All the chemical reactions in a cellular environment take place in solution. Before a substance can enter a cell it must dissolve. The term solution can refer to the situation in which a substance is dissolved more or less evenly in a liquid. If, for example, the carbohydrate glucose is mixed thoroughly with water, the solid crystals of the substance dissolve and are dispersed throughout the liquid. The resulting mixture - a solution - is composed of the liquid solvent and the dissolved solute. Substances that dissolve are soluble, while those that do not dissolve are insoluble. The digestive process in humans and animals generally involves turning a variety of large compounds into small compounds that are soluble in water and can be carried in and out of cells.

Substances move into and out of cells by several physical processes including:

1. Diffusion, resulting from a concentration gradient (movement of molecules from an area of higher concentration to a lower one),
2. Osmosis, the diffusion of water molecules across a semipermeable membrane; and
3. Filtration, produced by hydrostatic pressure differences.

Within cells numerous minute particles appear to move in an erratic, zigzag motion known as Brownian movement. The movement occurs as small particles suspended within the cytoplasm are bumped about by the molecules of the cell's internal environment.

A cell membrane consists mainly of protein and lipid molecules compactly arranged into a delicate, thin bilayer. Most of the functions are associated with regulating the passage of materials and information into and out of the cells. A membrane that allows molecules to pass through it is called permeable. Since cell membranes allow certain ions and certain molecules to pass through them, but do not allow the passage of others, they are said to be selectively permeable, differentially permeable, or semipermeable.

Movement of dissolved inorganic and organic substances within solutions or through the cell membrane involves a natural physical process call diffusion. Diffusion can be demonstrated easily by placing a small dye crystal (the solute) in a volume of water (the solvent). The solute moves from an area of higher solute concentration to an area of lower concentration.

In osmosis, a type of diffusion, a solven5t (the water in which substances are dissolved) moves from a solution of lower solute concentration to a solution of higher solute concentration when the solutions are separated by a semipermeable membrane.

Filtration is described as the movement of solvents and dissolved substances across a semipermeable membrane under the influence of mechanical pressure. Such movement is always from an area of higher pressure to an area of lower pressure and continues as long as a difference in pressure exists.

The laboratory exercise is designed to demonstrate these various phenomena and processes. (Dialysis, a process involving the separation of small molecules from larger ones by diffusion of the smaller molecules through a semipermeable membrane, will also be utilized.)

1. Brownian Movement

Observe the demonstration set up on the counter. Record in notebook and on worksheet.




2. Filtration

Chemical compounds in solution may behave in one of two ways. The molecules may remain intact, that is, undissociated, or they may dissociate, that is, separate into components called ions. This process is called ionization. Compounds that behave in this manner are known as electrolytes; compounds made up of molecules that do not dissociate are called nonelectrolytes.




Ions carry an electrical charge. For example, when the electrolyte sodium chloride is dissolved in water, it provides positively charged sodium ions (Na⁺) and negatively charged chloride ions (Cl^-).




Both nonelectrolytes and electrolytes are used in this exercise to show how dissolved substances can be forced through physical barriers.




Materials.

1. Per four students
1. One funnel (65 mm diameter)
2. One ring stand and clamp
3. One 50 ml beaker
4. One sheet of filter paper
5. Two test tubes
2. For class use
1. 3% starch solution
2. 3 - 5 % sodium chloride solution
3. Iodine reagent (Lugols solution)
4. Silver nitrate solution 0.1 molar
5. Powdered wood charcoal
6. 10 ml pipette with pipetter
7. Wooden tongue depressor

Methods

1. Measure 2 ml of sodium chloride solution. Pour the solution into a test tube. Add 2 drops of silver nitrate. Observe and record you findings in your notebook and worksheet. This is a test for the presence of chloride ions.
2. Put 3 ml of the starch solution into the other test tube. Add 3 drops of the iodine reagent. Observe and record your findings in your notebook and on the worksheet. This is a test for the presence of starch.
3. Clean both test tubes.
4. Flute one piece of filter paper and place it into the funnel.
5. Set the funnel in the ring stand such that it will drain into the 50 ml beaker.
6. Prepare a mixture for filtering by mixing the following in the small beaker.
1. 4.5 ml starch solution
2. 2 ml sodium chloride solution
3. A small amount of the powdered charcoal
4. 10 ml of water
7. Place a test tube under the funnel and slowly pour 10 ml of water through the system.. Collect the water and discard it. The filter paper should now be firmly in place.
8. Place another test tube temporarily under the funnel, and pour the mixture through the system.
9. Collect the filtrate (the solution that passes through) in a beaker.
10. Divide the filtrate between two tubes. Perform the test for starch with one tube and the test for chloride ions with the other.
11. Record you findings in your notebook and answer the worksheet questions.
3. Diffusion

Materials

1. Per four students
1. One Petri plate with a layer of agar
2. One pair of forceps
3. One millimeter ruler
2. For class use
1. 5 mm filter paper discs
2. 1 % crystal violet solution
3. 1 % methylene blue solution
4. 8 wax pencil
5. Paper towels

Methods

1. Measure the diameter of the filter paper discs and record.
2. Obtain and mark the bottom of a Petri plate with a wax pencil into two sections.
3. Holding a filter paper disc with the forceps, touch it to the surface of the crystal violet solution until the dye penetrates throughout the disc.
4. Remove the Petri plate top and place the disc in the center of one of the marked sectors. Press the disc gently with the forceps so that a good contact is made with the agar surface. Close the plate.
5. Wipe the forceps free of the dye solution. Repeat steps 3 and 4 with the methylene blue solution, placing the disc in the center of the second sector of the Petri plate.
6. Examine the discs on the plate at the beginning of the experiment (0 time) and at 15, 30, 45, and 60 minutes. Measure the diameters of the developing diffusion zones.
7. Enter your findings in your notebook and on the worksheet.
4. Osmosis

The membrane used in this portion of the exercise is a piece of synthetic material often used as a substitute for a true biological membrane. Such synthetic materials contains pores or holes that are approximately the same size as those in biologic membranes but more uniform. The membranes you are using are actually a tube.

Materials

1. Per four students
1. One 5 ml pipette
2. 15 cm length of dialysis tube
3. One rubber band
4. One 250 ml beaker
5. One 500 ml beaker
6. One ring stand and clamp
2. For class use
1. Scales
2. 1 lbs box of sucrose
3. Tape
4. String

Methods

1. Each group of eight students will create 100 ml of the assigned sucrose solution.
• Group 1 - 1 % - 1 gm sucrose
• Group 2 - 10 % - 10 gm sucrose
• Group 3 - 20 % - 20 gm sucrose
• Group 4 - 40 % - 40 gm sucrose
• add enough methylene blue to color the solution.
2. Construct an osmometer system.
   1. Open the dialysis tubing
   2. Tie an overhand knot in one end of the tube.
   3. Place 50 ml of the assigned concentration solution into the dialysis tube.
   4. Place the end of a 5 ml pipette into the tube and secure with a rubber band; the solution should just reach the markings at the bottom of the pipette.
3. Fill the 500 ml beaker with water and place it under the ring stand and clamp.
4. Clamp the pipette in the clamp and the dialysis tube in the beaker.
5. Allow five minutes for the system to equilibrate and then locate the top level of the solution and record it.
6. Read the values at 15, 30, 45, 60, and 90 minutes, record, and plot the data.
7. Determine the diffusion rate of your solution, ml/min, record and write on the board for the rest of class.

This page last updated: 2/10/01