Type of Document Dissertation Author Tedjo, Chrysanty Author's Email Address email@example.com URN etd-03252011-151048 Title Controlling protein-surface interactions in chromatography using mixed self-assembled monolayers Degree PhD Department Chemical Engineering Advisory Committee
Advisor Name Title Paul E. Laibinis Committee Chair David E. Cliffel Committee Member G. Kane Jennings Committee Member M. Douglas LeVan Committee Member Scott A. Guelcher Committee Member Keywords
- self-assembled monolayers
- protein separation
Date of Defense 2011-01-05 Availability unrestricted AbstractHigh performance liquid chromatography (HPLC) has become an essential tool in biotechnology. Numerous proteins have been separated by HPLC for analytical and preparative separation purposes. The main challenge in protein chromatography is the ability to isolate the protein of interest in its active form with high purity. The goal of this research is to provide a generic chromatographic approach for separating proteins efficiently with high retention of their biological activity. Here, I utilized mixed self self-assembled monolayers (SAMs) of Cl3Si(CH2)11(OCH2CH2)3OCH3 (EG3OMe) and n-octyltrichlorosilane (C8) to modulate protein-silica support interactions in a controlled manner. The oligo(ethylene glycol) tail groups in the EG3OMe SAM display a surface hydrophilicity that minimizes interactions with proteins. The C8 SAM displays a hydrophobic surface that adsorbs proteins. With mixed SAMs, the wetting properties of their surface and their level of interactions with proteins can be controlled.
Mixed SAMs of EG3OMe and C8 were deposited on SiO2/Si substrates and silica supports. Analysis using x-ray photoelectron spectroscopy showed that the surface compositions of mixed SAM-coated particles were similar to those of mixed SAMs formed on flat substrates. The critical surface tensions of the mixed SAM-coated silica particles, estimated by a developed flotation method, were similar to those of the same mixed SAMs on SiO2/Si substrates, as measured using contact angle goniometry.
The performances of these mixed SAM coated-supports were examined in a HPLC system using several model proteins. The results from chromatographic experiments showed that protein retention and separation in a chromatographic column was controllable by selecting the appropriate level of column hydrophobicity and the concentration of salt in the mobile phase. Analysis of protein mass recoveries after chromatography revealed that the rate loss of protein in the column was primarily a function of the protein residence time.
The described methods provide a systematic way for controlling protein retention during chromatography by tuning the hydrophobicity of the support. With proper selection, a surface hydrophobicity can be produced so that a protein can be effectively retained by a support without resulting in its permanent loss during chromatography.
Filename Size Approximate Download Time (Hours:Minutes:Seconds)
28.8 Modem 56K Modem ISDN (64 Kb) ISDN (128 Kb) Higher-speed Access tedjoc.pdf 3.33 Mb 00:15:25 00:07:56 00:06:56 00:03:28 00:00:17