Where Z (i, a) is the retention index of the new solute, i, on solvent, a ; C(i,a)are constants calculated for each solvent, a, from the factor analysis reproduction scheme; the proper set of solvents,j, are selected using the method given in the section in solvent choice above, and Z (i,j ) are the retention indices of solute i determined on the eight test solvents, j . In order to test the accuracy of the resulting equations, we purposely left four solutes out of the original data matrix: toluene, nitroethane, methyliodide, and 1-propanol. Then, choosing the best group of eight test solvents: I) D, 11) G, 111) H, IV) M, V) P, VI) Q, VII) S, and VIII) W, where the Roman numerals refer to values of j in Equation 1, we can determine the C ( j , a)’s from factor analysis. The predicted values of the C ( j , a)are shown in Table V. As an illustration of the method, we will calculate the retention index of methyl iodide on column K : a
Z(Me1, K)
C(j, K)Z(MeI,j) = 0.3824Z(MeI, D)
= j=l
0.0881Z(MeI, G) 0.1357Z(MeI, P)
The coefficients are taken from Table V, and the Z(Me1, j ) are experimental values reported by Rohrschneider ( I ) . The experimental value for Z(Me1, K) reported by Rohrschneider is 685. In Table VI, the retention indices for the four solutes calculated on all columns (not including the eight test solvents) are given. For the eight test solvents, C(j, a) = 1 f o r j = a and C(j, a) = 0 f o r j # a. Therefore, for the test solvents, Equation 1 reduces to Z(i, a) = 1 .Z(i, a). This predicative ability represents a quite useful application of factor analysis.
+
- 0.0034Z(MeI, H) + 0.4248Z(MeI, M)
-
+ 0.2010Z(MeI, Q) - 0.0046Z(MeI, S) + 0.0603Z(MeI, W) = 691 index units
ACKNOWLEDGMENT
The authors thank J. F. Parcher of The University of Mississippi for his helpful assistance. We also thank the computer center personnel at Brooklyn College for their help and for the use of their facilities. RECEIVED for review December 13, 1971. Accepted February 22, 1972. This work was supported by a grant from the City University of New York Faculty Research Award Program. One of the authors (PHW) received partial support from Grant Number GP-27999 from the National Science Foundation. This work was presented at the 162nd National Meeting, American Chemical Society, Washington, D.C., September 1971.
Characterization of Gas Chromatographic Effluents via Scanning Fluorescence Spectrometry D. J. Freed’ and Larry R. Faulkner Coolidge ChemicaI Laboratory, Harvard University, Cambridge, Mass. 02138 The direct coupling of a fast-scanning fluorescence spectrometer to a gas chromatograph has permitted fluorometric detection and characterization of effluents in the outflow stream. Typical detection limits are 1 x 10-11 grarn/sec for anthracene, 5 x 10-12 gram/ sec for pyrene, and 2 x lO-llgram/sec for naphthalene. Fluorescence emission and excitation spectra which are sufficiently structured for use in identification have been obtained with as little as 10 ng of sample. A scan rate as high as 150 nm/sec renders these spectra free from distortions due to changing effluent concentrations during peak passage, and repeated scans of the entire 200-800 nm wavelength range can be carried out continuously at 5-sec intervals. Qualitative and quantitative aspects are compared to other detectors and the vast simplification that wavelength discrimination affords to complex chromatograms is illustrated.
