Loren Davis
Wahkiakum Middle School
Cathlamet, WA
Fall 1994
Chromatography is a separations technique that has been widely utilized in the middle school classroom. The procedure is basic, the student would place a small amount of ink on a piece of filter paper and place one end of the paper in water. As the water is wicked through the paper by means of capillary action, the constituent colors of the ink would separate out into bands. The student could observe the different colors of ink used to produce the desired color of the sample. The process by which the colors separate is less clear. The student must first understand that the molecules that make up the different colors of ink have slightly different physical and chemical properties. The differences in these properties will determine how fast the molecules will move across the paper. For example, the molecules that make up red ink might have more of a tendency to adhere to the filter paper, therefore, it would take longer for it to move across the paper. The size of the molecule also has an effect on the movement of the substance.
Electrophoresis, on the other hand, may be a term that is intimidating to some educators unfamiliar with the topic. The techniques involved, however, are simple and can be easily adapted to the middle school age group with only the minimum of supplies. Electrophoresis, put simply, is a tool used to separate particles of matter based on molecular size and ionic charge. The procedure uses an electric field to move solutions through a gel, or other electrophoretic substance. Because molecules of different substances have different sizes and charges, the particles will move across the gel at different speeds. Thus, all of the like molecules will separate from the others forming a band in the gel that will slowly move toward one of the electrodes. Therefore, if more than one electrically charged substance is put on the gel, they will separate into bands of like particles and move across the plate.
At the present time electrophoretic procedures are used in the labs for two main purposes: (1) As an analytical tool to determine the nature or type of proteins or dyes that are in an unknown sample, and (2) to purify substances by separating the unwanted particles from a sample. Efforts are currently under way to upscale the electrophoretic process so that it can be utilized by industry as a purification technique.
Even though these processes seem abstract at first glance, if properly used they can be a tool for motivating students towards science investigations at the middle school level. Labs and other "hands-on" activities are good for student learni~g. "Hands-on" activities, however, are not an "end all" to student motivation and knowledge acquisition. It is true that, by and large, students would rather do activities than "book work". Real motivation comes when the young people can come up with the questions and hypothesis and follow through with testing these guesses themselves. They are also highly motivated when they are given a scenario to study themselves and solve the problem. This separations module is an effort to do just that.
Chromatography and electrophoresis are procedures that students can easily do and be successful at without really understanding what is going on at the molecular level. With careful planning and questioning, the educator can lead the students to make guesses about what is happening, and then guide the class into the procedures to test the these theories. When the students have this "vested interest" in the outcome of the activity, they will be more interested in the completion of the lab that they themselves had a hand in designing. In the activity outline later in this module there are "Key Questions" that will help direct the student toward developing hypotheses that lead to the next activity.
One of the advantages of doing a separations unit is that it is an integrative process. The students will use information and knowledge that they have learned from several different scientific disciplines to do the activities of this unit. Here are some of the basic principles that the student should have an understanding of before this module is presented:
In doing the activities in this unit the students will learn:
The procedures used in chromatography are simple, straight forward, and safe to use. All that is needed are some samples of colors (dyes, food colors, or other colorful samples), toothpicks, filter paper (paper towels if filter paper is not available), and water.
After the students have worked through the setup procedures once, they are ready to start testing some other colored solutions. They can test anything that will make solution, for example they can try drinks, candy and other foods. They will have to figure out how to make the solutions concentrated enough to use, yet this too is a learning process. They will start to see that the certain colors will always be in the same order so they will understand the analytical nature of chromatography.
If the students have troubles getting a solution that will work suggest crushing the sample and then straining it with filter paper. Boiling the solution to concentrate will also work. In this part of the investigation really try to challenge them to try as many different things as they can so they can start to see the patterns that evolve with trial and error.
The procedures involved with doing electrophoresis in labs are a little more difficult for the students to master, as well as having a few more safety concerns. The safety concerns stem mostly from the use of a power supply. The laboratory activities call for a direct current of 100 volts. While the system is running, the students will need to stay clear. Even though 100 volts DC will not seriously injure the students, it is always good practice to keep a "hands off" policy when using a power supply. If you do not have a power supply that will give you 100 V, I have included in the appendices an address where you can borrow one, as well as other equipment.
To set up an electrophoresis station in your room you need three main apparatuses. (1) system to pour the gel on a microscope slide, (2) electrophoresis testing apparatus, and (3) power supply.
1. The gel pouring apparatus. The labs in this unit are designed so that an entire class can be involved. A pouring system that is shown here can keep the gel hot so that the entire class can get a gel slide that they can work with. The apparatus consists of a loaf pan with a hole drilled on the end towards the bottom and a small piece of tygon tubing sealed into the hole with silicon rubber sealant. The gel can be mixed in the loaf pan and set directly on a hot plate to heat. The tygon tubing is closed off with a clamp. When the students are ready to pour the gel on the slide they undo the clamp and pour the gel onto the slide and set it aside to cool and harden. They can reclamp it when they are finished and the gel in the pan will stay hot and ready for the next group.
