Analysis of ascorbate in plant tissues by high-performance capillary zone electrophoresis.
We describe here a simple and rapid capillary electrophoresis method for the determination of ascorbic acid (L-AA) and isoascorbic acid (D-AA) in vegetative tissues. For optimal yields and stabilization, samples are extracted with cold 3% metaphosphoric acid. Hydrophobic contaminants are then removed by passage through a C18 solid-phase extraction cartridge. The analysis itself is performed on a fused silica capillary with 200 mM borate, pH 9, as the carrier electrolyte, using on-line diode array detection over the range 190-350 nm. Quantitation was performed at 260 nm, the uv-absorption maximum for ascorbate at this pH. This method has a minimum detection limit of 84 fmol/injection and linearity of detector response was observed up to at least 12 pmol/injection. We also describe the influence of electrolyte concentration, pH, and the presence of detergent on separations of L-AA, D-AA, and L-galacturonic acid-1,4-lactone. The protocol has been demonstrated to be suitable for the analysis of L-AA in Arabidopsis, parsley, and mushroom. The method has superior resolution to comparable HPLC separations, a comparable analysis time, but lower sensitivity because of the concentration limitations of the detection system.
Davey
M W
MW
Department of Genetics, Flanders Interuniversity Institute for Biotechnology, Universiteit Gent, Gent, Belgium.
Bauw
G
G
Montagu
M V
MV
D017360:Arabidopsis / Q000737:chemistry; D001204:Ascorbate Oxidase / Q000494:pharmacology; D001205:Ascorbic Acid / Q000032:analysis*; D001487:Basidiomycota / Q000737:chemistry; D002851:Chromatography, High Pressure Liquid; D019075:Electrophoresis, Capillary / Q000379:methods*; D006863:Hydrogen-Ion Concentration; D010944:Plants / Q000737:chemistry*; D015203:Reproducibility of Results; D012967:Sodium Dodecyl Sulfate / Q000494:pharmacology
C005056:isoascorbic acid; D012967:Sodium Dodecyl Sulfate; D001204:Ascorbate Oxidase; D001205:Ascorbic Acid