July 18, 2025

Thin layer chromatography - Huaqiang Electronic Network

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Principle

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Fundamental

Chromatography is a technique that separates mixtures based on the differences in their adsorption, solubility, or affinity properties within a given medium. As the mixture flows through this medium, repeated interactions such as adsorption and partitioning occur, leading to the separation of individual components. Thin-layer chromatography (TLC) is a simple, fast, and efficient method used for analyzing small quantities of substances. Due to varying polarities among compounds, they exhibit different affinities for the stationary phase (such as silica gel), causing them to move at different rates when exposed to a mobile phase (developing agent). The Rf value, calculated by dividing the distance traveled by the compound from the origin by the distance traveled by the solvent front, serves as a unique identifier for each compound under specific conditions. Higher polarity compounds tend to be more strongly adsorbed, resulting in lower Rf values. This makes TLC particularly useful for qualitative and quantitative analysis in organic chemistry, especially for non-volatile or thermally sensitive compounds that cannot be analyzed via gas chromatography.

Experimental Procedure

To begin, prepare the TLC plate by using clean glass slides measuring 7.5 cm x 2.5 cm. Wash and dry them thoroughly. In a 50 mL beaker, mix 3 g of silica gel G with 8 mL of 0.5% sodium carboxymethylcellulose (CMC) solution to create a smooth paste. Spread the paste evenly over the slides, ensuring uniform thickness. Let the plates air dry for 30 minutes, then heat them in an oven at 110°C for 30 minutes before storing them in a desiccator for future use. For sample application, use a capillary tube with a diameter less than 1 mm. Dissolve the sample in a low-boiling solvent such as acetone, ethanol, or tetrahydrofuran. Before spotting, draw a straight line 5 mm from the bottom of the plate using a pencil. Apply the sample carefully onto the line, ensuring spots are no larger than 2 mm in diameter and spaced at least 5 mm apart. If multiple samples are to be tested on the same plate, maintain even spacing between them. Choose a suitable developing solvent based on the polarity of the sample and the activity of the adsorbent. Place the solvent in a sealed chamber to allow vapor saturation for 5–10 minutes. Then, carefully insert the TLC plate into the chamber, making sure the sample spot remains above the solvent level. Allow the solvent to rise until it reaches 5–10 mm below the top edge of the plate or until all components are clearly separated. Remove the plate and let it dry. Use a pencil to mark the solvent front. If the compound is colorless, observe under UV light to detect fluorescent spots. For non-fluorescent compounds, expose the plate to iodine vapor, which often causes visible yellowish-brown spots. Once developed, mark the positions of the spots with a pencil. Measure the distances from the origin to the center of each spot and from the origin to the solvent front to calculate the Rf value.

Precautions

1. Ensure the plate is laid smoothly and evenly; avoid shaking or moving the board during preparation. 2. When applying samples, keep them aligned in a straight line, with appropriate size and spacing. 3. Do not reuse the same capillary for different samples. 4. If the sample concentration is too low, reapply after the previous spot has dried to avoid spreading or tailing. 5. Dry the sample before development to prevent damage to the thin layer. 6. Handle the plate carefully to avoid punctures or uneven spots. **Equipment and Reagents:** - 50 mL beakers - Glass slides - Glass rods - 0.5% CMC aqueous solution - Silica gel G - Deionized water **Notes:** - Avoid high humidity and ensure proper temperature control. - Adjust the ratio of silica gel to water (typically 2.5–4.5) based on sample viscosity. - Mix the silica gel thoroughly and patiently. - During activation, avoid disturbing the plate. - Ensure the thin layer is free of bubbles and maintains even thickness.

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