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Microcolumn high-performance liquid chromatography—thin-layer chromatography—Fourier transform infrared spectrometry
Journal of Chromatography,
438 (1988) 329-337
Elsevier Science Publishers B.V., Amsterdam -
Printed in The Netherlands
CHROM. 20 323
MICROCOLUMN HIGH-PERFORMANCE LIQUID CHROMATOGRAPHYTHIN-LAYER CHROMATOGRAPHY-FOURIER TRANSFORM INFRARED SPECTROMETRY
CHUZO FUJIMOTO*,
TOMOHIRO
MORITA and KIYOKATSU
Materials Science, Toyohashi University of Technology, Tempaku-cho,
JINNO Toyohashi 440 (Japan)
(First received November 2nd, 1987; revised manuscript received December 30th, 1987)
SUMMARY
A method is described for acquiring identifiable Fourier transform infrared (FTIR) spectra of well resolved chromatographic components. The technique involves the combination of high-performance liquid chromatography (HPLC) and thin-layer chromatography (TLC), each utilizing a different separation mode. In this way, the full identification power of FTIR can be realized for mixture analysis. In this particular work, microcolumn size-exclusion HPLC was followed by conventional silica gel TLC. Subsequently, the infrared spectra of each spot immobilized on the plate were measured by diffuse reflectance spectrometry. Spectral features of the eluates could be readily distinguished when 8 pg of each component were injected into the column. The feasibility of measuring submicrogram amounts of materials directly on a TLC plate was demonstrated.
INTRODUCTION
The analysis of highly complex mixtures places stringent demands upon analytical methodology and in such instances separations are generally unavoidable. Numerous separation techniques are available, but chromatography is the most widely and frequently used. High-performance liquid chromatography (HPLC) is an obvious choice for the separation of non-volatile or thermally unstable samples. Although the separation efficiencies cannot match those obtainable with gas chromatography, the expanding number of packing materials permits the separation of most classes of compounds. However, it is important to realize that no single HPLC separation mode may be completely adequate for the separation of a complex mixture. Mostly, a full separation requiries more than one separation mode and a combination of separation modes is required. A simple method of extending the potential of HPLC is to couple it with thin-layer chromatography (TLC). Boshoff et al. l investigated TLC plates as a transport mechanism for conventional HPLC. The column effluent was split to reduce the amount of liquid reaching the TLC plate. Regular application of the effluent to the plate was achieved by maintaining a continuous liquid bridge between the effluent transfer line and the adsorbent surface. More recently, Hofstraat and co-workers2J 002 l-9673/88/%03.50
0
1988 Elsevier Science Publishers B.V.
330
C. FUJIMOTO,
T. MORITA, K. JINNO
described the coupling of microcolumn HPLC and TLC. Because of the reduced flow-rate, microcolumn HPLC has great promise for this type of coupling. The effluent from a microcolumn (0.7 mm I.D.) was sprayed on to a TLC plate through a Camag Linomat III spray-jet assembly. Both of these studies involved the use of spectrofluorimetric detection, which is popular in TLC because of its high sensitivity and selectivity. However, there are only a limited number of naturally fluorescing compounds, which necessitates either derivatization of various solute types with fluorescent agents or the use of fluorescent TLC plates. Infrared spectrometry has several important characteristics that make it a potentially ideal detection method for any chromatographic technique. Most organic compounds (and many inorganic compounds) have strong, relatively narrow absorption bands in the mid-infrared region. These absorptions are highly specific and give a wealth of structural information about a compound. Several methods of identifying the components separated by TLC using Fourier transform infrared (FTIR) spectrometry have been suggested. Percival and Griffiths4 described a method of obtaining spectra from TLC spots directly using transmission spectrometry. This approach requires the preparation of the TLC plate on an infrared-transparent material, such as silver chloride. FTIR photoacoustic spectrometry has also been employed to analyse substances deposited on silica gel TLC plates5,‘j. Although good spectra are often obtained, the availability of such an attachment is limited to very few laboratories. Diffuse reflectance (DR) measurements of TLC spots were accomplished in situ’s8 or after the spots had been transferred to an infrared-transmitting substrateg-12. The sample transfer approach is superior to in situ measurement because the spectrum obtained allows a comparison to be made directly with absorbance spectra. However, the transfer of the spot is time consuming and also introduces the possibility of loss, decomposition or contamination’l,l 2. There is no doubt that the above FTIR techniques offer an effective means of identifying TLC spots, but they are of limited value as they do not involve HPLC separation prior to TLC separation. In this paper, we propose the coupling of microcolumn HPLC, TLC and DR-FTIR spectrometry. Here, the HPLC trace stored on the TLC plate serves as a starting point’for TLC separation. Chromatographic resolution is increased by combining two different separations, viz.,size-exclusion HPLC and silica gel adsorption TLC. Using currently available FTIR instrumentation, high-quality spectra can be obtained from 8 fig of each component injected into a microcolumn. EXPERIMENTAL
Reagents
Methanol, tetrahydrofuran (THF) and benzene were of Cica-Merck Uvasol quality (Kant0 Chemical, Tokyo, Japan). The model compounds Irganox 1010 {tetrakis[methylene-3-(3’,5’-di-tert.-butyl-~-hydroxyphenyl)propionate]methane~, oleamide, Antioxidant 2246 [2,2’-methylenebis(6-tert.-butylcresol)] and Cyasorb UV-9 (2-hydroxy-4-methoxybenzophenone) were analytical-reagent grade materials from Tokyo Kasei Kogyo (Tokyo, Japan).
