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Amino acids are the building blocks of proteins -- one of the most important classes of biological molecule. At A-Level, you need to understand the structure of amino acids, their acid-base behaviour, how they link together to form proteins, and how proteins can be broken down and identified.
An amino acid contains two functional groups attached to the same carbon atom (the alpha-carbon):
The general structure is:
H2N-CH(R)-COOH
where R is the side chain (also called the R group). The identity of R is what distinguishes one amino acid from another. There are 20 naturally occurring amino acids used by living organisms to build proteins, each with a different R group.
| Amino Acid | R Group | Notable Feature |
|---|---|---|
| Glycine (Gly) | -H | Simplest; no chiral centre |
| Alanine (Ala) | -CH3 | Simplest chiral amino acid |
| Serine (Ser) | -CH2OH | R group contains -OH |
| Cysteine (Cys) | -CH2SH | Can form disulfide bridges (S-S) |
| Aspartic acid (Asp) | -CH2COOH | R group is acidic |
| Lysine (Lys) | -(CH2)4NH2 | R group is basic |
| Phenylalanine (Phe) | -CH2C6H5 | R group is non-polar, aromatic |
The nature of R determines the amino acid's properties: acidic R groups have extra -COOH, basic R groups have extra -NH2, non-polar R groups make hydrophobic regions in proteins.
Because amino acids contain both an acidic group (-COOH) and a basic group (-NH2), they undergo an internal acid-base reaction in which the -COOH donates a proton to the -NH2:
H2N-CH(R)-COOH --> +H3N-CH(R)-COO-
The result is a zwitterion -- a species that carries both a positive and a negative charge simultaneously, but has an overall charge of zero.
In the solid state and in solution at a specific pH, amino acids exist predominantly as zwitterions. This explains several of their properties:
The charge on an amino acid depends on the pH of the solution:
| pH Condition | NH2 Group State | COOH Group State | Overall Charge | Species |
|---|---|---|---|---|
| Low pH (acidic) | +H3N- (protonated) | -COOH (protonated) | Positive (+1) | Cation |
| Isoelectric point | +H3N- (protonated) | -COO- (deprotonated) | Zero | Zwitterion |
| High pH (alkaline) | H2N- (deprotonated) | -COO- (deprotonated) | Negative (-1) | Anion |
The isoelectric point (pI) is the pH at which the amino acid exists predominantly as the zwitterion and has no net charge. At this pH, the amino acid will not migrate in an electric field (important for electrophoresis).
| pH relative to pI | Charge on amino acid | Direction of migration |
|---|---|---|
| pH < pI | Positive | Toward cathode (negative electrode) |
| pH = pI | Zero (zwitterion) | Does not migrate |
| pH > pI | Negative | Toward anode (positive electrode) |
All amino acids except glycine are optically active. This is because the alpha-carbon is bonded to four different groups:
When four different groups are attached to a single carbon, that carbon is called a chiral centre (or asymmetric carbon), and the molecule can exist as two non-superimposable mirror images -- called enantiomers.
The two enantiomers are designated D and L (or R and S in IUPAC nomenclature). In biological systems, almost all naturally occurring amino acids are the L-form. The D-form is extremely rare in nature.
Enantiomers have identical physical properties (melting point, solubility, boiling point) except that they rotate the plane of plane-polarised light in opposite directions. They can also interact differently with biological molecules such as enzymes (which are chiral themselves), which is why the D/L distinction matters in biology.
Glycine (R = H) is the exception -- it has two hydrogen atoms on the alpha-carbon, so there is no chiral centre and no optical isomerism.
Amino acids link together through peptide bonds -- a type of amide bond formed by a condensation reaction between the amino group of one amino acid and the carboxyl group of another, with the loss of water:
H2N-CH(R1)-COOH + H2N-CH(R2)-COOH --> H2N-CH(R1)-CO-NH-CH(R2)-COOH + H2O
The bond formed (-CO-NH-) is the peptide bond. The product of two amino acids joining is called a dipeptide. Further amino acids can be added to form tripeptides, and eventually polypeptides (chains of many amino acids).
If you have amino acids A and B:
So from two different amino acids, four dipeptides are possible. But if the question says "one molecule of each," only A-B and B-A are possible (two dipeptides). Read the question carefully.
Key features of the peptide bond:
A polypeptide is a chain of amino acids linked by peptide bonds. A protein is one or more polypeptide chains folded into a specific three-dimensional shape.
