Thin layer chromatography

Time-lapse of a thin-layer chromatography of a pigment filtrate of the common ivy ( Hedera Helix L. )

TLC of a dye mixture from a permanent marker

The thin layer chromatography or -chromatografie ( DC or TLC , English thin layer chromatography ) is a physically - chemical separation process which is used to study the composition of samples. Particularly advantageous with this chromatographic method are the low expenditure on equipment, the speed, the high separation efficiency and the low substance requirement. It is used, for example, to quickly prove the purity of a substance or to check its identity with a reference substance. It is also possible to follow the course of chemical reactions in the laboratory with little effort.

history

NA Izmailov and MS Shraiber, two Russian researchers, carried out a chromatographic separation in 1938 with a horizontal thin-layer plate on which they dripped the solvent. But her method was hardly noticed. It was only when Justus G. Kirchner (1911–1987) at the Fruit and Vegetable Laboratory of the US Department of Agriculture in Southern California and his colleagues (including B. Harnischmacher) started working with it in 1951 that the interest of others in the method was aroused. Egon Stahl helped her breakthrough when he described the production of high-performance panels. The name thin-layer chromatography also comes from him.

Theoretical foundations

Basic principle of chromatographic separation

The basic principle of chromatography applies to all chromatographic methods and can be briefly summarized as follows: Particles to be analyzed (molecules, ions) are distributed over two phases in a ratio that is characteristic of the type of particle. How the ratio varies with the type of particle also depends on the physical properties of the two phases. The relationships are established as dynamic equilibria ( diffusion due to the movement of heat) and are converted into speed differences in chromatography by the movement of a mobile against a stationary phase. The speed of a particle type results from the product of the speed of the mobile phase by the amount of time that the particles spend in the mobile phase. It is assumed that the particles in the mobile phase (statistically speaking) have the same speed as the solvent molecules. If they are bound to the stationary phase, the speed is zero (“stop and go” model).

To achieve a separation with small differences in distribution is not only a question of the length of the route; it is also crucial that the particles change between phases very often. Then the law of large numbers applies and the proportion of time that the individual particle spends in the mobile or stationary phase corresponds exactly to the proportion of particles of this type in both phases.

TLC is one of the liquid chromatographic methods . This means that all samples that are sufficiently stable and can be brought into solution can be examined. In TLC, a solvent migrates upwards through capillary forces in a solid, fine-pored carrier material (e.g. silica gel ).

Stationary phase

The stationary phase (separating layer) consists of a thin layer of a very fine-grained material (e.g. silica gel , kieselguhr , aluminum oxide , cellulose ). This separating layer is applied very evenly to a carrier film or carrier plate made of plastic, aluminum sheet or glass and is commercially available in different layer thicknesses. As a rule, silica gel is used as the stationary phase (normal phase chromatography), which, due to the free terminal hydroxyl groups, serves as a polar adsorbent for the sample molecules. The average pore diameter of the silica gels is usually 4 to 100 nm, the pore diameter of 6 nm (silica gel 60, Merck) being the most common. Silica gels contain siloxane or silanol groups.

Alternatively, TLC materials with other functional groups (e.g. amino groups) can also be used. They differ from standard silica gel not only in their polarity, but also in their basicity and thus lead to completely different separation results. Also, surface modified silica gels with nonpolar traps (by coupling with organochlorosilanes) are used (reverse phase chromatography, reversed phase ). The order in which the different sample molecules are separated is then reversed - the polar molecules run faster, the non-polar molecules are held more firmly. Among other things, it is advantageous that very polar samples can also be examined. Aluminum oxide, magnesium silicate, kieselguhr, polyamide and cellulose are also suitable as further stationary phases for the TLC.

The separation of geometric and positional isomers with double bonds is achieved by means of silver nitrate thin layer chromatography. To separate chiral samples, reversed phase DC plates are used which are coated with the copper complex of a chiral derivative of the amino acid L - proline and which allow the direct thin-layer chromatographic separation of enantiomers according to the principle of chiral ligand exchange chromatography .

