Improved separation of C12-C22 fatty acid phenacyl esters by reversed phase column liquid chromatography

Phenacyl esters of C12-C22 fatty acids were separated on Separon SGX C18 column, using a gradient elution with methanol-acetonitrile-water. The proposed gradient showed better resolution of the critical pairs C18:3-C14:0, C16:1-C20:4, and C16:0-C18:1 than the gradient elution with methanol-water or acetonitrile-water, or than the isocratic elution with methanol-acetonitrile-water. The optimum volume concentration (83%) of the sum of both methanol and acetonitrile was maintained constant for 35 min; in this period the acetonitrile concentration decreased linearly from the initial 42-60% to 0% while the methanol concentration increased from the initial 41-23% to 83% at the same rate. After 35 min the elution was completed with a methanol-water gradient. The whole analysis can be performed within 63 min at a flow rate 1 ml/min.