Optional Procedures

In addition to the basic steps of thin-layer chromatography - sample application, chromatogram development, derivatization, chromatogram evaluation - there are optional procedures that complement the technique. Which of these are needed depends on the nature of samples and/or the tasks of the particular laboratory.

These are

• UV-inspection
• In-house preparation of TLC plates

UV-INSPECTION

Hardly any TLC laboratory can be without the use of UV light for inspecting chromatograms, not even where only dyes are analyzed, because these may contain colorless but UV active components. Of course, a Reprostar instead of a UV lamp may serve this purpose. Two types of ultraviolet light are required for inspecting thin-layer chromatograms:

Long-wave UV light 366nm
Under 366 nm UV substances with either inherent or reagent induced fluorescence appear as bright spots, often differently colored, on a dark background. The sensitivity of this detection method increases with the intensity of the long-wave UV light and also as more visible light is eliminated. A fluorescent indicator F254 contained in the layer neither contributes to nor interferes with this detection method.

Short-wave UV light 254nm
Under 254 nm UV substances absorbing at that wavelength become visible, provided the TLC layer contains a fluorescent indicator F 254. These substances appear as dark spots on a bright background. For this detection method, UV intensity and complete elimination of visible light are less critical.

IN-HOUSE PREPARATION OF TLC PLATES

To day, in-house preparation of TLC plates is of interest for laboratories which, for economical or logistic reasons, cannot rely on precoated plates exclusively.

The self-coating of TLC plates should also be considered when special layers are required that are not available in the form of precoated plates, e.g. layers containing silver nitrate, buffer substances or other reagents, layers consisting of adsorbent mixtures, or in the exceptional cases where the binder contained in commercial precoated plates might interfere with detection.

Adsorbents for In-House Preparation of TLC Layers

For the self preparation of chromatographic layers, adsorbents in the form of powder are mixed with water or with aqueous solutions of salts or buffering compounds to form a thick slurry which is spread onto glass plates by means of a coating device.

Adding calcium sulfate as a "binder" makes it easier to achieve the correct consistency of the slurry for coating; the calcium sulfate contributes very little to the mechanical strength of the layer.

A fluorescence indicator is required to visualize substances which absorb UV light of a wavelength (254 nm), by which the indicator is excited to emit visible light. Such substances appear as dark spots on a bright background. The fluorescence indicator does not interfere with the chromatographic separation, with any derivatization reactions, nor with densitometric evaluation. Most of these type indicators, however, lose their fluorescence on contact with acids. Fluorescence indicators which are stable against acids are only available in certain precoated layers.

PRECOATED LAYERS

In most cases modern planar chromatography utilizes precoated plates. Not only are they more convenient, their quality is superior to that of layers available for self-coating. Several types of phases, HPTLC layer, etc. are only available in the form of precoated plates.
The following list contains the more common types of precoated plates and sheets manufactured by E. MERCK. Other MERCK precoated plates not listed here may also be ordered through CAMAG.

Test Dye Mixtures

Test dye mixtures are useful for functional checks on individual steps in the TLC procedure and for studying the influence of specific parameters. Dye mixtures are convenient as their fractions are easy to track.

Basic Assemblies

All CAMAG TLC and HPTLC basic kits have been carefully composed so that you can immediately start planar chromatography work. These assemblies are configured to make it possible for you to form a complete system for quantitative TLC analysis by adding items at any time without the fear of duplication or redundancy of tools.
Also the transition from using conventional TLC material to high-performance (HPTLC) layers is straight forward.
Using HPTLC material can offer considerable advantages. It does, however, require the consistent use of appropriate instrumentation.