Anal. Chern. 1988, 6 0 , 386-390
388
Interferogram-Based Method for the Correlation of Gas Chromatography/Fourier Transform Infrared Spectral Components Paul T. Richardson' and James A. de Haseth* Department of Chemistry, School of Chemical Sciences, Uniuersity of Georgia, Athens, Georgia 30602
A method Is presented that correlates gas chromatography/Fourler transform Infrared (GC/R-I R) interferograms from separate GC/FT-IR analyses. The method Involves dlrect Fourler transform Infrared spectrometer Interferograms, whkhareontypmwssed by phase cortectkn and conelated wlth other similarly processed Interferograms by uslng a dot product algorlthm. The correlatlon of these data Is useful In maklng mole poslthre Mentlfkatbns d unknown components, especlally In low signal-to-nolse sltuatlons. Thls Is because the correlation operation Is more reliable than the search operatlon. Furthermore, It Is posslble to recognlze the cornponents of a serles of cOmpOnents as separated, or partlaily separated, by the gas chromatograph. An addltknalappYcaknkpre8entedInwhichagaschromatogaphic eluate that is comprlsed of two components Is recognlzed.
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A majority of the time required for an analysis of a complex mixture using gas chromatography/Fourier transform infrared (GC/FT-IR) spectrometry is associated with the isolation and identification of eluate peaks. The sample turn-around-time can be extremely long when numerous components are not identified through automated library search procedures. This, of course, is a common occurrence when environmental or fragrance samples are analyzed. Once the first of a series of similar samplea has been evaluated, however, it is quite feasible that the subsequent samples can be evaluated more rapidly. Complex samples of environmental origin have often been shown to contain numerous members of a homologous series. Sometimes a homologous series elutes as a partially resolved cluster of peaks that may appear as an elevated section of a chromatogram (1). More often, homologues elute as separate peaks,which contribute to the complexity of a chromatogram. In practice, infrared spectrometry is not generally a practical tool toward differentiating individual homologues of the same series. For this reason, if a rapid means were available by which the members of a homologous series could be related, sufficient structural information for the homologues might be provided by processing only one representative member. This would contribute to an increased efficiency in data processing since the number of spectral computations and library searches could be minimized. Often an eluate peak from one sample can be correlated to one of the same compound that is isolated and identified from a different sample through the comparison of retentions data. Therefore, accurate peak correlation is another means of obtaining a substantial reduction in the necessary number of spectral computations and library searches required during an analysis. Because of the numerous factors that influence the precision of absolute retention times, the use of reference IPresent address: E. I. du Pont de Nemours & Co., Agricultural Products Department, Experimental Station E402/3108,Wilmington, DE 19898.
compounds and subsequent computation of relative retention times is preferred (2). Unfortunately, the typical reference compounds employed in gas chromatography are not strong IR absorbers and cannot be easily discriminated from their spectral data. In addition to this problem, complex environmental samples can contain various chemical compounds that exhibit nearly identical retention behavior. Therefore, two eluate peaks of different chromatograms do not correspond to the same compound necessarily just because their retention data are similar. Also, the coelution of two or more compounds producing a single peak will not be detected without additional information such as that derived from the infrared absorbance data. In order to circumvent the limitations associated with retention data-based peak correlation, a method was devised that bases comparisons upon the similarities of eluate peak interferometric data. Because the interferometric data provide qualitative information about eluate peaks, t h i s technique also c o n f i i the presence of homologues within the same sample as well as detects coeluting components.
