Ultra-high pressure liquid chromatography in separation of botanicals and natural products: Retention mechanism studies

Liquid chromatography with C₁₈-bonded packings and aqueous-organic eluents is a mature method for the separation and analysis of botanicals and natural products. Recent improvements in pump technology (1,200bar pressure) and silica chemistry (very stable, bridged-ethylene hybrids) have allowed the use of very small particles (<2µm) to achieve high efficiency separations. However, the role of the stationary phase has not been thoroughly investigated mostly due to the complexity of the commonly used chemically-bonded reversed phase liquid chromatography (RPLC) packings. The systems are physically complex and heterogeneous since 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 analytes as a function of eluent composition. In this way, the excess amount of eluent sorbed by the stationary phase can be determined. The effect of such eluent uptake on the separation of botanicals and natural products was investigated.