Anal. Chem. 1982, 5 4 , 549-552
549
Fourier Transform Infrared Photoacoustic Spectroscopy in Thin-Layer Chromatography Lindsay B. Lloyld, Randall C. Yeates, and Edward M. Eyrlng"
Department of Chemt/stty,Un/vers/@of Utah, Salt Lake C/@, Utah 84 7 12
Two procedures flor obtalnlng Fourler transform Infrared photoacoustlc spectra (PAS) of samples In situ on thin-layer chromatography i(TLC) plates are described. One Involves a microphonic detection system. The other requires a plezoelectrlc transducer for the detection. Relative merits and limltations of each are dlscussed. Wlth both methods PA spectra of tetraphenyicyclopentadlenonewere produced on three types of commercial TLC plates. An extension of the photoacoustlc dettrction system to quantitative analysls of TLC adsorbed samples Is also considered.
A significant advantage of thin-layer chromatography (TLC) over column chromatography is the simplicity and speed with which many components of a mixture may be separated simultaneously ( I ) . However, if qualitative information concerning these separated components is desired using infrared spectrometry, the advantages of TLC are lost by the time required to removie the sample from the TLC support so that transmission spectra may be obtained. A component may also be altered or destroyed by removal from the TLC plate. Photoacoustic spectroscopy (PAS) has been shown to be a versatile technique for obtaining spectral information from highly scattering, opaque, or very weakly absorbing condensed phase samples ( 2 ) . It is thus a particularly suitable method for obtaining spectra of samples separated on TLC plates. Qualitative and quantitative information has been obtained previously on TLC adsorbed samples by dispersive PAS at visible and ultraviolet wavelengths (3-7). These experiments all utilized microphonic P14S cells to obtain the spectra. The recent development of Fourier transform infrared photoacoustic spectroscopy (FT-IR/PAS) (8, 9) suggested the extension of the TLC-PAS technique to the more useful infrared wavelength region. Described below are two FT-IR/PAS methods for obtaining infrared spectra in situ of a compound adsorbed on a TLC plate. The first method is a simple extension to the infrared of the TLC microphonic PAS techniques already demonstrated a t visible and ultraviolet wavelengths (3-7). The second technique, which is novel for FT-IR/PAS, uses a piezoelectric transducer (PZT) as a PAS detector in a manner previously demonstrated a t visible wavelengths on a variety of samples (10-12). A FT-IR/PAS method for quantntative analysis of TLC adsorbed samples is also described. EXPERIMIZNTAL SECTION Samples were prepared by spotting solutions of sample and solvent onto the TLC plate using a micropipet. The TLC plates used in these experiments were E. M. Reagents aluminum backed precoated TLC sheets with silica gel F-254 in a 0.25-mm layer, E. M. Reagents TLC sheets with alumina (neutral Type E) in a 0.20 mm thick layer, and plastic (poly(ethy1eneterephthalate)) backed Eastman Chromatogram sheets with 13181 silica gel support. In the microphonic detection method 1cm diameter disks were excised from the TLC plates and placed in the sample compartment of a P A microphonic cell. The cell was sealed in a glovebag after purging for 15 min with pure He. The cell itself
was constructed of polished stainless steel with a NaCl windovv and had a total gas volume of -0.5 cm3. The cell was isolated from ambient vibrations by placing it on Lord Kinematics isolation mounts. The infrared output of a Nicolet 7199 Fourier transform infrared spectrometer (FT-IR) was focused through the NaCl window in the cell onto the desired portion of the TLC plate. Acoustic waves arising from the periodic heat transfer from the sample to the He gas were detected by a Bruel & Kjoer 4165 microphone whose diaphragm was exposed through a 1 mm diameter, 10 mm long passage to the sample compartment of the cell. The signal from the microphone was then passed to a locally designed interface where the signal level was adjusted appropriately and presented to the normal signal processing electronics of the Nicolet FT-IR. In rapid scan interferometry the modulation frequency