Thin-Layer Chromatography

Gregor Kos and Rudolf Krska

Thin-layer chromatography (TLC) is a chromatographic technique that is useful for the separation, purity assessment and identification of organic compounds. Because of the simplicity and rapidity of TLC, it is also used to monitor the progress of organic reactions.

TLC has found widespread use in mycotoxin determination, but has now largely been replaced by other chromatographic methods (e.g. high performance liquid chromatography [HPLC] and gas chromatography [GC]), because of superior analytical performance. In countries, which do not have expensive equipment at their disposal, but which often produce and export agricultural commodities, TLC remains important. It is also in use for screening purposes prior to labour intensive and time consuming HPLC and GC methods.

Basic Principle

Thin-layer chromatography consists of a stationary phase immobilised on a glass or plastic plate and a solvent as mobile phase. The sample, either liquid or dissolved in a volatile solvent, is deposited as a spot on the stationary phase. The constituents of a sample can be identified by simultaneously running standards with the unknown spot. One edge of the plate is then vertically placed in a solvent reservoir and the solvent moves up the plate by capillary action. When the solvent front reaches the other edge of the stationary phase, the plate is removed from the solvent reservoir. The separated spots are then visualized with ultraviolet light or by a suitable reaction procedure.

The different components in the mixture move up the plate at different rates due to differences in their partitioning behaviour between the mobile liquid phase and the stationary phase. The R_F value for each spot should be calculated. R_F stands for "ratio of fronts" and is characteristic for any given compound on the same stationary phase using the same mobile phase for development of the plates. Hence, known R_F values of standards can be compared to those of (unknown) samples for identification purposes:

 

Figure 1: Typical DC plate with substance spots and solvent front.

Stationary phases are well known from other chromatographic techniques (e.g. normal and reversed phase material). Other principles of separation include ion exchange and size-exclusion mechanisms. The materials are applied as finely dispersed powder to the supporting plate resulting in a coating with a thickness of 200-250 microns.

Additional information about the sample is gained by using 2-dimensional TLC with 2 different mobile phases. After the first separation and evaporation of the mobile phase, the plate is turned by 90 Deg C and a second TLC run is performed using a different type of mobile phase.

A further development is the use of high performance thin layer chromatography (HPTLC). Reduction of layer thickness (down to 100 microns) and particle size (down to 5 microns) of the stationary phase, leading to an improved separation within a shorter time. A HPTLC procedure takes approximately 10 min (conventional TLC up to 15-20 min). A major disadvantage is the smaller sample capacity.

Practical Considerations

Most samples need an extraction and clean-up step for the removal of potential interferences and matrix components prior to analysis (see also fact sheets on Extraction and Clean-up). The analyte concentration, which is spotted onto the plate should be in the range of 0.01-0.1%.

Spotting is usually performed using a capillary. Volumes vary between 0.5 to 5 mL. The spot should be as small and compact as possible with a distance of 1 to 2 cm from the edges of the plate and between the spots. The chromatography chamber is closed and therefore saturated with mobile phase for stable conditions. After removal and drying, the sample spots are detected and, if a standard was used, the sample spots can then be identified.

Locating spots can be performed with the following techniques:

• Exploiting available luminescence characteristics of the sample spot
• Impregnation of the fluorescent indicator substance in the layer bed
• Oxidation of organic compounds by spraying a strong oxidant such as H₂SO₄ or KMnO₄ creating black spots
• Spraying of group or substance specific reagents that create visible/UV active reaction products

Automation is possible with commercially available spotters and plate readers. The most common method for detection is the use of a densitometer, which measures diffusely reflected light from the sample spot in the UV-VIS range.

Application in Mycotoxin Analysis

TLC methods are available for a large number of mycotoxins. Detection and identification procedures have been specifically developed for each single mycotoxin making use of molecular properties or reactions with spray reagents. A short summary for the determination of the most common toxins can be found here, but methods for other toxins are also available (see references):

Aflatoxins

Numerous methods have been developed for the determination of aflatoxins by TLC. Silica plates are most frequently employed with a number of solvent mixtures. Most solvent systems are based on chloroform and small amounts of methanol or acetone, but a shift can be observed to less toxic and more environmentally friendly mixtures (e.g. toluene/ethylacetate or acetone/iso-propanol).

Aflatoxins can easily be detected by fluorodensiometry, because they are strongly fluorescent themselves without any further treatment (  (excitation) = 365 nm,   (detection) = 430 nm). Detection limits are in the lower microgramme/kg range.

A- and B-Trichothecenes

Extraction and clean-up methods have to be applied prior to detection of trichothecenes. Most TLC methods use silica gel as adsorbent. Detection is not an easy task because trichothecenes are either non-emitters or very weak emitters of fluorescent radiation without additional treatment. Chromogenic reagents used for visualisation are sulphuric acid, p-anisaldehyde (for both type A and B trichothecenes). Deoxynivalenol has a characteristic bright blue emission pattern after heat treatment with aluminium chloride and viewing at 365 nm. Apart from the above mentioned spray reagents, other reagents can be used for the detection of different groups of toxins (e.g. cerium(IV)sulphate for the detection of T-2 toxin and roridin). Some toxins also show colouring with visible light.

Ochratoxin A

Adsorbents for TLC for the detection of OTA include silica gel, rice starch and modified silica gel (impregnation with oxalic acid). Solvent systems again focus on the use of chlorinated solvents (mixtures of chloroform and methanol or ethanol), but the use of toluene/ethylacetate has also been reported.

OTA can be viewed as a greenish spot by UV irradiation at 366 nm. The colour changes to purple blue after treatment with aqueous NaHCO₃ or NaOH. Ammonia is also used for de-protonation of the acidic function also resulting in the characteristic purple blue colour of the spot.

Patulin

This toxin is frequently found in apple juice. Liquid samples still require extraction and clean-up procedures before the sample can be analysed using TLC. Solvent mixtures use chloroform and methanol/ethanol, but the use of ethanol/water or ethylacetate/hexane has also been reported. Silica gel plates are most common.

Detection limits are in the microgramme/kg range (down to 25 microgramme/l depending on the solvent system). Detection is performed by utilising yellow fluorescence after excitation with long-wave UV light.

Zearalenone

Samples containing zearalenone are subjected to TLC with silica plates as adsorbent. Solvent systems again employ mostly chloroform with mixtures of methanol, n-hexane and acetone. Toluene/ethylacetate mixtures are also employed. Zearalenone exhibits greenish blue fluorescent light after excitation with UV light at 274 nm. A yellow and brown spot can be observed after treatment with 50% sulphuric acid in methanol. A blue spot is observed after irrigation with a 1% aqueous solution of K₃Fe(CN)₆ and Fe(III)chloride, followed by 2 M HCl. Detection limits are in the microgramme/kg range.

References

J.-M. Mermet, M. Otto, R. Kellner (Eds.), Analytical Chemistry, Wiley-VCH, Weinheim/Germany (1998) 204-208.

V. Betina, Thin Layer Chromatography of Mycotoxins, Journal of Chromatography 334 (1985) 211-276