THEORY The interferograms collected by a GC/FT-IR system are digitized representations of interference patterns that result from the modulation of infrared radiation by an interferometer. Each datum of an interferogram represents the sum of intensities of all frequencies of radiation that reach the detector for a corresponding movable mirror displacementwithin the interferometer. The appearance of certain regions of an interferogram becomes significantly altered when an IR-absorbing eluate is present within the lightpipe. This effect is illustrated in Figure 1,which shows a segment of raw interferograms collected when a lightpipe contains a sample eluate and when the carrier gas alone is passing through the lightpipe. The interference patterns change as a result of the loss, or significant attenuation of the intensities at certain frequencies of radiation that are absorbed by the eluting compound. In principle the raw interferograms associated with different sample eluates should exhibit unique patterns that allow a discrimination or correlation of eluate peaks. Unfortunately, it is not possible to compare raw interferometric data with a high degree of accuracy because of the presence of spectrometer-dependent features known as the instrument function. The instrument function dominates the Fourier domain signal since it is dependent upon the band-pass of the spectrometer, the dynamic range of the detector, the sampling frequency of the spectrometer, and the instrument line shape. It is necessary to remove the instrument function and other background effects from a sample interferogram in order to obtain a unique representation (Le. a corrected spectrum) of an eluate component. Information from a reference interferogram,collected when no eluate is present in the lightpipe, may be used for the removal of most of the instrument function from a sample interferogram. This operation is most efficiently achieved through the use of the Gram-Schmidt vedor orthogonalization
0003-2700/88/0360-0386$01.50/00 1988 Arnerlcan Chemical Society
ANALYTICAL CHEMISTRY, VOL. 60, NO. 5, MARCH 1, 1988
387
Table I. Dot Products with and without Phase Correction
compound name methanol acetone methylene chloride methyl acetate chloroform
dot product values not phase phase corrected corrected 0.992 0.998
0.974 0.987 0.993
0.851 0.872 0.970 0.946
0.788
:5
8 $2
3.75 2.5 1.25
a 0
Figure 1. Sections corresponding to the same region of raw interferograms when (a) a component was in the lightpipe and (b) the lightpipe contained carrier gas only. method which performs a vector subtraction between the sample and reference interferograms. Historically, the first application of the Gram-Schmidt method on interferometric data was for the constructionof chromatograms for GC/FT-IR systems (3). The techniques later was adopted for the successful removal of the instrument function from interferometric data used for an interferogram-based search system (4). A similar method has been adopted for the processing of interferometric data for the peak correlation algorithm. In this case, 100 consecutive points of the interferometric signal beginning with the 55th point from the centerburst are extracted for the subsequent comparisons. The actual comparison of interferometric data can be achieved by using various vector techniques. The use of dot product computations is straightforward and is particulary attractive since a computed value of 1.000 (for normalized vectors) corresponds to a perfect match between two vector representations. Since both positive and negative data points are associated with interferograms,possible dot product values range between -1.000 and 1.OOO. Generally if a value of 0.950 or greater is obtained, the two peaks involved are likely to be of the same chemical compound. Other match algorithms are possible, and the relative merits of these are discussed elsewhere (5). Following the correlation of a peak with an entry from a reference chromatogram, information available for the compound may be extracted from a corresponding header file. In this way, the identity of a compound may be obtained provided that the same chemical compound has been encountered previously. An option allows the vector reference and chemical information files to be updated automatically whenever a chemical species is detected and identified for the first time.
EXPERIMENTAL SECTION All interferometric data were collected using a Digilab FTS14/C/D or a Digilab F"I'S-SO/E/D spectrometer (Digilab Division of Bio-Rad, Cambridge MA) with a mercury-cadmium telluride (MCT) detector (Infra-Red Associates, Inc., New Brunswick, NJ). All separations were achieved by using a Varian 3700 gas chromatograph fitted with a fused-silica capillary column (50 m X 0.53 mm i.d.) coated with a 5 pm thick SE-54 stationary phase (Quadrex, New Haven, CT). Samples were injected directly onto the column. The computer programs were written in either FORTRAN or Data General Assembler language. Data processing was done on
SCAN NUMBER Figure 2. Constructed chromatogram for a perfume sample (a)from which the correlation reference file was generated and (b) for which the correlation technique was performed.
a Data General NOVA 4/X minicomputer, which is dedicated to the Digilab spectrometer, or on an Information Now Incorporated (INI) 300-MI0 microcomputer (Information Now Incorporated,Salt Lake City, UT). The IN1 300-MI0 microcomputer is a NOVA 4/X clone.
RESULTS AND DISCUSSION In earlier work performed by de Haseth and Azarraga, it was shown that a much higher accuracy occurred for an interferogram-based search system if the interferograms were phase corrected prior to use of the Gram-Schmidt operation (4). Nevertheless, it was not clear if phase correction would be necessary for the peak correlation algorithm as all data are collected by the same interferometer under similar conditions. The technique was evaluated both with and without the use of the Forman phase correction procedure (6). Two GC/ FT-IR analyses were performed on mixtures containing the same components. Dot products were calculated for interferogram vectors of identical components from the two analyses. The dot product results, which are given in Table I, clearly indicated that the use of phase-corrected interferograms would be necessary in order to obtain consistently accurate peak correlation. The utility of the technique toward the rapid identification of eluate peaks that are encountered in other samples was illustrated through the analysis of various perfumes. A comparison of the eluates associated with the two chromatograms shown in Figure 2 resulted in the correlation program output given in Table 11. The correlation results accurately showed that six of the sample components were common to both perfumes. Most of the components that were separated from the first sample were identified by a spectral library search prior to the correlation of the second sample. This information was stored in a header file and later used to specify the identities of the six eluates that correlated with those of the first sample.
