Determining the purity of samples from natural products by coupling HPLC and CCD spectrometry

Isolation and identification of natural products is a very important and active research field. However, establishing the purity of the samples during the isolation process is quite difficult, especially when the retention times are similar for two desired components in HPLC. Although some technologies, e.g. MS and NMR, offer effective ways of obtaining purity information about the samples, the expensive instrumentation required or the off-line nature of coupling (generally speaking) make purity analysis somewhat inconvenient. In this paper, an on-line analytical system coupling HPLC and a CCD spectrometer for determination of purity for each eluate was developed in a thin layer spectrometric cell. The effectiveness of the system was demonstrated by differentiating Tanshinone I, Tanshinone IIA, and their mixture. The time-resolved UV-Vis spectra promptly revealed significant differences between the three samples while conventional single wavelength detection (CSWLD) could not. The system was then used to distinguish two steroid compounds which behaved as a single component in CSWLD. The compounds were isolated from a Chinese marine invertebrate animal, a marine annelid, Arenicola cristata, referred to here as Stimpson. The method reported here provided an efficient, convenient, fast, and inexpensive approach holding promise for on-line determination of the purity of samples isolated from natural products.