MICROCOLUMN HPLC-TLC-FTIR
331
TLC
The TLC plate used was an Art. 5553 from E. Merck (Darmstadt, F.R.G.) (aluminium-backed silica gel 60 with a 0.2-mm coating). Plates (7 cm x 5 cm) were cut from standard 20 cm x 20 cm plates. Prior to their use, the plates were continuously developed with methanol for 24 h to remove possible organic impurities. Throughout this work, spots were detected by gently treating the TLC plates with iodine vapour. HPLC
The HPLC pump was an MF2 Microfeeder (Azumadenki Kogyo, Tokyo, Japan) fitted with a 5009 microsyringe. The HPLC column consisted of TSK G-2000H (Tosoh, Tokyo, Japan) packed into a fused-silica tube (100 cm x 0.53 mm I.D.). The sample solutions were introduced into the column with an ML-422 micro-loop injector (0.0X-$ volume) (Jasco, Tokyo, Japan). THF was used as the mobile phase at a flow-rate of 2.67 pl/min. FTIR
An Analect Instruments (Irvine, CA, U.S.A.) AQS-20 FTIR spectrometer fitted with a TLX-30A automated diffuse reflectance sampler was used for in situ measurement of reconstructed infrared chromatograms. The TLX-30A is equipped with DR optics, a TLC Optitrain moved by a stepper motor and a narrow-range mercurycadmium telluride (MCT) detector. The reconstruction was calculated by numerically summing the square of the reflectance of the Fourier transform spectra (4 cm-l resolution, 64-scan coaddition) over the selected window after a baseline had been generated by linear fitting of the points within this range. For the measurement of the DR spectrum of each component, a Spectra-Tech (Stamford, CT, U.S.A.) Collector diffuse reflectance device was mounted within the FTIR’s auxiliary output beam sample compartment (Analect Instruments, AQG505). All DR spectra were calculated by measuring the single-beam spectra of the TLC spots and rationing this against the blank spectrum. Spectra of the separated components were obtained at a resolution 4 cm-l with 500-scan coaddition using HapppGenzel apodization. Procedure
The interface used for HPLC-TLC coupling was similar to that developed originally for “buffer-memory” HPLC-FTIR 13-z*. The HPLC effluent was transferred with a fine stainless-steel capillary tube (10 cm x 0.13 mm I.D. x 0.31 mm O.D.) attached to the outlet of the column to the surface of the TLC plate placed on a translation table, which was driven at 1 mm/min by a variable d.c. motor. A flow of warm nitrogen was applied around the tip of the capillary in order to facilitate the evaporation of the eluent. The flow-rate and temperature of the nitrogen were chosen to provide about 100% transfer efficiency and were 0.3 l/min and 40°C respectively. Under these conditions, a small droplet (less than 0.5 mm in diameter) of the HPLC effluent was formed at the tip of the transfer tube. For comparison purposes, a Uvidec 100-11 (Jasco) ultraviolet detector was included in the chromatographic set-up. After the solutes had been collected, any solvent remaining was evaporated by
332
C. FUJIMOTO,
T. MORITA, K. JINNO
gently heating the TLC plate with a hot-air gun below 50°C (this treatment may not necessarily be required, because the presence of trace amounts of the HPLC solvent on the plate does not have any effect on the subsequent TLC separation). The solute-immobilized plate was then developed with benzene as the solvent in a direction perpendicular to the deposition direction. After detection of the TLC spots, each spot was cut out from the plate and placed on the standard cup of the Collector accessory. Prior to the DR measurements, the TLC spots were subjected to about a 5-min purge within the IR sample compartment. RESULTS AND DISCUSSION
All the sample materials in which great interest is shown are made up of a number of components of different molecular size and polarity. In this instance, a preliminary sample fractionation might be carried out by size-exclusion chromatography (SEC) and the components of similar molecular size further resolved by adsorption or bonded-phase liquid chromatography. Therefore, size-exclusion HPLC followed by silica gel TLC is likely to be one of the most suitable combinations for the separation of highly complex mixtures. A mixture of four polymer additives (Irganox 1010, oleamide, Antioxidant 2246 and Cyasorb UV-9) was separated on a TSK G-2000H microcolumn with THF as solvent. The chromatogram recorded with a UV detector is shown in Fig. 1. It is seen that the peaks due to oleamide and Antioxidant 2246 are not resolved because of the similarity of their molecular sizes. 4
om TIME
(MIN)
Fig. 1. Microcolumn size-exclusion separation of a four-component
mixture using THF as the mobile phase at a flow-rate of 2.67 p/min and UV absorption detection at 230 nm. (1) Irganox 1010, 8 pg; (2) oleamide, 8 pg; (3) Antioxidant 2246, 8 pg; (4) Cyasorb UV-9, 8 pg; (5) sample solvent (chloroform).