For special applications, it may also be necessary to “wash” the plate before applying the sample or to dry it in a desiccator or drying cabinet at an elevated temperature. The plates are washed by placing them in a chromatography chamber with the appropriate solvent until the solvent front has reached the top edge of the plate.

Mobile phase

In normal-phase TLC, non-polar organic solvents are used as mobile solvents, usually as a mixture with moderately polar solvents (e.g. petroleum ether and ethyl acetate ); in reverse-phase TLC, however, polar solvents (e.g. acetonitrile and water ). The polarity of the solvent can be controlled via the mixing ratio.

The adsorption capacity of silica gel for the functional groups decreases in the sequence -COOH> -OH> -NH ₂ > -SH> -CHO> R ₂ C = O> CO ₂ R> - OCH ₃ > -HC = CH-.

Organic carboxylic acids or alcohols therefore have a very high adsorption on silica gel and thus low R _F values. If the solvent is not very polar, these substances can possibly remain at the starting point.

Isolating distance

Different types of diffusion counteract a good separation. The decisive factor is the rapid change of the particles between the two phases. It is therefore also beneficial to have the smallest possible running distances and fine, uniform grain sizes of the layer material. Too low and too high speeds of the mobile phase have a negative effect. Too low a speed favors an enlargement of the zones in which the sample molecules are located. The more time is available, the greater the role that diffusion processes play within the mobile phase. If the speed is too high, there is less of a change in the particles between the mobile and the stationary phase. This leads to a greater statistical spread and is also undesirable. In all chromatographic methods there is accordingly an optimal speed of the mobile phase, which is described by the Van Deemter equation . The finer the grain sizes (or the dimensions), the higher the speed can be. This also has economic advantages.

When the desired DC spatial separation between the different sample components of the entire travel distance is proportional (- solvent front distance from starting line). The enlargement of the individual zones due to statistical effects is smaller (not proportional to the running distance, but the root of the running distance). Therefore, it makes sense to use larger DC foils and running distances for difficult separations.

With thin layer chromatography, a separation efficiency of approx. 400–3000 theoretical plates can be achieved over a 15 cm long run.

Practical implementation

Sample application

Glass capillaries with ring mark

The substance to be examined is dissolved in a suitable solvent and applied in dots or lines with the help of a capillary . With the one-dimensional TLC this happens on the starting line of the foil or plate, with the two-dimensional TLC (see below) in a corner. The amount of substance to be separated is approx. 5–20 micrograms. It is crucial to keep the application areas as narrow as possible (a few millimeters). TLC foils with so-called concentration zones below the start line, coated with a material with particularly low adsorption, are also commercially available. The substances in the possibly imprecisely placed or large sample spots then dissolve immediately upon passage through the solvent front and with it reach the starting line, compressed in the direction of travel. For a particularly even, quantitatively reproducible application, machines are also available which spray the solution with the aid of compressed air or nitrogen. After application, the plate must be dried, as residual solvent can change the result.

In addition to the samples, solutions of pure reference substances or reference mixtures are also applied on the starting line in many cases.

Separation

Schematic representation of the process of thin-layer chromatography

After application, the plate is placed vertically in a chromatography chamber with a suitable flow agent (mobile phase). In order to prevent the results from being influenced (excessively ) by the evaporation of the solvent, the separation is carried out in an atmosphere saturated with the solvent in a closed vessel. A filter paper can be inserted for better saturation of the vapor space with solvent. ${\ displaystyle R_ {f}}$

The flow agent is now sucked up into the stationary phase by capillary forces . As soon as the liquid reaches the starting line, the substances in it dissolve. The molecules are now exposed to the forces of attraction of the stationary phase on the one hand and the forces of attraction of the mobile phase on the other. Depending on the balance of forces, a particle tends to stay at the starting point or it moves upwards with the mobile phase. The more non-polar the flow agent and the more polar a substance, the less the substance migrates. The solvent polarity is analogous to that in column chromatography .

