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ANALYTICAL CHEMISTRY, VOL. 51, NO. 13, NOVEMBER 1979
Quantitative Examination of T hin-Layer Chromatography Plates by Photoacoustic Spectroscopy S. L. Castleden," C. M. Elliott, G. F. Kirkbright, and D. E. M. Spillane Department of Chemistry, Imperial College, London SW7 2AY. U.K.
A technique Is described for the nondestructive quantitative determination of materials on thin-layer chromatography plates utilizing photoacoustic spectroscopy with minimal sample preparation and without the need for solvent extraction. Linear calibration Is obtained for the determination of fluorescein in the range 0.2-2 pg following development of separated fluorescein spots on silica gel substrates, supported on aluminum and glass plates. A limit of detection of 20 ng of fluorescein in 4.5 mg of silica gel Is reported; relative standard deviations on the measurements made were In the range 0.08-0.1.
A number of techniques exist for the quantitative determination of analyte species separated on thin-layer chromatograms (1,2). These methods may be divided into two distinct types, those which involve measurements being made in situ and techniques by which the material is removed from the TLC plate and then determined by, for example, gas chromatography, UV-visible spectrophotometry, gravimetry, or titrimetry. Difficulties are commonly encountered when either type of technique is used. Deviant spots and the stringent control of conditions required to produce reference standards make in situ densitometric determinations exacting ( 3 ) . Nonquantitative recovery of eluted fractions and the pre-treatment required before measurements can be made complicate further any determinations carried out upon material removed from TLC plates (I). One of the reasons for the current interest in photoacoustic spectrometry (PAS) is the usefulness of the technique for samples, such as opaque or light-scattering solids and turbid liquids, which are difficult to determine satisfactorily using conventional spectrophotometric techniques ( 4 , 5). The light-scattering nature of TLC substrates suggests that PAS might have advantages over other techniques for qualitative and quantitative determinations. Rosencwaig and Hall (6) have shown that absorption spectra may be obtained from thin-layer chromatograms and qualitative identification achieved using PAS. This paper reports the application of PAS in the quantitative determination of analyte species on TLC plates with the minimum of sample pre-treatment, excepting that necessary to remove the sample from the plate and place it in the PAS cell. EXPERIMENTAL Instrumentation. The double-beam photoacoustic spectrometer used in this study has been described in detail elsewhere (7). A pyro-electric detector (Eltec type 404CM, Rofin Ltd., Egham, U.K.) was used in the reference channel of the spectrometer t o correct for the variation of the output power of the source with wavelength. The source used was a 300-W compact xenon arc lamp (Varian-Eimac,type VIX-300). The signals from the microphone (Bruel and Kjaer, type 4166), fitted with a type 4169 preamplifier, and from the pyro-electric detector were taken t o lock-in amplifiers (Model 124A, Princeton Applied Research Corporation; and Model 9502, Brookdeal Electronics Ltd., Bracknell, U.K., respectively). The two outputs from the lock-in 0003-2700/79/0351-2152$01.OO/O
amplifiers were taken to a ratiometer (Brookdeal, Model 5047) and the normalized output was displayed on a digital voltmeter or chart recorder. An aluminum insert was made to fit the sample cell described in earlier work (7). The insert was polished and contained a central depression (diameter 10 mm, depth 1 mm), which was used to contain the small samples (ca. 10 mg) scraped from TLC plates. The use of the insert achieved the two objectives of reducing the dead volume of the cell and positioning the sample reproducibly when the sample material available was insufficient to cover the whole of the base of the cell. Chromatography. Studies were carried out using glass-backed and aluminum foil-backedthin-layer chromatographyplates. The aluminum-backed plates (20 cm X 20 cm Silica gel 60 FZM,TLC aluminum sheets, Merck type 5554, BDH Chemicals Ltd., Pool, U.K.) were used by cutting out a 20-mm diameter disk containing the analyte spot at its center. The disk was then placed directly into the PAS cell. The glass-backed plates (20 cm X 20 cm Silica gel 60, Merck type 5721) were used by removing the analyte spot from the developed plate. A circle (diameter 9.5 mm) was marked out containing the analyte spot and the material within this area removed using a vacuum collection device of the type described by Mulhern (8). This consisted of a glass probe, containing a sintered disk, attached to a vacuum line. The sample from the TLC plate was collected on the sintered disk and then transferred to the PAS cell. The average weight of sample removed from the plate by this method was 9 mg. Procedure. Aluminum-backed and glass-backed TLC plates were cleaned by development with ether followed by activation by heating for 2 h in an oven at 120 "C. Solutions of fluorescein were spotted onto the TLC plates using a microsyringe. Development of the chromatograms was carried out using a solvent mixture containing ethyl acetate (33% v/v) in acetone. After development the chromatograms were allowed to dry in a desiccator.
