The Effect of Alcohol on Biological Membranes

1. Obtain and wear goggles, an apron, and gloves.

2. Obtain the following materials:

a. Place about 10 mL of methanol in a medium sized test tube. Label this tube M.

b. Place about 10 mL of ethanol in a medium sized test tube. Label this tube E.

c. Place about 10 mL of 1-propanol in a medium sized test tube. Label this tube P.

d. Place about 30 mL of tap water in a small beaker.

3. Prepare five methanol solutions (0%, 10%, 20%, 30% and 40%). Using Beral pipets, add the number of drops of water specified in Table 1 to each of five wells. Use a different Beral pipet to add alcohol to each of five wells in the microwell plate. See Table 1 to determine the number of drops of alcohol to add to each well.

4. Clean the pipet used to transfer alcohol. To do this, wipe the outside clean and empty it of liquid. Draw up a little ethanol into the pipette and use the liquid to rinse the inside of the pipette. Discard the ethanol.

5. Prepare five ethanol solutions. To do so, repeat Step 3, substituting ethanol for methanol. Place each solution in the second row of wells. See Figure 1.

6. Prepare five 1-propanol solutions. To do so, clean your pipette and repeat Step 3, substituting 1-propanol for methanol. Place each solution in the third row of wells. See Figure 1.

7. Now, obtain a piece of beet from your instructor. Cut 15 squares, each 0.5 cm X 0.5 cm X 0.5 cm in size. They should easily fit into a microwell without being wedged in. While cutting the beet, be sure:

• There are no ragged edges.

• No piece has any of the outer skin on it.

• All of the pieces are the same size.

• The pieces do not dry out.

8. Rinse the beet pieces several times using a small amount of water. Immediately drain off the water. This will wash off any pigment released during the cutting process.

9. Set the timer to 10 minutes and begin timing. Use forceps to place a piece of beet into each of 15 wells, as shown in Figure 1. Stir the beet in the alcohol solution once every minute with a toothpick. Be careful not to puncture or damage the beet. While one team member is performing this step, another team member should proceed to Step 10.

10. Connect the Colorimeter to the computer interface and prepare the computer for data collection by opening the file “08 Alcohol and Membranes” from the Biology with Vernier folder of LoggerPro.

11. Prepare a blank by filling a cuvette 3/4 full with distilled water.

12. Calibrate the Colorimeter.

13. After the 10-minute period is complete, remove the beet pieces from the wells. Remove them in the same order that they were placed into the wells.

14. Discard the cuvette contents into your waste beaker. Remove all of the solution from the cuvette. Use a cotton swab to dry the cuvette. Fill the cuvette with the 10% methanol solution from Well 2 using a Beral pipet. Wipe the outside with a tissue and place it in the device (close the lid if using a Colorimeter). Wait for the absorbance value displayed in the meter to stabilize.

15. Repeat Step 14, using the solutions in Wells 3, 4, and 5.

16. In the data table, record the absorbance and concentration data pairs listed in the data table.

17. Repeat Steps 14–16, measuring the five ethanol solutions.

18. Repeat Steps 14–16, measuring the five propanol solutions.

Conclusion

Some trends or patterns that can be seen in Figure 1 are that as alcohol concentration increased, in general so did light absorption, except for the propanol solutions at 30-40%. There was a decrease in light absorption for all alcohol solutions from 20% to 30%.

From figure 1, it can be seen that membranes are most affected when alcohol is at a higher concentration in the solution, as when the solution is more concentrated the integrity of the beet membrane decreases and more of the betacyanin leaks out.

Figure 2: Image showing the hydrophobic and hydrophilic areas of a phospholipid bilayer

The reason that the integrity of the beet membrane decreases is that alcohols are permeable to cell membranes, causing a change in the internal environment of the cell. Osmosis, the movement of water, is highly affected by the introduction of alcohol to a cell. If the cell gets too ‘full’ it will cause the membrane to expand and stretch, or vice versa if too much is released from the cell, the membrane would collapse and no longer maintaining its structure; alcohol disrupts this flow. Alcohol also has the ability to make hydrogen bonds, which affects the organization of water molecules. This then disrupts the interaction between the hydrophilic areas of the plasma membrane and the water molecules which in turn can change the structure of the membranes. Between the disruptions of proper flow of ions and water through the membrane, alcohol creates a very instable environment for the cell. The changes it causes are usually enough to break the membrane open and thus dehydrate the cell.

Alcohol

Structure

Formula

Ethanol

CH3CH2OH

Methanol

CH3OH

Propanol

CH3CH2 CH2OH

Table 5: Table showing information about the different types of alcohol tested

As seen in Table 4, Methanol is the smallest molecule with the least number of hydrogen atoms, followed by Ethanol and then Propanol, which is the largest molecule. Alcohols are able to form hydrogen bonds due to the fact that they have a non-polar end. The longer the alkyl chain becomes the less polar and less water soluble the compound becomes.[1] 1-Propanol is the most non-polar of all of the alcohols that were tested, and it was observed to have the highest effect on the beet root’s membrane. From this information it can be assumed that the lower the polarity the alcohol, the higher the effect that alcohol has on the membrane.

Evaluation

There were many instances and opportunities for human error in this experiment. It is possible that in step 3, someone may have miscounted the number of drops of alcohol or water required in each well. This would have negatively impacted the results, as there would be a different alcohol concentration in some of the wells. It is also possible that the beetroot cubes varied in size slightly. A larger beetroot cube would have a larger surface area and thus have more accessible cells to become dehydrated. Furthermore, it is possible that the beetroot cubes were not thoroughly rinsed to rid them of the betacyanin pigment that was released during the cutting. This would give the solution in the well a higher level of pigment absorbance regardless of the damage done by the alcohol in the solution. Step 9, which requires stirring the beet in the alcohol solution, cautions us to not puncture the beetroot during stirring. If the beetroot was punctured it would also release pigment into the solution that was not caused by alcohol damage to the membranes.

My lab partner and I were responsible for finding the data for the methanol alcohol. We pooled our data with the other groups to obtain the information for the other two alcohols. It is impossible for me to know what errors were made in the other two groups’ data collection as I was not a part of it.

The investigation could be improved if the experimenters were extremely vigilant during the setup process, especially any steps involving the handling of the beetroot to ensure it is not damaged and doesn’t negatively impact the results. Using more types of alcohol would give us a more precise representation of the effect of alcohol on the biological membrane. It is also important to ensure that the Colorimeter is calibrated properly to ensure precise results are given.

References:

[1] Polarity vs. Chain Lenglths in Organic Compounds. Virtual Chembook Elmhurst College. Web. 03.04.12. www.elmhurst.edu/~chm/vchembook/214organicchain.html