Shortly before the solvent front reaches the upper end of the plate, the plate is removed from the chromatography chamber and dried as quickly as possible.

evaluation

Schematic representation of a TLC plate

TLC separation of lichen components

In the simplest case, the separated substances are visible as dots when viewed under UV light. Alternatively, they can be derivatized with chromophores prior to chromatography to make them UV-active. Spraying with or immersion in reagent solutions are further options.

Many layer materials contain additives that fluoresce in UV light and show dark fluorescence quenching at those points where the separated substances are located. These fluorescent dyes must not migrate during the chromatographic separation. Used mainly are manganese activated zinc silicate (with UV light of wavelength 254 nm is irradiated), and calcium tungstate (with UV light of wavelength 366 nm is irradiated). In fact, the method is not about fluorescence quenching in the strict sense. Sample molecules become visible when they absorb UV light in the range of 254 nm or 366 nm . Less UV light then reaches the fluorescent dye molecules (dark spots can be seen on a green or blue background). For this there must be a sufficient number of functional groups or sufficiently large systems with conjugated double bonds . Saturated hydrocarbons and many amino acids can therefore not be detected with this method, aromatic compounds z. B. very light at 254 nm.

The self-fluorescence of certain substances or other properties such as radioactivity can also be used for detection.

When using spray or immersion reagents (e.g. NBD-Cl , molybdophosphoric acid or 2,7-dichlorofluorescein ), color reactions occur that are sensitive and specific enough to be used to detect certain functional groups . The information content of the TLC can be increased significantly by selecting the color reaction. Alternatively, reactions are used that are generally effective (for example, oxidation with the aid of nitric acid solutions or iodine vapor). With a number of color reactions, it is necessary to heat the film after spraying or immersion.

Another very simple method is vapor deposition with molecular iodine . All you have to do is put a few iodine crystals in a glass vessel. They sublime , that is, they evaporate directly at room temperature, forming a violet vapor of diiodine molecules. By placing a TLC film in such a trough, loose complex compounds (purple or brown) are formed within a short time via diffusion and reaction with the molecules of the substance stains. Advantage or disadvantage of the method: the iodine compounds disintegrate relatively quickly.

In biochemistry , an acidic ninhydrin solution is a common spray reagent used to detect amino acids. Here, the ninhydrin becomes Ruheman's violet via the Schiff base and through decarboxylation and hydrolysis . By applying reference samples that migrate to the same extent as the corresponding sample components under the same conditions, the qualitative occurrence of substances can be demonstrated. For this purpose, the position of the various points is compared with the position of the reference samples.

In order to be able to compare different DC, the so-called -values (retention factor , retention factor , ratio of fronts ) are calculated. This is the ratio of distance run by the substance ( ) for the running track of the eluent ( ) . The values are material constants for the same panel material and the same solvent composition. The value is also used, the distance traveled by the substance stain being set in relation to the distance traveled by a standard. The standard is usually a pure substance. A qualitative evaluation is therefore possible on the basis of the value. ${\ displaystyle R _ {\ mathrm {f}}}$ ${\ displaystyle S}$ ${\ displaystyle L}$ ${\ displaystyle R _ {\ mathrm {f}} = {\ frac {S} {L}}}$ ${\ displaystyle R _ {\ mathrm {f}}}$ ${\ displaystyle R _ {\ mathrm {st}}}$ ${\ displaystyle R _ {\ mathrm {st}}}$

The quantitative evaluation can be carried out with a densitometer . As so-called TLC scanners, these devices offer the option of measuring in the visible and ultraviolet spectral range. They can also be used to measure fluorescence. Regular flatbed scanners can also be used for a densiometric evaluation in the DC.

New device developments also enable the direct coupling of thin-layer chromatography with mass spectrometry . In this way, the chromatographically separated components can also be reliably identified using their mass spectrum .

