Plan
Apparatus
• Cork Borer
• Scalpel
• Ceramic white tile
• Sieve
• Filter paper
• Weighing Boats
• Balance
• Ruler
• 4 Stopwatches
• Marker pen
• 6 Boiling tubes
• Test tube rack
• Glass rod
• Beaker
• Graduated Syringe
• Laboratory coat
• Safety goggles
• Safety mask
• Calculator
• 10 cm3 Measuring Cylinder
• Gloves
• Thermometer
• Electronic Thermostatically Controlled Water Bath
• Distilled Water
• 2 eggs
• Pepsin Powder
• pH 2 buffer solution
• pH 3 buffer solution
• pH 4 buffer solution

Method:
1. Prepare the control tests for each pH (the fixed independent variable) of pH 2, pH 3 and pH 4:

(i) Using a syringe, withdraw 10cm3 pH 2 buffer solution into a boiling tube.

(ii) Label with a ‘C’ for control and ‘pH 2’ with a marker pen.

(iii) Repeat for two more boiling tubes for pH 3 and 4.

2. Add 10cm3 distilled water to each boiling tube containing buffer solution.

3. Put all the boiling tubes into a provided 40°C thermostatically controlled water bath


4. Allow the boiling tubes to reach 40°C, checking them with a thermometer, in the water baths, stirring and checking the temperature with a thermometer occasionally. Rinse and dry the thermometers in between boiling tubes.

5. Whilst they are heating up, prepare the egg white:

(i) Make a water bath to poach the eggs in: bring some water to the boil in a large beaker by heating with a Bunsen burner until bubbles start appearing.

(ii) Break two eggs (preferably from the same hen, to control the source of egg) into a smaller (in cm3) beaker and place this into the water bath containing boiling water.

(iii) Simmer for five minutes and then take the eggs out with a spoon- careful around the hot water.

(iv) Leave the eggs to cool for a few minutes.

6. When the eggs have cooled, cut around the yolk in the middle using a scalpel, leaving you with a sample of egg white. Discard the egg yolk responsibly.

7. Extract a few 1cm (in diameter) cylinders from the cooked egg white using a cork borer. (The cylinder of egg should come out easily by blowing down the cork borer.) The cork borer means all the cylinders will have the same diameter, controlling the surface area.

8. Using a scalpel on the ceramic white tile, cut from the cylinders 9-10 discs, each one should be 3mm (in width) using a ruler- take care when using the scalpel as it is very sharp.

9. Put 3 of the egg white discs into a weighing boat and place the boat containing the discs onto the balance- first press ‘T’ to clear the balance.

10. Record the weight (g) in a suitable table (as mass is the dependent variable to be measured).

11. Using a spoon, add the 3 egg white discs you have weighed into the boiling tube containing buffer solution of pH 2. Stir with a glass rod. Rinse and dry rod. Immediately start one stopwatch.

12. Repeat steps 9-11 for the other two boiling tubes for pH 3 and 4 in the water bath. Time the other two boiling tubes on the same stopwatch and three minute intervals, for accurate timing.

13. Prepare 3% pepsin solution:

(i) Put on lab coat, safety goggles, safety mask and gloves.

(ii) Pour 100cm3 of distilled water into a beaker.

(iii) Weigh out 3g of pepsin powder using a weighing boat and balance and add to the distilled water.

(iv) Mix thoroughly with a glass rod to make a 3% pepsin solution.

14. Syringe out 10cm3 of pH 2 buffer solution into a 10cm3 measuring cylinder and shake. Pour into a boiling tube.

15. Syringe out and add 10cm3 of previously prepared pepsin into the boiling tube containing 10cm3 of pH 2 buffer solution. Label the boiling tube with ‘pH 2’.

16. Put the boiling tube into the water bath and measure the temperature with a thermometer, until it reaches 40°C.

17. Syringe out 10cm3 of pH 3 buffer solution into another boiling tube. Rinse and dry the syringe.

18. Syringe out another 10cm3 of previously prepared pepsin. Label this boiling tube with ‘pH 3”. Place the tube into the water bath.

19. Repeat for pH 4, using pH 4 buffer solution. Allow them all to reach 40°C, measuring the temperature with a thermometer, in the water baths.

20. If there is no egg white left after the control tests, extract a few more 1cm (in diameter) cylinders from the cooked egg white using the cork borer and cut 9-10 more discs with the scalpel.

21. Weigh 3 discs in a weighing boat on the balance. Record the weight (g) in a suitable table.

22. Add the 3 egg white discs, using a spoon, into the boiling tube containing pepsin solution buffer solution of pH 2. Stir with a glass rod. Rinse and dry rod.

23. Immediately start stopwatch. Use a different stopwatch each time, for accurate results. You should end up with three stopwatches started a few minutes after each other, (e.g. the stopwatch for boiling tube containing pH 2 buffer solution will be started first, then the one for pH 3, then pH 4.) This will determine the order they are taken out.

24. Repeat steps 21-23 for boiling tubes containing pepsin solution and pH 3 and pH 4 buffer solutions.

25. Throughout the fixed time:

(i) Agitate the boiling tubes frequently with a glass rod- approximately every 20 minutes.

(ii) Check on the temperature at the same time with a thermometer and stir, being careful not to contaminate between the three boiling tubes by washing and drying equipment between uses. This keeps the temperature constant.

(iii) Use a pH testing meter to check on the pH. Dip the pH sensitive rod of the meter into each boiling tube (wash and dry in between tubes). The meter will measures and display a pH reading- the pH of each tube should remain constant to the pH they have been labelled with.

26. After four hours has passed on the first stopwatch (for boiling tube containing pH 2 buffer solution), stop it and take out the pH2 boiling tube from the water bath.

27. Pour the contents of the boiling tube labelled ‘pH 2’ into a sieve to get the discs and dry them carefully with filter paper- in a patting motion rather than a rubbing motion.

28. Place the discs into a weighing boat and onto the balance. Record the weight (g) in a suitable table.

29. Repeat steps 27-28 for the rest of the boiling tubes, as the stopwatches reach 4 hours. This means the egg discs will be taken out in the order they were put in for accurate timing: so after pH 2 (explained in step 27, 28);

(i) do the same for boiling tube labelled ‘pH 3’ when the stopwatch for this reaches 4 hours;

(ii) then boiling tube labelled ‘pH 4’, when the stopwatch for this reaches 4 hours.

(iii) the control tests (labelled ‘C pH 2’, ‘C pH 3’, ‘C pH 4’) in the order that they reach four hours on their stopwatch: There should be three minute intervals between each tube, allowing sufficient time to take out the discs, dry and weigh them.

30. Record all the masses in the table.

31. Calculate the percentage difference in mass for all the tests, including the controls, using the equation: (change in mass ÷ original mass) x 100

32. Record all the percentage differences which will show the effect pepsin has had on the protein in the egg white in the table.

33. Repeat the tests at each pH (2, 3, 4) 30 times, to get representative results.

34. Carry out statistical tests on the results (to compare means from all the data):

(i) calculation of standard error:
calculated by the equation: standard deviation ÷ √n
(where n is the number of samples and standard deviation is the standard deviation of the mean of all the samples)

(ii) and then application of 95% confidence limits.