ANALYTICAL CHEMISTRY, VOL. 60, NO. 5, MARCH 1, 1988
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Table 11. Correlation of Two Perfume Samples peak no.
status
dot product
1 2
matches no entry matches no entry matches entry 4 matches entry 5
0.968 0.977
3 4
matches entry 6 matches no entry matches entry 9
5
6 7
matches no entry matches no entry matches no entry matches no entry matches no entry matches no entry matches no entry matches no entry matches no entry matches entry 13 matches no entry matches entry 18
8 9 10 11 12
13 14 15
16 17 18
19
0.996
identity
linalool o-methylphenylethanol benzyl acetate WAVE-RS
0.996
4-methylpentyl acetate
1
54 0.997
diethyl phthalate
0.980
5-isopropyl-2-nitroto1u ene
Table 111. Benzyl Acetate Search Results from Two Different Samples hit entry no.
quality index
1 I
I
i
WAKWHBERS
Figure 3. Two absorbance spectra obtained for benzyl acetate that was isoiated from different samples: (a) hlgh signaCtMldse ratio (SNR) spectrum that was correctly Mentlfied by the spectral library search: (b) iow SNR spectrum that was incorrectly identified by the same procedure. 25 21.675
CAS no.
A
+I I
I
'
"
"
"
"
'
'
identity
t
t
Search Results, Sample No. 1 1 2
3 4
5
54 1672 3249
0.1470 0.1968 0.2597
568 2701
0.2806 0.2811
hit entry no.
quality index
140-11-4 benzyl acetate 103-54-8 cinnamyl acetate 30614-73-4 3,4-bis(acetoxymethyl)furan 623-17-6 furfuryl acetate 6963-44-6 1,5-pentadioldiacetate
3.125 0
SCW
CAS no.
identity
NUreER
F b r e 4. Constructed chromatogram for a sample containing a peak plateau that corresponded to a homologous series of components.
Search Results, Sample No. 2 1
3249
0.3971
2
1672
3
54
4
568 616
0.4022 0.4045 0.4159 0.4266
5
30614-73-4 3,4-bis(acetoxymethyl)furan 103-54-8 cinnamyl acetate 140-11-4 benzyl acetate 623-17-6 furfuryl acetate 591-87-7 allyl acetate
The correlation technique alternatively may be used to provide complementary information for library search results. For example, if two peaks from separate chromatograms are shown to be of the same chemical compound through the correlation procedure, that information may be used as a means to detect erroneous library search results. An example of differing spectral library search results obtained for benzyl acetate, which was isolated from two perfume samples, is given in Table 111. Search results were obtained by using the GIFTS package (7). The tabulated library search results indicated which library spectra were the most similar to the sample spectrum, which was most probably benzyl acetate. In one case the closest matching spectrum corresponded to benzyl acetate, while in the other a different compound was indicated as the most probable possibility. The spectra for the eluate peaks exhibited significantly different signal-tonoise ratios (SNR) as shown in Figure 3. The search results were erroneous for the spectrum having the lower SNR because some of the absorbance bands for that compound were hidden beneath the base-line noise. The correlation dot product value computed between the two eluate peaks, however, had a high value of 0.996, which indicated that they were
very likely the same chemical compound. This is almost a perfect correlation. In most cases, the margin of error associated with the correlation technique is considerably smaller than one found in a general purpose search system. This is particularly important when the data are extremely noisy or when a reference spectrum of a compound is not contained in the spectral library. When used for comparing eluates that are separated from the same sample, the correlation algorithm indicates which components are similar in structure. Therefore it is possible to detect groups of eluates that comprise a homologous series. Figure 4 represents a chromatogram obtained during the separation of the higher molecular weight components of a perfume sample. The elevated region of the chromatogram beginning near scan number 4600 was shown to consist of a number of partially resolved ester homologues. A reference vector was generated for the interferogram of scan number 4600. Dot products were computed between the reference and sample vectors taken from later scans. A gradual decrease in dot product values occurred with increasing scan number as illustrated in Figure 5. The one exception is the dot product for scan number 5000. This spectrum is shown in Figure 6b and a sinusoidal base line is evident. This was probably caused by disturbing the interferometer during the scan. It was apparent from the correlation data that the eluates were of approximately the same structure. Some of the corresponding spectral data, shown in Figure 6, indicate gradual changes in the absorbance bands associated with C-H stretches occurring in the vicinity of 3000 cm-l. Further
ANALYTICAL CHEMISTRY, VOL. 60, NO. 5, MARCH 1, 1988 1.000
,
0
0
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Table IV. Interferogram Matching from a Single-Component Peak
displacement from peak
0
a
max 1 2 3
a 0
4 5
displacement from peak
dot product
max
0.982 0.990 0.994 0.976 0.900
6 7
dot product 0.941 0.862 0.803 0.741 0.611
8 9 10
0 900
5000
b5OO
is00
6000
Scan number
Flgure 5. Plat of dot product values computed between interferogram number 4600 (Figure 4) and Interferogramsat 100 interferogram intervals across the homologous series of components.
