Enzymes+Lab+Report

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TITLE: INVESTIGATING THE EFFECT OF TEMPERATURE AND pH ON ENZYME ACTION**__

__**AIM**__ The aim of this experiment is to find out how enzyme action is affected by different temperatures and different pHs of solvents.

__**PREDICTION**__ We think that the rate of enzyme action and starch digestion will be the highest at room temperature of 37 O C and for acidic solvents with pH below 7. This is near to the body temperature and thus, enzyme action will take place the fastest in the condition similar to its natural habitat, that is the human body.

__**APPARATUS**__ 1. Bunsen Burner 2. 3 Water Baths 3. Thermometer 4. 12 Test-Tubes and Racks 5. Measuring Cylinder 6. 4 250ml Beakers 7. 2 White Tiles 8. 2 Droppers 9. Ice Cubes 10. Distilled Water 11. Test-Tube Holder 12. Stop Watch 13. Goggles 14. 2% Starch Solution 15. 1% Amylase Solution 16. 1 M Hydrochloric Acid Solution 17. 1 M Ammonium Hydroxide Solution 18. Iodine Solution

__**PROCEDURE** PART A__ 1. Label 8 test-tubes A1, A2, B1, B2, C1, C2, D1 and D2.
 * 1) Add 5cm 3 of starch solution into tubes A1, B1, C1, and D1. Add 3cm 3 of distilled water to D2 and then 3cm 3 of amylase solution into tubes A2, B2 and C2.
 * 2) Place A1 and A2 into a beaker of ice water, B1 and B2 into a water bath at 37 O C, and D1 and D2 into another water bath at 37 O C.
 * 3) Place C1 and C2 in boiling water.
 * 4) Leave the tubes for 10 minutes to allow the solutions in the tubes to reach the temperature of the water bath.
 * 5) Pour the contents of A2, B2, C2 and D2 into the starch tube, A1, B1, C1 and D1, next to it. Record the time immediately. Keep tubes A1, B1, C1 and D1, containing the mixture in the respective water bath throughout the experiment and ensure that the temperatures are maintained.
 * 6) After 10 minutes, test the mixture in each tube with iodine solution, on a white tile. Draw table 1 with appropriate headings and record the results and conclusion.

__Part B__
 * 1) To each of the 4 test-tubes, W, X, Y, Z, add 2cm 3 of starch solution. Place the tubes in the water bath at 37 O C.
 * 2) Make up the contents of the four test-tubes as shown in the table below.
 * 3) Test the solution with iodine solution at the beginning of the experiment and after 10 minutes.
 * 4) Record your observations and conclusions in table 2 below.

__**RESULTS**__ **Table 1 Results of the Effect of Temperature on the Enzyme Action**


 * **Test-Tube** || **Contents** || **Temperature** || **Iodine Test Before Experiment** || **Iodine Test After Experiment** || **Conclusion** ||
 * A1 + A2 || 5 cm 3 starch + 3cm 3 amylase || 0 O C || Blue-black || Brown-black || Starch digestion has occurred. ||
 * B1 + B2 || 5 cm 3 starch + 3 cm 3 amylase || 37 O C || Blue-black || Brown-black || Starch digestion has occurred. ||
 * C1 + C2 || 5 cm 3 starch + 3 cm 3 amylase || 100 O C || Blue-black || Black || Starch digestion has occurred. ||
 * D1 + D2 || 5 cm 3 starch + 3 cm 3 distilled water || 37 O C || Blue-black || Blue-black || Starch digestion has not occurred. ||


 * Table 2 Results of Starch Digestion Using Amylase in Different Conditions**


 * **Test-Tube** || **Contents** || **Iodine Test** || **Conclusion** ||
 * W || 2 cm 3 of starch solution + 2cm 3 of HCl + 2cm 3 of amylase || Blue-black || Starch digestion has not occurred. ||
 * X || 2 cm 3 of starch solution + 2cm3 of distilled water + 2cm 3 of amylase || Brown-black || Starch digestion has occurred. ||
 * Y || 2cm3 of starch solution + 2cm3 of NH 4 OH+ 2cm3 of amylase || Violet-black || Starch digestion has occurred. ||
 * Z || 2cm3 of starch solution + 4cm3 of distilled water || Blue-black || Starch digestion has not occurred. ||

__**DISCUSSION QUESTIONS + CONCLUSION** Tubes A1, B1 and C1 show that starch digestion has occurred. When the mixtures were tested with iodine before the experiment, the iodine turned from a yellow to a dark blue-black colour, indicating the presence of starch. However, after the experiment, when the iodine test was carried out for the second time, the mixtures in test tubes A1, B1 and C1 changed colour while the mixture in test tube D1 remained blue-black. This showed that starch digestion had occurred in A1, B1 and C1, while not in D1.
 * Part A**__
 * 1) __Which tube shows that starch digestion has occurred? Give your reasons.__

