The column eluate was oxidized with chloramine-T to give a pyrrole that condensed with Ehrlich’s aldehyde (p-dimethylaminobenzal-

line tending to appear as a shoulder on the aspartic acid peak. Resolution could be improved, but at the expense of analysis time. To overcome these problems found with the ninhydrin method, we decided to adopt the column conditions of Callahan et al. (4), but to use a different chromogen (6, 7). The column eluate was oxidized with chloramine-T to give a pyrrole that condensed with Ehrlich’s aldehyde (p-dimethylaminobenzaldehyde) to form a complex with a maximum absorbance at 560 nm (Figure 1). The technique described provides analysis of total hydroxyproline in urine hydrolysates in a complete cycle time of 50 mm. An automated continuous-flow method is described for estimating total hydroxyproline in urine. The method utilizes column chromatography on sulfonated polystyrene resin, the eluate being reacted with chloramine-T and p-dimethylaminobenzaldehyde to produce a colored complex that is measured at 560 nm. The method is sensitive and specific, and requires 50 mm per analysis.