Comparison of normal and reverse phase column in phytosterol detection by high performance liquid chromatography(HPLC)

Abstract

Phytosterols are natural bioactive compounds,usually including stigmasterol,sitosterol,campesterol,and rapeseed sterol.They have attracted growing attention owing to their beneficial effects.High performance liquid chromatography(HPLC)is the most frequently used method to quantify phytosterols.Although several detectors such as ELSD(evaporative light scattering detector)and UV(ultraviolet detector)are available for HPLC analysis,but UV detector is believed to be much more effective and always preferred in laboratories.In the recent literature available,the reverse phase column was mostly applied to determine phytosterols in HPLC-UV system.However,the performance of normal phase column in phytosterol analysis has not been well studied and compared.In this study,the performances of both reverse phase and normal phase columns in HPLCUV system for phytosterol analysis were compared,and their respective advantages were illustrated.Both normal phase and reverse phase columns in HPLC-UV were applied to detect phytosterols,respectively.The normal phase chromatographic conditions were as follows:using hypersil SiO2(4.6mm×250mm,5μm)as column and V(hexane)∶V(isopropanol)=99∶1as mobile phase,column temperature of 35℃,flow rate of 1.0 mL/min,and detection wavelength of 205nm.Reverse phase chromatographic conditions were as follows:using Merck RP-18(4.6mm×250mm,5μm)as column and pure methanol as mobile phase,column temperature of 35 ℃,flow rate of 1.0mL/min,and detection wavelength of 205nm.As a result,high sensitivity was observed for phytosterol detection using UV detector in both systems.It was revealed that the normal phase system could not separate individual phytosterol compounds,while the reverse phase system exhibited good separation capacity.In terms of retention time,the normal phase system needed much less time as compared with reverse one.In addition,phytosterols were much more soluble in the eluent of the normal phase system.It is concluded that the normal phase column is more suitable than reverse one for quantification of total phytosterols despite of lower separation ability.Furthermore,an efficient method for quantification of phytosterols employing the normal phase column and the benzoate internal standard is developed.