Anal. Chem. 1986, 58, 3249-3254
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Capillary Gas Chromatography/Fourier Transform Infrared Microspectrometry at Subambient Temperature Sir: Increasing the differentiating power of chromatographic and spectroscopic techniques by interfacing them together has been a long-recognized goal of analytical chemists (1).However, the marriage of gas chromatography (GC) and Fourier transform infrared (FT-IR) spectrometry has not always been synergistic because capillary GC has often required lower detection limits than FT-IR has been able to provide ( 2 , 3 ) . The development of the light-pipe interface has been largely responsible for the success of gas chromatography/Fourier transform infrared spectrometry. The key component of this interface is usually a light-pipe gas cell through which the effluent from a capillary column flows continuously during the IR measurement. Typical sample quantities required to yield identifiable spectra using a light pipe with an internal diameter of 1 mm are between 5 and 25 ng, although a light-pipe interface based on the "counter-Jacquinot advantage" holds promise in reducing these identification limits by a factor of 2 or 3 (4). The need for a further reduction of the injected amount required to yield an identifiable spectrum by a t least an order of magnitude has resulted in the development of alternative approaches to the light-pipe interface. Possibly the best approach to high-sensitivity GC/FT-IR has involved trapping each eluate in a matrix of argon a t about 12 K ( 5 , 6 ) . A device which is based on this principle, known as the Cryolect, was introduced commercially in 1984 (7,8). With this interface, identifiable spectra of compounds with average absorptivities can be obtained in the 200-400-pg range. Such low limits of identification have been achieved primarily because of the very small area over which the sample is deposited, the effective thickness of a given amount of a GC eluate being inversely proportional to the area the sample occupies. Let us compare the band intensities of a given amount of material measured by matrix isolation and using a light pipe. With the Cryolect, each eluate can be deposited in a spot the diameter of which is as small as 0.25 mm (8). Neglecting the effect of the low-temperature matrix on the widths and peak absorbances of spectral bands, it can be readily seen that the intensity of the bands due to a given quantity of a GC eluate trapped in an argon matrix as a 0.25-mm-diameter spot should be 16 times greater than that of bands due to the same amount of the eluate held in a 1-mm-i.d. light pipe. For samples of this size, the 1-mm-diameter image a t the sample focus of a conventional 6 X beam condenser is too large and the use of an infrared microscope is indicated (9). In an earlier note (IO), we showed that it is possible to trap a GC eluate of low volatility on a stationary infrared window held a t ambient temperature. When this window was transferred to an infrared microscope, spectra of deposited materials were measured with 1-ng detection limits. In this paper, we report the feasibility of constructing an interface for real-time GC/FT-IR measurements based on this principle. Eluates are trapped on an infrared-transparent plate which is either held at ambient temperature or is thermoelectrically cooled to temperatures as low as -45 "C. This window is located in the focal plane of an FT-IR microscope so that each eluate passes through the beam shortly after it is deposited. Although the sensitivity of this device for GC/FT-IR is not quite as high as that of the Cryolect, detection limits appear to be approximately 1 order of magnitude lower than those of corresponding spectra measured with a light-pipe interface. The fact that this device operates near ambient conditions
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means that its cost should be considerably less than that of matrix-isolation GC/FT-IR interfaces.