f, in Hz, of a given light frequency, 2, in cm-l, is determined by the velocity in cm/s, u, of the moving mirror of the interferometer by the equation f = 2eu (11 In this case f is analogous to the chopping frequency in dispersive PAS. Generally, in PAS the smaller f is, the larger the photoacoustic signal. Thus the mirror velocity of the Nicolet FT-IR was set at 0.122 cm/s which corresponded to a low range of chopping frequencies for the mid-infrared. For the 400-4000 cm-l incident radiation the range was 97.6-976 Hz which also corresponded to the narrowest band-pass setting of the Nicolet FT-IR high andl low pass electronic filters encompassing these frequencies. This frequency range was also sufficiently free of synchronous noise that PA spectra could be obtained. No gain in signal to noises was achieved, even with the PZT detectors, by using higher mirror velocities. The PA interferograms were then averaged as needed and transformed by the Nicolet FT-IR standard software to produce the FT-IR/PAS spectra. For PZT detection an Edo Western Corp. barium titanate piezoelectric transducer disk was attached with an epoxy cement to the back of the TLC plate or other sample. These disks were -10 mm in diameter by -0.25 mm thick. The PZT-epoxy sample sandwich was then mounted in the spectrometer and the output of the interferometer was directed onto the desired portion of the sample. Care was taken to isolate the PZT as much as possible from ambient vibrations and acoustic noise by using thin lead wires, isolation mounts, and foam rubber insulation. The PZT tended to pick up electromagnetic noise from the interferometer's linear drive motor and therefore was located in the light path within the spectrometer as far as possible from the motor. The signal from the PZT was presented to a PARC 184 current sensitive preamp connected to the signal channel of a PARC 124A lock-in amplifier operating in a high pass mode above 90 Hz with a Q setting of 1. The signal was taken from the signal monitor jack on the lock-in and fed into the Nicolet FT-IR interface. The PA interferogramswere then averaged and transformed as before.
RESULTS AND DISCUSSION FT-IR/PA spectra were obtained in the microphonic cell of the three types of TLC plates both with and without 300 Mg of the sample of tetraphenylcyclopentdienone (I) spotted on the plates (Figures 1-3). This sample was chosen because of availability, its convenient infrared absorption spectrum, and its dark purple color which made it easy to locate visually on the TLC plate. A KBr pellet transmission spectrum of I is shown in Figure 4. The signal to noise ratio of these spectra was such that most spectral features could be seen after only 0 1982 Amerlcan Chemical Society
ANALYTICAL CHEMISTRY, VOL. 54, NO. 3, MARCH 1982
550
/ 4000
I
3200
2400
,
,
iaoo
,
,4co
,
,
IC00
,
,
600
WPVENUMBERS
Flgure 4. A transmission spectrum of the ketone sample in a KBr Figure 1. The FT-IR/PA spectra obtained by using mlcrophonlc detection of a portion of an aluminum backed TLC plate with silica support and 300 hg of adsorbed ketone (tetraphenylcyclopentadienone)sample (-), the same type of TLC plate wlth no sample (---), and the scaled difference spectrum (- - -).
I
“OCO
1
I
3200
,
, 2403
I
,
iaoo
,
, 40C
I
, I300
,
, 600
,
VIAVEVUMBERS
Figure 2. The same as Figure 1 except uslng poly(ethy1ene terephthalate) backed TLC plate wlth silica support.
400i
3200
2403
1eo0
1400
l0OC
600
W EULNSERS
Flgure 3. The same as Figure 1 except using aluminum backed TLC plate wlth alumina support.
50 scans had been averaged and transformed in approximately 3 min. However, it required the averaging of about lo00 scans, depending on the sample, to obtain PA spectra of equivalent quality to conventional 8 cm-l resolution infrared spectra. To obtain these PA spectra it was necessary to purge the cell with He for 15 min to remove all of the TLC solvent and most of the ambient COz and water vapor. Any infrared absorbing gas in the sample cell produces photoacoustic signals at least 100 times greater (depending on the amount of gas) than the
pellet produced on the Nicolet FT-IR using the TGS detector, and ratioed to the background response of the TGS detector.
