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Anal. Chem. 1887, 59, 411-415
Determination of Amino Acids at Subfemtomole Levels by High-Performance Liquid Chromatography with Laser-Induced Fluorescence Detection Mark C. Roach' and Marlin D. Harmony* Department of Chemistry and Center for Bioanalytical Research, University of Kansas, Lawrence, Kansas 66045
Laser-induced fluorescence detection methods have been used for the determination of o-phthaialdehyde (0PA)- and naphthalenedialdehyde (NDA)-derivatlzed primary amino acids by high-performance liquid chromatography. The UV lines of an argon-Ion laser were utilized for the OPA studies, while the visible 457.9-nm line was used for the NDA work. With the cell design of Yeung and co-workers, detectlon llmits obtained by using OPA were found to be In the range of 4-15 fmol, while the detection limits obtained by using NDA were In the 200-500 amol range. Additional sensitivity (down to at least 100 amol) has been achieved by using a l-mm microbore column. Dipeptides have also been detected at low levels.
Amino acid determinations have played an important and varied role for many years in the characterization of polypeptides and proteins as well as in the areas of food and clinical chemistry among others. Prior to the advent of reverse-phase liquid chromatography, ion-exchange chromatography with postcolumn derivatization by ninhydrin was the standard method of choice (1,2). The sensitivity of the ninhydrin method, utilizing UV absorption for detection purposes, was limited to the subnanomole range (3). More recently a wide variety of more sensitive precolumn derivatization methods have been developed for use with liquid chromatography. Phenyl isothiocyanate has been shown to provide picomole sensitivities for both primary and secondary amines when using UV absorption detection (4). Numerous useful reagents have also been developed to take advantage of the intrinsically high sensitivity of fluorescence detection. Fluorogenic reagents such as dansyl chloride (5),fluorescamine (6),nitrobenzoxadiazoles (7,8), and o-phthaladehyde (9) have all served as relatively sensitive derivatization reagents for amino acid analyses. o-Phthaladehyde (OPA), in the presence of a thiol such as mercaptoethanol, has been the most widely used reagent in this class for primary amines because of its low cost, rapid reaction, and highly fluorescent adducts (absorption maxima a t 230 and 340 nm and emission maximum at 455 nm). When precolumn derivatization methods are used the reported detection limits with OPA and conventional (nonlaser) fluorescence detection are in the vicinity of 100 fmol (10, 11). In an effort to push the amino acid detection limits still lower, researchers at the University of Kansas have been seeking and exploring synthetic methods for producing new and improved fluorogenic reagents. One such reagent which appears to have excellent potential for primary amino acids and peptides is 2,3-naphthalenedialdehyde (NDA), which when used in the presence of cyanide as the nucleophile, yields adducts of excellent thermal stability and high quantum yields (12-14). In addition to an intense absorption band in the 'Present address: Department of Chemistry, Stanford University, Stanford, CA 94305. 0003-2700/87/0359-0411$01.50/0
a
CH=NR
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-
UV at approximately 250 nm, the resulting 1-cyano-2-alkylbenz[flisoindoles exhibit two weaker excitation maxima in the visible region a t approximately 420 and 440 nm with a fluorescence maximum a t 490 nm. The visible absorption bands are especially desirable since excitation in this range eliminates many of the potential interferences caused by naturally fluorescent contaminants present in biological fluids. On the other hand, the relatively short range between the absorption and emission bands makes Raman band interferences somewhat more troublesome. In any case, with conventional non-laser-fluorescence detection in HPLC separations, detection limits in the range of 10 fmol have been realized (14). The excellent characteristics of the laser as a fluorescence excitation source have provided the opportunity for obtaining much improved detection limits in a variety of HPLC experiments (15-18). However, the latest technology in laserinduced fluorescence (LIF) detection has apparently not been applied to extending the detection limits of amino acids to still lower levels. Therefore, in the present work, we report state-of-the-art HPLC-LIF studies of amino acids using both the well-known OPA-derivatization method and the recently developed NDA-derivatization procedure.