MICROCOLUMN
HPLC-TLC-FTIR
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20 MM Fig. 2. Appearance of chromatographic spots. (A) After HPLC separation and deposition on the TLC plate; (B) after development of the chromatogram with benzene.
TLC results obtained for immobilization of the SEC column effluent on a silica gel plate are shown in Fig. 2A. The deposition was started 25 min after sample injection into the microcolumn. Three apparent spots, having band widths of l-l.5 mm, can be seen in the chromatogram (infrared beam positioning within the overlapping spots and the infrared subtraction technique were unsuccessful for the analysis of each of the spots). In the HPLC-TLC couling, the crucial aspect is the maintenance of the chromatographic integrity during the deposition process. The immobilized thin-layer chromatogram was scanned with the TLX-30A automated diffuse reflectance sampler 14.000
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Fig. 3. FTIR chromatogram of the four-component mixture in Fig. 1 produced after deposition on the TLC plate. Identification of components as in Fig. 1. Window region, 1500-1400 cm-r.
Fig. 4. FTIR spectra of the separated components.
76.6936
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at a spatial resolution of 0.5 mm in the direction of the scan (or deposition). An infrared chromatogram was reconstructed from the series of reflectance spectra stored in the FTIR’s data system. The result is shown in Fig. 3. It is noteworthy that the data represent the first HPLC-FTIR results to be obtained using a TLC plate as a substrate. A comparison of the IR chromatogram with the UV chromatogram shows that no loss of chromatographic resolution had occurred on transfer of the eluates. Subsequently, the TLC plate was developed with benzene at 90” to the deposition direction (as in two-dimensional chromatography). The TLC development was useful for the separation of oleamide and Antioxidant 2246, which had not been resolved by the SEC separation. The RF values of Irganox 1010, oleaide, Antioxidant 2246 and Cyasorb UV-9 were cu. 0.47, 0, 0.78 and 0.65, respectively. After development, the spot diameters were 2-4 mm (Fig. 2B), whereas the infrared beam diameter of the TLX-30A at focus was approximately 0.8 mm. To obtain the maximum signal collection efficiency from the TLC spots, the TLX-30A was replaced with the Collector, which provides an infrared beam diameter of about 3 mm at the focus. However, it should be noted that the collection efficiency is still not optimized for the spots of Irganox 1010 and Antioxidant 2246, as their diameters are larger than 3 mm. The TLC plate size (7 cm x 5 cm) did not allow for direct insertion of the plate into the Collector’s sample chamber, and therefore each spot was clipped out of the TLC plate for the DR measurement with the Collector. Fig. 4 shows the in situ DR spectra of the spots. The broad band that appeared around 1280 cm-l is the result of the superimposition of the intense IR band due to the background silica gel on the sample’s IR bands. As pointed out by several workers7,8, interpretation of DR spectra is complicated by the presence of reststrahlen features in the region between 1300 and 1200 cm-l where silica gel absorbs strongly. Nevertheless, the signal-to-noise
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Fig. 5. FTIR spectrum of Cyasorb UV-9 for a O.S-pg amount spotted on the TLC plate.
