Rapid screening and target‐guided isolation of antioxidants from German chamomile by 2,2‐diphenyl‐1‐picrylhydrazyl‐ultra‐high‐performance liquid chromatography‐mass spectrometry coupled with off‐line two‐dimensional high‐speed countercurrent chromatography

German chamomile is one of the most effective herbal elements used in anti‐allergic products and as an antioxidant. Herein, the antioxidant activity of different extract fractions of German chamomile was initially evaluated using an off‐line 2,2‐diphenyl‐1‐picrylhydrazyl spectrophotometric assay. The ethyl acetate extract demonstrated the highest efficacy in scavenging free radicals. Based on this, a rapid screening and separation method using ultra‐high‐performance liquid chromatography combined with the 2,2‐diphenyl‐1‐picrylhydrazyl assay was implemented to identify antioxidants in the ethyl acetate fraction of German chamomile flowers. Ten potential radical scavengers were tentatively screened from German chamomile using a target‐guided isolating approach with off‐line two‐dimensional high‐speed countercurrent chromatography and the structures of the compounds were analyzed and identified. Ultimately, 10 radical scavengers were obtained from the ethyl acetate extract with a purity quotient exceeding 90%. The results demonstrated the effectiveness and reproducibility of this method for isolating potential antioxidants from complex mixtures in a targeted manner. This strategy can be applied to the target‐guided isolation of complex mixtures of natural products with broad K‐values and similar structures.