Performing Thin-Layer Chromatography to analyze organic samples

Summary

In this instructional video, Dr. Frank demonstrates how to perform thin-layer chromatography (TLC) in a lab setting at the University of Ottawa. The video covers the necessary materials, including a developing jar, eluent, TLC plate, and samples. It outlines the steps for preparing the TLC plate, making a baseline, and applying sample spots. Dr. Frank also explains the importance of using a proper capillary for sample application and adjusting spot concentration. The video further details the development process, emphasizing the need to keep the baseline above the eluent level and concludes with analyzing the plate under UV light to determine compound purity and reaction progress.

Takeaways

• 🧪 **TLC Overview**: The video is a tutorial on thin-layer chromatography (TLC), a technique used in laboratories, particularly for undergraduates at the University of Ottawa.
• 📝 **Preparation**: Before starting, ensure familiarity with TLC concepts by watching part one of the series and gather necessary materials like a developing jar, eluent, TLC plate, capillaries, and samples.
• 💧 **Eluent Setup**: Pour a small amount of eluent into the developing jar, seal it, and swirl to allow solvent vapors to fill the jar before proceeding.
• 🔍 **Plate Inspection**: Check the TLC plate for smoothness and minimal chipping, as imperfections can affect solvent migration and spot analysis.
• 📏 **Baseline Marking**: Use a sharp pencil to lightly draw a baseline on the plate, which is where samples will be spotted and start their migration.
• 🧬 **Sample Preparation**: Dissolve the sample or reference compound in a volatile solvent to create a homogeneous solution for spotting.
• 🌡️ **Capillary Selection**: Choose a clean capillary that is neither too thin nor too wide, ensuring a smooth end for precise spotting.
• 💧 **Spotting Technique**: Dip the capillary in the sample solution, wick off excess, and deposit a small spot on the baseline with minimal contact time.
• 🔬 **UV Visualization**: Examine the spotted plate under UV light before development to assess the quality of the spots and avoid wasting time on poor samples.
• 📈 **Spot Adjustment**: Adjust the spotting based on the initial UV visualization, either respotting or diluting/concentrating the solution as needed.
• 🧑‍🔬 **Development Process**: Insert the plate into the developing jar with the eluent level below the baseline, and allow the solvent to migrate the compounds upwards.
• 🌟 **Final Analysis**: After development, mark the solvent front, use UV light to reveal compounds, and calculate Rf values to analyze the plate's results.

Q & A

What is the purpose of the video by Dr. Frank?

The purpose of the video is to demonstrate how to perform thin-layer chromatography (TLC) in an undergraduate lab setting at the University of Ottawa.

What is assumed about the viewer before watching this video?

It is assumed that the viewer is familiar with the technique of TLC and its terminology, and has watched part one of the series for foundational concepts.

What are the essential items needed to perform TLC as described in the video?

The essential items needed include a developing jar, eluent mixture, TLC plate, clean capillaries, a pencil, tweezers, a ruler, sample and reference compounds, and a solvent to dissolve them.

How much eluent should be poured into the developing jar and why?

Approximately 10 milliliters of eluent should be poured into the developing jar to allow the solvent vapors to fill the jar, which is important for the TLC process.

Why is it important for the TLC plate to be smooth and homogeneous?

A smooth and homogeneous TLC plate is important because imperfections like creases or chipping can affect the migration of the solvent and thus the spots' resolution.

How should the baseline be marked on the TLC plate?

The baseline should be marked lightly with a sharp pencil across the short axis of the plate, approximately one centimeter from the bottom, without chipping the silica.

What is the typical amount of compound used for preparing the solution in TLC?

About five milligrams of the compound is typically used, which corresponds to the tip of a small spatula for solids or one or two drops for liquids.

How should the capillary be prepared for spotting the sample on the TLC plate?

The capillary should be clean, of good quality, not too thin or wide, and the extremity used for dipping should be smooth, which can be achieved by rubbing it with brown paper.

What is the correct method for depositing a spot on the TLC plate?

The spot should be deposited by quickly dipping the capillary into the solution, wicking off excess liquid, and touching the plate very briefly to leave a small spot, roughly a millimeter or two in diameter.

Why is it recommended to visualize the TLC plate under UV light before developing it with the eluent?

Visualizing the plate under UV light before development helps to identify if the plate is already showing spots, which can save time and resources by not developing a useless plate.

How should the TLC plate be inserted into the developing jar during the elution process?

The plate should be inserted into the jar vertically as possible with the baseline below the level of the liquid eluent to ensure the solvent drags the compound upwards.

What is the significance of marking the solvent front on the TLC plate after elution?

Marking the solvent front is significant as it provides a reference point for calculating the Rf values of the spots, which is crucial for the analysis of the plate.

What is the Rf value and how is it calculated?

The Rf value is a measure of the distance a compound travels relative to the solvent front. It is calculated using the formula: Rf = (distance of the compound from the baseline) / (distance of the solvent front from the baseline).