Experimental conditions for complete separation and quantification of mixtures containing lactobionic acid, sorbitol, lactose and fructose are discussed for the first time. These mixtures appear in the enzymatic bioconversion of fructose and lactose catalyzed by glucose-fructose oxidoreductase (GFOR) and glucono-delta-lactonase (GL) enzymes of Zymomonas mobilis cells. The high-performance liquid chromatography (HPLC) separation was carried out in a strong cation ion exchange resin (hydrogen form) based on a copolymer of styrene divinylbenzene (PS-DVB). A stationary phase of beta-cyclodextrin was also evaluated. An efficient separation was obtained with PS-DVB column eluted with sulfuric acid 0.450 mM solutions (pH 3.0-3.2) at 75 degrees C. The formation of lactones was observed, which is associated with the dissolution of lactobionic acid crystals; however, by dissolving the lactobionic acid crystals on alkaline calcium hydroxyde solution in equimolar ratio a single lactobionic acid chromatographic peak without lactobionolactone is obtained.