Grape Soda Column Chromatography

Introduction

Separate different colored dyes in grape soda using column chromatography, a popular method used in research and industry to separate, isolate and purify components of mixtures.

Concepts

• Polarity
• Column chromatography

Materials

Safety Precautions

Isopropyl alcohol solution is a flammable liquid; keep away from open flame. Wear chemical splash goggles, chemical-resistant gloves and a chemical-resistant apron. This activity requires the use of hazardous components and/or has the potential for hazardous reactions. Please review current Safety Data Sheets for additional safety, handling and disposal information.

Disposal

Please consult your current Flinn Scientific Catalog/Reference Manual for general guidelines and specific procedures, and review all federal, state and local regulations that may apply, before proceeding. All solutions may be flushed down the drain with excess water according to Flinn Suggested Disposal Method #26b.

Prelab Preparation

1. To prepare 500 mL of a 25% isopropyl alcohol solution, add 180 mL of 70% isopropyl alcohol to a 600-mL beaker. Dilute to the 500-mL mark with distilled or deionized water.
2. To prepare 500 mL of a 5% isopropyl alcohol solution, add 35 mL of 70% isopropyl alcohol solution to a 600-mL beaker. Dilute to the 500-mL mark with distilled or deionized water.
3. If the syringe has a tip cover, remove it before performing this demonstration.

Procedure

1. Pretreat the column by drawing 10 mL of the 70% isopropyl alcohol solution into the syringe. Place the Sep-Pak^® C₁₈ cartridge snugly on the luer lock tip of the syringe. Expel the alcohol out of the syringe through the column.
2. Repeat step 1 using 10 mL of distilled or deionized water in place of the 70% isopropyl alcohol solution.
3. Place the 6-well microplate on the overhead projector and pour grape soda into one of the wells. Remove the cartridge from the syringe and draw 10 mL of the grape soda from the microplate into the syringe.
4. Place the cartridge back on the syringe and force the soda through the column and into a clean well on the microplate. Notice the clear solution that elutes (or exits) from the column.
5. Again, remove the cartridge from the syringe. If there is any grape solution left in the syringe, rinse the syringe with 5% isopropyl alcohol first. Draw 10 mL of 5% isopropyl alcohol solution into the syringe and place the cartridge back on the syringe.
6. Force the 5% isopropyl alcohol solution through the column into a clean well on the microplate. Note the red-colored solution that elutes from the column.
7. Remove the cartridge from the syringe and draw 10 mL of 25% isopropyl alcohol solution into the syringe. Replace the cartridge.
8. Force the 25% isopropyl alcohol solution through the column into a clean well on the microplate. Note the blue-colored solution that elutes from the column.

Teacher Tips

• Grape Kool-Aid^® works well for this experiment. Prepare the Kool-Aid according to the package instructions. The resulting concentration is approximately 0.3 g of Kool-Aid powder per 100 mL of distilled or deionized water. Do not add sugar.
• The Sep-Pak^® C₁₈ cartridge had a short and a long end. The cartridge can be used either direction. It is important to keep the flow going in one direction.
• The Sep-Pak C₁₈ cartridge can be used to separate many mixtures of varying polarity. The experiment becomes more visually appealing if the mixture has at least two colors of different polarities.
• An air pocket in the syringe will not affect the outcome of the demonstration.
• To store or reuse the Sep-Pak C₁₈ cartridge, first clean the column by rinsing it with 10 mL of 70% isopropyl alcohol solution,then rinsing with 10 mL of distilled or deionized water. If cleaned properly after each use, the Sep-Pak C₁₈ cartridge can be reused indefinitely.

Discussion

The ingredients of grape soda include carbonated water, sugar, citric acid, red dye, ascorbic acid, and blue dye. As the soda passes through the very non-polar Sep-Pak C18 column, the polar molecules, such as citric acid, preferentially adhere to the polar solvent—water. The non-polar molecules, such as the dyes, spend very little time adhering to the polar solvent and therefore stay in the column. The 5% isopropyl alcohol solution is slightly non-polar. As the dilute alcohol solvent is passed through the column, the red dye is more attracted to the solvent than it is to the column. The blue dye, however, is more non-polar than the red dye and is still attracted more strongly to the column than it is attracted to the solvent. Because of these two factors, only the red dye is eluted from the sample by the 5% alcohol. The 25% isopropyl alcohol solution is more non-polar than the 5% isopropyl alcohol solution. The more non-polar mixture now attracts the blue dye away from the column, allowing it to flow out of the cartridge with the solvents.

Column liquid chromatography (LC) is often used in industry to separate mixtures and detect trace components of a mixture. High performance liquid chromatography (HPLC) has become the instrument of choice for many quantitative analyses. This demonstration and the processes involved can be compared directly to HPLC. As with HPLC there is a solvent delivery system (the syringe), an injector (the syringe), a column (Sep-Pak cartridge) and a detector (the human eye).

References

Bidlingmeyer, B. A.; Warren, F. V. J. Chem Educ. 1984, 61, 716–720.

Vonderbrink, S. A. Laboratory Experiment for Advanced Placement Chemistry. Flinn Scientific: Batavia, IL, 1995; pp 149–153.