ポストカラム還元法による植物キノン類の測定

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Measurements of naphthoquinones and plastoquinones are carried out by high performance liquid chromatography (HPLC) with a spectrophotometric detector. However, this method lack both selectivity and sensitivity, and quinones measurements require concentrating extract and removing substances strongly absorbing ultraviolet light as chlorophylls and carotenoids from quinone extract. Therefore, HPLC method with fluorescence detection after post-column chemical reduction was developed for measurements of phylloquinone (PK), menaquinone (MK), plastoquinone A-45 (PQ)and tocopheryl quinone (TQ) in leaf segments without the pretreatment as removing chlorophylls from quinone extracts. The quinone derivatives extracted from leaves were separated by reversed-phase C18 column at 35℃ with a mixture of equivalent volume of methanol and ethanol as a mobile phase at a flow rate of 1.0ml/min. Separated naphthoquinone derivatives were detected by monitoring fluorescence intensity at 430 nm of quinoles excited at 320 nm with a fluorescence detector after reaction with ethanolic sodium borohydride of 0.045% (W/V) at a flow rate of 1.0 ml/min in a reaction coil (0.5mm i.d. x 200cm) kept at 35℃ connected online chromatographic column. Tocophery quinone and plastoquinone were detected by measuring fluorescence(Ex=290nm, Em=320nm)of the quinone derivatives, the other separation and reduction conditions except for fluorescence detection is the same as in naphthoquinone analysis. The calibration curves constructed by plotting the peak area against the amount of standard quinone/-ol were linear over the tested range from 7.5 to 2000 pmol. The proposed method is simpler, more specific snd sensitive than other conventional methods.
長岡工業高等専門学校の論文
2000-11-30

ポストカラム還元法による植物キノン類の測定

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