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    Home > Biochemistry News > Biotechnology News > Selection and preparation of thin layer chromatography solvent/developing agent/color developer and matters needing attention

    Selection and preparation of thin layer chromatography solvent/developing agent/color developer and matters needing attention

    • Last Update: 2020-10-20
    • Source: Internet
    • Author: User
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    Related topics Thin layer chromatography (TLC) technology Summary of Thin Layer Chromatography Methods1. Method principle (1) The mobile phase uses capillary force to carry the sample through the stationary phase. (2) The interaction between the sample and the stationary phase refers to the dipole- (induced)-dipole interaction of the component during the migration process, The role of hydrogen bonds and van der Waals forces produces different degrees of separation mechanisms such as delay, adsorption, dispersion, ion exchange and complexation.2. Solvent The solvent used must be "analytical pure" or "chromatographic pure", and the solvent composition adopts a volume ratio (such as n-butanol-glacial acetic acid-water = 4:1:1, V/V/V), or absolute amount (such as 18ml toluene + 2 ml methanol). The total amount should be sufficient to make the immersion depth of the TLC/HPTLC board approximately 5mm. The spreading agent must be freshly prepared and should not be used repeatedly. If layering is required, place the layering agent as required and take the required phase (upper or lower layer) for use.1. Solvent selection rules: 1. Consider the polarity, solubility, and adsorption of the separated components. 2. First add a solvent with less polarity, if not, add a small amount of solvent with a large polarity. 3. Generally, according to the principle of similar compatibility, it is necessary to pay attention to the immiscibility with large differences in polarity. 4. Mixed solvents usually use a mixed solvent composed of high polarity and low grade solvents. 5. The ratio of the expander depends on experimentation. Generally, according to what kind of expander is used for this type of compound reported in the literature, first try to use this type of expander, and then keep trying the ratio until a good separating effect is found. 6. Generally, when mixing two solvents, use a high-polarity/low-polarity mixed solvent with a volume ratio of 1/3. If there are signs of separation, adjust the ratio (or add a third solvent) to achieve the best Effect; if there is no sign of separation (the spots are more "drag"), it is best to change the solvent.2. Conditions for selecting the developing agent: ① Good solubility for the required components; ② Separate the components;② The Rf of the component to be tested is between 0.2 and 0.8, and the quantitative determination is between 0.3 and 0.5; ③ not send the adsorbent component to be measured or biochemical chemical reaction; ⑤ moderate boiling point, a small viscosity; ⑥After unfolding, the spots of the components are round and concentrated; ⑦The mixed solvent should be prepared fresh.3. Solvent polarity parameter table Cyclohexane: -0.2, petroleum ether (class I, 30~60℃), petroleum ether (class II, 60~90℃), n-hexane: 0.0, toluene: 2.4, xylene : 2.5, benzene: 2.7, dichloromethane: 3.1, isopropanol: 3.9, n-butanol: 3.9, tetrahydrofuran: 4.0, chloroform: 4.1, ethanol: 4.3, ethyl acetate: 4.4, methanol: 5.1, acetone: 5.1 , Acetonitrile: 5.8, Acetic acid: 6.0, Water: 10.2 1. Generally speaking, the basic two phases of a weakly polar solvent system are composed of n-hexane and water, and then methanol, ethanol, and ethyl acetate are added as needed to adjust the solvent system. Polarity, in order to achieve a good separation effect, suitable for the separation of alkaloids, flavonoids, terpenes, etc.; 2. The medium-polar solvent system is composed of chloroform and water basically two phases, composed of methanol, ethanol, ethyl acetate, etc. Adjustment, suitable for the separation of anthraquinone, coumarin, and some polar lignans and terpenes; 3. Strong polar solvent, composed of n-butanol and water, and also adjusted by methanol, ethanol, ethyl acetate, etc., suitable for the separation of alkaloids with high polarity.Fourth, the choice of spreading agent The chemical structure of the substance molecule usually consists of two parts, a more polar part and a non-polar part. For example, phenylpropane is