Practical 8 - Investigation into the effect of temperature or pH on enzyme activity Flashcards
Why do we use milk in this practical?
Contains fats/lipids that lipase will break down
What contains fats/lipids in this practical and what happens to this?
Milk, which is broken down by lipase
What does lipase break down in this practical?
The milk
What does phenolphthalein do at different pH levels?
Pink in alkaline (pH of about 10)
Colourless in a pH less than 8.3
What’s our indicator that we’re using and the colour change we’re expected to see as we stir?
Phenolphthalein
Pink (alkali) to colourless (acidic)
What makes the phenolphthalein turn pink at the start of the reaction and why?
Sodium carbonate - is an alkali
What is sodium carbonate?
An alkali
How much lipase do we need in total for this practical?
5g/100cm^3
What does lipase do in this practical?
Catalyses the hydrolysis of triglycerides into fatty acids and glycerol
What catalyses the hydrolysis of triglycerides into fatty acids and glycerol in this practical?
Lipase
What is lipase?
An enzyme
What is our enzyme in this practical?
Lipase
What causes the pH of the solution to change and how?
Fatty acids from the hydrolysis of the triglycerides in the milk, which contribute H+ ions to the solution, making it more acidic
Which ions make a solution more acidic?
H+
What do H+ ions do to a solution?
Make it more acidic
What do fatty acids do to the solution here and where have they come from?
Turn it colourless
From the hydrolysis of triglycerides by lipase to the milk
Concentration of sodium carbonate used
0.05moldm^-3
What are the temperatures of the water baths used in this practical?
15, 25, 35, 45, 55 (degrees Celsius)
Which one of the temperatures used in the practical is different and why?
15 degrees Celsius - is below room temperature, so we need ice
What do we need to do to the 15 degrees Celsius water bath and why?
Add ice as it’s below room temperature
What’s the first thing we do during this practical?
Place a breaker of lipase solution in the 25 degrees Celsius (will be at different temperatures to match the other ones) water bath
Why do we place the beaker of lipase solution in the water bath that’s to be used?
To equilibrate the enzyme to the right temperature
How do we equilibrate the enzyme to the correct temperature?
Place a breaker of lipase solution in the water bath at the correct temperature
How much milk do we use in the test tube?
5cm^3
How much phenolphthalein do we use in the test tube?
5 drops
How much sodium carbonate solution do we add to the test tube?
7cm^3
What do we do with the test tube once we’ve added all of the solutions?
Place in the water bath for 10 minutes to equilibrate
Why do we place the test tube in the water bath for 10 minutes?
To equilibrate
How much of the lipase from the beaker do we add to the test tube?
1cm^3
What do we do once we’ve added the lipase from the beaker into the test tube?
Start the stop clock - stir the contents of the test tube until the solution loses its pink colour and record the time taken
What are we recording the time taken for?
The solution to turn from pink to colourless by stirring it
What do we repeat this whole practical for?
All of the different temperatures (remember that the lipase solution temperatures is to be kept the same too)
What’s the result of this practical?
The higher the temperature, the less time taken for the colour change
Why did we receive the result we did - the higher the temperature, the less time taken for the colour change - ?
Increased temperature = more kinetic energy = probability of successful collisions to form enzyme-substrate complexes increases = less time to change colour as milk is catalysed faster to release fatty acids which lower the pH, causing the colour of the phenolphthalein to change
Under which conditions is milk catalysed faster to release fatty acids that lower the pH and why?
Higher temperatures, as there’s more kinetic energy, meaning that the probability of successful collisions to form enzyme-substrate complexes increases
Why does milk being catalysed faster cause the colour of the phenolphthalein to change quicker?
Releases fatty acids faster, which lower the pH, causing the colour change
Does the rate of the reaction continue to increase? Why?
No - the enzyme starts becoming denatured
Why does having some denatured enzymes decrease the rate of a reaction?
Less available active sites for the substrate to collide with to catalyse the reaction
Does denaturing happen immediately?
No - it’s a process
How do we actually get the lipase in the first place for this practical?
Extract it from a living cell and purify it for laboratory use
What must lipase be after being extracted from a cell before laboratory use?
Purified
What type of range bars on a graph show more reliable data?
-smaller
-no overlap with next set of results
Which other variables which were not controlled SHOULD have been controlled?
Enzyme concentration
Milk concentration
pH of milk
Type of milk used
What are the sources of inaccuracy in this practical?
Maintaining a constant temperature
Difficulty determining when the milk has changed colour
How do we maintain a constant temperature in this practical?
Use a thermostatically controlled water bath
How do we solve the problem of having difficulty determining when the milk has changed colour?
Use a colourimeter
Why should we have used a colourimeter in this practical?
As we had some difficulty determining when the milk had changed colour
