This Application Note presents different online 2D-LC concepts for the high?resolution analysis of complex N-linked glycans in erythropoietin (EPO) using the Agilent 1290 Infinity 2D-LC Solution with fluorescence detection. EPO has a complex glycosylation pattern with differently branched and charged glycans. A combination of hydrophilic interaction chromatography (HILIC) with weak anion exchange chromatography (WAX) enables highly orthogonal separation. Comprehensive 2D-LC analysis with HILIC in the first and WAX in the second dimension facilitates high-resolution 2D chromatography together with simultaneous charge profiling. In addition, multiple heart-cutting 2D-LC analysis, combining WAX and HILIC separation, provides a flexible alternative whereby the user can select multiple peaks to be analyzed in the second dimension and, moreover, run longer gradients.