Separation of di- and trisaccharide mixtures by comprehensive two-dimensional liquid chromatography. Application to prebiotic oligosaccharides.

Carbohydrates are one of the most important ingredients in foods. They are normally present as complex mixtures with different glycosidic linkages, monomeric units and degrees of polymerization. This structural heterogeneity impairs their comprehensive characterization and requires the use of analytical techniques with high resolving power and sensitivity. The use of chromatographic techniques, especially liquid chromatography (LC), has been extremely helpful for the analysis of carbohydrates. However, in many cases, the use of monodimensional LC is not enough to resolve these complex mixtures; then, the use of techniques with a higher resolving power, as multidimensional LC, could be a good alternative. To the best of our knowledge, our findings are pioneer in applying online LC × LC for the analysis of carbohydrate mixtures. For this purpose, different conditions such as stationary phases (BEH amide, C18 and PGC columns) and chromatographic conditions for the separation of di- and trisaccharide mixtures were optimized. The BEH amide × C18 combination was selected for the LC × LC analysis of carbohydrate standards with different degrees of polymerization, linkages and monomeric units. In order to allow their proper UV detection, carbohydrates were previously derivatized using p-aminobenzoic ethyl ester. This method also resulted to be successful for the separation of commercial prebiotic mixtures of galacto-oligosaccharides and gentio-oligosaccharides. This is the first time that LC × LC has been applied for the separation of bioactive carbohydrate mixtures and it could be considered as a powerful analytical technique for the characterization of other oligosaccharide complex mixtures.