Table V. Interferogram Matching from a Two-Component Peak
displacement from peak maximum
dot product 0.928 0.776 0.605 0.464 0.351
-75
I
1
62.5 $50 g 37.5
6%
g 4
p
-
12.5
0 100
67.5
75
= 62.5 p a
2 37.5 825 22 -
12.5 0
HAVENLMBERS
Flgure 8. Absorbance spectra computed from various interferograms corresponding to a homologous series of components (Flgure 4): (a) interferogram number 4700; (b) interferogram number 5000;(c) interferogram number 5300.
evidence of these changes can be seen in the methylene symmetric bend near 1450 cm-l. As expected, the library search results obtained for each spectrum were equivalent. By applying the interferogram-basedcorrelation for the examination of chromatographic regions consisting of partially resolved peaks, the degree of similarity between eluates can be established quickly. In those cases where some or all of the eluates are of the same homologous series, the number of spectral computations and additional postprocessing procedures required to obtain qualitative information may be substantially reduced. In certain cases the correlation algorithm may be used to detect the presence of a coeluting component in an eluate peak. This is easily achieved if two coeluting components exhibit significantly different infrared absorbance patterns and their maximum concentrations in the lightpipe do not occur at the exact same time. In such a situation, the interferograms on opposite sides of the peak maximum tend to differ from one another more significantly than do those corresponding to a pure component eluate peak. The dot product values computed between background-corrected interferograms extracted from opposite sides of a pure component eluate peak are given in Table IV. The displacement
WAVENUMBERS Flgure 7. Absorbance spectra computed from various interferograms collected during the coeiutlon of two components. The bottom spectrum corresponds to the leading edge of the peak and the top spectrum corresponds to the tailing edge of the peak.
values indicate the number of scans that separated the leading and trailing edge interferogramsfrom the peak maximum. As larger displacementswere used, the dot product values became smaller because much smaller eluate concentrations were involved. The same operation was performed for an eluate peak that contained two chemical components. The dot product results shown in Table V illustrate how the interferometric data from opposite sides of the peak maximum differ significantly at small displacements. The absorbance spectra associated with various interferograms comprising the peak are shown in Figure 7. The spectral data confirmed the presence of an additional chemical component within the eluate peak. Through the use of the correlation technique, it is possible to detect a coeluting component without the
Anal. Chem. 1900, 60,390-394
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necessity of examining the spectral profile of a peak. Furthermore, the detection of a coeluting component that might otherwise be missed may be achieved automatically.
CONCLUSIONS The utility of this method is that recurring components can be identified, or at least recognized. As more samples of similar components are analyzed, the selectivity of this approach improves. This technique does not replace spectral searching, but it does provide confirming evidence to the identity of a component. If the signal-to-noise ratio of a component is consistently low, a good spectrum of that compound may never be recorded from a single GC/FT-IR analysis. Nonetheless, if successive samples are shown to contain the same unidentified component, the spectra from all the analyses can be coadded to produce a high signal-tonoise ratio spectrum. The identity of the unknown compound can then be determined, either by searching or by interpretation. Clearly, if a component is repeatedly found in a series of analyses, its identity is important. It is interesting to note that all the searches presented in this paper were done by using spectral libraries. The searches could have been accomplished by using interferometric data. Based on other work in this laboratory, the identity of a few
more components may have been found by using such a search, but the value of the correlation method is in no way diminished by a better search. The correlation method serves to confirm the identity of components from analysis to analysis, regardless of the original method by which the components were identified.
