In the Classroom
Simulating Chromatographic Separations in the Classroom Charles A. Smith* and F. Warren Villaescusa Department of Chemistry, Our Lady of the Lake University, San Antonio, TX 78207; *
[email protected] Several articles in this Journal have dealt with simulations of chromatographic separations; however, all utilize computers or spreadsheet programs (1–5). A common difficulty for students studying chromatography is understanding that, upon elution, all analytes have spent the same quantity of time in the mobile phase. Also, students have difficulty visualizing why peak shapes sharpen with faster flow rates or shorter column length. As instructors, we can tell them that this broadening, or lack thereof, is due to longitudinal diffusion; however, it is a totally different experience for students to discover or observe these facts for themselves. This chromatographic simulation allows students to control chromatographic parameters and be an active participant in a separation process. Method
Classroom Preparation Locate a classroom with a tiled floor and a chalkboard that runs across one side of the room. If such a room is not available, the chalkboard may be replaced by taping paper to a wall and the tiles may be replaced by marking a grid on the floor with masking tape. Move all the desks to one end of the classroom so that there is a rectangular open space from one end of the room to the other. Count the number of tiles that lie across the width of the open space. Adjust the width of the open space such that there is one tile per student in the class. For example, if you have fifteen students, then your open space should be fifteen tiles wide. The data from the simulation will be recorded on the chalkboard using meter sticks as a scale. Place the meter sticks in the tray of the chalkboard. Line the meter sticks end-to-end in increasing fashion with the metric side facing out (i.e., 0 to 100 cm from left to right for each meter stick). Draw a long horizontal arrow on the chalkboard beginning directly above the left end of the left-most meter stick and ending at the right end of the right-most meter stick. Below the arrow place the label “time (min)”. On the left end of the arrow make a vertical mark for the zero position and label it as such. Be sure the zero position is directly above the 0-cm mark on the leftmost meter stick. Student Preparation Before performing this exercise, students should be familiar with some fundamental chromatographic concepts, including flow rate, longitudinal and eddy diffusion, mobile and stationary phases, and analyte affinity for the mobile and stationary phases. Students are told that they are the sample consisting of compounds A, B, and C where compound B has the highest affinity for the stationary phase and compound C is an unretained species. One student is chosen to be compound C and a piece of tape labeled C is placed on the student’s shirt. Half the class is given pieces of tape labeled A and the other half of the class is given pieces of tape
labeled B. The students place their labels on their shirts. Each student is provided with a die and a flap cut from a cardboard box. The cardboard flap provides a surface for the die to be rolled upon. Students are also told that the open space before them is the column. The ‘injection’ on the column consists of the students forming a straight line across the width of the column top. Each student stands on a different tile. Remind the students that they are a homogenous sample so they should line themselves in a random order across the top of the column. Ask the students to develop rules to simulate a chromatographic separation. After some discussion and guidance from the instructor, the class should develop the following rules: Rule 1. Since compound B has the highest affinity for the stationary phase, species B will have a lower probability of entering the mobile phase and moving through the column. For example, species A can move through the column when a 1, 2, 3, or 4 is rolled and species B can only move through the column when a 5 or 6 is rolled. Rule 2. To simulate flow rate, everyone rolls his or her die at the same time. Also, for those allowed to move through the column, all moving species move ahead at the same rate. For example, after the roll of the die, all species allowed to move, move ahead the same number of tiles. Usually students choose an initial flow rate of one tile per roll of the die. Rule 3. Each molecule of a species is independent and moves independently of the other species present; hence, each person rolls his or her own die and moves accordingly. Rule 4. The unretained species is always moving and never has to stop regardless of what is thrown on its die. However, the unretained species travels at the same rate through the column as the other species. Also, the unretained species cannot travel through obstructions.
To enhance the effect of the simulation, chromatograms of each separation are recorded for comparison purposes. This allows the students to perform the chromatography with the goal of optimizing resolution and minimizing dead time in the separation. Chromatograms of all the separations are recorded on the chalkboard one after the other without erasing. The instructor tells the students when to roll the die and keeps track of the number of rolls. When a student reaches the end of the column, the instructor tells the student how many rolls occurred and the student records his or her label on the line on the chalkboard. The student’s label is placed above the corresponding mark on the meter stick. For example, assume a scale of 1 roll = 1 min = 1 cm is chosen. If a student with label A comes off the column after 25 rolls, then he or she places a mark with the label A on the time line just above the 25-cm mark on the meter stick that corresponds
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In the Classroom
to 25 minutes. After all students have come off the column, the instructor draws a general chromatogram on the chalkboard over the student marks. The instructor draws more intense peaks where there are higher concentrations of student marks and less intense peaks in places where there are fewer marks. The instructor may draw separate peaks for species A, B, and C. The purpose of sketching the chromatograms is to allow students to easily observe the effects of modifying the chromatographic parameters in the different experiments. However, it should be noted to the students that the actual chromatogram would appear as a sum of the individual peaks.
