ABSTRACT
Post column reaction (PCR) detection employs an on-line derivatization system to convert chromatographically-separated polypeptides to fluorescent products prior to transfer to a fluorimetric detector. PCR detection offers advantages when compared to detection of polypeptides by native absorbance, by native fluorescence, or by precolumn derivatization with fluorogenic reagents. Post column reaction systems are most cost-effective when operated routinely on a daily basis with a rigorous preventive maintenance program. Post column reactions were performed with the Varian System I or Varian System II post column reaction systems, including the Fluorichrom fluorescence detector in both systems. Reaction of fluorescamine with polypeptide amino groups is pH-dependent. Reaction of O-Phthalaldehyde (OPA) with ammonia produces a fluorescence signal comparable to that for primary amines, indicating that high reagent purity and reduced environmental contamination will be critical for high sensitivity OPA detection. Like fluorescamine, OPA forms a fluorescent product only with primary amino groups.
