The basic principles of thin-layer chromatography and the choice of flow/fixed phase.

Related TopicsThin Layer Analysis (TLC) TechnologyThin Layer
Chromatography
, like other chromatography techniques, is a technique that separates samples using the different physical properties of their components. These physical properties include the size, shape, charge, volatility, solubility, and adsorption of molecules. Thin-layer chromatography separation is generally the result of several separation factors, most of which are adsorption and distribution, as well as ion exchange or gel penetration. In view of the fact that in the process of thin-layer chromatography, the characteristics of fixed and flow phases vary greatly from one to the other, in order to facilitate the narrative, the basic principles of each type of chromatography and the technical requirements of the relevant fixed and flow phases are summarized below.A first section adsorption chromatography 1, the principle of adsorption chromatography adsorption chromatography dissolves a single component of a mixture in one phase will show a change in concentration on the other phase interface, the other phase surface often appears component concentration, this phenomenon is called adsorption. Adsorption thin layer chromatography is the adsorbent on the surface of the smooth, such as glass, metal or plastic and other surfaces evenly spread into a thin layer, and then on the top point of the sample, to expand the flow phase, so that the parts are constantly absorbed by the adsorbent, and then dissolved by the flow phase, desorption and forward movement.
Because the adsorbent has different adsorption capacity for different parts, the flow phase also has different desorption capacity, and the parts that bind tightly with the adsorbent are more difficult to be de-absorbed by the expander, while the looser parts combined with the adsorbent are easier to be de-absorbed by the expander. Therefore, in the process of flow phase forward, different components move at different distances, the original mixture can be separated. The degree of separation is generally expressed by the ratio shift value RF value, whose value can be calculated by the distance from the center of the speck of the separated group from the origin to the distance from the front of the expander., the requirements of the fixed phase in the adsorption chromatography the fixed phase of the adsorption chromatography is often referred to as the adsorbent. At present, the most commonly used adsorbents are silicone and aluminum oxide, followed by polyamide, magnesium silicate, and other substances such as calcium oxide (magnesium), calcium hydroxide (magnesium), calcium sulfate (magnesium), calcium phosphate (magnesium), starch, sucrose, etc., but because the alkalinity is too large or adsorption is too weak, resulting in limited use; The specific requirements are as follows:(1) has a large surface area and sufficient adsorption capacity;(2) have different adsorption properties to different components, so that different chemical components can be better separated;(3) insoluble in the solvents and expanders used;(4) does not destroy or decompose the components in the test, does not react with solvents and expanders in the test;(5) particle size is uniform, generally requires a diameter of less than 70u.m (less than 250 eyes) and will not break during use;(6) has reversible adsorption, which can absorb sample parts and is easy to desorption;(7) for easy observation of separation results, preferably white solids., the requirements of the flow phase in the adsorption chromatography the choice of the flow phase must be considered in the light of the combination of the separated substance and the nature of the adsorbent selected. When chromatography separation is carried out with polar adsorbent, when the separated substance is a weak polar substance, the weak polar solvent is generally chosen as the expander, and when the separated substance is a strong polar component, the polar solvent is selected as the flow phase. If a less adsorption agent (e.g. silicone is replaced by diatomite or talcum powder) for a polar substance, the polarity of the flow phase must also be reduced accordingly.In a thin layer chromatography, when the adsorbent activity is at a certain value (e.g. GRADE II.grade or III.grade), the ability to obtain satisfactory separation of the multi-part samples depends on the selection of the expander. The principle of the selection of the expander is mainly to choose the corresponding polarity of the expander according to the polarity of the separated substance.A second section, distribution chromatography 1, the basic principle of the distribution chromatography distribution chromatography is a chromatography method that uses the different distribution coefficients of the components of the mixture in two-phase solvents to achieve separation. However, one phase of the two-phase solvent needs to be fixed to a solid substance. Such solid substances, such as diatom soil, cellulose powder, etc., are often referred to as carriers. A solvent that is absorbed by a carrier, called a fixed phase.
phase flows slowly on the fixed phase surface, so it is called the flow phase. However, the two phases must be balanced and saturated with each other, otherwise the concentration of the solvent system used will change during chromatography separation. In the chromatography process, when the expander flows through the origin, the different substances in the mixture under test are distributed between the two phases, and each time a little distance is expanded, the separated substances are distributed repeatedly and repeatedly.
