Anal. Chem. 1987, 59, 415-418 (17) Hershberger, L. W.; Caliis, J. 6.; Christian, 0 . D. Anal. Chem. 1979, 57, 1444-1446. (18) Green, R. B. Anal. Chem. 1983, 55, 20A-32A. (19) Sepaniak, M. J.; Yeung, E. S. J . Chromatogr. 1980, 790, 377-383. (20) Niemczyk, T. M.; Ettinger, D. G.; Barnhart, S. G. Anal. Chem. 1979, 57, 2001-2004. (21) Vo Dinh, T.; Wild, U. P. J . Lumin. 1973, 6 , 296-303. (22) Savitzky, A.; Golay, M. Anal. Chem. 1964, 3 8 , 1627-1639. (23) McGuffin, V. L.; Zare, R. N. I n Chromatography and Separation Chemlstfy: Advances and Development; Ahuja, S., Ed.; ACS Symposium Series NO. 297; American Chemical Society: Washington, DC, 1986.
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(24) Wong, O.,Oread Laboratories, University of Kansas, personal communication. (25) Chen, R. F.; Scott, C.; Trepman, E. Biochlm. Biophys. Acta 1979, 578, 440-445.
RECEIVED for review June 2,1986. Accepted October 1,1986. Support of this work by the Kansas Commission on Advanced Technology and Oread Laboratories, Inca,is gratefully acknowledged.
Characterization of Thin-Layer Chromatographically Separated Fractions by Fourier Transform Infrared Diffuse Reflectance Spectrometry John M. Chalmers,* Moray W. Mackenzie, and John L. Sharp
Imperial Chemical Industries PLC, Petrochemicals and Plastics Division, Research and Technology Department, P.O. Box 90, Wilton, Middlesbrough, Cleveland TS6 8JE,England Roger N. Ibbett
School of Chemical Sciences, University of East Anglia, Norwich NR4 7 T S , England
A slmpie and convenlent procedure for characterlzlng thinlayer chromatographkalty separated fractions is described by use of an example taken from the plastlcs Industry. The method Is based upon the transfer of material from the thinlayer chromatographic plate to a potassium chloride pellet, followed by examination by diffuse reflectance Fourier f r a r t s f m Infrared spectrometry. The spectra recorded corn pare favorably wlth thelr absorbance counterparts. However, care must be taken with some substances slnce they may voiatliire readily in the absence of a strong Interaction with the substrate.
Diffuse reflectance (DR) and photoacoustic (PA) measurement techniques in combination with Fourier transform infrared (FTIR) spectrometry are becoming increasingly popular methods of examining powders or solids with matte surfaces. However, neither DR nor PA FTIR spectrometry has proved particularly well-suited to the in situ characterization of thin-layer chromatographically (TLC) separated spots. In both cases a significant proportion of the “fingerprint” region is obscured due to the presence of the substrate. In the DR spectrum this may be further complicated by the presence of reststrahlen features in regions where the substrate absorbs strongly, for example, the SiOSi stretching region of silica (1,2). PA FTIR spectrometry fares no better with silica since photoacoustic saturation occurs over the same region, that is 1300-900 cm-’ (3), and additionally some broadening of the adsorbate bands can be expected (3). Moreover, care must be taken to ensure that the TLC solvent is completely removed from the plate, otherwise residual solvent in the gaseous phase may interfere with the PA condensed phase spectrum (4). Unfortunately with both techniques difference spectrometry has proved to be of limited value, since strong interactions between the separated material and the substrate frequently result in significant wavenumber shifts in peak maxima. These interactions mean that spectral 0003-2700/87/0359-0415$01.50/0
libraries containing adsorbed species on a variety of substrates would be required (2, 3) before direct examination of TLC plates could be undertaken routinely with confidence. Griffiths et al. (5, 6)have described an elegant method of obtaining spectra from TLC spots directly using conventional transmission spectrometry. However, this approach uses specially prepared infrared transparent TLC plates and is likely to be both costly and labor intensive. One obvious method of overcoming the problem of limited spectral information is to remove the TLC spot from the plate and either deposit the separated material onto a suitable alkali halide (e.g., KC1) disk or concentrate the eluate at the tip of a Wick-Stick. Griffiths (7) and Chalmers and Mackenzie (8) have shown that this approach generally produces better results than in situ measurement. There is no doubt that removal of the TLC spot from the plate increases the chances of contamination (8)and can, for certain materials, result in further reaction (2,4). However, in our opinion the advantages of obtaining a spectrum which covers the whole region 4000-500 cm-’ and allows a comparison to be made directly with absorbance spectra outweigh those of in situ measurement. In this paper we report a simple procedure for characterizing TLC spots based on transferring the eluate from the TLC plate into a KCl pellet and recording its diffuse reflectance FTIR spectrum. This approach can be used routinely with most commercially available TLC plates. As an example of the method, a series of polypropylene additives have been identified following conventional extraction procedures from granular samples and subsequent TLC separation.
