What are the disadvantages of chromatography

Thin layer chromatography



The Thin layer chromatography, often simple DC abbreviated, is a physico-chemical separation process that is used to investigate the composition of samples, for example to quickly prove their purity and identity. Solutions of known comparison components are often examined at the same time as the actual sample and are used to reliably identify the chemical compounds in the sample. The method is based on migration processes in a liquid that flows through a layer of powder. Different molecules mostly show different migration behavior (e.g. amino acids). The method can be used in many ways and is widespread. All samples that are sufficiently stable and can be brought into solution can be examined. The success depends on how great the differences in the migration behavior of the molecules of interest are. It is also necessary that methods are available that make the molecules, which are usually colorless, visible. For example, sample solutions of natural substances, drugs, reaction mixtures and reaction products are examined in the chemical laboratory. On a larger scale, the method is also used to obtain and process sample components: "preparative thin-layer chromatography" (or thick-layer chromatography).

General terms are liquid chromatography and chromatography.

history [1]

N. A. Izmailov and M. S. 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 J. G. Kirchner and his colleagues (including B. Harnischmacher) started working with it in 1951 that others became interested in the method. E. Stahl helped her breakthrough when he described the manufacture of high-performance panels. The name thin-layer chromatography also comes from him.

Cutting process

Instruct

A small amount of the diluted sample solution to be examined is given as punctiform drops or a short line from a capillary onto a thin layer of very fine-grained material (e.g. silica gel, kieselguhr, aluminum oxide, cellulose) Separating layer or stationary phase is called. This separation 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.

The mixture to be examined is applied to the starting line of the TLC slide (usually once or twice at the lower edge of the slide; in the case of 2-dimensional TLC once in the lower left corner) and placed in the TLC chamber with the mobile phase. (After an initial chromatography process, after evaporation of the solvent, turning the film 90 degrees, changing the solvent and renewed chromatography, a so-called 2-dimensional separation can be achieved. Advantage: better separation of multi-component mixtures, disadvantage: identification more difficult.) In many cases, solutions of pure reference substances or reference mixtures are also applied to the starting line (solutions diluted, about one percent). It is crucial to keep the application zones as narrow as possible (one to three millimeters maximum). TLC foils with so-called concentration zones are also commercially available. This means that particularly narrow application zones can be achieved. As a result, the sample spots are compressed in the direction of the path after the start of the chromatography (zone with low adsorption is connected upstream).

Separation

Now you place this separation system vertically in a vessel with a suitable solvent, the mobile phase, in which the TLC foil is immersed to a small extent, and seals it. A mixture of a non-polar and a moderately polar organic solvent is usually used as the mobile phase (e.g. petroleum ether and ethyl acetate). The polarity of the superplasticizer can thus be easily controlled via the mixing ratio. The superplasticizer is now sucked up into the stationary phase via capillary forces. As soon as the liquid has reached the mixture spot, it is dissolved; its 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 tends to move upwards with the mobile phase. In general, the following applies: the more polar the flow agent, the further polar substances migrate, and vice versa.

The mixture separates due to different interactions of the components of the applied mixture with the stationary and mobile phases. In order to prevent the results from being influenced by the evaporation of the solvent, the separation is carried out in an atmosphere saturated with the solvent in a closed vessel.

The forces and thus the migration behavior of a particle depend both on the type of layer material and the flow agent, as well as on the type of particle. In most cases, layer materials and superplasticizer mixtures can be combined in such a way that the different types of particles in a mixture migrate to different distances so that they can be separated from one another.

Phase relationships

When analyzing this term more precisely, one should speak of a competition between the various sample molecules and the molecules of the solvent (solvent) for the points of adhesion of the adsorbent. As a rule, these are polar points of adhesion (e.g. in the case of silica gel). The use of TLC material is also very interesting, in which adsorbents are used that have non-polar points of adhesion (“reversed phase” as opposed to “normal phase”). The order in which the different sample molecules are separated is usually reversed (the polar molecules come faster). Among other things, it is advantageous that very polar samples can also be examined or samples taken directly from aqueous solution (the polarity of the flow agent is generally also higher). There is also DC material with special chemical groups in the adsorbent (so-called chemically bound phases) for special applications.

Basic principle of separation

The Basic principle of chromatography can be understood and summarized as follows: Molecules are distributed over two phases in a certain ratio (state of equilibrium). It is crucial that they individually migrate very quickly from one phase to the other (due to the movement of heat, diffusion and the rapid exchange processes) and back again (more dynamic Equilibrium). The proportion of time that the individual molecule spends in the mobile (or stationary) phase also corresponds exactly to the proportion of molecules of this type of molecule in the two phases. These relationships also apply if the "mobile" phase is not moved. The "trick" of chromatography is simply to convert the differences that exist in these exchanges between the different types of molecules (compounds, including ions) into speed differences. The speed is simply the product of the speed of the mobile phase and the proportion of time that the sample molecules spend in the mobile phase. It is assumed that the molecules 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). So distribution differences (distribution in the general sense) are transformed into speed differences. Often the differences in distribution are only minor. Separations could hardly be achieved with other methods. At the moment when there are differences in speed, it is only a question of the time available for the experiment or the length of the distance until a sufficient separation has been achieved.

