The amino acid/peptide analyzer was sold as a kit by the Dionex Corporation. In 1980, Thressa's research assistant, Joe Davis, and her postdoctoral fellow, Greg Dilworth, assembled it, like an erector set, by attaching equipment to a frame of connected rods on a work cart in the laboratory. They used the analyzer for identifying amino acids in selenium-containing proteins. Earl's group also took advantage of it for analyzing amino acids in oxidized proteins. These days, high pressure liquid chromatography (HPLC) systems are available from many manufacturers, and any of them can be configured to do the job of analyzing amino acids and peptides.
The instrument consists of several parts. First, it is controlled by a Dionex Chromatograph Programmer, a controlling computer, which programs the times at which different solvents are pumped and the temperatures at which they are pumped. Second, solvents are kept in glass bottles on top of the rack, and their temperature is usually kept at 40o to 65oF by the Dionex Column Heater Control. Third, connected to the mixing column is the filtering and detecting device, the Gilson Spectra/flo Fluorometer. Finally, all the information-the pH level of solvents and the program of solvents/temperatures/time is recorded in the Shimadzu Chromatopac C-R3A Recorder. This particular recorder was a later addition to the instrument.
The operation of the amino acid/peptide analyzer is largely automated but requires some experience. A mixture of amino acids is injected into the system and pumped into the mixing column. Next, each amino acid is then eluted or washed out from the column in a specific order. This is accomplished by pumping in solvents of increasing pH and by varying the column temperature. The eluted amino acid in buffer is then mixed with the detecting materials at the bottom of the column. Subsequently, the mixture goes into the detecting device, in which the filter controls the specificity of the fluorometer. As it passes through the filter, the emission of fluorescence is detected.
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Image Modified The instrument set up in Thressa's laboratory.
