Amino Acid Analysis by High Performance Liquid Chromatography of Phenylthiocarbamyl Derivatives
Reliable and sensitive amino acid analyses are important steps in studies of protein structures. In this respect, high performance liquid chromatography (HPLC) has greatly increased speed and sensitivity. The use of ortho-phthalaldehyde  and subsequent fluorimetric detection is applicable also to HPLC. Thus, separation of underivatized amino acids by ion exchange HPLC and subsequent detection by post-column derivatization , as well as pre-column derivatization and subsequent separation of amino acid derivatives by reverse phase HPLC have been widely used. We have tested both methods and find, like others, that they are suitable. However, base line drift due to ammonia contamination is a serious problem when maximal sensitivity is attempted in the post-column mode of the ortho-phthalaldehyde method. Similarly, the lack of direct detection of proline requires one further step in this derivatization procedure  both in the pre- and post-column derivatization mode. Therefore, additional methods for analysis are of value. In this respect, pre-column derivatization with phenylisothiocyanate (PITC) to produce the phenylthiocarbamyl (PTC)-amino acids for subsequent separation by reverse phase HPLC has proved highly efficient and valuable [4, 5]. We have tested this method extensively and find it reliable, sensitive, and easy to use.
KeywordsHigh Performance Liquid Chromatography Amino Acid Derivative Reverse Phase High Performance Liquid Chromatography Coagulation Factor VIII Artifact Peak
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