Interpret electrophoresis of amino-acid and dipeptide mixtures
Predict the relative movement of amino acids and dipeptides at different pHs using each species' isoelectric point and net charge.
Section 34 · Nitrogen compounds
A Level; Cambridge International Chemistry 9701, 2028–2030 syllabus, Topic 34. Use the stated reagents, conditions and product scope. Written practice is self-checked This online lesson cannot demonstrate supervised laboratory skills.
Before you begin
- Recall nucleophilic substitution, reduction, acyl-chloride addition–elimination, acid–base equilibria and covalent bonding.
Work on paper. Try each question before revealing help, and explain why your method works.
Quick readiness check
At a fixed pH, what should you compare for each component in an amino-acid mixture?
What you will learn
- Compare several species with their pI values
- Predict directions at a stated pH
- Interpret zero movement and relative migration without assuming equal speed
Apply the same pH–pI rule to each species
For each amino acid or dipeptide, compare the buffer pH with that species' pI. Below pI it is net positive; above pI it is net negative; at pI it has no net movement.
A mixture can therefore separate because different components have different pI values and charge states at the same pH.
Interpret only what the data support
Use anode/cathode polarity to assign direction. A component with zero net charge remains near the origin; charged components migrate toward the opposite electrode.
Do not infer exact migration distances or speeds from charge alone unless the question supplies the relevant information. The syllabus excludes assembling the apparatus but includes predicting and interpreting results.
Pause and explain
At pH 6, species B has pI 8. Which way does B move?
Worked example
At pH 6, species A has pI 4 and species B has pI 8. Predict their directions.
Show the worked solution
- For A, pH is above pI, so A is negative.
- For B, pH is below pI, so B is positive.
- Match each sign to the electrode with opposite charge.
Answer A moves toward the positive anode; B moves toward the negative cathode.
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Test this topic →From guided practice to a new situation
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.
At pH 7, amino acid A has pI 5, amino acid B has pI 7 and dipeptide C has pI 9. Predict each charge and direction.
Give me a hint
Compare each pI separately with pH 7.
Compare my reasoning
- A: pH above pI, so negative.
- B: pH equals pI, so zero net charge.
- C: pH below pI, so positive.
A moves to the anode; B has no net movement; C moves to the cathode.
Look for these in your work
- A comparison
- B comparison
- C comparison
A mixture at pH 4 contains two species with pI values 3 and 6. Which moves toward the anode and which toward the cathode?
Give me a hint
The species with pI below pH is negative.
Compare my reasoning
- pI 3 species is negative at pH 4.
- Negative species moves to the positive anode.
- pI 6 species is positive and moves to the cathode.
pI 3 species moves to the anode; pI 6 species moves to the cathode.
Look for these in your work
- First charge
- Anode
- Second charge/direction
A student claims the pI 7 component at pH 7 must disappear from the electrophoresis result because it has no charge. Refine the interpretation.
Give me a hint
Zero net charge predicts movement, not visibility or presence.
Compare my reasoning
- The component is still present in the mixture.
- At pH equal to pI it has no net movement.
- It remains near the origin rather than disappearing.
It remains present but shows no net migration from the origin under that condition.
Look for these in your work
- Presence distinguished
- Charge condition
- Movement described
Common mistakes
- Using one shared pI for all mixture components
- Reversing anode and cathode
- Assuming zero charge means no polar groups
- Claiming exact travel distances without evidence
What can you explain now?
At a fixed pH, what should you compare for each component in an amino-acid mixture?
Compare with the explanation
Its pI with the buffer pH
That comparison predicts its net charge.
After trying it yourself, choose your next review. This is your self-assessment.
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Make it stick
How can different pI values separate amino acids and dipeptides in one electrophoresis run?
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.