THEPAST SEVERAL YEARS have seen the development of a class of chromatographic detection systems designed to cope with increasingly complex mixtures by yielding qualitative characterizations of effluents in addition to the usual quantitative data. Relying ordinarily upon such means as infrared (1-4), Present address, Bell Laboratories, Murray Hill, N. J. 07974. (1) P. A. Wilks and R. A. Brown, ANAL.CHEM.,36,1896 (1964). (2) R. A. Brown, J. M. Kelliher, J. J. Heigl, and C. W. Warren, ibid., 43,353 (1971). (3) R. P. W. Scott, D. A. Fowlis, D. Welti, and T. Wilkens in “Gas Chromatography 1966,” A. B. Littlewood, Ed., Institute of Petroleum, London, 1967, pp 318 ff. (4) S. K. Freeman in “Ancillary Techniques of Gas Chromatography,” L. S. Ettre and W. H. McFadden, Ed., Wiley, New York, N. Y., 1969,pp 227 ff,and references contained therein. 1194
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far-ultraviolet (5), and mass spectrometry ( 6 4 , these detection systems have been coupled directly to the outlet of the gas chromatograph in attempts to generate spectra for emerging components. Although there is no lack of sensitive gaschromatographic detectors, the scarcity of those which can provide qualitative information from submicrogram samples often renders auxiliary methods such as sample collection and subsequent analysis almost a necessity for reliable identification. Indeed, most systems developed to date, except those based on mass spectrometry, require tens of micrograms for reliable spectra, thereby militating against the use of capillary or open tubular columns, with consequent loss to the analyst of their high resolving power. However, even with mass-spectrometric characterization, which is capable of producing spectra for samples in the 10-100 ng range, interfacial problems often limit sensitivity, and the great cost effectively puts the technique out of the reach of many laboratories. In contrast, fluorescence spectrometry, with its inherent high sensitivity, seems well-suited as the basis for a simple, inexpensive spectrometric detection system. Yet, to date only two reports have described its utilization. Bowman and Beroza developed a system in which gas chromatographic (5) W. Kaye, ANAL.CHEM., 34,287 (1962). (6) L. P. Lindeman and J. L. Annis, ibid., 32,1742(1960). (7) W. H. McFadden in “Advances in Chromatography,” J. C. Giddings and R. A. Keller, Ed.,Vol.4, Marcel Dekker, New York, N. Y . ,1967,pp 265 ff, and references contained therein. (8) J. T. Watson in “Ancillary Techniques of Gas Chromatography,’’ L. S.Ettre and W. H. McFadden, Ed., Wiley, New York, N. Y . ,1969, pp 145 ff, and references contained therein.
effluents were extracted into a flowing liquid solvent (9). Their apparatus displayed good sensitivity (ranging down to the nanogram level), but it suffered from complex sample transfer and the need for large quantities of costly, high grade solvents, Burchfield, Wheeler, and Bernos eliminated the liquid stream and detected fluorescence directly from the gas phase (IO), but their system was considerably less sensitive than that of Bowman and Beroza. A major drawback to both previous approaches was the lack of a useful ability to generate spectra of the emerging components. Since both emission and excitation spectra are obtainable from a fluorescing molecule, two qualitative aids are available for the identification of unknown compounds. These two pieces of information, coupled with retention time(or temperature), considerably enhance the ability of the analyst to characterize components of a complex mixture. The only requirement for this type of detection is that the molecule of interest fluoresce in the vapor phase. Admittedly this requirement excludes a large number of compounds; but by virtue of this exclusion, chromatograms are often drastically simplified. This simplification often reduces or eliminates the need for time-consuming sample pretreatment, certainly a desirable goal for any analysis. The present report describes the construction and evaluation of a scanning fluorescence detection system for gas chromatography. Although similar in many respects to the detector constructed by Burchfield et al. (IO),the present system differs importantly in its ability to generate both the emission and excitation spectrum of a fluorescing component. The detector is compared with others and advantages and disadvantages are critically examined. EXPERIMENTAL Apparatus. The basic building blocks of the analysis system consisted of an Aminco-Bowman Spectrophotofluorometer (SPF), modified as described below, and a Bendix Instruments Model 2100 gas chromatograph. The total cost of the interface was under $1000, and its design can be adapted to fit a variety of commercially available instruments. Heated Transfer Line. The line was constructed of Type 304 stainless steel tubing of '/kin. 0.d. and 14-in. length. A 400 "C heating tape was wrapped lengthwise along the line, and the whole was insulated with several layers of asbestos tape alternated with aluminum foil. Triac phase control devices are used to control the heating tape, and an iron-constantan thermocouple is employed to monitor the temperature via the pyrometer mounted on the chromatograph. The upper temperature limit is 400 "C and control is precise to =k5 "C. Swagelok l/S-in. stainless steel fittings were used to connect the line to the chromatograph and the heated cell. No undue peak broadening or mixing problems have been encountered. Heated Flow Cell. The cell proper was obtained from American Instrument Company (Part No. B369-62140) and is constructed entirely of fused silica with an internal volume of 1.5 ml. The cell rests in an aluminum flow-through cell holder (Aminco part No. A367-62140), which has provision for standard Aminco slits. Thus the instrument bandpass can be selected over the range from 1 to 30 nm exactly as in normal operation (11). The cell holder is covered, as shown in Figure 1, by a specially machined aluminum block, which contains channels for two 75-W cartridge heaters (Chromalox, Inc.) and a gas flow tube. The latter is 1/8-in.stainless steel tubing (9) M. C . Bowman and M. Beroza, ANAL. CHEM., 40,535 (1968). (10) H. P. Burchfield, R. J. Wheeler, and J. B. Bernos, ibid., 43,
1976 (1971). (11) American Instrument Co., Silver Spring, Md., Instruction
Booklet No.904-A (1968).
