Amino acids

· Amino acids contain both an amine group and a carboxylic acid group.
· General structure: H₂N–CHR–COOH, where R = side chain.
· Because they contain both acidic and basic groups, amino acids are amphoteric.
· The –COOH group can act as an acid by donating H⁺.
· The –NH₂ group can act as a base by accepting H⁺.

This diagram shows how an amino acid can form a zwitterion by internal proton transfer. The amine group becomes –NH₃⁺ and the carboxyl group becomes –COO⁻. Source

Zwitterions and isoelectric point

· A zwitterion has both a positive charge and a negative charge in the same molecule.
· Amino acid zwitterion form: ⁺H₃N–CHR–COO⁻.
· A zwitterion has no overall charge because the charges cancel.
· The isoelectric point, pI, is the pH at which the amino acid has no overall charge.
· At pH = pI, the amino acid exists mainly as a zwitterion and shows no net movement in electrophoresis.
· At low pH, amino acids are more protonated and tend to have a positive overall charge.
· At high pH, amino acids are more deprotonated and tend to have a negative overall charge.

This diagram links pH to the overall charge of an amino acid. It clearly shows the cation, zwitterion and anion forms used to predict electrophoresis movement. Source

Amide (peptide) bond formation

· Amino acids join by condensation to form an amide bond, also called a peptide bond.
· The bond forms between the –COOH group of one amino acid and the –NH₂ group of another.
· During formation, H₂O is eliminated.
· Peptide bond linkage: –CONH–.
· Dipeptide = formed from 2 amino acids joined by 1 peptide bond.
· Tripeptide = formed from 3 amino acids joined by 2 peptide bonds.
· Exam focus: identify the atoms removed as H₂O and draw the new –CONH– bond correctly.

This structural diagram shows two amino acids undergoing condensation to form a peptide bond. It highlights the removal of water and formation of the –CONH– linkage. Source

Electrophoresis of amino acids and dipeptides

· Electrophoresis separates amino acids/dipeptides because they can have different overall charges at a given pH.
· Species with positive overall charge move towards the negative electrode / cathode.
· Species with negative overall charge move towards the positive electrode / anode.
· Species with no overall charge stay at or near the origin / baseline.
· Key rule: compare pH with pI.
· If pH < pI, the amino acid/dipeptide is more positive → moves to the cathode.
· If pH > pI, the amino acid/dipeptide is more negative → moves to the anode.
· If pH = pI, the amino acid/dipeptide has no overall charge → little/no movement.
· The syllabus requires interpreting/predicting electrophoresis results, but assembling the apparatus is not tested.

These diagrams show how the charge of an amino acid changes with pH. They are useful for predicting whether a substance moves to the positive electrode, negative electrode, or remains near the origin. Source

This image demonstrates how amino acids with different net charges separate in an electric field. It reinforces the rule that positive ions move to the negative electrode and negative ions move to the positive electrode. Source

Exam method: predicting electrophoresis movement

· Step 1: identify the pH of the buffer and the pI of each amino acid/dipeptide.
· Step 2: decide whether each species is positive, negative or neutral.
· Step 3: predict direction: positive → cathode, negative → anode, neutral → no movement.
· Step 4: if comparing distances, larger charge usually means greater attraction to the opposite electrode.
· Step 5: remember that dipeptides can also form charged species, so apply the same pH vs pI logic.

Common exam traps

· Do not say zwitterions are “uncharged”; they have charges but no overall charge.
· Do not confuse peptide bonds with ester bonds; peptide bonds are amide links, –CONH–.
· Do not forget that condensation releases water.
· Do not reverse electrode movement: positive → negative electrode, negative → positive electrode.
· Do not assume all amino acids move at neutral pH; movement depends on pH compared with pI.