LABORATORY # 6: DIFFUSION & OSMOSIS
Required Material - Diffusion
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Petri dish
Millimeter ruler
Potassium permanganate crystals
Sodium hydroxide in dropper bottle
Agar in petri dish with basic indicator
0.15 mg/ml potassium permanganate
solution
Thermometer
Dialysis tubing & string
Grease pencil
Stirring rods
5 ml pipettes & pipet bulbs
Spectrophotometer
Scissors
14. Test tubes and test tube racks
15. 2x250ml beakers
Required Materials – Osmosis
1. Osmometer
2. Potatoes
3. Sucrose solutions : 0.0M, 0.4M, 0.6M,
0.8M and 1.0M
4. Beam balance
5. Cork borer
6. Scalpel
7. 5x250ml flasks
8. 10% salt solution
9. Normal saline
10. Elodea
11. 50ml graduated cylinders
Introduction
Diffusion is a random molecular movement which has a net flow of molecules from an area of greater
concentration to an area of lower concentration. This net movement will continue passively until
equilibrium is reached (i.e. the molecules are evenly distributed between the two areas). Diffusion can
occur in gases, liquids and even solids. And it occurs across living membranes
The rate at which diffusion reaches equilibrium varies with certain factors. The three most important
factors are: concentration gradient, temperature and molecular weight of the solute that is diffusing.
The steeper the concentration gradient, the higher the temperature and the smaller the solute – the
faster diffusion will occur.
This lab will study the diffusion of solutes. In the first experiment, you will study the diffusion of a solute
in water. Diffusion to equilibrium will eventually result in a homogenous solution. A solution is
comprised of small particles, usually less than 1 nm. In the second experiment, you will also study
diffusion within a colloid. A colloid is comprised of larger particles , usually between 1 and 100nm in
diameter. The large particles in this case will be agar suspended in water. You will calculate the diffusion
rate in both of these experiments and compare them.
Like diffusion, osmosis can also occur across living membranes. But it is the movement of water across a
membrane from high water concentration to low water concentration - or, in other words, the
movement of water from low solute concentration to high solute concentration. Osmosis is an
important factor when considering cells. Taking the human body as an example, the majority of water in
your body is found in the cytosol (i.e. intracellular fluid). Although water also makes up the majority of
water found in tissues (i.e. interstitial fluid) and in your extracellular fluids, like blood plasma. Therefore,
osmosis can occur across the plasma membrane between intracellular and extracellular fluids.
Hypertonic and hypotonic are terms we use to describe the solute concentration of a solution in
comparison to another. Hypertonic solutions have higher solute concentrations in relation another,
while hypotonic solutions have lower solute concentration. Isotonic solutions have the same solute
concentrations.
Solute concentration results in the movement of water across a membrane, or osmosis. This driving
force is known as tonicity. To determine whether water will move, one has to consider the
concentration of solutes between the two solutions – i.e. the number of osmotically active particles.
When dissolving NaCl in water, it produces two osmotically active particles – Na+ and Cl-. The
concentration of each of these particles can influence the movement of water. If you were to measure
the driving force of water movement that is created by these osmotically active particles, you would be
measuring Osmotic Pressure. In other words, if you wanted to place pressure on a solution to prevent
water from moving into the solution, the pressure you would be exerting would be called osmotic
pressure.
Osmotic pressure can be measured and is expressed with the units Osmoles (OsM). If you dissolve 1
mole of NaCl in 1 liter of water, the osmotic pressure would be 2 OsM. One also should remember that
when dissolving a substance in water, it may dissociate into more than two osmotically active particles.
And some complex molecules held together by strong, covalent bonds may only dissociate as one active
particle. The sugar glucose is a good example of this.
The general rule is, the more osmotically active particles in water (i.e. the higher the solute
concentration in the water), the greater the osmotic pressure. The less the number of active particles,
the lower the osmotic pressure. Another way of stating it is this - increased osmotic pressure can be
found in hypertonic solutions, while decreased osmotic pressure can be measured in hypotonic
solutions. In the human body, when you increase osmotic pressure, it “attracts” water to that area to
lower back down into the normal range. When you decrease osmotic pressure, water will flow away in
order to elevate it.
Procedure – Diffusion
I.
Diffusion in a solution: Observe and measure the diffusion of potassium permanganate in
water.
Place one large crystal in a petri dish full of room temperature water. BE CAREFUL NOT TO BUMP
THE DISH!! Wait for 5 minutes and carefully measure the radius of the spreading violet color cloud.
Calculate in millimeters per second the diffusion rate. If you assume that a cell is 100 microns (um)
in length and is composed of a water solution, how quickly would the potassium permanganate
move across the cell?
II.
Diffusion in a colloid: Observe and measure the diffusion of NaOH in a gel colloid.