EXPERIMENTAL SECTION Apparatus. A Harrick “Praying-Mantis” diffuse reflectance accessory was used for the infrared measurements. Spectra were recorded on a Nicolet 170SX FTIR spectrometer fitted with a broad band mercury cadmium telluride (MCT-B) liquid-nitrogen-cooled detector. Reagents. Merck Art 5554 aluminum-backed silica gel TLC plates containing F254 fluorescing agent for spot detection was 0 1987 American Chemlcai Soclety
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ANALYTICAL CHEMISTRY, VOL. 59, NO. 3, FEBRUARY 1, 1987 N
used in all cases. Alkali halide pellets were made from dried ball-milled Anal& potassium chloride. Four additives commonly present in commercial polypropylene were used in these investigations. These included three hindered phenol antioxidants, viz., 1,3,5-tris(3’,5’-di-tert-butyl-4’-hydroxybenzyl)-2,4,6-trimethylbenzene (Irganox 1330, Ciba-Geigy), pentaerythrityl tetrakis(3,5-di-tert-butyl-4-hydroxyphenylpropionate) (Irganox 1010, w (Topanol OC, ICI), Ciba-Geigy),3,5-di-tert-butyl-4-hydroxytoluene Z? and the slip additive cis-13-docosenamide,commonly referred to ’1 00 3600 Si00 2eoo 21100 2600 le00 1600 I400 li00 1600 800 600 URVENUMBERS as erucamide. A mixture of 98.5% volume HPLC grade toluene ? with 1.5% volume HPLC grade ethyl acetate was used for the chromatographic elution. AnalaR grade chloroform was used as the solvent for the subsequent spot removal. Procedures. Alkali halide pellets were made by compressing 0.7 g of dried KC1 powder in a 13-mm die to a pressure of approximately 500 psi. The resulting pellets were about 4 mm high and could be made reproducibly to within f0.5 mm. ?11 00?3600b 3200 JU ~~” To transfer an eluted material into a KC1 pellet a TLC spot 2600 P h O Zdca I600 I600 140 UWENUMBERS was cut out and placed metal backing downward in a 17 mm diameter, 50 mm high flat-bottomed glass sample tube. A KC1 Figure 1. Diffuse reflectance spectra of TLC separated fractions on pellet was then placed centrally on top of each spot and about KCI pellets: (a) 200 pg of erucamide (500 scans): (b) 200 pg of 2 mL of chloroform carefully pipetted down the inside of each Irganox 1010 (200 scans). tube. The tubes were than left at room temperature and the chloroform was allowed to evaporate. After about 1h the pellets were carefully removed for infrared examination. In preliminary experiments using colored materials it was observed that this procedure resulted in most of the material collecting in the top one-third (i.e. 1-1.5 mm) of the pellet. (These colored materials l a were methyl red and 3,3’,5,5’-tetra-tert-butyl-4,4’-stilbenequinone, the latter having solubility and elution properties similar to Topanol OC.) This method of spot removal was carried out on test mixtures URVENUMBERS containing the polypropylene additives at concentrations of 200, Nrn 50, and 5 pg per 5 p L of chloroform. Five microliters of a test mixture was micropipetted onto the base line of a TLC plate and eluted using the toluene/ethyl acetate solvent mixture. After elution, the plate was dried and examined under a UV lamp and the boundaries of the separated components were marked with a metal scribe. These materials were then transferred into KC1 pellets and their diffuse reflectance infrared spectra recorded. A mixture of two granular polypropylene (PP) samples was chosen as a representative test material since it contained three additives with a range of retention properties. One PP grade Flgure 2. Diffuse reflectance spectra of TLC separated fractions on contained 0.07% of Irganox 1010 and the other contained 0.11% KCI pellets: (a) 50 pg of erucamide (500 scans);(b) 50 pg of Irganox of Irganox 1330 and 0.16% of erucamide. Thirty grams of the 1010 (500 scans). PP