\
Retaining Spring
Figure 1. Schematic diagram of the heated fluorescence cell, as viewed from the emission monochromator. For clarity, the facing slit and the thermal insulation have not been shown
which has been silver soldered to a steel adapter, which, in turn, has been force fitted into the block. The flow tube connects to the transfer line by a stainless steel union, and its other end extends through a Teflon (Du Pont) collar into the cell tubulation about 5 mm. A stainless steel retaining spring forces the cell against the collar to ensure against leaks. This whole assembly was painted on the outside with Pittsburgh flat black high temperature paint, then it was insulated with four layers of asbestos tape alternated with aluminium foil. An outer coat of asbestos retort cement (Grant Wilson Co.) serves as final insulation. This cell assembly is mounted on an accurately machined Transite base designed to replace the regular base plate for the cell holder. Temperature control and readout is exactly as described above. In use, temperatures as high as 450 "C could be achieved with only slight (2030 "C) heating of the nearest parts of the SPF. Spectrophotofluorometer. The standard Aminco-Bowman SPF with the ellipsoidal condensing system was used, but two important modifications were effected. The usual wavelength scanning motors were replaced with Bodine 10 RPM motors, thereby enabling the entire spectrum (200-800 nm) to be scanned repeatedly at 5-sec intervals. The maximum scan rate is 150 nmjsec. To accommodate this increased rate, the microphotometer (Aminco No. 10-213 with 2-sec time constant) output filter was altered by the addition of a rotary switch which enables the substitution of several alternate capacitances to give time constants ranging from 0.05 to 2 sec. A 200-watt high pressure mercury-xenon lamp was used for excitation spectra. The widths of the resolution-defining slits used in various applications can be calculated from the bandpass values reported below by means of the instrument's 6 nm/ mm dispersion relation (11). The output from the Hammamatsu IP21 photomultiplier was recorded via the microphotometer either on a Tektronix Type 564 storage oscilloscope (for spectra) or on a Sargent Model MR recorder equipped with a Disc Integrator (for chromatograms). Gas Chromatography. A Bendix Instruments Model 2100 gas chromatograph with thermal conductivity detection and linear temperature programming was employed. Six-foot, 1j8-in.0.d. stainless steel columns were packed with 5 OV-1 on 60j80 Chromosorb W, DCMS. Conditions for analysis are given in the Results section. ANALYTICAL CHEMISTRY, VOL. 44, NO. 7,
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Table I. Detection Limits for Fluorescence Chromatography.
Detection limitb Peak width at Excitation Emission Compound half-height, sec wavelength, nm wavelength, nm Weight basis, ng Flow basis, ng/sec Naphthalene 10 26 5 3 50 0.2 0.02 350 10 0.5 Phenanthrene 20 26 5 Anthracene 20 313 400 0.2 0.01 100 313 400 0.5 0.005 Pyrene Flow rate, 60 ml/min; column temperature, programmed from 180-240 "Cat 4 "C/min. Other temperatures: injection port and thermal conductivity detector oven, 275 f 1 "C; transfer line, 340 & 5 "C; fluorescence cell, 300 f 5°C. Excitation bandpass, 18 nm (3-mm slits); emission bandpass, 12 nm (2-mm slits). Signal/Noise = 211. Q
I I
300
I
I I
400
500
WAVELENGTH (nm)
Figure 2. Fluorescence excitation (left) and emission (right) spectra for ( a ) anthracene, ( b ) pyrene, and ( c ) phenanthrene Solid lines show gas phase spectra obtained with 100-ng injections of each species at the system conditions given in the caption to Figure 5. Dashed lines show comparison spectra for cyclohexane solutions at room temperature Reagents and Chemicals. The solvents used in this work were Eastman spectrograde materials, Aromatic hydrocarbons were obtained as Gold Label reagents from Aldrich Chemical Co., and Matheson Gas products supplied the high purity (99.995%)helium used as a carrier for chromatography. The coal tar extract examined as an illustration was Wright's Liquer Carbonis Detergens.
