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Chromatography of Dyes

Chromatography is one of the most common and important laboratory methods in the separation and identification of components of a mixture. All chromatographic separations are based on the same principle — that components of a mixture have different affinities for a stationary phase (paper or alumina, in this experiment) as well as different affinities for a mobile phase (the solvent). These different affinities, in turn, may be related to the polar nature of the various molecules involved.

Part 1: Paper chromatography of food dyes

This experiment is designed to illustrate the process of chromatography. We will be separating and possibly identifying the food dyes in some drink mixes and food colors. The effect of solvent polarity on the ability to separate and elute compounds will also be demonstrated.

Procedure

Obtain a piece of filter paper approximately 10x15 cm in size. Draw a pencil line parallel to one of the longer edges, about 1 cm from that edge. Make 9 "tic-marks" on the pencil line, about 1.5 cm apart. At this point, your chromatogram (the piece of filter paper) should look like this:

Spot each of the 5 standards on separate tic-marks, using toothpicks to spot each sample, as shown in pre-lab. Be sure to record which standard is spotted on which tic-mark. Choose two food colors and two drink mixes, and spot them on the remaining 4 tic-marks. Curl the filter-paper into a cylinder (colored spots on the outside), and staple the edges together. Do not allow the edges of the filter paper to overlap too much, if at all.

Add about 10 mL of a 7:3 isopropanol:water mixture to your 400 mL beaker, then carefully place the chromatogram (spotted edge down) into the beaker — the line of spots must be higher than the level of the solvent. Cover the beaker, and allow the solvent to rise within 1-2 cm of the top edge of the chromatogram. Remove the chromatogram from the beaker, carefully detach the stapled edges, and allow it to dry.

Prepare another chromatogram exactly the same as the one above, but develop it using 0.1% NaCl in water as the solvent. You can do this while the other chromatogram is running, if you use your 600 mL beaker as a developing chamber.

By comparing the colors of the spots in the standards and the unknowns, and the distances the spots moved, you should be able to tell what compounds are present in the food colors and drink mixes. Some of the food colors and drink mixes may contain only one dye, and some may contain more than one. One solvent system may separate a mixture better than the other. Some of the food colors and drink mixes may contain dyes that are not in the standards. If so, note these.

Part 2: Column chromatography of unknown dye mixture.

In this experiment a column is packed with a finely-divided solid of uniform particle size and a liquid phase is allowed to percolate downward through the solid. The common solid absorbant, alumina (aluminum oxide), will be used.

Procedure

Obtain two disposable pipettes. Insert a small plug of cotton in the bottom of one pipette (do not ram it in!), and then add about 3 mm of clean sand. Add about 4 cm of alumina to the pipette, and then add another 3 mm of clean sand. Obtain about 10 mL of 95% ethanol, and add ethanol to the top of the column to completely wet the alumina: ethanol will be dripping out of the bottom when it is completely wetted. Allow the ethanol to drain until the level of liquid is almost down to the top of the upper sand layer: NEVER LET THE LIQUID LEVEL GO BELOW THE LEVEL OF THE TOP SAND LAYER! Add 5 drops of dye solution to the top of the column, and allow the dye to absorb into the sand layer, then IMMEDIATELY add 95% ethanol slowly. The ethanol will begin to elute the less polar component of your dye mixture. Keep adding ethanol until the first dye has been completely eluted. At this point, begin adding water to the top of the column to elute the second, more polar dye, and continue adding water until all of the second dye is removed. Report the colors of the dyes that elute with each solvent, as well as the color of any dye that remains in the alumina.


Date: ______Name: ______

Part 1: Paper Chromatography

Tape your chromatogram that you developed in 7:3 isopropanol:water here.

Record your data in the following table. Label your samples 1-9 from left to right.

Sample # / Sample Identity / Color(s) / Distance(s) spot(s) rose
1
2
3
4
5
6
7
8
9


Tape your chromatogram that you developed in 0.1% NaCl in water here.

Record your data in the following table. Label your samples 1-9 from left to right.

Sample # / Sample Identity / Color(s) / Distance(s) spot(s) rose
1
2
3
4
5
6
7
8
9


Questions

1. Which standards were in your two food dyes? Explain your reasoning. Were there any colors in the food dyes which were not in the standards?

2. Which standards were in your two drink mixes? Explain your reasoning. Were there any colors in the drink mixes which were not in the standards?

Part 2: Column Chromatography

Unknown Number?
Original color of dye unknown?
What color dye eluted with ethanol?
What color dye eluted with water?
What color dye remained on the column, if any?