Zone broadening

Certain statistical circumstances counteract a good segregation. The decisive factor is the rapid change of the sample molecules between phase 1 and phase 2. It is therefore also advantageous to have the smallest possible spacing (and fine, uniform grain sizes of the layer material). This is also beneficial in order to keep the differences in the total path that occur for a certain type of molecule to a minimum ("zigzag path"). 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. - One can think, for example, of increasing the volume of an ink solution that is surrounded by pure water. The more time elapses, the greater the expansion (even if there are no vibrations. - If the speed is too high, the sample molecules change less frequently between phases 1 and 2. This leads to a greater statistical spread and is also In all chromatographic methods there is an optimal speed of the mobile phase (see also Van Deemter equation). The finer the grain size (or the dimensions), the larger it can be. This is also an economic advantage.

In TLC, the desired spatial separation between the various sample components is proportional to the entire running distance (distance from the start line to the "solvent front"). 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.

proof

 

The separated substances usually have to be made visible (detected) by spraying them with reagent solutions. Alternatively, immersion in solutions is also possible during the application. Often these are color reactions that are sensitive and specifically suitable for the detection of certain functional groups. The information content of the TLC can be significantly increased by selecting the color reaction. Alternatively, reactions are used that are generally effective (for example, oxidation using nitric acid solutions). In the case of a number of color reactions, it is necessary to heat the film after spraying or immersion.

Many layer materials contain additives that fluoresce in UV light and show dark fluorescence quenching ("fluorescence quenching") at those points where the separated substances are located. These fluorescent dyes must not migrate during the chromatographic separation. Mainly manganese-activated zinc silicate (irradiated with UV light with a wavelength of 254 nm) and calcium tungstate (irradiated with UV light with a wavelength of 366 nm) are common. In fact, the method is not a quenching of fluorescence in the strict sense. Sample molecules become visible when they absorb UV light in the range of 254 nm or 366 nm (depending on the UV lamp used). Less UV light then reaches the fluorescent dye molecules (dark spots can be seen on a green or blue glowing 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 e.g. very easily (at 254 nm).

The self-fluorescence of certain substances or other properties such as radioactivity can also be used for detection (irradiation with UV light at 254 or 366 nm).

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, i.e. 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.

A combination of methods is sensible and common: first examination under the UV lamp, then chemical reaction methods.

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

In addition to this qualitatively fast detection method for amino acids, there are also other chromatographic detection methods for amino acids such as ion exchange chromatography and reversed phase HPLC.

In order to be able to compare different DCs, the so-called R.fValues ​​(retention factor) calculated. This is the ratio of the migration distance of the substance stain (S.) to the migration path of the solvent (L.): . The R.fValues ​​are material constants for the same plate material and the same solvent.

Preparative TLC

The DC (TLC Thin Layer Chromatography, French: CCM) can also be used preparatively. For this purpose, relatively large amounts of the substance mixture to be separated are applied in one line to thicker stationary phases. After the separation run, ideally two or three completely separate lines are formed at different heights, which are then distributed into different vessels, for example by mechanical scraping, and eluted separately.

For certain, particularly sensitive analysis methods (mass spectrometry, infrared spectroscopy), the usual thin (analytical) TLC foils can also be used preparatively.

The so-called is also recommended circular thin layer chromatography. Here, circular panes of glass are coated (a circular zone). The layer material contains a fluorescent dye (which fluoresces in UV light with a wavelength of 254 nm or 366 nm). The sample solution is fed to the inner edge of the layer with the help of a pump, before and after the corresponding solvent.

The disc sits on a rotor and is set in rapid and evenly controlled rotation with the help of an electric motor. The separation process is monitored under irradiation with 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. Over time, the sample breaks down into a series of rings that migrate outward.

When a sample component has reached the outer edge of the layer, it is centrifuged together with the solvent in a plastic tire (due to the rapid rotation) that surrounds the guide of the disk. The axis of the disk is inclined during the chromatography, the "eluate" runs together at the lower end of the plastic tire and through an opening and a hose into the appropriate collecting container.

