FAQ – Frequently Asked Questions

TLC is an acronym for Thin Layer Chromatography. It’s called Thin Layer because the stationary phase is formed into a thin layer coated onto an inert backing material which can be rigid or flexible. A typical rigid backing is glass, and flexible backings can be either plastic or aluminum. Coatings on a rigid backing are usually referred to as TLC plates whereas those on a flexible backing are referred to as TLC sheets.

Chromatography is the science of separation of things in a mixture. It is a multi-phase system with a stationary phase, a moving phase, and a phase in which the analyte mixture is separated. The stationary phase, as its name implies, is fixed in place. The moving phase, also called the mobile phase, moves past the stationary phase. The analyte components interact with the two phases to different degrees. This differential interaction results in a physical separation of the components of the mixture.

Chromatography can be subcategorized in various ways. If the moving phase is a liquid, it is called Liquid Chromatography or LC. If the moving phase used is a gas, it becomes Gas Chromatography or GC. Liquid Chromatography operated under high pressure is called High Pressure Liquid Chromatography or HPLC.

The distinction might be based on the type of analyte being analyzed, such as Ion Chromatography. The name could reflect the form of the stationary phase, such as a thin layer, hence Thin Layer Chromatography or TLC. Or the name could derive from the mechanism of the interaction between stationary and mobile phase, such as Size Exclusion Chromatography.

HPTLC is an acronym for High Performance Thin Layer Chromatography. It is a form of TLC utilizing a smaller particle adsorbent, which improves resolution.

High Performance plates are also smaller in size, typically 10×10 cm or even 5×5 cm. Smaller plates necessitate shorter development distance, which means quicker development times.

HP plates also consist of a thinner coating layer than analytical TLC plates. HPTLC layer thickness is typically 150-200 microns, versus 250 microns for standard TLC plates.

A thinner coating requires a reduction in the sample size. Sample sizes begin to range into nanoliters of applied sample versus microliters for analytical plates. Larger sample volumes must be divided into aliquots during sample application.

The combination of smaller particle adsorbents with tighter distribution, thinner coatings and shorter development distances, along with smaller sample size combine to decrease diffusion and improve performance. The result is an increase in resolution and detectability of the sample components.

TLC can be performed to accomplish a variety of goals. The purpose could be simply observing the presence or absence of a particular chemical component in a mixture. In this case, a thinner layer of adsorbent coating is preferred. A typical layer thickness for this use would be 250 microns (0.25 mm). TLC plates with a coating thickness of 250 microns are referred to as analytical layers.

Layers thinner than 250 microns are usually termed High Performance TLC plates or HPTLC. These layers in a general sense are also analytical layers, but the goal begins to shift from a qualitative observation to determining how much of a material is in the sample. HPTLC layers are usually preferred for quantitative determinations.

On the other end of the thickness spectrum are plates with layers thickness greater than 250 microns, namely layers 500 microns (0.50 mm) to 2000 microns (2.0 mm). These thicker coatings are termed preparative plates. They are intended for separating larger amounts of sample, usually so that one or more components can be recovered for subsequent analysis or use.

The division of uses of TLC plates by the coating thickness generally holds true. Layers below 250 microns are typically used for quantitative work, 250 microns are useful for qualitative analyses, and preparative layers 500 microns or thicker find the most use isolating multi-milligram to even gram quantities of material.

However, there is lots of overlap between the goals and the plate thickness chosen for the job. In other words, it’s not outlandish for quantitative determinations to be made using 250-micron analytical layers. Nor is it impossible to achieve preparative separation and recovery of significant amounts of material from a 250-micron analytical plate.

A5 TLC is in its basic form an inexpensive technique to use in the laboratory. It has low capital requirements for equipment, and most labs have the necessary glassware and incidental items already on hand. Some solvents and reagents for development and visualization of TLC plates may be required, but they tend to be stocked in most labs.

