Accelerated Articles Anal. Chem. 1994,66, 1937-1940
Development of Microchannel Thin-Layer Chromatography with Infrared Microspectroscopic Detection S. P. Bouffard, J. E. Katon,. A. J. Sommer, and N. D. Danlelson' Molecular Microspectroscopy Laboratow, Department of Chemistw, Miami Universiw, Oxford, Ohio 45056
A new thin-layer chromatography (TLC) technique termed microchannel TLC, is described. Channels, having the dimensions 400 pm X 200 pm X 5 cm ( W X D X L), have been packed with a zirconia stationary phase and used for TLC. Subsequent infrared microspectroscopic detection of organic dyes separated in these channels provided excellent diffuse reflectance spectra and an improvement in the minimum identifiable quantity by a factorof about 500 times over previous TLC work usingmicroscope slides. This technique also requires smaller amounts of sample and stationary phase compared to conventional TLC techniques. A practical application of the separation and identificationof four polyaromatichydrocarbons (two of which are isomers) is also shown. Thin-layer chromatography (TLC) can be an efficient, inexpensive, and effective means of separating components in a given mixture. Likewise, Fourier transform infrared spectroscopy can offer a simple and effective means to identify the components after separation. A variety of methods has been developed over the years to link TLC with FT-IR detection. The most common method involves the use of diffuse reflectance infrared Fourier transform spectroscopy (DRIFTS) for detection of separated components, and it has been summarized in a review article.' One of the biggest problems with in situ DRIFTS-TLC is the strong background absorbance that common TLC stationary phases, such as silica and alumina, exhibit in the mid-infrared region. This background interference greatly decreases the breadth of application of the technique since only small regions of the infrared spectrum remain suitable for compound identification. TLC on silica with near-infrared spectroscopy detection from 2000 to 2500 nm is one approach that avoids this background interferencea2 A variety of techniques involving the transfer of analyte from the TLC plate to a KC1 or KBr pellet for ( I ) Brown, P. R.; Beauchemin, B. T. J. Liq. Chromutogr. 1988, 11, 1001. (2) Ciurczak, E. W.; Murphy, W. R.; Mustillo, D. M. Specrroscopy 1990,6,34.
0003-2700/94/0366-1937$04.50/0 0 1994 Amerlcan Chemical Society
transmission infrared analysis3 or to a diffuse reflectance cup for DRIFTS e x a m i n a t i ~ nhave ~ * ~been reported. A transfer TLC accessory has also been developed to examine analyte spots upon removal to cups containing IR-transparent glass.6 However, for all TLC spot removal methods, the chance of contamination is increased and sample handling can lead to irreproducible results. An approach taken in our laboratory has been to eliminate the strong background interference by the stationary phase through the employment of zirconia instead of silica or alumina. This work clearly showed that zirconia as a stationary phase for TLC-DRIFTS has distinct spectral advantages over silica and alumina stationary phases. In situ DRIFTS-TLC using zirconia as a stationary phase provides a means to obtain DRIFTS spectra of a quality comparable to transmission spectra without sacrificing the chromatographic efficiency found with either silica or alumina.' Our approach has now turned to the optimization of TLC for infrared microspectroscopicdetection. Our goal was to develop a TLC technique which resulted in a smaller analyte spot size than that obtained in conventional TLC so that the power of infrared microspectroscopicdetection and identification could be fully utilized. If TLC was performed in small channels, it was found that sample spreading could be kept to a minimum, yielding a smaller analyte spot size. We report the use of zirconia-packed channels having the dimensions of 400fim X 200 pm X 5 cm for in situ identification of separated TLC analytes with diffuse reflectance infrared microspectroscopy. Using microchannel TLC, we have been able to enhance our detection limits (now about 1-10 ng) by Issaq, M. J. J . Liq. Chromarogr. 1983, 6, 1213. Chalmers, J. M.; Mackenzie, M. W.; Sharp, J. L.; Ibbett, R. N. Anal. Chem. 1987, 59, 415. Iwaoka, T; Tsutsumi, S.; Toda, K.; Suzuki, F. Sunkyo Kenkyusho Nenpo 1988, 40, 39; Chem. Abstr. 1989, 111, 36035q. Shafer, K. M.; Griffiths, P. R.; Shu-Qin, W. Anal. Chem. 1986, 58,2708. Danielson, N. D.; Katon, J. E.; Bouffard, S. P.; Zhu, 2 . Anal. Chem. 1992, 64, 2183.
