The Effect of Detergent Concentration on Membrane Permeability of Cells

Introduction:

There is often a rule in cooking that says; do not rip off the skin of the beetroot or wash the tail of the beetroot when you cook*1, unless you want to dye the cooking water red. This red pigment that comes out of the beetroot is caused by a substance called betalaine, which is located within the tonoplast, the membrane of plant cells which usually contains liquid, of the beetroot cell. Normally, these pigments do not leak out of the cell because they are surrounded by the cell’s plasma membrane, which is a phospholipid bilayer. Since these membranes are soluble in water, not in liquid, they do not leak out, unless the cell’s plasma membrane is somehow disrupted or damaged. If a beetroot cell leaks out betacyanin, the cell is presumably dead because its membrane and vacuole are damaged.

The plasma membrane of a cell is made up of a phospholipid bilayer. These layers are composed of hydrophilic heads that attract water and the hydrophobic tails repel the water. The hydrophilic heads are always on the outside of the cell and the hydrophobic tails are always on the inside. This membrane controls the transportation of particles into and out of the cell. With this arrangement of membrane in a beetroot cell, the membrane prevents the pigments from leaking off of the cell because they are not soluble.

Question:

What is the effect of detergent on organic membrane?

Aim:

To be able to use the colorimeter to measure the amount of absorbance and transmittance of the betalaine pigment in the beetroot and find out the effect of different concentration of detergent on the cell.

Hypothesis:

Null Hypothesis:

1. If the percent increment of detergent increases when applied on the beetroot, the light absorption rate from the colorimeter will remain the same because there is no relationship between the concentration of detergent to the absorption rate

Alternative Hypothesis:

1. If the percent increment of detergent increases when applied on the beetroot, the light absorption rate from the colorimeter will increase because the increase in detergent concentration will cause more damage to the plasma membrane of the beet root. This will cause the membrane to be less permeable, thus, allowing more betalaine pigments to be released.

Variables:

Variables

How measured or determined?

Independent Variable

Concentration of Detergent

Given

Dependent Variable

Absorption Rate

From readings of the colorimeter

Controlled Variables (at least 3)

Why is it controlled?

How is it controlled?

The amount of light(photons) in the colorimeter

In order to make the experiment a fair test, only one variable must be changed at a time. The amount of light must not be changed to maintain the percent transmittance of each solution. The readings would not be accurate and reliable if the absorption rate and percent transmittance are changed for every solution.

There is an option to change the amount of light in the colorimeter

Volume of Solution

To observe the effects of different increment. If the volume of the solution is different, the concentration of betacyanin in the sample solution may change (Concentration = Amount of Solute/Volume of Solvent)

Measure the amount of solution with a graduated cylinder before transferring them into the wells.

Size of beet root(amount of betalaine)

To make sure that there is equal amount of pigment available in each well. If a beet root is bigger in a certain well, that well may have more betacyanin dissolved into the solution, changing the absorption rate and percent transmittance

Only one person will do the cutting because every person has different skills. Only one ruler will be used to avoid any misreading.

Duration

There should be a fair amount of time for the membrane to be penetrated. If a sample cell is left in the solution longer than other cells, the amount of betacyanin leaked from the tonoplast may be different because there is more time for the plasma membrane to be penetrated

Every sample beetroot pieces stay in the solution for an equal amount of time; 10 minutes. Only one person does the timing to avoid difference in reaction times between humans.

Dropping Pipette

Observation:Qualitative –

Cutting Knife

Distilled Water

Microwell

Detergent Solutions

Toothpicks

Graduated cylinder

In the experiment, we were given 5 types of solutions, as depicted in the picture above, distilled water, and 4 solutions with different concentration of detergent; 4%, 8%, 12% and 16%. When we measured and placed each solution into the microwell, we noticed that the detergent that was settled in the solution were diffusing, thus, causing the solution to be cloudy, and not as transparent as the distilled water. Therefore, we placed an extra row of the original solution into the top row of the microwell to compare and find the difference in absorbance that the translucency of the detergent causes.

