200 ri detector

Soluble carbohydrates were extracted and analyzed following the protocol of Tattini et al.^{25 (link)}. Briefly, 5 ml of 75% ethanol was added to 200 mg of powdered fresh leaves. Samples were sonicated for 20 min. and centrifuged for 30 min at 10,000 g/min at room temperature to pellet the insoluble material. The supernatant was removed and the pellet was extracted twice as above. The ethanol fraction was reduced to dryness under vacuum and finally rinsed with 10 ml of water (pH 7.0). The aqueous extract was purified through –CH and –SAX Bond-Elute cartridges (Varian, Harbor City, CA, USA), and the eluate was reduced to dryness under vacuum at 35 °C. Samples were rinsed with 2 ml of ultrapure water and injected in a Series 200 HPLC equipped with 200-RI detector (Perkin Elmer), and separated on an 8 × 300 mm SC1011 column (Showa Denko, Tokyo, Japan) maintained at 88 ± 1 °C. Eluent was ultrapure water at a flow rate of 0.8 ml min⁻¹. Sorbitol was added as internal standard.

Soluble carbohydrates as sucrose, glucose, fructose and galactose were identified and quantified by a high-performance liquid chromatography (HPLC) analysis. In detail, 75 mg of lyophilized tissue were extracted with 3 × 5 ml of 75% ethanol, the ethanol fraction reduced to dryness under vacuum, and finally rinsed with 2 ml of water (pH 7). The extract was purified using -CH and -SAX Bond-Elute cartridges (Agilent, Santa Clara, CA, USA) and the eluate reduced to dryness. Samples were rinsed with ultrapure water, injected in a Series 200 HPLC equipped with 200-RI detector (all from Perkin-Elmer, Bradford, CT, USA) and separated on an 8 × 300 mm SC1011 column (Showa Denko, Tokyo, Japan) maintained at 88 ± 1°C. Eluent was ultrapure water at a flow rate of 0.8 ml min⁻¹.