Amino acid composition analysis

The number of every kind of amino acid in a protein sample can be determined through amino acid composition analysis. Purifled protein sample is hydrolyzed into its constituent amino acids through heating it at 110°C in 6 M HCl for 24 h in a sealed and evacuated tube. The resulting  mixture (hydrolysate)  of amino acids is subjected  to ion  replace  chromatography on a column  of sulfonated  polystyrene  to separate  out the 20 standard  amino acids.  The separated amino acids are then quantifled and detected    through reacting them with ninhydrin.  The α-amino acids generate a blue color while the imino acid proline generates a yellow color. The amount of every amino acid in an unknown sample can be determined through comparison of the optical absorbance with  a  known  amount  of  each  of  the  individual  amino  acids  in  a  ordinary sample. With ninhydrin as little as 10 nmol of an amino acid can be detected. A more sensitive  detection  system which detecting  down to 10 pmol of an amino acid uses  fluorescamine   to  react  with  the  α-amino  group  to  form  a  fluorescent product.  The Amino acid composition analysis indicates the number of every amino acid residue in a peptide but it does not give information on the sequence of the amino acids. For instance, the amino acid composition of the oligopeptide:

                                      Val-Phe-Asp-Lys-Gly-Phe-Val-Glu-Arg
would be:
                                     (Arg, Asp, Glu, Gly, Leu, Lys, Phe₂, Val₂)

where the commas and the parentheses  among  each amino  acid indicate  in which this is the amino acid composition not the sequence.