Use a colorimeter to follow enzyme reactions
A colorimeter can quantify a colour change over time when the enzyme reaction produces or removes a coloured substance.
Section 3.1 · Mode of action of enzymes
AS Level · Cambridge International Biology 9700, 2028–2030. These lessons teach enzyme concepts and experimental reasoning. Written practice is self-checked; it does not demonstrate supervised practical competence or replace teacher-reviewed assessment.
Before you begin
- Recognise that proteins have specific three-dimensional shapes and that reaction rates can be compared using controlled measurements.
Work on paper. Try each question before revealing help, and explain why your method works.
Quick readiness check
What does a colorimeter measure in a coloured reaction mixture?
What you will learn
- Outline how a colorimeter follows a colour-changing enzyme reaction
- Explain the purpose of a blank, consistent wavelength and timed readings
Measure light through a coloured sample
A colorimeter passes selected-wavelength light through a sample and measures transmitted light or absorbance. As the concentration of a coloured substance changes during a reaction, the reading changes under consistent conditions.
Choose an appropriate filter or wavelength for the colour being measured. Use a blank containing the other relevant reagents without the coloured substance, so the instrument is zeroed for the background and cuvette.
Make comparable progress readings
Mix enzyme and substrate using a defined start time, then take readings at regular intervals. Keep the same cuvette orientation, volume, path length and wavelength; wipe the cuvette and avoid bubbles or fingerprints in the light path.
Plot the colorimeter reading against time. Interpret the direction of change from the assay: absorbance may rise or fall depending on whether coloured product forms or coloured substrate disappears. A reading is not automatically a direct concentration unless the relationship is calibrated.
Pause and explain
Why is a reagent blank used?
Worked example
A coloured product forms during an enzyme reaction and absorbance rises over time. State what should be plotted and one control.
Show the worked solution
- Plot absorbance against time.
- Use a reagent blank and hold the wavelength and cuvette conditions constant.
Answer Plot absorbance against time to follow product formation. Zero the colorimeter with a suitable blank and keep wavelength, sample volume and cuvette orientation consistent.
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Write your answer and reasoning first. Use a hint only if you are stuck. The model solution is for self-checking; your written work is not automatically marked.
Outline how to use a colorimeter to follow a colour-changing enzyme reaction.
Give me a hint
Describe baseline, regular readings and controlled optical conditions.
Compare my reasoning
- Select a suitable wavelength and zero with a blank.
- Measure the reaction mixture at regular times under consistent cuvette conditions.
Select a suitable wavelength or filter, zero the colorimeter with an appropriate reagent blank, then record readings at regular intervals after a clearly timed enzyme–substrate mix. Keep cuvette, volume and orientation consistent.
Look for these in your work
- Blank and wavelength included
- Timed readings and fair conditions included
Absorbance falls from 0.80 to 0.32 during an assay in which a coloured substrate is used. Interpret the direction without claiming a product measurement.
Give me a hint
Use what the instrument reading indicates about the coloured substance.
Compare my reasoning
- Less light is absorbed as the reading falls.
- The coloured substrate may be disappearing.
The absorbance decreased, consistent with a decrease in the coloured substrate under this assay. The reading alone does not identify the product or give its concentration without calibration.
Look for these in your work
- Reading direction interpreted
- Calibration limit stated
Bubbles are trapped in the cuvette and readings vary. Explain one likely effect and a method improvement.
Give me a hint
Consider how bubbles affect the light path.
Compare my reasoning
- Bubbles scatter or block light inconsistently.
- Prepare a bubble-free sample and handle the cuvette consistently.
Bubbles can alter the light path and scatter light, making readings inconsistent. Prepare a bubble-free sample, wipe the cuvette and keep its orientation and volume consistent.
Look for these in your work
- Optical effect identified
- Practical improvement given
Common mistakes
- Assuming absorbance always rises during every enzyme reaction
- Using a blank that contains the coloured product being measured
- Treating absorbance as concentration without a calibration or stated relationship
What can you explain now?
What does a colorimeter measure in a coloured reaction mixture?
Compare with the explanation
Light transmitted through or absorbed by the sample
The optical reading changes as the coloured mixture changes.
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Make it stick
What does the colorimeter reading track, why is a blank needed, and which conditions must stay consistent?
If you needed the solution, close it and solve the problem again from a blank page. A correct answer is stronger when you can explain the reason for each step.