Date of Award


Document Type


Degree Name

Doctor of Philosophy (PhD)

Legacy Department

Food Technology

Committee Chair/Advisor

Chen, Feng

Committee Member

Dawson, Paul

Committee Member

Pometto III, Anthony

Committee Member

Wells, Christina


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 µm 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.

Included in

Food Science Commons



To view the content in your browser, please download Adobe Reader or, alternately,
you may Download the file to your hard drive.

NOTE: The latest versions of Adobe Reader do not support viewing PDF files within Firefox on Mac OS and if you are using a modern (Intel) Mac, there is no official plugin for viewing PDF files within the browser window.