RESULTS AND DISCUSSION Although luminescing species in the liquid and solid phases exhibit both fluorescence and phosphorescence, gas phase 1196
ANALYTICAL CHEMISTRY, VOL. 44, NO. 7, JUNE 1972
emission at high temperature is almost always restricted to fluorescence from the first excited singlet state and, furthermore, to molecules with some degree of aromatic character. Because analytical interest in aromatic hydrocarbons is high and because the proposed method seemed well-suited to their detection, virtually all our investigations have been carried out with such substances. In addition, a series of high-temperature, gas-phase studies (12-16) have suggested that the fluorescence quantum yields for aromatic hydrocarbons are not greatly different from their solution phase values (17). Since one can detect lo-'* to gram/ml of many substances in the solution phase (13,it was therefore not unreasonable to expect similar sensitivity for detection in the chromatographic outflow. This early expectation has been encouraged by the ultimate performance of the instrument. In studying the detector response as a function of injected sample size, we employed an internal standard technique to minimize effects of random changes in the lamp output with time. Thus the quantity plotted in calibration curves was the ratio of the integrated peak area from the fluorescence chromatograms of the sample and standard. In practice, 1methylnaphthalene and 9-methylanthracene were used, respectively, as internal standards for naphthalene and phenanthrene and for anthracene and pyrene. These plots were similar in character and precision to those presented by Burchfield et al. (IO), and they were linear from subnanogram or nanogram levels up to about 10 pg. Typical detection limits (signal/noise = 2 : l ) are displayed in Table I together with the wavelength employed for excitation and observation. These limits are comparable to those reported by Bowman and Beroza (9) for their liquid flow detector, but they show considerable improvement over the limits given by Burchfield et al. for their gas phase system. As with any chromatographic detector, the operating parameters which determine the system sensitivity may be conveniently divided into those characteristic of the detected molecules (hence different for each species) and those pertaining to the apparatus itself (hence common to all substances). Given a fluorescent species, the two most important molecular parameters bearing on the sensitivity are the fluorescence efficiency and the molar absorptivity at the wavelength of excitation, The instrument variables governing the sensitivity are the light-gathering-power of the emission monochromator, the sensitivity of the photomultiplier (and its varia(12) W. R. Ware and P. T. Cunningham, J. Chem. Phys., 43, 3826
(1965). (13) G. L. Powell, ibid., 47,95(1967). (14) B. Stevens and P. J. McCartin, Mol. Phys., 8,597 (1964). (15) G. B. Kistiakowsky and C . S. Parmenter, J . Chem. Phys., 42,
. 2942 (1965). (16) R. B. Cundall and L. S. Davies, Trans. Faraday Soc., 62, 1151 (1966). (17) C . A. Parker, "Photoluminescence of Solutions," Elsevier, Amsterdam, 1968,and references contained therein.
