Thin-layer chromatography

TLC or chromatography (TLC or TLC, thin layer chromatography English ) is a physico- chemical separation method is used to investigate the composition of samples. A particular advantage of this chromatographic method is the low expenditure on equipment, the speed, high separation efficiency and low sample requirements. It is, for example, for the rapid detection of the purity of a substance or of verifying the identity of a reference substance. The persecution of the reaction process of chemical reactions in the laboratory is possible with little effort.

• 3.1 Sample order
• 3.2 separation
• 3.3 Evaluation

• 4.1 Two-dimensional DC
• 4.2 Circular thin-layer chromatography
• Preparative thin layer chromatography 4.3

History

NA Izmailov and MS Shraiber, two Russian researchers, led in 1938 a chromatographic separation with a horizontal thin-layer plate by to which they auftropften the solvent. But their approach was hardly observed. Only when JG Kirchner and his staff ( including B. Harnischmacher ) is from 1951 dealing with her, the interest was aroused in the other method. For Her breakthrough helped Egon Stahl, as he described the production of high-performance disks. He also provided the name of thin layer chromatography.

Theoretical foundations

Basic principle of the chromatographic separation

The basic principle of chromatography is applicable to all chromatographic methods and can be summarized briefly as follows: particles ( molecules, ions ) are divided into two phases in a certain ratio ( equilibrium ). In the DC a solvent migrates by capillary forces in a solid, microporous support material ( eg silica gel) upwards. In which solvents are various organic substances contain various functional groups, there are adsorptive interactions of the various substances with the carrier material. A strong interaction attenuates the migration rate of each component with respect to the solvent front. The higher the proportion of a highly polar solvent ( water is particularly bad) in the plot, the more surface areas of the coating are ineffective for the adsorption, the worse the separation result in TLC ( Rf values ​​changed, spot broadening). It is crucial that the particles rapidly migrate individually from one phase to the other (due to the thermal motion, the diffusion and the rapid exchange processes ) and back again (dynamic equilibrium ). The proportion of time the individual particle spends in the mobile and the stationary phase corresponds exactly to the proportion of particles of this species in the two phases. These conditions also apply if the mobile phase is not moving.

In chromatography, the differences that exist in the exchange operations between the different types of particles, into the speed differences are. The speed results from the product of the velocity of the mobile phase and the percentage of time the particles spend in the mobile phase. It is believed that the particles ( by definition) in the mobile phase velocity are the same as the mobile phase molecules. Are they bound to the stationary phase, the velocity is zero ( "stop and go " model). Thus differences in distribution ( distribution in the general sense ) are transformed into speed differences. Often the distribution differences are small. Separations would be so hard to achieve with other methods. In the moment in which it is but the speed differences, it is only a question of length of the path, until it has come to a sufficient separation.

With liquid chromatographic methods, including thin-layer chromatography counts can be all the samples that are sufficiently stable and can be brought into solution, investigate.

Basis of thin layer chromatography are thus transport processes in a liquid ( " mobile phase "), which flows through a layer of powder ( " stationary phase "). Different molecules usually show different migration behavior. How large these differences are, depends on the chemical and physical properties of the stationary and mobile phase used.

Stationary phase

The stationary phase (interface ) consists of a thin layer of very fine-grained material (eg, silica gel, diatomaceous earth, alumina, cellulose). This release layer is very uniformly applied to a carrier foil or support plate made ​​of plastic, aluminum or glass and is commercially available in different thicknesses. Typically comes as the stationary phase silica is used ( normal phase ) serving as a polar adsorbent for the sample molecules, due to the free terminal hydroxyl groups. The average pore diameter of the silica is usually 4 to 100 nm, wherein the pore diameter of 6 nm (Kieselgel 60, Merck), is used most commonly. Silica gels containing siloxane or silanol groups.

Alternatively, DC - materials come with other functional groups (eg, amino groups ) were used. They differ from standard silica gel not only in their polarity, but also in the basicity and thus lead to completely different separation results. Also, surface-modified silica gels with non-polar traps ( by coupling with organochlorosilanes ) are used ( reversed phase chromatography, reversed phase ). The order in which the various sample molecules are separated, then reverses itself - the polar molecules run faster, the non-polar molecules are strongly held. It is advantageous, inter alia, that even very polar samples can be examined. As a further stationary phases for TLC are also suitable alumina, magnesium silicate, diatomaceous earth, polyamide, cellulose.

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

For special applications, the "washing" of the plate prior to the application of the sample, or drying in a desiccator oven or at elevated temperature may be necessary. The plates are washed by placing them in a chromatography chamber with the appropriate solvent until the solvent front reaches the top edge of the plate.

