An Invesitgation of a Novel Monolithic Chromatography Column, Silica Colloidal Crystal Packed Columns

Persistent Link:
http://hdl.handle.net/10150/193936
Title:
An Invesitgation of a Novel Monolithic Chromatography Column, Silica Colloidal Crystal Packed Columns
Author:
Malkin, Douglas Scott
Issue Date:
2010
Publisher:
The University of Arizona.
Rights:
Copyright © is held by the author. Digital access to this material is made possible by the University Libraries, University of Arizona. Further transmission, reproduction or presentation (such as public display or performance) of protected items is prohibited except with permission of the author.
Abstract:
Many researchers have investigated ways to improve the separation power of conventional chromatography, most notable is the development of ultra-high performance liquid chromatography (UHPLC). However, only slight improvements in separation efficiency have been achieved up to this point, and unfortunately, modern reversed phase liquid chromatography (RPLC) methods do not have high enough resolving power to analyze complex proteomic mixtures.Uniformly sized silica particles from 10 nm to 1 micron are known to self-assemble into a highly ordered face centered cubic crystal. Silica colloidal crystals have shown recent promise in biological applications such as permselective nanoporous membranes, DNA sieving, reversed phase separation of small molecules on planar substrates, protein sieving, microarrays, total internal reflection fluorescence microscopy of live cells, and 3-D scaffolds for supported lipid films. In this work, silica colloidal crystals packed in capillaries are explored for their potential improvement in the efficiency of reversed phase chromatography.The silica colloidal crystal columns were chemically stabilized by with trichlorosilanes. The trichlorosilanes form chemical bonds between the particles and the particles and the substrate creating an increase in mechanical stability, and at the same time, providing an excellent chromatographic monolayer. After stabilization the fritless columns were able to withstand the pressure limit of the commercial UHPLC. Next, the columns were characterized using a small dye molecule, 1,1' - Didodecyl - 3,3,3',3' - tetramethylindocarbocyanine (DiIC12). The dye was run under capillary electrochromatography (CEC), and sub-micron plate heights were achieved. Further, a van Deemter plot of the dye molecule indicates that the plate height is largely due to the molecule's diffusion. This result suggests that the plate heights for proteins would be even smaller, since proteins have diffusion coefficients an order of magnitude smaller. The analysis of proteins by CEC yielded nanometer plate heights. Finally, pressure driven flow separations coupled with nano-electrospray ionization (n-ESI) MS have also been explored. The Poiseuille flow profile has been shown not to perturb the low plate heights. Gradient elution of peptides was also achieved, and the results demonstrate the highest chromatographic peak capacities for short analysis times to date.
Type:
text; Electronic Dissertation
Keywords:
Chromatography; High Efficiency LCMS; Peptide; Plate Height; Protein; Resolution
Degree Name:
Ph.D.
Degree Level:
doctoral
Degree Program:
Chemistry; Graduate College
Degree Grantor:
University of Arizona
Advisor:
Wirth, Mary J.; Pemberton, Jeanne E.
Committee Chair:
Wirth, Mary J.; Pemberton, Jeanne E.

Full metadata record

DC FieldValue Language
dc.language.isoenen_US
dc.titleAn Invesitgation of a Novel Monolithic Chromatography Column, Silica Colloidal Crystal Packed Columnsen_US
dc.creatorMalkin, Douglas Scotten_US
dc.contributor.authorMalkin, Douglas Scotten_US
dc.date.issued2010en_US
dc.publisherThe University of Arizona.en_US
dc.rightsCopyright © is held by the author. Digital access to this material is made possible by the University Libraries, University of Arizona. Further transmission, reproduction or presentation (such as public display or performance) of protected items is prohibited except with permission of the author.en_US
dc.description.abstractMany researchers have investigated ways to improve the separation power of conventional chromatography, most notable is the development of ultra-high performance liquid chromatography (UHPLC). However, only slight improvements in separation efficiency have been achieved up to this point, and unfortunately, modern reversed phase liquid chromatography (RPLC) methods do not have high enough resolving power to analyze complex proteomic mixtures.Uniformly sized silica particles from 10 nm to 1 micron are known to self-assemble into a highly ordered face centered cubic crystal. Silica colloidal crystals have shown recent promise in biological applications such as permselective nanoporous membranes, DNA sieving, reversed phase separation of small molecules on planar substrates, protein sieving, microarrays, total internal reflection fluorescence microscopy of live cells, and 3-D scaffolds for supported lipid films. In this work, silica colloidal crystals packed in capillaries are explored for their potential improvement in the efficiency of reversed phase chromatography.The silica colloidal crystal columns were chemically stabilized by with trichlorosilanes. The trichlorosilanes form chemical bonds between the particles and the particles and the substrate creating an increase in mechanical stability, and at the same time, providing an excellent chromatographic monolayer. After stabilization the fritless columns were able to withstand the pressure limit of the commercial UHPLC. Next, the columns were characterized using a small dye molecule, 1,1' - Didodecyl - 3,3,3',3' - tetramethylindocarbocyanine (DiIC12). The dye was run under capillary electrochromatography (CEC), and sub-micron plate heights were achieved. Further, a van Deemter plot of the dye molecule indicates that the plate height is largely due to the molecule's diffusion. This result suggests that the plate heights for proteins would be even smaller, since proteins have diffusion coefficients an order of magnitude smaller. The analysis of proteins by CEC yielded nanometer plate heights. Finally, pressure driven flow separations coupled with nano-electrospray ionization (n-ESI) MS have also been explored. The Poiseuille flow profile has been shown not to perturb the low plate heights. Gradient elution of peptides was also achieved, and the results demonstrate the highest chromatographic peak capacities for short analysis times to date.en_US
dc.typetexten_US
dc.typeElectronic Dissertationen_US
dc.subjectChromatographyen_US
dc.subjectHigh Efficiency LCMSen_US
dc.subjectPeptideen_US
dc.subjectPlate Heighten_US
dc.subjectProteinen_US
dc.subjectResolutionen_US
thesis.degree.namePh.D.en_US
thesis.degree.leveldoctoralen_US
thesis.degree.disciplineChemistryen_US
thesis.degree.disciplineGraduate Collegeen_US
thesis.degree.grantorUniversity of Arizonaen_US
dc.contributor.advisorWirth, Mary J.en_US
dc.contributor.advisorPemberton, Jeanne E.en_US
dc.contributor.chairWirth, Mary J.en_US
dc.contributor.chairPemberton, Jeanne E.en_US
dc.contributor.committeememberWysocki, Vicki H.en_US
dc.contributor.committeememberGhosh, Indraneelen_US
dc.identifier.proquest11347en_US
dc.identifier.oclc752261207en_US
All Items in UA Campus Repository are protected by copyright, with all rights reserved, unless otherwise indicated.