used as the small polar part below. As the polar group part increases, the overall polarity increases, and the polarity of the spreading agent also increases. The polarities of different compounds and their corresponding developing agents are discussed separately below.1. Borneol: petroleum ether (30~60℃)—ethyl acetate (17:3), magnolol: benzene-ethyl acetate (9:1.5), α-carbohydrate Ketone: benzene-acetate ethyl ester-glacial acetic acid (92:5:5), paeonol: cyclohexane-ethyl acetate (3:1), Conclusion: Petroleum ether, n-alkane and benzene are used as solvents with relatively large volume percentages, which usually dissolve and separate compounds, while ethyl acetate is used as the solvent to adjust Rf (ratio shift value). To reduce the impact of other than like dissolves like tailing principle, suitable additives, such as organic acids or organic bases.2. β-sitosterol, a non-volatile substance with less polarity : cyclohexane-ethyl acetate-methanol (6:2.5:1) or cyclohexane-acetone (5:2), ursolic acid: toluene -Ethyl acetate-glacial acetic acid (12:4:0.5), oleanolic acid: chloroform-methanol (40:1), hyodeoxycholic acid: chloroform-diethyl ether-glacial acetic acid (2:2:1), emodin :Benzene-ethyl acetate-methanol (15:2:0.2) or benzene-ethanol (8:1), TanshinoneⅡA: benzene-ethyl acetate-formic acid (40:25:4), Andrographolide: chloroform-none Water ethanol (9:1) Indirubin, indigo chloroform-ethanol (9:1) or benzene-chloroform-acetone (5:4:1)Conclusion: The polarity of this kind of substance is stronger than that of the less polar volatile substance, because this kind of substance has a small polarity and a large nucleus, and a group with a large polarity can usually form hydrogen bonds, such as Carboxylic acid, hydroxyl. For the above substances, the molecular weight of the core decreases, the unsaturated bond in the core structure increases (especially the appearance of a benzene ring), and the increase of polar groups increases the polarity, and the polarity of the spreading agent also increases. . There are many substances in this range. Generally, the solvent with a large percentage of the developing agent can be selected from the order of cyclohexane -> toluene -> xylene -> benzene -> chloroform according to the polarity requirements. Note here that the polarity index of isopropanol and n-butanol is also relatively small. Compounds in this range are rarely used because they are highly viscous and slow to spread, causing spots to spread; in addition, the hydrogen bonding force of the hydroxyl group is also disadvantageous. The solvent for adjusting the Rf value is from ethyl acetate -> methanol -> acetone -> ethanol. There are many volatile substances with carbonyl and hydroxyl groups, but from its volatility, it can be understood that the intermolecular force is not strong. In addition, the structure difference between the mother nucleus and petroleum ether, n-alkane and benzene is small, and it is estimated that it is easier to break away. Silica gel adsorbs and enters the solvent faster without increasing the polarity of the developing agent.Due to the polyhydroxy structure of sugar, the structure of aglycon has less influence. A polar organic solvent (chloroform, ethyl acetate, methanol, n-butanol) and water are used in the developing agent. The use of acetic acid and formic acid, on the one hand, increases the polarity of the developing agent, on the other hand, it can also inhibit the effect of the silica gel hydroxyl group and reduce tailing. Due to the limitation of miscibility and acid resistance of silica gel, the use of water and acid is limited.3. Small-molecule organic acids with large polarities: gallic acid: chloroform-ethyl acetate-formic acid (5:4:1), ferulic acid, caffeic acid, cichoric acid, chlorogenic acid, isochlorogenic acid. For example, phenylpropane is used as the small polar part below. As the polar group part increases, the overall polarity increases, and the polarity of the spreading agent also increases. The order is cinnamic acid, ferulic acid, caffeic acid, cichoric acid, and chlorogenic acid. The corresponding developing reagents are: n-hexane-diethyl ether-glacial acetic acid (5:5:0.1), benzene-glacial acetic acid-methanol (30:1:3), chloroform-methanol-formic acid (9:1:0.5), petroleum ether -Ethyl acetate-formic acid (3:6:1), butyl acetate-formic acid-water (7:2.5:2.5). in conclusion: Most of these substances are styrene cores, and the polarity of this structure itself is relatively