TLC with other Techniques

TLC may be combined with column chromatography and vapour-phase chromatography as discussed here briefly  

After development of TLC plates, the next important step is to detect the separated components so as to determine their respective Rf values. 

Example (/ ) Coloured Substances e.g.,Xanthophylls, Chlorophylls, Carotenes, etc., may be located visually. 

---

One of the biggest issues of TLC is the visualization and characterization of compounds resolved on the TLC plate. Although there is no problem with visualization of naturally colored or fluorescent compounds, these compounds are rare in the laboratory. Other compounds can be visualized with UV light or by using appropriate TLC-visualization reagents. These methods, however, provide only little information abont the nature of the compounds resolved on the TLC plate. For this reason, coupling of TLC with other techniques permitting better detection and characterization of componnds has become a subject of many research projects and scientific papers [5-6]. 

One of the attractive features of SFE with CO2 as the extracting fluid is the ability to directly couple the extraction method with subsequent analytical methods (both chromatographic and spectroscopic). Various modes of on-line analyses have been reported, and include continuous monitoring of the total SFE effluent by MS [6,7], SFE-GC [8-11], SFE-HPLC [12,13], SFE-SFC [14,15] and SFE-TLC [16]. However, interfacing of SFE with other techniques is not without problems. The required purity of the CO2 for extraction depends entirely on the analytical technique used. In the off-line mode SFE takes place as a separate and isolated process to chromatography extracted solutes are trapped or collected, often in a suitable solvent for later injection on to chromatographic instrumentation. Off-line SFE is inherently simpler to perform, since only the extraction parameters need to be understood, and several analyses can be performed on a single extract. Off-line SFE still dominates over on-line determinations of additives-an... 

TLC-Raman laser microscopy (X = 514 nm) in conjunction with other techniques (IR microscopy, XRF and HPLC-DAD-ESI-MS) has been used in the analysis of a yellow impurity in styrene attributed to reaction of the polymerisation inhibitor r-butylcatechol (TBC) and ammonia (from a washing step) [795]. Although TLC-FT-Raman did not allow full structural characterisation, several structural elements were identified. Exact mass measurement indicated a C20H25O3N compound which was further structurally characterised by 1H and 13C NMR. 

As with other techniques, a single instrument is usually not sufficient for full analysis of organophosphorus residues. HPLC is often combined with other instrumental methods, mostly GLC, TLC or mass spectrometry. A recently suggested procedure for the analysis of residues found in potatoes requires the use of three different HPLC and three different GLC instruments for the study of one sample183. 

GC, MS, NMR, IR, HPLC, TLC and other techniques, used bofli alone and in combination can analyze, authenticate and identify F F materials. Table II lists the pros and cons of using various analytical techniques to authenticate F F materials, GC using element specific detectors can detect sulfur or nitrogen compounds with high odor impact that occur at low concentration. It is not uncommon for F F extracts of natural materials to contain hundreds of components. GC analysis of off-line rough cut LC separations of a complex extract can help simplify the identification of the many individual components. 

The results obtained by TLC-spectrophotometry have been compared with those obtained by atomic absorption spectrometry using a sample prepared separately or scraped off from the separate TLC plate. Other techniques (55,143,164,186,191) have also been suggested to confirm the TLC results before recommending the method for analysis of real samples. 

In addition to the aforementioned methods, TLC in combination with other instrumental techniques have also been used for quantification of inorganic species. For example, two-dimensional TLC coupled with HPLC has been utilized for the separation and quantification of REEs in nuclear fuel fission products using silaiuzed silica gel as layer material [60]. In another interesting method, REEs in geological samples have been determined by ICP-AAS after their preconcentration by TLC on Fixion plates [32]. TLC in combination with neutron activation has been used to determine REE in rock samples on Eixion 50 x 8 layers with the sensitivity limit of 0.5 to 10 pg/g for 10- to 30-mg samples [41]. A combination of TLC and A AS has been utilized for the isolation and determination of zinc in forensic samples [27]. 

Section 6.4 deals with other EI-MS analyses of samples, i.e. analyses using direct introduction methods (reservoir or reference inlet system and direct insertion probe). Applications of hyphenated electron impact mass-spectrometric techniques for poly-mer/additive analysis are described elsewhere GC-MS (Section 7.3.1.2), LC-PB-MS (Section 7.3.3.2), SFC-MS (Section 13.2.2) and TLC-MS (Section 7.3.5.4). 

