To study the effect of three diferent pH on the activity of salivary amylase on starch | Biology Practical Class 12 | Gaurav Kumar

 To Study The Effect of Three Different pH on 

the Activity of Salivary Amylase on Starch.

Aim: -     

To study the effect of three different pH on the activity of salivary amylase on starch.

REQUIRMENTS: - 

Test tubes, test tube stands, beakers, pipette, funnel, measuring cylinder, cotton, starch, iodine, potassium iodide, sodium chloride, different pH tables etc.

PREPARATION OF REAGENTS: -

1.    Lugol's iodine solution:-     Take iodine 1 g. potassium iodide 2 g and 100 ml. distilled water; mix and allow to stand. Deep this solution in glass bottle.

2.    1% NaCl Solution:-     Dissolve 1 g NaCl in 100 ml distilled water.

3.    1% starch solution:-     Take about 0.5 g of starch in a 100 ml. beaker and add enough water to make a paste dilute the paste by adding 50 ml. water and boil for about 5 minutes. Leave the solution overnight and filter it to get 1% starch solution.

PROCEDURE: -

1.    Take two sets of test tubes (8 - 10 test tubes in each set in two separate test tube stand each containing 1 ml. of iodine solution to act as indicator tubes.

2.    Switch on the electric water bath or oven. Set the temperature at 37\C. Maintain uniform temperature (37'C) of the water in water bath or water in a beaker inside incubator throughout the experiment.

3.    In a test tube, take 10 ml of starch solution, 2 ml of 1% NaCl solution and 2 ml of buffer solution.

4.    Mix them well and transfer half of the solution into another test tube. Mark one test tube as experimental tube and the other as a control tube.

5.    Prepare three sets of indicator test tubes (8 to 1- in each set) in three separate test tube stands. Label test tubes stands as A (for 6.8 pH), B (for 5 pH) and C (for 9 pH). In each test tube take 0.5 ml of iodine solution.

6.    In a test tube, take 5 ml of 1 % starch solution, 1 ml of 1 % NaCl solution and 1 ml of 6.8 buffer solution, mark it as control tube or "A".

7.    In another test tube, take 5 ml of 1 % starch solution, 1 ml of 1% NaCl solution and 1 ml of pH 4 buffer solution, mark it as experimental tube "B".

8.    In the third test tube, take 5 ml of 1 % starch solution, 1 ml of 1 % iodine solution and 1 ml of pH 9 buffer solution. Mark it as experimental tube "C".

9.    Transfer 1 ml of dilute saliva into each test tube and mix the two thoroughly. Place the all three tubes in water bath set at 37'C.

10.    Take a drop form each of the experimental tubes with the help of dropper and add to the corresponding indicator tubes containing iodine solution. Note this time as zero minute reading.

11.    After 2 minutes, again take a drop from each tube and pour it to the iodine tubes and note the change of color of iodine.

12.    Keep on repeating it after 2 minutes till the color of iodine does not change.

13.    Note the total time taken for different experimental tubes till the do not give any color with iodine.

OBSERVATION: -

Time required to reach the achromic point (end Point). In digesting 5 ml of 1 % starch at different pH by the diluted enzyme.

OBSERVATION TABLE 

RESULT: -

It will be observed that in tube A, achromatic point is reached first of all. This is so because optimal activity for most of the enzymes is generally observed between pH 5.0 and 9.0. However, a few enzymes, e.g. pepsin are activity at pH values well outside this range. Above and below this range, the reaction rate reduces an enzyme to get desaturated.

PRECAUTIONS: -

1.    Always filter the saliva through a wet cotton thin film and not through a filter paper.

2.    While measuring the saliva, there should be no air bubble.

3.    Maintain the temperature i.e. 37'C thoroughly the experiment.

4.    All the glassware used should be thoroughly cleaned and dried.

5.    All apparatus and measuring should be very accurately.

6.    Zero minute and achromic point reading should be taken very carefully.

7.    All solutions should be make accurately.

8.    Iodine solution should be freshly prepared.