To improve the separation of monosulfate glycosaminoglycan disaccharide isomers by microchip electrophoresis, we found that addition of 1,4-dioxane (DO) dramatically improved analyte resolution, probably due to solvation effects. Methylcellulose (MC) was tested for the ability to suppress EOF and analyte adsorption to the chip. To improve analyte resolution, buffer pH, P-CD, and DO were systematically investigated. Fast separation was achieved by increasing the electric field strength, and field-amplified sample stacking occurred with increasing buffer concentrations. Therefore, based on our findings, we describe an efficient method for the separation of monosulfate and trisulfate unsaturated disaccharides (Delta Di-UA2S, Delta Di-4S, Delta Di-6S, and Delta Di-triS) derivatized with 2-aminoacridone hydrochloride. A mixture of monosulfate disaccharide isomers (Delta Di-UA2S, Delta Di-4S, and Delta Di-6S) was baseline-separated within 75 s on a poly(methyl methacrylate) chip using a mixed buffer (DO/running buffer 57:43 v:v), 0.5% MC, pH 6.81, with an E-sep of 558 V/cm. The theoretical plate was in the range of 5 x 10(5) to 1 x 10(6) m(-1).