Thin layer chromatography - Huaqiang Electronic Network

principle

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Fundamental

The basic principle of chromatography is to use the difference in the adsorption or dissolution properties of the components in the mixture in a certain substance, or the difference in the performance of other affinity, so that the solution of the mixture flows through the substance for repeated adsorption or Assignment and other functions to separate the components. Thin layer chromatography is a small, fast and simple chromatographic method. Due to the different polarities of the various compounds, the adsorption capacity is different, moving on the developing agent, and performing different degrees of analysis. Calculating the specific shift value (Rf) based on the distance from the origin to the center of the main spot and the front of the developing agent: adsorption of the compound The ability is proportional to their polarity, and compounds with larger polarities are more strongly adsorbed, so the Rf value is smaller. Under the given conditions (adsorbent, developer, thickness of the layer, etc.), the ratio of the distance the compound moves and the distance the developer moves is constant, ie the Rf value is the physical constant of the compound, which is only the size of the compound itself. The structure is related so that the compound can be identified based on the Rf value. Thin layer chromatography is suitable for the separation of small samples (several to tens of micrograms or even 0.01 μg): it can also be used for the separation of up to 500 mg of sample, which is an important means for qualitative and quantitative determination in modern organic chemistry. It is especially suitable for compounds with low volatility and substances that are susceptible to chemical changes at high temperatures and cannot be analyzed by gas chromatography.

experiment process

Plan the plate to develop the color and calculate the Rf value. Take 5 pieces of 7.5x2.5cm slides, wash and dry. In a 50 mL beaker, place 3 g of silica gel G, gradually add 8 mL of 0.5% sodium carboxymethylcellulose aqueous solution (CMC), make a uniform paste, apply to the above clean glass slides, and slide the slide with the hand by hand. Make a slight up and down movement on the horizontal tabletop to make a thin layer with a uniform thickness and a smooth surface. The thin layer of the coated silica gel G is placed on a horizontal glass plate and placed at room temperature for 0.5 h. Into the oven, slowly warmed to 110 ° C, heated at 0.5 h, and then taken out, slightly cooled and placed in a desiccator for use. The spotted capillary is the inner diameter Factors to consider. The unwinding of the thin layer takes place in a closed container. First, put the selected developing agent into the jar, so that the air in the jar is saturated for 5-10 minutes, and then put the thin layer of the sample into the jar for expansion. The spotting position must be expanded. Above the liquid level, when the developing agent rises to the front edge of the thin layer (5~10mm from the front end) or the multi-component has been clearly separated, take out the thin layer plate and let it dry, and draw the solvent front edge with a pencil. Can be colored. Color development If the compound itself has a color, it can be observed directly. If it is colorless, you can first observe the presence or absence of fluorescent spots (all substances with benzene ring) under ultraviolet light. Use a pencil to mark the spot on the thin layer; for non-coloring under UV light, check the color point in a container containing a small amount of iodine vapor (because many compounds can be yellowish brown with iodine) Spot), immediately after color development, mark the position of the spot with a pencil. Calculate the Rf value to accurately find the origin, the solvent front and the center of the spot after the sample is unfolded, and measure the distance between the solvent front and the sample on the thin plate to determine the Rf value.

Precautions

1. Because it is a dry board, the hand should be stable when laying the board, otherwise it will be uneven. 2. When sampling, several samples should be on a straight line, the size should be suitable (the spot diameter is generally less than 2mm), and the spacing is 5~6mm. 3. The capillary used for spotting cannot be used interchangeably. 4. Because the solution is too thin, if it is not enough to repeat the sample, it should be focused on the solvent before the previous spotting, so as to prevent the sample from being too large, causing tailing, diffusion and other phenomena, affecting the separation effect. 5. After the sample is finished, the sample is dried before it can be expanded. The spot should be light, and the thin layer should not be pierced. 6. Keep your hands steady when you put the board, otherwise walk out the diagonal line. Equipment and reagent beakers (50mL), glass slides, glass rods, 1% aqueous sodium carboxymethyl cellulose (CMC) solution, silica gel G, deionized water required for the preparation of thin-layer plates Note: 1) Indoor temperature, especially Can not be too moist 2) The ratio of silica gel to water should be suitable: 2.5-4.5 best. Adjust according to the viscosity of your own sample 3) Grinding, uniform, patience, don't be afraid to spend time 4) Activation, pay attention not during the activation process Disturbed 5) Grinding should be fine and uniform, and the board should be careful to avoid air bubbles and uniform thickness.