EXPERIMENTAL SECTION Separations were performed on a Hewlett-Packard Model 5880 gas chromatograph equipped with a 0.25-mm4.d. X 30-m fused silica column coated with a 0.25-pm film of DB-5 (5% phenyl methyl siloxane). Nitrogen carrier gas was passed through the column at a flow rate of 1 mL/min. One-microliter quantities of sample were delivered to the column by direct injection. The effluent from the column was split, with 60% flowing to the interface and 40% passing to a flame ionization detector (FID). The splitter was constructed from a Valco '/'&. union in which the column was connected to one end and the transfer lines for the FID and interface were connected to the other end using a ferrule drilled with two holes. A 12-cm length of 50-pm4.d. fused silica served as transfer line to the FID. The transfer line between the chromatograph and interface consisted of a 1.5-m length of 100-pm4.d.fused silica (with no internal coating) which was passed through externally heated 1/16-in.stainless-steel tubing. A 4-cm length of fused silica (50-pm-i.d.) was joined at the end of the transfer line with a silicone rubber adhesive (Gasket Seal, Dow Corning). By analogy to supercritical fluid chromatography, this will be termed a restrictor, although it imposes only a very small reduction in flow rate. The end of the restrictor was located about 50 wm from the surface of a ZnSe window (39 X 19 X 2 mm) which was translated at a speed of 4.5 mm/min unless specified otherwise. The 50-pm4.d. tubing was held at the same temperature as the transfer line. The FID was connected to the end of the transfer line of the interface to determine the split ratio. The split ratio did not change significantly with changes in temperature of the GC oven or transfer line. The effluent from the gas chromatograph is sprayed onto a ZnSe window which is then moved to position the trapped sample into the beam of an FT-IR microscope. The interface is similar to one which we reported previously for supercritical fluid chromatography/Fourier transform infrared spectrometry (SFC/FT-IR) (I1,12),except that for GC/FT-IR the window is held below ambient temperature. Infrared spectra were measured at 4-cm-' resolution on an Analect fX-6200 FT-IR spectrometer equipped with an Analect AQM-515 microscope module. Analect's GC/FT-IR software package was used to collect spectra to disk every 1.25 s and to reconstruct chromatogramsfrom the integrated absorbance in specified spectral regions. A micrometer was attached to the outside of the microscope to position the restrictor. A purge box was constructed to encase the restrictor, the microscope, and one end of the stage so that all the optics and interface were purged with dry air. After the deposition was completed,the window was repositioned and the IR measurements performed. The window assembly was moved by a rotating screw (1.1threads/mm) fastened to a variable dc motor. The temperature of the window was controlled by two Peltier coolers cemented to a brass plate in which the window was set with a silicone rubber adhesive (Gasket Seal, Dow Corning). The cold side of the Peltier cooler was placed in contact with the brass holder of the window. Heat was transferred from the face of the Peltier coolers to a brass block, which was cooled by circulating methanol. In turn, the methanol was cooled to 0 "C or -25 "C by an ice-water bath or a dry ice/methanol bath, respectively. The temperature of the window could be reduced to 25 "C below the temperature of the circulating fluid by changing the voltage applied to the Peltier coolers. The variable aperture of the microscope optics defined the area of the IR beam which was focused onto the detector element. The element of the narrow-band mercury cadmium telluride (MCT) detector was 250 wm square. With an adequate purge of dry air, measurements could be performed at a window temperature of -45 "C without ice forming on the window. When the temperature of the window was checked by attaching a thermocouple directly
0003-2700/86/0358-3249$01.50/0 0 1986 American Chemical Society
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ANALYTICAL CHEMISTRY, VOL. 58, NO. 14, DECEMBER 1986
to the surface of the window, it was found to be the same as that of the brass plate surrounding the window. Consequently, a thermocouple was glued onto the brass plate to monitor the temperature of the window. All chemicals were used as received from Aldrich Chemical Company. Solutions were prepared in dichloromethane (Mallinckrodt, Inc.). The standard solution of Neutral Extractables V was obtained from the Quality Assurance Division, Environmental Monitoring Systems Laboratory, Las Vegas, NV. This sample contained 1,4-dichlorobenzene, 1,2-dichlorobenzene, N-nitrosodi-rz-propylamine, nitrobenzene, isophorone, acenaphthene, dibenzofuran, fluorene, hexachlorobenzene, anthracene, pyrene, bis(2-ethylhexyl) phthalate, and benzo[k]fluoranthene in dichloromethane at a concentration of 2 mg/mL per component.