The method has a number of advantages (compared to normal TLC and compared to the usual preparative column chromatography): 1. Larger amounts can be separated than with normal thick-layer chromatography (in less time). 2. The surface of the layer can be sanded well (uniform layer thickness). 3. The costly layer material is used well for the separation process ("geometry" is ideal). 4. It is easily possible to increase the polarity of the solvent (solvent) during the chromatography. People like to make use of this. This means that mixtures can also be broken down which contain groups of substances that differ greatly from one another in terms of polarity. 5. At the end it is easy to clean the layer again by washing it out for a long time with a polar solvent. The cleaning process can be easily followed (UV lamp). In contrast to the regeneration with column chromatography methods, this "washing liquid" can easily be removed: Let it evaporate, dry and activate in an electric oven (approx. 50 to 60 degrees Celsius). 6. The progress of the chromatography can be followed continuously. This makes it easier to collect the sample components ("cutting" the fractions). The separation process can be adapted according to the ongoing observation. 7. The task of the sample is easier to design than with normal preparative TLC. A particularly narrow ("sharp") start zone can be achieved by using a solvent with a low polarity. Disadvantages: 1. There can be problems with connections that are sensitive to air and UV light. Under certain circumstances it is also possible to work under protective gas. 2. A number of advantages are lost if the compounds cannot be made visible with the fluorescent dye method. 3. The acquisition costs of the device are relatively high (compared to simple DC troughs). The plastic parts are made of Teflon.

Advantages and disadvantages of thin layer chromatography

In contrast to the more powerful chromatography methods such as gas chromatography and HPLC, 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. There are few restrictions on liquid chromatography. A way to dissolve a sample can almost always be found. Compared to column chromatographic methods, there is 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 obtain a quantitative Perform analysis. For certain tasks, however, it is sufficient to estimate the proportions (progress of a chemical reaction, for example).

Example experiment: thin layer chromatography of leaf dye extract

Thin-layer chromatographic analysis of leaf pigment extract shows which color components it consists of: carotenes, chlorophylls and xanthophylls.

Test material

  • Aluminum or plastic film (s) or glass plate (s) coated with an adsorbing powder (e.g. silica gel), leaf dye extract (for preparation see below), superplasticizer (best suited: gasoline-isopropanol mixture, mixing ratio 10: 1) , Capillary tubes (very small, or alternatively: micro-pipette or thin brush), applesauce or other glass
  • for the leaf pigment extract: fresh, green leaves, e.g. from spinach or lettuce (in winter also winter lettuce), acetone (20ml), calcium carbonate (spatula tip), (possibly cleaned) sea or sea sand (spatula tip), mortar, pestle, Erlenmeyer - Flask, funnel, folded filter, aluminum foil (not absolutely necessary: ​​hair dryer, pencil, ruler)

Production of the chlorophyll extract / isolation of the leaf pigments

Approx. 5 grams of leaf material are torn into small pieces while the strong leaf veins and the stems are warped and placed in the mortar. Calcium carbonate is added to the crushed leaf material, this serves to neutralize the acidic cell sap. Then sea sand is put into the mortar to break open the leaf pieces. After adding acetone, the material is ground in a mortar for a few minutes. When a bright dark green solution has formed, it is filtered off into an Erlenmeyer flask.
Danger: The resulting solution is sensitive to oxygen, light and heat, so it must be stored in a container in the refrigerator, wrapped in aluminum foil. Now the extract can be used (do not drink, it contains solvents!).

Carrying out the experiment

The leaf pigment extract obtained is applied sparingly with a capillary tube, a micro-pipette or a very fine brush to a coated (rough) side of the foil (or plate) that is placed on edge. Depending on the method, this can be point-like (distance from below: about 1.5 cm) or linear (about 1.5 cm parallel to the lower edge). The order line can also be marked with a fine pencil. The silica gel layer should be damaged as little as possible. After the extract has dried on the foil (this can be accelerated with a hair dryer - with a cold air stream), this application process can be repeated. This should happen about 10 to 15 times. Once the green dot (or line) on the foil has dried again, the foil is placed in the applesauce jar filled with a little superplasticizer, then it is closed. The glass should no longer be touched, or should only be touched very carefully, as the flow process now begins. As soon as the liquid has risen to the end of the sheet (or earlier, depending on the desired result), it is removed from the glass and dried on a few sheets of paper.
However, the splendor of colors does not last long: after drying, the yellow components have already faded. After a while, only different shades of green remain on the film.
Note: The longer the dye extract stood before the experiment, the greater the gray portion on the coated film. This is due to the fact that the gray part consists of breakdown substances of the chlorophylls, which are only formed after the extract has been produced and with the supply of oxygen, heat or light (pheophytin). It is therefore advisable to store the solution as described above.

Explanation of the flow process

The flow agent is absorbed by the film layer towards the top. It carries the leaf pigment extract with it. The different adsorption capacities of the individual components of the leaf ensure that the mixture is broken down into its components. Thanks to this method, it is possible to determine which components the leaf pigments consist of (see left figure).

Gallery: The attempt in 7 pictures



literature

  1. Joseph C. Touchstone: Practice of Thin Layer Chromatography, WILEY, 3rd edition, 1992, pp. 3-4

Category: Chromatography