Unit 24: Organic Nitrogen Compounds — Long Questions

Order of basicity ethylamine > ammonia > phenylamine (pKb: ethanamine 3.29 < ammonia 4.75 < phenylamine 9.38; the smaller the pKb, the stronger the base).

Ethylamine is a stronger base than ammonia because the ethyl group is electron-donating (+I effect); it pushes electron density onto the nitrogen atom, increasing the availability of the lone pair for accepting a proton, so the equilibrium CH₃CH₂NH₂ + H₂O ⇌ CH₃CH₂NH₃⁺ + OH⁻ lies further to the right than for ammonia.

Phenylamine is a much weaker base than ammonia. In phenylamine, the nitrogen is attached directly to the benzene ring; the nitrogen is essentially planar (sp²-like) with its lone pair in a p-orbital that overlaps and delocalises with the π electron system of the ring. This delocalisation makes the lone pair far less available to bond to a proton, so phenylamine is about a million times less basic than a comparable alkylamine, reflected in its much larger pKb (9.38) and much smaller Kb (4.2 × 10⁻¹⁰).

Electrophoresis is a technique used to separate a mixture of amino acids (or dipeptides) based on their electric charge. A few drops of the mixture are applied to a filter paper or gel placed in a buffered solution between two electrodes, and an electric field is applied across it.

Each amino acid has a characteristic isoelectric point (pI), the pH at which it exists predominantly as the neutral zwitterion (no overall charge). If the pI of an amino acid is greater than the pH of the buffer, the amino acid carries a net positive charge and migrates towards the cathode (negative electrode); if its pI is lower than the buffer pH, it carries a net negative charge and migrates towards the anode (positive electrode). The greater the difference between pI and the buffer pH, the faster the amino acid migrates. For example, at pH 6, lysine (pI = 9.74) migrates towards the cathode, glutamic acid (pI = 3.22) migrates towards the anode, and alanine (pI = 6.02, close to the buffer pH) barely moves. If two amino acids have similar pI values, the one with the larger molecular weight moves more slowly, since the same charge must carry a greater mass.

Since amino acids are colourless, ninhydrin is sprayed onto the paper/gel as a locating agent; it reacts with amino acids to give a purple-coloured product, revealing the spots.

Electrophoresis is only an analytical method, used to determine the number and identity of amino acids in a mixture — it cannot separate large (bulk) quantities. For actual bulk separation, other techniques such as column chromatography are used.