My Compounds are Colorless; How Can I See Them After the Plate is Developed?

TLC plates with an inorganic fluorescent indicator are available. Activate the indicator with a 254nm UV light, and the background turns green - with some compounds giving a dark spot against this background. If this still does not allow the compounds to be seen, then placing in a tank of iodine crystals for a I2 complex to form. Next, special visualizers based on the compounds structural components which might react (NH₂ groups will react with ninhydrin, etc.). Lastly, the compounds can be charred with some combination of reagents and heat to carbon itself. Details are found in TLC books.

What are the Best Selling TLC Plates?

Just like the silica gel used in column chromatography, the silica gel used on a TLC plate has 60Å pore sizes. It is just crushed more to give about a 15µm average particle (like talcum powder). This silica is mixed with a monomeric organic binder and coated (0.25mm) on a glass (or other) support. The binder crosslinks on drying, giving a hard layer. The standard TLC plate is 20x20cm in size.

What are the Differences Between Hard and Soft Layer TLC Plates?

Hard layer TLC plates are better in terms of resolution, more resilient to shock in transport. However, when charring these plates with sulfuric acid (vapors), they will darken and make small, isolated substances less visible.

Soft layer TLC plates show spot diffusion and band spreading, which makes it more difficult to see/identify small amounts of isolated components. Soft layer TLC plates are also very brittle and the silica gel layer separates easily from the glass. Soft layer plates are easier to scrape to remove compounds of interest and, because the binder is inorganic, will not discolor on charring with sulfuric acid.

Typically, the attributes of hard layer TLC plates outweigh those of soft layer plates. However, some methods specify soft layer TLC plates.

What are the Recommended Storage Conditions for TLC Plates?

TLC plates can be stored in their original container. Once opened, they tend to absorb impurities from the air and will discolor especially around the edges. They need only be prewashed overnight (by just developing in a tank as usual) with methanol to wash these absorbed material to the top of the plate. Then reactivate (100°C for 30min) and spot as usual. Once activated, the plates can be stored in an dry box or air tight container or desiccator over phosphorus pentoxide until used.

What Causes the Solvent to Move Up the TLC Plate?

Capillary action is the force moving the solvent up the plate. Because of gravity, this process slows down as the solvent moves higher on the TLC plate. The more viscous the solvents, the slower is the capillary action, so lower viscosity solvents are recommended for TLC work.

What is the Binder in TLC Plates?

Binder composition is proprietory. It is an acrylate polymer type material.

What is the Difference Between a Hard and Soft Layer TLC Plate?

The binder in hard layer plates is organic (see above). In soft layer plates it is gypsum - calcium sulfate.

What is the Difference Between Silica on Glass, Plastic, or Aluminum Backing?

The only difference in these products is the thickness. The flexible supports (aluminum or plastic) are made a little thinner (0.2mm vs 0.25 on glass). This does not affect the performance at all. The advantage of these layers is that they can be cut either to allow using only what is needed, or after development they allow the spots to be cut from the rest of the plate. This can be done with scissors or a razor blade. Once cut, the compound can be eluted easily or can be added to a scintillation cocktail for counting if they had been labeled.

What is the Difference Between TLC and HPTLC?

A smaller particle silica gel is used for HPTLC (an average of 5µm; it has an average of 15µm for TLC). The spots stay more compact on this silica gel - increasing limits of detection. Shorter development distances are used with this type of plate. The standard size for an HPTLC plate is 10x10cm, or as small as 5x5cm. Obviously, very short developing distances are all that are needed on this type of plate.

What is Geduran^® Silica Gel 60?

Product #11567, or Geduran^® Silica Gel 60, is a more economical alternative to item #9385.

What is the Fluorescent Indicator? What is the 254 After the F Letter?

It is an inorganic compound that is added during plate manufacture. It will not dissolve and will not interfere with any other detection method. Three different F indicators are used. The most used is the F254 - the 254 is the wavelength which makes the indicator fluoresce - , but its crystal structure will be destroyed (and its fluorescence) if a strong mineral acid is used during development (acetic, phosphoric acids are fine, sulfuric is not). If a strong acid is to be used, order the plate with the F254s indicator. A few products contain F366, an alternative to the other two indicators The F254 fluoresces green, the F254s fluoresces a blue white, the F366 fluoresces a blue white, too.

What is a Prescored TLC Plate?

This is a glass plate that has been scored on the back side, so that it can be broken to a smaller size. Usually there are a number of score marks to allow the plate to be broken down into various sizes.

What Kind/Type of Silica Gel is Used for Column Chromatography?

Product #9385 is the most frequently used silica gel - silica gel 60, particle size: 40-63 µm. The next most popular silica gel is product #7734. It is a silica gel 60 with a particle size of 63-200 µm.

Note: The "60" in silica gel 60 refers to the pore size in Angstroms (Å). One inch is 254,000Å, or 60Å is 0.0002 inch.

Why are There so Many Different Sizes of Plates?

Although the 20x20cm plate is the best seller, you may opt to order a smaller size because you are only doing a few sample. The 20x20 plate will be able to analyze about 19 samples and standards, spotted about 1cm apart. If only 3 or 4 sample are to be analyzed a 5x20 or 5x10cm plate will suffice. Merck KGaA, Darmstadt, Germany, makes sizes to microscope slide size (2.5x7.5cm) - this would be good for one or two spots. The height of the solvent development is also the other parameter to be considered. Although some developments are taken 10-12cm above where the samples were spotted, a 8 or 9cm development is also often enough.

Why is it a Good Idea to Activate TLC Plates? How do you activate?

Silica gel plates can absorb moisture easily. Thus to get better reproducibility, activation is recommended. This is done by heating to about 100°C for 30min. (put the plastic plate on a glass or metal plate so it heats up evenly). Heating also helps the binders do a better job under more solvent conditions.

Why Might I Want to Pre-Wash a TLC Plate?

A silica gel plate besides absorbing water, can also absorb other airborne vapors in a lab (or other chemicals). By prewashing (developing overnight in methanol or other solvent mixtures), then any of these impurities can be removed. This technique also gives a cleaner plate for better reproducibility and quantitative work. It is also recommended if any separated compounds are to be removed for other work or characterization. The prewash (also called predevelopment) is often done with chloroform/methanol (1:1).

Will the Organic Binder or Fluorescent Indicator Change My Chromatography?

The binders and indicators have been chosen so they affect the chromatography as little as possible. They are considered inert for most applications, and comparisons with silica gel G (with the Gypsum binder) plates will show little or no difference.