An Experiment to determine the lowest concentration of copper (2) sulphate that brings about full denaturation of egg albumen.
An Experiment to determine the lowest concentration of copper (2) sulphate that brings about full denaturation of egg albumen
INTRODUCTION
Egg albumen is also known as egg white. It surrounds the yolk in the egg. It is a globular protein which is colloidal. This means that it cannot be filtered
Egg albumen is has a protein structure. This is important as protein structure is extremely complicated and precise and has to be exact for it to carry out its function.
Firstly, proteins are made at a basic level by chains of amino acids bonded with peptide bonds. An amino acid looks like
The R is a side chain that differs in amino acids.
They bond in a peptide bond which is a condensation reaction which forms between the carbon of one and the nitrogen of another forming a compound like this
The difference between the all the amino acids is the R group which defines the function of the protein. The overall structure of a protein (primary, secondary, tertiary and quaternary) is very precise and is held together by hydrogen bonds, ionic bonds, disulphide bonds, van der Waals forces and hydrophobic interactions. It is necessary for a protein to do its job to have its exact structure other wise it could not do it. Any slight change in the structure of protein could drastically change its properties, which is called denaturation.
Copper sulphate is a toxic chemical. This is because copper sulphate is a compound of copper ions and sulphate ions. The copper ions will form strong bonds with carboxylate anions which form a part of the peptide bond. This creates a new set of ionic bonds hence disrupting the peptide links that join the amino acids together and changes the secondary structure. Furthermore, the positive copper ions affect the protein's electrical polarity which increases its insolubility and causes it to precipitate out of the solution.
This denatures the protein as the protein has a specific unique secondary structure so a slight change would cause a change in function. However, the fact that the albumen will be precipitated out of solution is very useful. This is because albumen is usually soluble in water as it is a globular protein, which are usually soluble in water, so it will be precipitated out of solution, as it re-forms into a fibrous protein which are insoluble in water, so you can test it with a colorimeter as the precipitate is opaque.
There are many other factors that could affect this experiment. These are;
Variable
How it affects the experiment
How to control
pH
pH is a measure of hydrogen ion concentration i.e. higher pH lower Hydrogen conc.
Buffer solution
The hydrogen ions can interact with the R groups causing a change in its overall 3D structure,ie denatures it
Concentration of egg albumin
The more egg albumin, the more that can react with the Copper Sulphate until it reaches the V-max where there is too
Measure out ...
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There are many other factors that could affect this experiment. These are;
Variable
How it affects the experiment
How to control
pH
pH is a measure of hydrogen ion concentration i.e. higher pH lower Hydrogen conc.
Buffer solution
The hydrogen ions can interact with the R groups causing a change in its overall 3D structure,ie denatures it
Concentration of egg albumin
The more egg albumin, the more that can react with the Copper Sulphate until it reaches the V-max where there is too
Measure out volumes carefully with a syringe
many egg albumin to react with the Copper Sulphate therefore different rates of reaction
Temperature
Temperature supplies energy to the molecules of egg albumin causing them to move around more therefore collide
Conduct at room temperature, not in
with Copper Sulphate and react. However, too much energy causes them to break their
the presence of anything that could provide a lot of heat
bonds as they vibrate rigorously and fight free of them. this causes denaturation
The ways they could be controlled are
Variable
How to control
pH
Buffer solution
Concentration of egg albumin
Measure out volumes carefully with a syringe
Temperature
Conduct at room temperature, not in
the presence of anything that could provide a lot of heat
Preliminary Work
Using egg albumin as sample and no filter (white light) with a 1to 1 ratio for copper sulphate solution to egg albumin
Concentration of Copper Sulphate
Colorimeter reading
0.10 mol
.93
0.08 mol
.65
0.06 mol
.23
0.04 mol
0.84
0.02 mol
0.56
What I found from my preliminary work, was that firstly that a 1to 1 ratio for copper sulphate solution to egg albumin gives the best results on the calorimeter as I found that any more would show to much denaturation as even the 0.02 mol copper sulphate solution with a 1 to 5 ratio of copper sulphate solution to egg albumin would show complete denaturation (a reading of 1.00 on the Colorimeter). Using this ratio, it is easier to distinguish the differences between concentrations of copper sulphate.
I also confirmed that when egg albumin is denatured, an opaque precipitate is formed. I did this by boiling 5cm³ of egg albumin which went white and was easy to see.
I also found the volumes that I am going to suggest to be tested. Do to finding that there is denaturation at 0.06 mol dm³, my values are 0.01mol/dm³,0.02mol/dm³, 0.03mol/dm³, 0.04mol/dm³ ,0.05mol/dm³ and 0.06mol/dm³). From my preliminary work, I have found how to create these values using the 0.1mol dm³ solution and the distilled water.
Concentration (molsdm³)
Parts distilled water
Parts Copper Sulphate
0.01
4.5
0.5
0.02
4.0
.0
0.03
3.5
.5
0.04
3.0
2.0
0.05
2.5
2.5
0.06
2.0
3.0
The best volumes to use were 3.5cm³ of egg albumin and 3.5cm³ copper sulphate solution as it is easy to see if any precipitate is formed, to verify your results and it is a test tube value.
