Complete chromatographic separation of steroids, including 17alpha and 17beta-estradiols, using a carbazole-based polymeric organic phase in both reversed and normal-phase HPLC.
Poly(2-N-carbazolylethyl acrylate) with terminal trimethoxysilyl groups was prepared as an organic phase and immobilized onto silica. The retention behavior of the column packed with this carbazole-based polymer-immobilized silica (Sil-CEA) was investigated by using various estrogenic steroids and corticoids in both reversed-phase and normal-phase liquid chromatography. As a result, complete separation was confirmed for eight kinds of steroids with Sil-CEA. The most specific separation with Sil-CEA can be emphasized by the high separation factor (e.g., alpha = 1.39 in methanol-water (7:3, v/v) at 35 degrees C) for 17alpha and 17beta-estradiols, one of the most difficult pairs of isomers in chromatographic separation, whereas for two kinds of commercially available polymeric ODS columns as references alpha = 1.01, only, under the same conditions. Because the excellent separation and retention order with Sil-CEA was maintained even in a normal-phase mobile phase such as a hexane-2-propanol, it is estimated that the CEA phase has multiple interaction mechanisms through stronger interactions such as dipole-dipole, carbonyl-pi, and hydrogen bonding interactions than the hydrophobic effect expected with ODS.
Mallik
Abul K
AK
Department of Applied Chemistry and Biochemistry, Faculty of Engineering, Kumamoto University, 2-39-1 Kurokami, Kumamoto, Japan.
Shingo
Kaori
K
Gautam
Usha Ghimire
UG
Sawada
Tsuyoshi
T
Takafuji
Makoto
M
Ihara
Hirotaka
H
D002227:Carbazoles / Q000737:chemistry*; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D056148:Chromatography, Reverse-Phase / Q000379:methods*; D004958:Estradiol / Q000032:analysis / Q000302:isolation & purification*; D013256:Steroids / Q000032:analysis / Q000302:isolation & purification*
D002227:Carbazoles; D013256:Steroids; C041514:carbazole; D004958:Estradiol