tion with wavelength), the level of blank emission from quartz windows and other sources, the level of scattered and stray light, the intensity and wavelength dependence of the excitation beam, and the noise levels of the lamp and the photomultiplier readout system. Most of these factors are governed by the instrument design and construction, so the main concern in planning a given determination is usually to maximize the product of the excitation beam intensity and the molar absorptivity of the compound to be determined. All other factors being equal, this maximum will occur at the excitation wavelength of a greatest sensitivity. Most aromatic substances feature extremely high absorptivities (typically lo4 to 105 l./mole-cm) in the 250-265 nm region; therefore excitation there with a source having appreciable output in the region can often offer greatly increased sensitivity over the more commonly used 366 nm mercury line. It is for this reason that we employed a mercury-xenon lamp for excitation whenever sensitivity was a prime concern. Short wavelength excitation offers, additionally, very low scattered light levels because the excitation wavelengths are not passed by the glass envelope of our phototube. The use of the mercury-xenon lamp in conjunction with the high light-gathering power of the ellipsoidal condensing system probably accounts for the improved sensitivity of the present system over that devised by Burchfield et al. However, their comment that they could not use the most sensitive scales of the microphotometer may suggest a high blank as a limiting factor. In contrast, we have had no difficulty working on the lowest level of the most sensitive scale. The difference may involve the level of scattered light, or it may concern the level of emission from the silica cell itself. Perhaps it is worth noting that we have observed reversible inducement of fairly strong emission capability in samples of optical quality silica and quartz upon heating to the operating temperature. We have been able to avoid this problem only by selecting the silica pieces for low emission at high temperature, but fortunately one can essentially eliminate the effect altogether by so doing. It is an interesting observation that the liquid phase detector of Bowman and Beroza ought to have an inherent sensitivity about ten times greater than the present gas phase system, because their extraction process concentrates the sample from a large gas volume to a small solution volume. The fact that our detection limits are comparable to theirs may be explained by a more efficient excitation, but their theoretical advantage is probably also compromised somewhat by incomplete extraction at the gas-liquid interface (9). In connection with this comparison, it probably is worth noting that the gas-phase system will never be limited by the solvent blank, which is an important consideration in many liquid-phase determinations. Since the spectral characteristics of each compound are unique, it is not generally possible to state unambiguously the lower limits of detection which might be expected. However, it probably is not unreasonable to anticipate an ultimate lower limit of about gram/sec by the use of improved excitation sources and signal-to-noise enhancement techniques, If sensitivity were the most important factor, it could probably be increased considerably by exciting the sample with a closely-placed low-pressure mercury lamp, which has almost all its output at 254 nm. Although sensitivity is indeed an important consideration, the real value of fluorescence detection lies perhaps in its ability to furnish qualitative records at fairly low levels of concentration. Figure 2 shows several representative spectra,
I
I
I
I
I
I
A
0
2
6
8
10
12
TIME, min
Figure 3. Chromatograms of a coal tar extract (1 p1 injections. 511 mg extract/l ml cyclohexane): (a) thermal conductivity detection; (6) fluorescence at 400 nm excited by 313 nm light; (c) fluorescence at 350 nm excited by 265 nm light Operating conditions: flow rate, 50 ml/min; column temperature, 180-220 "C programmed at 3 "Clmin; detector, 310 "C; injection port, 275 "C; transfer line, 340 " C ; fluorescence cell, 300 "C. Components identified with spectroscopic characterization: A, naphthalene; B, 1-methylnaphthalene; C, phenanthrene; D , carbazole; E, anthracene; F, pyrene. Excitation bandpass, 30 nm; emission bandpass, 24 nm
which were obtained with the present system on 100 ng of each compound emerging in the effluent from the chromatograph. As one expects from temperature considerations, the gas phase spectra show somewhat poorer resolution than those obtained from the liquid phase, but there is still much qualitative information in the inflection points, locations of peak maxima, and the band widths at half-height. The gas phase excitation spectra, showing relatively greater structural features, are probably the more valuable of the two as an aid for identification, especially since two bands are often present. Neither set of spectra shows distortions due to changing effluent concentrations during peak passage. The chromatographic peak width at half height was usually about 20 sec, the component concentration in the cell could change but slightly during the 1-sec period required to scan the pertinent wavelength region. All spectra shown in Figure 2 are uncorrected for the nonuniformity with wavelength of the excitation source and the photomultiplier sensitivity; however for comparison with a reference library obtained with the same instrument, they proved sufficient for identification. Although the spectra of Figure 3 were recorded with slits adjusted for 6 nm band pass, it was found that with wider slit openings (24 nm), one could obtain characteristic emission spectra from as little as 10 ng of pyrene or anthracene. Because each compound exhibits unique spectral features, the relative response of the system to two compounds may ANALYTICAL CHEMISTRY, VOL. 44, NO. 7, JUNE 1972
