Experiment 6: Paper Chromatography In this experiment you will explore the technique of chromatography, a very important method for the separation and identification of substances. There are many different forms of chromatography, but they are all based on the same principles. A mixture is generally dissolved in a solution or absorbed onto a solid surface. The mixture is then pushed across a stationary surface, causing it to separate into its components. The different components demonstrate a differing level of attraction for the stationary surface; those that are more attracted to the surface tend to be pulled more slowly across it. In this experiment you will carry out paper chromatography, which uses paper as the stationary surface and a solvent as the mobile phase. The compounds you will be separating are the colored dies commonly used in foods and in inks. PROCEDURE: Part A: Chromatography of Ink from Felt-Tip Markers 1. Cut a strip of chromatography paper from the roll. It should be approximately 15 cm long. Fold the paper about 3 to 4 cm from the bottom into an L shape. The paper should be able to stand up on the short side. 2. Draw a line (in pencil) about 1 cm from the fold. On the line, make three small dots with felt-tipped pens of different colors. These dots should be about equally-spaced from each other, with none of them too close to the edge of the paper. 3. Pour a small amount of water into your 150 ml beaker. You should add only as much water as is necessary to completely cover the glass at the bottom of the beaker. 4. Place the bottom of the chromatography paper into the beaker. The water-level must be lower than the dots on the paper. The water will push the ink up the paper. Try to keep the paper from touching the sides of the beaker. 5. Remove the paper from the water once the water level has reached about 1 cm from the top of the paper. Do not allow the water to rise all the way to the top! Sit the paper aside and allow it to dry. 6. Answer the questions on the report sheet. Part B: Separation of Colored Mixtures (Read all the way through before beginning!) 1. Using a scrap piece of chromatography paper and a capillary tube, practice making small spots of dye solution on the paper. A good spot should be round and no more than 2 mm wide. You may use any of the five dye compounds to practice this. 2. On the 14 cm 10 cm chromatography paper, draw a straight baseline in pencil--do not write on the paper with ink-approximately 1.5 cm up from the bottom of the sheet. (Consider the 14- cm edges to be the top and bottom of the sheet.). Refer to Figure 6-1 for an example. 3. Using separate capillary tubes for each solution, place one spot of each of the following solutions along your baseline: tartrazine (label as T ) erioglaucine (label as E ) allura red (label as A.R. ) erythrosin B (label as E.B. ) sunset yellow (label as S.Y. ) Page 6-1
In applying the spots you should follow these guidelines: Use a fresh capillary tube for each solution. Place the first spot at least 2 cm in from the left edge of the paper and the last spot at least 2 cm in from the right edge of the paper. Leave about 2 cm of space between neighboring spots. Write the labels for each spot along the bottom edge of the paper. Figure 6-1: Preparing the Chromatograph 5. Holding the paper upright with the spots facing you, curve the two vertical edges away from you and toward each other until they just touch each other (make sure they do not overlap ). Staple the touching sides together at the top and bottom of the cylinder you ve created. See the figure below. Figure 6-2: Chromatograph Folded and Stapled 6. Add a small amount of the ammonia solution to an 800 ml or a l-l beaker (the liquid should not be more than 0.5 cm deep). Stand your paper cylinder alongside the beaker and check to see that the level of the ammonia in the beaker is below the baseline on the cylinder. If the ammonia level is above the baseline, pour out some of the solution and check again. Refer to Figure 6-3 for this and the next two steps.. Page 6-2
Figure 6-3: Developing the Chromatograph 7. Once you are sure your spots won't be submerged, place the cylinder in the beaker, baseline towards the bottom. You may ask your instructor for help if you are unclear about what to do. 8. Cover the beaker with plastic wrap or aluminum foil. Leave the setup undisturbed once the solvent has begun moving up the paper. Excessive motion can ruin the chromatograph. 9. When the solvent has traveled to just below the top of the paper cylinder (about 2 cm from the top) remove the cylinder from the beaker and allow it to dry. 10. Repeat this experiment, using the four unknown mixtures (A, B, C, and D) instead of the pure colorings. Label each spot with the letter of the unknown. 11. Once you have finished developing the chromatograph, it should be possible to identify which of the five colorings were present in each mixture. Answer the questions on the report sheet. Page 6-3
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Report Sheet Name: Paper Chromatography Attach your dry chromatograms to this page with a stapler or paper clip. 1. Which of the inks (blue, green, and black) appear to be mixture of other colors? Which individual colors appear to be in each ink? 2. Which of the colorings were present in each of the four mixtures? Mixture A Mixture B Mixture C Mixture D 3. Why did you use pencil instead of pen when writing on the chromatography paper? 4. All of these colorings were made and purified in a chemical laboratory. Alternatively, they could have been extracted from natural sources such as plants and then purified. The Food and Drug Administration directs food suppliers to label color additives as natural colors if they are obtained from natural sources, and artificial colors if they are man-made. Which (if either) is safer for you to consume? Explain your reasoning. Page 6-5
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Preparation for this Experiment Felt-tip pens (blue, black, and green) 1 800-mL or 1-L beaker per student Roll of chromatography paper and scissors Capillary Tubes (preferably mini or prepared by me) 1.0 M NH 4 OH (about 500 ml-1 L) Label this as ammonia solution Solutions of each of the dyes in small bottles and unknowns A, B, C, and D (should already be prepared see me if you need to make new solutions) Plastic wrap (or aluminum foil) to cover beakers 2 pieces of chromatography paper per student plus several extra for those who must redo experiment(each cut to 14 cm 10 cm) Page 6-7