mixture was Soxhlet extracted with diethyl ether for 15 h. The ether was then evaporated off and the residue refluxed with and Irganox 1010, respectively. Both materials can be iden5 mL of ethanol to precipitate any residual polymer. Finally the tified readily; however, at the 5-pg level we were unable to liquid was cooled and centrifuged. Twenty microliters of this obtain good “fingerprint” spectra of these materials. ethanol extract was transferred by a micropipet to the base line Of more concern at this stage was our inability to obtain of the TLC plate. Assuming quantitative extraction of the ada diffuse reflectance spectrum from a Topanol OC fraction, ditives from the polymer, the spot contained 42 pg of Irganox 1010, despite the fact that we were able to record spectra with good 66 pg of Irganox 1330, and 96 pg of erucamide. The plate was signal-to-noise ratios from TLC spots of Irganox 1330 which then eluted and dried and the areas of the separated additives were located as described above. After the spots were transferred has a similar R, value. We believe this can be attributed to into KCl pellets, their diffuse reflectance infrared spectra were the volatility of Topanol OC on the particle surfaces of the recorded. KC1 substrate since it is clearly retained on silica gel. This The R values of erucamide, Irganox 1010, Irganox 1330, Tois supported by the observation that providing diffuse repanol Od, and the stilbenequinone were 0, -0.3, -0.95, -0.9, flectance spectra are recorded soon after transfer of the and -0.8, respectively. fraction from the TLC plate, then spectra characteristic of All infrared spectra were recorded at 4 cm-’ resolution. A Topanol OC may be obtained readily. Figure 3 shows KCl freshly prepared hC1 pellet was used t o provide the single beam pellet diffuse reflectance spectra recorded from 200 pg of background for FTIR diffuse reflectance measurements. Topanol OC and 50 pg of Irganox 1330 TLC fractions. The RESULTS AND DISCUSSION diffuse reflectance spectra shown in Figure 4 were recorded successively over a period of about 20 min from a 50-pg ToIn the development of our preferred procedure, use was panol OC spot transferred into a KCl pellet as described above made of a test mixture containing 200 Mg of erucamide, Irganox but without allowing 1 h to ensure complete evaporation of 1010, and Topanol OC per 5 pL of solvent. Figure l a shows the chloroform. These spectra show how rapidly the Topanol the diffuse reflectance spectrum recorded from a 200-pg OC volatilizes off the KC1. At the time of recording Figure erucamide spot following transfer to a KC1 pellet, while Figure 4a a trace of the chloroform transfer solvent was evident in l b shows the diffuse reflectance spectrum recorded from a the infrared spectrum, while after 25 min most of the Topanol 200-pg Irganox 1010 spot. Surprisingly, we were unable to OC had disappeared and the spectral detail was poor (see obtain a spectrum from the Topanol OC spot (see later). below). This does, however, highlight a limitation of the Figure 2 shows the spectra recorded from KC1 pellets contechnique. taining material transferred from BO-pg spots of erucamide
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ANALYTICAL CHEMISTRY, VOL. 59, NO. 3, FEBRUARY 1, 1987
Flgure 3. Diffuse reflectance of TLC separated fractions on KCI pellets: (a) 200 pg of Topanol OC (500 scans): (b) 50 pg of Irganox 1330 (1000 scans).
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Figure 4. Diffuse reflectance spectra of 50 pg of Topanol OC TLC fraction on a KCI pellet: (a) 1000 scans; (b)300 scans: (c) 100 scans: (d) 100 scans. Time Intervals between start of recording spectra were as follows: a-b, - 6 mln; b-c, -9 min; c-d, - 7 mln.
