What is the purpose of the sand layer in column chromatography?

The sand layer, placed 0.5 cm above the cotton plug, prevents silica gel from passing through the plug into your collected fractions. This protective barrier ensures that only the separated dyes are collected in pure fractions, maintaining the integrity of your samples throughout the separation process.

Why does column chromatography use two different eluents for separating food dyes?

Using ethanol first elutes erioglaucine, which has lower affinity for silica gel, while water elutes tartrazine more effectively due to its higher polarity. This two-solvent approach saves time and minimizes solvent waste while maintaining fraction purity, compared to using a single eluent throughout the entire separation.

How do you prepare the silica gel slurry for column chromatography?

Weigh 5 grams of silica gel powder and mix it with 18 mL of 95% ethanol in a beaker. Stir with a glass rod until the texture is consistent and resembles batter with no dry patches or clumps. The slurry should flow easily to avoid clogging the funnel or trapping air bubbles in the column.

What flow rate should you maintain when eluting dyes through the column?

Adjust the stopcock so the solvent level decreases approximately 1 mL every 15 to 20 seconds. Maintain at least 1 cm of solvent above the silica gel at all times by periodically refilling the column. This controlled solvent flow rate ensures proper separation and prevents band broadening during the elution process.

How do you apply the green food dye to the silica gel column?

Use a clean pipette to gently apply 5 to 7 drops of the dye solution evenly across the top of the silica gel. If necessary, add a few drops of ethanol to keep the silica gel covered. Allow the dye to soak in before beginning the elution process with your chosen eluent.

What do the different colors of fractions tell you about dye affinity for silica gel?

The blue erioglaucine elutes with ethanol, indicating lower affinity for silica gel, while yellow tartrazine requires water, indicating higher affinity. This separation demonstrates that tartrazine binds more strongly to silica gel than erioglaucine does, which is why different solvents are needed to elute each dye effectively.

Why is it important to avoid stopping the column chromatography for extended periods?

Stopping for more than 1 minute at a time causes compound bands to start broadening, which reduces separation quality and makes it harder to collect pure fractions. Continuous flow maintains sharp band definition and ensures efficient separation of the dyes throughout the elution process.

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.

Print Procedure Steps

Source: Lara Al Hariri and Ahmed Basabrain at the University of Massachusetts Amherst, MA, USA

