Chromatography 16 Sept 2008 Section 2307 Chemistry 1 TA: Alison Baierl Peter Bolgert

Purpose: The purpose of the experiment is to introduce the technique of chromatography, which is a way to physically separate mixtures into individual substances. The goals of the experiment are to extract pigments from a leaf and to test the purity of food dyes used in candy.

Procedure: Part 1 of the experiment is about column chromatography. First the column was set up. A wad of cotton was placed in one end, and then the plastic piece and stop cock were attached. The column was filled two-thirds full with hexane, making sure there were no bubbles. Then 23 grams of solid sodium bicarbonate were added to the column, making sure there were no bubbles or air spaces. Then the hexane was drained out until the level was right above the solid. Next the leaf sample was prepared. A sample of thawed spinach leaves was placed in a beaker and swirled with several mL of acetone. Using a pipet, approx. 1 mL of acetone extract was placed on top of the solid column. After that 12 test tubes were marked with lines at approx. 1 mL. In preparing for the addition of the first solvent, the glass tube was drained of excess liquid, until the level of liquid was right above the solid. Then 3.0 mL of hexane were added to the column and the stop cock was opened so the solvent could start flowing. The speed of the flow (# of drops in the first 30 seconds of each mL test tube) was measured. After the first test tube (1mL) was filled, it was removed and the next empty test tube was added. The speed of the flow was recorded for 9 test tubes. When the level of hexane was just above the top of the solid, the next solvent (acetone, 3.0 mL) was added. The procedure continued in the same

manner with two more solvents: 70% isopropyl alcohol (3.0 mL) and saturated sodium bicarbonate (3.0 mL). Part 2 of the experiment dealt with Paper Chromatography. A line of dots spaced 1 cm was drawn along the edge of a piece of chromatography paper. Each dot was labeled for a color of M&Ms. The color from an M&M was extracted by swirling several candies of a certain color in a dish with a few drops of water. Small of dots of green, orange, and blue color were added to the paper using a capillary tube. A drop of commercial food coloring (positive control) was also added. Then the bottom edge of the paper (with the colored dots) was dipped into a 0.1% sodium chloride solution, which slowly moved up the paper. Once the salt water nearly reached the top of the paper, the paper was removed, and the solvent front was marked with a pencil. The distances that the solvent traveled and that the dyes traveled were measured and the retention factors for each dye were calculated (see results section for details).

Results/Observations: Column Chromatography Data:

# of drops in Test tube first 30 s 1 50 2 50 3 50 4 5 6 7 8 9 40 40 35 32 50 42

Observations Pale Green Leafy Color - darkest of all tubes Pale, like #1 Nearly clear, a touch of green Clear Clear Very pale yellow green Clear w/ a hint of yellow Same as #8

Sodium Bicarbonate

e ne an xa ex He o on et Ac e h h co co All ol

Color Intensity of Test Tubes

7 6 Intesnity (0 low - 6 high) 5 4 3 2 1 0 1 2 3 4 5 Test Tube # 6 7 8 9

Additional qualitative observations: The solute flowed out much more quickly than was expected. In fact, 1 mL test tubes were being filled well before 30s. For example, in test tube 8, there were 37 drops in 22 seconds. At that rate there would have been about 50 drops in 30 seconds.

Paper Chromatography Data:

Distance Sample Traveled (cm) Green blue component yellow component Orange Blue primary spot secondary spot Food Coloring yellow component pink component 4.40 0.00 4.05 2.50 2.50

Distance Solvent Traveled (cm) 4.70 4.70 4.92

Retention Factor (Rf) 0.86 0.53 0.51

4.85 4.85

0.91 0.00

3.52 1.50

4.93 4.93

0.71 0.30

Additional Qualitative Observations: In the blue sample, a streak of blue traveled up the paper, but a blue dot also remained at the starting notch.