The problem experienced at the 5-pg level did not appear to be one of transfer of material from the TLC plate into the KC1 pellet, since the preliminary experiments using colored compounds had shown that most of the material was transferred to the upper third of the pellet. Also, little difference in signal intensity was observed between the spectrum shown in Figure 1b and that of an equivalent quantity of Irganox 1010 deposited directly onto the surface of a KC1 pellet. Although, it was possible to observe many of the absorption bands, material identification was made difficult due mainly to overlapping impurity bands (mostly aliphatic hydrocarbon). Preconcentration on the TLC plate prior to transfer might improve the signal-to-noise characteristics of the diffuse reflectance spectra; however, this may be accompanied by a corresponding increase in impurity levels. During the development of the procedure for removing spots from the plate, several other techniques were evaluated. These included (1)a second elution at 90" to the separation stage during which the material was transferred to a strip of KC1 powder along one edge of the TLC plate and (2) overcoating the TLC plate with KC1 followed by solvent drenching. Both
417
Figure 5. Absorbance spectra of room temperature capillary layers prepared from the melt between KBr plates: (a) erucamlde (500 scans); (b) Irganox 1010 (500 scans).
of these procedures suffered from excessive spreading of the spot material. In an attempt to avoid cutting the plate, we also investigated placing a pellet on the eluted spot and surrounding it with a filter paper ring with a hole which just fitted around the pellet. Several milliliters of solvent was pipetted around the ring periphery and allowed to evaporate through the KC1 by capillary action. Although good quality diffuse reflectance spectra could be recorded from material transferred in this manner, it was less effective than the method described above. At this point in the discussion, two other practical observations are worthy of note. These concern the possible effect of sample preparation on the spectral Characteristics used for identification and library-searching purposes and the influence of water vapor imbalance on Kubelka-Munk spectra. The effect of sample preparation on spectral characteristics is important when considering libraries of spectra for identification purposes. The procedure outlined in this paper is more likely to yield spectra which may be compared directly with transmission data in commercially available spectral libraries than if in situ DR examination was undertaken. Indeed White (3) found that PA FTIR spectra of in situ TLC spots bore little resemblance to KBr disk spectra and proposed that meaningful comparisons could only be made with a library of difference spectra of adsorbed substances on silica gel. Fuller and Griffiths (7)have shown that material deposited from solution as a thin layer on KCl produces a DR spectrum whose relative band intensities can be markedly different from the DR spectrum of the solid material prepared by grinding the sample with KC1. Similar effeda are, of c o w , well-known in transmission spectroscopy (9). The abosorbance spectra of erucamide and of Irganox 1010 shown in Figure 5 are typical of those recorded from a liquid paraffin mull, a thin film cast from solution, or a room temperature capillary layer prepared from the melt as shown here. However, if neat erucamide and Irganox 1010 are prepared as alkali halide disks, their absorbance spectra can look quite different. Therefore, apart from minor changes in the relative intensities of bands, the diffuse reflectance spectra shown in Figure 1 are much more like their absorbance counterparts shown in Figure 5, since elution into the pellet yields thin layers of solute on the KC1 crystals. Our second practical observation highlights the effect of a slight change in spectrometer purging between collecting the background and sample spectra. In a transmission measurement this would normally be accommodated by simply subtracting a water vapor spectrum from the absorbance spectrum of the sample. However, it should be noted that
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ANALYTICAL CHEMISTRY, VOL. 59, NO. 3, FEBRUARY 1, 1987 N.
