Diffusion and Osmosis
In this laboratory you will investigate the processes of diffusion and osmosis. You will investigate these events through cell models (dialysis tubing) and through living plant cells.
At the completion of this laboratory you should be able to:
* Describe the mechanisms of diffusion and osmosis.
* Describe how solute size and molar concentration affect the process of diffusion through a selectively permeable membrane.
* Describe the effects of a selectively permeable membrane on diffusion and osmosis between
two solutions separated by a membrane.
* Relate osmotic potential to solute concentration and water potential.
* Measure the water potential of a solution in a controlled experiment.
* Describe the effects of water gain or loss in animal and plant cells.
* Experimentally determine the water potential of an unknown solution.
* Calculate the water potential of living plant cells from experimental data
Many aspects of the life of a cell depend on the fact that atoms and molecules are constantly in motion (kinetic energy). This kinetic energy results in molecules bumping to and rebounding off each other and moving in new directions. One result of this molecular motion is the process of diffusion.
Diffusion is the random movement of molecules, atoms, or ions from an area of relative high concentration to an area of relative low concentration. When the concentration between two areas is the same, there is no NET movement of molecules from one area to another and diffusion will appear to stop. In these situations, the two systems are said to be in equilibrium.
Osmosis is a special case of diffusion. Osmosis is the diffusion of water through a selectively permeable membrane (a membrane that allows for diffusion of selected solutes and water). Water potential is the measurement of free energy of water in a solution and indicates the tendency of water to move from one area to another.
Diffusion and osmosis are important processes to life because they can be one of the methods for moving materials in and out of cells. This laboratory will focus on the factors that affect the processes of diffusion and osmosis.
Diffusion: Part A
In this experiment you will measure diffusion of small molecules through a selectively permeable membrane called dialysis tubing. Small solute molecules and water can move freely through a selectively permeable membrane, but larger molecules will pass through more slowly, or not at all. The movement of a solute through a selectively permeable membrane is called dialysis. The size of the minute pores in the dialysis tubing determines which substances can pass through.
A solution of glucose and starch will be placed inside a bag of dialysis tubing. Distilled water will be placed in a beaker outside the dialysis bag. After about 30 minutes, the solution inside the dialysis tubing and the solution in the beaker will be tested for glucose and starch. The presence of glucose will be tested with a special tape. The presence of starch will be tested with Lugol's solution (IKI) which forms a dark color in the presence of starch.
You will be assigned to work in groups. Each group should do the following:
1. Obtain a 15 cm dialysis tubing that has been soaking in water. Cut the tubing into two equal pieces. Save the second piece for use later in the lab. Tie off one end of the tubing with string to form a bag. Open the other end of the bag by rubbing the end between your fingers until the edges separate.
2. Place 10 ml of the 15% glucose/1% starch solution in the bag. Tie off the other end of the
bag, leaving sufficient space for the possible expansion of the contents in the bag. Record the
color of the solution in the bag in Table 1.
3. Test the 15% glucose/1% starch solution with the glucose test tape and record the results in Table 1.
4. Fill a 250 ml beaker or cup two-thirds full with distilled water. Add approximately 2 ml of the IKI solution and record the color of the solution. Test this solution with the glucose test tape and record the results in Table 1.
5. Rinse the dialysis bag with distilled water, then place the bag in the beaker with the IKI/water solution.
6. Allow the experiment to run for approximately 30 minutes or until you see a distinct color change in the bag or in the beaker. Record the final color of the solutions in the bag and the beaker in Table 1.
7. Empty the contents of the bag into a clean beaker and test with the glucose test tape. Record the results in Table 1.
Table 1. Diffusion Observations and Results.
Initial Solution Color Glucose Test Tape
Contents Initial Final Initial Final
15% glucose & 1% starch
H20 + IKI
Analysis of Results
On a separate sheet of paper, answer the following questions:
1. Which substance(s) are entering the bag and which are leaving the bag? Support your answers with experimental evidence.
2. Explain the results you obtained. Include the concentration differences and the membrane
pore size in your discussion.
3. How could this experiment be modified so that quantitative data could be collected to show that water diffused into the dialysis bag?
