Development and application of scalable two-dimensional high performance liquid chromatography for aromatic selective separations of biologically active phytochemicals from Oplopanax horridus.

摘要

The high demand for more efficient purification processes with increased automation and throughput pushes the development of more advanced preparative, pilot, and process scale HPLC instrumentation that is capable of achieving higher purities in a shorter amount of time than are currently achieved using one dimensional separations. A preparative scale 2D HPLC system was designed and reduced to practice in order to demonstrate the capacity for scalability of on-line comprehensive 2D HPLC separations of basic compounds from a challenging natural product extract of Oplopanax horridus. The methodology and instrumentation design herein permits direct method transfer from analytical to preparative scale purifications to alleviate resolution and throughput problems with traditional reversed phase separations. The incorporation of aromatic selective phases (C6-phenyl and biphenyl) increases the resolution of a two dimensional HPLC system that follows a hydrophobic subtraction approach to achieve orthogonality between dimensions. The two dimensional separations herein demonstrate the utility and application of long columns (250 mm) packed with 5 microm fully and superficially porous particles which enable direct scalability of the HPLC separations from analytical to preparative scale and beyond where the instrumentation limitations of 5,000 psi are already factored in. This approach enables equipment already in place in either a lab or a processing environment to be retrofit with a modulation mechanism to incorporate multidimensional chromatography without the capital investment of entirely new instrumentation resulting in a huge cost savings. Utilizing a hyphenated purification approach, a 2D HPLC-ESI-MS system incorporating a C6-phenyl and biphenyl in the first and second dimension respectively was able to successfully resolve more than 90% of detected analytes with a resolution of 1.0 or more in 11 distinct subfractions of the ethyl acetate liquid-liquid extraction layer of the crude methanol extract of O. horridus. The incorporation of pi selective phases such as C6-phenyl and biphenyl offers increased selectivity of aromatic molecules and was demonstrated as a powerful screening phase. Furthermore, using a stronger retaining phase in the second dimension enabled for large (67% of the void volume) loops to be used for transferring eluent from the first to the second dimension without breakthrough.