58
Anal. Chem. 1986, 58,58-64
Supercritical Fluid Chromatography/Fourier Transform Infrared Spectrometry with an Automatic Diffuse Reflectance Interface Kenneth H. Shafer, Stephen L. Pentoney, Jr., and Peter R. Griffiths* Department of Chemistry, University of California, Riverside, California 92521
An automated interface Is demonstrated for supercritical fluid chromatography/Fourier transform infrared spectrometry (SFCIFT-IR) using a microbore column in which the mobile phase Is rapidly eliminated and the diffuse reflectance (DR) spectrum of the solute is measured. Because the mobile phase in SFC can be a gas at atrnospherlc pressure, sample deposltlon is better accomplished in SFC/FT-IR than in hlgh-performance liquid chromatography/Fourier transform infrared spectrometry (HPLCIFT-IR). The result is an SFCIFT-IR Interface that not only is mechanlcaily more slmpie than the HPLCIFT-IR interface but also has the potential of lower limits of spectral ldentlflcation (acenaphthenequlnone at an injected quantity of 50 ng). I n addition, the continuous deposition accompilshed In SFCIFT-I R, not yet possible with a DR interface for HPLC/FT-IR, has afforded the capability of reconstructing chromatograms from infrared spectra.
Every hyphenated technique of chromatography/spectrometry shares an important commonality in that identification is accomplished with unique spectral information rather than retention-time data. The identification of a sample using a hyphenated technique is achieved by spectral interpretation or computerized spectral searching. The purpose of the spectrometry is then identification, even though detection is also possible. A number of absorption bands (at least five) is generally necessary for identification by infrared spectrometry. A complex mixture can be analyzed in detail with little, if any, prior chemical information about the sample using a hyphenated technique ( I ) , whereas identification by chromatography alone depends on a knowledge of the exact chemical structure of the components to be analyzed in the sample and the availability of authentic reference standards. A minimum detection limit (MDL) in chromatography is an indication of the quantity needed for detection of an eluting component. Identification is accomplished solely by measurement of retention time and comparison with standards. Because hyphenated techniques are different from purely chromatographic techniques in the way identification is made, an MDL provides little indication of the identification capability of a hyphenated technique; instead minimum identification limit (MIL) should be defined. A MIL is the quantity of compound required for identification by spectral interpretation or computer search. Indeed, spectral identification by computer may well be found to serve as an objective means of determining the MIL. The development of supercritical fluid chromatography/Fourier transform infrared spectrometry (SFC/FT-IR) has been performed with the intent of improving the capability to identify nonvolatile organic compounds in complex mixtures. To demonstrate the success of SFC/FT-IR in this regard, spectroscopic identifications are presented and MIL’S rather than MDL’s are discussed in this paper. The development of SFC/FT-IR has closely paralleled that of high-performance liquid chromatography/Fourier transform infrared spectrometry (HPLC/FT-IR). In both cases the first
demonstration of the technique was made by using an interface that consisted of a transmission flow cell (2-5), while the solvent elimination techniques of “buffer memory” transmission (6-9) and diffuse reflectance (DR) infrared spectrometry (10-12) were described somewhat later. The impetus to the development of the solvent elimination techniques has been the desire to improve the identification capability of the hyphenated techniques by eliminating the interfering solvent absorption bands present in the sample spectra and increasing the concentration of the analyte in the infrared beam. For SFC/FT-IR with transmission flow cells, supercritical COz has been most widely used as the mobile phase because the strongest absorption bands of COz occur in spectral regions where few organic compounds absorb (4, 5). However, spectral interference in the important fingerprint region is still observed due to the Fermi resonance bands of COZ, which not only absorb (at 1284 and 1384 cm-l) but also change intensity with changes in pressure (4,5). Furthermore the low polarity of COz limits separations to relatively nonpolar compounds. To increase the solvent strength of the mobile phase, small amounts of a polar compound (modifier) must be added so that intermediate-polarity compounds can be separated. When a flow cell is used for SFC/FT-IR, the addition of small percentages of methanol to COz can result in major spectral interferences due to the absorption of the modifier. In contrast to flow cell interfaces, the development of the solvent elimination approaches of “buffer memory” transmission ( 9 ) and DR (12) have resulted in the capability to obtain spectra that are free of solvent