To get started in TLC, a development chamber is required to contain the mobile phase. It must be enclosed with a lid to retain the solvent mixture used for development. It also must be large enough to contain the TLC plate, so some thought must be given as to what size TLC plate will be used. Conventional size for a TLC is 20×20 cm, although many smaller sizes are available.

The next basic required item is a sample application device. Most labs have microliter pipettors, which would work, but better yet are disposable microliter capillary pipettes. Disposable capillaries are easier to handle and control the flow and position of the liquid sample.

Saturation pads are used inside the developing chamber to equilibrate the vapor space of the chamber with the bulk solvent mixture reservoir. It works by increasing the surface area of the mobile phase to hasten and maintain the vapor equilibrium between the gaseous and liquid states. A simple way to implant vapor saturation is to place one pad against the rear wall of the developing chamber. The bottom should be in contact with the mobile phase reservoir. More elaborate saturation schemes may be required for critical equilibrium situations.

More than likely, the final items a lab might need to procure are methodology for visualizing the separated sample components after developing a TLC plate. This is usually a necessary step unless your samples are visually colored. A reagent sprayer and UV light will cover most visualization tasks. The TLC sprayer is used to derivatize a colorless sample component into something visually colored. A hand-held UV light can be used to examine the developed TLC plate under UV254, a wavelength that many molecules of interest absorb.

The last item needed is, of course, some TLC plates. A variety of adsorbents are available to separate different compound classes. By far the most popular is silica gel, which offers wide-ranging use from nonpolar samples to fairly polar materials. It’s usually the best choice to start with in a new or unknown situation. As in all chromatography, choosing the proper mobile phase is key to coaxing the separation of the desired sample components.

The letter “F” means that UV254 fluorescent phosphor is in the coated layer. It is used in the detection of UV254 absorbing sample components that have been separated on the TLC plate.

Miles Scientific recommends a shelf life of approximately nine months after the receipt of the product.

Technically, TLC plates don’t expire, but they tend to do their job of adsorbing organic vapors and water vapor even when sitting in the box. Usually this doesn’t interfere with the separation but could have two other effects. First, the adsorbed organic content might alter the chromatographic properties of the adsorbent. This you will see because the Rf’s of the sample components are not what they should be. Second, adsorbed water influences the activity of adsorbent, which may alter the Rf’s as well.

Fortunately, you can restore the plate to its “like new” condition by a two-step process: prewashing and heat activating before use. Prewashing is the process of running a middle-of-the-road polarity solvent (something like chloroform:methanol ~1:1 ratio) up to the top edge of the plate. This will wash any adsorbed organic junk to the top, where it will be out of the way of actual chromatography. After this, heat activate the plate by drying at 90-100 C for 45-60 minutes. This will drive off reversibly bound water from the silica gel (and ensure complete removal of organic prewash solvents). The clean TLC plate is now ready to use. A rugged TLC method will include both steps, but they are oftentimes overlooked by the original authors of the method.

NOTE: Make sure to mark the top of the plate so you can orient your “fresh” TLC plate correctly.

It’s best to store TLC plates in their own cabinet away from chemicals and other sources of vapor. If a cabinet isn’t available, a shelf removed from chemical storage will suffice. Exact temperature control is not necessary, although storage is best in an office or lab environment rather than warehouse space. A warehouse or outlying storage space is subject to greater temperature swings as well as potentially damaging humidity extremes.

Adsorbent materials used to coat TLC plates have very active surfaces. They tend to do their job of adsorbing stuff even while sitting in storage. Vapors causing issue can be either organic in nature or water vapor, i.e. humidity in the air.

Organic vapor contamination is cumulative. The longer the storage interval, the more likely the plate surface will pick up contaminants from the air. It manifests itself as a yellowing of the layer surface, growing more intense with age. After development of the plate, it appears as a garbage band extending below the solvent front.

Unlike organic vapors, water vapor expressed through relative humidity of the air will seek equilibrium with water on the surface of the adsorbent layer. If the storage area is higher in relative humidity than the place of analysis, the layer surface will have too much adsorbed water relative to the environment. Given enough time, it will come to equilibrium, but problems could occur. This sometimes explains why a given separation changes over time, because the remaining quantity of plates are slowly coming into equilibrium with the new environment.