Performing Experiments with the Simulation Before each experiment begins, students form a single line across the top of the column with each student standing on a different tile. The instructor tells the students when to roll their die and the students follow the rules that they have developed. When students move through the column they only travel forward toward the end of the column. When students reach the end of the column they record their position on the line drawn on the chalkboard using the meter sticks as a scale. Typical results for the initial experiment following the rules described earlier are shown in Figure 1. After discussion of the effects of longitudinal diffusion, students devise two ways to minimize this type of diffusion: shorter column length and increased flow rate. Allow the students to decide how to perform these experiments and ask them what the effect should be on the chromatogram in comparison to the chromatogram obtained from the first experiment. The shorter column simulation may be performed by repeating the first experiment but shortening the total distance the students move to reach the end of the column. Decreasing the distance traveled through the column by a factor of two will dramatically illustrate the effect of a shorter column length. An increased flow rate may be simulated by repeating the first experiment but with a modified Rule 2. Rule 2 should be modified such that students move more than one tile when moving through the column. In Rule 2, an increase from 1 to 6 tiles per movement will strongly illustrate the effect of increased flow rate on the chromatogram. Typical chromatograms of shortened column length and increased flow rate are illustrated in Figures 2 and 3, respectively. Eddy diffusion can be discussed next. Students are asked how they can simulate this type of diffusion. Students typically place books, backpacks, or any other obstruction in random locations in the column. Discuss with the students whether they can move diagonally around these objects or not. Can more than one student occupy a tile? Usually a textbook is about the size of a single tile; hence, it requires the same number of moves to go diagonally around the textbook as it does if the textbook were not there. Thus, students usually decide that diagonal moves are not allowed to go around an obstruction. Also, it seems that chromatograms for these experiments are best if you allow more than one student to occupy the same tile during the separation. A typical chromatogram obtained with obstructions placed in the column is depicted in Figure 4. After performing the eddy diffusion experiments, students are asked to devise a gradient separation to further improve the chromatograms drawn on the chalkboard. Their
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C
A A A
A
B BB
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Figure 1. A typical chromatogram for Experiment 1 is obtained by using the four rules specified in the text. The peaks drawn by the instructor are shown with dashed lines.
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CA AA B B B A B
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Figure 2. Typical chromatogram obtained by repeating Experiment 1 but with a column length decreased by a factor of two. In comparison to Experiment 1, faster retention times and narrower peaks are observed with the shorter column.
0C
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Figure 3. Typical chromatogram obtained by repeating Experiment 1 but with a flow rate increased by a factor of six. Comparison with Experiment 1 illustrates faster retention times and unresolved peaks.
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Figure 4. Typical chromatogram obtained by repeating Experiment 1 but with obstructions placed in the column. Comparison with Experiment 1 illustrates longer retention times and wider peaks.
goal is not only to achieve baseline resolution but also to minimize the time between resolved peaks (i.e., to reduce the excess resolution). Students first plan how the flow rate of the mobile phase will evolve with time. For example, with the original rules used to describe the initial parameters of the run, students may decide that after 3 minutes (i.e., 3 rolls of the die) the flow rate changes from 1 to 6 and after 25 min-
Journal of Chemical Education • Vol. 80 No. 9 September 2003 • JChemEd.chem.wisc.edu
In the Classroom B Affinity Ratio
Flow Rate
A 6
1 3
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2:1 25
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Figure 5. Gradient changes in the (A) flow rate and (B) affinity ratio (i.e., likelihood of being in the mobile phase/stationary phase) were chosen by the students to improve the chromatogram obtained in Figure 1.
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Figure 6. Typical gradient chromatogram obtained by initially using parameters of Experiment 1 and then following the gradients described in Figure 5.
Typical flow rate and solvent composition or temperature plots determined by students are illustrated in Figure 5. The flow rate described in Rule 2 changes from 6 to 1 after three rolls of the die (i.e., 3 minutes). The affinity for the mobile phase is greatly increased after 25 rolls of the die. For the first 25 rolls Rule 2 is followed and species B only moves forward in the column when a 5 or 6 is rolled. However, after 25 rolls of the die, species B moves forward in the column whenever 2, 3, 4, 5, or 6 is rolled. The resulting chromatogram is illustrated in Figure 6. Discussion This simulation has proven to be an effective classroom tool in illustrating to students the effects of flow rate, column dimension, and mobile兾stationary phase affinity on chromatographic separations. It may also be used to illustrate the differences and operations of isocratic and gradient (or isothermal and temperature programmed) chromatographic separations. Modifications to the described simulation may include the use of more than one die per student. This would allow a much greater range of affinities to be illustrated. Another possible modification could be the placement of tape on one side of the die that reads “go back one tile” to illustrate that longitudinal diffusion also opposes flow through the column. Literature Cited
utes the solvent composition or temperature (i.e., Rule 1) changes to allow compound B to come off the column faster. Have students sketch solvent composition or temperature and flow rate plots on the chalkboard to aid in following the gradient they have selected before performing the simulation.
1. 2. 3. 4. 5.
Haigh, J.; Lord, J. R. J. Chem. Educ. 2000, 77, 1528. Armitage, D. B. J. Chem. Educ. 1999, 76, 287. Sundheim, B. R. J. Chem. Educ. 1992, 69, 1003. Rittenhouse, R. C. J. Chem. Educ. 1988, 65, 1050. Ghosh, A.; Morison, D. S.; Anderegg, R. J. J. Chem. Educ. 1988, 65, A154.
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