interferences. Recently, we reported preliminary studies of the first solvent elimination interface for SFC/FT-IR using diffuse reflectance infrared spectrometry (12). In this work, a synthetic mixture of three quinones was separated on a microbore column packed with 5-pm silica using a mobile phase of 5% methanol in COz at supercritical conditions. Each peak was collected at different positions on a plate on which a layer of KCl powder had been deposited. The plate was kept stationary as each peak was collected. From the signal-to-noiseratio (SNR) of the infrared spectra measured for an injected quantity of 350 ng/component, it was estimated that the detection limits were approximately 10 ng. With the “buffer memory” technique demonstrated by Jinno et al. (9),an analysis of OV-17 (a series of methylsiloxane oligomers) has been demonstrated in which a 5-pg sample (total) injected was separated on a 0.35 mm i.d. X 60 cm fused silica column packed with 10-pm ODS particles using supercritical n-hexane with an ethanol modifier as the mobile phase. By use of pressure programming, a series of oligomers was nicely separated, with spectra shown for less than 1 pg/oligomer. Sample deposition while the solvent is being eliminated must be accomplished with a minimum of spreading or chemical change, while leaving no trace of the solvent. Because of the effect of capillarity in HPLC/DR-FT-IR, the eluates have been collected in discrete cups. The smallest cup diameter reported to date (13) has been 2 mm, resulting in a relatively low concentration of the deposited solute. Because
0003-2700/86/0358-0058$01.50/00 1985 American Chemical Society
ANALYTICAL CHEMISTRY, VOL. 58, NO. 1, JANUARY 1986
the mobile phase in SFC can be gaseous at STP, sample deposition is better accomplished in SFC/FT-IR than in HPLC/FT-IR. For SFC/FT-IR, the sample can be continuously deposited in a smaller area than in the corresponding HPLC/FT-IR interface. The result is an interface for SFC/FT-IR that is mechanically more simple than its counterpart in HPLC/FT-IR and one that also has the potential of lower identification limits and allowing chromatograms to be reconstructed from the series of spectra measured from a given KC1 strip, since diffuse reflectance is one of the most sensitive microsampling techniques in IR spectrometry (14). The combination of high sensitivity and freedom'from solvent interferences makes DR an excellent sampling technique for the SFC/FT-IR analysis of low levels of nonvolatile compounds in complex mixtures. In this paper we will describe an SFC/DR-FT-IR interface in which the eluates from the chromatograph are deposited on a continuously moving strip. Identification of analytes injected into the chromatograph at levels as low as 50 ng was possible, and the first demonstration of IR reconstructed chromatograms for either an HPLC/FT-IR or SFC/FT-IR interface by diffuse reflectance is reported.
)-(I-[ )-( INFRARED TRANSMISSION
HEATED
PREHEATER
EXPERIMENTAL SECTION Infrared measurements were performed on an Analect fX-6200 FT-IR spectrometer. The optical module consists of a Transept interferometer with double-beam capability and a narrow-band MCT detector (1mm2detector element). The control and display terminal is comprised of an ASCII keyboard, independent abscissa and ordinate expansion and cursor controls, and a display oscilloscope. A multiprocessor data system and firmware program have been constructed as part of the fX-6200 FT-IR spectrometer. Data acquisition was effected by using Analect's GC/FT-IR software. Each single-beam spectrum of the sample was ratioed against a background single-beam spectrum of 64 coadded scans. Interferograms were apodized by using a Happ-Genzel function. Every 1.3 s a 4-cm-' reflectance spectrum was stored to a floppy disk. As soon as one floppy disk in the dual drive was filled, storage was switched to the second disk. The fiist disk containing the collected spectra can then be replaced with an empty disk without interrupting the run. IR reconstructed chromatograms were computed from the integrated absorbance in specifed regions of the spectra. In turn, these IR reconstructed chromatograms were used to determine which spectra to coadd over a peak in order to obtain spectra with optimum signal-to-noise ratios. Spectral identification was performed manually because of the lack of library search software for the fX-6200 for condensed-phase spectra. Spectral library searches were performed by using Analect's software written for the CP/M operating system on their Model AQS-20 FT-IR spectrometer. Results were inconclusive because of the relatively small number of quinones in the condensed-phase spectral library. Separations were performed with a Brownlee Labs MPLC Micropump equipped with a lecture bottle of liquid COz (solvent reservoir A) and methanol (solvent reservoir B). Injections were made with a Rheodyne 7413 injector with a 0.5-pL sample loop in place. Detection at 254 nm was accomplished by a modified Tracor Model 960 ultraviolet detector (UVD), vide infra. The mobile phase was passed through a length