Chromatography

Modern TLC (Part 2 of 2)

High Performance Thin Layer Chromatography

Although HPTLC plates have been commercially available since the end of the 1970s, only few TLC users have experience with HPTLC.

Previous generation TLC used silica with a particle size distribution of 5–17 µm.  The use of narrowly fractionated silica – particle size 2–10 µm – in HPTLC yields separation efficiencies that are an order of magnitude higher than the previous generation of TLC.

Summary of HPTLC Advances:

  • Shorter migration distances,
  • Shorter separation times,
  • Higher resolution,
  • Lower diffusion and
  • Smaller 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.

Comparison of TLC and HPTLC plates
for the separation of anthraquinone dyes

Modern TLC 4

HPTLC silica layers, too, are available on aluminum 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.

AMD  Procedure

A rapid and efficient analysis at ultra-trace level requires methods that allows to checking on as 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. For example, Burger [5,6] developed an 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, such as our AMD SIL glass plates with a thickness of layer of 0.05 or 0.10 mm (conventional HPTLC glass plates: 0.20 mm).

Efficient modified TLC and HPTLC silica layers

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 either required or are more efficient. For this purpose, modified silicas, like those known from HPLC, are available.

Modern TLC 5Nano-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 their elution properties differ.

  • Nano-SIL C18-100: complete 100 %,
  • Nano-SIL C18-50: partial 50 %,
  • RP-18 W: partial, wettable with water)

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.

Conclusion

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.

Literature

[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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Modern TLC (Part 1 of 2)

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