July 18, 2025

Thin layer chromatography - Huaqiang Electronic Network

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Principle

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Fundamental

The basic principle of chromatography involves using the differences in adsorption or solubility properties of components within a mixture when interacting with a specific substance, or through other affinity differences. This allows the mixture to flow through the medium, undergoing repeated adsorption, distribution, and other processes to separate individual components. Thin-layer chromatography (TLC) is a simple, fast, and efficient method that leverages the varying polarities of compounds. These differences in polarity lead to different levels of adsorption on the stationary phase and movement with the mobile phase. By calculating the retention factor (Rf), which is determined by measuring the distance from the origin to the center of the main spot and comparing it to the distance traveled by the solvent front, we can assess the adsorption capacity of each compound. The Rf value is directly related to the polarity of the compound—compounds with higher polarity tend to be more strongly adsorbed, resulting in lower Rf values. Under consistent experimental conditions, such as the same adsorbent, developing agent, and layer thickness, the Rf value remains constant for a given compound, making it a physical constant used for identification. TLC is particularly effective for small sample quantities, ranging from several micrograms up to 500 mg. It plays a crucial role in both qualitative and quantitative analysis in modern organic chemistry, especially for substances that are not suitable for gas chromatography due to low volatility or thermal instability.

Experiment Process

To begin the experiment, prepare five 7.5 x 2.5 cm glass slides. Wash and dry them thoroughly. In a 50 mL beaker, mix 3 grams of silica gel G with 8 mL of 0.5% sodium carboxymethylcellulose (CMC) aqueous solution, stirring until a uniform paste forms. Apply this paste evenly onto the clean slides, using gentle up-and-down movements on a flat surface to ensure an even and smooth layer. After drying at room temperature for 30 minutes, place the plates in an oven and gradually heat to 110°C for 30 minutes. Cool slightly and store in a desiccator for future use. For spotting, use a capillary tube with an inner diameter less than 1 mm and a smooth tip. Dissolve the sample in a low-boiling-point solvent such as acetone, ethanol, or tetrahydrofuran. Before applying the sample, draw a light horizontal line 5 mm from the bottom of the plate using a pencil. Carefully spot the sample on the starting line, ensuring that the spot size is no larger than 2 mm in diameter and that spots are spaced at least 5–6 mm apart. If multiple samples are applied on the same plate, repeat the process only after the previous spot has dried completely. Choose an appropriate developing solvent based on the polarity, solubility, and adsorbent activity of the sample. The TLC plate should be placed in a sealed chamber containing the developing agent. Allow the chamber to saturate with vapor for 5–10 minutes before inserting the plate. Ensure the spotting line remains above the solvent level. Once the solvent front reaches 5–10 mm from the top of the plate or when all components are clearly separated, remove the plate and allow it to dry. Visual inspection can be done under UV light if the compounds are colorless. Some substances may fluoresce, while others may show yellowish-brown spots when exposed to iodine vapor. 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. Handle the plate carefully during preparation to avoid uneven layers. 2. Ensure that spots are aligned in a straight line and not too large (less than 2 mm in diameter). 3. Avoid using the same capillary for different samples to prevent cross-contamination. 4. If the sample is too dilute, re-spot after the previous spot has dried to avoid spreading or tailing. 5. Dry the plate fully before development to prevent smearing. 6. Keep your hands steady when placing the plate into the developing chamber to avoid diagonal lines.

Equipment and Reagents

- 50 mL beakers - Glass slides - Glass rods - 0.5% CMC solution - Silica gel G - Deionized water **Notes:** - Maintain a stable indoor temperature, avoiding high humidity. - Adjust the ratio of silica gel to water (ideally 2.5–4.5) depending on sample viscosity. - Mix thoroughly and patiently to avoid air bubbles. - Ensure even layering and proper activation during heating. - Always handle the thin layer with care to maintain uniformity.

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