Spherisorb c18 column

As previously mentioned [75 (link)], glucosesinolates were examined. High performance liquid chromatography (HPLC) was used to examine desulfoglucosinolates. The Agilent 1260 HPLC apparatus with a variable wavelength detector (VWD) was used for the analysis. Acetonitrile, water, and a Waters Spherisorb C18 column (250 × 4.6 mm) were used to separate the samples at a temperature of 30 °C and a flow rate of 1.0 mL/min⁻¹. On a 250 × 4.6 mm Waters Spherisorb C18 column, the separation was carried out. At 226 nm, the absorption was found.

Glucosinolates were extracted and analyzed as previously described^{2 (link)}. Briefly, freeze-dried samples (100 mg) were boiled in 4 ml water for 10 min. The supernatant was collected after centrifugation (5 min, 7000 g), and the residues were washed once with water (4 ml), centrifuged, and then combined with the previous extract. The aqueous extract was applied to a DEAE-Sephadex A-25 (40 mg) column (pyridine acetate form) (GE Healthcare, Piscataway, NJ). The column was washed three times with 1 ml pyridine acetate (20 mM) and twice with 1 ml water. The glucosinolates were converted into their desulpho analogues by overnight treatment with 100 μl of 0.1% (1.4 units) aryl sulphatase (Sigma), and the desulphoglucosinolates were eluted with 2 × 0.5 ml water. HPLC analysis of desulphoglucosinolates was conducted using a Waters HPLC instrument equipped with a Model 2996 PDA absorbance detector (Waters, USA). Samples (20 μl) were separated at 30 °C on a Waters Spherisorb C18 column (250 × 4.6 mm i.d.; 5 μm particle size) using acetonitrile and water at a flow rate of 1.0 ml min⁻¹. The procedure employed isocratic elution with 1.5% acetonitrile for the first 5 min; a linear gradient to 20% acetonitrile over the next 15 min followed by isocratic elution with 20% acetonitrile for the final 10 min. Absorbance was detected at 226 nm.