2. The electrophoresis apparatus. The easiest apparatus to use and set up is the gel and plate method. In the appendices, I will describe a simple column apparatus that you can choose to make and run also. The plate apparatus consists of two wells and a plate that is placed between them. This plate will support the gel slides. The easiest way to set this up is to obtain two containers that are at least 42 cm long. (The width is less important.) The plate that is set between the two wells should be 42 cm long and 8.5 cm wide. This plate can be made out of Plexiglas or glass. The dimensions of the plate are more important because on the plate the gel slides will be placed for the actual electrophoresis to take place. You should also obtain two stainless steel bolts that will attached with alligator clips to the electrodes leading to the power supply.
3. The power supply should be able to provide 100 volts of DC.
Procedures for setting up and running the gels.
Microscopic Slide with gel
Provided are two basic lab sheets, one for chromatography, and one for electrophoresis. These lab sheets provide the students with the necessary set up information for the two types of separations activities. With some direction they will want to explore other separations with solutions derived from food and beverages that they use every day. Flexibility on the part of the instructor is important because as the students explore the different items that they electrophoresed, they will start to discover patterns and trends in the solutions they work with. A complete record of the labs that they do ought to be required so that they can get into the habit of writing everything down.
As the students do the lab they should record the beginning and ending time, as well as the voltage of electricity used. They should use a ruler to measure the distance of each color band from the sample slot and record this information because they can later identify the food dyes used in an unknown sample by the time and the distance data. Molecules of the same substance will always move across the gels at the same rate.
When the studentsı are working with the food dyes they can usually find the names of these artificial colors on the side of the packaging. They should write down the colors and the names as they work with each substance. Encourage them to mix some of their samples together and electrophorese them to compare the rates of different colors. One extension activity would be for the students to plot a distance time graph for each of the color's so the colors velocity across the gel could be more visual.
To evaluate the students comprehension of the procedures the instructor should prepare some unknown samples of color dyes used in the foods that the students tested. The teacher could evaluate the students by requiring them to separate the solution using one of the two separation methods and then identify the names of the colors in the unknown samples.
Electrophoresis is used by scientists and engineers as a way to separate proteins found in living organisms. In the recent O.J. Simpson case, electrophoresis was used to identify some of the proteins that match the same group of individuals of which O.J. is a part. Parts of blood samples can be electrophoresed, and some of them can be purchased and mixed together in the class and separated out using the above techniques. Parts of plants may be ground up and electrophoresed by the students. You may also wish to try staining some proteins and electrophoresing them to find their velocities.
The pH of the buffer solution also effects the rate at which the molecules move across the gel. A controlled experiment could be done with the students in which you do two runs with the same dyes, but by changing the pH of the solution on the second run to see if it speeds up or slows down the color separation. The amount of electricity used also effects the outcome. With caution you could turn up or down the voltage of the system and observe the changes. Be careful when upping the voltage, as you increase the voltage, the heat of the system also increases. If the gel gets too hot it will melt and ruin the run. (You may wish to do this on purpose once to demonstrate it to the students and then question them on why it got too hot. Challenge them to design a system to keep the gel cool while increasing the voltage.)
When you do your separations unit, remember the students are now applying the scientific knowledge that they have been learning over the past few years. Constantly question and remind them what is going on at the molecular level. As they start to have the freedom to do their own electrophoretic and chromatographic experiments some of them will get exited about the results and want to do more. Tell them that these are the types of procedures that are going on in labs all around the country on many different types of molecules. They should realize that the knowledge that they are receiving in class this year, and in other classes in years to come, are important tools to understanding the world in which we live. Reemphasize the different scientific disciplines that where used in understanding these processes and show them how scientific knowledge builds on itself.
Always require them to keep understandable lab records and grade them on their work often, they will appreciate this structure and see the value in organized records. Don't tell them the answers, instead make them write hypotheses and try to have them figure it out for themselves. Only give them the solutions if all else fails to get them on the right track. Above all, be as creative as you can to get the students thinking about separations. This module is just a guideline for getting you started, let the students take it where they can, and have fun along the way.
The students will be evaluated on their lab work. As mentioned earlier, the students will keep accurate records of their hypotheses and lab activities. Their lab notebooks and additional graphs will be collected at least once a week for evaluation by the instructor. The final lab will be with an unknown solution in which they will separate using both chromatography, and electrophoresis. They will evaluate the unknown substance for the types of artificial colors found in it. Finally they will predict it's contents. The instructor should prepare several different unknown solutions so not all of the groups are doing the same thing. The instructor should not only grade the outcome and the student's final prediction, but also how well the student recorded the data and followed the proper procedure.
To observe the contents of colored dyes using chromatographic procedures.
| Filter paper |
| Metric ruler |
| 250 ml beaker |
| Toothpick |
| Test tube |
| Food colors |
To investigate separations of unlike molecules in a solution.
| Electrophoresis Setup |
| Food colors |
| Knox Unflavored Gelatin |
| Microscope Slides |
| Eye Dropper |
| Test tube |
Microscopic Slide with gel
If you do not have access to a power supply you may be able to borrow one through Washington State Universityıs Equipment Loan Program.
Contact:
John Paznokas
Associate Professor and Chair
Program in Biology
Washington State University
Pullman, WA 99164-4235
(509)335-8649
If you would like to do further studies in electrophoresis here is a easy to assemble electrophoresis column you can do for either a independent project for a student or a classroom demonstration.
| A 16 or 20 ounce pop bottle with a plastic lid |
| Clear plastic drinking straw |
| 150 ml beaker |
| Agarose Type VIII gel |
| Buffer solution |
| Power supply |
| Vacuum grease |
| Color sample |
| Glycerol |