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336
C. FUJIMOTO,
T. MORITA, K. JINNO
ratios of the DR spectra are sufficient to permit discrimination of each of the four polymer additives. The sample size used in this experiment, i.e., 8 pg per component, is compatible with those previously reported for in situ measurements by DR spectroscopy6 (10 pg per component), but is significantly smaller than the sample levels for photoacoustic (50 fig per component) and sample transfer DR measurements’ l,l 2 (20-50 pg per component). Dilute solutions of Cyasorb UV-9 were spotted directly on a silica gel TLC plate using a microsyringe, to determine the minimum identifiable limit. Even after a 0%,ug sample of Cyasorb UV-9 had been developed by 2.5 cm using benzene, all the strong bands could still be observed, as shown in Fig. 5. One thousand scans were required to be coadded before this spectrum was obtained. CONCLUSION
The example shown in this paper has demonstrated the feasibility of analysing multi-component mixtures using one sample application and two separation methods (HPLC followed by TLC) by in situ DR-FTIR spectrometry. As a TLC plate is used as the substrate instead of a potassium bromide plate, this work can be also considered as an extension of the buffer-memory technique13-20, which was developed in this laboratory for HPLC-FTIR and supercritical fluid chromatography-FTIR. However, as the TLC plate used in this work can serve as the stationary phase in the second separation, it becomes possible to resolve the components that were unresolved in the first separation. It is noteworthy that infrared spectral comparison with references requires that TLC-FTIR spectra of these standards be available, as differences often exist between TLC-IR reference spectra and transmittance spectra, as pointed out by Zuber et al. 8. In spite of this limitation, we believe that a new dimension has been added to “hyphenated techniques”. If it is desirable that spectra are obtained without the customary spectral changes for measurements made in situ, then the analytes should be transferred to an IR-transparent substrate. ACKNOWLEDGEMENTS
We gratefully acknowledge the loan of an AQS-20 spectrometer and diffuse reflectance accessories from JEOL and Analect Instruments. C. F. acknowledges partial support from the Japanese Ministry of Education, Science and Culture, Grantin-Aid for Developmental Science Research, under Grant number 61750743. REFERENCES 1 P. R. Boshoff, B. J. Hopkins and V. Pretorius, J. Chromatogr., 126 (1976) 35. 2 J. W. Hofstraat, M. Engelsma, R. J. van de Nesse, C. Gooijer, N. H. Velthorst and U. A. Th. Brinkman, Anal. Chim. Acta, 186 (1986) 247. 3 J. W. Hofstraat, M. Engelsma, R. J. van de Nesse, U. A. Th. Brinkman, C. Gooijer and N. H. Velthorst, Anal. Chim. Acta, 193 (1987) 193. 4 C. J. Percival and P. R. Griffiths, Anal. Gem., 47 (1975) 154. 5 L. B. Lloyd, R. C. Yeates and E. M. Eyring, Anal. Chem., 54 (1982) 549. 6 R. L. White, Anal. Chem., 57 (1985) 1819. 7 M. P. Fuller and P. R. Griffiths, Anal. Chem., 50 (1978) 1906. 8 G. E. Zuber, R. J. Warren, P. P. Begosh and E. L. O’Donnel, Anal. Gem., 56 (1984) 2935.
MICROCOLUMN 9 10 11 12 13 14 15 16 17 18 19 20
HPLC-TLC-FTIR
M. P. Fuller and P. R. Griffiths, Appl. Spectrosc., 34 (1980) 533. J. M. Chalmers and M. W. Mackenzie, Appl. Spectrosc., 39 (1985) 634. J. M. Chalmers, M. W. Mackenzie. J.L. Sharp and R. N. Ibbert. Anal. Chem.. 59 (1987) 415. K. H. Shafer, P. R. Griffiths and W. Shu-Qin, Anal. C/rem., 58 (1986) 2708. K. Jinno, C. Fujimoto and Y. Hiram Appl. Spectrosc., 36 (1980) 313. K. Jinno, C. Fujimoto, M. Ideriha, T. Takeuchi and D. Ishii, Bunseki Kugaku, 29 (1980) 612. K. Jinno and C. Fujimoto, J, High Resolut. Chromatogr. Chromatogr. Commun., 4 (1981) 532. K. Jinno, C. Fujimoto and D. Ishii, J. Chromatogr., 239 (1982) 625. C. Fujimoto, K. Jinno and Y. Hirata, J. Chromatogr., 258 (1983) 81. C. Fujimoto, T. Oosuka and K. Jinno, Anal. Chim. Acta, I78 (1985) 159. C. Fujimoto, T. Morita, K. Jinno and S. Ckhiai, Chromatographia, 23 (1987) 512. C. Fujimoto, Y. Hirata and K. Jinno, J. Chromatogr., 332 (1985) 47.
