How do you prepare a green food dye solution in ethanol for column chromatography?

Tare a clean 5-mL volumetric flask and add 2.5 g of green food dye. Pour approximately 1.5 mL of 95% ethanol into the flask, stopper it, and invert several times to mix thoroughly. Then fill to the mark with 95% ethanol, reseal, and invert until the color appears uniform throughout. Label the flask with its name and concentration.

What safety equipment and waste containers are required before starting a column chromatography lab?

Put on a lab coat, safety glasses, and nitrile gloves before beginning. Ensure the lab has labeled waste containers for organic waste, silica gel, and glass. Fill a plastic wash bottle with acetone and place it near the organic waste container for post-lab column cleaning by students or teaching assistants.

What materials and equipment should be set up at each student workstation for column chromatography?

Each pair of students needs five 50-mL beakers, one 100-mL beaker, graduated cylinders (one 5- or 10-mL and two 50-mL), two powder funnels, a glass stirring rod, laboratory tape, a marking pen, a ruler, and two pipette bulbs. Additionally, ensure a lab stand or scaffold with an appropriate clamp secures the column at each workspace.

How should the stopcock on a chromatography column be tested and prepared?

Test each stopcock to ensure it fits securely but still turns easily. After testing, close the stopcocks and distribute the columns to student workspaces. Proper stopcock function prevents solvent leakage during the chromatography procedure and ensures accurate separation results throughout the experiment.

What materials should be placed in the central lab area for student access?

Place the prepared dye solution, a container of silica gel powder, sand, laboratory wipes, clean spatulas, weighing boats, a bottle of 95% ethanol in a nearby hood, glass or cotton wool, at least one long blunt-ended metal or wooden rod, and a box of Pasteur pipettes in the central area for all students to access.

Why is a well-ventilated area important when setting up materials for column chromatography?

A well-ventilated area is essential when placing silica gel powder, sand, spatulas, and weighing boats near the balance because these materials can generate dust. Additionally, 95% ethanol must be stored in a nearby hood to minimize vapor exposure and ensure safe handling of volatile organic solvents during lab preparation and execution.

How much dye solution should be prepared for a typical undergraduate lab with 10 students working in pairs?

Prepare 5 mL of green food dye solution in ethanol for 10 students working in pairs, with some excess. This quantity is sufficient for the standard lab setup. Adjust quantities proportionally if your class size differs or if additional practice runs are needed for student learning outcomes.

Column Chromatography

Concepts Instructor Prep Student Protocol

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Column chromatography is a technique that uses a packed column to separate compounds based on their interactions with a stationary phase, which is usually in the form of microscopic beads. The spaces between the beads are filled with solvent. Opening the column allows the solvent to flow through the stationary phase. When a mixture is applied to the top of the packed column followed by more of the solvent, the mixture moves into the mobile phase and flows through the stationary phase.

Each component of the mixture interacts with the stationary phase in a different way. Some components have weak interactions with the stationary phase and so move quickly through the column, while others have strong interactions with the stationary phase and thus move more slowly. This separates the different compounds into bands, which are collected in small fractions. This enables the purification of each compound.

So, what kind of properties can be used to separate mixtures? One of the most commonly used properties is polarity. For this, silica gel, which is a form of silicon dioxide, is often used as the stationary phase. Silica gel interacts with compounds by dipole-dipole interactions and hydrogen bonding through the -OH groups that form on its surface. Thus, polar compounds will interact strongly with the stationary phase, while non-polar compounds will interact weakly.

Other properties that can be exploited for separation include size, charge, and hydrophobicity. A common way to load the stationary phase into the column is as a slurry of the stationary phase and solvent. Then, the stationary phase is packed by flowing more solvent through the column to compact the slurry. It is essential that the stationary phase is packed uniformly without air bubbles, empty channels, or dry patches. These can disrupt flow and cause mixing of bands.

When choosing a column, the diameter is based on the volume of the sample to be separated. The sample should cover the top of the column in a thin, even layer. A thicker sample layer leads to broader bands. The column length depends on how well the compounds separate on the stationary phase. Compounds with similar affinities for the stationary phase require a long column for adequate separation. However, a mixture of compounds with very different affinities for the stationary phase can be separated on a shorter column.

In this lab, you will explore column chromatography by first packing and preparing a silica gel column. Then, you'll use your column to separate the colored components in green food dye.

At the end of this lab, students should know...

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What are the two phases in column chromatography?

Column chromatography involves a mobile phase and a stationary phase.

How are mixtures separated in column chromatography?

Compounds separate based on their interactions with the stationary phase.

What compounds elute first?

Compounds interacting weakly with the stationary phase elute first because they interact stronger with the mobile phase and thus travel through the stationary phase faster than those with strong stationary phase interactions.

What physical properties are used to separate mixtures in column chromatography?

The most common properties utilized to separate mixtures are based on size, charge, polarity, and hydrophobicity of the constituent substances.

How do column properties such as the diameter and length affect the separation of mixtures?

The diameter of the column determines the amount of sample that can be loaded on to the column. A small thin layer of sample provides the most efficient separation; therefore, a smaller diameter means less sample can be loaded and vice versa. The length of the column is dependent on the affinity of the substances to the stationary phase of the column. A longer column is required for substances with similar affinity, while a shorter column can be used for substances with very different affinities.

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