PA signals arising from the condensed phase and thus can completely bury the desired spectrum. The interferometer compartment was also purged with N2 to remove C 0 2 and water vapor from the light path outside the cell. In addition, the TLC plates were left in an oven at 80 O C for several days prior to use to remove loosely bound water from the TLC support which could be desorbed by the modest cumulative heating of the sample from incident infrared radiation during production of the P A spectra. For each TLC plate type, the spectrum of the TLC plate without sample was subtracted from the TLC plate spectrum with sample after scaling the former so that the absorption peaks ascribable to the TLC support in both spectra exactly canceled in the difference. Scaling and subtracting spectra are readily accomplished by the standard Nicolet FT-IR software. Resulting difference spectra are also shown in Figures 1-3. Though these microphonic FT-IR/PA spectra compare well in terms of peak location with the KBr pellet transmission spectrum of I in Figure 4, the peak intensities are not identical principally because of the small, varying amount of vapor-phase water desorbed from the TLC support in the cell (despite the attempts described above to reduce this problem). This caused the spectral subtractions to be mis-scaled in the regions from 3900 to 3550 cm-l and 1900 to 1400 cm-l where gaseous water strongly absorbs. This problem was most severe for the TLC with alumina support. The high temperatures required to desorb all the water from the TLC support materials were impractical. Quantitative analysis of samples adsorbed on TLC plates has been accomplished by using dispersive PAS with visible-ultraviolet microphonic detectors (4,6, 7). Quantitative information may also be obtained from the broad-band FTIR/PA spectra of the TLC plates. Though the absolute magnitudes of absorption peaks in the FT-IR/PA spectra may vary from spectrum to spectrum because of changes in the intensity of the incident light or position of the sample in the cell, the relative peak amplitues should remain the same for a given sample. By use of a TLC support material absorption peak as an internal standard (13),a calibration curve may be constructed by measuring the ratio of the magnitudes of an absorption peak of the sample to an absorption peak of the TLC support material over a range of sample concentrations. This ratioing was done for I adsorbed on a piece of TLC plate with alumina support material in the microphonic cell. Since the PA signal in a microphonic cell depends strongly on the surface area of the sample, care was taken to spot each successive sample concentration as uniformly as possible onto the TLC plates using micropipets, Variations in spot size and sample distribution may be expected to be a source of deviation from the analytical curve. As stated, the alumina sup-
ANALYTICAL CHEMISTRY, VOL. 54, NO. 3, MARCH 1982
5!51
a
4000
Flgure 5. The FT-IR/PA spectra obtained using PZT detection of 300 pg of the sample ketone adsorbed on an aluminum backed TLC plate with sllica support (-), the same TLC plate with no sample (- -), and
the scaled difference spectrum (- - -).