331
438 (1988) 329-337
Elsevier Science Publishers B.V., Amsterdam -
Printed in The Netherlands
CHROM. 20 323
MICROCOLUMN HIGH-PERFORMANCE LIQUID CHROMATOGRAPHYTHIN-LAYER CHROMATOGRAPHY-FOURIER TRANSFORM INFRARED SPECTROMETRY
CHUZO FUJIMOTO*,
TOMOHIRO
MORITA and KIYOKATSU
Materials Science, Toyohashi University of Technology, Tempaku-cho,
JINNO Toyohashi 440 (Japan)
(First received November 2nd, 1987; revised manuscript received December 30th, 1987)
SUMMARY
A method is described for acquiring identifiable Fourier transform infrared (FTIR) spectra of well resolved chromatographic components. The technique involves the combination of high-performance liquid chromatography (HPLC) and thin-layer chromatography (TLC), each utilizing a different separation mode. In this way, the full identification power of FTIR can be realized for mixture analysis. In this particular work, microcolumn size-exclusion HPLC was followed by conventional silica gel TLC. Subsequently, the infrared spectra of each spot immobilized on the plate were measured by diffuse reflectance spectrometry. Spectral features of the eluates could be readily distinguished when 8 pg of each component were injected into the column. The feasibility of measuring submicrogram amounts of materials directly on a TLC plate was demonstrated.
INTRODUCTION
The analysis of highly complex mixtures places stringent demands upon analytical methodology and in such instances separations are generally unavoidable. Numerous separation techniques are available, but chromatography is the most widely and frequently used. High-performance liquid chromatography (HPLC) is an obvious choice for the separation of non-volatile or thermally unstable samples. Although the separation efficiencies cannot match those obtainable with gas chromatography, the expanding number of packing materials permits the separation of most classes of compounds. However, it is important to realize that no single HPLC separation mode may be completely adequate for the separation of a complex mixture. Mostly, a full separation requiries more than one separation mode and a combination of separation modes is required. A simple method of extending the potential of HPLC is to couple it with thin-layer chromatography (TLC). Boshoff et al. l investigated TLC plates as a transport mechanism for conventional HPLC. The column effluent was split to reduce the amount of liquid reaching the TLC plate. Regular application of the effluent to the plate was achieved by maintaining a continuous liquid bridge between the effluent transfer line and the adsorbent surface. More recently, Hofstraat and co-workers2J 002 l-9673/88/%03.50
0
1988 Elsevier Science Publishers B.V.
330
C. FUJIMOTO,
T. MORITA, K. JINNO
described the coupling of microcolumn HPLC and TLC. Because of the reduced flow-rate, microcolumn HPLC has great promise for this type of coupling. The effluent from a microcolumn (0.7 mm I.D.) was sprayed on to a TLC plate through a Camag Linomat III spray-jet assembly. Both of these studies involved the use of spectrofluorimetric detection, which is popular in TLC because of its high sensitivity and selectivity. However, there are only a limited number of naturally fluorescing compounds, which necessitates either derivatization of various solute types with fluorescent agents or the use of fluorescent TLC plates. Infrared spectrometry has several important characteristics that make it a potentially ideal detection method for any chromatographic technique. Most organic compounds (and many inorganic compounds) have strong, relatively narrow absorption bands in the mid-infrared region. These absorptions are highly specific and give a wealth of structural information about a compound. Several methods of identifying the components separated by TLC using Fourier transform infrared (FTIR) spectrometry have been suggested. Percival and Griffiths4 described a method of obtaining spectra from TLC spots directly using transmission spectrometry. This approach requires the preparation of the TLC plate on an infrared-transparent material, such as silver chloride. FTIR photoacoustic spectrometry has also been employed to analyse substances deposited on silica gel TLC plates5,‘j. Although good spectra are often obtained, the availability of such an attachment is limited to very few laboratories. Diffuse reflectance (DR) measurements of TLC spots were accomplished in situ’s8 or after the spots had been transferred to an infrared-transmitting substrateg-12. The sample transfer approach is superior to in situ measurement because the spectrum obtained allows a comparison to be made directly with absorbance spectra. However, the transfer of the spot is time consuming and also introduces the possibility of loss, decomposition or contamination’l,l 2. There is no doubt that the above FTIR techniques offer an effective means of identifying TLC spots, but they are of limited value as they do not involve HPLC separation prior to TLC separation. In this paper, we propose the coupling of microcolumn HPLC, TLC and DR-FTIR spectrometry. Here, the HPLC trace stored on the TLC plate serves as a starting point’for TLC separation. Chromatographic resolution is increased by combining two different separations, viz.,size-exclusion HPLC and silica gel adsorption TLC. Using currently available FTIR instrumentation, high-quality spectra can be obtained from 8 fig of each component injected into a microcolumn. EXPERIMENTAL
Reagents
Methanol, tetrahydrofuran (THF) and benzene were of Cica-Merck Uvasol quality (Kant0 Chemical, Tokyo, Japan). The model compounds Irganox 1010 {tetrakis[methylene-3-(3’,5’-di-tert.-butyl-~-hydroxyphenyl)propionate]methane~, oleamide, Antioxidant 2246 [2,2’-methylenebis(6-tert.-butylcresol)] and Cyasorb UV-9 (2-hydroxy-4-methoxybenzophenone) were analytical-reagent grade materials from Tokyo Kasei Kogyo (Tokyo, Japan).
