Category Archives: TLC

Getting a Grip on Your Chromatography: “Why Me?” – Part I

Have you ever pulled your TLC plate out of the developing tank, only to realize that your Rfs are out of whack? Or the analyte you were expecting to see is missing or still overlapping another component? Time is wasted. Emotions flare. Did you forget something? Are the plates any good? Did you pick the wrong profession?

Many factors affect your results in chromatography. They include:

  • layer thickness
  • particle size and pore size of adsorbent
  • activation of adsorbent
  • mobile phase (quantity and freshness)
  • equilibrium inside the developing chamber
  • sample (size and application technique)
  • accurate Rf measurement

All of these controllable variables are important. To save time, you may be tempted to focus on the most influential and leave the others alone. However, some applications are more sensitive to slight variations. It’s better to invest time in the beginning to make sure every variable is just right. You’ll save time and energy—and will rest assured you’re indeed in the right profession. 

That’s why we’re publishing a series of articles to explain these contributing factors in detail. (We know how much our analytical audience loves details!) Our goal is to broaden your overall knowledge in chromatography and help you achieve more consistent results.

Stay tuned…and happy chromatographing!

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


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).

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.


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!

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 20×20, 10×20, 5×20, 10×10, 5×10, and 2.5×7.5 (microscope slide). Of course, all measurements are in centimeters. Additional specialty sizes are 2.5×10, 5×5, 2.5×5, and, our favorite, 20×40. 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!

Chromatography reveals 19th century fashion trends

Our friends at ChemistryWorld recently published a piece about how thin layer chromatography Victorian-fashion.jpgwas used in connection with other analytical methods to determine the use of synthetic dyes in the clothing industry throughout the 1800s.

Here’s a few excerpts:

‘This analysis reveals important information for conservators and curators of fashion, raising interesting observations and questions,’ saysJeffrey Church, a molecular spectroscopist at the Commonwealth Scientific and Industrial Research Organisation (CSIRO) in Australia who worked on the study with colleagues from the National Gallery of Victoria…

After taking small samples from each dress, the researchers separated and analysed the dyes and mordants, which are used to bind the dyes to the material, using various techniques, including thin layer chromatography, surface enhanced Raman spectroscopy and energy dispersive x-ray spectroscopy

You can see the complete article by clicking here.

TLC and Flash: Two great methods that work well together

Bob Bickler is a Technical Specialist with 38 years of chromatography experience and writes for The Flash Purification Blog.TLC-sep

Recently, Mr. Bickler wrote a post that outlines something we’ve been telling people for years:

Invest 10 minutes on TLC and save a day of grief

Here’s an excerpt:

I know your time is valuable but investing 10 minutes of it running TLC prior to your flash purification will give you much better separations, increased fraction purity, and reduce the amount of wasted solvent. You really have nothing to lose and everything to gain.

You can read the full post here.

In addition to Flash Chromatography, we believe the same principle applies to HPLC and other separation methods.

For a wide variety of TLC plates, click here.

Some Applications of Thin Layer Chromatography 薄层层析法的一些应用



1. 样品的提纯:样品的提纯可以通过薄层层析法来完成。直接比较可在随机样品和标准或真实样品间进行;如果样品经检测不纯,那么一定有额外斑点存在,而这个很容易被发现。

2. 混合物的识别:薄层层析法可以被应用于天然产品的提纯、隔离和鉴定,其中包括挥发油、精油、固定油、蜡、萜烯、生物碱、苷类、类固醇,等等。

3. 反应监测:薄层层析法可以对反应混合物进行检测从而判断其反应是否完全。这个方法也可用于检查其他分离过程或提纯进程,比如蒸馏、分子蒸馏等等。

4. 生物化学分析:薄层层析法对于生物化学代谢物以及体液、血浆、血清和尿液构成物的隔离或分离检测特别有效。

5. 在化学中的应用:薄层层析法在相似成分的分离和识别实验中的应用等化学领域中越来越普及。同时,该方法也应用于无机化学中阴阳离子的鉴别。

6. 在医药学领域的应用:不少药典已经采用薄层层析法来检测化学药物的纯度。

7. 像催眠药、镇定剂、抗惊厥剂、抗组胺剂、镇痛剂、局部麻醉剂、类固醇等药物都可以用薄层层析法有效检测。

8. 薄层层析法另一个重要应用是对多成分药物配方的分离。

9. 食物和化妆品领域的应用薄层层析法也用于颜色、防腐剂、甜味剂以及各种化妆产品要素的分离和鉴别。