EXPERIMENTAL SECTION HPLC-LIF System. The HPLC system consisted of two LKB Model 2150 dual piston pumps (Bromma, Sweden), an LKB Model 2152 controller with a 2040-203 low-pressure mixing valve, and a 150 X 4.6 mm Hypersil ODS column packed at the University of Kansas. All connections between injector and column and between column and detection cell were made by using short pieces of stainless steel tubing (0.01 mm i.d.). The fluorescence detector, modeled after that of Yeung et al. (19), utilized fiber optics for collection of the laser-induced fluorescence. The detector cell consisted of a 2-cm length of 1-mm4.d. quartz capillary inserted into the bottom of a stainless steel tee. One end of a 1-m length of fused-silica optical fiber (1-mm core diameter, selected) was inserted into the quartz cell through the top of the tee, and the column effluent flowed into the cell via the third (central) arm of the tee. The opposite end of the optical fiber was terminated at the focus of a collimating lens interfaced to a photomultiplier tube (PMT) housing by an interference filter. For the OPA studies the selected filter was centered at 450 nm with AA = 35 nm, while for the NDA studies the filter was centered at 490 nm with AA = 10 nm. A cooled EM1 9558A photomultiplier tube was used for fluorescence detection, and the PMT signals were amplified and shaped by using a Model 1121A amplifier-discriminator (EG&G Princeton Applied Re@ 1987 American Chemical Society
412
ANALYTICAL CHEMISTRY, VOL. 59, NO. 3, FEBRUARY 1, 1987
search). Pulses from the 1121A were counted with a Model 1109 photon counter (EWG), whose output was fed to a chart recorder using a digital to analog (D/A) converter (Model 1109/99, EG&G) or to a Zenith 2-100 computer using a digital option board (constructed by the Instrument Design Lab, University of Kansas). Voltage and temperature settings of the PMT and the amplifier-discriminator threshold voltage were chosen by using the procedure of Niemcyk et al. (20). The optimum values for the three parameters were as follows: PMT voltage, -1650 V dc; temperature, -16.3 "C; discriminator threshold voltage, 1.25mV. Counts were normally accumulated for a period of 1s before being output to the computer or recorder. The column effluent flowing through the quartz detector cell was irradiated with the appropriate output of a Spectra-Physics 171-17 argon-ion laser. For the OPA-amino acid studies the UV multiline output (334-363 nm) was utilized with available power levels in the range of 0-350 mW, while for the NDA studies the 457.9-nm line was used with available power up to 900 mW. Superradiant emission accompanying the UV lines was reduced by a Schott UG-11 filter, while amplified stimulated emission was eliminated from the visible l i e by means of an interference filter centered at 460 nm. After passage through the appropriate filter, the laser beam was focused into the quartz capillary cell by a 100-nm focal-length cylindrical lens mounted in an X, Y, Z pasitioner. The distance of the focused laser beam from the optical fiber was adjusted by a Model FP-1 fiber-optic positioner (NRC, Fountain Valley, CA) in which the quartz capillary tube was inserted. The laser beam was focused into the quartz capillary at a distance of -3 mm from the tip of the inserted fused-silica optical fiber. Final adjustments of the focusing lens and the optical-fiber position were made to optimize the signal to noise (S/N) ratio of the chromatographic peaks. A conventional Hypersil ODS column (150 X 4.6 mm i.d.) was used in most of the experiments, although a few microbore column experiments were performed using a Spheri-5 RP-18 column (250 x 1mm i.d., Brownlee Labs). In this case the 1-mm4.d. detector cell described was replaced with a 320-pm4.d. fused-silica capillary cell, and the 1-mm optical fiber was replaced by a 200-pm fiber. Also, the column effluent was run directly into the top of the cell and the optical fiber was inserted vertically through the bottom of the cell. Laser irradiation was accomplished as before by focusing the laser beam into the cell at right angles to the cell axis. Flow rates were maintained at 1mL/min for the microbore column. Reagents. HPLC grade acetonitrile and methanol (Fisher Scientific, Fairlawn, NJ) were used without further purification. Deionized, distilled water was used for all purposes. Amino acids, o-phthalaldehyde, and 2-mercaptoethanol were purchased from Sigma Chemical Co., while reagent grade sodium cyanide was obtained from Aldrich Chemical Co. Naphthalenedialdehyde was synthesized locally by published methods (12-14). Reagent grade sodium acetate and boric acid were used to prepare buffers. Preparation of Amino Acid Standards. Stock solutions of amino acids were prepared by weighing out 1-mg amounts and