LITERATURE CITED (1) Azarraga, L. V.; Potter, C. A. HRC CC, J. Hhh Resolut. Chromatogr. Chromatogr. Commun. 1981, 4 , 60-69. (2) Wehrli, A.; Kovats, E. Helv. Chlm. Acta 1959, 42, 2709-2736. (3) de Haseth, J. A.; Isenhour, T. L. Anal. Chem. 1977, 49, 1977-1981. (4) de Haseth, J. A; Azarraga, L. V. Anal. Chem. 1981, 53, 2292-2296. (5) Lowry, S. R.; Huppler, D. A. Anal. Chem. 1981, 53, 889-893. (6) Forman, M. L.; Steel, W. H.; Vanasse, G. A. J. Opt. SOC.Am. 1986, 56, 59-63. (7) Azarraga, L. V.; Hanna, D. A. "GIFTS Gas Chromatography Fourier Transform Infrared Software Package and User's Guide"; U.S. EPA/ ERL, Athens, GA, 1979.
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RECEIVED for review January 27, 1987. Resubmitted October 12,1987. Accepted October 27,1987. The support of the U.S. Environmental Protection Agency under Cooperative Agreement No. CR-807302 is gratefully acknowledged. This work was presented in part at the 34th Annual Pittsburgh Conference on Analytical Chemistry and Applied Spectroscopy, Atlantic City, NJ, March 1983.
Direct Monitoring of Supercritical Fluids and Supercritical Chromatographic Separations by Proton Nuclear Magnetic Resonance L. A. Allen, T. E. Glass, and H. C. Dorn* Department of Chemistry, Virginia Polytechnic Institute and State University, Blacksburg, Virginia 24061
The direct coupling of 'H nuclear magnetic resonance ('H NMR) as a detector to a flowing supercritical fluid (SF)apparatus is described. The SF/'H NMR system is capable of elevated temperatures (- 100 "C) and pressures (-3500 psi). One application of this apparatus is the measurement of 'H NMR relaxation t h e (T,'s) over a wide range of temperatures and pressures. A second application is the monitorhg d supercrttlcaifluids contalnhg quadrupdar nudel (e.g., "N) where reduced line widths are obtained. Another application described in this paper Is supercritical fiuld chromatography dlrectly coupled to 'H NMR (SFC/'H NMR). The direct Identlkatbn of mnponats present in a model system Is demonstrated and comparison Is made to resuits obtained by using normal phase HPLCINMR conditions.
Supercritical fluids (SF) are becoming increasingly important in a number of different applications because of their unique properties. For example, supercritical fluids have relatively high densities in comparison with normal gases, while solute diffusion coefficients place them intermediate to liquids and gases, as illustrated by the data in Table I (I). The separation of high molecular weight and/or thermally labile species and the coupling of extraction-separation analysis are important applications of supercritical fluid chromatography 0003-2700/88/0380-0390$0 1S O / O
Table I. Typical Values for Fluid Physical Properties property
gas
SCF
diffusivity, cm2/s viscosity, g / ( c m s)
lo-'
2X 2x 0.4-0.9
density, g / m L
liquid
5
X
lo6
1.0
(2). The solvating ability of supercritical fluids is related to the density of the fluid, which is readily varied by changes in temperature and pressure. This increased flexibility can be an advantage of SFC over normal liquid and gas chromatography. To operate in the supercritical region, temperatures and pressures above the critical point (Tc,p,) must be used. A common SFC solvent, COP,has the advantage of relatively low critical point parameters, T, = 31 O C and P, = 1072 psi. 'H nuclear magnetic resonance has been shown to be an effective detector for liquid chromatography (LC/'H NMR) [3-10). Solvent systems such as halocarbons (e.g., Freon 113) and deuteriated solvents have been used in normal-phase chromatography to help alleviate 'H NMR background signals. However, for the case of reverse-phase chromatography, a large residual 'H Nh4R background signal is usually present for the solvents typically used (e.g., D,O, CD,OD, etc.) (8). Multipulse techniques can be used to suppress this residual 'H signal; however, certain spectral regions of interest are normally not @ 1988 American Chemical Society