of stainless-steel tubing to bring its temperature to that of the column. The mixer, preheater, injector, column, and detector were all held at 50 OC. The SFC/DR-FT-IR interface was applied to the analysis of a synthetic mixture of 2-methyl-174-naphthoquinone,anthrone, acenaphthenequinone, and phenanthrenequinone (Aldrich Chemical Co., Milwaukee, WI). All solutions were prepared in dichloromethane and injected in 0.5-pL volumes. Separations were performed on a 1mm i.d. X 25 cm microbore column packed with a 5-pm silica. These columns were packed in our laboratory by preparing a slurry mixture of 0.25 g of the stationary phase in 4 mL of 12% CH30H in CCll and then pumping the packing material at 7000 psi into the column with a driving solvent of methanol using a Haskel (Burbank, CA) Model DSTV-122 pneumatic amplifier pump. All separations were performed with 5% methanol in COPheated to 50 "C and pressurized to 3000 psi.
59
, Flgure 1.
INFRARED DIFFUSE REFLECTANCE ACCESSORY
Schematic diagram of fluid-elimination SFC/FT-IR instru-
mentation. -
FLOW PATH I N
/
T i i l l /
FIBER OPTIC
\
FERRULE
f L O W PATH OUT
Diagram of the fiber optic UVD cell used with the fiuidellmination SFCIFT-IR interface. Figure 2.
Although the Brownlee Labs MPLC Micropump had the capability to program changes in pressure or composition, all separations described in this paper were performed at constant composition, temperature, and pressure. The flow rate of the mobile phase was approximately 200 pL/min as determined from the value displayed by the pump. The configuration of the instrumentation is shown as a block diagram in Figure 1. The composition of the mobile phase was monitored from its infrared spectrum using a transmission flow cell (Harrick Scientific, Ossining, NY), which was constructed to withstand high pressures. This cell was equipped with ZnSe windows and had a path length of 150 pm. The cell was placed in the front beam and the DR interface in the back beam of the IR spectrometer. All components of the SFC/DR-FT-IR interface were constructed in a configuration that minimized the dead volumes imposed by transfer lines and also made temperature control efficient. A fiber optic UVD cell was constructed from a Valco zero dead volume union as shown in Figure 2. The cell (0.8 mm i.d. X 3 mm path length) had a volume of approximately 1.5 pL. The flow paths were drilled on an angle to favor laminar flow and thereby reduce the adverse effect that mixing has on chromatographic resolution. The cell was constructed by silver soldering a 0.8 mm i.d. X 3 mm length of 1/16-in.stainless steel inside the union. The dog-leg corners of the flow path were made by cutting the union in half and drilling a 0.3 mm diameter hole at a 45O angle in each piece of the union. These pieces were then pressed together, and
80
ANALYTICAL CHEMISTRY, VOL. 58, NO. 1, JANUARY 1986 Injector
5
b
de
Reference, Photodiode
TOGSTRICTOR Flgure 3. Diagram of optical configuration of the UVD constructed for the fiber optic cell.
a '/la in. diameter hole was drilled on either side of the union until contact was made with the 0.3-mm holes. Stainless-steeltransfer lines (l/16 in. 0.d.) of 0.1 mm i.d. were then inserted into each hole, and the entire unit was silver soldered together. Because the UVD cell was located inside a heated aluminum block, fiber optics were employed to transmit the ultraviolet light into and out of the block. The fiber optics were directly sealed to the cell by the pressure fittings of the union. Vespel-graphite ferrules were seated against the stainless-steelcover of the fiber optics. The fiber optics were silver soldered into the stainless steel jackets. The optical configuration of the UVD was changed to fit around the aluminum block as shown in Figure 3. The restrictor assembly consisted of a low dead volume union, one end of which was attached to the minimum dead volume stainless steel tubing (0.1 mm i.d.) coming from the UVD cell, and the other to a 1.5-2.0 mm length of 10 pm i.d. fused silica. Although the union could be heated, results presented in this paper were obtained with the restrictor at ambient temperature. The restrictor was placed approximately 1 mm away from the surface of the KC1 strip on which each solute was deposited. This strip was prepared by depositing a slurry of finely ground KC1 mixed with methanol into a slot (4mm wide X 2 mm deep X 25 crn long) formed on the upper,surface of a glass strip and allowed to dry. The excess KCl was then scraped away, resulting in a very smooth KCl surface. The surface of the KCI strip prepared in this manner was less susceptible than the surface of dry packed KCl to being disturbed during the deposition of the sample. The IR beam was focused onto the strip of KC1, and the diffusely reflected light was collected with an Analect DR optical accessory. During deposition the strip was moved under the restrictor by a stepping motor and IR measurements were made in real time as the deposition was being effected. The distance between the point at which deposition occurred and the focus of the IR beam was 1 cm.