I also found that : too vigorous mixing breaks hydrogen bonds so I must not over shake in the main practical, denaturation was almost instantaneous, rendering time as a factor irrelevant, different colorimeters give different readings so you must stick to using one, the same for the different batches of copper sulphate can vary slightly in concentration
Apparatus:
Colorimeter: I have chosen to use this as a way to measure if the solution is opaque or not as it will give me numbered results which I could compare quantitatively.
50cm³ 0.1mol/dm³ of Copper sulphate solution: From this concentration of copper sulphate, I can make the concentrations already quoted
Distilled water: this is used instead off tap water to ensure there are no impurities which could affect the results during the experiment. It can also be used to clean equipment.
50cm³: this should provide me with the egg albumin I need for the experiment
8 test tubes and 2 beakers (200ml) and 2 test tube racks: to be used to contain solutions during experiment
0 Cuvettes: many of these are needed because it is important to use a different one each time, to ensure that the results remain accurate and fair. They should be cleaned after use with distilled water.
Pipettes and syringes: these can be used to measure accurately to 0.5cm³ the volumes involved in making up the solutions. Therefore both a 10cm³ and a 1cm³ syringes is needed.
Buffer solution: this is needed to keep the pH constant.
Tweezers: to be used in removing the chalaza
Method
. Open the 4eggs and split the yolks from the albumin by rolling it to one side of the shell of the egg and pour the albumin into the beaker.
2. Using a set of tweezers, remove the chalazas from the albumin as they are cloudy and would give an increased reading off precipitate on the colorimeter. Store it away from the egg in another beaker.
3. Take 5cm³ of the egg albumin using the 10cm³ syringe and place it in a test tube. Put 5cm³ samples in each of the other test tubes in this fashion. Then label all test tubes so you have 3 test tubes with 0.01, 3test tubes with 0.02 etc. After using the syringe, clean it with distilled water to avoid contamination.
4. Place 3.5cm³ of the buffer solution in each test tube
5. Make up the 0.01-0.06 mol/dm ³ concentrations of copper sulphate using the table I provided. Do this using the 1cm³ syringe into the beakers. After using the syringe, clean it with distilled water to avoid contamination.
6. Then, take a 5cm³ sample from each beaker using the 10cm³ syringe and put it in its corresponding test tube. After using the syringe, clean it with distilled water to avoid contamination.
7. Now you must shake the test tubes gently. This is done to make sure the reaction is complete and that the precipitate spreads evenly through the test tube. It has to be done gently though as otherwise it can cause the bonds in the protein to break by supplying them too much energy.
8. Then, pipette your result in the test tube into a cuvette and then place it in the calorimeter. This is done to take the reading off how much light can pass through the cuvette as the higher the amount of light that can pass through the cuvette, the lower the amount of precipitate. As the cuvettes cannot be labeled, make sure that they stay in order to stop any errors taking place there.
9. You take a reading in the colorimeter without a filter. This is done as I would like to test the affect of white light and not any other colours as the egg albumin is closer to a clear colour than any other. The standard we use is the egg albumin, which is placed in a cuvette on its own and is used to reset the colorimeter each time.
0. Then, place your test sample in the calorimeter and press the test button. Take the result to 2 decimal places to increase accuracy. Using a 0-2 scale, 1 is full denaturation. Also make sure that the clear sides of the cuvette are placed in the right direction, as the arrows on it show, because the colorimeter measures the amount of light that passes through solution, therefore the other side will affect the reading, reducing the result.
1. Record your results in the table provided, and draw a result of concentration of copper sulphate against calorimeter reading. As a precaution for an incorrect colorimeter reading, note down if there is much precipitate formed as in the preliminary, there were some difficulties with the colorimeters.
2. Repeat this experiment twice more to gain check readings. This will indicate any anomalous results, and therefore increase reliability of the readings.
3. Then plot a graph as I stated before for the average.
Analysing results
Concentration (mols/dm³)
Calorimeter Reading (amps) 1
Calorimeter Reading (amps) 2
Calorimeter Reading (amps) 3
0.02
0.03
0.04
0.05
0.06
Further Work
To find the a more accurate concentration which is needed to denature the egg albumin, you would look at your graph and pick out where on it, it dips under one. You then take more reading with concentrations which are to more decimal points. For example, this sample graph suggests that 0.05 mol/dm³ is the concentration where the value lies around. Therefore, I would repeat the method I did earlier, but do it with the values, 0.045 mol/dm³, 0.046mol/dm³,0.047mol/dm³, 0.48mol/dm³, 0.49mol/dm³,0.50mol/dm³, 0.51 0.52 ,0.53 ,0.54 and 0.55 . This would give your final result a higher level of accuracy.
Safety
Wear a lab coat through experiment and also wear safety goggles as copper sulphate is toxic.
Also be careful to wash your hands thoroughly when you finish as there is a salmonella risk with the raw eggs.
Take care when using your equipment, especially syringes which can squirt out unexpectedly if you are not paying attention.
Reference
Biology 1- Cambridge
Advanced Biology- Jones and Jones
www.canadaegg.ca/english/educat/egg-anatomy.html
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