Introduction Differently branched and charged glycans are attached to erythropoetin(EPO),resulting in a highly complex and heterogeneous glycan profile.The complexity of the EPO glycans requires state-of-the-art analysis techniques that deliver high resolution with little expenditure of time.Although current hydrophilic interaction chromatography(HILIC)columns for glycan separation have a high resolving power,the EPO glycans cannot be resolved within one dimensional HILIC separation.Methods Comprehensive and multiple heart-cutting online 2D-LC with a combination of HILIC and weak anion exchange chromatography(WAX)was used for the analysis of EPO glycans.This combination provides a totally orthogonal separation setup for highest peak capacity and resolution.One of the biggest advantages of online 2D-LC is the enormous amount of time which can be saved because there is no need for fraction collection and re-injection.Results and Novel Aspects High resolution as well as simultaneous charge profiling was possible with a comprehensive 2D-LC analysis with HILIC in the first and WAX in the second dimension.If higher flexibility is desired,especially allowing WAX as first dimension separation media,multiple heart-cutting 2D-LC is the method of choice.With this 2D separation method,the user is able to select several specific peaks for re-analysis in the second dimension.In addition,because the peaks are stored in ten loops,there is no longer the need for super short gradients.Depending on the distribution of the peaks within the first dimension chromatogram,the cycle time of the second dimension can be adjusted as needed.Online 2D-LC analysis in combination with fluorescence detection and online QTOF analysis and the combination of HILIC and WAX separation media enable excellent resolving power for highly complex glycans from biopharmaceutically used glycoproteins.