dissolving in water. These solutions were stored at 4 O C and used for up to a week. Standard mixtures were prepared by mixing the appropriate volumes of the various amino acids followed by dilution with water to yield concentrations of 50 pmol/pL for each amino acid. OPA-Derivatizing Reagent. Fifty milligrams of ophthaladehyde was dissolved in 1.25 pL of HPLC-grade methanol. Fifty microliters of 2-mercaptoethanol (MCE) and 11.2 mL of 0.4 M borate buffer (pH 9.5) were added and the solution was thoroughly mixed, sparged with helium, and stored in the dark at 4 OC. The solution was allowed to stand for 24 h before use to minimize fluorescence from impurities present in the reagents. The OPA-MCE reagent was used for 3 days before replacement with freshly prepared reagent. OPA-Derivatization Procedure. Thirty microliters of the standard amino acid mixture was mixed thoroughly with 30 pL of the derivatizing solution. After a 1 min reaction time, 30 fiL of sodium acetate (0.1 M, pH 7.0) was added and the solution was diluted t o 3 mL. The solution was mixed and a 20-fiL sample was injected onto the column. Naphthalenedialdehyde Solution. A stock solution of NDA (12-14) was freshly prepared each day by dissolving 5 mg of the
reagent in 1 mL of HPLC-grade methanol. Sodium Cyanide Solution. An aqueous stock solution of sodium cyanide at 0.1 M was prepared from reagent grade material and used for periods of up to a month before replacement. NDA-Derivatization Procedure. Aliquots of the amino acid standards were mixed with a 100-fold excess of cyanide, and the solution made basic by adding borate buffer (0.1M, pH 9.5). A 100-fold excess of NDA was then added and thoroughly mixed. The resulting solution was allowed to stand for 15 min before diluting with distilled water to achieve the final desired concentration. This particular order of reagent addition is important in minimizing possible benzoin condensation reactions. Twenty microliters of the derivatized solution was injected in the HPLC runs when usingthe standard column, while only 1p L was injected on the microbore column. In a few experiments with dipeptides, the derivatization procedure was identical except that the pH was lowered to 8.5 to increase the reaction rate. RESULTS AND DISCUSSION Optimization a n d System Performance. Considerable effort was placed upon optimizing the optical irradiation and detection system for maximum sensitivity. The black glass filter inserted into the irradiation beam in the UV (OPA) studies and the interference fiiter used similarly in the visible (NDA) studies played important roles in reducing background radiation at the detection wavelength. A comparison of cylindrical and spherical focusing lenses showed that the former provided S/N improvements of at least a factor of 2 for the standard column. For the microbore column there appeared to be no advantage in using the cylindrical lens, so the more easily focused spherical lens was utilized. A comparison of monochromator vs. interference filter for fluorescence signal processing indicated that the more elaborate monochromator system provided no significant advantages. Indeed, because of the broad emission band and high available excitation power, the filter system yielded somewhat enhanced detection limits. Careful focusing of the laser beam into the detection cell and precise positioning of the optical fiber were important in decreasing scattered radiation from the cell walls and in enhancing the fluorescence signal detection efficiency. This matter has been discussed previously by other workers (19).
System S/N ratios were investigated with UV power levels up to 350 mW and visible (457.9 nm) power levels up to 900 mW for the OPA and NDA experiments, respectively. In both cases, optimum S / N ratios (and detection limits) were generally found near the upper level of the possible ranges. In all the experiments reported here, the irradiation power levels were fixed at 250 mW in the UV region and 800 mW in the visible region. In the light-stabilization mode the long-term amplitude stability of the laser was considerably better than 1 %. The detection system operated linearly except at very high signal levels, in which case pulse pileup effects became significant. Since this occurred only at high analyte concentrations, this effect caused no problems. In any case, the problem could be easily avoided by use of a neutral-density filter placed in front of the PMT or by operating at lower laser power levels. Chromatographic runs were performed with a variety of solvent mixtures under both isocratic and gradient conditions. The HPLC-LIF system was found to be reliable and trouble-free and was run day-to-day with no substantial adjustments. Reproducibilities in peak heights and retention times for multiple injections were generally better than 1%. Chromatography of OPA-Derivatized Amino Acids. Figure 1 presents a chromatogram for a sample injection of 15 amino acids (see Table I) containing 10 pmol of each amino acid. The conventional 4.6-mm column was used with UV irradiation of 250 mW. Note that the complete run lasted less than 30 min with excellent resolution of all of the components except for glycine and threonine. The particular solvent