RESULTS AND DISCUSSION The efficiency of trapping an eluate on the surface of the ZnSe window was examined with respect to changes in window temperature, distance between restrictor and window surface, and the volatility of eluates. Nitrobenzene and acenaphthenequinone were chosen to represent relatively volatile and nonvolatile compounds, respectively. For all molecules tested, sample absorbance increased as the distance between the end of the 50-pm-i.d. restrictor and the window decreased. In agreement with matrix isolation GC/FT-IR data (8), the greatest band intensities are observed when the distance between the window and the restrictor is less than or equal to the internal diameter of the restrictor. The fact that the interface was not evacuated did not significantly increase the diameter of the spot. With a 50-ym-diameter restrictor, a reduction of about 10% in absorbance was noted when the microscope aperture was increased to 100 pm. Increasing the aperture to 200 pm resulted in a reduction of the absorbance of each band by more than a factor of 2. For depositions of acenaphthenequinone, which is a particularly nonvolatile analyte, band absorbances were the same whether the window was held a t 22 "C or -10 "C. For more volatile compounds this was not found to be the case (vide infra). A typical concentration profile of acenaphthenequinone deposted on a KBr window held 100 pm from the restrictor was reported previously ( I O ) . Similar profiles were measured for the same compound deposited on a cooled ZnSe window held at several distances from a 5O-pm restrictor. With 67 ng of acenaphthenequinone delivered to the window, over 90% of the integrated absorbance occurs within f l O O pm of the center of spot when the separation of the window and restrictor is 50 pm. At a separation of 100 gm, the spot has enlarged so that 90% is deposited within f200 pm of the center. At a separation of 350 pm, the diameter of the spot is approximately 1 mm, and band absorbances are reduced by an order of magnitude in comparison to measurements made with a 50-pm separation. If the window temperature was decreased while the nominal temperature of the restrictor was held constant at 260 "C, the absorbance of acenaphthenequinone spectrum was observed to decrease. Similarly when the temperature of the restrictor was increased, band intensities were found to increase. Thus, partial condensation of the eluate onto the end of the restrictor was apparently being caused by convective cooling of the restrictor because of the close proximity of the cold window. Insulation of the end of the restrictor, which is presently directly exposed to the air surrounding the cold window, should significantly decrease the sample loss. It may be noted that convective cooling of the restrictor in matrix-isolation GC/FT-IR is minimized by the vacuum which surrounds the restrictor and the cooled mirror surface on which the eluates are deposited, although some radiative cooling may be expected. A plot of the absorbance of acenaphthenequinone at 1720
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Figure 1. Effect of window temperature on quantity of nitrobenzene deposited on the surface of the window monitored using the 1524-cm-' band. The quantity delivered to the interface was 150 ng. Window temperatures were (A) -30 "C, (B) -20 "C, and (C) -10 "C.
cm-' vs. sample quantity split to the interface is nonlinear for quantities greater than 30 ng. This nonlinearity results mainly from the area of the depositions increasing slightly with increasing sample quantities. Since these measurements were performed with a 50-pm-i.d. aperture, the percentage of the sample being interrogated decreases as the total amount injected is increased. Another potential cause of the nonlinearity is that the hot mobile phase emerging from the restrictor could evaporate larger quantities of sample off the window as the sample quantity increases due to the low thermal conductivity of the window and/or sample, permitting a local increase in temperature at the point of deposition. A higher temperature would therefore result from a decrease in the rate of heat transfer caused by the thicker sample. In view of the low thickness of the deposit even when microgram quantities are injected, we do not consider the thermal conductivity of the sample to be a major contributing factor. If the sample was held stationary in the infrared beam for about 1 min, detection limits for molecules of low volatility and high band absorptivities were approximately 100 pg. The amount of sample required to give a spectrum suitable for identification was about 500 pg (see for example Figure 2 of ref 10). Achievement of detection limits in the subnanogram regime for more volatile eluates proved to be difficult because of the effect of evaporation. The effect of window temperature on sample loss of nitrobenzene is shown in Figure 1. For a quantity of 150 ng deposited onto the window, the temperature of the window had to be lowered to -30 "C before sample loss was appreciably retarded. Even at this temperature 4 ng of nitrobenzene evaporated away in 4 min. Further cooling of the window to -45 "C resulted in the same quantity of the compound being present for an hour before sample loss was apparent. The temperature of the transfer line for this measurement was 200 "C. When this temperature was increased, convective (and possibly radiative) heating increased the rate of evaporation of nitrobenzene. In order to compare the effect of an eluate's volatility on quantitation, a calibration plot was made for nitrobenzene deposited -30 "C and -45 "C, see Figure 2. As the quantity of sample injected is increased, a marked difference is observed between the calibration plots for nitrobenzene depositions performed at different temperatures. Nitrobenzene appeared completely crystalline at -45 "C, whereas at -30 "C the deposit was still in the liquid state. The spectrum of nitrobenzene measured at -45 "C has a sloping base line, indicative of light scattering by the crystalline sample; see Figure 3A. In comparison, the spectrum measured at -30 "C has a straight
ANALYTICAL CHEMISTRY, VOL. 58, NO. 14, DECEMBER 1986
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baseline as a result of the sample being in the liquid state, see Figure 3B. The sample in the liquid state has a greater effective path length than in the crystalline state. This results from the more uniform coverage of the window surface by the liquid than the crystals. The effect is greatest at higher injected quantities as seen by the noticeable difference in the calibration curves obtained for the -30 O C (liquid state) and -45 "C (crystalline state) shown in Figure 2. An identifiable spectrum of nitrobenzene, measured by coadding 64 scans, was obtained for a quantity of 2.4 ng, as shown in Figure 4A. From a comparison of this spectrum to the reference spectrum shown in Figure 4B, it is evident that several of the less intense bands of nitrobenzene can be observed. The effect of scattering on spectra of molecules which are crystalline at ambient temperatures (such as acenaphthenequinone) is similar to the spectrum of nitrobenzene in the crystalline state. The base-line slope increases as the quantity of each component on the window surface increases. Less than 30 ng of sample was required to minimize band distortions and sloping base lines.
