Abstract: The invention concerns a process of chromatographically separating glycosylated proteins from non-glycosylated proteins by subjecting a solution comprising glycosylated and non-glycosylated proteins to chromatography using a Ca++ containing eluant. By using this process a fraction comprising non-glycosylated proteins substantially free from glycosylated proteins is obtained. The process may be applied to the separation of proteins used in the medical industry, such as insulin.