DETERMINATION OF RETENTION FACTORS OF PHOSPHOLIPIDS BY GRADIENT ELUTION

초록

To predict the relationship of retention factor and mobile phase composition, the retention times of samples at known mobile phase composition are required in isocratic elution. However, some samples are not separated in isocratic elution, but they are separated in gradient elution. Retention factor in gradient elution might be calculated by more complicated equation of the retention factor and the mobile phase composition. In isocratic elution, the retention factor is often expressed in terms of binary mobile phase compositions (F, G as volume percentages) by the following: ln k = A + B F + C F2 + D G + E G2 , where A, B, C, D, and E are the experimentally determined constants. However, in gradient elution the factor can be correlated by a function of mobile phase composition, gradient time, and gradient mode. In this work, the three phospholipids of phosphatidylethanolamine (PE), phosphatidylinositol (PI), and phosphatidylcholine (PC) extracted from soybean were experimented. The retention times of PE and PC were individually measured in different isocratic elutions, while those of PI and PC were determined in gradient elution of a mixture of PI and PC. It means the retention time of PC could be calculated by both isocratic and gradient elution. Purpose of this work is to propose a methodology of determination of retention relationship in gradient elution, and compare the retention times of PC by isocratic and gradient elution to prove the validity of the methodology.