Column Chromatography of Food Dye
In this experiment, you'll use column chromatography to separate green food coloring into its component blue and yellow dyes, called erioglaucine and tartrazine. First, you'll use 95% ethanol to elute the dye with a weaker affinity for silica gel. Then, you'll flush the second dye from the column with water. To recover the purified dyes, you will collect each band of dye as it leaves the column. These collected volumes are called fractions.
- To begin, put on a lab coat, safety glasses, and a pair of nitrile gloves.
- Bring a column or burette to the central supply area. Obtain a piece of cotton or glass wool and press it into the top of your column. Use a long, blunt-ended wire or rod to push it down to plug the column just above the stopcock. Note: The cotton must be stable, but don't pack it too densely, because the solvent needs to pass through it easily. Make sure that the stopcock can turn freely without catching on the plug.
- Attach a burette clamp to a lab stand and clamp the column upright with about 4 in of space underneath it. Make sure that the stopcock is closed.
- Label a 100-mL beaker as ‘waste’ and place it under the column.
- Next, measure 1 g of sand in a tared weighing boat and bring it back to the bench.
- Then, mark the column about 0.5 cm above the top of the cotton plug.
- Place a small funnel in the top of the column and pour enough sand through the funnel so that there is a layer 0.5 cm deep over the plug. This will keep the silica gel from passing through the plug into your fractions.
- Remove the funnel and gently tap the column to evenly distribute the sand.
- Now, prepare your eluents. Label a 50-mL beaker and a 50-mL graduated cylinder as ‘water’.
- Measure 50 mL of deionized water with the graduated cylinder and pour it into the beaker.
- Then, label another 50-mL beaker and a 50-mL graduated cylinder as ‘ethanol’. Measure 50 mL of 95% ethanol and pour it into this beaker.
- Now, obtain 4 or 5 clean Pasteur pipettes and a pipette bulb. Gently pipette 7 – 8 mL of ethanol along the inner walls of the column. This layer of solvent will let you pour this silica gel slurry into the column without disturbing the sand.
- Open the stopcock and drain ~1 mL of ethanol into the waste beaker to flush air from the stopcock and to confirm that the solvent flows easily through the plug. Make sure that there are no dry patches in the sand and the plug and gently tap the side of the column to remove trapped air bubbles.
- Next, place a clean funnel in the top of the column and label a third 50-mL beaker ‘silica gel slurry in ethanol’.
- Carefully weigh 5 g of silica gel powder and pour it into your labeled beaker.
- Then, bring the silica gel, a glass rod, and your ethanol graduated cylinder to the ethanol dispensing hood.
- Measure 18 mL of 95% ethanol and pour it into the beaker. Stir the mixture with the glass rod until the texture is consistent and no dry patches or clumps of powder remain. This usually takes ~1 min. Note: The slurry should be thick, like batter, but it should still flow easily. If the slurry is too stiff, it could clog the funnel or trap air bubbles in the column.
- Once the powder is completely suspended in ethanol, bring it to your bench. Stir the slurry until its texture is consistent again and immediately pour it into the column.
- Gently tap the sides of the column as the silica gel settles to get rid of air bubbles.
- Rinse the beaker with 1 or 2 mL of ethanol, and pour the rinse into the column. Then, remove the funnel and set it aside.
- Pipette ethanol along the inside of the column to wash down particles stuck to the walls. Then, drain the solvent until the meniscus is just barely above the top of the silica gel.
- Now, use a 5- or 10-mL graduated cylinder to obtain about 0.8 mL of the green food dye solution.
- Use a clean pipette to gently apply 5 to 7 drops of the dye solution evenly across the top of the silica gel. If necessary, add a few drops of ethanol to keep the silica gel covered.
- While you wait for the dye to soak in, label two clean 50-mL beakers as ‘ethanol fraction‘ and ‘water fraction’. Exchange the waste beaker under the column with the ethanol fraction beaker.
- Next, fill the column with ethanol, pipetting the first mL along the inner walls to avoid disturbing the silica gel.
- Then, open the stopcock partway and adjust it until the solvent level decreases ~1 mL every 15 – 20 s.
- Periodically refill the column to keep the ethanol flowing. Make sure that there is at least 1 cm of solvent above the silica gel at all times.
- Monitor the yellow and blue bands as they separate. Note: If you need to step away, close the stopcock so that the column won't run dry while you're gone. Avoid stopping for more than 1 min at a time because the compound bands will start broadening. It usually takes about 20 – 35 min for the faster-moving dye to leave the column.
- Once you have collected all of the faster-moving dye in the ethanol fraction beaker, close the stopcock, set the ethanol fraction aside, and put the waste beaker under the column.
- Drain the solvent until the meniscus is just above the top of the silica gel. Then, exchange the waste beaker for the water fraction beaker.
- Use a clean pipette to fill the top of the column with deionized water. Then, open the stopcock just enough to let water flow through the column at a rate of about 1 mL every 15 – 20 s.
- Refill the column with water as needed until the second band of color has completely left the column. This usually takes another 15 – 25 min. Then, close the stopcock and exchange the water fraction beaker for the waste beaker.
- Now, drain the column completely and leave the stopcock open.
- Record the colors of the ethanol and water fractions in your lab notebook.
  Table 1: Column Chromatography of Food Dye
  
  Color Volume (mL)
  Ethanol fraction
  Water fraction
  Click Here to download Table 1
- Pour the fractions into the corresponding 50 mL graduated cylinders and record the volume of each fraction. Then, close the stopcock and bring the column to a fume hood.
- Clamp the column upright in the hood so that the silica gel can dry. Your instructor will either tell you how to clean the column or take care of it after the lab.
- Now, flush the collected fractions and leftover ethanol, water, and dye solution down the drain and clean the rest of your glassware as usual.
- Discard used Pasteur pipettes in the glass waste. Throw out used paper towels and weighing boat in the lab trash and clean the benchtop before you leave.
Results
In this experiment, you saw that ethanol eluted the blue erioglaucine in a timely manner, but it wasn't an effective eluent for the yellow tartrazine. Paper chromatography of the green dye shows similar results. Thus, we conclude that erioglaucine has a lower affinity for silica gel than tartrazine does.

Now, consider the advantages of using two solvents to elute the dye separately. Water is more polar than ethanol, so we assume that it would elute both dyes faster than ethanol alone would. While you also would have used less solvent overall if you had only used water, the separation would have been worse, making it harder to collect pure fractions.

Alternatively, you could have kept using ethanol after you collected the erioglaucine, but it would have taken a long time and a lot of ethanol to elute tartrazine that way. Switching the eluent to water after eluting erioglaucine saved time and minimized solvent waste without sacrificing purity.