1 ml of iodine was inverted and inserted into the spectrophotometer. The spectrophotometer was then set at 560 nm. Two experiments were conducted:.
The effects of pH:.
Six different starch solutions were prepared in different flasks by mixing a 35 ml stock starch solution and 35 ml of buffered distilled water at pHs: 4.0, 4.5, 5.0, 5.5, 6.0 and 6.5. 0.1 ml of iodine indicator was added to 66 tubes (11 for each reaction flask).
It's important to note that each pH solution sample was added to a tube immediately before placing it into a spectrophotometer. 5 ml from the first reaction flask (4.0 pH starch solution) was mixed into the first tube. Tube one was immediately mixed and placed into the spectrophotometer. The absorbance was read and recorded for time zero. Tube one was removed and 1 ml amylase extract was mixed into tube 2. Tube 2 was mixed thoroughly and placed in the spectrophotometer 2-miin after the first tube was placed in the spectrophotometer. The absorbance was read, recorded and repeated at 2-min intervals to each of the nine remaining tubes. This procedure was repeated to every reactant flask containing a different pH level (66 data entries).
The effects of temperature:.
Five different water baths were prepared at the following temperatures: 15, 30, 45, 55, 60, and 70 deg C. Each water bath had a separate reaction flask containing 35 ml of starch solution and 35 ml of distilled water. When reaction flasks reached the appropriate temperature the enzyme was added. The basic procedure (pH procedure) was followed for each temperature, and the reactions at the different temperatures were run in succession. The absorbance however was read on the spectrophotometer at 1-min intervals.
Results.
Figure one shows the absorbance values of each of the temperature-controlled reactions compared to time in minutes. This is an indication of the rate at which starch molecules turn to glucose in each tube.