Mobile phase

The eluent nonpolar organic solvents are usually used as a mixture with moderately polar solvents (eg petroleum ether and ethyl acetate) in the normal phase TLC; in the reverse phase TLC contrast polar eluant (e.g. acetonitrile and water). One can control the polarity of the eluent, the mixing ratio.

The adsorption ability of the functional groups with silica gel takes > -OH > - NH2 > SH > -CHO > CO > CO 2 R > in the sequence -COOH - OCH3 > -HC = CH- off.

Organic carboxylic acids or alcohols therefore have a very high silica to adsorb and, thus, low RF values ​​. In a less polar eluent, these substances may be able to remain at the starting spot.

Isolating distance

Different types of diffusion counteract a good separation. The decisive factor is the rapid change of particles between the two phases. Therefore, it is also convenient to have the lowest possible running routes and fine, uniform grain size of the bed material. Too low and high speeds of the mobile phase to have a negative effect. Too low a speed favors an increase in the zones in which to stay the sample molecules. The more time is available, the greater the role of diffusion processes in the mobile phase. If the speed is too high, it is less common to a change of the particles between the mobile and the stationary phase. This leads to a large statistical variation, and is also undesirable. In all chromatographic methods there is a corresponding optimum velocity of the mobile phase is described by the Van Deemter equation. The finer the grain sizes (or dimensions ), the higher can be the speed. From this follow -economic benefits.

When DC is the desired spatial separation between the different sample components of the total running distance proportional to (distance from starting line - solvent front ). Is the magnification of each zone on the basis of statistical effects low (not the running track but is proportional to the root of the distance). Therefore, it is useful for difficult separations to use larger DC film and running trails.

With the thin layer can be applied to a 15 cm long running track a separation efficiency of about 400-3000 theoretical plates achieved.

Practical implementation

Sample application

The test substance is dissolved in a suitable solvent and applied point-or line-shaped with the aid of a capillary. This happens in the one-dimensional TLC on the start line of the sheet or plate in the two-dimensional TLC ( see below) in a corner. The substance to be separated amount is approximately 5-20 micrograms. It is crucial to keep the order zones as closely as possible (a few millimeters). Are commercially available and DC films with so-called concentration zones. Here a zone upstream with particularly low adsorption. The sample spots are thereby pushed together after the beginning of the chromatography in the direction of running distance and thus very narrow application zones are achieved. For a particularly uniform, quantitatively reproducible application also machines are available that spray the solution by means of compressed air or nitrogen. In addition to the samples, solutions of pure reference substances or mixtures are applied compared to the starting line in many cases.

After application, the plate must be dried, as residual solvent may change the result.

Separation

After coating, the plate is set perpendicular to a chromatography chamber with a suitable solvent ( mobile phase). To avoid influencing the results by the evaporation of the eluent, the separation is carried in a saturated with the developing solvent atmosphere in a closed vessel. For better saturation of the vapor space with eluent a filter paper can be inserted. The saturation preventing evaporation of the eluent from the plate and therefore a change in the composition on the disc.

The flux now attaches itself via capillary forces in the stationary phase to the top. Once the liquid has reached the starting line, the substances dissolve in it. The molecules are then the attractive forces of the stationary phase on the one hand and the forces of attraction of the mobile phase on the other hand exposed. Depending on the balance of forces, a particle remains rather at the starting point or it migrates with the mobile phase upward. In general, the non-polar is the flux, the less polar wander substances and vice versa. But it is also true that polar substances with polar stationary phases interact strongly. The polarity of the solvent is similar as in column chromatography.

Just before the solvent front reaches the top of the plate, the plate is removed from the chromatography chamber and dried as quickly as possible.

Evaluation

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

Many coating materials contain additives that fluoresce under UV light, indicating those places dark fluorescence quenching, where there are the separate substances. These fluorescent dyes shall not wander during the chromatographic separation. In use are mainly of 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 ). It is actually in the method is not a fluorescence quenching in the strict sense. Sample molecules are visible when they absorb in the range of 254 nm or 366 nm UV light. It then passes less UV light by the fluorescent dye molecules ( dark spots on green or blue illuminated background is visible ). For this purpose, a sufficient number of functional groups or sufficiently large systems with conjugated double bonds must be present. Saturated hydrocarbons and many amino acids are therefore not detectable by this method, aromatic compounds, for example, very light at 254 nm

The intrinsic fluorescence of certain substances or other properties such as radioactivity may be used for detection.

With the use of spray or dip reagents (eg NBD -Cl, molybdophosphoric acid or 2,7- dichlorofluorescein ) proceed color reactions which are sensitive and specific enough to be used for the detection of certain functional groups. About the choice of the color reaction can be the information content in the DC increase significantly. Alternatively, reactions can be used, which are generally effective (for example, oxidation using nitric acid solutions or iodine vapor ). In a number of color reactions, it is necessary to heat the film by spraying or dipping.