ANALYTICAL CHEMISTRY, VOL. 59, NO. 3, FEBRUARY 1, 1987 150001
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Table I. Detection Limits of OPA- and NDA-Derivatized Amino Acids detection limits," fmol OPA-
amino acid aspartic acid glutamic acid asparagine histidine glutamine serine arginine glycine threonine alanine
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"Detection limits based on SIN = 2. bReportedin ref 11. cThis work. Recomputed from original data of ref 14 on same statistical basis as present work. Reported detection limits in ref 14 are somewhat higher. system used in Figure 1was chosen to enhance the resolution of this pair of amino acids, and phosphate buffer was avoided since it quenches the fluoresence of some of the OPA-amino acid adducts (21). Calibration curves for five of the amino acids (aspartic acid, asparagine, alanine, valine, and phenylalanine) appearing at various retention times showed excellent linearity below 10 pmol (injected),with correlation coefficients of 0.998. From detailed investigations of this type, detection limits for the various amino acids have been determined and are summarized in Table I. The best detection limit realized, -4 fmol for glutamic acid, could undoubtedly be improved to approximately 1fmol by using lower flow rates and longer signal processing time constants. As also shown in Table I, the HPLC-LIF detection limits (based on SIN = 2) are 1-2 orders of magnitude lower than previously reported results for the same amino acids obtained when using conventional (nonlaser) fluorescence detection with excitation a t 330 nm (11). Thus it is clear that the present LIF results with the cell design of Yeung (19)show greatly increased detection limit capabilities. Chromatography of NDA-Derivatized Amino Acids. Amino acid mixtures were analyzed extensively with the
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457.9-nm argon-ion laser line and standard column. Figure 2 presents a typical chromatogram of a mixture of 16 amino acids derivatized with NDA before injection on the column. The injected sample contained 500 fmol of each amino acid in a total volume of 20 pL. The chromatographic run of Figure 2 used a 30-min linear gradient of 15-40% phosphate buffer (solvent B, 0.05 M, pH 6.9) with acetonitrile (solvent A). Retention times and peak heights were reproducible to within 1% for triplicate runs,and the system could be operated from day to day with no substantial tuning of the laser or the detection system. Resolution of the various amino acid peaks of Figure 2 is very good for all compounds except glycine and threonine, whose resolution could be improved by changing the organic phase to methanol with tetrahydrofuran as modifier. The chromatographic data were routinely smoothed by using the Savitsky-Golay procedure (22) to enhance signal-to-noise ratios. Calibration curves for the amino acids showed excellent linearity from the low picomole range downward, with typical correlation coefficients of 0.998. At higher concentrations, nonlinearities due to inner filter effects and pulse pileup became apparent. From studies of this type, detection limits were obtained for each of the 16 amino acids as summarized in Table I. For comparison purposes we list also the detection limits obtained when using a commercial HPLC system with a conventional fluorescence detector operating at 246-nm UV excitation (14). The LIF detection limits of this work are nearly 2 orders of magnitude lower than the conventional source results, even though the molar absorptivity at 246 nm is approximately 50 times larger than at the laser wavelength (457.9 nm) used in our experiments. The NDA-amino acid detection limits are also seen to be lower by over an order of magnitude compared to the LIF results for OPA-amino acids. Thus, the use of LIF detection and NDA derivatization lowers the detection limits some 3 orders of magnitude compared to conventional determinations utilizing OPA derivatization. Microbore Column Chromatography. The capabilities of LIF microbore column chromatography have been investigated in a preliminary fashion by performing studies of the NDA-alanine adducts. Figure 3 shows a chromatogram obtained from a 1-pL injection of 25 fmol of the adduct. The isocratic run used a flow rate of 50 pL/min and a mobile phase of 25:75 acetonitrile/phosphate buffer (0.05 M, pH 6.9). A detection limit of approximately 100 am01 was estimated based on S I N = 2. Although this result is no more than a factor of 2 lower than that obtained with the conventional column, it shows clearly that the same experimental methodology can be extended with high sensitivity to microbore column chromatography. Thus, the many advantages of microbore work, such as enhanced resolution and decreased solvent utilization, are available under very high sensitivity conditions,