More complex techniques and methods

In addition to the linear and one-dimensional thin layer chromatography described so far, techniques have been developed for special applications and separation tasks in order to be able to separate more complex substance mixtures, for example. The high performance thin layer chromatography represents such a development.

Two-dimensional DC

In two-dimensional TLC, after the first development, the solvent is evaporated, the plate rotated 90 ° and - usually in a different solvent - a second development is carried out. This enables better separation of multicomponent mixtures. However, the identification is more complex, since no reference substances can run along. Before the second development, the TLC plate can also be processed (e.g. irradiation with UV light) before the second development takes place in the same solvent ( transport-reaction-transport technique , or TRT technique for short)

Circular thin layer chromatography

An alternative technique to linear TLC is what is known as circular thin layer chromatography (abbreviated to CLC from Centrifugal Layer Chromatography or RPC from Rotary Planar Chromatography ). Round glass panes are used that are coated with the stationary phase in a ring. The disc is set in rapid and evenly controlled rotation with the help of an electric motor. The sample solution is fed to the inner edge of the layer with the help of a pump, before and after the corresponding solvent.

Since the stationary phase usually contains a dye that fluoresces in UV light with a wavelength of 254 nm or 366 nm, the separation process can be controlled by exposure to a UV lamp. At the beginning of the separation process, the sample is located in a circular zone a few millimeters thick on the inner edge of the disc. As the separation progresses, the substance mixture in the sample is split up into a series of rings which, driven by centrifugal force, migrate outwards.

Preparative thin layer chromatography

The TLC can also be preparative, i.e. H. can be used to clean small amounts of substance. Then it is also called PLC ( Preparative Layer Chromatography ). As a rule, larger quantities (up to 100 mg) of the substance mixture to be separated are applied in a line-like manner to glass plates with thicker stationary phases (up to 2 mm). After the separation run, the separated substances are located as lines at different heights. The target substance can then be mechanically scraped off together with the carrier material. It is eluted from the stationary phase by simple filtration with a suitable solvent and thus kept pure.

Sufficient amounts of pure substance can also be obtained from the usual (analytical) TLC foils with a thin stationary phase in order to be able to use them for sensitive analysis methods such as mass spectrometry or infrared spectroscopy .

The circular DC can also be used preparatively. Here, the desired sample component, after it has reached the outer edge of the separating layer, is collected together with the solvent in a corresponding collection container.

In order to purify larger amounts of substance with far less expenditure on equipment, column chromatographic techniques such as flash chromatography are used today .

Advantages and disadvantages of thin layer chromatography

In contrast to the more powerful chromatography methods such as gas chromatography and high-performance liquid chromatography , TLC requires little equipment and is a fast, versatile and inexpensive analysis method.

Gas chromatography can only be used for samples that can be vaporized without being decomposed. In the liquid there are few restrictions. A way to dissolve a sample can almost always be found. Compared to column chromatographic methods, thin-layer chromatography has the advantage that samples that contain groups of components that differ greatly in polarity are easier to detect. Changing the eluent is not as easy as with column chromatography. However, it is possible to develop first in one solvent and after intermediate drying in another (which differs greatly in polarity).

The disadvantage of the analytical application of TLC is that it is more difficult to carry out a quantitative analysis. For certain tasks, however, it is sufficient to estimate the proportions (progress of a chemical reaction). The previous problems of reliable quantification have been overcome in recent years by developing high-performance densitometers - as mentioned above. The quality criteria of the quantitative evaluation now meet the guidelines for good laboratory practice .