The mixture in test-tube D1 contained mixtures that gave a blue-black colour after an iodine test was conducted after the experiment.
 * 1) __Which of the test-tubes contained mixtures that gave a blue-black colour?__

This was because in test-tube D1, starch digestion had not taken place, and therefore, the starch molecules caused the iodine to turn blue-black. As a result, no colour change was observed after the experiment.
 * 1) __Why did the mixtures in the test-tubes stated in (2) remain blue-black colour?__

D1 serves the purpose of a control. In a scientific experiment, a control is important in order to eliminate all possible variables except the one being tested and also for the purpose of comparison.
 * 1) __What purpose does tube D1 serve?__

When starch digestion takes place, the starch is broken down by enzyme action into glucose molecules. Therefore, I can test it with Benedict’s solution.
 * 1) __Using your knowledge of the structure of starch, describe the test you would use to detect the presence of the substance produced as a result of the reaction in the test-tube stated in (1) above?__

When the temperature is low (0 O C), enzyme action is slowed down and the starch is not completely digested. 37 O C is the optimum temperature for enzyme action to take place and therefore, enzyme action takes place the fastest at this temperature. In boiling water (100 O C), the enzyme denatures and dies and therefore, enzyme action stops.
 * 1) __What conclusion(s), based on the observations in this experiment, can be made about the effect of temperature on enzyme reaction?__

__**Part B**__ Tubes X and Y show that starch digestion has occurred. After the experiment, when the iodine test was carried out, the mixtures in test tubes X and Y changed colour, showing that a reaction had taken place while the mixture in test tubes W and Z remained blue-black. This showed that starch digestion had occurred in X and Y, but not in W and Z. In X and Y, the starch molecules had been broken down into simpler particles and caused the blue-black colour of the iodine to change.
 * 1) __Which tube shows that starch digestion has occurred? Give your reasons.__

The mixtures in test-tubes W and Z contained mixtures that gave a blue-black colour after an iodine test was conducted after the experiment.
 * 1) __Which one of the test-tubes contained mixtures that gave a blue-black colour?__

This was because in test-tubes W and Z, starch digestion had not taken place, and therefore, the starch molecules caused the iodine to turn blue-black. As a result, no colour change was observed after the experiment because no reaction had taken place.
 * 1) __Why did the mixtures in tubes stated in (2) remain blue-black?__

It can be observed from the results that the pH at or above 7 is favourable for enzyme action to take place. The mixtures in test-tubes X and Y contain distilled water (pH 7) and ammonium hydroxide solution (pH above 7) respectively. Starch digestion occurs in these two test-tubes, as can be seen from the colour change. However, the mixture in test-tube W contains hydrochloric acid (pH below 7). Starch digestion does not take place here as there is no colour change observed.
 * 1) __What conclusion(s) based on the observations in this experiment, can be made about the effect of pH on enzyme action?__

The pH at or above 7 is favourable for enzyme action to take place. Enzyme action takes place the fastest at 37 O C, which is the optimum temperature for enzyme action.
 * 1) __How does pH and temperature affect enzyme action?__

__**ERRORS** __**Things Done Wrongly**__
 * Precautions Taken**__
 * We labelled the test-tubes correctly so that the experiment could be carried out in a more organised manner.
 * Before using any apparatus, we made sure to wash them thoroughly to wash off any remaining solvent that might be in it. This helped minimise the error caused due to the contamination of chemicals.
 * We wore goggles during heating process and during the handling of acid and alkalis, for safety purposes.
 * Thermometers were used to ensure that the temperatures of the water baths were maintained at the stated temperatures.
 * The thermometers were not used as stirrers in the test-tubes or left inside the beaker during the heating of the water bath.
 * The water in the beakers did not overflow when the test-tubes were placed in them.
 * The level of water in the water baths was higher than the levels of mixtures in the test-tubes.
 * The droppers for the respective test-tubes were not mixed during the experiment to prevent contamination of chemicals.
 * Temperature was taken by placing the thermometer at eye level to avoid parallax error.
 * A test-tube holder was used to hold tube C2, and this was for to maintain safety in the laboratory.
 * After the experiment, when we were supposed to carry out the iodine tests on the mixtures, we did not use the white tile in order to save time. Instead, we dropped the iodine inside the test-tubes themselves and this might have added errors to our experiment.
 * Although the temperatures of the water baths were maintained, they were not constant all the time and fluctuated slightly, giving rise to errors.
 * The tile that was supposed to be used for the iodine tests was not entirely dry.
 * We did not take out the test-tubes out of their water baths, beaker of boiling water and beaker of ice all the same time, therefore, a prolonged period of time in that temperature could have affected the results of our experiment.

**Done By: Reetaza Chatterjee (19)** 
 * Class 311**