Successful Transfer of Size-Exclusion Separations between HPLC and UPLC

Library Number:
APNT134822077
Part Number:
720005214EN
Author(s):
Stephan Koza, Susan Serpa, Edouard Bouvier, and Kenneth J. Fountain
Source:
Waters
Content Type:
Application Notes
Content Subtype:
Application Notes
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Successful implementation of SE-UPLC separation technology requires high efficiency columns and low dispersion LC systems capable of operating at high back pressures. The advantage in doing so is the capability to dramatically improve sample throughput while decreasing sample requirements and mobile phase use. However, there are several situations where it is beneficial to be able to transfer between SE-UPLC and SE-HPLC separations, including instrumentation limitations in a method transfer, or in the event that a UPLC separation needs to be scaled up in order to facilitate the structural or functional characterization of low abundance species. However, in some cases, a direct method transfer using the same mobile phase and temperature conditions is not possible between SE-UPLC Columns packed with BEH-based particles and SE-HPLC columns packed with traditional silica-based particles due to the differences in the surface characteristics of the two particle types. These differences can necessitate the re-optimization of the method in order to get comparable results. Waters’ recent development of HPLC compatible 200Å and 450Å, 3.5 μm particles based on the same diol-coated BEH-particle chemistry as that used in ACQUITY UPLC Protein BEH SEC Columns provides the capability to readily transfer between SE-UPLC and SE-HPLC for the first time. This application note details and provides examples of the methodology that can successfully employed for this method transfer.


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