RESULTS AND DISCUSSION For this investigation, separations were performed on microbore (1.mm i.d.) packed columns instead of conventional bore (4.6mm id.) columns because of their lower volume flow rates. Through the use of the Brownlee Laboratories Micropump, binary mixtures of COzand methanol could be used as the mobile phase with a flow rate as low as 10 pL/min. Even at a flow rate through the microbore column of 200 pL/min, the surface of the KCl powder was not disturbed during deposition. As a result, spectral base lines were relatively straight, so weak absorption bands could be observed in ordinate-expanded spectra. A synthetic mixture of 2-methyl-1,4-naphthoquinone, anthrone, acenaphthenequinone, and phenanthrenequinone was prepared at a concentration of 4 pg/pL per component in dichloromethane. The volume of the injection loop was 0.5 pL. The SFC/FT-IR analysis of this mixture resulted in the separation and identification of five components (the four standards and anthraquinone, a contaminant of phenanthrenequinone). Based on calculated peak areas of the anthraquinone and phenanthrenequinone peaks in the IR reconstructed chromatogram shown in Figure 4,anthraquinone
WINDOW REGION (crn-1)
I400 - I200
2
4
6
8
TltlE WIlNS)
Figure 4. Chromatograms of the SFC/FT-IR separation of the synthetic quinone mixture with detection by (A) UVD at 254 nm and (B) IR spectrometry. Peaks are identified as (1) 2-methyi-1,4naphthaquinone, (2) anthraquinone, (3)anthrone, (4) acenaphthenequinone, and (5) phenanthrenequinone.
was estimated to be present a t approximately 33% of the injected quantity of 2 pg/component, or about 700 ng. In turn, the quantity injected of phenanthrenequinone was estimated to be approximately 1.3 pg. IR reconstructed chromatograms were obtained by software that displays the integrated absorbance of IR bands in specific spectral regions as a function of time. A comparison of the UV and IR chromatograms shows the effect of the DR interface on chromatographic resolution, see Figure 4. A small decrease in the chromatographic resolution is evident when the separation of peaks 1 and 2 is compared. The shape of peak 1 in the UV chromatogram is due to the solvent (dichloromethane) peak partially coeluting with the first component. This effect is not seen in the IR chromatogram because the solvent has evaporated away during deposition of peak 1. A high signal-to-noise ratio (SNR) is observed for the IR reconstructed chromatogram at the 2-pg level of sample injected. Spectra shown in Figure 5 were acquired from a single scan of approximately 0.25-9 duration at each IR peak maximum. Each eluate was easily identifiable after comparison to reference spectra. These spectra were not scale expanded, but were plotted to full-scale. The high SNR of these spectra are indicative of the sensitivity of the DR interface. Unlike the case for a flow cell interface, no spectral interferences result from the mobile phase (even the methanol modifier). Thus gradient elution could be used to effect chromatographic separation without spectral intereference. It should be stressed that the capability of identifying the structure of an unknown is very dependent on the entire IR spectrum from 4000 to 700 cm-l being free of spectral interferences. Of particular interest are the absorption bands near 1690 cm-l in the spectra of 2-methyl-l,4-naphthoquinone, anthraquinone, anthrone, and phenanthrenequinone; see spectra A,
ANALYTICAL CHEMISTRY, VOL. 58, NO. 1, JANUARY 1986 WINDOW REGION
(ern-')
I755 - 1655
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Figure 5. Infrared spectra of each peak of chromatogram A in Figure The identificationof spectra A-E is given in the caption of Figure
4.