RESULTS Figure 1 shows the PAS spectrum obtained for 1 pg of fluorescein developed under the conditions described above on a silica gel TLC plate and the corresponding spectrum for the silica gel substrate. The maximum PAS signal is observed a t 447 nm for fluorescein; the signal a t 600 nm is the same for both fluorescein and the silica gel blank and the difference in the signal amplitudes a t 600 and 447 nm may be employed to determine the mass of fluorescein present in the samples. A calibration graph for the mass of fluorescein present on disks cut from aluminum-backed TLC plates was prepared by placing on the plate 1-pL aliquots of a series of standard solutions containing fluorescein in the concentration range The calibration graph was found to 0.2 pg pL-l to 2 pg be linear over the weight range studied and reproducibility studies yielded a relative standard deviation on each point of 0.1. On cutting an aluminum-backed plate into a series of strips and placing a 1-pL aliquot of one of a series of the standard fluorescein solutions upon each strip, it was found to be possible to obtain a linear calibration graph by developing each strip separately without any precise control of the development time. A calibration graph for the weight of fluorescein present in scrapings obtained from silica coated glass-backed TLC 0 1979 American Chemical Society
ANALYTICAL CHEMISTRY, VOL. 51, NO. 13, NOVEMBER 1979
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Spectrum of fluorescein on a silica gel-coated aluminumbacked TLC plate
Figure 1.
plates was obtained by spotting a 1-pL aliquot of each of a series of standard fluorescein solutions, having concentrations in the range 0.2 hg pL-' to 1.2 pg pL-l. Under these conditions the area of the spot obtained on the TLC plate was maintained approximately constant as the mass of fluorescein present was varied. This technique resulted in a linear calibration graph being obtained for weights of fluorescein in the range 0.2 to 1.2 pg. The reproducibility of the results obtained using material removed from glass-backed TLC plates was investigated and the relative standard deviation was found to be 0.08. A limit of detection of 20 ng of fluorescein in a sample of silica gel weighing 4.5 mg was obtained. This result corresponds to a fluorescein concentration in silica gel of 4.4 pg g-1.
DISCUSSION The relative standard deviations of measurements made in the determination of fluorescein using both glass-backed and aluminum-backed TLC plates have been found to be in the range 0.084.1. The majority of the errors incurred in the results appear to be caused by problems in transporting the sample from the TLC plate to the PAS cell. It was found to be difficult to remove a 20-mm diameter disk from aluminum-backed plates without some silica gel becoming detached from the perimeter of the disk. Any exposed aluminum produced in this way was found to have a considerable enhancement effect upon the magnitude of the PAS signal observed. As a result of some spatial variation in the intensity of the radiation illuminating the sample holder in the PAS cell, the location of the analyte spot on the disk cut from a TLC plate was found to require careful identification and reproducible sample positioning in the cell. In the case of sample spots being removed from glass-backed TLC plates, difficulty was experienced initially in preventing loss of analyte material during the transfer process involved. This problem became less severe as the experience of the operator increased.
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It has been shown that the square root of the area of the sample spot, after development, is a linear function of the logarithm of the weight of the material it contains (9). Although this implies that the area of the eluted spot is only a very weak function of the weight of silica present, nevertheless this function was not found to affect the linearity of calibration graphs over the range of weights of fluorescein studied. The amount of silica gel containing the analyte spot was found to be an important parameter. Importance is, therefore, attached to the thickness of the silica gel coating being uniform over the whole of the plate. An increase in the amount of silica gel transferred with the analyte spot has a dilution effect upon the sample and consequently reduces the magnitude of the PAS signa: obtained. All the points on the calibration graphs presented in this paper were obtained from a single TLC plate.
CONCLUSIONS The technique employed with the glass-backed TLC plates is superior to the use of disks cut from aluminum-backed plates. With a chromatogram containing several different spots, it is unlikely that adjacent analyte spots would be sufficiently well separated to allow a disk to be cut containing only one spot at its center. The current wavelength range of the PAS system employed is from the UV (250 nm) to the near-IR (2.5 pm). The method described in this paper for the quantitative and nondestructive determination of substrates on thin-layer chromatograms is potentially applicable to the determination of a wide range of analyte species with absorption bands in this range. The plate-to-plate reproducibility for quantitative measurements is no less than for any densitometric technique and may be overcome simply by adequate standardization. The immunity to interference effects caused by light-scattering solids, such as silica gel, and the capability of the technique for the examination of opaque substrates makes the quantitative study of thin-layer chromatograms H particularly promising application for PAS.
LITERATURE CITED (1) "Chromatography", 3rd ed.,E. Heftmann, Ed., Van Nostrand-Reinhold, New York, 1975,pp 164-186. (2) "Quantitative Thin Layer Chromatography",J. C. Touchstone, Ed., Wiley Interscience, New York, 1973. (3) S. Sarnueis, J . Chromatogr., 1, l(1974). (4) A. Rosencwaig, Anal. Chem., 47, 592A (1975). (5) W. R. Harshbarger and M. B. Robin, A c c . Chem. Res., 8, 329 (1973). (6) A. Rosencwaig and S. S. Hall, Anal. Chem.. 47, 546 (1975). (7) M. J. Adams, B. C. Beadle, and G. F. Kirkbright, Analyst(London), 102, 569 (1977). (8) B. M. Mulhern, J . Chromatogr., 34, 558 (1968). (9) S. J. Purdy and E. V. Truter, Analyst(London), 87, 802 (1962).
RECEIVED for review May 7, 1979. Accepted ,July 23, 1979. We are grateful to the Laboratory of the Government Chemist (Department of Industry) and to EDT Research Limited, U.K., for the provision of studentships for two of us (C.M.E. and D.E.M.S., respectively).
