Anhydrous sodium sulfate

For cutin depolymerization, delipidated leaves of C. colocynthis and leaflets of P. dactylifera were transesterified with boron trifluoride in methanol (Fluka) at 70 °C overnight to release methyl esters of cutin acid monomers. Sodium chloride-saturated aqueous solution (AppliChem), chloroform, and n-dotriacontane (C₃₂; Sigma-Aldrich) as an internal standard were added to all reaction mixtures. From this two-phase system, the de-esterified cutin components were extracted three times with chloroform. The combined organic phases were dried over anhydrous sodium sulfate (AppliChem). All extracts were filtered, and the organic solvent was evaporated under a continuous flow of nitrogen.
Derivatization and subsequent gas chromatographic analysis with mass spectrometric detection were performed according to Leide et al. (2011) (link). Separation of cutin mixtures was carried out at 50 kPa for 60 min, increased by 10 kPa min^–1 to 150 kPa, and at 150 kPa for 30 min using a temperature programme of 50 °C for 1 min, raised by 10 °C min^–1 to 150 °C, held at 150 °C for 2 min, and then raised by 3 °C min^–1 to 320 °C and held at 320 °C for 30 min. The quantitative composition of the mixtures was studied using capillary gas chromatography and flame ionization detection under the same gas chromatographic conditions.

Anhydrous sodium sulfate was purchased from AppliChem GmbH (Darmstad, Germany); dichloromethane (99.9%) was supplied by Honeywell Riedel-de Haën (Steinheim, Germany) and silanized glass wool by Supelco (Steinheim, Germany). 2-Octanol (≥99.5%), used as an internal standard was purchased from Sigma-Aldrich (St. Louis, MO, USA).
GC-FID and GC-MS used standards: acetic acid was purchased from Riedel-de-Haen (Seelze, Germany); isobutanol, 2-methyl-1-butanol, 3-methyl-1-butanol,benzyl alcohol, 2-phenylethanol,propanoic acid, butanoic acid, isovaleric acid, octanoic acid, hexanal, ethyl propanoate, isobutyl acetate, ethyl 2-methylbutyrate, ethyl hexanoate, ethyl decanoate, phenethyl acetate, ethyl dodecanoate, eugenol were acquired from Fluka (Buchs, Switzerland);1-propanol, 2,3-butanediol, 1,2-propanediol, glycerol, isobutyric acid, ethyl butyrate, ethyl octanoate were purchased from Merck (Darmstadt, Germany); e-ethoxy-1-propanol, methional, ethyl isobutyrate, ethyl isovalerate, isoamyl acetate, ethyl hydrogen succinate, furfural were bought from Aldrich (Steinheim, Germany) and 2,3 butanedione was acquired from Sigma-Aldrich (Saint Louis, MO, USA).

The enzymatically isolated and dewaxed fruit cuticle was transesterified with boron trifluoride in methanol (Sigma-Aldrich) at 70°C overnight. After cooling, n-dotriacontane (C₃₂; Sigma-Aldrich) as internal standard and trichloromethane (1:1, v/v; Roth) were added to the extracts. Transmethylated cutin monomers were washed three times with sodium-chloride saturated aqueous solution, dried with anhydrous sodium sulfate (AppliChem), and filtered. Afterward, the organic solvent was evaporated under a gentle flow of nitrogen. Derivatization with N,O-bis-trimethylsilyl-trifluoroacetamide in pyridine was performed at 70°C for 60 min.
Separation of cutin monomers was carried out at 50 kPa for 60 min, 10 kPa min⁻¹ to 150 kPa, and at 150 kPa for 30 min using a temperature program of 50°C for 1 min, raised by 10°C min⁻¹ to 150°C, held at 150°C for 2 min, and, subsequently, increased by 3°C min⁻¹ to 320°C and maintained at 320°C for 30 min. The qualitative and quantitative composition was studied using capillary gas chromatography with mass spectrometric and flame ionization detection under the same chromatographic conditions.