4.
B, C, and E, respectively, of Figure 5. This absorption band is characteristic of quinones or aromatic ketones and results from a carbonyl stretching mode that usually absorbs below 1700 cm-* because of the delocalization of the ?r electrons of the carbonyl group into the aromatic ring. However, the carbonyl stretch of acenaphthenequinone occurs at about 1730 cm-I because of strain of the five-member ring containing the carbonyl groups. These differences in the spectra of the quinones can also be observed in the IR reconstructed chromatograms shown in Figure 6. Since all five compounds have carbonyl absorptions, an IR chromatogram reconstructed from the 1755-1655-~m-~ region of the spectra results in five peaks being observed as shown in chromatogram A. The 16901655-cm-l spectral region is characteristic of most quinones, so four major peaks and one minor peak are seen in chromatogram B. Because the fourth and minor peak is due to acenaphthenequinone selective monitoring of that compound can be accomplished by changing to a spectral region between 1755 and 1690 cm-l, indicated by the single peak in chromatogram C. Because all of the compounds are aromatic, the 900-750-~m-~region, which is characteristic of aromatic compounds (the out-of-plane bending mode of the C-H aromatic groups), can be monitored resulting in chromatogram D. The use of IR chromatograms to selectively monitor a separation according to compound functionality has been previously demonstrated for HPLC/FT-IR (3, 8, 15) and GC/FT-IR (16, 17). The first report of an IR reconstructed chromatogram using the solvent elimination technique known as "buffer memory" was reported by Jinno for HPLC/FT-IR (6). Very recently Jinno has applied similar techniques to a buffer memory interface for SFC/FT-IR but with a somewhat lower sensitivity than the DR results reported above (9). A GramSchmidt real-time chromatogram has also been shown for
D
I
2
'
4
6
c 0
to
Tint ( N l N S )
Figure 6. Infrared reconstructed chromatograms (integrated absorbance of selected spectral regions vs. time) of SFC/FT-IR separation of synthetic quinone mixture. SFC/FT-IR using a flow cell ( 5 ) . The base line of the Gram-Schmidt chromatogram had a significant slope, which was a result of the increasing intensity of the COz Fermi resonance bands as a consequence of the pressure program. No drift in base line is observed with the solvent elimination approaches because the IR measurement is made after elimination of the mobile phase. A 50 ng injected quantity of acenaphthenequinone has been identified from the seven bands appearing in spectrum C of Figure 7. Spectra A and B were obtained for 1 pg and 300 ng injected quantities, respectively, and are easily identified by comparison to reference spectra. Spectral identification at this low level of injected sample has not been reported in work performed with flow cells even though MDL's of this magnitude have been claimed, vide supra. An MDL for 2,6-
62
ANALYTICAL CHEMISTRY, VOL. 58, NO. 1, JANUARY 1986
1
WINDOW REGION lcrn-'l
Y
e
o'O1
t
0
2
4
6
B
T l r C Iil N"'
Infrared reconstructed chromatograms obtained (A) immediately alter separation of synthetic quinone mixture and (B) 15 h later. Figure 8.
il
C
i
J
WAVENUMBER (cm-')
Infrared spectra of acenaphthenequlnone for injected quantities of (A) 1 kg, (B) 300 ng, and (C) 50 ng. Spectra A and B were measured with a single scan while C necessitated 64 coadded scans of the sample interferogram. Figure 7.