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a factor of approximately 500 over previous work using zirconia-coated microscope slides. Analyte spot sizes are typically 400 pm X 600 pm with the 400 pm corresponding to the channel width. An example of a separation and identificationof four polyaromatic hydrocarbons (twoofwhich are isomers) is also shown. EXPERIMENTAL SECTION Instrumentation. All infrared spectra were acquired using either a Perkin Elmer (Norwalk, CT) Model 1800 Fourier transform infrared spectrometer equipped with a SpectraTech (Stamford, CT) IRPLAN microscope or a Perkin Elmer Model 1600 Fourier transform infrared spectrometer equipped with a Perkin Elmer infrared microscope. Both systems were purged with dry nitrogen and utilized liquid nitrogen-cooled HgCdTe detectors. In all cases, apertures were used to define the sampling area. For samples which could be visualized, the area defining the sample size was apertured. For samples which could not be visualized, apertured areas with the dimensions of 160pm X 240pm wereutilized. Threechannels, 400 pm X 200 pm X 5 cm ( W X D X L)spaced 0.64 cm apart, were grooved into a brass plate by the Miami University machine shop. Reagents. The crystalline monoclinic zirconia microspheres, a gift from the 3M Co. (Minneapolis, MN), were approximately 5-10 pm in size with a surface area of 25 m2/ g, a pore diameter of 220 A, and a 45 vol 9% porosity. Rhodamine B was supplied by Eastman Kodak (Rochester, NY) while the other dyes were from a variety of sources. Phenanthrene, 1,lO-phenanthroline,phenanthridine,and 7,8benzoquinoline were supplied by Aldrich (Milwaukee, WI). The methanol solvent was HPLC grade; the hexane solvent was UV grade; and the chloroform solvent was reagent grade (99%).
Procedure. The TLC stationary phase was packed in the grooves by pouring a zirconia-methanol slurry, about 0.2 g of zirconia to about 3 mL of methanol, onto the face of the brass plate containing the channels. After methanol evaporation, the edge of a razor blade was passed over the face of the plate to eliminate the excess zirconia that overfilled the grooves. Typically, 0.1 pL of sample was spotted into one end of a channel with a 1-pL syringe. The brass plates were placed vertically into a covered container for development. After development, a period of at least 2 h was allowed for the evaporation of a particular mobile phase. For colorless analytes, visualization via ultraviolet light was necessary to locate the spots in the channels after TLC development. Diffuse reflectance spectra were taken by coadding 256 scans (typically) at 4-cm-l resolution using a medium BeerNorton apodization function. The ratios of spectra of the analyte spots on zirconia to a background spectrum collected from another region in the channel were determined. Transmission spectra were gathered by placing a flattened sample onto a KCl window.8 RESULTS AND DISCUSSION
In this work, 400-ccm channels were employed for easy monitoring of the chromatographythrough a stereomicroscope (8) Katon. J. 25, 173.
E.;Sommcr, A. J.; Lang, P.L.Appl. Spectrmc. Rev. 1989-1990.
1938 Ana&ticaIChemktry, Vol. 66, No. 13, July 1, 1994
in the early stages of the experiments using the dyes and indicators. The 200-pm channel depth corresponds to the typical thickness of stationary phase on conventional TLC plates. Future work could utilize narrower and shallower channels, which should further enhance detection limits. Practical limitations using diffuse-reflectance microspectroscopic detection for either of the two dimensions are on the order of approximately 100 pm. In order to characterize the microchannel TLC technique, our first step was to repeat the initial TLC work performed on zirconia-coated microscope slides.’ This work examined the separation and identification capabilities for some common dyes and indicators using TLC-DRIFTS. The mobile phase used for development in the channelswas a 90: 10 chloroformmethanol mixture and the development time was 20 min. Methyl orange and fluorescein gave Rfvalues of 0, indicating strong retention on the zirconia stationary phase. Rhodamine B and methyl red gave Rfvalues of 0.1 and 0.4, respectively, indicating moderate retention. Methylene green and methylene blue gave Rf values of 1, indicating weak retention. These results compare well to previous except that rhodamine B and methyl red were more weakly retained using standard zirconia-coated TLC plates by a factor of about 10 and 2.5, respectively. Retention seems to be dependent on the acidic functional groups (either a carboxyl or a sulfonate) present in the structure of all of these compounds. The spot sizeof thedye mixture, utilizinga 0.1-pL injection volume, was approximately 400 pm X 600 pm. After elution, the methyl orange and fluorescein spots, which show no movement in the zirconia-packed channels, retained the initial dimensions. Rhodamine B, which did elute, showed no signs of streaking and also retained its initial dimensions. Methyl red did show signs of streaking, and its dimensions changed to approximately 400 pm X 800 pm after elution. Methylene blue and methylene green, which eluted with the solvent front, actually showed a more concentratedspot size with dimensions of approximately 400 pm X 400 pm. Spot sizes will vary with different injection volumes. The use of an automatic analyte spotter would also contribute to more accurate and reproducible spot volumes. It should be noted that Rf values are not as simple to calculate in microchannel TLC as they are in standard TLC because of the inability to visualize the solvent front in the channels. This problem can be remedied by the use of an internal standard that is known to elute with the solvent front. This internal standard need not be present in the sample mixture but can be spotted in an adjacent channel on the same TLC plate. The standard microscope slide has a width of 2.54 cm, and one can easily envision up to 25 channels in that amount of space. The use of microchannel TLC significantly enhanced the detection limits of analytes compared to standard TLC. Figure 1a-c shows three diffuse reflectance spectra of differing amounts of rhodamine B detected on zirconia in the channels. Figure l a presents a spectrum of 400 ng of rhodamine B after elution. This is a very clean spectrum which closely matches the transmission spectrum previously published.’ The limit of detection (LOD) given previously using standard TLC with a zirconia stationary phase was 300 n g 7 The spectrum in Figure 1b is of 50 ng of rhodamine B, and it is similar to the
