Editing Protein sequencing (section)

=== Separation and quantitation ===
The amino acids can be separated by [[ion-exchange chromatography]] then derivatized to facilitate their detection. More commonly, the amino acids are derivatized then resolved by [[High-performance liquid chromatography#Reversed-phase chromatography .28RPC.29|reversed phase HPLC]].

An example of the ion-exchange chromatography is given by the NTRC using sulfonated polystyrene as a matrix, adding the amino acids in acid solution and passing a buffer of steadily increasing [[pH]] through the column. Amino acids are eluted when the pH reaches their respective [[isoelectric point]]s. Once the amino acids have been separated, their respective quantities are determined by adding a reagent that will form a coloured derivative. If the amounts of amino acids are in excess of 10 nmol, [[ninhydrin]] can be used for this; it gives a yellow colour when reacted with proline, and a vivid purple with other amino acids. The concentration of amino acid is proportional to the absorbance of the resulting solution. With very small quantities, down to 10 pmol, fluorescent derivatives can be formed using reagents such as [[phthaldehyde|ortho-phthaldehyde (OPA)]] or [[fluorescamine]].

Pre-column derivatization may use the Edman reagent to produce a derivative that is detected by UV light. Greater sensitivity is achieved using a reagent that generates a fluorescent derivative. The derivatized amino acids are subjected to reversed phase chromatography, typically using a C8 or C18 [[Column chromatography|silica column]] and an optimised [[Eluent|elution]] gradient. The eluting amino acids are detected using a UV or fluorescence detector and the peak areas compared with those for derivatised standards in order to quantify each amino acid in the sample.