MICROCOLUMN HPLC-TLC-FTIR
331
TLC
The TLC plate used was an Art. 5553 from E. Merck (Darmstadt, F.R.G.) (aluminium-backed silica gel 60 with a 0.2-mm coating). Plates (7 cm x 5 cm) were cut from standard 20 cm x 20 cm plates. Prior to their use, the plates were continuously developed with methanol for 24 h to remove possible organic impurities. Throughout this work, spots were detected by gently treating the TLC plates with iodine vapour. HPLC
The HPLC pump was an MF2 Microfeeder (Azumadenki Kogyo, Tokyo, Japan) fitted with a 5009 microsyringe. The HPLC column consisted of TSK G-2000H (Tosoh, Tokyo, Japan) packed into a fused-silica tube (100 cm x 0.53 mm I.D.). The sample solutions were introduced into the column with an ML-422 micro-loop injector (0.0X-$ volume) (Jasco, Tokyo, Japan). THF was used as the mobile phase at a flow-rate of 2.67 pl/min. FTIR
An Analect Instruments (Irvine, CA, U.S.A.) AQS-20 FTIR spectrometer fitted with a TLX-30A automated diffuse reflectance sampler was used for in situ measurement of reconstructed infrared chromatograms. The TLX-30A is equipped with DR optics, a TLC Optitrain moved by a stepper motor and a narrow-range mercurycadmium telluride (MCT) detector. The reconstruction was calculated by numerically summing the square of the reflectance of the Fourier transform spectra (4 cm-l resolution, 64-scan coaddition) over the selected window after a baseline had been generated by linear fitting of the points within this range. For the measurement of the DR spectrum of each component, a Spectra-Tech (Stamford, CT, U.S.A.) Collector diffuse reflectance device was mounted within the FTIR’s auxiliary output beam sample compartment (Analect Instruments, AQG505). All DR spectra were calculated by measuring the single-beam spectra of the TLC spots and rationing this against the blank spectrum. Spectra of the separated components were obtained at a resolution 4 cm-l with 500-scan coaddition using HapppGenzel apodization. Procedure
The interface used for HPLC-TLC coupling was similar to that developed originally for “buffer-memory” HPLC-FTIR 13-z*. The HPLC effluent was transferred with a fine stainless-steel capillary tube (10 cm x 0.13 mm I.D. x 0.31 mm O.D.) attached to the outlet of the column to the surface of the TLC plate placed on a translation table, which was driven at 1 mm/min by a variable d.c. motor. A flow of warm nitrogen was applied around the tip of the capillary in order to facilitate the evaporation of the eluent. The flow-rate and temperature of the nitrogen were chosen to provide about 100% transfer efficiency and were 0.3 l/min and 40°C respectively. Under these conditions, a small droplet (less than 0.5 mm in diameter) of the HPLC effluent was formed at the tip of the transfer tube. For comparison purposes, a Uvidec 100-11 (Jasco) ultraviolet detector was included in the chromatographic set-up. After the solutes had been collected, any solvent remaining was evaporated by
332
C. FUJIMOTO,
T. MORITA, K. JINNO
gently heating the TLC plate with a hot-air gun below 50°C (this treatment may not necessarily be required, because the presence of trace amounts of the HPLC solvent on the plate does not have any effect on the subsequent TLC separation). The solute-immobilized plate was then developed with benzene as the solvent in a direction perpendicular to the deposition direction. After detection of the TLC spots, each spot was cut out from the plate and placed on the standard cup of the Collector accessory. Prior to the DR measurements, the TLC spots were subjected to about a 5-min purge within the IR sample compartment. RESULTS AND DISCUSSION
All the sample materials in which great interest is shown are made up of a number of components of different molecular size and polarity. In this instance, a preliminary sample fractionation might be carried out by size-exclusion chromatography (SEC) and the components of similar molecular size further resolved by adsorption or bonded-phase liquid chromatography. Therefore, size-exclusion HPLC followed by silica gel TLC is likely to be one of the most suitable combinations for the separation of highly complex mixtures. A mixture of four polymer additives (Irganox 1010, oleamide, Antioxidant 2246 and Cyasorb UV-9) was separated on a TSK G-2000H microcolumn with THF as solvent. The chromatogram recorded with a UV detector is shown in Fig. 1. It is seen that the peaks due to oleamide and Antioxidant 2246 are not resolved because of the similarity of their molecular sizes. 4
om TIME
(MIN)
Fig. 1. Microcolumn size-exclusion separation of a four-component
mixture using THF as the mobile phase at a flow-rate of 2.67 p/min and UV absorption detection at 230 nm. (1) Irganox 1010, 8 pg; (2) oleamide, 8 pg; (3) Antioxidant 2246, 8 pg; (4) Cyasorb UV-9, 8 pg; (5) sample solvent (chloroform).
