Check the effect of increasing concentration of acid and alcohol on the amount of denaturation of the albumen protein.

Aim: The aim of my experiment is to check the effect of increasing concentration of acid and alcohol on the amount of denaturation of the albumen protein.

I will be carrying out my experiment by using the below conditions as variables.

Concentration of acid
Concentration of alcohol
Temperature

For this experiment to work there are certain factors that have to be controlled like temperature and amount of egg white to be added. In all the experiments the amount of egg white used will be 10ml and the temperature maintained will be room temperature. One air conditioner will be left on all throughout the day to make sure the temperature remains the same. If there is an increase in the temperature the protein will denature faster thus the temperature must be maintained.

The amount of egg white to be added is 10 ml. This is done by accurately measuring it by first putting the egg white is a measuring cylinder of 10 ml capacity and then adding it to the test tubes.

Equipment/ Apparatus

Round Bottom Flasks. These will be required after the acid and alcohol solutions are prepared. These are better suited than beakers or other flasks (conical) because these have a bung that prevents the alcohol and acid to vapourise and escape. This can also be done using a beaker with a lid but since these flasks come with stoppers of perfect size to plug the hole, there is less chance of this happening.

Pipette. The pipette is used to add the alcohol and the acid into the measuring cylinders.

Measuring Cylinders. These will be used to measure the amount of water added to the acid and the alcohol to adjust the concentrations. The measuring cylinders come in different volume ranges and are thus more helpful.

Gloves. This is more of a safety feature more than anything. Even if by accident the acid were to drop onto my hands, there would be no damage such as itching or burning of the skin.

Glass rods. To stir the albumen for easy addition.

Rubber Bungs. These are to be used once the alcohol and acid solutions are added to the albumen to prevent atmospheric oxygen from reaction with the mixture.

Colorimeter. For some of the readings the length of the coagulated protein formed cannot be easily measured because the denaturation is not complete. Therefore the better way would be to pass light through the suspension and then check the intensity of light that passes through. This is preferred to a spectrophotometer. The operation of a spectrophotometer is basically to illuminate the sample with white light and to calculate the amount of light that is reflected by the sample at each wavelength interval. They are preferred because of their low cost of manufacture and portability.

Chemicals used

Acid – 0.1 M, 0.05 M, 0.025M

Alcohol – 10%, 50%, 80%

I have planned to use the following concentrations of acid and alcohol so that I can test the effect of the most concentrated, least concentrated and the middle concentration. This can give me a better idea of the change taking place in the albumen.

To prepare the acid solutions I will use the formula C1V1=C2V2

I was provided with 0.1 M of HCl. So I just added 10cm3 of this to the measuring cylinder and then check the lower meniscus and then ...

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Chemicals used

Acid – 0.1 M, 0.05 M, 0.025M

Alcohol – 10%, 50%, 80%

To prepare the acid solutions I will use the formula C1V1=C2V2

I was provided with 0.1 M of HCl. So I just added 10cm3 of this to the measuring cylinder and then check the lower meniscus and then pour it into the round bottom flask.

The next concentration requires me to us the above formula.

C1 = 0.1 M of HCl already provided to me.

C2 = 0.05 M of HCl required

V2 = 10 ml of 0.05 M HCl required

V1 = The volume of 0.1 M HCl required to make 10 ml of 0.05 M HCl.

C1V1=C2V2

V1 = 0.05 x 10

V1 = 5 ml

So this I need 5 ml of 0.1 M of HCl and I will need to add 5 ml of water to it to make 0.05 M. The ratio of acid to water is 1:1.

Similarly to make 0.025 M of HCl, the same formula is applied and the answer for V1 is 2.5 ml. This is the volume of 0.1 M acid to be added and then diluted by adding 7.5 ml of water to it to obtain 10 ml of 0.025 M solution.

To prepare the alcohol solutions the content of alcohol will be according to the percentage specified. Since I will be using 10cm3 of each solution, the percentage will depict the volume of alcohol or water accordingly.

Procedure

The egg will be broken and the egg white will be separated from the yolk using an egg separator. The egg white contains the protein albumen. Inside the egg separator, the egg white will be stirred well using a glass rod because then otherwise the jelly (the egg white is in the form of a jelly) will not be easy to measure and add into the test tube. 10 ml of the egg white is taken in different test tubes using a pipette. The acid and the alcohol solutions, which were previously prepared will have to be added to the albumen proteins, which when added have a yellowish colour. They will be then left for 24 hours for denaturation to occur completely. My control will be water where the albumen protein was just dissolved in water. For each of the three concentrations of acid and alcohol, there will be 3 readings so that once I take my result; I can take the average to increase the accuracy of the answer. The time period allotted was 24 hours and then I decided to check on the status of the experiment.

