V, 0.60 Torr, and an electrode spacing of 2.184 cm. The results from all three of these sets of conditions are combined to determine if a greater distinction between analytes can be abtained using the combined responses compared to the 1088 Analytical Chemistry, Vol. 67, No. 6, March 15, 1995
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individual responses. The data set becomes sixdimensional and requires a multidimensional data analysis. An exploratory principal component analysis (PCA) was conducted, and the eigenvalues are presented in Table 1. A cumulative percent variation of greater than 96%at the third principal component indicates that the sixdimensional data set can be effectively reduced to three dimensions. A two-dimensional projection of a three-dimensional scores plot in Figure 9 shows nine distinct clusters representing the nine analytes investigated. None of the two-dimensional plots of the original ratios showed as much distinction between all the
analytes as is observed in the scores plot. Thus, the combined results provide greater analyte identification information. A clearer visualization of clustering may be obtained from a dendrogram from hierarchical cluster analysis (HCA), a second exploratory technique independent of PCA. For each set of conditions, the height ratio was found to be highly correlated with the area ratio, as expected. However, the correlations between the responses from dif€erentconditions were weak. Since HCA is sensitive to highly correlated variables, the area ratios are removed from the data set. Analytical Chemistry, Vol. 67, No. 6, March 15, 1995
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Table 1. PCA Eigenvalues.
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Figure 9. PCA scores plot where the xaxis is principal component number 1 and the yaxis is principal component number 2. Triplicate injections of nine compounds under three sets of discharge conditions form nine distinct groups: (a) 1-hexene, (b) 1-heptyne, (c) 2-pentanone, (d) 1-decene, (e) 1-decyne, (f) +hexane, (h) rrdecane, (i) 1-pentanol, and [j) heptanal. De-ran
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A dendrogram for the frequency-to-current peak height ratios for one set of operating conditions is shown in Figure 10. Horizontal line lengths indicate (for a given data set) the relative distances between points or groups of points in multidimensional space (or one-dimensional space for this single set of operating conditions). For example, the two points at the top of Figure 10, heptanal and ldecene, are very close to each other. At the bottom of the list, the first two n-decane points are adjacent but not quite as close to each other as the two points at the top of the list. The last n-decane point is even farther from the first two n-decane points, but all three n-decanes are closer to each other than to any of the other points. Figure 10 shows that only three (pentanone, n-hexane, and n-decane) of the analyte triplicates are clustered together without interference from other analytes. This should improve when data from additional operating conditions are included, as we will now see. Dendrograms obtained using the frequency-to-current peak height ratios from the three sets of operating conditions are shown in Figure 11. The relatively short horizontal branches within clusters on the dendrogram indicate that the responses from replicate injections of each analyte are closer to each other than to any other analyte response. One exception is 1-pentanol,where one of the replicate responses falls closer to the 2-pentanone cluster than to the other two 1-pentanol responses. Improving the reproducibility of replicate injections should eliminate this overlap. However, separate clusters corresponding to individual analytes are clearly being observed. These HCA runs used the 1090 Analytical Chemistry, Vol. 67, No. 6, March 15, 7995
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Figure 10. HCA dendrogram using the complete link procedure for triplicate injections of nine organic compounds for one set of discharge conditions. The lengths of the horizontal lines indicate the spatial distances between adjacent points or groups of points.
most conservative linking procedure, called a complete link, to calculate the spatial distances between points and groups. The fact that the same clusters are also observed using the simplest linking procedure (single link) indicates that the clusters are well separated.14J5 A true test of the signjjicance of clusters can be determined only by running the multivariate classification algorithms on unknown samples using predefmed clusters. The classification algorithms are not planned at this time because the system has yet to be optimized to provide the maximum amount of distin(14) Pirouette Multivariate Data Analyskfor IBM PC Systems, Version 1.1; Users Manual; Mometrix Inc.: Seattle, WA, 1992. (15) Haswell, S.J., Ed. Practical Guide to Chemometrics; Marcel Dekker, Inc.: New York, 1992.