MICROCOLUMN
HPLC-TLC-FTIR
)r
333
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20 MM Fig. 2. Appearance of chromatographic spots. (A) After HPLC separation and deposition on the TLC plate; (B) after development of the chromatogram with benzene.
TLC results obtained for immobilization of the SEC column effluent on a silica gel plate are shown in Fig. 2A. The deposition was started 25 min after sample injection into the microcolumn. Three apparent spots, having band widths of l-l.5 mm, can be seen in the chromatogram (infrared beam positioning within the overlapping spots and the infrared subtraction technique were unsuccessful for the analysis of each of the spots). In the HPLC-TLC couling, the crucial aspect is the maintenance of the chromatographic integrity during the deposition process. The immobilized thin-layer chromatogram was scanned with the TLX-30A automated diffuse reflectance sampler 14.000
r
I’
3.2
’
A.0000
0.000$
Fig. 3. FTIR chromatogram of the four-component mixture in Fig. 1 produced after deposition on the TLC plate. Identification of components as in Fig. 1. Window region, 1500-1400 cm-r.
Fig. 4. FTIR spectra of the separated components.
76.6936
76.966
86.666
65.080
65.000
M-l)
I
19ee Spectra A, B, C and D were obtained from the spots 1, 2, 3 and 4, respectively, in Fig. 2B.
WAVENUMBER
62.666
74.060
66.006
98.006
165.06
55.0046
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MICROCOLUMN
335
HPLC-TLC-FTlR
at a spatial resolution of 0.5 mm in the direction of the scan (or deposition). An infrared chromatogram was reconstructed from the series of reflectance spectra stored in the FTIR’s data system. The result is shown in Fig. 3. It is noteworthy that the data represent the first HPLC-FTIR results to be obtained using a TLC plate as a substrate. A comparison of the IR chromatogram with the UV chromatogram shows that no loss of chromatographic resolution had occurred on transfer of the eluates. Subsequently, the TLC plate was developed with benzene at 90” to the deposition direction (as in two-dimensional chromatography). The TLC development was useful for the separation of oleamide and Antioxidant 2246, which had not been resolved by the SEC separation. The RF values of Irganox 1010, oleaide, Antioxidant 2246 and Cyasorb UV-9 were cu. 0.47, 0, 0.78 and 0.65, respectively. After development, the spot diameters were 2-4 mm (Fig. 2B), whereas the infrared beam diameter of the TLX-30A at focus was approximately 0.8 mm. To obtain the maximum signal collection efficiency from the TLC spots, the TLX-30A was replaced with the Collector, which provides an infrared beam diameter of about 3 mm at the focus. However, it should be noted that the collection efficiency is still not optimized for the spots of Irganox 1010 and Antioxidant 2246, as their diameters are larger than 3 mm. The TLC plate size (7 cm x 5 cm) did not allow for direct insertion of the plate into the Collector’s sample chamber, and therefore each spot was clipped out of the TLC plate for the DR measurement with the Collector. Fig. 4 shows the in situ DR spectra of the spots. The broad band that appeared around 1280 cm-l is the result of the superimposition of the intense IR band due to the background silica gel on the sample’s IR bands. As pointed out by several workers7,8, interpretation of DR spectra is complicated by the presence of reststrahlen features in the region between 1300 and 1200 cm-l where silica gel absorbs strongly. Nevertheless, the signal-to-noise
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Fig. 5. FTIR spectrum of Cyasorb UV-9 for a O.S-pg amount spotted on the TLC plate.