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FIgure 6. P((0H) and a(-) regions of Irganox 1010: (a) absorbance spectrum of a capillary layer; (b) diffuse reflectance spectrum of TLC fraction on a KCI pellet after water vapor absorptions have been removed using an absorbance mode manipulation; (c) absorbance spectrum of Irganox 1010 in a KCI disk; (d) diffuse reflectance spectrum of TLC fraction on a KCI pellet after attempted removal of water vapor absorptions using a Kubetka-Munk generated water vapor spectrum.
in the Kubelka-Munk mode cancellation is difficult to achieve since the Kubelka-Munk algorithm has the effect of enhancing the intensities of water vapor bands overlapping strong absorption bands of the sample relative to those occurring where the sample exhibits essentially no absorption. For example, at first glance it might appear that the diffuse reflectance spectrum of Irganox 1010 in Figure 1 shows some evidence of structure on the low wavenumber side of P(OH)and similar splitting of P(C=O) to that observed in the KC1 disk absorbance spectrum shown in Figure 6c. However, these observations are misleading since they occur as a result of the presence of water vapor absorptions. Good compensation can be obtained if the subtraction is carried out in the absorbance mode (Le., log,, R,/R, where Ro and R are the reflectance of the background and sample, respectively), this being a linear function. The sample reflectance spectrum is then regenerated prior to application of the Kubelka-Munk algorithm. This procedure was applied to the reflectance data used to produce the Irganox 1010 diffuse reflectance spectrum shown in Figure l b , resulting in the spectrum shown in Figure 6b. It can be seen that the diffuse reflectance spectrum of Irganox 1010 corrected as described above and the absorbance spectrum of Irganox 1010, prepared as a capillary layer from the melt (Figure 6a), are essentially identical in both the s(C=O) and s(0H) regions. It was observed that for both Irganox 1010 and Irganox 1330 the method of sample preparation had little effect on the position of the phenolic P(OH) band. However, with Topanol OC a shift of about 24 cm-' to higher wavenumber was observed in the KC1 pellet diffuse reflectance spectrum when compared to either the melt capillary layer or the KC1 disk spectrum, this being consistent with a decreased interaction with the substrate and presumably accounting for the observed volatility from the KC1 pellet. These digressions to discuss the effects of sample preparation and water vapor imbalance were felt important since
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Flgure 7. Diffuse reflectance spectra (500 scans) of TLC separated fractions on KCI pellets of extract from polypropylenegranules: (a) erucamide; (b) Irganox 1010; (c) Irganox 1330.
they highlight areas where misinterpretation might arise. We believe the reproducibility of the technique described here is better than simply using KC1 powder since the surface presented to the incident radiation is more uniform. Moreover, the technique appears to be as sensitive as the WickStick method (8) but less prone to contamination. The success of the technique is evident when the spectra of the additives extracted from the polypropylene sample and separated by TLC (Figure 7) are compared with the spectra shown in Figures 1-3 and 5. There is no doubt that this technique offers a simple and effective means of identifying TLC fractions for quantities of material in excess of about 40 Fg, and probably 10-20 Fg in favorable circumstances.
ACKNOWLEDGMENT The authors are indebted to I. D. Newton for help given with the thin-layer chromatography and to N. Sheppard and D. H. Chenery (UEA) for helpful discussions. LITERATURE CITED (1) Fuller, M. P.;Grlffis, P. R. Anal. Cbem. 1978. 50, 1906-1910. (2) Zuber, G. E.; Warren, R. J.: Begosh, P. P.: O'Donnell, E. L. Anal. Cbem. 1984, 56, 2935-2939. (3) White, R. L. Anal. Chem. 1985, 5 7 , 1819-1822. (4) Lloyd, L. 6.; Yeates, R. C.; Eyring, E. M. Anal. Cb8m. 1982, 5 4 , 549-552. (5) Perclval, C. J.; Griffiths, P. R. Anal. Chem. 1975, 4 7 , 154-156. (6) Gomez-Taylor, M. M.; Griffiths, P. R. Appl. Spectrosc. 1977, 3 1 , 528-530. (7) Fuller, M. P.; Grlffkhs, P. R. Appl. Spectrosc. lS80, 3 4 , 533-539. (8) Chalmers, J. M.; Mackenzle, M. W. Appl. Spectrosc. 1985, 39, 634-641. (9) Beaven, G. H.; Johnson, E. A.; Willis, H. A,: Miller, R. G. J. Molecular Spectroscopy; Heywood: London, 1961; Chapter 8.
RECEIVED for review June 23,1986. Accepted October 2,1986. R.N.I. thanks the SERC for a CASE award during the tenure of which this work was carried out.
