■ Development of an HPLC method for the determination of phenolic by-products: optimisation of the separation by means of the experimental designs methodology

The separation of nine phenolic by-products was realized by Reverse Phase HPLC using a binary gradient water-acetic acid/acetonitrile whose elution program was optimized by means of the experimental design methodology. Significant factors are: the initial isocratic elution time, the gradient running time and the gradient curvature. Modelization was made by using a central composite plane in 17 experiments. Responses were measured by the resolution between four couples of components: p-hydroxybenzaldehyde, acetovanilone - acetosyringone and acetosyringone - benzoic acid. According to the resulting quatraic model, an optimized program of gradient elution was set up and successfully tested as it gives satisfactory separations (isocratic elution time: 8 min; gradient running time from 1 % to 25 % acetonitrile: 40 min; linear gradient; room temperature).