Hyperclone 3u ods c18 120 150 4.6 mm column

For quantification of amino acids 50 mg of plant material was frozen into liquid nitrogen and homogenized using a ball mill. The amino acids were extracted with 80% ETOH and the extract was diluted 1:10 with water. Twenty microliters of the diluted extract were injected into the HPLC system. Amino acid were separated using a HyperClone 3u ODS (C18)120 150 × 4.6 mm column (Phenomenex) connected to the Dionex UltiMateTM 3000 system (Thermo Fisher Scientific). The HPLC system held a discontinuous flow gradient comprising solvent A (8.8 mM NaPO4, pH 7.5, and 0.2% (v/v) tetrahydrofuran) and increasing proportion of solvent B (18.7 mM NaPO4, pH 7.5, 32.7% (v/v) MeOH and 20.6% (v/v) acetonitrile), at a flow rate of 0.8 ml/min. Quantification was performed by pre-column derivatisation with ortho-phtalaldehyde (OPA; Grace Davison Discovery Science) and subsequently quantified by fluorescence detection of the obtained derivatives (Lindroth and Mopper 1979 (link)).
Extraction and quantification of desulpho-glucosinolates was performed as described previously (Gigolashvili et al. 2007b (link)).

For quantification of amino acids, lyophylized plant material was extracted with 80% ETOH and the extract was diluted 1:10 with water. Twenty microliters of the diluted extract were injected into the HPLC before HPLC analysis. Amino acids were separated using a HyperClone 3u ODS (C18)120 150 × 4.6 mm column (Phenomenex) connected to the Dionex UltiMateTM 3000 system (Thermo Fisher Scientific). The HPLC system held a discontinuous flow gradient comprising solvent A (8.8 mM NaPO4, pH 7.5, and 0.2% (v/v) tetrahydrofuran) and increasing proportion of solvent B (18.7 mM NaPO4, pH 7.5, 32.7% (v/v) MeOH and 20.6 % (v/v) acetonitrile), at a flow rate of 0.8 mL/min. Quantification was performed by pre-column derivatization with ortho-phtalaldehyde (OPA; Grace Davison Discovery Science) and subsequently quantified by fluorescence detection of the obtained derivatives according to Lindroth and Mopper (1979) (link).
Starch was isolated from lyophylized plant material as described by Lin et al. (1988) (link) and the content was determined with a coupled enzymatic assay as described earlier by Stitt (1989) (link) using a Spectrafluor Plus plate reader (TECAN, Austria) in the absorbance mode.