In our last article, we described the first four of eight factors in choosing a TLC plate. Surely, you’ve been waiting with bated breath for the last four factors—and we don’t blame you! In this article, let’s look at these final considerations. Hold onto your seats…
Fluorescent indicator – UV254
This is an additive to the adsorbent which you may need depending on how your samples are visualized after the chromatography process. Present in only 2-4% by weight, this special material makes the entire plate glow blue or green (pretty!) when exposed to short wave, 254 nm UV light.
Why are fluorescent indicators important? Most sample types separated by TLC are not visible to the naked eye. This is usually because of the relatively small sample size or because there is no inherent coloring or shading in the compound. That’s why another method is needed to locate the sample components.
Some materials have an excited state that can be seen when exposed to 366 nm UV light. In this case, the components will fluoresce a characteristic color. In some cases, you need to spray the TLC plate with a solution before exposing it to UV light. The sample components will then be temporarily or permanently visible.
Back to UV 254 plates: Many compounds can absorb light around 254 nm. These include most compounds with aromatic rings or conjugated double bonds and some unsaturated compounds. When exposed to 254 nm UV light, absorbing compounds will fade (quench) the uniform fluorescence of the TLC plate, thus showing as dark spots on the otherwise bright background. This method is popular because it doesn’t modify or destroy the compounds, so it’s suitable for preparative TLC.
Glass plate scoring
The most popular size of TLC plate is 20×20 cm. Just as in life, being popular has its advantages; for TLC plates it means they’re usually the best value. Adding to that value, glass-backed TLC plates may be purchased in a pre-scored format. A pattern of scoring is etched onto the backs of plates, allowing you to “snap” them into smaller sizes as needed. Therefore, a pre-scored 20×20 cm plate can be snapped into four 5×20 cm or two 10×20 cm plates (kind of like Select-A-Size™ paper towels). It would cost you a lot more to purchase these same plates already in the smaller size. Other commonly ordered plates are the pre-scored 10×20 cm plates; they are popular for method development. They can be snapped into eight slide-sized plates (2.5×10 cm).
The preadsorbent zone is typically a non-reactive material coated adjacent to the regular TLC layer at the bottom of the plate. This special zone has two great properties. First, it allows you to apply a larger than average sample since it effectively compresses the sample into a tight “band” before entering the regular separation zone. This helps with separation power by improving vertical resolution of closely chromatographing sample components. Second, it allows you to apply samples quickly, without strict attention to the vertical location in the preadsorbent zone. In other words, the preadsorbent zone cleans up sloppy sample application. Note that you should always apply samples above the level of the mobile phase.
Channeled TLC plates have adsorbent tracks (sample lanes) that are separated by channels where the adsorbent layer is removed from the glass backing. These specially made plates limit the horizontal spreading of sample components during development, thus preventing cross-contamination. In general, unless an adsorbent is coated in a way that causes a non-uniform layer, samples will always travel vertically and should never cross-contaminate an adjacent sample lane. However, for Type-A folks who crave absolute certainty—or in cases where clear evidence is needed for court—channeled plates are the way to go.
There you have it. You now know the eight key factors in selecting a TLC plate—you should feel like a pro now! Of course, if you need any additional guidance, our friendly team is here to help you.
Have a great TLC day!