-
port contained bound water, some of which was released into the transducing gas in the PA cell, with the result that the FT-IR/PA spectrum contained both the broad and sharp absorption features of bound and unbound water, respectively, in unpredictable amounts. Fortunately, the ketone sample has a strong absorption peak at 1286 cm-l, where the alumina has an absorption minimum and vapor-phase water does not strongly absorb. The alumina has an absorption maximum around 951 cm-l and this was used as the internal standard. The ratios of the 951-cm-l peak to the 1286-cm-l peak over a range of several hundred micrograms of ketone sample per square centimeter of TLC plate yielded a linear plot (correlation coefficient of 0.998) which gave a sensitivity (from the negative slope) of 0.001 change in the ratio per microgram of ketone sample. The limit of detection by this method was -90 pg of sample/cm2. This is to some extent determined by the SIN which improves as the square root of the number of scans averaged. The detection limit is also dependent on the absorption coefficient of the sample absorption peak used in the ratio. In the absence of water absorptions, a lower detection limit could, in principle, be achieved by using the more strongly absorbing 1705-cm-' sample absorption peak in the ratio. The presence of an absorbing species in the transducing gas imposes limitations in both qualitative and quantitative FTIR/PAS using microphonic detection as noted above. In contrast, PZT detmtors directly coupled to the sample require no transducing gas and therefore provide at least one advantage for use in FT-IIE/PAS. The use of PZT detectors in rapid scan FT-IR/PAS is new and required a verification of their performance. A bare PZT was placed so that the output of the interferometer impinged upon it. C02and water vapor were purposely introduced into the interferometer compartment by shutting off the N2purge gas and having the operator exhale several times into the compartment. The resulting spectrum clearly showed the presence of C02 at -2350 cm-'. The spectrum of the bare PZT is very similar to the background spectrum obtained using the Nicolet TGS aletector with COz and water vapor present. The signal to noise ratio of the bare PZT was such that the presence of COz could be detected after only 10 scans had been averaged in a total time, including Fourier transformation, of -40 s. A neat pellet -3 mm thick of the tetraphenylcyclogentadienonewas then attached to the PZT with a thin layer of epoxy glue. The pellet was thick enough to prevent infrared light from being transmitted through the sample and absorbed by the glue. The absorption peaks in this -15 min spectrum corresponded well with the trans-
3200
2400
IBOC 1400 WAVENUMBEQS
1000
600
Flgure 6. The same as Figure 5 except using a poly(ethy1ene terephthalate) backed TLC plate with sillca support.
4000
3200
2400
I800 1400 WAVENUMBERS
1000
600
The same as Figure 5 except using an aluminum backed TLC plate with alumina support. Flgure 7.
mission spectrum of the same ketone in the KBr pellet. FT-IR/PA spectra representing the average of several thousand scans of TLC plates with and without 300-pg ketone samples attached were then sought using PZT detection. Thle resulting spectra involving TLC plates with silica support material (Figures 5 and 6) were appropriately scaled and subtracted as before. It will be noted that these represent transmission spectra of the sample rather than absorption spectra. (One obtains absorption spectra with the microphoniic cell.) One explanation for this transmission behavior is that ordinarily the PZT signal is produced by localized thermal expansion of the sample where light absorption occurs with consequent deformation of the rigidly attached PZT (14). However, in the present case with the silica gel TLC support materials the support was not coupled rigidly enough to the substrates to deform the PZT after thermal expansion of the sample/support material. The small signal that was obtaineld resulted from what little light penetrated through the support and sample to the substrate where the resulting absorption could be transferred as a deformation to the PZT. In contrast, the alumina support TLC plate glued on a PZT produced interferograms with a signal to noise ratio comparable to the sample pellet on a PZT. Transformation yielded the absorption type spectra of the TLC plate and ketone shown in Figure 7. Evidently, the alumina support was rigid enouglh to transfer deformations of the PZT. Regardless of the mechanism of production, these spectra in all cases provide qualitative information about the ketone samples on the TLC plate.