The sequence of amino acids in a polypeptide is called the primary structure. This sequence determines the protein's overall shape and function because the R groups interact with each other through:
| Interaction Type | Between Which R Groups | Strength | Example |
|---|---|---|---|
| Hydrogen bonds | Polar R groups (-OH, -NH2, -CONH2) | Moderate | Serine-Asparagine |
| Ionic interactions | Oppositely charged R groups | Strong | Lysine (+) and Aspartic acid (-) |
| Disulfide bridges | Cysteine residues (-SH groups) | Strong (covalent) | Cys-S-S-Cys |
| London forces | Non-polar R groups | Weak individually | Valine-Leucine |
These interactions fold the chain into secondary structures (alpha-helices and beta-pleated sheets), tertiary structure (the overall 3D shape), and sometimes quaternary structure (multiple polypeptide chains assembled together, e.g., haemoglobin has four subunits).
Proteins can be broken down into their constituent amino acids by hydrolysis -- the reverse of peptide bond formation. This can be achieved by:
After hydrolysis, the amino acids produced can be identified using thin-layer chromatography (TLC) or paper chromatography:
Rf = distance moved by the amino acid spot / distance moved by the solvent front
Both distances are measured from the baseline. Rf values are always between 0 and 1.
Pen ink contains dyes that would dissolve in the mobile phase solvent and move up the chromatogram, interfering with the results. Pencil (graphite) is insoluble in the solvents used and stays put.
Forgetting that glycine is not chiral. Glycine has R = H, so the alpha-carbon has two identical groups (two H atoms) and is not a chiral centre.
Writing the wrong form at a given pH. At low pH, the amino acid is positively charged (both groups protonated). At high pH, it is negatively charged (both groups deprotonated). At the isoelectric point, it is a zwitterion.
Confusing the number of dipeptides. From amino acids A and B (one molecule of each), you can form A-B or B-A -- two dipeptides, not one. The peptide bond has directionality.
Drawing the peptide bond incorrectly. It is -CO-NH-, not -CO-N= or -C-NH-. The carbonyl (C=O) must be shown bonded to the nitrogen.
Forgetting to mention ninhydrin. When asked how to visualise amino acids on a chromatogram, the answer is ninhydrin spray followed by heating. Not iodine, not UV light (unless the amino acid is aromatic).
Amino acids are bifunctional molecules that exist as zwitterions at their isoelectric point. All except glycine show optical isomerism due to the chiral alpha-carbon. They link together through peptide bonds (condensation) to form polypeptides and proteins. Proteins can be hydrolysed back to amino acids, which can be identified by chromatography. These concepts connect organic chemistry directly to biology and are frequently tested at A-Level.
Edexcel 9CH0 specification, Topic 18 — Organic nitrogen compounds, sub-strands 18.5–18.7 covers the structure of α-amino acids (general formula H2N-CHR-COOH; R is the side-chain), the zwitterion form predominant in the solid state and at the isoelectric point (pI), the pH-dependence of amino acid charge (cation at low pH, zwitterion at pI, anion at high pH), peptide-bond formation by condensation between the -NH2 of one amino acid and the -COOH of another (loss of H2O), and primary protein structure (sequence of amino acids linked by peptide bonds) (refer to the official specification document for exact wording). The specification also covers the chirality of all natural amino acids except glycine — natural amino acids are L (S configuration at Cα). Examined directly on Paper 2 with synoptic appearances on Paper 3 and CP16 (analytical identification of biological molecules).
Question (8 marks):
(a) Draw the zwitterion form of alanine (CH3CH(NH2)COOH) and explain why amino acids exist predominantly as zwitterions in the solid state and at their isoelectric point. (3)
(b) The pKa values of alanine are pKa1 = 2.34 (-COOH) and pKa2 = 9.69 (-NH3+). Calculate the isoelectric pH (pI) of alanine, and predict the predominant form at pH 1, pH 6 and pH 12. (5)
Solution with mark scheme:
(a) Zwitterion structure: H3N+-CH(CH3)-COO⁻
The carboxylic acid group has lost its H+ to give -COO⁻; the amine group has gained that H+ to give -NH3+. Net charge = 0.
M1 — correct zwitterion structure with -NH3+ and -COO⁻.
A1 — explanation: -COOH (pKa ≈ 2) is a stronger acid than -NH3+ is conjugate acid of -NH2 (pKa of -NH3+ ≈ 9–10). The proton naturally transfers from the more acidic -COOH to the more basic -NH2, giving the zwitterion as the most thermodynamically stable form.
A1 — link to physical properties: the zwitterion is ionic and so amino acids have high melting points (>200 °C, often with decomposition), high water solubility and low solubility in non-polar solvents.
(b) Step 1 — calculate pI.
For an amino acid with two ionisable groups (no ionisable side chain), pI is the average of the two pKa values:
pI = (pKa1 + pKa2) / 2 = (2.34 + 9.69) / 2 = 6.02
M1 — formula stated.
A1 — pI = 6.02 (or 6.0).
Step 2 — predict forms at pH 1, 6, 12.
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