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Is Thin Layer Chromatography still up to date?
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The principle of Thin Layer Chromatography (TLC) has already been described more than 100 years ago [1]. For this method the adsorbent is coated as a thin layer onto a suitable support (e.g., glass plate, polyester or aluminium sheet). The substance mixture is separated in this layer by elution with a suitable eluent. The real break-through as an analytical method came in the 1960s as a consequence of the pioneering work of E. Stahl [2]. ... and today?
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Today Thin Layer Chromatography is a widely used technique in routine analyses. Already during training as lab assistant or during studies in natural science young scientist may have contact with this chromatographic procedure, either with introductory kits for scientific education (TLC mikro-sets) or during qualitative analyses of organic syntheses with TLC sheets (e.g., ALUGRAM® Xtra SIL G, 4 x 8 cm).
Due to its convenient and economical handling this method is very often used in scientific laboratories, but also for clinical, pharmaceutical, natural product and food analyses. Although the method is very well known, the scope of efficient application is frequently not recognized. For this reason we wish to present below some powerful techniques and up-to-date products.
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State-of-the-art TLC silica layers |
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Often TLC plates or sheets are labeled with a lead pencil prior to application of the sample and the analytical result is marked by outlining the substance spot. However, conventional silica layers may be damaged by this procedure, rendering the plate useless. Due to the improved binder system ADAMANT glass plates exhibit excellent hardness of the layer, allowing labeling of the plates without any difficulty. The optimized particle size distribution results in an increased separation efficiency, a beneficial feature, e.g., for the separation of pharmaceuticals like analgesics.
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A UV indicator with increased brilliance combined with a low-noise background allows application of the plates for trace analyses. |
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Especially when used in instrumental Thin Layer Chromatography (also called Planar Chromatography [3]), these properties can increase the analytical efficiency. Automation of the steps sample application, chromatographic separation, derivatization with visualization reagents (if necessary), photometric direct evaluation and archiving allows to increase the sample throughput in quantitative analyses. Due to further advantages instrumental TLC can compete with HPLC as quantitative method. Often the efforts for sample preparation are lower. Irreversible damage caused by matrix constituents, which may destroy an HPLC column, is irrelevant for TLC plates, since they are disposed of after one-time application. In addition there are no restrictions for the choice of solvents due to their inherent adsorption. Unlike in HPLC the solvent is evaporated prior to detection (in UV light).
Planar Chromatography with ADAMANT glass plates is mainly used for identity and purity assays, quantitative analyses and stability checks.
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Cost-saving application of aluminium sheets, especially appreciated in universities, has also been optimized in recent years. Thus, ALUGRAM® Xtra SIL G allows easy and reliable cutting without flaking of silica, due to an improved binder system. Use of aluminium sheets does not mean to forgo excellent separation efficiency as the separation of nutmeg ingredients shows.
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Separation of nutmeg ingredients
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Application of large volumes of very dilute samples can cause irregular spreading of substance spots, which makes evaluation difficult. To solve this problem, silica-coated glass plates with a concentrating zone of the inactive adsorbent kieselguhr are known for decades. Of late this layer is also availabe as aluminium sheet. The following figure shows concentration of a sample on an ALUGRAM® Xtra SILGUR.
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Concentration of samples
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High performance HPTLC silica layers |
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Although HPTLC silica layers are commercially available since the end of the 1970s, only few TLC users have experience with High Performance Thin Layer Chromatography (HPTLC).
Due to use of narrowly fractionated silica with particle size 2–10 µm (TLC: 5–17 µm) HPTLC silica layers reach plate heights, which are an order of magnitude lower than for TLC silica layers. This results in a higher resolution, lower diffusion and thus a lower peak width. Shorter migration distances and separation times – typical pharmacopeia applications of 45–60 min only take 8–20 min on HPTLC plates [4] – are not only of interest for users of instrumental TLC, but also for TLC applications in other fields, especially since they require less solvent.
Nano-ADAMANT combine the advantages of HPTLC with the properties of ADAMANT plates (e.g., hardness and abrasion resistance). A comparison of both plates for the separation of anthraquinone dyes shows more narrow bands in half the elution time.
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Comparison of TLC and HPTLC plates
for the separation of anthraquinone dyes
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HPTLC silica layers, too, are available on aluminium sheets like the new ALUGRAM® Xtra Nano-SIL G and the new ALUGRAM® Xtra Nano-SILGUR with concentrating zone. The increase in efficiency compared to conventional TLC sheets is shown in the analyses of peppermint oil and Japanese herbal oil and in the separation of dexpanthenol in wound and healing ointment, both performed in the classical TLC chamber.
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A rapid and efficient analysis at ultra trace level requires methods, which allow to check a large number of samples simultaneously for as many active ingredients as possible. Such multi-methods considerably increase the sample throughput and lower the cost per analysis. Burger [5,6] developed a HPTLC procedure with automated multiple development of the same plate, the AMD procedure. Contrary to conventional multiple development of TLC plates the migration distance of every single run is slightly longer than the previous one (10–30 runs, constant increment of 1–3 mm). In most cases a step gradient with eluents of different polarity is used. On silica plates one starts with a polar, strongly eluting solvent and finishes the multiple development with a non-polar, weakly eluting eluent. Depending on their different polarity the analytes migrate at the front until the weaker elution strength of a development is no longer sufficient and thus causes a separation. After every single development the plate is dried under vacuum and prior to the following development it is conditioned via the gas phase.
The AMD procedure requires HPTLC plates with very thin layers, like our AMD SIL glass plates with a thickness of layer of 0.05 or 0.10 mm (conventional HPTLC glass plates: 0.20 mm).
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Efficient modified TLC and HPTLC silica layers |
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A great number of separations can be performed with the selectivity of silica phases in normal phase mode. However, for certain applications layers with different selectivity, sometimes also in the reversed phase mode, are required or more efficient. For this purpose modified silicas, like those known from HPLC, are available.
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Nano-SIL C18 and RP-18 W/UV254 are octadecyl modified silica layers used for reversed phase separations with polar eluents (e.g., methanol – water or acetonitrile – water). Depending on the degree of modification (Nano-SIL C18-100: complete 100 %, Nano-SIL C18-50: partial 50 %, RP-18 W: partial, wettable with water) their elution properties differ. |
Elution properties of RP plates
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Other modified TLC and HPTLC silica layers available are RP-2, cyano, amino, and diol. For an economical selection of the most suitable layer for a specific separation we recommend our HPTLC method development kits.
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Conclusion |
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Although the principle of Thin Layer Chromatography is known for more than a century, recent developments of TLC silica layers like ALUGRAM® Xtra SIL G and ALUGRAM® Xtra SILGUR as well as the versatile features of HPTLC silica layers (e.g., Nano-ADAMANT) evidence that TLC is still an up-to-date chromatographic technique.
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Literature |
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[1] Beyerinck, Z. Phys. Chem. 3 (1889), 110
[2] Stahl, E., Dünnschicht-Chromatographie, 2. Auflage, Springer-Verlag, Berlin (1967)
[3] Zieloff, K., GIT Labor-Fachzeitschrift (5/2004), 497–500
[4] Majors, R. E., LCGC North America, Volume 23, Number 5 (May 2005), 458–469
[5] Burger, K., Fresenius Z. Anal. Chem. 318 (1984), 228
[6] Burger, K., Pflanzenschutz-Nachrichten-Bayer 41,2 (1988), 173
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