Part 5: Amino acids, zwitterions and proteins

Use two functional groups in the same molecule to predict charge, optical activity and peptide chemistry, then interpret amino-acid chromatography.

Find the amino and acid groups on the same molecule

An amino acid contains an amino group and a carboxylic acid group. In a 2-amino acid, H₂NCH(R)COOH, NH₂ is bonded to carbon 2, the carbon next to the carboxyl carbon. Glycine has R = H; alanine has R = CH₃. Extra acidic or basic groups may occur in R, so inspect the whole structure.

The conventional neutral formula makes connectivity easy to see but is not the dominant representation in many aqueous and solid-state conditions. Proton transfer from the acid group to the amino group gives a zwitterion, H₃N⁺CH(R)COO⁻, with both formal charges on one molecule and zero net charge.

Strong electrostatic attractions between zwitterions help explain their high melting temperatures and low volatility. Net zero charge does not mean there are no charged sites. The distribution of forms in solution depends on pH and the ionisation equilibria.

The reagent chooses which charged group changes

In strongly acidic solution the carboxylate accepts H⁺, giving H₃N⁺CH(R)COOH for a simple one-amine/one-acid amino acid. In strongly alkaline solution OH⁻ removes a proton from NH₃⁺, giving H₂NCH(R)COO⁻ and water. Around an appropriate intermediate pH the zwitterion is prominent.

Amino acids are amphoteric: they can react with both acids and bases. Explain this using the groups that actually transfer a proton. Starting from a zwitterion, acid protonates COO⁻; it does not add a fifth bond to an already protonated nitrogen.

Worked extra-group example: H₂NCH(CH₂COOH)COOH has two carboxyl groups. At sufficiently high pH its limiting form is H₂NCH(CH₂COO⁻)COO⁻, with net −2 rather than −1. At sufficiently low pH the simple limiting form has NH₃⁺ and two COOH groups, net +1.

Check both structure and enantiomeric composition

Alanine’s carbon 2 has NH₂, COOH, CH₃ and H, so it is a chiral centre; a single enantiomer in solution rotates plane-polarised monochromatic light. Glycine has two H atoms attached to carbon 2 and is achiral there. Do not assert that all amino acids are optically active.

Protonation changes the groups’ charge but does not ordinarily remove the four-different-groups arrangement. A racemic solution of a chiral amino acid has zero net rotation because the two enantiomers cancel. Optical activity therefore depends on sample composition as well as molecular structure.

If a supplied amino acid has additional stereocentres or symmetry, inspect each centre and the whole molecule. The simple glycine/alanine comparison explains the single-centre requirement without making a universal claim about all conceivable amino acids.

Make the C(=O)–NH bond and keep the end groups

A peptide bond is an amide link formed when the carboxyl group of one amino acid reacts with the amino group of another, with water eliminated. A dipeptide has one peptide bond, a free amino end and a free carboxyl end unless otherwise stated.

Glycine followed by alanine gives H₂NCH₂CONHCH(CH₃)COOH. Alanine followed by glycine gives H₂NCH(CH₃)CONHCH₂COOH. These have the same overall formula but different sequences. The carbonyl carbon of one residue bonds directly to the N of the next.

Proteins are polypeptides: many amino-acid residues joined by peptide bonds. Interactions between carbonyl O and N–H groups help determine folded structures, but formation of a hydrogen bond is distinct from formation of the covalent peptide backbone. Heating or pH changes that alter shape are not automatically complete peptide hydrolysis.

Hydrolyse first, then separate the amino acids

Heating a protein with acid or alkali hydrolyses peptide links. Acidic hydrolysis leaves amino groups protonated; alkaline hydrolysis leaves carboxyl groups deprotonated. The starting sequence is lost when every link is cleaved, so complete hydrolysis reveals composition but not the original order of residues.

Amino acids in the hydrolysate can be separated by chromatography. Spot a small sample and standards on a pencil baseline above the solvent level, develop under controlled conditions and mark the solvent front promptly. Amino acids are usually colourless, so use a suitable locating reagent such as ninhydrin under supervised conditions.

Rf = distance travelled by spot centre from baseline ÷ distance travelled by solvent front from baseline. Matching a standard under the same conditions supports an identity; Rf is not a universal constant independent of solvent and stationary phase. Two amino acids can co-migrate, so one spot is not absolute proof of one substance.

Original interpretation: three spots at Rf 0.25, 0.50 and 0.75 match three standards run on the same plate. This supports three detectable amino-acid components. It does not show a three-residue protein, equal amounts, or a particular sequence: each residue may occur repeatedly and staining responses may differ.

Quick checks

Original Finesse questions. Reveal the indicative worked solutions after attempting each question; these are not official Edexcel mark allocations.

Sources