Design of Bonded Phase to Increase LC Resolution and MS Sensitivity
Abstract
Histones are strongly cationic, making them challenging to overcome electrostatic interactions with surface silanols in reversed-phase liquid chromatography coupled to mass spectrometry (RPLC-MS) separation, where trifluoroacetic acid (TFA) cannot be used. In this work, two novel bonded phases were designed to minimize or avoid the need for TFA while also increasing chromatographic resolution, as compared with conventional mono-functional alkylsilane bonded phases. The tri-functional horizontal polymerized C18 surface achieved 3.5-fold resolution increase for histone RPLC/MS separation compared with traditional mono-functional C18 in MS-compatible 0.1% difluoroacetic acid (DFA). It is attributed to less exposed silanols and a larger slope of ln k vs. %B for proteins in gradient elution. It is further applied to ubiquitin post-translational modifications RPLC/MS. On another novel polymer-shell on silica bonded phase, resolution higher than commercial alkyl bonded phase column has been observed for IgG1 free thiols RPLC analysis in 0.5% formic acid. The reduction of detrimental electrostatic interaction between proteins and stationary phase is shown to be the key to narrower peak widths. With change of monomer the polymer-shell on silica bonded phase can be applied in HILIC/MS separation mode for IgG1 glycosylation analysis.
Degree
Ph.D.
Advisors
Wirth, Purdue University.
Subject Area
Analytical chemistry
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