di-tert-butyl phenol eluting from a capillary SFC column was calculated by Olesik et al. (5)to be 38 ng injected for a SNR of 5, monitoring the 1426 cm-l band (CH3 bend). Whether peak-to-peak (p-p) or root mean square (rms) noise was used for these calculations was not stated. In addition, a flow cell constructed with a lightpipe (1mm i.d. X 5 mm) has recently been reported by Taylor et al. for separations performed on a 4.6 mm i.d. HPLC column (18). A MDL of 155 ng (SNR of 3) was reported using this interface. However, this value was calculated as the amount of injected sample required to give a signal-to-rms-noise ratio of 3 for the most intense band in the spectrum. Although an exact comparison of the spectral identification capability of existing SFC/FT-IR interfaces with the system described in this paper is difficult to make due to the fact that MDL's rather than MIL'S have been reported for flow cell interfaces, an approximation can be made in comparing sensitivities. An MDL of 11ng (SNR of 5 for peak-to-peak noise measured at 2000 cm-') can be extrapolated from the strongest band (1730 cm-') of spectrum C in Figure 7. This might appear to be an order of magnitude lower than the MDL reported by Taylor et al. and comparable to the value reported by Olesik et al. However, values reported in this paper are calculated by using the rather conservative criterion of 5 X NPp,as opposed to 3 x N,,, used by Taylor et al. Had we
used the latter criterion, reported detection limits would have been 8 times lower; however, the band would not have been observable. It should also be noted that both the MDL's and MIL'S in this work were calculated from spectra of peaks deposited sometime before the infrared spectrum was measured, since signal averaging of spectra measured from a given region of the KC1 strip is usually performed after the chromatogram has been completed and sample loss caused by evaporation has sometimes been observed for certain compounds. Unless the analytes are very volatile, the effect is usually small. To demonstrate the effect of evaporation, the strip used to obtain the reconstructed chromatograms shown in Figures 4 and 6 was left standing for 15 h, and then remeasured. The resulting chromatogram is shown in Figure 8. The complete loss of peak 1 (2-methyl-1,4-naphthoquinone) and partial loss of peak 3 (anthrone) were observed, while no loss of peaks 2,4, and 5 could be detected. Although loss of peak 4 (acenaphthenequinone) is not evident when the sample was injected a t the 2-kg level, a nonlinearity is noted when band absorbance or Kubelka-Munk function is plotted against injected sample amounts. The complete cause of this effect is the subject of a more complete study currently under way in our laboratory, but it could be related to sample loss by evaporation either during deposition or in the short time taken to transport the sample to the infrared beam. With the DR interface, every separated component is deposited onto the surface of a strip of KC1 powder. The strip must be moved continuously at a rate that is sufficiently high to ensure that chromatographic resolution is maintained during deposition. A strip speed that is too high results in an unnecessary decrease in the concentration of each deposited eluate. Based upon the data shown in Figure 9, a value of 0.25 mm/s was chosen as being the best rate of movement of the strip. Chromatogram A shows a loss in chromatographic resolution because of the slow rate of movement of the strip. However, chromatograms B and C, obtained a t faster strip velocities, show only a very small loss in chromatographic resolution upon comparison to the UV chromatogram of Figure 4. Because the entire chromatographic separation in effect resides on the surface of the strip of KC1 powder, the SNR of a spectrum can be increased by coaddition, either by stopping the strip during IR measurement, or (preferably) by returning the peak to the IR beam or moving the strip at a slower rate of travel after the separation is complete. The resulting increase in SNR, which is a consequence of the signal averaging without moving the strip, is shown in Figure 10 for spectra of acenaphthenenquinone at a level of 50 ng injected. Spectrum A was obtained from a single scan, whereas spec-
ANALYTICAL CHEMISTRY, VOL. 58, NO. 1, JANUARY 1986
4
K 6
E
6
E
10
12
63
14
TIME (MINS) Flgure 11. An infrared reconstructed chromatogram of three consecutive 2-pg injections of acenaphthenequinone. 10
TiME (MINS) Figure 9. Infrared reconstructed chromatograms of a twMomponent separation measured with strip speeds of (A) 0.125 mm/s, (B) 0.25 mm/s, and (C) 0.417 mmls.
WAY EW UMBER Figure 12. Infrared absorbance spectrum of 15% methanol in carbon dioxide measured at 3000 psi and ambient temperature for a c d i path length of 150 pm. The reference spectrum was pure COP measured under the same conditions.
I
B
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500 A O W
.~...o
'
A 0
'
A 9
,k
WAVENUMRER (cm-')
Figure 10. Infrared spectra of acenaphthenequinone for an injected quantity of 50 ng: (A) a single scan and (B) a 64-scan coaddition of the sample.
trum B was acquired from a 64-scan coaddition. The improvement in the capability to identify is well worth the slightly longer analysis time. All that is required is for one band to be observed above the noise level of a single-scan spectrum and the position of that peak is known. Thus at the end of the run, the strip can be retraced to that position and the spectrum measured with an appropriate amount of signal averaging. Several injections of acenaphthenequinone were made to study the reproducibility of band intensity after elimination of the mobile phase and sample deposition. The resulting chromatogram, shown in Figure 11, indicates that good reproducibility is achieved for injections of 2 Kg of a relatively nonvolatile component.
