Polypeptides are polymers of amino acids

Polypeptides are formed through condensation reactions between amino acids. The carboxyl group of one amino acid reacts with the amine group of another, forming a peptide (amide) link.

A dipeptide is formed when two amino acids are linked by a peptide bond, whereas three linked amino acids create a tripeptide.

For example, the formation of a dipeptide, where R represents the amino acid side group, is illustrated below:

Long chains of polypeptides, with more than 50 amino acid units, are called proteins. The sequence and shape of proteins are determined by the order of amino acids in their polypeptide chain.

Hydrolysis of polypeptides

Polypeptide chains can be broken back down into amino acids through hydrolysis reactions. This process typically involves the use of hot aqueous acids, such as concentrated hydrochloric acid. The hydrolysis reaction adds a water molecule to break the peptide bonds between amino acids.

Once the polypeptide has been hydrolysed, the resulting mixture of amino acids can be analysed using chromatography to identify the specific amino acids present.

Thin layer chromatography of amino acids

Thin layer chromatography (TLC) is a technique used to separate and identify amino acids in a mixture.

The process involves the following steps:

1. Sample application - The amino acid mixture is placed as a small spot on a TLC plate coated with a silica or alumina adsorbent, which serves as the stationary phase.
2. Developing chamber - The plate is positioned in a developing chamber containing a solvent or solvent mixture, known as the mobile phase.
3. Separation - As the solvent ascends the TLC plate by capillary action, it carries the amino acids upwards. The amino acids travel at different speeds based on their solubility in the solvent. Less soluble amino acids adhere more strongly to the silica, slowing their ascent, resulting in the separation of amino acids into distinct spots.
4. Visualisation - Once the solvent has nearly reached the top of the plate, it is removed and dried. The amino acid spots are made visible using developing agents such as ninhydrin or ultraviolet light.
5. Identification - The distance travelled by each amino acid spot (A) relative to the solvent front (B) is used to calculate the retention factor (R_f) values using the following equation:

${\text{R}}_{\text{f }}=\frac{A}{B}=\frac{\text{distance moved by amino acid spot}}{\text{distance moved by solvent}}$

The R_f values of the sample spots can be compared to those of known amino acid standards run on the same plate under the same conditions to identify the amino acids present in the mixture.