The influence of the organic modifier in hydro-organic mobile phase on separation selectivity of steroid hormones separation using cholesterol-bonded stationary phases

► Separation of steroid hormones on cholesterol-phases were performed. ► The influence of the organic modifier on separation selectivity were investigated. ► Steroid retention depends on coverage density of cholesterol moiety. ► Cholesterol-bonded phases allow to separate estradiol isomers.