Laboratory # 6: Diffusion

LABORATORY # 6: DIFFUSION

Required Material - Diffusion

1. Petri dish

2. Millimeter ruler

3. Potassium permanganate crystals

4. Sodium hydroxide in dropper bottle

5. Agar in petri dish with basic indicator

6. 1.5 mg/ml potassium permanganate solution

7. Thermometer

8. Dialysis tubing & string

9. Grease pencil

10. Stirring rods

11. 5 ml pipettes & pipet bulbs

12. Cuvettes & Spectrophotometer

13. Scissors

14. Test tubes and test tube racks

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.

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. Wait for 5 minutes and measure the radius of the spreading violet color cloud. Calculate in millimeters per second the diffusion rate. If you assume that a cell is 100um 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 100um?

III. 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.

1. Label two 250 mL beakers with grease pencil. Place 150 mL of tap water in each beaker.

2. Place one beaker in a cold bath of ice and water (i.e. at 0°C) and keep the other at room temperature (i.e. on your lab bench). Use your thermometer to verify their temperatures before proceeding with the next step.

3. 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 8.0 mL of 1.5 mg/ml potassium permanganate and tie off the open end with more string.

4. Place one bag into each beaker and record the time as time 0.

5. 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 5.0 mL 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 5.0 mL to the correct beaker after reading. Record the time and absorbance for each sample in your notebook.

6. Construct a graph, using two different colors, to plot the absorbance versus time for each temperature.

7. Prepare a 3-fold sample serial dilution using 1.5 mg/ml potassium permanganate standard. Prepare a total of 5 test tubes using the method you learned in the spec lab. Determine the absorbance for each dilution and construct a standard curve. 8. Using the standard curve and your absorbance values, determine the concentration of each of your test samples. Prepare a second table, recording the time interval and the concentration of each sample.

9. 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?