Amino acid analysis refers to a variety of methodologies which are used to determine the amino acid content of peptides, proteins and other samples. AA’s, of course, are organic compounds which contain an amino group and a carbolic acid group as well as any of many possible side groups. These side groups are typically linked by peptides, forming proteins or compounds which are employed as intermediates in the metabolic process or as chemical messengers within living organisms.
Proteins and peptides are organized as linear polymers; these macromolecules are composed of covalently bonded AA residues. The properties of a given organic molecule (such as a peptide or protein) are determined by the sequence of AA’s present – data which is gathered through amino acid analysis. A peptide is a smaller molecule, often consisting of only a few amino acids. Proteins, by comparison are large and are generally folded into a specific structural model containing a larger number of AA’s.
Identification and quantification of proteins and peptides can be determined through analysis; it is also used to detect atypical AA’s present in a peptide or protein analyzed as well as for the evaluation of fragmentation strategies in peptide mapping applications. Before the analysis proper can be performed, proteins and peptides must be hydrolyzed to separate their constituent amino acids. After hydrolysis, amino acid analysis can be performed in the same manner as is used for free amino acids (such as is done in preparing pharmaceuticals).
The most common methodology for the analysis of AA’s in a sample involved chromatographic separation of the AA’s present. Automated chromatographic instruments with post column derivation are the most commonly used technologies at present; most analysis of amino acids is most commonly done with a liquid chromatograph (low or high pressure) which can generate mobile phase gradients. This procedure separates the AA analytes in the column.
Background contamination is always a concern when performing AA analysis. High purity reagents are absolutely necessary. For instance, low purity hydrochloric acid can contribute to glycine contamination. Analytical reagents are changed routinely every few weeks using only high-pressure liquid chromatography (HPLC) grade solvents. Potential microbial contamination and foreign material that might be present in the solvents are reduced by filtering solvents before use, keeping solvent reservoirs covered and not placing instruments in direct sunlight.
The accuracy and reliability of the analysis process can be ensures through basic best laboratory practices. The lab must be sterile, instruments installed in a relatively low traffic area and pipettes cleaned (or replaced) and calibrated regularly. Vials containing samples must be opened only when absolutely necessary; contamination by dust can cause elevated glycine, alanine and serine.
Accuracy in AA analysis depends on proper maintenance of the instruments, which should be checked for leaks daily if the equipment is in regular use. The stability of the lamp, detector and the column’s ability to provide proper resolution of individual AA’s should all be checked and filters and other consumables replaced regularly.