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400-ng spectrum. Figure ICshows a spectrum of 12.5 ng of rhodamine B and many of the infrared bands are still evident. This spectrum represents the minimum identifiable quantity (MIQ) with about a 500-fold enhancement over our previous work. Clearly this indicates a possible LOD in the 1000100-pg range. Figure 2a-c illustrates that low detection limits for a variety of organic compoundsare possible using zirconia microchannel TLC. Panels a and b of Figure 2 present a reference transmission spectrum of methyl orange and a DRIFTS spectrum of 50 ng of methyl orange after development, respectively. The DRIFTS spectrum can easily be identified as that of methyl orange by comparison to its reference transmission spectrum. Figure 2c represents an LOD for methyl orange of 9 ng. To examine the practical nature of zirconia microchannel TLC, the separation and identification of two isomers, 7,8benzoquinoline and phenanthridine, as well as two similarly structured compounds,phenanthrene and 1,lo-phenanthroline, was studied. The structures for these compounds are shown in Figures 3 and 4. Hexane was used as the mobile phase and the development time was 15 min. Initial spot sizes, for 0.1pL injection volumes, were again approximately 400 pm X 600 pm. These dimensions did not appear to change for any analyte upon elution. 1,lO-Phenanthroline was retained the strongest on zirconia, yielding an Rfvalue of 0. The two isomers, phenanthridine and 7,8-benzoquinoline, show moderate retention but were still easily separated, yielding Rf values of 0.1 and 0.32, respectively. Phenanthrene was unretained (Rf value of 1) and used as an internal standard,to calculate the Rfvalues. Reproducibility of the Rfvalues is at least within f0.03 units
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for three different separations using a micro-TLC plate containing three channels. Separation in these cases is based on the interaction of the nitrogen in the compounds with the zirconia substrate. 1,lo-Phenanthroline, with two nitrogen atoms, did not move from the initial spot location. This was expected since both nitrogen atoms can interact simultaneously with the zirconia hydroxyl groups. The two isomers, with one AnaWcal Chemism, Vd. 66,No. 13, Ju& 1, 1994
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nitrogen atom each, did move and steric hindrance of the nitrogen in 7,8-benzoquinoline with the zirconia causes it to be retained less than phenanthridine. Figure 3a-d shows the reference transmission spectra of all four compounds. Though all spectra have similar bands in the infrared region shown, each can be differentiated from the others upon close examination. The spectrum of phenanthroline indicates only two major bands between 1400 and 1500 cm-l. The presence of the doublet at 1450 cm-l and the band at about 1250 cm-l in the phenanthridine spectrum (Figure 3c) allows the distinction between this spectrum and that of its isomer, 7,8-benzoquinoline,which has a strong band at 1400 cm-l.
Figure 4a-d shows the DRIFTS spectra of the separated compounds. These spectra match fairly well with their corresponding reference transmission spectra. The transmission spectrum of 1,IO-phenanthrolineis of the monohydrate, so a band at about 1650 cm-l due to the OH bending motion of the water in this molecule is not apparent in the DRIFTS spectrum of the TLC-developed compound (Figures 3d and 4d). The band at about 1400 cm-I for 7,8-benzoquinoline and the doublet at 1450 cm-* for phenanthridine clearly distinguishes the two isomers. These spectra represent the MIQ (2-3 pg) for each compound using zirconia microchannel TLC. Detection limits for these compounds differ by almost 3 orders of magnitude from those of the indicators in the above experiments. A partial explanation for the higher detection limits could stem from the use of the apertures for this experiment which were intentionally smaller than the analyte because it could not be visualized under the microscope. The detection limits are still respectable because the compounds are known to be rather weak infrared absorbers. This application represents a simple, efficient, and practical method to separate and identify isomeric components in a mixture.
CONCLUSION This work represents a significant development in the area of TLC with DRIFTS detection. TLC separations in small channels provide for a smaller analyte spot size and thus a more concentrated area of analyte compared to conventional TLC. This enables a much improved detection limit for TLC with infrared microspectroscopic detection. The employment of zirconia as a stationary phase provides a much wider infrared detection window when compared to common TLC substrates. Zirconia microchannel TLC with infrared microspectroscopic detection also requires smaller amounts of sample and stationary phase than does conventional TLC. The channels are reusable and can be easily packed with stationary phase. Zirconia microchannel TLC should be amenable to infrared mapping of separated components via a computer-controlled stage. Recelved for review March 29, 1994. Accepted Apdi 22, 1994.8 Abstract published in Aduance ACS Absrracrs, May 15, 1994.