4. Based on your observations, rank the following by relative size, beginning with the smallest:
glucose, water, IKI, starch, membrane pores
5. What results would you expect if the experiment started with a glucose and IKI solution inside the bag and only starch and water outside? Why? Explain.
6. Why was it necessary to rinse the dialysis bag before placing it in the beaker?
7. If the experiment was repeated using sucrose (a disaccharide) instead of glucose (a monosaccharide), do you think the sugar would have moved through the dialysis tubing? Explain.
8. Why was it necessary to test the solutions with the glucose test tape before and after the solution?
Osmosis: Part B
In this experiment you will use dialysis tubing to investigate the relationship between solute concentration and the movement of water through a selectively permeable membrane by the process of osmosis. The dialysis tubing will create a barrier between two areas or systems. If the concentration of water is not equal between the two areas, water should move from the area of relative high concentration (of water) to the area of relative low concentration.
When the water concentration outside the bag is higher (i.e. has less solutes) than the water concentration inside the bag, the solution outside the bag is said to be hypotonic (hypo = below) relative to the solution in the bag. The solution inside the bag has a higher solute concentration or lower water concentration and is said to be hypertonic (hyper = above). Water can be expected to move from the area of high concentration (of water) to the area of low concentration in these cases. If the water and solute concentrations between the outside and inside are equal, the solutions are said to be isotonic (iso = equal) relative to each other. In this case, no NET water movement will occur.
A laboratory assistant prepared solutions of 0.8M, 0.6M, 0.4M, and 0.2M sucrose but forgot to label them. After realizing the mistake, the assistant randomly labeled the flasks containing these four solutions as flask A, flask B, flask C, and flask D.
Design an experiment, based on the principles of diffusion and osmosis that the assistant could use to determine which of the flasks contained each of the four unknown solutions.
Determining the Water Potential of Potato Cells: Part C
In this exercise you will use cores of potato tissue placed in different molar concentrations of sucrose in order to determine the water potential of potato cells. First, however, we must discuss what is meant by the term "water potential".
In animal cells, movement of water into and out of cells is influenced by the relative concentration of solute on either side of the cell membrane. If water moves out of the cell, the cell will shrink or crenate because the cell volume has been decreased. If water moves into the cell, the cell volume will increase causing swelling or even bursting of the cell. In plants, the cell wall prevents cells from bursting as water enters the cells, but pressure eventually builds up inside the cell and affects the process of osmosis.
In predicting which direction water will diffuse through living plants cells, a quantity know as water potential is used. Water potential is abbreviated by the Greek letter psi (y). Water potential is the chemical potential of water (free energy per mole of water) and has two major components:
y = yp + yr
Osmotic potential = Yp
Pressure potential = yr
Osmotic potential is dependent on solute concentration while Pressure potential results from the exertion of pressure - either positive or negative (tension) - on a solution.
The water potential of pure water in a beaker open to the atmosphere is zero (y = 0) because both the osmotic and pressure potentials are zero. An increase in positive pressure raises the pressure potential (makes yr more positive) and therefore raises water potential The addition of solute to water lowers the osmotic potential (makes yp more negative) and lowers the water potential. Therefore, a solution at atmospheric pressure will always have a negative water potential due to the solute. For instance, a 0.1 M sucrose solution has an osmotic potential of -2.3 bars due to the solute. If the solution is open to the atmosphere (pressure potential = 0), then the water potential is -2.3 bars.
When a solution, such as that inside a potato cell, is separated from pure water by a selectively permeable membrane, water will osmosis from the surrounding area where the water potential is higher, into the cell where the water potential is lower due to the presence of solutes. The movement of water into the cell causes the cell to swell and the cell membrane pushes against the cell wall to produce an internal cell pressure known as turgor pressure.
Eventually, enough positive turgor pressure builds up to oppose the more negative osmotic potential of the cell. This process will continue until the water potential of the cell equals the water potential of the pure water outside the cell. At this point, equilibrium is reached and the NET movement of water will cease.
If solute is added to the water outside the potato cells, the water potential of the solution surrounding the cells will decrease. It is possible to add just enough solute to the water so that the water potential outside the cell is the same as the water potential inside the cell. In this case, there will be no net movement of water. This does not mean, however, that the solute concentrations inside and outside the cell are equal, because the water potential inside the cell results from the combination of both solute concentration and pressure potential.
