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Absorption spectra are rarely sufficient for identification, except when directly compared with the spectrum of a standard substance, measured on the same chromatoplate. If possible, preference should be given to the fluorescence mode, which has a much higher spectroscopic selectivity, because two different wavelengths, an excitation and an emission wavelength, are used for each measurement.
Depending on the nature of the compound, the IR absorption of a TLC spot can be measured either after transfer to an IR-transparent substrate or in situ (74). The off-line method involves elution of ca. 5 ÷-g substance, evaporation of the solvent, and pressing into a micropellet suitable for recording spectra. IR spectra can also be recorded in situ by DRIFT-IR spectroscopy (75). To obtain suitable IR spectra, all solvents must be carefully removed prior to measurement, and a background correction must be applied for absorption by the chromatoplate. Generally, more than 1 ftg of compound is necessary for detection of individual functional groups and at least 10 (ig for partial spectral recording. Recently Kovar et al. (76,77) reported a new method for on-line coupling of TLC and FTIR spectroscopy. Direct HPTLC-FTIR measurement in combination with AMD was also used successfully (78).
Analysis of TLC spots by photoacoustic spectrometry (TLC/PAS) has been preferred over DRIFT analysis of strongly IR-absorbing samples (79). The spot containing 1-50 jug of the sample must be physically removed from the chromatoplate. After some preparation, it is placed in the photoacoustic cell for measurement.
Reasonable spectra can also be obtained by surface enhanced Raman spectroscopy (SERS) (80,81). Recently, a new method was reported for preparing SERS-active surfaces, in which colloidal silver spheres are deposited on HPTLC plates. The sensitivity of these activated HPTLC plates is so high that in situ vibrational investigations of spots are possible at the picogram level (82). Comparison of the relative intensities of HPTLC-SERS spectra and normal Raman spectra was reported by Koglin (83). The results clearly demonstrate that varying electric charge density and hydrophillic character of the silver sol-activated nano-TLC plate may strongly influence the HPTLC-SERS detection limit. The size and shape of the silver colloids are, furthermore, also important.
Pharmaceuticals and Drugs
Different methods have been described for obtaining mass spectra ofTLC spots (84-91). The zone of the compound to be identified can be scraped from the stationary phase, and after elution and solvent evaporation the residue is inserted into the mass spectrometer. Alternatively, the sample, together with the stationary phase, can be inserted directly. In fast atom bombardment (FAB) and secondary-ion mass spectrometry (SIMS), a high-energy ion or atom beam is used to sputter molecules from the condensed phase into the gas phase for mass spectrometric analysis. However, most mass spectrometric measurements are destructive in nature; FAB and SIMS spectrometry are surface-sensitive methods in which the material actually consumed in the analysis is sputtered only from the top layers of the sample spot. The sample required for FAB and SIMS is between 1 ng and 1 ng (85-86). Bush et al. (87,88) described a new interface for the combination ofTLC and MS, which makes possible a successful coupling between these techniques. Also a charge coupled device for optical detection of sample bands was applied (89). Wilson et al. (90-92) described the application of TLC-MS-MS to drugs and their metabolites. They showed that FAB-MS alone gave spectra which were dominated by ions from the matrix. When MS-MS was applied to the same samples the resulting spectra were devoid of matrix interferences and contained only ions from the compounds of interest.
For the quantification of separated radioactive substances, autoradiography, liquid scintillation counting, and direct scanning with radiation detectors can be used. Recently, a new detector for radiochromatography was reported (93) that measures position and intensity of ionizing radiation on a two dimensional TLC plate. Digital autoradiography (DAR) offers higher sensitivity than contact autoradiography, which makes possible significant reductions in the time needed for detection of radiolabeled compounds, and/or reduction of the quantity of the analyte (94) as was shown for metabolite products in dog urine samples.
A general approach to quantitation in the pharmaceutical analysis using TLC may be made on the basis of methods used forevaluation. Two basic approaches can be distinguished: direct methods, in which the separated spots are evaluated in situ on the plate, and indirect methods, in which quantitative measurements are carried out after elution of the spots on the chromatoplate. Although the importance of direct methods has increased considerably, the spot elution technique is also being used (e.g. some assay methods in USP Pharmacopoeia).