TLC/SERS

The main features of PC are low cost, need for small sample amount, high level of resolution, ease of detection and quantitation, simplicity of apparatus and use, difficult reproducibility (because of variation in fibres) and susceptibility to chemical attack. Identification of the separated components is facilitated by the reproducible Rj values. Detection methods in PC have been reviewed [368]. Fluorescence has been used for many years as a means of locating the components of a mixture separated by PC or TLC. However, also ATR-IR and SERS are useful. Preparative PC is unsuitable for trace analysis because filter paper inevitably contains contaminants (e.g. phthalate esters, plasticisers) [369]. For that purpose an acceptable substitute is glass-fibre paper [28]. 

The sensitivity limitations of TLC-FT-Raman spectroscopy may be overcome by applying the SERS effect [782]. Unlike infrared, a major gain in Raman signal can be achieved by utilising surface activation and/or resonance effects. Surface-enhanced Raman (SER) spectra can be observed for compounds adsorbed on (rough) metahic surfaces, usually silver or gold colloids [783,784], while resonance Raman (RR) spectra... 

Since SERS and SERRS are substance specific, they are ideal for characterisation and identification of chromatographically separated compounds. SE(R)R is not, unfortunately, as generally applicable as MS or FUR, because the method requires silver sol adsorption, which is strongly analyte-dependent. SE(R)R should, moreover, be considered as a qualitative rather than a quantitative technique, because the absolute activity of the silver sol is batch dependent and the signal intensity within a TLC spot is inhomogeneously distributed. TLC-FTIR and TLC-RS are considered to be more generally applicable methods, but much less sensitive than TLC-FT-SERS FT-Raman offers p,m resolution levels, as compared to about 10p,m for FTIR. TLC-Raman has been reviewed [721],... 

A soln of Boc-Ser(Bzl)-ONbz in THF was treated with 1 M TBAF-3H2O (2.4 equiv) at rt. After completion of the reaction (TLC), cold H2O was added and the soln was concentrated under reduced pressure. The residue was dissolved in EtOAc and washed with 5% KHSO4 and with brine. The organic layer was dried (Na2S04) and concentrated to dryness. The residue was converted into the cyclohexylamine salt yield not reported mp 158-160 °C. 

Compared with normal Raman spectroscopy, the SERS technique gives a considerably lower limit of determination of the substance to be analyzed. This is achieved by bringing colloidal noble metal atoms into close proximity to the sample molecules after they have been separated by TLC. Further information can he found in [90, 91]. 

FIGURE 5 TLC plate marked for separation of phosphoamino acids in two dimensions. Four samples can be analyzed on one TLC plate. The positions of the sample origins, indicated as 1,2, 3, and 4, are shown. Marker dye can be spotted on the fifth origin (upper right corner) before electrophoresis in the first dimension at pH 1.9 in the direction shown. The plate is rotated 90° for electrophoresis in the second dimension at pH 3.5 in the direction shown. The positions of phosphoserine (P.Ser), phosphothreonine (P.Thr), phosphotyrosine (P.Tyr), orthophosphate P, and partial hydrolysis products (phosphopeptides) after two-dimensional separation are shown for a sample spotted on origin 4. Phosphoamino acids migrate from the cathode toward the anode during electrophoresis at pH 1.9 and 3.5. 

The release of free [ P]phosphate at a particular cycle, which results from P elimination during cyclization, indicates the presence a P.Ser or P.Thr residue. P.Tyr is stable to cyclization and is released as the anilinothiazolinone derivative of P.Tyr. This can be converted to the phenylthiohydantoin (PTH) derivative of P.Tyr by incubation in 0.1 N HCl for 20 min at 80°C. Marker PTH-P.Tyr is readily synthesized by reacting P.Tyr with phenylisothiocyanate as described above in step 2, and can be detected as a dark spot when the TLC plate is examined under a hand-held UV light. In addition to the cycle at which free p P]phosphate or p P]PTH-P.Tyr is released, information on the sequence of the peptide may be obtained by the electrophoretic mobility shifts detected at each cycle. Thus, if a positive or negatively charged amino acid is removed at a cycle before the phosphoamino acid, there will be a corresponding shift in the mobility of the peptide. Remember that if the peptide contains a C-terminal lysine this will react with phenylisothiocyanate at cycle 1, which will cause the loss of a positive charge. It may be difficult to determine the position of a second, more C-terminal phosphorylated residue present in the same peptide. 

Mroczek T (2008) TLC of tropane alkaloids. Chnnnatogr Sci Ser 99 (Copyright (C) 2012 American Chemical Society (ACS). All Rights Reserved.) 685-699... 

Kai.ser, R. E. (1977). Simplified theory of TLC. In HPTLC—High Performance Thin Layer Chromatography, A. Zfatkis and R. E. Kaiser (Eds.). Elsevier, New York, and Institute of Chromatography, Bad Durkheim, Germany, pp. 15-38. 

Wippo and Stan, 1997). Other on-line couplings have included TLC with gas chromatography, supercritical fluid extraction, and the thermal separation technique (Kovar and Morlock, 1996). Somsen et al. (1995) have described couplings of colunm liquid chromatography with TLC and spectrometric methods (FTIR, SERS, and fluorescence spectrometry). 

Caudin, J. P., Beljebbar, A., Sockalingum, G. D., Angiboust, J. F., and Manfait, M. (1995). Coupling of FT-Raman and FT-SERS with TLC plates for in situ identification of chemical compounds. Spectrochim. Acta Part A 51A 1977-1983 Caudin, J. P., Beljebbar, A., Sockalingum, G. D., Nabiev, I., Angiboust, J. F., and Manfait, M. (1995). NIR FT-Raman and FT-SERS microspectroscopy combined with thin layer chromatography for biological applications. Spectrosc. Biol. Mol, Ear. Conf., 6th pp. 31-32. 

Koglin, E. (1989). Combining HPTLC and micro-surface-enhanced Raman spectroscopy (micro-SERS). J. Planar Chromatgr.—Mod. TLC 2 194—197. 

Koglin, E. (1996). Combination of thin layer chromatography (TLC) and surface-enhanced Raman scattering (SERS). CLB Chem. Labor Biotech. 47 257-261. 

Furthermore, a trend in direction of coupling TLC with spectroscopic methods (e.g. FTIR, RAMAN, SERS, and MS) is recognizable to enlarge the analytical possibilities. For this purpose tailor-made precoated layers are in preparation. 

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