The ratio of the concentration of substances in a fixed phase to the concentration of substances in a fixed phase is often referred to as the distribution coefficient, and substances with a small distribution coefficient, i.e. substances that dissolve much more in the flow phase, move at a larger distance with the flow phase, with a large Rf value; Therefore, after a certain distance of expansion, the distribution coefficient of different substances gradually pull apart the distance, and then achieve the purpose of separation. Generally used in polar components, such as sugar,
amino acids
,
acids
phenols and so on. two-phase system in the distribution chromatography, such as one phase contains more machine solvents, while the other phase contains more water. The water phase is usually fixed to solid hydro-hydrocarbial carriers, such as silicone, diatom soil, starch, hydro-hydrogel, powder fiber rope, filter paper, etc. Organic phases are usually mobile phases, and this method is called positive chromatography. If the carrier of hydrophobic material is saturated with organic phase impregnation, and the water phase is the flow phase, then this method is called reverse chromatography. In some special cases, a better separation effect can be obtained by this method. , the requirements for fixed phase in the distribution chromatography the fixed phase of the distribution chromatography is often referred to as the carrier, the most commonly used carriers are cellulose and diatom soil. As a carrier for the distribution of chromatography, the following conditions are required: (1) should be a neutral porous powder, no adsorption to the sample parts or very weak adsorption. Insoluble in the expander system during the chromatography process, the chemical reaction with the expander and sample components is not suitable. (2) can absorb a certain amount of fixed phase, the fixed liquid is inert, does not change its composition, and the flow phase can pass freely. (3) surface area is large, suction of the fixed corresponding as much as possible, it is best to reach more than 50% of the carrier weight. Such as diatom soil as a carrier of the distribution of chromatography is very good, because diatom soil can absorb 100% of its weight of water, and almost no adsorption properties, they are mainly used to separate
protein
, nucleic acids, enzymes, sugar and other substances, in
bio-chemical
applications more. 3. The requirements for the flow phase in the distribution chromatography there are a wide variety of solvent systems that make up the distribution chromatography, and are generally selected for the properties of the separated compounds, most of which are composite solvent systems, which can be adapted to the properties of the separated compounds by regulating the properties of the solvent system. For example, a strong polar solvent A is mixed with a weak polar solvent B, and by changing its composition ratio, a series of medium polar strength flow phase systems can be obtained. Table 2-1 is the relationship between fixed and flow phases based on the properties of the separated compounds. . Section III Ion exchange chromatography 1, the basic principle of ion exchange chromatography ion exchange chromatography is suitable for amino acids, proteins, protein hydrolytes and other ion compounds. The ion exchange resin is used as a fixed phase, and the liquid phase is used with water or a solvent mixed with water. The ionized substance present in the flow phase is adsorbed in ion exchange reaction with the resin, replacing the adsorption effect produced by the suction surface activity. The separation principle of ion exchange chromatography is mainly manifested in the following three aspects: (1) is separated by different selectivity coefficients of sample members. (2) using the difference between the groups of decomposition degrees to separate; ion exchange separation after the formation of a network ion at the same time (3). When the type, properties and experimental conditions of the resin are maintained to a certain level and the ion strength of the flow phase is maintained to a certain amount, the ratio of concentration of the separated group separators between the fixed phase (resin) and the flow phase (solution) is a constant, which is called the distribution coefficient. The size of the distribution coefficient value determines the retention time of the group separators on the resin, and if the distribution coefficient value is large, the retention time of the solute is long. , the requirements of fixed phase in the ion exchange chromatography ion exchange resins are divided into two categories, namely, cation exchange resin and anion exchange resin. The resin nuclei of the ion exchange resin are made of styrene polymerized by a dvinyl styrene chain. The percentage of weight of ethylene benzene in ion exchange resins is called "crosslinking". The cross-linking degree reflects the mesh size of the resin. The greater the cross-linking, the smaller the holes in the resin, and the more difficult the large molecular material is to enter the resin particles.
