Practical: Investigating pH & Enzyme Activity (Edexcel IGCSE Biology)

Practical: Enzymes & pH

• Amylase is an enzyme that digests starch (a polysaccharide of glucose) into maltose (a disaccharide of glucose)

• The effect of different pH levels on the activity of amylase can be investigated

Apparatus

• Spotting tile

• Measuring cylinder

• Test Tube

• Syringe

• Pipette

• Stopwatch

• Buffer solutions

• Iodine

• Starch solution

• Amylase solution

Method

• Add a drop of iodine to each of the wells of a spotting tile

• Use a syringe to place 2 cm³ of amylase into a test tube

• Add 1cm³ of buffer solution (at pH 2) to the test tube using a syringe

• Use another test tube to add 2 cm³ of starch solution to the amylase and buffer solution, start the stopwatch whilst mixing using a pipette

• Every 10 seconds, transfer a droplet of the solution to a new well of iodine solution (which should turn blue-black)

• Repeat this transfer process every 10 seconds until the iodine solution stops turning blue-black (this means the amylase has broken down all the starch)

• Record the time taken for the reaction to be completed

• Repeat the investigation with buffers at different pH values (ranging from pH 3.0 to pH 7.0) 

Investigating the effect of pH on enzyme activity

Results and Analysis

• Amylase is an enzyme which breaks down starch

• When the iodine solution remains orange-brown, all the starch has been digested

• This investigation shows:

  • At the optimum pH, the iodine stopped turning blue-black and remained orange-brown within the shortest amount of time

    • This is because the enzyme is working at its fastest rate and has digested all the starch

• At higher or lower pH's (above or below the optimum) the iodine took a longer time to stop turning blue-black or continued to turn blue-black for the entire investigation

  • This is because on either side of the optimum pH, the enzymes are starting to become denatured and as a result are unable to bind with the starch or break it down

Limitations

• The starch and amylase solutions that need to be used should be placed in a water bath at optimum temperature before being used

• A colorimeter can be used to measure the progress of the reaction more accurately by measuring the absorbance/transmission of light through the coloured solution

  • A control of iodine solution would be used for comparison

A graph showing the optimum pH for an enzyme from a region of the small intestine

Applying CORMS to practical work

• When working with practical investigations, remember to consider your CORMS evaluation

CORMS Evaluation

• In this investigation, your evaluation should look something like this:

  • C - We are changing the pH of the environment

  • O - This is not relevant to this investigation as we aren't using an organism

  • R - We will repeat the investigation several times to ensure reliability

  • M1 - We will measure the time taken for

  • M2 - the iodine to stop turning black

  • S - We will control the concentration and volume of the amylase, iodine and starch solution used in the investigation