1197
I
I
I
I
A
I
I
I
I
0
2
4
6
TIME, min
Figure 4. Chromatograms for a known hydrocarbon mixture in cyclohexane: solid line, fluorescence at 350 nm excited with 265 nm light; dashed line, fluorescence at 410 nm excited with 250 nm light Each injection was 1 pl and contained about 1 pg of each compound. Compounds: A , l-methylnaphthalene; B, anthracene/phenanthrene; C, 9-methylanthracene; D,Ruoranthene; E , pyrene. Conditions: Row rate, 60 ml/min; column, 180240 "C programmed at 6 "Cjmin; detector oven, 275 "C; injection port, 275 "C; transfer line, 340 "C; fluorescence cell, 300 "C. Excitation bandpass, 30 nm; emission bandpass, 24 nm often be varied at will from zero to unity simply by proper selection of excitation and emission wavelengths. In this respect, the present system is similar to those described previously, but it differs in that the operator need have no prior knowledge of the sample composition. In practice, the 200800 nm spectral range is continuously scanned at a 5-sec repetition rate, and the spectra are displayed on a storage oscilloscope until the chromatogram is complete. If subsequent quantitation is desired, it is a simple matter to set the instrument to the proper wavelengths indicated for the peak of interest and to measure the peak area on a fluorescence chromatogram for a newly injected sample. However, there are two practical facts worth noting in this connection. First, it is rare that an analyst has no inkling of the sample makeup; hence proper wavelength selection is often predetermined. In addition, even though compounds may have different peak emission wavelengths, a factor of two or three in sensitivity is rarely the limiting factor for analysis. Thus, two or three monitoring wavelengths may suffice for most compounds. The power of wavelength discrimination is a convenient contrast and complement to the greater universality of such detectors as thermal conductivity, flame ionization, and electron capture, because it can enormously simplify a complex
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ANALYTICAL CHEMISTRY, VOL. 44, NO. 7, JUNE 1972
chromatogram, This property is displayed in Figure 3, wherein the uppermost trace is a thermal conductivity recording of a sample of coal tar extract and the lower two traces are 400-nm and 350-nm fluorescence chromatograms of the sample. Comparing the latter to each other and to Figures 3a, one can readily see the dramatic simplification that fluorescence detection can afford. Many of the peaks in the fluorescence chromatogram are spectrometrically resolved even though they are not individually discernible in the thermal conductivity recording. In addition, the figure points out several components that could be identified on the basis of their fluorescence spectra and retention parameters. Figure 4, which displays 350-nm and 410-nm fluorescence chromatograms of an artificial mixture of hydrocarbons representative of those contained in the coal tar extract, perhaps illustrates the utility of wavelength discrimination in greater detail. At 350 nm, the only compounds exhibiting appreciable response are 1-methylnaphthalene and phenanthrene. Only at 410 nm are anthracene, 9-methylanthracene, fluoranthene, and pyrene readily evident. It should be mentioned that, even though anthracene and phenanthrene elute simultaneously, they may be spectrometrically resolved and quantitated if their relative concentrations are not overwhelmingly different, It is also noteworthy that the coal tar sample was simply diluted with cyclohexane, rather than carried through the timeconsuming and laborious procedures used by earlier workers. Obviously most of the aromatic hydrocarbons occurring in the sample could have been determined quantitatively if the need had arisen. Because this technique is limited by its need for fluorescence in the detected effluent, a fluorescence detection system would probably be used ordinarily as an auxiliary unit in conjunction with a more universal detector such as the hydrogen flame. Many of the other drawbacks, such as the need for manual wavelength selection and multiple injections, are peculiar to the present design, and they could presumably be automated if necessary. Indeed, one of the method's more intriguing aspects is its computer adaptability. One can easily envision direct memory storage and conversion of spectra, with complete control of all operations by a computer. Additional valuable features are the ability of the detector to operate with any carrier gas and the absence of base-line instability due to temperature or flow fluctuations. The combined considerations of cost and effectiveness should render this technique useful for characterization and determination in certain gas phase applications, but perhaps most intriguing is the promise that the concept holds for liquid chromatography, for which sensitive characterizing tools are all but nonexistent. RECEIVED for review January 10, 1972. Accepted March 1, 1972. The financial support of this project by a departmental grant from the E. I. du Pont de Nemours Co., and by grants from the William F. Milton Fund of Harvard University is gratefully acknowledged. In addition, we wish to thank the National Science Foundation and the National Institutes of Health for fellowship support to D. J. F.