e.g. separation of amino acids or small molecule peptides (ddipeptides or tripeptides) is appropriate for resins with 8% cross-linking, while for peptides with a larger molecular weight, it is appropriate to select resins with a small crosslinking degree of 2% to 4%. Generally as long as it does not affect the completion of separation, to use a higher cross-linking resin is more suitable. Because of the high degree of cross-linking, small mesh, tight composition, more stable nature, not easy to destroy, long life. However, because the mesh is small, the molecule small ions can go in, the molecular large ions can not enter, that is, the exchange can not, so the performance of the exchange capacity is small, and the selectivity is strong. , the ion exchange resin is divided into three levels according to the size of crosslinking, according to different needs to choose the application. If the manufacture of ion exchange water to select high cross-link resin is better, the separation of large molecular substances need to apply low cross-link resin, see Table 2-2. . Third, the requirements of the flow phase in the ion exchange chromatography ion exchange chromatography expander selection should be based on the acidity of the separated compound, can be used various types of buffer or salt solution as an expander. The premise of the expander is to choose whether the isolated compound is acid or alkali, anion or cation. In order for these ionized compounds to be better separated, the thin sheet must be expanded once with water or 0.1mol/L sodium chloride solution before the sample can be used to regenerate or regenerate the thin layer of ion exchange. because water is an excellent solvent and ionized, most chromatography separation with ion exchange resins is carried out in aqueous solutions. Sometimes water/methanol mixture solvents are also used, as the addition of a small amount of organic solvents that can be mixed with water in the water phase expander can increase the solubility of the sample parts and change the separation selectivity of the solvent, but also reduce the drag of certain parts.
buffer is used to control the pH of the expander, for example, in cation exchange resin, acetic acid, radonic acid, phosphoric acid buffer is often used, and in anion exchange resin, ammonia, radon and other buffers are used. The ion concentration of the expander can also be adjusted by adding certain salt solutions. commonly used ion exchange chromatography expanders are available in four types: (1) pH, a buffer solution of various ion strengths from 3.0 to 8.0, such as pH8.0's 0.005mol/L phosphate buffer or pH3.4's 2mol/L potassium chloride or sodium chloride solution; (2) salt solution, such as 0.1 to 2.0mol/L potassium chloride or sodium chloride solution; (3) distilled water; (4) to add a small amount of organic solvents such as methanol, tethydrofuran, acetylene and so on to the above-mentioned various expanders. fourth section of the gel chromatography 1, the basic principle of the gel chromatography the gel as a fixed phase of the chromatography, called the gel chromatography. The separation principle of glucosaccharose gel is that glucosamine gel forms gel particles after absorbing water, and there are many meshes of a certain size in the skeleton of its crosslinking key. The mesh is large, the large molecular material can enter the mesh, the mesh is small, only small molecular material can enter the mesh, and the large molecular material beyond a certain limit, is blocked outside the gel particles and can not enter the mesh.
makes it possible to enter the inside of the gel and not into the inside of the gel, as if the size of the molecule has been screened, so called a molecular sieve. When operating, the gel is first soaked in a suitable solvent, so that it fully expands, and then spread into a thin layer, add the sample, and then wash away with the same solvent, then the sample of large molecular compounds can not enter the gel particles, only between the gel particles, so the resistance is small, fast, walking in front; After a period of time of escape, the components in the mixture are separated according to the size of the molecule. requirements for fixed phases in the gel chromatography the gel commonly used in the gel chromatography is a glucosaccharin gel. Glucosamine gels are often divided into three categories: hydrophobic glucosamine gels, pro-lipid glucosamine gels and glucosaccharide gel ion exchange agents. hydrophobic glucosaccharide gel is composed of a certain molecular weight of glucosaccharides (right-handed glucosin, dextran) suspended in the organic phase, the addition of cross-linked chlorool to make glucosaccharides cross-linked polymerization, its commodity named Sepha-dex. Different cross-linking doses can be added to make gels with different cross-linking degrees, so their application range is also different. The degree of water absorption and application of different cross-linking commodity gels.