If enough solute is added to the water outside the cells, water will leave the cells, moving from an area of higher water potential to an area of lower water potential. The loss of water from the cells will cause the cells to lose turgor. A continued loss of water will eventually cause the cell membrane to shrink away from the cell wall, an event known as plasmolysis.
Continue to work in groups. Each group should prepare the following:
1. Pour 100 ml of the assigned liquid into a labeled 250 ml beaker.
2. Use a cork borer to cut four potato cylinders. Cut each cylinder to approximately 3 cm in length. Do not include any skin on the cylinders.
3. Determine and record the initial mass (grams) of the four potato cylinders.
4. Place the cylinders in your beaker and cover with plastic wrap to prevent evaporation. Let stand overnight.
5. Remove the cylinders from the beaker. Blot dry and weigh. Record the final mass.
6. Calculate the % mass change for your group's data. Give the result to the instructor so that a class data set can be obtained.
Note - the % mass change may be positive or negative. Be sure to give the correct sign with your group's result.
Analysis of Results
Graph - Prepare a graph of the class data. Graph the independent variable (sucrose molarity) as the X axis and the dependent variable (% mass change) as the Y axis. The Yaxis will need positive and negative regions because the potato cylinders will have gained mass in some solutions while losing mass in others. Place a "best-fit" line on the graph. Be sure to label the axis and give their units.
Answer the following questions on a separate sheet of paper:
1. Determine the osmolarity of the sucrose solution in which the mass of the potato cylinders
does not change (Y = 0). Drop a line from this point to the X axis. This point represents the sucrose molarity with a water potential that is equal to the potato tissue water potential. What is the concentration of this sucrose solution?
If a computer or graphing calculator is used to calculate the best fit line (or trendline), the
osmolarity of the solution can be found where Y = 0.
2. The osmotic potential of this sucrose solution can be calculated using the following formula:
yp = -iCRT
Where: i = ionization constant (1 for sucrose)
C = osmotic molar concentration (as determined from your graph in question 1).
R = pressure constant (0.0831 liter bars/mole oK)
T = temperature oK (273 + oC of solution)
The units of measure will cancel out giving a final answer in bars.
Example: a 1.0 M sucrose solution at 22o C
yp = -(1)(1.0 M/liter)(0.0831 liter bar/M oK)(295 oK)
= -24.51 bars
What is the osmotic potential of the sucrose solution in which the mass of the potato cylinders does not change? (show your calculations)
3. If a potato cylinder is allowed to dehydrate by sitting in the open air, would the water potential
of the potato cells become more negative or more positive? Explain.
4. If a potato cell has a higher water potential than its surrounding (less negative) and if pressure
is equal to zero, is the cell hypertonic (in terms of solute concentration) or hypotonic to its environment? Explain.
5. Produce departments at grocery stores often spray the fruits and vegetables with water to help maintain their "freshness". Use your knowledge of diffusion and osmosis to explain if this technique will work and why.
6. Plant fertilizers are a combination of various mineral salts. Adding too much fertilizer often causes the plant to wilt (a decrease of turgor pressure). Why? Explain.
7. Your instructor may have had one group use the ? M sucrose solution from the Osmosis experiment as the test solution on a potato sample. If the data is available, what was the M sucrose concentration of the unknown solution? How did the value calculated for the potatoes compare to value calculated in the Osmosis experiment?
Onion Cell Plasmolysis: Part D
Plasmolysis is the shrinking of the cytoplasm of a plant cell in response to diffusion of water out of the cell and into a hypertonic solution (high solute concentration) surrounding the cell. During plasmolysis the cellular membrane pulls away from the cell wall. In the next lab exercise you will examine the details of the effects of highly concentrated solutions on diffusion and cellular contents.
1. Prepare a wet mount of the epidermis of an onion. Observe under 100X magnification.
Sketch and describe the appearance of the cells. Note the distribution of the red pigment.
2. Add two or three drops of 15% NaCl solution to one edge of the cover slip. Draw this salt solution across the slide by touching a piece of paper towel to the fluid under the opposite
edge of the cover slip. Sketch and describe the onion cells. Explain what has happened.
3. Remove the cover slip and flood the onion epidermis with fresh water. Observe under 100X magnification. Describe and explain what happened.