Hyphenated TLC techniques

Linking TLC with a tandem instrument differs from combining GC or LC with an appropriate spectrometer. Hyphenation of planar chromatographic techniques represents a niche application compared to HPLC-based methods. Due to the nature of the development process in TLC, the combination is often considered as an off-line in situ procedure rather than a truly hyphenated system. True in-line TLC tandem systems are not actually possible, as the TLC separation must be developed before the spots can be monitored. It follows that all TLC tandem instruments operate as either fraction collectors or off-line monitoring devices. Various elaborate plate extraction procedures have been developed. In all cases, TLC serves as a cleanup method. 

Many methods can be used for spot location and sample identification, including visual analysis, UV/VIS, 

Recently, various excellent reviews have been published on hyphenated and multidimensional TLC techniques [720-722]. 

Principles and Characteristics Many of the planar chromatography methods rely on fluorescence detection to achieve the required identification limits exploitation of sensitive and selective derivatisation reactions is of considerable importance. Most TLC scanning densitometers can be operated in the fluorescence mode and are able to record in situ excitation spectra of TLC 

Rather high concentrations are needed to afford in situ TLC-XRF elemental images. TLC-XRF is mainly used for element qualification rather than quantification. The major difficulty in using XRF for detection in TLC is the strong emission background of the (cellulose and silica gel) support, which seriously interferes with particular spectral regions. In practice, TLC plates with moderately low background, i.e. 0.5-mm-thick cellulose plates and 0.25-mm-thick silica gel plates, are used [735], 

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Hyphenated TLC techniques. TLC has been coupled with other instrumental techniques to aid in the detection, qualitative identification and, occasionally, quantitation of separated samples, and these include the coupling of TLC with high-pressure liquid chromatography (HPLC/TLC), with Fourier transform infra-red (TLC/FTIR), with mass spectrometry (TLC/ MS), with nuclear magnetic resonance (TLC/NMR) and with Raman spectroscopy (TLC/RS). These techniques have been extensively reviewed by Busch (1996) and by Somsen, Morden and Wilson (1995). The chemistry of oils and fats and their TLC separation has been so well established that they seldom necessitate the use of these coupling techniques for their identification, although these techniques have been used for phospholipid detection. Kushi and Handa (1985) have used TLC in combination with secondary ion mass spectrometry for the analysis of lipids. Fast atom bombardment (FAB) has been used to detect the molecular species of phosphatidylcholine on silica based on the molecular ion obtained by mass spectrometry (Busch et al, 1990). 

Applications of hyphenated LSIMS techniques are described elsewhere, e.g. TLC-MS (Section 73.5.4). 

Postcolumn hyphenated and multidimensional TLC techniques have been reviewed [721,996,1004]. 

Various analytical methods exist for flavonoids. These range from TLC to CE. With the introduction of hyphenated HPLC techniques, the analytical potential has been dramatically extended. Gas chromatography (GC) is generally impractical, due to the low volatility of many flavonoid compounds and the necessity of preparing derivatives. However, Schmidt et al. ° have reported the separation of flavones, flavonols, flavanones, and chalcones (with frequent substitution by methyl groups) by GC. 

Recent advances in hyphenated analytical techniques, where a separation device is coupled online with detectors generating spectral information, have remarkably widened the analysis field of complex biological matrices. During the last few years covered by this chapter, a number of papers describing the application of TLC, GC-MS, HPLC-UV, HPLC-UV/MS, CE-MS, and NMR to the qualitative and quantitative analysis of tropane alkaloids in toxicological, physiological, forensic, phytochemical, and chemotaxonomical studies have been published. 

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). 

In the deformulation of PE/additive systems by mass spectrometry, much less fragmentation was observed with DCI-MS/MS using ammonia as a reagent gas, than with FAB-MS [69]. FAB did not detect all the additives in the extracts. The softness and the lack of matrix effect make ammonia DCI a better ionisation technique than FAB for the analysis of additives directly from the extracts. Applications of hyphenated FAB-MS techniques are described elsewhere low-flow LC-MS (Section 7.3.3.2) and CE-MS (Section 7.3.6.1) for polar nonvolatile organics, 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... 

XRF has also been hyphenated to various chromatographic techniques, cf. TLC-XRF (Section 7.3.5.1). For process XRF, the stream interface is a simple by-pass flow the window material that allows the X-rays to enter the product stream-a thin film of polycarbonate-confines pressure and temperature. 

