Thin-layer plates

The plates are usually prepared by applying a slurry of the adsorbent to an inert glass plate. The preparation of the slurry may differ according to the adsorbents used. Some adsorbents are available in various mesh sizes and types, e.g., silica gel, alumina, cellulose, Kieselguhr and polyamide. The adsorbents often contain a binder such as calcium sulfate or starch which makes the finished layer more resistant to abrasion during spotting or spraying. However, the presence of these additives often affects the separations and thus they cannot always be used. 

Bio-Rad Labs., 32nd Griffin Aves, Richmond, Calif. 94804, U.S.A. 

MC/B Manufacturing Chemists, 2909 Highland Ave., Norwood, Ohio 45212, U.S.A. E. Merck, Darmstadt, G.F.R. 

The adsorbent slurry may be applied to the TLC plates by means of several methods. For analytical work, the plates are best prepared with a special TLC adsorbent applicator which provides uniform layers and can be often adjusted to various thicknesses from 250 pm (normal) up to 500 or 1000 pm for preparative separations. The preparation of plates with a typical applicator is shown in Fig.3.1. These applicators are available from a number of suppliers of TLC equipment including most of those mentioned in Table 3.2. After the slurry has been applied, the plates are dried in air overnight or in a warm oven at 80-90°C for ca. 30 min. The dry plates are stored in a dust-free cupboard for further use. Portable cabinets for plate storage are available from a number of suppliers. 

The activation of silica gel, alumina or similar adsorbents is often necessary for the chromatography of many types of compound, especially lipids and related materials. It involves heating of the TLC plate at 110- 130°C for 2 h in order to remove all of the water that is not strongly chemically bound to the adsorbent. Such plates must be cooled and stored in desiccators and should be covered when taken out for spotting. The elution solvents should also be free from water or preferably should contain a well defined content of water which might permit a reproducible equilibrium. Chromatography is often difficult to reproduce from day to day, since the highly activated layer rapidly adsorbs water and 

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Gas-flow counting is a method for detecting and quantitating radioisotopes on paper chromatography strips and thin-layer plates. Emissions are measured by interaction with an electrified wire in an inert gas atmosphere. AH isotopes are detectable however, tritium is detected at very low (- 1%) efficiency. 

Purity can be checked by chromatography (on thin-layer plates, Kieselguhr, paper or columns), by UV or NMR procedures. 

SERS has also been applied as a sensitive, molecule-specific detection method in chromatography, e.g. thin layer, liquid, and gas chromatography. SERS-active colloids were deposited on the thin layer plates or mixed continuously with the liquid mobile phases. After adsorption of the analytes, characteristic spectra of the fractions were obtained and enabled unambiguous identification of very small amounts of substance. 

This is an oversimplified treatment of the concentration effect that can occur on a thin layer plate when using mixed solvents. Nevertheless, despite the complex nature of the surface that is considered, the treatment is sufficiently representative to disclose that a concentration effect does, indeed, take place. The concentration effect arises from the frontal analysis of the mobile phase which not only provides unique and complex modes of solute interaction and, thus, enhanced selectivity, but also causes the solutes to be concentrated as they pass along the TLC plate. This concentration process will oppose the dilution that results from band dispersion and thus, provides greater sensitivity to the spots close to the solvent front. This concealed concentration process, often not recognized, is another property of TLC development that helps make it so practical and generally useful and often provides unexpected sensitivities. 

Figure 2. The Elution of Solute (a) Along a Thin Layer Plate...

Referring again to the retention of a solute on a thin layer plate depicted in figure 2 and comparing this with a normal GC or LC chromatogram, then the distance (y - x)... 

Dispersion equations, typically the van Deemter equation (2), have been often applied to the TLC plate. Qualitatively, this use of dispersion equations derived for GC and LC can be useful, but any quantitative relationship between such equations and the actual thin layer plate are likely to be fraught with en or. In general, there will be the three similar dispersion terms representing the main sources of spot dispersion, namely, multipath dispersion, longitudinal diffusion and dispersion due to resistance to mass transfer between the two phases. 

The multipath dispersion on a thin layer plate is the process most likely to be described by a function similar to that in the van Deemter equation. However, the actual mobile phase velocity is likely to enter that range where the Giddings function (3) applies. In addition, as the solvent composition is continually changing (at least in the vast majority of practical applications) the solute diffusivity is also altered and thus, the mobile phase velocity at which the Giddings function applies will vary. 

The complex distribution system that results from the frontal analysis of a multicomponent solvent mixture on a thin layer plate makes the theoretical treatment of the TLC process exceedingly difficult. Although specific expressions for the important parameters can be obtained for a simple, particular, application, a general set of expressions that can help with all types of TLC analyses has not yet been developed. One advantage of the frontal analysis of the solvent, however, is to produce a concentration effect that improves the overall sensitivity of the technique. 