large. In addition, there are phenolic hydroxyl groups and carboxylic acid groups, and some have polyhydroxyl ligands. The spreading agent of saponins is similar, and the polarity is large. Note that formic acid usually refers to a concentration of about 85%, containing water.4. Nitrogen-containing organic matter: berberine hydrochloride: benzene-ethyl acetate-methanol-isopropanol-concentrated ammonia test solution (12:6:3:3:0.6) (saturated with ammonia vapor) or n-butanol-ice Acetic acid-water (7:1:2), ephedrine: chloroform-methanol-concentrated ammonia test solution (20:5:0.5) or n-butanol-glacial acetic acid-water (8:2:1) ammonium glycyrrhizinate: acetic acid Ethyl acetate-formic acid-glacial acetic acid-water (15:1:1:2). Conclusion analysis: Due to the strong effect of NH2 silanol group, organic acid and organic base are added to the strong polar developing agent to sweep the tail. For polar compounds, the use of n-butanol has less effect on spot spreading because the compound and silica gel have a strong effect. When the separated substance is a weakly polar substance, a weakly polar solvent is generally used as the eluent; when the separated substance is a strong polar component, a polar solvent must be selected as the eluent. If a relatively weak adsorbent is used for a polar substance (such as diatomaceous earth or talc instead of silica gel), the polarity of the eluent must be reduced accordingly.3. TLC general color rendering method is ideal for color rendering, high sensitivity, stable spot color, good contrast between spot and background, spot size and color depth are directly proportional to the amount of substance, and the composition of the sample is not completely known Under the circumstance of circumstance, the general color rendering method is particularly important. The general color rendering methods mainly include: E/m T2K gV"p Fg4244131, ultraviolet irradiation method: convenient and non-destructive of the sample; Pl o;E r"m‑zs? d T $V4244132, iodine vapor method: strong versatility, combined with the ultraviolet method, the sensitivity is higher than the two methods used alone; $b8M"}?(P [+e4244133, fluorescent reagent : create a fluorescent background, so that no fluorescent substance under ultraviolet For identification, it is more obvious if there are fluorescent substances; the small woodworm academic blog JM0D6{Rf;r 4. Sulfuric acid solvent: effective for most organic substances, but destructive.4. Precautions for TLC and Paper Chromatography 1. The spotting device is used to spot the sample on the thin-layer plate, usually as a round spot, the baseline of the spot is 1.0~1.5cm from the bottom edge, and the diameter of the sample spot is generally not more than 2mm , The distance between the dots can be seen as the spread of the spots so as not to affect the detection. If the sample solution is too dilute, spotting can be repeated, but the spotting should be repeated after the solvent of the previous spotting has evaporated to prevent the sample spot from being too large, causing tailing, diffusion, etc., and affecting the separation effect. Be careful not to damage the surface of the thin layer when spotting the sample. 2. Expand the spotted thin layer plate into the spreading agent of the spreading cylinder. The depth of the spreading agent should be 5mm from the origin. Seal it and unfold it to the specified distance ( Generally 8-15cm), take out the thin-layer board, dry it, and wait for testing. Note: If the expansion tank needs to be pre-balanced with expansion agent in advance, you can add an appropriate amount of expansion agent to the tank and keep it tightly closed. Generally, keep it for 15-30 minutes. 3. Color development and inspection The test product contains components that are colored under visible light and can be inspected directly in the sun. It can also be developed with a suitable color developer by spraying or dipping, or by heating , and inspected in the sun. Fluorescent substances or substances that can excite fluorescence in the presence of certain reagents can be observed under 356nm ultraviolet light. For components that are colorless under visible light, but absorb under ultraviolet light, use a silica gel plate with fluorescent agent (such as GF254 plate), and observe the chromatogram formed by the fluorescence quenching substance on the fluorescent plate under a 254nm ultraviolet lamp. Tell you my ancestral secret recipePE(60-90)EtAc/PE=1:2 EtAc/PE/AcOH=15:5:1 