JCE Classroom Activity: #61
Instructor Information
Checkerboard Chromatography Charles A. Smith Department of Chemistry, Our Lady of the Lake University, San Antonio, TX 78207;
[email protected] This Activity simulates column chromatography separations using a grid, colored pieces of paper, and a six-sided die. Students observe the effects of changing flow rate, column length, and mobile phase composition. All types of chromatography have mobile and stationary phases of different polarities. The mobile phase transports the mixture to be separated through the stationary phase. Separation of the mixture into its pure components occurs when the components have different solubilities in the phases. For example, if the stationary phase is a very polar material, then nonpolar components will pass unhindered through the stationary phase while polar components will spend more time in the stationary phase. The difference in time spent in the stationary phase separates the mixture: the least polar components elute first and the most polar components elute last.
Integrating the Activity into Your Curriculum A recent Journal article (1) describes how to convert a classroom into an interactive chromatographic simulator. This Activity is a board game based on the same idea. The procedure is easy to follow and offers insights into column chromatography and how separations are achieved. As squares come off the grid, the separation (or lack thereof ) of the colors is noticeable. The Activity is best performed after the class has discussed solubility and polarity. Terminology including resolution, dead time, isocratic, gradient, and diffusion, as described in “About the Activity”, can be introduced. Other Journal articles describe column chromatography experiments suitable for the high school level (2–4).
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About the Activity In one or more of the simulations in this Activity you may find that the components of the mixture are well separated and a long time (i.e., many rolls of the die) may be needed before the last component completely comes off the stationary phase. When this occurs, there is too much resolution (separation between the components), or dead time. Chromatographers improve this situation by changing the mobile phase composition during the separation. A separation with a constant mobile phase composition is called an isocratic separation. One that has a changing mobile phase composition is a gradient separation. In this Activity, a gradient separation is achieved by modifying rule (i) and is described in “More Things to Try”. For example, many turns may be needed for the blue compound to elute after the red compound has eluted during the first twenty turns. A gradient separation is created by modifying rule (i) to something like: From 0–20 turns, blue moves when a 1 or 2 is rolled, red moves when 3, 4, 5, or 6 is rolled, and yellow moves when anything is rolled. After 20 turns, blue moves when anything is rolled. In other cases components may co-elute. Separating them requires increasing resolution, which can be done by increasing the length of the stationary phase, using a gradient separation, or decreasing the flow rate. In this Activity, increasing the stationary phase length is accomplished by adding a second grid. Diffusion increases the time for all the molecules of a component to reach the end of the stationary phase. Eddy and longitudinal diffusion are two types of diffusion that occur in chromatography. In this Activity, eddy diffusion can be represented by blocking off regions of the grid and causing molecules to take different paths. Longitudinal diffusion is enhanced when the column length is doubled or by placing a negative number on the die. When the negative number appears, the molecule must go backward. For more information on these types of diffusion see reference 5.
Answers to Questions
This Classroom Activity may be reproduced for use in the subscriber’s classroom.
fold here and tear out
Background
1. The mixture will mostly be well separated. A good separation results in separate peaks for each color. 2. The separation is not as effective. The colors tend to come off the column mixed; resolution is lost. 3. Doubling the length of the stationary phase enhances the separation but it also increases the time needed for all components to reach the end of the stationary phase.
References, Additional Related Activities, and Demonstrations 1. Smith, Charles A.; Villaescusa, F. Warren. Simulating Chromatographic Separations in the Classroom. J. Chem. Educ. 2003, 80, 1023–1025. 2. Kimbrough, Doris R. Supermarket Column Chromatography of Leaf Pigments. J. Chem. Educ. 1992, 69, 987–988. 3. Reynolds, Robert C.; O’Dell, C. Allen. A Thin-Layer and Column Chromatography Experiment Adapted for Use in Secondary Schools. J. Chem. Educ. 1992, 69, 989–991. 4. Wigman, Larry S.; Kelsch, Catherine T. Separation Science and Chromatography: A Colorful Introduction. J. Chem. Educ. 1992, 69, 991–992. 5. Chromatography: introductory theory. http://www.shu.ac.uk/schools/sci/chem/tutorials/chrom/chrom1.htm (accessed January 2004). JCE Classroom Activities are edited by Erica K. Jacobsen
www.JCE.DivCHED.org •
Vol. 81 No. 3 March 2004 • Journal of Chemical Education
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JCE Classroom Activity: #61
Student Activity
Checkerboard Chromatography Chromatography is a powerful tool that separates a chemical mixture into its pure individual components. It can be used to identify drugs, purify plant extracts, and discover new compounds. Chromatography uses a mobile and a stationary phase. The mobile phase carries the mixture through the stationary phase to a detector. A common column chromatography experiment separates the pigments in plant leaves. The column, formed from a chemical packed into a glass tube, is the stationary phase. The mobile phase, a liquid solvent containing the mixture to be separated, is poured onto the top of the column. Separation occurs in the stationary phase due to the rule “like dissolves like”. The components that best dissolve in the stationary phase spend the most time in the stationary phase. For example, with a polar stationary phase, the most polar components reach the detector last and the least polar reach the detector first. In this Activity, you will construct a board game to investigate column chromatography.