Because of the low polarity of COz, it has been suggested that small amounts of polar compounds can be added to vary the solvent strength without substantially compromising SFC/FT-IR measurements made with flow cells (5). However, this is not generally found to be the case. For example, the addition of 15% methanol, a commonly used modifier, to Cop at 3000 psi and 23 "C results in appreciable absorption bands in the spectrum, as shown in Figure 12. If the optical path length of 150 pm used to obtain this spectrum were increased to 1mm (the path length used by Olesik et al.), the absorbance of the methanol bands would increase by a factor of 6.7. The resulting solvent interference would make most of the important regions of the IR spectrum opaque and therefore useless for spectroscopic identification. It may be noted that the path lengths employed by Taylor et al. (18) and Shafer and Griffiths (4) were even longer (5 and 10 mm, respectively), making the use of a methanol modifier, even at a level of 1-2%, unfeasible for SFC/FT-IR measurements using flow cells. On the other hand, we have found flow cells to be very useful for monitoring the composition of the mobile phase, especially during gradient elution when the percentage of a less compressible modifier in C02 is being increased. A short IR flow cell was mounted between the pump and injector, as shown in Figure 1. A plot of the intensity of the methanol absorption band at 1060 cm-l with respect to the percentage of methanol in COz delivered by the pump is linear, indicating the pump is working to specification despite the fact that one component is much more compressible than the other. The acceptance of SFC/FT-IR as a viable analytical technique depends on the development of the separation capability of SFC. More polar supercritical fluids that are gaseous at STP need to be studied as mobile phases for SFC. Pumping
Anal, Chem.
64
systems are also necessary that will provide the chromatographer with all of the capabilities of SFC (e.g., automated pressure and gradient programming). Finally, an analogous SFC/FT-IR interface to the one described in this paper for packed microbore columns should be built for capillary SFC in view of the improved resolution that can be obtained by using wall-coated open tubular columns. A system based on this principle is currently being constructed in our Iaboratory and results will be reported at a later date.
ACKNOWLEDGMENT We wish to thank Analect Instruments and Brownlee Laboratories for the loan of the spectrometer and pump, respectively, used in this work. Registry No. 2-Methyl-1,4-naphthaquinone, 58-27-5;anthraquinone, 84-65-1; anthrone, 90-44-8; acenaphthenequinone, 82-86-0; phenanthrenequinone, 84-11-7.
LITERATURE CITED Hirschfeld, T. L. Anal. Chem. 1980, 52, 197A. Klzer, K. L.; Mantz, A. W.; Bonar, L. C. Am. Lab. (Fairfieid, Conn.) 1975, 7, 85. Vldrine, D. W.; Mattson, D. R. Appl. Spectrosc. 1978, 32, 502. Shafer, K. H.; Griffiths, P. R. Anal. Chem. 1983, 55, 1939. Oleslk, S. V.; French, S . B.; Novotny, M. Chromatographia 1984, 18, 489.
64-68 Jinno, K.; Fujimoto, C. HRC CC, J . High Resoiut. Chromatogr. Chromatogr. Commun. 1981, 4,532. Jinno, K.; Fujimoto, C.; Hirata, Y. Appl. Spectrosc. 1982, 36, 67. Jinno, K.; Fujlmoto, C.; Ishii, D. J . Chromatogr. 1982, 239,625. Fujimoto, C.; Hirata, Y.; Jenno, K. J . Chromatogr. 1985, 332, 47. Kuehl, D.; Griffiths, P. R. J . Chromatogr. Sci. 1979, 17, 471. Conroy, C. M.; Grifflths, P. R.; Duff, P. J.; Azarraga, L. V. Anal. Chem. 1984, 56, 2636. Shafer, K. H.; Pentoney, S . L.; Griffiths, P. R. HRC CC, J . High Reso/ut. Chromatogr . Chromatogr . Commun . 1984, 7, 707. Conroy, C. M.; Griffiths, P. R.; Jinno, K. Anal. Chem. 1985, 57, 822. Griffiths, P. R.; Fuller, M. P. "Advances in Infrared and Raman Spectroscopy"; Clark, R. B., Hester, R. E., Eds.; Heyden: London, 1979;p 63. Vldrlne, D. W. J . Chromatogr. Sci. 1979, 17, 477. Shafer, K. H.; Hayes, T. L.; Brasch, J. W.; Jakobsen, R. J. Anal. Chem. 1984, 56, 237. Smlth, S.L.; Garlock, S . E.; Adams, G. E. Appl. Spectrosc. 1983, 37,
192. Johnson, C. C.; Jordan, J. W.; Skeiton, R. J.; Taylor, L. T. Pittsburgh Conference on Analytical Chemistry and Applied Spectroscopy, New Orleans, LA, 1985;paper 538.