EtAc/AcOH/n-Butanol/H2O=2:1:1:1 I have used these four systems for 8 years without any problems. I have been using ethyl acetate: cyclohexane and keep adjusting the ratio until a satisfactory Rf value is obtained. If there is tailing, it may be necessary to add acid or alkali. , I generally use acetic acid and triethylamine.I have used it for several years, and it is okay. Give it a try! Plank The homogenate used for paving should not be too thick or too thin: too thick, the plate is prone to stratification caused by drag or pause; too thin, the surface of the plate will be rough after the water evaporates. The homogenate ratio is generally silica gel G: water = 1:2 to 3, silica gel G: sodium carboxymethyl cellulose aqueous solution = 1:2. The time for grinding and homogenization is determined by experience and is related to air humidity. It is generally judged by the dripping of the homogenate when picking up the pestle. The thicker the slurry, the more difficult it is to drip. The thinness of the homogenate not only affects the smoothness of the board, but also affects the thickness of the board coating, which further affects the sample amount. The coating is thin and spotting is easy to overload; the coating is thick, the color development is not so obvious. Generally, the quality of the plate does not have a great influence on the identification of the thin layer. The most important influence is the preparation of the spreading agent and the saturation of the spreading system. Spotting Try to use a small sample tube. If you have enough patience, it is best to use only 1 microliter spotting tube. In this way, the spots of the dots are smaller, and the unfolded chromatogram has good resolution and clear colors. The smaller the water content of the sample solution, the better, the larger the water content of the sample solution, the greater the spread of spotting spots. The solvent of the sample solution is generally absolute ethanol, methanol, chloroform, and ethyl acetate. Good lamina point drying with hot air dryer or into the drying Lane dry. Developing agent preparation Select a suitable measuring device to transfer the solvents of each composition into the separatory funnel, shake vigorously to mix the mixture thoroughly, and place it. If stratified, use a larger layer as a developing agent. It is absolutely not necessary to pour each component solution into the unfolding tank and shake the unfolding tank to prepare the developing agent. Inhomogeneous mixing and undispersed developing agent will cause complete failure of chromatography. The ratio accuracy of each component of the solvent has different requirements for different analysis tasks, try to achieve the highest accuracy of laboratory instruments , such as: take 1ml of solvent, should use 1ml single-standard pipette, pipette should meet the measurement Certification requirements, although most of the time this is not required. Saturation of the expansion system generally uses a double tank expansion tank, one tank is used to put the expansion agent, and the other tank can be added with ammonia or sulfuric acid. Put the plate to be unfolded into the platform between the two grooves, stand it diagonally, and cover the unfolding cylinder cover. Let the vapor of the spreading agent fill the spreading cylinder, and make the thin-layer plate adsorb the vapor to reach saturation to prevent edge effect. The saturation time is about half an hour. When unfolding, it is inevitable to open the lid and put the thin-layer board into the spreading agent, but it has little effect on the adsorption balance of the thin-layer board and steam. Of course, the action should be as light and fast as possible. Temperature and humidity control Temperature and humidity have a great influence on the thin layer. Under the premise of not freezing, usually the lower the temperature, the better the separation, and the more difficult separation needs to be separated at low temperature, such as ginsenosides. The influence of humidity is estimated to mainly affect the adsorption capacity of the thin-layer plate, leading to changes in selectivity (capacity factor). The humidity should be determined according to the actual situation. The temperature control uses an air conditioner or a freezer, and the humidity control is by placing a corresponding concentration of sulfuric acid in another expansion tank. Color rendering
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