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C. FUJIMOTO,
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ratios of the DR spectra are sufficient to permit discrimination of each of the four polymer additives. The sample size used in this experiment, i.e., 8 pg per component, is compatible with those previously reported for in situ measurements by DR spectroscopy6 (10 pg per component), but is significantly smaller than the sample levels for photoacoustic (50 fig per component) and sample transfer DR measurements’ l,l 2 (20-50 pg per component). Dilute solutions of Cyasorb UV-9 were spotted directly on a silica gel TLC plate using a microsyringe, to determine the minimum identifiable limit. Even after a 0%,ug sample of Cyasorb UV-9 had been developed by 2.5 cm using benzene, all the strong bands could still be observed, as shown in Fig. 5. One thousand scans were required to be coadded before this spectrum was obtained. CONCLUSION
The example shown in this paper has demonstrated the feasibility of analysing multi-component mixtures using one sample application and two separation methods (HPLC followed by TLC) by in situ DR-FTIR spectrometry. As a TLC plate is used as the substrate instead of a potassium bromide plate, this work can be also considered as an extension of the buffer-memory technique13-20, which was developed in this laboratory for HPLC-FTIR and supercritical fluid chromatography-FTIR. However, as the TLC plate used in this work can serve as the stationary phase in the second separation, it becomes possible to resolve the components that were unresolved in the first separation. It is noteworthy that infrared spectral comparison with references requires that TLC-FTIR spectra of these standards be available, as differences often exist between TLC-IR reference spectra and transmittance spectra, as pointed out by Zuber et al. 8. In spite of this limitation, we believe that a new dimension has been added to “hyphenated techniques”. If it is desirable that spectra are obtained without the customary spectral changes for measurements made in situ, then the analytes should be transferred to an IR-transparent substrate. ACKNOWLEDGEMENTS
We gratefully acknowledge the loan of an AQS-20 spectrometer and diffuse reflectance accessories from JEOL and Analect Instruments. C. F. acknowledges partial support from the Japanese Ministry of Education, Science and Culture, Grantin-Aid for Developmental Science Research, under Grant number 61750743. REFERENCES 1 P. R. Boshoff, B. J. Hopkins and V. Pretorius, J. Chromatogr., 126 (1976) 35. 2 J. W. Hofstraat, M. Engelsma, R. J. van de Nesse, C. Gooijer, N. H. Velthorst and U. A. Th. Brinkman, Anal. Chim. Acta, 186 (1986) 247. 3 J. W. Hofstraat, M. Engelsma, R. J. van de Nesse, U. A. Th. Brinkman, C. Gooijer and N. H. Velthorst, Anal. Chim. Acta, 193 (1987) 193. 4 C. J. Percival and P. R. Griffiths, Anal. Gem., 47 (1975) 154. 5 L. B. Lloyd, R. C. Yeates and E. M. Eyring, Anal. Chem., 54 (1982) 549. 6 R. L. White, Anal. Chem., 57 (1985) 1819. 7 M. P. Fuller and P. R. Griffiths, Anal. Chem., 50 (1978) 1906. 8 G. E. Zuber, R. J. Warren, P. P. Begosh and E. L. O’Donnel, Anal. Gem., 56 (1984) 2935.
MICROCOLUMN 9 10 11 12 13 14 15 16 17 18 19 20
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M. P. Fuller and P. R. Griffiths, Appl. Spectrosc., 34 (1980) 533. J. M. Chalmers and M. W. Mackenzie, Appl. Spectrosc., 39 (1985) 634. J. M. Chalmers, M. W. Mackenzie. J.L. Sharp and R. N. Ibbert. Anal. Chem.. 59 (1987) 415. K. H. Shafer, P. R. Griffiths and W. Shu-Qin, Anal. C/rem., 58 (1986) 2708. K. Jinno, C. Fujimoto and Y. Hiram Appl. Spectrosc., 36 (1980) 313. K. Jinno, C. Fujimoto, M. Ideriha, T. Takeuchi and D. Ishii, Bunseki Kugaku, 29 (1980) 612. K. Jinno and C. Fujimoto, J, High Resolut. Chromatogr. Chromatogr. Commun., 4 (1981) 532. K. Jinno, C. Fujimoto and D. Ishii, J. Chromatogr., 239 (1982) 625. C. Fujimoto, K. Jinno and Y. Hirata, J. Chromatogr., 258 (1983) 81. C. Fujimoto, T. Oosuka and K. Jinno, Anal. Chim. Acta, I78 (1985) 159. C. Fujimoto, T. Morita, K. Jinno and S. Ckhiai, Chromatographia, 23 (1987) 512. C. Fujimoto, Y. Hirata and K. Jinno, J. Chromatogr., 332 (1985) 47.
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