Demonstrated Calculations: Retention Factor Calculation

B 2 3 4 5 6 7 8 9 10 11 12 13 14 15 C Distance Sample Traveled (cm) D Distance Solvent Traveled (cm) 4.7 4.7 4.92 E Retention Factor (Rf) =C4/D4 =C5/D5 =C7/D7

Green blue component 4.05 yellow component 2.5 Orange Blue primary spot 4.4 secondary spot 0 Food Coloring yellow component 3.52 pink component 1.5 4.85 4.85 =C10/D10 =C11/D11 2.5

4.93 4.93

=C14/D14 =C15/D15

Discussion: In Part 1, column chromatography, the objective was to isolate plant pigments from a spinach leaf. As shown by the Color Intensity Graph, two pigments eluted from the spinach sample. The first sample was a moderately dark green color. The second pigment took longer to drip down and was a pale yellow-green color. This color of the second pigment was not as intense as the first one. The two main pigments in spinach leaves are chlorophyll a and b. If these are the most common pigments in plant cells, it stands to reason that the two pigments that were isolated were chlorophyll a and b. The ratio of the amount of chlorophyll a in plant cells to the amount of chlorophyll b is 3:1. It makes sense that the more plentiful pigment is the more intensely colored one, because there is more pigment concentrated together. The paler pigment would then be chlorophyll b. This pigment is paler in the test tubes because there is less of it to begin with. The color is spread out in the solvent. Also it is known that chlorophyll a is green and that chlorophyll b is more of a yellowish green, which adds more evidence to support this hypothesis (http://food.oregonstate.edu/learn/pop/plant3.html). In Part 2, paper chromatography, the objective was to determine if food dyes in M&Ms are pure. The objective was also to determine the presence or absence of yellow dye #5 in each M&M. The food coloring was the positive control because it was known to contain yellow #5. Each substance has a unique retention factor (Rf), which is the distance sample has traveled divided by the distance the solvent has traveled. The green dye was not pure, because it was made up of blue and yellow components with Rfs of 0.86 and 0.53, respectively. The orange dye did not separate, indicating that it was

a pure substance (Rf=0.51). The blue dot may or may not be pure. Finding a definite answer will take further experimentation. One blue streak traveled up the page and had a retention factor of 0.91, and another blue dot stayed at the beginning. This could be due to improper preparation of the chromatography paper, or it is possible that the blue dye is made up of two dyes, one with a strong affinity for salt water, and another with absolutely no affinity for salt water. Since most dyes will have some affinity with a solvent, the blue dot was probably caused by a mistake in the preparation. The food coloring was not pure. It separated into a yellow streak (presumably yellow #5) and a pink streak. To determine if any of the M&Ms contain yellow #5, compare the retention factor of yellow #5 (0.71) to any other yellowish dyes found. The only dye that fits this category is the yellow component dye of the green M&M, which has a retention factor of 0.53. This is a 25% difference from yellow #5.

This is too much of a difference to be taken lightly. The green dot was very faint to begin with, so a result, the yellow component is hardly visible, and it is hard to determine exactly where the streak ends. It is very possible that the streak is longer than was originally measured. If that is the case, then the retention factor would be closer to that of yellow #5. The M&Ms packaging lists Yellow #5, Yellow #6, Yellow #5 Lake, and Yellow #6 Lake as ingredients. It is possible that one of the other three dyes found in M&Ms is the yellow component of the green M&M. Further experimentation is necessary in this case.

Conclusion Using column chromatography, two plant pigments in a spinach leaf were isolated and determined to be green chlorophyll a and yellow-green chlorophyll b with moderate confidence. The goal for part 1 was accomplished. Using paper chromatography, it was determined that of the colors that were tested, only the orange M&Ms contained pure dye. The other colors were made up of several component dyes. The blue M&M may be pure; however, further experimentation is necessary. Furthermore, it is uncertain whether or not the green M&M contains yellow dye #5. Further experimentation is also necessary in this case. Further experiments should also include test on yellow M&Ms. The goal for part 2 will not be accomplished until more data is obtained.