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diation leads to much increased background noise levels from Raman and Rayleigh scattering (which vary as Ad) and from extraneous fluorescence which is largely absent when using 457.9-nm irradiation. These factors are largely independent of the fiber-optics cell design and, consequently, the intrinsic improvement factor of the Yeung cell and optical system (19) is probably best reflected by the OPA improvement factor of 44.5. It is interesting also to compare the average amino acid LIF detection limit improvements in going from OPA to NDA. From Table I, we find an average improvement factor of 37.4. To understand this result, several parameters must be considered. First, fluorescence quantum yields for NDA-amino acids (12,13)tend to be about twice as large as those for the OPA-amino acids (25). On the other hand, the OPA-amino acid e values (at 246 nm) are in the range of 50000 M-'cm-', while the e values (at 457.9 nm) for the NDA-amino acids are in the vicinity of lo00 M-' cm-'. Thus, with similar detection efficiencies at the two emission wavelengths (450 nm for OPA, 490 nm for NDA), the relative fluorescence detection efficiencies should be in the ratio OPA/NDA = 25. We conclude, then, that the observed detection limit improvement factor, NDAIOPA = 37.4, is some 900 times larger with 458-nm irradiation than would be expected purely on the basis of the analytical spectroscopic absorption-emissionparameters. This result parallels and is of the same order of magnitude as that just deduced by comparing the LIF and conventional fluorescence experiments and has its origin in all of the same factors. Note that the difference in the laser power levels does not play a major role, since in both the UV and visible experiments the power is sufficiently large to produce S/N ratios which are relatively insensitive to the power levels. We can now summarize our results rather concisely. First, detection limits for precolumn NDA-derivatized amino acids can be obtained in the subfemtomole range by using the HPLC-LIF cell design of Yeung et al. (19) with 457.9-nm irradiation. These results are approximately 3 orders of magnitude better than earlier reported results with the widely used OPA reagent. Second, the NDA-derivatization scheme is especially efficacious relative to OPA derivatization primarily because it permits irradiation with visible light, which leads to substantially reduced background (noise) levels. Third, when used for amino acid analyses, the HPLC-LIF cell design of Yeung et al. (19) leads to an intrinsic sensitivity increase of 1-2 orders of magnitude compared to conventional commercial fluorescence detectors.
ACKNOWLEDGMENT Advice and consultation from Edward S. Yeung in the early stages of system design were greatly appreciated.
LITERATURE CITED (1) Moore, S.; Spackman, D. H.; Stein, D. H. Anal. Chem. 1958, 30, 1 185-1 190. (2) Hamilton, P. B. Anal. Chem. 1983. 3 5 , 2055-2084. (3) Dong, M. W.; DiCesare, J. L. LC Mag. 1983, 7 , 222-228. (4) Bldlingmeyer, B. A,; Cohen, S.A.; Tarvin, T. L. J . Chromatogr. 1984, 336, 93-104. ( 5 ) Gray, W. R.; Hartley, B. S. Biochem. J 1963, 8 9 , 59P. (6) Udenfriend, S.; Stein, S.; Bohlen, P.; Dairman, W.; Leimgruber. W.; Weigeie, M. Sclence (Washlngton, D . C . ) 1972, 778, 871-872. (7) Imai, K.; Watanabe, Y. Anal. Chlm. Acta 1961, 730. 377-383. (8) Ghosh, P. B.; Whltehouse, M. W. J . Bimhem. 1988, 708, 155-168. (9) Roth, M. Anal. Chem. 1971, 43, 880-882. (10) Fleury, M. 0.; Ashley, D. V. Anal. Blochem. 1983, 733, 330-335. (11) Umagat, H.; Kucera, P.; Wen, L. F. J . Chromatogr. 1982, 239, 463-474. (12) Carison, R. G.; Srinivasachar. K.; Givens, R. S.; Matuszewski, B. K. J . Org. Chem. 1986. 57, 3978. (13) Matuszewski, B. K.; Glvens, R. S.; Srinivasachar, K.; Carlson, R. G.; Higuchi. T., submitted for publication in Anal. Chem. (14) De Montigny, P.; Stobaugh, J. F.; Givens, R. S.;Carlson, R. G.; Srinivasachar, K.; Sternson, L. A,; Higuchi, T., submltted for publication in Anal. Chem. (15) Yeung, E. S.; Sepaniak. M. J. Anal. Chem 1980, 52, 1465A-1470A. (16) Zare, R. N. Science (Washington, D . C . ) 1984. 226, 298-303.
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 frartsfm 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
