Effect of Immobilized Eluent on the Retention of Artemisinin (Qinghaosu) in Reversed-Phase Liquid Chromatography

Artemisinin is an effective drug for the treatment of falciparum malaria. It is a sesquiterpene lactone commonly isolated from the plant Artemisia annua. Despite the peroxide functionality, this molecule is quite stable and amenable to chromatographic analysis. A study of the retention mechanism(s) controlling the retention of this model compound was undertaken to determine what properties of the stationary phase influenced the retention mechanism.

Most commonly, mobile phase (eluent) composition has been used to manipulate the retention and resolution of analytes by a technique known as gradient elution or solvent programming. However, the role of the stationary phase has not been thoroughly investigated mostly because of the complexity of the commonly used chemically-bonded RPLC packings. The systems are physically complex and heterogeneous because in a RPLC column, the aqueous-organic components of the mobile phase interact with, and thus influence the chemical properties of, the stationary phase. Mass spectrometric tracer pulse chromatography is one of the few experimental techniques that is capable of accurately measuring the uptake of binary eluents by chemically bonded RPLC stationary phases. This experimental technique was used to measure the uptake of eluent and, at the same time, the retention volume of artemisinin as a function of eluent composition. The objective was to evaluate the retention model proposed by Kazakevich el al. [1]. The proposed model is illustrated in the Figure. Kazakevich proposed that the immobilized eluent formed a separate phase on the surface of the C₁₈-bonded phase rather than dissolving in it. A solute, such as arteminisin, could then be retained on both the surface of the C₁₈ by adsorption and the immobilized eluent layer by absorption. Thus, the immobilized eluent would act as stationary phase and the retention of solutes should increase as the amount of immobilized eluent increased. This hypothesis proved impossibe to verify because the amount of eluent adsorbed was determined by the eluent composition which in turn influenced te retention of the test solute. Isolation of the proposed retention mechanism from the concommitant effect of eluent solubility in the eluent is the overall goal of this continuing research project.

Acknowledgements: This research was supported by Grant CHE-0715094 from the National Science Foundation.

References: [1] Kazakevich YV, LoBrutto R, et al. (2001)J Chromatogr A, 913: 75–87.