The aim of this work was to develop a new method for the separation of arsenolipids, based on the separation by supercritical fluid chromatography (SFC) and identification by ESMS. For method development, the certified reference material CRM 7405-a (Hijiki), that is a rich source in arsenic-containing hydrocarbons and arsenosugar-phospholipids, was used. Nine different stationary phases, including RP- and NP- HPLC columns and one SFC column were tested with different mobile phase combinations, i.e. supercritical carbon dioxide combined with MeOH or EtOH as modifiers and water, ammonium acetate, ammonium formate, ammonia and formic acid as additives. The elution of all compounds was achieved on the RP-HPLC columns with MeOH and ammonium acetate or formate, or ammonia as additive. Although the AsHCs could be separated from the AsPLs, sufficient separation within the compound classes was not observed, especially not for the AsPLs that coeluted under all conditions tested. Furthermore, the peaks were severely tailing. However, in almost all cases the arsenolipids could be detected in less than 20 minutes. With the SFC column best results were obtained with MeOH and ammonium acetate or ammonium formate as modifiers. The peak widths could be reduced to less than 1 minute for both compound classes, a result that was never obtained with HPLC columns. The separation of the AsHCs and the AsPLs could be achieved, but again, a separation within the AsHCs and the AsPLs was not possible. Although, optimum results were never obtained in terms of resolution, peak shapes and selectivity, it was shown in this study that it is possible to elute polar compounds with this technique.