The plant photoreceptor phytochrome senses light quality and quantity in the red region of the spectrum, directing adaptation and development. The functional holo-protein is a dimeric chromoprotein which is formed by an autoassembly reaction between the translation product and the open chain tetrapyrroles phytochromobilin (PΦB) or phycocyanobilin (PCB). We are interested in structure/function relationships within the phytochrome molecule, in particular chromophore/protein interaction during the assembly and photoactivation, using IR and NMR spectroscopy. For this we use an automated F/HPLC system running in a darkroom to purify large amounts of protein and chromophore separately. To obtain highly pure PCB chromophore we developed improved extraction and purification methods in which the final step is RPC-18 HPLC. As there are many spectrally only slightly different tetrapyrroles in the extract, the triple-wavelength monitoring offered by the F/HPLC detector was inadequate for distinguishing between PCB and impurities. Furthermore, λ max for the phytochrome Pfr signalling state lies between 705 and 730nm, beyond the range of the detector. Also, as both holo-protein and chromophore are photoactive, we wished to minimize light exposure of the eluate. We therefore implemented a miniature CCD-based flow UV–vis spectrophotometer using a xenon flash and quartz fiber optics enabling us monitor the entire 250–800nm spectrum of the eluate to an accuracy of ±3×10 −3 A in real time. The instrumentation described can be added to any chromatographic system, thereby allowing the purification of any molecule to be monitored easily and efficiently.