Dendrogram Constructtan Method: Complete Link
1-dacme 1-decene 1-decene 1-decrJne 1-decyne 1-decune n-hexane 1-hexens 1-hexene 1-hexene heptyne heptyne heptyne pentano 1 pentano 1 pentanol pentanon pentanon pentanon
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Pirouette, by I n f o n e t r i x . Inc.
Figure 11. HCA dendrogram using the complete link procedure for triplicate injections of nine organic compounds under three sets of discharge conditions. Notice the improved clustering of the triplicates compared to Figure 10.
guishmg information. The discharge is rich in wave motion; perhaps with the use of additional discharge conditions an even greater distinction between analytes can be made. The ability to distinguish different analytes depends upon how closely together replicate points cluster, and this depends upon (16) Harrison, W. W.; Barshick, C. M.; Klingler, J. A; Ram,P. A; Mei, Y. Anal. Chem. 1990,62, 943A-948A. (17) Harrison, W. W. J. Anal. At. Spectrom. 1 9 9 2 , 7, 75-79. (18) Chapman, B. Glow Discharge Processes; Wiley: New York, 1980. (19) Marcus, R K Spectroscopy, 1992, 7(5), 12-16. (20) Smyth, K C.; Schenck, P. K Chem. Phys. Lett.,1978,55(3), 466-472. (21) Smyth, K C.; Bentz, B. L.; Bruhn, C. G.; Harrison, W. W.J. Am. Chem. SOC. 1979, 101, 797-799. (22) Hess, K. R; Harrison, W. W. Anal. Chem. 1988,60, 691-696. (23) Smith, R L.; Serxner, D.; Hess, K R Anal. Chem. 1 9 8 9 , 61, 1103-1108. (24) Sofer, I.; Zhu,J.; Lee, H.; Antos, W.; Lubman, D. M.App1. Spectrosc. 1990, 44, 1391-1398. (25) Levy, M . K; S e m e r , D.; Angstadt, A D.; Smith, R L.; Hess, K R Spectrochim. Acta, Pad B 1 9 9 1 , 46, 253-267. (26) Eisenhut, 0.;Conrad, R 2.Elektrochem. 1 9 3 0 , 36, 654. (27) Glockler, G.; Lind, S. C. The Electrochemisty of Gases and Other Dielectrics; Wiley: New York, 1939. (28) Howatson, A M. An Introduction to Gas Discharges, 2nd ed.; Pergamon Press: New York, 1976. (29) Hemng, C.J.; Piepmeier, E. H. Anal. Chem., in press.
the noise level and the reproducibility of sample injection. Automating our injection procedure should improve reproducibility. The mechanisms controlling the discharge current and oscillation frequency responses to analytes are not completely understood. Although the current and frequency mechanisms may be related, there are also significant differences in the way the discharge conditions influence each of these signals and the analyte detector response. The glow discharge is a complex combination of atomic and ionic species from the discharge gas and possibly from the cathode material, and many chemical reactions are possible.16J7 Although ionization is a prime feature of the glow discharge,18many discharge processes have been extensively studied for use in analytical in~trumentation.'~J~J~ The discharge conditions innuence the chemical reactions occurring in the discharge by controlling the absolute and relative abundances and energies of the species present in the disThe experimentally observed differences between the analyte responses may be due to different interactions between the analyte molecules and discharge species that change with discharge conditions. The presence of an analyte molecule may enhance, decrease, or even change the primary discharge reactions. The glow discharge detector is basically a small chemical reactor that the analyst can control.16 Once the mechanism or mechanisms responsible for providing the differences in the analyte responses are understood, the detector cell may be tuned to provide a maximum amount of distinguishing information. In the future, an array of detector cells in parallel or in series may be used in which each cell is selectively tuned to control the mechanisms to obtain the greatest amount of analyte identitication information. Toward this end, glow discharges of microscopic size are currently being studied as detectors for capillary gas chromatography and as detectors for liquid streams.29 ACKNOWLEDGMENT This research was supported by National Science Foundation Grant No. CHE-9013929. Received for review August 16, 1994. Accepted December 19, 1994.@ AC940809S @Abstractpublished in Advance ACS Abstracts, February 1,1995.
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