KROMATOGRAFI LAPIS TIPIS

Summary

This practical session demonstrates the process of Thin Layer Chromatography (TLC) for identifying compounds in a sample. The video guides viewers through the preparation of solvents, TLC plates, and the application of standards and samples. The chromatography process, UV visualization, and the use of aluminum chloride spray to enhance spot visibility are explained. Finally, the Rf values of standards and samples are calculated to confirm the presence of flavonoids in the sample, based on their similar behavior on the TLC plate.

Takeaways

• 😀 The experiment involves thin-layer chromatography (TLC) for the identification of flavonoids in a sample.
• 😀 The required equipment includes beaker glass, capillary tubes, TLC plates, and glass stir rods.
• 😀 The chemicals needed for the mobile phase are toluene, acetone, and formic acid, which are mixed in specific ratios.
• 😀 The mobile phase is prepared by combining toluene (10 mL), acetone (8 mL), and formic acid (2 mL) in a beaker.
• 😀 The TLC plate is prepared by marking a 1 cm baseline and applying standards and samples using capillary tubes.
• 😀 The plate is placed in a chamber saturated with the mobile phase, allowing the solvent to move up the plate.
• 😀 After solvent migration, UV light (254 nm) is used to visualize the spots on the TLC plate.
• 😀 The presence of flavonoids is confirmed by the appearance of yellow spots upon spraying with aluminum chloride (AlCl3).
• 😀 Rf values (retention factors) are calculated by measuring the distance traveled by the compound and the solvent front.
• 😀 The Rf values are compared to standard samples to confirm the identity of compounds in the sample.
• 😀 The TLC method is effective for identifying flavonoids based on their migration in the mobile phase and Rf comparison.

Q & A

What is the primary objective of this practical session?

The main objective of this practical session is to perform thin-layer chromatography (TLC) to identify a sample.

What are the key materials and tools required for this TLC experiment?

The key materials and tools include a beaker, glass chambers, tweezers, stirring rods, watch glasses, capillary tubes, solvents (formic acid, acetone, toluene), TLC plates, and filter paper.

How is the mobile phase prepared for the TLC experiment?

The mobile phase is prepared by mixing toluene, acetone, and formic acid in a 4:1 ratio. The specific amounts are 10 mL of toluene, 8 mL of acetone, and 2 mL of formic acid.

What is the purpose of saturating the chamber before performing the TLC?

Saturating the chamber ensures that the atmosphere inside is stable and conducive to proper development of the TLC plate, preventing evaporation of the solvents during the experiment.

How are the samples and standards applied to the TLC plate?

The standards and samples are applied to the TLC plate using a capillary tube. The application points are marked, and the samples are dried before additional applications are made.

Why is the TLC plate placed under UV light during the analysis?

The TLC plate is placed under UV light to visualize the fluorescence of the sample and standard, helping to identify the presence of specific compounds, such as flavonoids.

How is the Rf value calculated in this TLC experiment?

The Rf value is calculated by measuring the distance the compound traveled from the starting point to the final position and dividing it by the total distance the mobile phase traveled on the TLC plate.

What does a yellow stain on the TLC plate indicate?

A yellow stain on the TLC plate indicates the presence of flavonoid compounds in both the sample and the standard, as they fluoresce under UV light when reacted with AlCl3.

What solvent is used to spray the TLC plate after development?

After developing the TLC plate, AlCl3 (aluminum chloride) is sprayed to reveal the presence of specific compounds, such as flavonoids, which result in a yellow coloration.

What is the significance of calculating the Rf values for both the standard and the sample?

The Rf values for the standard and the sample help to confirm the identity of the compound in the sample by comparing it to known standards, allowing for identification based on similar Rf values.