Figure 4. (A) Spectrum of nitrobenzene for 2.4 ng delivered to interface and (B) reference spectrum of 75 ng of the same compound. The column temperature was held at 50 OC for 3 min, then increased at a rate of 25 OC/mln to 120 OC. The transfer line was maintained at 180 OC.
The separation of p-nitrophenol, 2,4-dinitrotoluene, and 4,6-dinitro-o-cresol using a rather fast temperature program demonstrated that the chromatographic resolution of the separation was maintained at the interface. The retention times of p-nitrophenol and 2,4-dinitrotoluene were within 5 s of each other, as shown in Figure 5. An injection of 200 ng/component resulted in 120 ng/component measured at the interface. The resolutions of the FID chromatogram (Figure 5A) and the reconstructed chromatogram (Figure 5B) are almost identical. The reconstructed gas chromatogram (RGC) is a plot of the integrated absorbance between 1200 and 1400 cm-* of each spectrum taken over the chromatographic separation. Single-scan spectra measured at each peak maximum are shown in Figure 6. These spectra were measured less than 1min after sample deposition and indicate the signal-to-noise ratio (SNR) that is possible for deposition and data collection in "real time". Each peak was identified by comparison of the sample spectrum with a library of reference spectra. The minimum detection limit (MDL) of the RGC is approximately 40 ng (SNR equal to 2 for peak-to-peak (p/p) noise), whereas for the FID chromatogram the MDL is at least 2 orders of magnitude lower. The SNR of the RGC is limited by fluctuations in the base line of the spectrum. We believe that Gram-Schmidt vector orthogonalization should yield significantly lower detection limits, since appropriate vectors can be selected to omit the lowest spatial frequencies. This software was not written for the early Analect data system installed on our spectrometer, but has been written for most contemporary FT-IR spectrometers, including the Analect instruments. The optimum speed at which to move the window during deposition was determined by comparing reconstructed chromatograms obtained at several different speeds. The speed of 4.5 mm/min which we found to be optimum is (not surprisingly) similar to the 3 mm/min speed used for the Cryolect (6).By collection of each eluate while the window
3252
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was stationary, a 4-fold decrease in identification limits was observed. When the window was translated only after each peak was eluted, the area in which each peak is deposited could be significantly decreased. (This approach was demonstrated for the analysis of the Neutral Extractables V sample described at the end of this paper.) To determine the optimum aperture size for the microscope, the absorbance of acenaphthenequinone a t 1720 cm-' was measured at several different aperture settings. The signal decreases after the aperture exceeds a value that is approximately equal to the full width at half-height of the deposited spot. The peak-to-peak noise at the base line reached a limiting value when the aperture was large enough that the SNR of the interferogram became limited by digitization noise. The maximum absorbance-to-noise ratio for injections of 17, 67, and 133 ng of acenaphthenequinone was found for an aperture diameter of approximately 110 pm in each case. The aperture was set to this value for all subsequent analyses. To illustrate the practicality of this interface, the identification of each peak observed during the chromatographic separation of a standard solution of Neutral Extractables V was attempted. This sample was chosen because the components are not only environmentally important but also represent a variety of compound types and polarities. The oven was held a t an initial temperature of 60 "C for 5 min, and the temperature was then raised at a rate of 10 OC/min to a final temperature of 280 "C. When the transfer line was
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