Try This You will need: pencil or marker, ruler, scissors, several sheets of white 8.5 × 11-in. paper, tape, three differently colored sheets of paper (e.g., red, blue, yellow), small container for mixing, and a six-sided die. __1. Mark 0.5-in. divisions on all four sides of the fronts of two sheets of white 8.5 × 1-in. paper with a ruler and pencil or marker. Connect the divisions with straight lines to make two grids that are each 17 boxes wide and 22 boxes high. Place one grid on a horizontal surface and label one of the shorter edges “bottom”. This grid represents the stationary phase where the separation will occur. __2. Cut sixteen 0.5 × 0.5-in. squares from the blue and red sheets of paper (eight squares from each sheet). Cut one 0.5 × 0.5-in. square from the yellow sheet. Mix the squares in a small container. __3. Randomly place each square in a different box at the bottom of the grid (the beginning of the stationary phase). __4. Each color represents a different compound; each square represents a molecule of that compound. Blue represents a very polar compound, red a slightly less polar compound, and yellow a nonpolar compound. Assume the stationary phase is polar. Thus the travel of the blue squares must be inhibited more than that of the red squares and the yellow square must be allowed uninhibited travel. Rule (i) reflects these assumptions: (i) Blue moves when 1 or 2 is rolled, red moves when 3, 4, 5, or 6 is rolled, and yellow moves when any number is rolled. A second rule reflects the fact that molecules move independently of one another: (ii) The molecules take turns rolling the die and only one molecule moves for each roll of the die. When a molecule moves through the stationary phase, it has dissolved out of the stationary phase and into the mobile phase. In the mobile phase, all molecules travel at the flow rate of the mobile phase. Hence, a third rule: (iii) When any molecule is allowed to move, it moves ahead at the mobile phase flow rate. Let’s set our mobile phase flow rate to four. When a molecule moves, it moves four grid boxes. __5. On a separate sheet of white 8.5 × 11-in. paper, draw and number a scale from zero to fifty using 0.5 cm divisions on the long side of the paper. __6. Roll the die for the leftmost square/molecule; follow the rules in step 4. Roll the die for each molecule from left to right. A single turn is finished after all molecules have had one roll. Keep a tally of the number of turns. When a molecule reaches the end of the grid, remove the square from the grid and determine the number of turns (including the current turn) that it took to move through the column. Record this value on the scale using marks labeled Y = yellow, R = red, and B = blue (see figure). Repeat until all molecules come off the grid. __7. Draw three peaks on the scale, one for each color, over the marks you have made. Draw higher where there are more marks for the chosen color and lower where there are fewer marks (see figure). This is a chromatogram. __8. Repeat steps 5–7, this time using a flow rate of eight. __9. Repeat steps 5–7 now doubling the length of the stationary phase by using the second grid 10 20 30 40 you made in step 1. Tape it to the end of the 0 Y R RRR R R B B B B B B B R R B first grid. Use a flow rate of four.
More Things To Try In high performance liquid chromatography (HPLC), the chemical composition of the mobile phase can be changed any time during a separation. You can change your mobile phase composition by changing rule (i). Try speeding up your separation by changing rule (i) after ∼20 rolls of the die and see what happens!
Questions 1. Was the mixture well separated in your first chromatographic run? How do you know? 2. What was the effect on the chromatogram when the flow rate was doubled? 3. What was the effect on the chromatogram when the length of the stationary phase was doubled?
Information from the World Wide Web (accessed January 2004) Chromatography. http://elchem.kaist.ac.kr/vt/chem-ed/sep/chromato.htm This Classroom Activity may be reproduced for use in the subscriber’s classroom.
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Journal of Chemical Education •
Vol. 81 No. 3 March 2004 •
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