| Diffusion | Osmotic Pressure Protocol | Transpiration |

We have seen how DIFFUSION is behind the movement of all molecules and small particles in the universe. We will now proceed on a pathway to cells. In particular we are interested in learning about membranes. So let us consider the effects of placing a simplified membrane between two different solutions and see what happens. Imagine the membrane as a sort of grill or fence with small holes in it. In the first case, the components of the solution are able to fit through the holes. Of course, this will slow down the diffusion of the components through the membrane. Additionally, if water is on both sides of the membrane, the Laws of Thermodynamics dictate that the total system - the solutions on both sides of the membrane - will strive to mix and reach equilibrium (i.e.: to become the same). The process of the diffusion of molecules through such a membrane is called OSMOSIS or DIALYSIS.

A very interesting and ultimately useful case arises in a multicomponent dialysis system when only certain components (including water) can pass through the holes of the membrane, while other, very large components cannot pass through. The system seemingly cannot obey the laws of thermodynamics, but, upon further inspection, it does. How? Well, wait for that answer until we get there further on. We will look at osmotic pressure inside of membranes shaped like sausages, which are closed systems - AND inside that sausage are molecules too large to squeeze through the membrane's holes.

So let's get started! Your instructor will demonstrate to you the mechanics of using dialysis tubing. This is a semi-permeable "cellophane" (cellulose acetate) membrane through which small molecules can pass but larger ones (approx. 10,000 dal and above) are unable to penetrate. This makes for a simplistic model of a cell membrane. (Real cell membranes differ in that their membrane surfaces are covered with charged molecules - usually negatively charged.) "Reverse osmosis" is also used for desalinization of sea water, and regular osmosis is used even with gases as "fuel cells" produce electrical energy. See stocks such as BLDP, FCEL and PLUG.)

DIALYSIS (Can be done in about 45 minutes.)

1. OUTSIDE of the tubing (see figure, below):
   1. Put these INSIDE the dialysis tubing (see figure below):
      1. 40 ml of distilled water
      2. add methylene blue until the solution is rather dark blue
      3. Dissolve in a pinch of NaCl
      4. Approx. 3 to 5 ml of Alpha-Amylase (ginger root aqueous extract); or alternatively one large glob of saliva
      5. Mix in 1/8 cup of polyethylene glycol (which should have a molecular weight of 8,000 or higher). Add this PEG at the very last moment before sealing the tubing (make sure to expel trapped air in the tubing before tying to allow for osmotic expansion.
      6. Thoroughly rinse bag under running water to wash away any exterior dribbled amylase
      7. Tie one end of a 2 ft long string to the knot at one end of the dialysis sausage, and tie a pencil to the other end of the string.

   2. To 375 ml of distilled water in a 500 mL graduated cylinder, add 75 ml of 1% starch solution (also made with distilled water (resulting in a solution that is 0.1% starch); test a very small amount of this for a positive iodine reaction. It should give a dark color.
   3. Place this solution in a liter graduated cylinder, and attach a bubbler to continuously mix the fluids in the cylinder. If excessive foaming results, add a small amount of ethanol.

   

2. Using the string, lower the filled and tied dialysis bag into the cylinder,
3. Either add to or subtract some of the exterior liquid so that the total volume (sausage plus liquid in cylinder) is exactly 500 ml.
4. Insert the bubbler and make sure that the rising bubbles are massaging the side of the dialysis bag. (Note that the pencil prevents the string from slipping into the cylinder.)

5. At timed intervals,* fish out the bag to read and record the volume of the cylinder.

6. At this point while you are not too busy and only taking occasional volume readings, begin reading the next part of today's lab assignment.

7. At the end of osmosis lab, test the fluids both inside and outside the bag for the iodine stain.

8. Data collection:
   • Plot the bag's volume with respect to time.
   • Follow the blue's diffusion from the bag
   • Did the NaCl come through the bag (use silver nitrate, which precipitates with chloride). This is such a sensitive test that distilled water must be used throughout this exercise.
   • Using tincture of iodine, is therch in the "outside"? What does that tell you about the amylase you put inside the sausage?
   • Use Benedict's solution on a small sample of the "outside". Use a glucose positive control. Again, what does this tell you about the interior amylase?

RAMIFICATIONS

Write a short summary of the experiment and discuss what happened and the ramifications.

| Supplies | Osmotic Pressure Protocol | VAST-2001 |