Trial 3

Trial 2

Trial 1

Solutions without Beet

16%%

12%%

4%%

8%%

0.125 cm3 beetroot

0%

Then, we cut the beet into 15 similar pieces, each with a size of 0.5 cm X 0.5 cm X 0.5 cm, and washed them to make sure that the extra betalaine that was leaked when the beet was cut will not diffuse in the water, since we only want to find out how much will be leaked after the membrane is penetrated.

After placing each beetroot pieces in their corresponding wells, we notice that the color of the solution started to change in the solutions that the beet were put in. After a period of 1 minute, we use a toothpick to stir the well to make sure that the betacyanin leaked from the tonoplast are completely dissolved and diffused in the solutions. In this process, we use different toothpicks for each set of wells with the same concentration to keep the concentration of detergent in each set of well constant. We also noticed that the beet in the well that contained solutions with concentration of 8% and higher float, presumably by the increase in density that the detergent causes

After 10 minutes, we removed the pieces of beet from the microwell. We noticed that the wells with 4% detergent solution had the clearest, yet darkest purple color, whereas other wells are more translucent with lighter color. This contradicts the result from other groups, where the solution with the highest concentration of other substances/chemical properties, such as pH, had the darkest color.

Quantitative – Data Collection

Solution

Absorbance (AU) (±0.001 AU)

Solution without Beet

Solutions with Beet

Trial 1

Trial 2

Trial 2

Mean of all Trials

Distilled Water (0% Detergent)

0.001

0.066

0.037

0.043

0.001

4% Detergent

0.775

0.682

0.627

0.560

0.775

8% Detergent

0.867

0.620

0.545

0.450

0.867

12% Detergent

0.982

1.041

0.653

1.042

0.982

16% Detergent

2.410

2.756

1.081

2.433

2.410

1. Calculations:

1. Mean

Solution

Mean (AU)

Distilled Water (0% Detergent)

0.049

4% Detergent

0.623

8% Detergent

0.538

12% Detergent

0.912

16% Detergent

2.090

Sample Calculation: 4% Detergent

Mean = Sum of a data set/Number of data

Mean = (0.682 AU + 0.627 AU + 0.560 AU)/3

Mean = 0.623 AU

1. Range

Solution

Absorbance (AU) (±0.0001 AU)

Trial 1

Trial 2

Trial 3

Range

Distilled Water (0% Detergent)

0.066

0.037

0.043

0.029

4% Detergent

0.682

0.627

0.560

0.122

8% Detergent

0.620

0.545

0.450

0.170

12% Detergent

1.041

0.653

1.042

0.389

16% Detergent

2.756

1.081

2.433

1.675

Sample Calculation: 4% Detergent

Range = Maximum Value – Minimum Value

Range = 0.682 AU – 0.560 AU

Range = 0.122 AU

Since the mixture of detergent in water causes the water to be translucent instead of transparent, less photon are able to travel though the solution, therefore, the colorimeter reads the data as having less absorbance than it normally should, if it was caused alone by the color leakage from the beetroot. Therefore, to measure how much the absorbance is changed when beet cells are placed in the original absorbance without the beet, we measured the amount of absorbance of each set solution with the beetroot and subtract them by the amount of absorbance of the original solution without the beet.

1. Change in Absorption Rate

Percentage of Detergent

Delta Absorption Rate (AU) ±0.001 AU

0% (Distilled Water)

0.048

4%

-0.152

8%

-0.329

12%

-0.070

16%

-0.319

Sample Calculation: 4% Detergent

ΔAbsorbance = Initial Absorbance – Final Absorbance

ΔAbsorbance = 0.775 AU – 0.623 AU

ΔAbsorbance = -0.152

Data Analysis & Graphs:

Graph 1

There is an outlier in the second trial in the 16% detergent concentration. This may cause error in other graphs and analysis

Graph 2

To our surprises, the absorbance of the solution decreases when a beetroot is placed into the solution.

Graph 3:

As depicted by the linear line that best fit, the change in absorbance increases (negatively) as the concentration of detergent increases

Graph 4:

Graph 5:

Contrary to graph 4, graph 5 depicts a direct proportional relationship between concentration of detergent and the absorption rate. This means that the absorption rate increases proportionally with the concentration detergent, without the beetroot. However when beetroot is added to the solution, the absorption rate of the solution increases exponentially. This means that the beetroot cells’ plasma membranes are being destroyed exponentially as the concentration of detergent increases.

