Simultaneous analyses of oxidized and reduced forms of photosynthetic quinones by high-performance liquid chromatography.
Plastoquinones and phylloquinones are the major plant quinones localized in chloroplasts, and they act as photosynthetic electron redox mediators in thylakoid membranes. These quinones are analyzed by two processes: extraction with organic solvents and quinone assay by high-performance liquid chromatography (HPLC) analysis. Solvent choice is very important from the viewpoint of stability of the redox state in the extraction processes and during storage of plant quinones. We introduce procedures and solvents to avoid changes in the redox state of quinones, in addition to achieving high extraction efficiency. Traditional methods have problems of low sensitivity and require preparation steps to remove interfering substances, such as plant pigments. HPLC systems have been developed utilizing the fluorescent properties of quinols (reduced forms) to measure quinones. Plastoquinones were detected by reversed-phase HPLC with dual detectors (ultra-violet and fluorescence detection). However, the peak of phylloquinone and plastoquinone isomers with shorter side chains often overlaps with a large peak of fast-eluting pigments. To address these issues, HPLC with fluorescence detection after post-column reduction to convert quinones to fluorescent quinol was applied for measurement of fast-eluting quinones (low hydrophobicity quinones and quinols) such as phylloquinone. Using post-column reduction methods with sodium borohydride or platinum black, not only the reduced forms (fluorescent) but also the oxidized forms (non-fluorescent) could be clearly measured by HPLC with a fluorescence detector.
Faculty of Education, Biological Institute, Yamaguchi University, 1677-1, Yoshida, Yamaguchi, Japan.
Masaru
M
Shibata
Hiroshi
H
Shimada
D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D010084:Oxidation-Reduction; D010788:Photosynthesis; D011809:Quinones / Q000032:analysis* / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D012997:Solvents / Q000737:chemistry; D013053:Spectrophotometry; D013997:Time Factors
D011809:Quinones; D012997:Solvents