One-dimensional multiple development and two-dimensional development Multiple developments through one or two dimensions can be applied to separate certain components in sequence, with detection at each step. This gives a theoretical increase in the capacity of the spots, so it is ideal for the separation of mixtures with a large number of components. In addition, it is a useful tool to confirm the purity of a given component. Though hyphenated HPLC could serve as a multiple separation technique, TLC takes the lead in this area by its faster separation and choice of different mobile phases and detection methods through each run. 

As indicated previously (see Section 1.2) pyrolysis must be associated with an analytical technique in order to provide information on a sample. Several common analytical techniques such as GC, GC/MS or GC/FTIR have been utilized either hyphenated with pyrolysis or off-line and were described previously. Less frequently, techniques such as HPLC, preparative LC, TLC, SFE/SFC, or NMR also have been used for the analysis of pyrolysates. These types of techniques are commonly applied off-line. They are used mainly for obtaining information on that part of the pyrolysate that is difficult to transfer directly to an analytical system such as a GC or for the analysis of materials associated with the char. However, the analysis of the non-volatile part of pyrolysates is frequently neglected, although this leads to an incomplete picture regarding the chemical composition of pyrolysates. 

The first exposure to spectroscopy for most scientists is ultraviolet/ visible absorbance. As virtually every HPLC chromatograph employed in the pharmaceutical industry uses UV absorbance as the detection method, it is no wonder that the most popular hyphenated technique is HPLC-DAD. DAD spectrographs have been coupled to all liquid-based chromatographic systems including HPLC (preparative, analytical, and microbore), capillary electrophoresis (CE) and supercritical fluid chromatography (SFC). There have been several successes with TLC plates,18 but it is more common for developed plates to be scraped and the sample analyzed offline. 

Because of the variety of interfaces and the low detection limits, hyphenated mass spectrometers are among of the fastest growing markets for scientific instrument manufacturers in the 1990s and beyond. Every available type of chromatographic separation has been analyzed by mass spectrometry from desorption methods for TLC plates26 to online CE-MS.27,28 Not all techniques are routinely used for the characterization of small organic molecules of pharmaceutical interest (Table 4). The two workhorse instruments for the characterization of small molecules are GC-MS and... 

Thin layer chromatography (TLC), also known as planar chromatography, is an invaluable method used in chemistry and biochemistry, complementary to HPLC while having its own specificity. Although these two methods are applied differently, the principle of separation and the nature of the phases remain the same. Cheap and sensitive, this technique that is simple to use, can be automated. It has become essential principally since it is possible to undertake several separations in parallel. The development of automatic applicators and densitometers have led to nano-TLC, also called HPTLC, a highly sensitive technique which can be hyphenated with mass spectrometry. 

Coupling of two (separation) techniques (hyphenated or orthogonal techniques, multidimensional chromatography) e.g. HPLC-GC, HPLC-TLC. HPLC-CE, HPLC-Elisa, HPLC-Westemblott. 

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. 

Several so-called hyphenated techniques have been developed, where the developed TLC plate is transferred to a modified spectrometer to record in situ the Fourier transform infrared, surface enhanced Raman, or mass spectra of the separated zones. This way more detailed structural information can be obtained to complement the data from densitometric evaluation. A true hyphenation is the direct application of the eluate from a microbore HPLC column onto an HPTLC plate, which is then developed by AMD. 

Silver-ion TLC is often used with GC as part of a hyphenated technique for analysis of CLA isomers as FAME and, to a lesser extent, TAG. Precht and Molkentin utilized Ag-TLC to isolate trans 18 1 and CLA isomers (as FAME) before analysis by GC (100 m CP Sil 88 capillary column) and applied this technology to determine the frequency distributions of CLA in European bovine milk fats (24,25) and German human milk lipids (26). Prefractionation of the milk lipids by Ag-TLC was utilized to remove such FA as 9c-20 l and 21 1, FA that would otherwise coelute with the CLA isomers (as FAME) during analysis by GC. The procedure thus simplified the calculation of the percentage of CLA in these samples. Robert Ackman (27) apphed a similar technique to analyze the CLA isomers present in raw seafood. After the lipids were extracted and converted to FAME, Ag-TLC was utilized to remove 18 4n-3 and 18 4n-l, FAME isomers that co-eluted with 9c, 11/-18 2 and 10/,12c-18 2, respectively, on polyglycol-based capillary GC columns. 

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