Color and Acid Diffusion

Developed by: Eric Burtson
© American Association of Immunologists 1995

Background
Immunologists study the immune system of animals and humans to try to figure out how the body defends itself against disease. Most diseases are caused by viruses or bacteria that infect the body. Any time the body recognizes a foreign protein like those found in the viruses or bacteria, it defends itself by producing antibodies and T-cells that help destroy the invaders and eliminate the proteins.

Before immunologists test an antibody, they need to purify it. In one common procedure, the purified antibody floats in a solution of sodium borate. This chemical solution must be replaced by a phosphate solution before the scientist may proceed with the next step of the test. They do this by a process called dialysis.

Dialysis relies on the principle of diffusion, which is the tendency of dissolved chemicals to migrate through a solution until equal amounts of the chemical can be found throughout the solution. It's kind of like what happens when a movie theater finally lets a crowd of people into the theater. Chairs are already spread equally throughout the room. As people enter, they spread out among the chairs and uniformly distribute themselves throughout the theater.

When immunologists replace the sodium borate with the phosphate solution, they put the antibodies in a small container and put the container in a large volume of the phosphate solution. The antibody and sodium borate solution is separated from the other solution by a semipermeable membrane covering one end of the small container. The membrane has tiny holes that are big enough for the water and chemicals to diffuse through, but small enough to keep the antibodies in the container. If everything is left to diffuse overnight, the sodium borate and phosphate will be spread equally both inside and outside the small container. But since the container is much smaller than the large volume of solution around it, most of the unwanted sodium borate will have left the container. If the scientist wants an extremely small amount of sodium borate in the antibody container, he or she can put the container in another large volume of phosphate solution and do the dialysis again.

In order to guarantee that the antibody doesn't diffuse through the membrane, but the chemicals do, the immunologist must select a membrane that has the right sized holes. So, when they shop for membranes immunologists look for the appropriate MWCO or molecular weight cut-off. For example, a particular antibody may have a molecular weight of 150,000 Daltons. Since sodium borate has a molecular weight of only 400, a good MWCO for the membrane would be 1000, or any number that is somewhat lower than 150,000 and somewhat greater than 400.

In this lab, we will do a similar dialysis with two chemical dyes, water, and an acid. The chemicals are alizarin yellow R and bromthymol blue. They are selected for three reasons:

  1. They have different molecular weights so that they may be separated by dialysis;
  2. They are different colors, which will allow you to see what happens to them; and
  3. They are both indicators; they change color in the presence of the acid.

Procedure - Day One

  1. Answer Processing the Data question number one now.
  2. Set up three tests tubes on a rack.
  3. To your small graduated cylinder add two drops of alizarin yellow. Use your wash bottle to bring the total volume to 1.0 ml. Pour the solution into one of the test tubes. Rinse the graduated cylinder out with lots of tap water followed by a small amount of distilled water.
  4. Repeat step three using bromthymol blue instead.
  5. For the third test tube, add two drops of each dye. Again, bring the total volume to 1.0 ml. Record the colors of each tube.
  6. Add two drops of hydrochloric acid to each tube. Record your observations. Dump the solutions in the waste beaker and clean your test tubes.
  7. Obtain about a 15 cm length of dialysis tubing. Prepare it by rinsing and soaking it in a small beaker of distilled water for one minute.
  8. In a test tube, mix 20 drops of each dye.
  9. Measure 20.0 ml of distilled water and pour it into a 100 ml beaker.
  10. Carefully tie a knot in one end of the tubing . To tighten the knot, grip the knot and the short end of the tube. You do not want to stretch the membrane.
  11. Use a pipette to transfer the dye mix into the tube. Tie a knot into the other end, as above. Hold one end of the bag with your tweezers as you rinse the bag with your wash bottle.
  12. Put the dialysis bag into the water bath and allow it to sit overnight. Label your beaker.

Hypothesis 1: What do you expect to happen by tomorrow, after the dialysis?

Procedure - Day Two

  1. Record your observations. Use your tweezers to lift the bag.

Hypothesis 2: What do you expect to happen after you add acid to the bath?

  1. Put on your goggles. Measure 0.7 ml of 1.0 M HCl and add it to the bath. Record your observations.
  2. Use your glass rod to occasionally and gently stir the beaker contents. Observe for about ten more minutes and record your observations. Dump the beaker contents in the beaker labeled "Waste Acid and Dyes."

Processing the Data

  1. Calculate the molecular weights of all chemicals involved: alizarin yellow R C13H9N3O5, bromthymol blue C27H28Br2O5S, hydrochloric acid HCl, and water H2O.
  2. What diffused across the membrane?
  3. Give a possible MWCO for the membrane you used in the experiment.
  4. What diffuses quicker, the dye or the acid? Why do you think?
  5. Even though companies advertise their membranes with specific MWCO's, they cannot guarantee that the membranes will not allow some larger molecular weight chemicals to pass slowly through the membranes. What could you do to demonstrate that molecules bigger than the cut-off size may migrate through the membrane?
  6. Propose one interesting use for semipermeable membranes.

Teacher Notes

Chemistry Concept: Molecular weight

  1. Alizarin yellow R does not store indefinitely, so you may have to prepare the solution from powder. It is 0.1% by in water. Bromthymol blue is 0.04% in water.
  2. Use 1.0 M HCl in dropper bottles.
  3. The full response to question two, "What diffused. . ..?" is "Alizarin Yellow, water, and acid." 
  4. Use 500 MWCO membrane. (Question 3)
  5. The acid diffuses in about seven minutes, while we wait for the dye overnight. (Question 4)
  6. If you have to scrimp on dialysis tubing, it will be difficult for students to tie knots. You can compensate by doubling the ends and tying them with thread.