552
ANALYTICAL CHEMISTRY, VOL. 54, NO. 3, MARCH 1982
A higher noise level, due primarily to ambient noise, caused the detection limit to be somewhat higher (-200 Mg cm-*) in the case of PZT detection of sample on alumina support TLC plates than in comparable microphonic experiments for the same number of scans averaged. However, the use of PZT detection reduces the number of parameters upon which the PA signal depends by reducing the PA signal dependence on sample spot size and surface area and thus simplifies the production of calibration curves. In listing the relative merits and limitations of the microphonic vs. the PZT detection techniques used here it should be noted that in applying either technique the Nicolet 7199 spectrometer used was a multiuser instrument to which only limited access was possible. This meant that the PAS modifications to the Nicolet FT-IR had to be easily effected and removed. With the techniques described above it was possible to convert the instrument to either PAS detection configuration in less than 5 min. A slight advantage in this adaptation scheme went to the microphonic detection system which in this multiuser situation was consistently less sensitive to ambient acoustic and electronic noise than the PZT detectors. Another advantage of microphonic detection is that it is a better known and documented PA method both in TLC detection and in FT-JR/PAS generally than PZT detection. The microphonic system does have, however, several limitations. With few exceptions (6) this system requires modification of the TLC plate either by excising a suitable sized portion or by removing the TLC support from the substrate. This requires the PA cdl to be reloaded for every sample run. The main problem with microphonic detection, as stated above, is the presence of an infrared absorbing species in the vapor phase such as the TLC solvent or volatile sample components which completely overwhelm the condensed phase PA signals. This latter problem is not confined to the TLC samples discussed here and represents a significant limitation to the production of PA infrared spectra of condensed phase samples using gas coupled microphonic detectors. The PZT photoacoustic detection method does not suffer from this limitation as the PZT is directly coupled to the sample. Even TLC samples saturated with solvent may be used in the PZT method as long as the spectrometer compartment remains well purged. Also, in principle, a PZT could be attached to the back of a single TLC plate bearing a large number of separated
sample components. An automated raster scan system could then be used to sweep the focused interferometer output from spot to spot while FT-IR/PA spectra were simultaneously produced. The limitation in using PZTs at infrared wavelengths is, as mentioned before, the necessity for a tightly coupled sample. The particular choice of either microphonic or PZT detector method depends ultimately on the properties of the sample and the availability and susceptibility of the spectrometer to modification. For instance, in the samples analyzed here it would be preferable to use the microphonic detector for the TLC plates with silica gel support because of the transmission problems described above when the PZT detectors were used. However, the elimination of the transducing gas by using PZT detectors may prove to be an overwhelming advantage for the use of PZT detection at infrared wavelengths.
ACKNOWLEDGMENT Use of the Lord Kinematics vibration isolation mounts was suggested by John B. Kinney, Massachusetts Institute of Technology. Materials and advice provided by Bany A. Lloyd, University of Utah, are also gratefully acknowledged.
LITERATURE CITED Fenlmore, B. C.; Davis, C. M. Anal. Chem. 1981, 5 3 , 252 A. Rosencwaig, A. Anal. Chem. 1975, 47, 592 A. Rosencwalg, A.; Hall, S. S.Anal. Chem. 1975, 4 7 , 548. Castieden, S. L.; Elliot, C. M.; Kirkbright, G. F.; Splllane, D. E. M. Anal. Chem. 1979, 5 1 , 2152. Lochmuller, C. H.; Marshall, S. F.; Wilder, D. R . Anal. Chem. 1980, 52, 19. Flshman, V. A,; Bard, A. J . Anal. Chem. 1981, 53, 102. Ikeda, S.;Murakaml, Y.; Akatsuka, K. Chem. Lett. 1981, 363. Rockiey, M. G. Chem. Phys. Lett. 1979, 68, 455. Vldrlne, D. W. Appl. Spectrosc. 1980, 3 4 , 313. Farrow, M. k4.; Burnham, R. K.; Auzanneau, M.; Olsen, S. L.; Purdle, N.; Eyrlng, E. M. Appl. Opt. 1978, 17, 1093. Lloyd, L. B.; Burnham, R. K.; Chandler, W. L.; Farrow, M. M.; Eyring, E. M. Anal. Chem. 1980, 52, 1595. Patel, C. K. N.; Tam, A. C. Appl. Phys. Lett. 1980, 3 6 , 7 . Rockley, M. G.;Davis, D. M.; Richardson, H. H. Appl. Spectrosc. 1981, 3 5 , 185. Jackson, W.; Amer, N. M. J . Appl. Phys. 1980, 5 1 , 2343.
RECEIVED for review June 19,1981. Accepted November 10, 1981. This research was financed by Grant HL23378-03 from the National Heart, Lung, and Blood Institute and by Biomedical Sciences Support Grant, PHS No. RR07092.