This chapter deals mainly with (multi)hyphenated techniques comprising wet sample preparation steps (e.g. SFE, SPE) and/or separation techniques (GC, SFC, HPLC, SEC, TLC, CE). Other hyphenated techniques involve thermal-spectroscopic and gas or heat extraction methods (TG, TD, HS, Py, LD, etc.). Also, spectroscopic couplings (e.g. LIBS-LIF) are of interest. Hyphenation of UV spectroscopy and mass spectrometry forms the family of laser mass-spectrometric (LAMS) methods, such as REMPI-ToFMS and MALDI-ToFMS. In REMPI-ToFMS the connecting element between UV spectroscopy and mass spectrometry is laser-induced REMPI ionisation. An intermediate state of the molecule of interest is selectively excited by absorption of a laser photon (the wavelength of a tuneable laser is set in resonance with the transition). The excited molecules are subsequently ionised by absorption of an additional laser photon. Therefore the ionisation selectivity is introduced by the resonance absorption of the first photon, i.e. by UV spectroscopy. However, conventional UV spectra of polyatomic molecules exhibit relatively broad and continuous spectral features, allowing only a medium selectivity. Supersonic jet cooling of the sample molecules (to 5-50 K) reduces the line width of their... 

Direct injection of blood serum (102) or sample extracts with little or no cleanup (53) is possible, which makes HPLC procedures comparable in speed with other rapid tests With increased use of solid-phase absorption in cleanup, automation of procedures is feasible TLC is also a useful and inexpensive technique and quantitative TLC methods have been described (30,63) The following chapter describes practical application of various procedures in a drug residue monitoring program ... 

Methods for the determination of herbicides usually involve solvent extraction of the sample with methanol, acetone or an acetonitrile followed by LC or GC. Other techniques include TLC, SCFC and enzyme immunoassay (Table 8.5). 

TLC is not the best chromatographic technique for quantitative analysis and, although it can provide quantitative results, the necessary procedure tends to be more cumbersome and tedious compared with other chromatographic methods. Furthermore, for accurate work, expensive scanning equipment is required, which, as already discussed, rather reduces the cost advantage of the technique. 

Because of the biological and pharmaceutical interest in phenothiazine derivatives, these compounds have been used in a variety of analytical studies and applications. For a number of years, several analytical methods have been proposed by various groups for the determination of phenothiazine and its derivatives in pharmaceutical formulations and biological fluids, including thin-layer chromatography (TLC) [51-54], high-performance liquid chromatography (HPLC) [55-58], spectrofluorimetry, either direct or with derivatization [59-64], flow-injection analysis (FIA) [10,65-73] and other techniques [11,74-76]. 

Although re versed-phase LC with various detectors is the dominant technique at the present stage for isocyanate derivative separations, other principles such as normal phase LC, thin layer chromatography (TLC) and GC, usually with derivatization to improve the volatility of the derivatives, have been explored for this purpose over the years. A detailed review of such methods for this purpose can be found elsewhere." For the most volatile isocyanates, however, GC separations have appeared in recent literature, and MIC and ICA have been determined by GC-MS as DBA and 2 MP derivatives. Furthermore, capillary electrophoresis (CE) has been explored for separation of isocyanate 2 MP derivatives. CE is a highly miniaturized technique, and provides the same attractive improved mass sensitivity as miniaffirized LC. However, in contrast to miniaffirized LC, the small injection volumes allowed with this technique make the concentration sensitivity somewhat limited. 

Miscellaneous detectors. TLC, as with other chromatographic methods, is a separation not an identification technique and thus for unambiguous identification of analytes the separated components must be examined by spectroanalytical techniques. Mass [67] and Fourier-transform infrared spectrometry [68] have both been used to good effect and considerable effort is currently being expended to develop robust methodologies and instrumentation in these areas. Instrumentation has recently been developed, for example, which elutes separated components directly onto a measured amount of potassium bromide which is then automatically pressed and introduced into an infrared spectrometer. 

Many FT-IR spectrometers have external ports for optical coupling to dedicated accessories. The IR radiation is conveniently directed to/from the external ports by computer-controlled flip mirrors. A large variety of accessories, like an IR microscope, interfaces for gas chromatography (GC/FT-IR), liquid chromatography (HPLC/FT-IR), thin layer chromatography FT-IR (TLC/FT-IR), etc., is commercially available. This type of method combination is usually called a hyphenated technique. FTIR spectrometers can even be supplemented by a FT-Raman accessory. The versatile combination of FT-IR spectrometers with other instruments has substantially contributed to their abundance in most analytical laboratories. 

---