literature

H.-P. Frey, K. Zieloff: Qualitative and quantitative thin layer chromatography. VCH, Weinheim 1993, ISBN 3-527-28373-0 .
F. Geiss: The parameters of thin layer chromatography. Vieweg, Braunschweig 1972, ISBN 3-528-08299-2 .
Elke Hahn-Deinstrop: Thin-layer chromatography - practical implementation and avoidance of errors. Wiley-VCH Verlag, Weinheim 1998, ISBN 3-527-28873-2 .
Justus G. Kirchner: Thin-layer chromatography. 2nd Edition. Wiley, New York 1978, ISBN 0-471-93264-7 .
Peter Pachaly: Thin-layer chromatography in the pharmacy , Wissenschaftliche Verlagsgesellschaft mbH, Stuttgart 1982, ISBN 3-8047-0624-X
Joseph Sherma, Bernard Fried (Ed.): Handbook of Thin-Layer Chromatography (= Chromatographic Science. Volume 55). Marcel Dekker, New York NY et al. 1991, ISBN 0-8247-8335-2 .
Lloyd R. Snyder, Joseph H. Kirkland, John W. Dolan: Introduction to Modern Liquid Chromatography. 3. Edition. Wiley-Interscience, Hoboken NJ 2010, ISBN 978-0-470-16754-0 .
Egon Stahl (Ed.): Thin-layer chromatography: A laboratory manual. Springer, Berlin et al. 1962.
Felix Schumm : Thin-layer chromatograms - also possible for the amateur. In: Current Lichenological Communications. No. 9, 2002, pp. 8-22.
Colin Poole: Instrumental Thin-Layer Chromatography. Elsevier, Oxford 2014, ISBN 978-0-12-417223-4 .

Web links

Wikibooks: Practical course in food chemistry / thin layer chromatography - learning and teaching materials

Thin layer chromatography of a crude chlorophyl extract

Individual evidence

^ Joseph C. Touchstone: Practice of Thin Layer Chromatography. 3. Edition. Wiley, New York NY et al. 1992, ISBN 0-471-61222-7 , pp. 3-4.
↑ Beate Breuer, Thomas Stuhlfauth, Heinrich P. Fock: Separation of fatty acids or methyl esters including positional and geometric isomers by alumina argentation thin-layer chromatography. In: Journal of Chromatographic Science . Volume 25, No. 7, 1987, ISSN 0021-9665 , pp. 302-306, doi: 10.1093 / chromsci / 25.7.302 .
^ Kurt Günther, Jürgen Martens , Maren Schickedanz: Thin-layer chromatographic separation of enantiomers by means of ligand exchange. In: Angewandte Chemie . Volume 96, 1984, pp. 514-515, doi: 10.1002 / anie.19840960724 .
^ Kurt Günther: Thin-layer chromatographic enantiomeric resolution via ligand exchange. In: Journal of Chromatography A . Volume 448, 1988, pp. 11-30, doi: 10.1016 / S0021-9673 (01) 84562-3 .
^ Kurt Günther, Maren Schickedanz, Jürgen Martens: Thin-Layer Chromatographic Enantiomeric Resolution. In: Natural Sciences . Volume 72, No. 3, 1985, pp. 149-150, doi: 10.1007 / BF00490403 .
↑ Teresa Kowalska , Joseph Sherma (Ed.): Thin Layer Chromatography in Chiral Separations and Analysis (= Chromatographic Science. Volume 98). CRC Press Taylor & Francis Group, Boca Raton FL 2007, ISBN 978-0-8493-4369-8 .
↑ H. Jork, W. Funk, W. Fischer, H. Wimmer: Thin layer chromatography. Volume 1 a: Physical and chemical detection methods: fundamentals, reagents. VCH Verlagsgesellschaft, Weinheim 1989, ISBN 3-527-26848-0 .
↑ H. Jork, W. Funk, W. Fischer, H. Wimmer: Thin layer chromatography - reagents and detection methods. Volume 1b, VCH Verlagsgesellschaft, Weinheim 1993, ISBN 3-527-26976-2 .
↑ Mitchell E. Johnson: Rapid, Simple Quantitation in Thin-Layer Chromatography Using a Flatbeted Scanner. In: Journal of Chemical Education. Volume 77, No. 3, March 2000, ISSN 0021-9584 , p. 368, doi: 10.1021 / ed077p368 .
↑ Paul Abu-Rabie, Neil Spooner: Direct Quantitative Bioanalysis of Drugs in Dried Blood Spot Samples Using a Thin-Layer Chromatography Mass Spectrometer Interface. In: Analytical Chemistry . Volume 81, No. 24, 2009, pp. 10275-10284. PMID 19919036 , doi: 10.1021 / ac901985e .
↑ Joseph Sherma, Bernard Fried (Ed.): Handbook of thin-layer chromatography. (= Chromatographic Science. Volume 89). 3., corr. and exp. Edition. CRC Press, Boca Raton FL et al. 2003, ISBN 0-8247-0895-4 , p. 323. ( Restricted preview in Google book search).

