The reversed phase HPLC (RP-HPLC) analysis is carried out using an Agilent 1100 HPLC system coupled to a diode array detector equipped with an ET250/2 Nucleosil 120-5 C18 column (250 x 2.1 mm, 5 µm particle size; Macherey & Nagel). A solvent system of methanol/water/acetic acid (90:10:0.1, v/v/v) and a flow rate of 0.18 ml/min are used.
For analysis of divinyl ethers, the absorbances at 250 nm and 268 nm indicating either one or two conjugated diene systems in conjugation to an ether bond of the fatty acids are recorded. All substances are identified by their characteristic UV spectra. For quantification, calibration curves (five point measurements) for CA, CnA, EA and EnA were established. (6Z,9Z,11E,13S)-13-hydroxy-6,9,11-octadecatrienoic acid is used as internal standard to determine the recovery of the divinyl ethers.
standards are available from Sigma (Germany) Larodan (Sweden), Cayman Chemical (USA), kindly provided by Mats Hamberg (Stockholm, Sweden) and Otto Miersch (Halle/Salle, Germany) or enzymatically synthesized, respectively
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