RECEIVED for review May 16,1985. Accepted August 20,1985. Partial support of this work by the Universitywide Energy Research Group of the University of California and by the U.S. Environmental Protection Agency under Cooperative Agreement CR812258-01 is gratefully acknowledged. S.L.P. wishes to thank the Shell Foundation for a Shell Analytical Fellowship while this work was being performed.
Fourier Transform Infrared Ellipsometry of Thin Polymer Films Robert T. Graf, Jack L. Koenig, and Hatsuo Ishida* Department of Macromolecular Science, Case Western Reserve University, Cleveland, Ohio 44106
Reflectlon spectra contalnlng phase as well as lntenslty Informatlon were collected on a FT-IR spectrometer, uslng two linear polarlrers. The sample conslsted of a poly(vlny1 acetate) (PVAc) fllm on a copper substrate. By the use of well-known principles of elllpsometry, the differential phase retardation (A) and differential amplltude ($) were calculated from these measurements. Given an Independent measurement such as the frequency of the peak maxlma of the carbonyl band of the PVAc, the thlckness and optical constants of the fllm were calculated from the elllpsometrlc measurements. A surface profller determined the fllm thlckness to be 40 nm, whlle I R ellipsometry produced a value of 55 nm. The PVAc optical constant spectra determined by I R elllpsometry compared favorably with reference spectra.
Infrared spectrometry has been increasingly used for surface studies in recent years. Improvements in IR detectors, the use of modulation techniques in the IR, and the inherent, high signal-to-noise ratio of FT-IR have all contributed to this trend. Furthermore, the wide variety of sampling techniques that exist for infrared spectrometry (e.g., diffuse reflectance, photoacoustic, reflection-absorption, etc.) has permitted surface studies on very difficult systems under ambient conditions. Traditional surface techniques such as ESCA, Auger electron spectroscopy, and SIMS, although extremely sensitive, require a high vacuum. The experimental flexibility of IR spectrometry and its huge literature base make it a competitive surface technique despite the sensitivity advantage of the other
surface spectrometries. The current sensitivity of IR surface spectrometry is such that for strongly absorbing molecules on metallic substrates monolayer studies can be considered routine (1). Ellipsometry has long been used in the UV-vis region of the spectrum for probing the thickness and optical properties of surface layers and films. Sensitivities on the order of angstroms are routinely achieved with visible light laser ellipsometry (2). For the rapid determination of the thickness of thin oxide and other layers on metals and dielectrics, UVvis ellipsometry is the technique of choice. Because of the great sensitivities achieved with UV-vis ellipsometry, much less work has been done on applying ellipsometry to other spectral regions. Nonetheless, some researchers have done ellipsometric measurements in the IR region. Dignam et al. have described an IR spectrometric ellipsometer with background noise approaching 2 X AU (3). Their use of IR ellipsometry allowed them to obtain the infrared dispersion properties of surface species as well as the absorption properties. For surface species that exhibit optical anisotropy, Dignam (4) has shown how to extract the IR absorption and dispersion properties normal and perpendicular to the surface. The wealth of information that an infrared spectrum contains makes such surface orientation measurements very desirable. Allen and Sunderland ( 5 ) used the 10.6-hm line from a COP laser to study the oxide thickness on aluminum substrates in the range of 20-200 nm. They chose infrared ellipsometry because of the limitations of visible light ellipsometry for layer thicknesses approaching 200 nm and because IR ellipsometry
0003-2700/86/0358-0064$01.50/00 1985 American Chemical Society
