- Look over Introduction the for this lab.
- Complete the pre-lab questions for this lab.
- Look at the INSTRUCTIONS
Laboratory
Introduction| Chromatography is a technique of separating a mixture of compounds into its various components. The mixture to be separated is dissolved in a solvent (called the mobile phase) and then passed through or over a substance (called the stationary phase) that causes the various components to move over it at different rates. The fastest component will be the first to emerge from the stationary phase. (Imagine a party of people walking a mountain path. The fastest moving will come to the top first, the slowest moving will come to the top last). In paper chromatography, paper is the stationary phase. |
| You will use first use paper chromatography to separate colored components of food dyes. You will spot your paper with the food dyes and then place the paper in solvent to allow the chromatograph to “develop” (i.e., to allow the components to separate). After the chromatograph has developed you will determine the ratio between the distance each colored component of the dye has moved and the distance the solvent has moved. This ratio is called the Rf value of the component. |
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| You will then use paper chromatography to separate a mixture of amino acids obtained from the hydrolysis of a protein. The various components of this mixture are colorless so you will spray the developed chromatograph with ninhydrin which allows you to see the amino-acid spots. |
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| You will again determine the Rf values of the individual amino acids and then will compare them to the Rf values of four known amino acids. This should allow you to identify some of the amino acids in the hydrolysis mixture. |
Instructions
| To place the samples on the chromatographic paper, you will need to prepare capillary tube droppers having a fine point as follows. Heat in the flame of a bunsen burner the center of a capillary tube which has both ends open. Just as the center of the tube becomes hot enough (this may take one practice trial), pull the ends of the tube to draw out the center. Let the tube cool and then break the tube in the center of the drawn-out region. This will provide you with two capillary droppers. |
| 2. You will need a dropper for each food dye (3). The droppers for the known amino acids, the mixture of amino acids, and the protein hydrolysate will be provided for you. |
| Chromatography of Food Dyes |
| 3. Take a 14 cm square piece of chromatography paper (or Whatman #1 filter paper) and lightly draw with a pencil (not a pen!) across the entire sheet approximately 3 cm up from the bottom edge. |
| 4. Draw small x's along this line at approximately 3 cm, 7cm, and l cm from one edge. |
| 5. Label under the line with your pencil the color of the dye you will be placing at each of the x’s. |
| 6. Using the droppers you have prepared, place a small drop (i.e., quickly remove the pipette once the sample has been applied to avoid a broad spot) of each food dye in the location you have labeled for it. |
| 7. Once the spots are dried (approximately 5 minutes), carefully make a cylinder out of the paper with the side you have marked and spotted on the outside. Staple the bottom, and the top of the paper together, leaving as little overlap as possible. |
| 8. Place approximately 1 1/2 cm, or 50 inches of the developing solvent into the developing tank (500 ml beaker). |
| 9. Cover tightly with saran wrap and swirl for a minute or so to saturate the atmosphere in the tank with solvent vapors. Remove the saran wrap and place your chromatography paper in with the spot side down. |
| 10. Do not allow the paper to touch the sides of the tank. Cover the tank with the saran wrap and allow the solvent to climb up the paper until the solvent front has moved approximately 8 cm up the paper from the pencil line at the bottom. Do not allow the solvent front to go all the way to the top of the paper! |
| 11. Stop the chromatography by removing the paper from the tank. |
| 12. Quickly mark the position of the solvent front for each lane containing a sample before it dries and is impossible to locate. |
| 13. Allow the paper to dry and measure the distance, to the nearest 0. 1 cm, between the pencil mark at the bottom and the center of the leading edge of each component and the solvent front. From these values you can calculate the Rf for each. |
| Chromatography of amino acids |
| 14. As in the above procedure, use a 14 cm square of paper. Be careful in this case only to handle the paper by the extreme edges. |
| 15. As before, draw a line 3 cm up from the bottom of the paper. At 2 cm intervals across the paper, draw x's for each of the 4 amino acids and for the two unknown mixtures. Under the line label each. Spot the samples at the labeled locations. Repeat the procedure for filling the tank but in this case using 50 ml of the acetic acid-butanol-water solvent. (What solvent did we really use here?) |
| 16. When the solvent has climbed approximately 8 cm from the pencil line, remove the paper and quickly mark the solvent front. Allow the paper to dry completely. |
| 17. In the hood, spray the paper lightly with the ninhydrin solution provided. |
| 18. Allow this to dry for 5 to 10 minutes using the hair dryer provided. This will develop the brown /purple color of the spots. |
| 19. When done heating, circle the spots with a pencil. Determine the Rf of all the known and unknown amino acids. |
| 20. Be sure to measure distances to the centers of the darkest part of the spots. |
| 21. Attempt to identify the unknown amino acids by comparison to the known amino acid Rf values. Be sure that you attach the chromatograms in your lab notebook along with your report for this experiment. |
Pre-lab Questions
1.) Why can't you use pen to mark the chromatography paper in this experiment).
2.) Why is it important to stop the chromatograph before the solvent reaches the top of the paper? What would happen if you allowed the paper to sit in the solvent for 10 minutes after the solvent had reached the top? Explain as specifically as possible.
3.) The solvent used in the second part of the experiment is very polar. Which amino acid would you expect to have the larger Rf value, alanine or aspartic acid? Explain.
4.) The solvent in the developing tank is only 1 1/2 cm high and the samples are spotted at about 3 cm. Why is it important to keep the spots above the solvent? Explain precisely.
5.) Why is it important, in step 14, to handle the paper only at the extreme edges when you did not have to take that precaution in step 6?
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