Conclusion:

We accept our alternative hypothesis and reject the null hypothesis because an increase in concentration of detergent causes the absorption rate to increase as well. Since detergent is used to bring fat and lipids into solution*2, it has the ability to break down plasma membranes. Since the detergent molecules repel the hydrophilic heads of the phospholipid, the detergent will cause the membrane to be disrupted. Once the cell’s plasma membrane is damaged, the detergent molecules will be able to enter the cell and reach the tonoplast, where the betacyanin is contained. If there are more detergent molecules available in the water, more phospholipid heads will be repelled, thus more cell membrane would be penetrated and more betalaine will be able to leak out of the cell exponentially.

However, as depicted by the graphs, the betacyanin leaked from the beet cell somehow causes the detergent to have less absorption rate and become more transparent. This is unlikely to be a systematic error because the results from all trials and every set of beet lead to the same result. Since the amount of change in absorbance increases as the concentration increases (graph3), and the amount of betalaine leaked from the beet increases as concentration increases (graph 4), we can assume that the betalaine leaked from the beet has less absorbance than the absorbance of detergent. Therefore, when the betalaine is diffused into the detergent solution, the absorbance of the solution slightly decreases.

Evaluation:

Although many variables of this experiments are controlled, keeping them the the same throughout the experiment is very difficult. In the experiment, we tried to keep the amount of betalaine in each piece of beet root the same by cutting them into pieces with the same size. This process causes many errors because the pieces do not have the exact same size. Even if they have the exact same size, they do not have to necessarily have the same amount of betacyanin, because they may vary between cells.

This greatly changes the result of the experiment because it changes the amount of membrane in the beet that is to be disrupted, as well as the amount of betalaine that would be leaked. Therefore, to solve this problem, we could have used a more precise method to make pieces of beet, such as using a cork bore, or cut the beet electronically, to make sure that they have the same size and shape. Then, we could measure its mass and volume, to make the result even more accurate. The temperature could have been lowered as well, because beetroot tends bleed in environment with high temperature, which will make the experiment unfair because 2 variables are changed. Due to the viscosity of the detergent, it was also hard to measure the amount of solution. We also do not know how the percentages of detergent in the solutions are measured. If they are not 100% accurate, they may cause error as well.

Since there are only 3 members in our group, we could not place the all the beet into the microwell instantaneously. Instead, we placed it in one by one, with approximately the same time interval between each one. This is also the case when we stirred and removed the beet from the wells. This causes the period in which each piece of beet is left in the solution to be different. This may cause some beet to have longer or shorter time for their membrane to be penetrated than others. This could be solved fairly easily if we had more people or a tool that would allow us to re-position all the pieces of beet instantaneously.

And as mentioned above, the detergent in the solution causes the solution to be translucent instead of being transparent like the distilled water. Even though we tried to eliminate the error by subtracting the absorbance of the original solution from the solution with beet, the error still persists because we do not know how much the betalaine leaked from the beet affect the translucency of the original solution without beet.

Since we have only 3 trials, and the data do not necessarily agree in every circumstance, the data collected are not 100% reliable. There is even an outlier in the data (as mentioned above). Therefore, the mean is not 100% accurate. We could have made the experiment more accurate by repeating the same procedure for more than 3 trials. They experiment would give a more accurate data if it was repeated for about 8-10 times.

Apart from the setup errors, we also noticed that the absorbance of the distilled water is not 0, even after the calibration. This would cause another slight systematic error in the experiment, because the data will likely be slightly above what they are supposed to be. It may even mean that the distilled water used in the experiment is not 100% transparent, which would be another slight error in the experiment. As with all electronic devices that uses electricity, the voltage that is needed to run the colorimeter would generate heat. This heat has the potential to affect and change the data that the colorimeter reads.

Citation

*1: http://www.sanitarium.com.au/health-and-wellbeing/superfood-beetroot

*2: http://www.madsci.org/posts/archives/2000-12/976555915.Cb.r.html

– Other resources

http://www.piercenet.com/browse.cfm?fldID=5558F7E4-5056-8A76-4E55-4F3977738B63

http://en.wikipedia.org/wiki/Cell_disruption