•Three EPG-containing pentafuorophenyl (PFP) stationary phases were synthesized.•The phases were evaluated by the Tanaka and Neue tests in RPLC and Tanaka test in HILIC.•One of the phases showed very high selectivity in RPLC and another in HILIC.•The phases were used successfully for the analysis of the real samples of chili pepper extract.•This work will open a new door for the analysis of complex samples both in 1D and 2D LC. Pentafluorophenyl (PFP) stationary phase is one of the most important phases after the C18 phase in terms of its applications. Three embedded polar groups (EPG)-containing stationary phases were newly synthesized to act the EPGs as additional interaction sites. The silica surface was initially modified with (3-aminopropyl)trimethoxysilane (APS). The APS-modified silicas were coupled with 2,3,4,5,6-pentafluorobenzoic acid, 2,3,4,5,6-pentafluorophenylacetic acid, and 2,3,4,5,6-pentafluoro-anilino(oxo)acetic acid to obtain Sil-PFP-BA, Sil-PFP-AA, and Sil-PFP-AN phases, respectively. The new phases were characterized by elemental analysis, ATR-FTIR, scanning electron microscopy (SEM), and thermogravimetric analysis (TGA). The phases were evaluated with the Tanaka and Neue tests in reversed-phase liquid chromatography (RPLC). In addition, they were characterized as hydrophilic phases by the Tanaka test protocol used in hydrophilic interaction chromatography (HILIC) separation mode. The Sil-PFP-AA phase showed the highest molecular shape selectivity in RPLC, while Sil-PFP-AN achieved the highest separability in HILIC compared to the commercial PFP reference column. The Sil-PFP-AA phase was successfully applied for the analysis of capsaicinoids from real samples of fresh chili peppers (Capsicum spp.) in RPLC and the Sil-PFP-AN phase for vitamin C (ascorbic acid) in HILIC.