Oftentimes no, but a rugged TLC method will include both a conditioning step as well as heat activation, although they are often overlooked by the original authors of the method.

TLC plates tend to do their job of adsorbing organic vapors and water vapor even during storage. Usually this doesn’t interfere with the separation but could have an effect in two ways. First, the adsorbed organic content might alter the chromatographic properties of the adsorbent. This you will see because the Rf’s of the sample components are not what they should be. In more severe cases, a yellowing of the adsorbent layer will be seen especially around the edges of plate. Second, adsorbed water influences the activity of adsorbent, which may alter the Rf’s as well.

Fortunately, you can restore the plate to its “like new” condition by a two-step process: prewashing and heat activating before use. Prewashing is the process of running a middle of the road polarity solvent (something like chloroform:methanol ~1:1 ratio) up to the top edge of the plate. This will wash any adsorbed organic junk to the top, where it will be out of the way of actual chromatography. After this, heat activate the plate by drying at 90-100 C for 45-60 minutes. This will drive off reversibly bound water from the silica gel (and insure complete removal of organic prewash solvents). The clean TLC plate is now ready to use.

Visualization techniques can be divided into two groups: destructive and nondestructive. As the name implies, destructive techniques chemically alter the molecule being investigated. Nondestructive visualization methods allow the location of a sample component on the TLC plate without acting on the identity of the material.

If the purpose of TLC separation is to isolate an amount of material for subsequent analysis, a nondestructive technique is required. Destructive visualization is usually used only for discerning the presence or lack of a material in a sample. There are several workarounds for using destructive visualization techniques for preparative TLC.

One common nondestructive approach is to locate the separated components using UV light. Most often, UV detection of TLC plates uses shortwave UV (254nm), which is sometimes called UVC. This technique uses a TLC plate which incorporates a UV254 fluorescent agent into the layer.

For this scheme to work, the materials being analyzed must absorb shortwave UV radiation. An aromatic ring in the molecule of interest is the most common route providing sufficient UV absorption. When a UV active plate is examined under UV254, the sample components absorb the UV light quenching the fluorescence of the indicator and thus appear as a dark area against a bright (usually green) background.

A less common use of UV detection is using longwave UV light at 366 nm, also referred to as UVA. Some compounds absorb longwave UV and emit in the visible portion of the spectrum. This process is known as fluorescence. It takes a fairly complex molecular structure for this to occur, which is why it is less common.

Most UV lights—whether portable, handheld units or those that are part of a UV viewing cabinet (darkroom)—have both wavelengths available. Separate switches will operate one or the other wavelength. It’s always a good idea to examine the TLC plate at both wavelengths.

Trying both UV wavelengths is part of more encompassing view of visualization. Sometimes it’s enough to simply locate the presence or lack of a sample component. Wanting to know how much material present is a natural extension to this need. But if the analytical reason expands into gleaning knowledge about the molecule, using a variety or series of different techniques, one can begin to build a framework of results. This might be used to piece together the structure if it’s unknown, give insight into solubility of the material, or uncover how the compound relates to others under investigation.

A non-UV active material will have to be derivatized after separation to induce visual contrast between the background and sample component. Derivatization is a destructive technique, meaning the material you started with no longer exists. Most texts concerning TLC, such as the CRC Handbook of Chromatography, will suggest visualization reagents or techniques for different compound classes.

Visualization reagents may be applied to the developed TLC plate by either spraying the reagent with an atomizer or dipping the plate into a jar of reagent. Dipping provides a more controlled application of reagent, which usually results in uniform colorization with a lower detection limit. However, dipping requires a greater volume of reagent, especially if larger plates are used. Also, some layers aren’t stable with regard to particular reagent solutions and can disintegrate. Spraying the reagent generally consumes less visualization solution, although it tends to produce grainy or spotty patterns.