| Research Abstract |
A combined analytical system for separation of bio-active large molecules in natural water by high performance liquid chromatography with detection by UV absorption, fluorescence and ICP-AES (inductively coupled plasma atomic emission spectrometry) has been developed. In ICP-AES detection the time-sequence measurement with 40 elements-simultaneous detection was explored, which allowed to measure multielement detection chromatograms. Metalloenzymes such as alkaline phosphatase (zinc enzyme), carboxypeptidase (zinc enzyme), catalase (iron enzyme), chlorophile compounds (magnesium-contained), and humic acid-like substances in pond water were of interest in the present research. In the case of metalloenzymes pond water was concentrated by ca. 10-1000 times as the analysis sample. Furthermore an on-line enzymatic activity measurement system was developed, where metalloenzymes were separated by size-exclusion chromatography and specific enzymatic activity was measured in the in-situ reaction column using some substrates. By this system 10^<-9>-10^<-12> g metalloenzymes in natural waters were determined successfully. As for chlorophile compounds (Chl), Chl-a, Chl-b, Chl-c and their derivatives were determined by fluorescence and ICP-AES detections after methanol extraction/reverse-phase chromatographic separation. In the study on metal-containing large molecules in natural water, the molecules with molecular weight larger than 10,000 were concentrated by 1000-1500 times with ultrafiltration, and separated by size exclusion chromatography. As the results, three kinds of humic acid-like substances were detected and it was found that some specific metal ions were combined in each molecular constituent. Further study is, however, required in order to elucidate the chemical forms of those large molecules.
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