A. Characterization of Standard Food Dyes.

1. Prepare a developing tank by pouring 10 mL of the mobile phase (0.10% wt/v solution of NaCl  in H₂O) into a 400 mL beaker and covering the beaker with Parafilm.  It is important that the air above the mobile phase become saturated with solvent vapor so that solvent does not evaporate from the stationary phase as the chromatogram develops. Therefore, be sure to keep the developing tank covered at all times.
2. [Check with your TA for the size and type of filter paper to be used] With a pencil, draw a horizontal line 1.5 cm from the bottom edge of the chromatography paper. Draw vertical tick marks along this line every 2 cm (see fig 2(a)). Using a capillary and one of the standard dye solutions, make a spot on the chromatography paper at one of the marks. Keep the capillary with the correct dye to avoid cross contamination and waste of capillaries.  Try to keep your spots less than 4 mm in diameter. Allow the dye to dry and reapply the same dye in the same spot 1 or 2 times or until a sufficiently dark spot has been achieved. With a pencil, note the name of the dye below the spot.
3. Repeat steps 2 and 3 for the remaining dyes across the bottom of the chromatogram.
4. When the spots have been applied, put your name in pencil across the top of the chromatography paper, form the chromatography paper into a cylinder, and staple the edges of the paper together making sure to leave a gap between the edges as shown in fig 2(b). If the edges come into contact, solvent will not travel at a uniform speed up the chromatography paper and the components of the mixture will not move in a straight line.
5. Place the chromatography paper into the developing tank, do not let it touch the sides of the tank and quickly replace the Parafilm cover. Make sure the level of the mobile phase is below the line of dyes on your paper. Allow the chromatogram to develop.
6. When the solvent front is approximately 1 cm from the top of the chromatography paper, remove the chromatogram and lay it flat on a paper towel. Immediately mark the position of the solvent front with a pencil. The front will continue to move as the paper dries so it is important that you mark this position now. Measure and note the distance  the solvent front traveled.

1. Draw an ellipse around each spot on the developed chromatogram and draw a horizontal line through the center of each spot. If a spot shows significant "tailing" make your horizontal line through the darkest part of the spot (see fig 3). Use the distance from the starting line (not the bottom of the paper!) to these horizontal lines to determine D_dye for each dye. Record distances and R_f values in your notebook. Recall:

R_f = D_dye / D_solvent

1. Place the chromatogram on edge, in the drying oven for 5 min or until dry. Take your dry chromatogram to the UV lamp area and observe the spots under illumination with a hand-held UV lamp on long-wavelength irradiation. Note the color of the observed fluorescence.

   WARNING: UV light can damage your eyes. Always point lamps away from you. Do not look into the UV lamps

   B.  Separation and Identification of Dyes Used to Coat M&M Candies.

2. Do the M&M candies contain any of the standard dyes above? To begin to answer this question, take one M&M of each color to the UV lamp and see if they fluoresce. Note the color of any fluorescence you observe in your notebook.[Three pairs of students will share making two solutions each.]  To separate the dyes used to coat M&Ms, you must first prepare a solution of the coating dyes. To do this, place 4 M&M's of the same color into a small beaker. Add 3 mL of a 50/50 mixture of water and ethanol and swirl the solvent until the candy coating has dissolved. Remove the M&M's from the solvent before the chocolate center is exposed. You and your partner may coordinate with two other pairs of experimenters so that each pair only has to prepare 2 solutions that can be shared with the group.

3. Prepare a second chromatogram like that in Part A using the 6 M&M solutions. The dye solutions prepared with the M&M candies are not as concentrated as the solutions of the standard dyes. You will need to spot these dye mixtures several (>5) times to obtain a sufficiently dark spot. [7-9 times may be necessary] Again, dry each spot between applications of the mixture to maintain as small and concentrated a spot as possible.

4. Develop the chromatogram. Characterize the R_f value, color, and fluorescence characteristics of each dye observed. Remember that unlike the previous chromatogram, you are now separating mixtures of dyes and there may be as many as four components each with its own R_f value, color and fluorescence characteristics. Use all three characteristics to identify all components found in each M&M coloring.