All Things Chromatography

Steven Miles

In August 2015, I purchased Analtech, the company I've worked for since college. Now as Miles Scientific (dba Analtech), the company continues to manufacture Thin Layer Chromatography (TLC) products for laboratories in more than 100 countries. We have the widest variety of TLC plates on the planet and have worked hard to earn our reputation for fast delivery and personalized service.
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Yes, You Can Pick the Right TLC Plate – Part II

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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 20x20 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 20x20 cm plate can be snapped into four 5x20 cm or two 10x20 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 10x20 cm plates; they are popular for method development. They can be snapped into eight slide-sized plates (2.5x10 cm).

Preadsorbent zone

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.

Channeling

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!

Topics: TLC, TLC Plates

Yes, You Can Pick the Right TLC Plate – Part I

TwoPlates.png“I’d like a box of TLC plates, please.” Sounds innocent enough, right? But when you have more than 500 plate options, things get a whole lot trickier. How do you dig through the layers (pun intended) to get the plates you need?

TLC plates come in lots of fun forms and sizes—at least we think they’re fun! You can differentiate TLC plates by:

• Adsorbent material that makes up the layer itself
• Type of plate backing
• Layer thickness
• Plate size
• Fluorescent indicator - UV254 (yes or no)
• Format of scoring (or none)
• Preadsorbent zone (yes or no)
• Channeling (yes or no)

All clear now? Didn’t think so! In this first article of our two-part series, let’s look at the first four factors to help you make the right choice.

Adsorbent material
Most often, the actual layer is silica gel, but it can also be aluminum oxide, cellulose, microcrystalline cellulose, Florisil®, as well as various forms of modified silica gel such as reversed phase layers (C2, C8, C18), amino, cyano, and others. Which layer should you use? It depends on the compound you’re separating. Your best bet is to check what others have done first so you’re not reinventing the wheel. Historical data matters!

Plate backing
The layer’s backing is most often glass, because it can tolerate the most chromatography solvents. Using a flexible backing—namely polyethylene or aluminum—makes sense only when you must quickly and easily cut the plate (or sheet) into smaller sized pieces. Glass can also be cut or purchased pre-scored, so really it comes down to preference, availability, and price.

Layer thickness
Thickness depends on the chromatographic separation you’re doing. For instance, the thickness for standard analytical TLC is 250um (1/4th of a millimeter). But for quantitative level analytical TLC, you’ll need a thinner plate—typically 200um, 150um, or even 100um. This category of TLC has a special name: high performance thin layer chromatography, or HPTLC. Meanwhile, thicker plates (500um, 1000um, 1500um, and 2000um) are best for preparative applications where you’re separating much larger samples. In this case, you’ll want to recover the separated sample components with a scraper or suction device.

Plate size
This is a lot easier to figure out—whew! You just need to know about how many samples you want to run at the same time and what distance you need to chromatograph your samples for adequate separation. Standard sizes are 20x20, 10x20, 5x20, 10x10, 5x10, and 2.5x7.5 (microscope slide). Of course, all measurements are in centimeters. Additional specialty sizes are 2.5x10, 5x5, 2.5x5, and, our favorite, 20x40. Yes, you do need a very large custom developing tank for that one!

We hope this gets you on the right track when you need TLC plates. For more info, stay tuned for part II, where we explain fluorescent indicator, scored plates, preadsorbent zone, and channeling. We find this stuff so exciting and can’t wait to share it with you!

Have a great TLC day!

Topics: TLC, TLC Plates