PITC sites are often among the very best sites in terms of least error, but are also well-represented among the highest error group. It is fair to conclude that there are no major obstacles to high quality pre-column techniques, and that they can be used with accuracy as high as any other technique. In this procedure, amino acids react with the AccQ reagent, yielding urea-type compounds. This reaction is much faster than the competing reaction of the reagent with water, and conditions for derivatization are very fast and specific. Reaction requires only 1 minute, after which samples are heated for 10 min at 55 C. Chromatography occurs on a reverse phase HPLC column at 37 C using gradient elution and buffers conveniently available from Waters.
Detection can be either with uv absorption or fluorescence.
Advantages of this newer method include the very simple derivatization procedure, stable derivatives, excellent separation, detection by either absorbance or fluorescence, and commercial availability of reagents. Since the goal of amino acid analysis is not only to identify but to quantitate each amino acid, good procedures for handling the data are required. While it is always necessary to pay careful attention to the instrumentation, reagents, and columns being employed, it is also necessary to process the data according to expected norms. Color values and calibration curves are the most important consideration, since all values derive from them. Calibration curves can be developed for external or internal standards.