Place one drop of NaOH in the center of a petri dish containing agar and a basic pH indicator. As the
NaOH diffuses through the agar (the colloid), the indicator will change color and show the amount
of diffusion. Wait 20 minutes and then measure the radius of the spreading color. Calculate the
diffusion rate as you did in experiment I. Compare the two rates. Which solution allowed for the
faster diffusion? Why? How long would the NaOH take to spread across a cell of 100 nm? Of 100
microns (um)?
III.
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Diffusion across a membrane at two different temperatures: The rate of diffusion is
dependent upon the number of collisions occurring between molecules over a period of
time. As the number of collisions increase, so does diffusion. If increasing temperature
increases the number of collisions, it will have an effect on diffusion rate. Observe and
determine the influence of temperature on diffusion rate.
Label two 250ml beakers with grease pencil. Place 150ml of tap water in each beaker.
Place one beaker in a cold bath at 0°C and keep the other at room temperature. Use your
thermometer to verify their temperatures before proceeding with the next step.
Obtain a sample of dialysis tubing and prepare two 13 cm ‘bags’. Dialysis tubing consists of
minute pores that allow the diffusion of molecules of a specific size. To prepare the tubing, wet
it in water, twist one end and tie it with string – securely, to prevent leaking. Fill both bags with
8.0ml of 0.15 mg/ml potassium permanganate and tie off the open end with more string.
Place one bag into each beaker and record the time.
At test intervals of 5, 10, 15, 20 and 40 minutes, gently stir the contents of the beakers (do not
hit the dialysis bags!). Remove 5.0ml of liquid with a pipette and pipette bulb. Place the liquid in
a test tube and measure its absorbance using the spec, set to 545nm. Be sure to use the same
procedures you learned in you lab on the spec! Return the 5.0mL to the correct beaker after
reading. Record the time and absorbance for each sample in your notebook.
Prepare a 2-fold sample serial dilution using the 0.15mg/ml potassium permanganate standard.
Prepare a total of 7 test tubes using the method you learned in the spec lab. Determine the
absorbance for each dilution and construct a standard curve.
Using the standard curve, determine the concentration of each of your test samples. Prepare a
second table, recording the time interval and the concentration of each sample.
Construct a graph, using two different colors, to plot the concentration versus time – one color
for each temperature.
Answer the following questions. In which environment was the rate of diffusion faster? Why?
For each temperature, was the diffusion rate greatest at the beginning or the end? Why? What
does the slope of your curve represent?
Procedure – Osmosis
NOTE: Due to time constraint, begin experiment #2 first.
1. Osmotic pressure: In this experiment, you will use an osmometer to measure the osmotic
pressure of a solution. An osmometer is a thistle tube with an expanded base, filled with a
hypertonic sugar solution and covered by a membrane, impermeable to all solutes but still
permeable to water. Water flowing in or out of the thistle due to differences in osmotic
pressure between the sugar solution and the surrounding experimental solution will cause a
change in the water mark.
Place the base of your osmometer into a container of pure water. Note your water level in your
notebook. Observe the thistle every 20 minutes after the water level is marked. Record the changes in
your lab notebook over the next few minutes. Which way did the water flow? Why? What is the osmotic
pressure of water?
2. Water potential and turgor pressure: Water potential is used by plant physiologists to discuss
water movement in plant cells. Unlike osmotic pressure, which is a function of solute/osmotic
concentration, water potential is a function of osmotic concentration and internal hydrostatic
pressure. Since plant cells have a strong cell wall, they can build up internal pressure without
bursting. This internal pressure is known as turgor pressure. When the turgor pressure plus
water potential in a plant cell is below the osmotic potential of the surrounding solution, there is
net water into the cell. When turgor pressure and water potential is equal to the osmotic
potential of the surrounding solutions, water movement into the cell is equal to water
movement out of the cell.
1. Cut 20 sections from a potato using the supplied cork borer
2. Cut each section into 40 mm lengths
3. Divide the sections into 5 groups of four pieces each and weigh each group, recording its
group
4. Place each group into a separate flask
5. Place 30ml each of 0.0M, 0.4M, 0.6M, 0.8M and 1.0M sucrose solution into the five flasks
and label them with a grease pencil
6. After 2 hours, remove the groups and reweigh. Record the new weight and determine the
change in weight in your lab notebook
7. Graph the weight change for each group against the molar concentration of sucrose.
What sucrose solution results in no water movement in or out of the potato pieces. Record and discuss
this in your notebook.
3. Responses of living cells to variations in water: Animal and plant cells respond differently to
water variations. Animal cells – which lack a cell wall – will either shrink (in hypertonic solutions
– known as crenation) or burst (in hypotonic solutions – known as lysis). Plant cells – which
have a cell wall – will shrink into a small section of the cell wall cavity in hypertonic solutions or
expand tightly against the wall in hypotonic solutions. When the protoplasm of a plant cell
shrinks, it is known as plasmolysis.
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Remove one leaf of Elodea and pat it dry with a paper towel.
Prepare a wet mount using 10% NaCl solution
Prepare another wet mount using normal saline
Observe and draw what you see. Compare the two cells and explain what has happened