Amino acid analysis

Abstract

Amino acid analysis is carried out to provide information on the different types and concentrations of amino acids in proteins and peptides from both natural and synthetic sources. It is also an important analysis technique in the determination of levels of free amino acids in foodstuffs and clinical specimens such as physiological fluids and tissues. In addition to the 20 well known amino acids some 700 unusual naturally occurring amino acids have been reported (1,2). The identification and quantification of amino acids can be divided into five constituent parts, sample purification and preparation, hydrolysis, derivatization, analysis, and data handling. a) Sample purification and preparation: In many types of samples the amino acids can be found in media not compatible with any of the analytical systems available. The presence of buffer salts, urea, detergents etc., can interfere with both the hydrolysis and subsequent derivatization so some purification and sample preparation steps are required before hydrolysis, derivatization and analysis. Methods that utilize postcolumn derivatization of amino acids are generally not affected by buffer component concentrations to the same extent as precolumn derivatization methods. Sample purification methods are outlined in Section 2. b) Hydrolysis of proteins and peptides: Purified peptides and proteins are subjected to elevated temperatures under acidic conditions to break the peptide bonds to produce free amino acids. Protein and peptide samples are first dried in a hydrolysis tube. In Liquid Phase hydrolysis the acid solution is added to the sample tube and each sample tube is then sealed under vacuum. In vapor phase hydrolysis the individual sample tubes are placed into a hydrolysis vessel and acid solution is added to the vessel before being sealed so only the vapor from the heated acid solution is in contact with the sample. Generally higher sensitivity is achieved with the vapor phase hydrolysis method because any possible contaminants from the acid hydrolysis solution are kept to a minimum. Hydrolysis methods are outlined in Section 3. c) Derivatization of free amino acids: Because amino acids do not have a natural chromophore some form of derivatization is required to aid their detection. In postcolumn derivatization, the derivatization occurs after the liquid chromatography which usually occurs on an ion exchange chromatography column whereas precolumn derivatization is carried out before the chromatography which is typically reverse phase liquid chromatography (RPLC). It is possible, however, to analyze amino acids without the need for derivatization but specialised detectors such as a mass spectrometer is required. Derivatization and Underivatized methods are outlined in Sections 4 and 4.8.1, respectively. d) Liquid chromatography: Typically, the derivatized amino acids are separated into the component parts usually by either ion exchange or reverse phase chromatography. The choice of generic chromatography column required is usually determined by the derivatization method. Capillary electrophoresis (CE) is considered a powerful technique owing to its high separation efficiency, small sample requirement, rapid analysis time and low running costs. It is used as an alternative to HPLC in the separation of free amino acids. e) Data Acquisition and Data Handling: The separated derivatized amino acids are identified by their peak retention times and quantified by measuring the area under the peak or by peak height. Comparison with known amounts of standards run under identical conditions will yield amount of sample. The use of internal standards will improve the accuracy of the method. There are many commercially available acquisition and data handling systems and software packages available. Basic Calculations are outlined in Section 5. © 2008 Humana Press.