Another very simple method is the vapor deposition of molecular iodine. For this purpose, it is enough to put a few iodine crystals in a glass jar. Sublimate, meaning they evaporate directly at room temperature, forming a violet vapor of Diiodmolekülen. Inserting a DC film in such a trough loose complex compounds are formed ( purple or brown) on diffusion and reaction with the molecules of the substance spots within a short time. Advantage or disadvantage of the method: the iodine compounds decompose relatively quickly.

In biochemistry, an acidic ninhydrin solution is a common spray reagent to detect amino acids. Here, the Ninhydrin on the Schiff base and by a decarboxylation and hydrolysis to rest Mans violet. By plotting of reference samples, which is equal to wander far under the same conditions as the corresponding sample components, one can detect the occurrence of qualitative materials. For this purpose the position of the various points with the position of the reference samples is compared.

In order to compare different DC, the so-called values ​​( retention factor, retention factor, ratio of fronts ) are calculated. It is the ratio of the migration distance of substance spot ( ) for migration distance of the solvent ( ). The values ​​are in the same plate material and the same eluent composition material constants.

The quantitative evaluation can be performed with a densitometer. These devices offer the possibility of measuring in the visible and ultraviolet spectral range as so-called TLC scanner. They can also be used to measure the fluorescence. And regular flat bed scanner can be used for evaluation in the densiometrische DC.

New devices developments also allow the direct coupling of thin-layer chromatography with mass spectrometry way also a reliable identification of chromatographically separated components are carried out on the mass spectrum.

More complex techniques and methods

In addition to the linear and one-dimensional thin layer chromatography as described previously techniques have been developed to unravel example, complex mixtures of substances can be used for special applications and separation tasks. The Hochleistungsdünnschichtchromatographie represents such a development

Two-dimensional DC

In the two-dimensional DC after the first development, the eluent is evaporated, the plate is rotated 90 ° and - by a second developing out - usually in a different eluent. This allows a better separation can be achieved in multi-component mixtures. The identification, however, is expensive, as can run with no reference substances.

Circular thin-layer chromatography

An alternative technique for linear DC, the so-called circular thin layer chromatography ( CLC abgek. from the English. Centrifugal Layer Chromatography or RPC Rotary Planar Chromatography) dar. Here, round glass panes used annularly coated with the stationary phase. The disk is rotated by means of an electric motor controlled in rapid and uniform rotation. The sample solution is fed by a pump to the inner edge of the layer before and after the corresponding fluid.

Since the stationary phase typically contains a dye which 254 nm or 366 nm UV light fluoresces in the wavelength, the separation process can be controlled by irradiation with a UV lamp. At the beginning of the separation process is the sample in a few millimeters thick circular zone at the inner edge of the disc. With progressive separation of the substance mixture of the sample is split into a series of rings that move driven by the centrifugal force to the outside.

Preparative thin layer chromatography

The DC can also synthetically, that is, be used for cleaning small amounts of substance. Then it is also called PLC (English for Preparative Layer Chromatography). In this case, (up to 2 mm) larger amounts (up to 100 mg) of the applied line-shaped substance to be separated in the mixture usually on glass plates with thicker stationary phases. After the separation run are the separate substances as lines at various heights. The target substance can then be mechanically scraped together with the support material. By simple filtration with an appropriate solvent it is eluted from the stationary phase and obtain pure.

Also from the usual ( analytic ) DC film with thinner stationary phase can be sufficient amounts of pure substance obtained in order to use them for sensitive analytical methods such as mass spectrometry or infrared spectroscopy can.

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

To clean larger amounts of substance at a far lesser apparatus complexity, today uses more column chromatographic techniques such as flash chromatography.

Advantages and disadvantages of thin layer chromatography

In contrast to the more powerful chromatographic methods such as gas chromatography and high performance liquid chromatography, the DC comes out with little equipment and arises dar. as a fast, versatile and inexpensive method of analysis

Gas chromatography can be applied only on samples that are vaporized without decomposition. In the liquid, there are few restrictions. Almost always can find a way to dissolve a sample. In comparison to the column chromatographic methods there is an advantage that samples containing the groups of components, each of which are very different in polarity, are easier to detect. Eluent change is not as easy as possible in the column chromatography. However, it is possible to first develop an eluent and after intermediate drying in another (which in polarity differs greatly ).

A disadvantage of the application of the DC analytical that it is difficult to perform a quantitative analysis. For certain tasks it is already sufficient, however, to estimate the proportions ( progress of a chemical reaction). The earlier problems of reliable quantification could in recent years by developing powerful densitometer - be overcome - as mentioned above. The quality criteria of quantitative evaluation meet the meantime the Guidelines for Good Laboratory Practice.