[geschichte-1] Joseph C. Touchstone: Practice of Thin Layer Chromatography. 3. Edition. Wiley, New York NY et al. 1992, ISBN 0-471-61222-7 , pp. 3-4.

[2] Beate Breuer, Thomas Stuhlfauth, Heinrich P. Fock: Separation of fatty acids or methyl esters including positional and geometric isomers by alumina argentation thin-layer chromatography. In: Journal of Chromatographic Science . Volume 25, No. 7, 1987, ISSN 0021-9665 , pp. 302-306, doi: 10.1093 / chromsci / 25.7.302 .

[3] Kurt Günther, Jürgen Martens , Maren Schickedanz: Thin-layer chromatographic separation of enantiomers by means of ligand exchange. In: Angewandte Chemie . Volume 96, 1984, pp. 514-515, doi: 10.1002 / anie.19840960724 .

[Günther_(a)-4] Kurt Günther: Thin-layer chromatographic enantiomeric resolution via ligand exchange. In: Journal of Chromatography A . Volume 448, 1988, pp. 11-30, doi: 10.1016 / S0021-9673 (01) 84562-3 .

[5] Kurt Günther, Maren Schickedanz, Jürgen Martens: Thin-Layer Chromatographic Enantiomeric Resolution. In: Natural Sciences . Volume 72, No. 3, 1985, pp. 149-150, doi: 10.1007 / BF00490403 .

[Kowalska-6] Teresa Kowalska , Joseph Sherma (Ed.): Thin Layer Chromatography in Chiral Separations and Analysis (= Chromatographic Science. Volume 98). CRC Press Taylor & Francis Group, Boca Raton FL 2007, ISBN 978-0-8493-4369-8 .

[7] H. Jork, W. Funk, W. Fischer, H. Wimmer: Thin layer chromatography. Volume 1 a: Physical and chemical detection methods: fundamentals, reagents. VCH Verlagsgesellschaft, Weinheim 1989, ISBN 3-527-26848-0 .

[8] H. Jork, W. Funk, W. Fischer, H. Wimmer: Thin layer chromatography - reagents and detection methods. Volume 1b, VCH Verlagsgesellschaft, Weinheim 1993, ISBN 3-527-26976-2 .

[9] Mitchell E. Johnson: Rapid, Simple Quantitation in Thin-Layer Chromatography Using a Flatbeted Scanner. In: Journal of Chemical Education. Volume 77, No. 3, March 2000, ISSN 0021-9584 , p. 368, doi: 10.1021 / ed077p368 .

[10] Paul Abu-Rabie, Neil Spooner: Direct Quantitative Bioanalysis of Drugs in Dried Blood Spot Samples Using a Thin-Layer Chromatography Mass Spectrometer Interface. In: Analytical Chemistry . Volume 81, No. 24, 2009, pp. 10275-10284. PMID 19919036 , doi: 10.1021 / ac901985e .

[Sherma-11] Joseph Sherma, Bernard Fried (Ed.): Handbook of thin-layer chromatography. (= Chromatographic Science. Volume 89). 3., corr. and exp. Edition. CRC Press, Boca Raton FL et al. 2003, ISBN 0-8247-0895-4 , p. 323. ( Restricted preview in Google book search).