Acid

0.025 M. It was difficult to find the length of the coagulant formed. Instead I observed that a thin cloudy coagulant was formed, white in colour. I then decided to measure the intensity of light that would pass through this.

0.05M. This concentration had a layer of coagulant at its surface unlike the 0.025 M in which the coagulant formed was like a straight line. In this concentration, some of the unreacted acid remained on top and the unreacted albumen remained down in a yellowish lump.

0.1M. According to my predictions, the higher concentration of acid had more of the coagulant formed. But I could not say this for certain because I didn’t know whether it was cloudier.

For this reason I decided that the best way was to remove some of the solutions formed from each of the test tubes and empty them into a cuvette. The test tube would have to be shaken properly to try to homogenize it. The coagulant would spread evenly throughout the ethanol and then this could be emptied into the cuvette. I would then place this cuvette into the colorimeter. The filter I selected was the blue one because the albumen that remained at the bottom of the test tubes and was unreacted had a shade of yellow. This was according to the colour wheel which shows that the colour on the opposite side of a particular colour would absorb it.

As can be seen from the above diagram, the circled colour shows the precise colour of the solution and the opposite would be a shade of blue.

Results

To record the results I will use the colorimeter to check for light transmittance and percentage absorbance.

In the above results, the numbers in brackets are the absorbance of light whereas the percentage is the percentage transmission of light.

The colorimeter passes light through the medium placed in the cuvette and the received light on the other end is measured and the values are then measured on the meter. Before I put the resulting solution into the cuvette, I made sure that I shook the test tube well so that the coagulant and the acid would form a suspension. This way I could also measure the amount of the coagulant formed by the amount of suspension formed. I added 10 ml to each of the cuvettes. The above results show that in the higher concentration of acid, the coagulant formed was thicker and cloudier because the light transmitted through it is less. As the concentration of acid increases there is a decrease in the amount of light that passes through it, showing that the coagulant has become thicker and has increased also. As the concentration of the acid increases, more H+ ions are available to disrupt the shape of the protein and break its bonds. This causes more coagulant to be formed. And because there is an increasing amount of coagulant formed, it becomes thicker.

As can be seen from this graph that the relationship is proportional but not directly proportional. The line does not pass through the origin. This could be because of many mistakes that might have occurred. If I were extend the line further as a forecast for further concentrations, it would show me that the line would cross the origin.

Alcohol

Ethanol was the chosen alcohol. For this I had to be very careful because ethanol is volatile and so I would have to make sure that I should not leave it exposed to the air for a long time. Once the solutions had been left for 24 hours, I measured the amount of coagulant formed. In this case it was easier to do so because the ethanol was found on the top, the coagulant below it and finally the rest of the albumen below it.

10% alcohol. In this, the coagulant formed was less thick and was found very little between the ethanol and albumen. The small droplets of coagulant formed were hardly even 0.2 cm in length.

50% alcohol. In this the coagulant formed was even thicker. The amount formed was also more because the height of it was now 3.5 cm.

80% alcohol. The coagulant formed in these test tubes was the greatest and more concentrated in one area. The amount of it formed was approximately 10cm.

Once I analysed them in a colorimeter, these were my results.

As seen in the table, the coagulant formed becomes thicker and increases in amount. This can be seen because less light travels through as the concentration increases. This shows that the amount formed also is more.

This also shows that the concentration of alcohol is proportional to the absorbance. This proves that more concentrated the alcohol the more of the protein will be coagulated.

Precautions

I will have to be extremely careful with the apparatus because most is made of glass.
I will make sure that the rubber bungs are tightly fitted on so that there is no foaming due to atmospheric interaction.
When measuring liquid volumes, I will always use the lower meniscus and keep the reading at eye level to avoid parallax error.
I have to be careful to ensure that when the hole is being made in the egg, only the white part of the egg is removed and the rest is not.
The ethanol should be emptied quickly and then covered so that it does not evaporate.

Factors controlled

The factors that I controlled were temperature and the amount of egg white to be added. Switching on only 1 air conditioner would control the temperature and the test tubes would be placed at the other end of the room so that the temperature would remain more or less the same.

The amount of egg white added should be the same so that there is no difference in the readings. This would be because I added the same mount of albumen using pipettes.

According to my hypothesis, this shows that the more concentrated the alcohol or acid is, the more the coagulant is formed. This is also proved through the graphs that have been shown here. This would prove that my hypothesis is correct and that the protein has coagulated. I would know that the protein has coagulated because it does not have a definite structure now and will not foam if kept in air. If it were in its native confirmation, it would foam in air because it is a good foaming agent.