HETP of a TLC plate is taken as the ratio of the distance traveled by the spot to the plate efficiency. The same three processes cause spot dispersion in TLC as do cause band dispersion in GC and LC. Namely, they are multipath dispersion, longitudinal diffusion and resistance to mass transfer between the two phases. Due to the aforementioned solvent frontal analysis, however, neither the capacity ratio, the solute diffusivity or the solvent velocity are constant throughout the elution of the solute along the plate and thus the conventional dispersion equations used in GC and LC have no pertinence to the thin layer plate. 

The moist cells were suspended in 750 parts of volume of ethanol and extracted by warming at 60°C for 1 hour. A total of 3 extractions were carried out in a similar manner and the extracts were pooled, diluted with water and further extracted three times with 1,000 parts of volume portions of n-hexane. The n-hexane layer was concentrated to dryness under reduced pressure to recover 4.12 parts of a yellow oil. This oily residue was dissolved in 6 parts by volume of benzene and passed through a column (500 parts by volume capacity) packed with Floridil (100 to 200 meshes). Elution was carried out using benzene and the eluate was collected in 10 parts by volume fractions. Each fraction was analyzed by thin-layer chromatography and color reaction and the fractions rich in ubiquinone-10 were pooled and concentrated under reduced pressure. By this procedure was obtained 0.562 part of a yellow oil. This product was dissolved in 5 parts by volume of chloroform, coated onto a thin layer plate of silica gel GF254 (silica gel with calcium sulfate) and developed with benzene. The fractions corresponding to ubiquinone-10 were extracted, whereby 0.054 part of a yellow oil was obtained. This oil was dissolved in 10 parts by volume of ethanol and allowed to cool, whereupon 0.029 part of yellow crystals of ubiquinone-10 were obtained, its melting point 4B°to 50°C. 

Thin-layer plates were made with silica gel-calcium sulfate and each contained a mixture of zinc silicate and zinc cadmium sulfide as phosphors. Separated components are generally visible under ultraviolet light by fluorescence quenching. This was true, in part, for the pyrethrins, except that some of the separated components possessed a natural fluorescence under the ultraviolet lamps. 

Analytical. The thin layer chromatog of MEDINA and the sepn from other expls and nitramines on thin layer plates (MEDINA spots tend to comet) are described in Ref 23... 

Analyses in the pg range, e.g., in water and oil, can be carried out with thin-layer chromatography [244]. After separation on the thin-layer plates, then by Dragendorff reagent, colored spots are measured with the help of a spectral photometer at 525 nm. 

In order to verify the minimum number of carotenoids in an extract or fraction, a very easy and fast test can be carried out with only one small piece of silica gel thin layer plate, consecutively developed with 10% diethyl ether or 4% acetone in petroleum ether for carotene separation, followed by 70% diethyl ether or 15% acetone in petroleum ether for visualization of monohydroxy xanthophyll separation, and finally with 30% acetone in petroleum ether for separation of di- and trihydroxy-carotenoids. ... 

FIGURE 6.7 Cross section of ES chamber with funnel distributor (modified by Wang et al.) 1 — spacing plate, 2 — base plate, 3 — distributor, 4 — glue, 5 — slide, 6 — thin-layer plate, 7 — cover plate. (From Lan, M., Wang, D., and Han, J., J. Planar. Chromatogr. 16, 402-404, 2003. With permission.)... 

Isolation of Sesquiterpene Lactones. The ether extract was evaporated and dissolved in 952 ethanol. Then an equal volume of 42 aqueous lead acetate was added. After 1 hour the mixture was filtered to remove precipitated chlorophyll and phenolic products and the ethanol removed under vacuum. The aqueous layer was extracted with chloroform giving a dark colored oil from which the sesquiterpenes were isolated by a combination of chromatographic procedures, i.e., LH-20 gel permeation, silica gel using both packed columns and thin layer plates. A variety of solvents were also used to purify the individual sesquiterpene lactones, e.g., benzene-acetone (1 1), ethyl acetate, chloroform-methanol (9 1). On thin layer chromatographic plates, spots were visualized by spraying with 22 aqueous KMn04 solution. 

Fluorescence in UV radiation is a frequently used method for detection of TLC spots, e.g. of Tinuvin 326 [42]. The fluorescence emitted by optical brighteners under UV light on a thin-layer plate has been utilised as a means of analysing these compounds [42]. On the whole, the use of fluorescence detection in poly-mer/additive analysis of extracts is certainly not overwhelming. Applied fluorescence has been described in a monograph [156]. 

Lipids were separated on the thin-layer plate with solvents of increasing polarity. 

Reagents which selectively retard certain chemical species can be incorporated into a thin-layer plate. Thus, silver nitrate, which forms weak 7t-complexes with unsaturated compounds, aids their separation from saturated compounds. 

The Rf value for dobutamine hydrochloride when chromatographed on a silica gel 60 F254 thin layer plate developed by ethyl acetate/n-propanol/water/acetic acid (100/40/15/5 v/v/v/v) in an unsaturated chamber is about 0.67. The spot of the drug may be visualized under short wavelength UV light (254 nm), or under white light after exposure to iodine vapors. 

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