High-performance liquid chromatography of nucleic acid constituents: Chromatographic examination of novel stationary phases |
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Authors: | Z El Rassi Cs Horváth |
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Institution: | (1) Department of Chemical Engineering, Yale University, 06520 New Haven, CT, USA |
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Abstract: | Summary Silica-bonded stationary phases were developed for the separation of nucleic acid constituents and their properties investigated
with homologous oligoriboadenylic acids in electrostatic interaction chromatography and with alkylbenzenes in reversed-phase
chromatography. Analysis of retention data confirmed the stratified molecular structure of the surface which consist of a
layer of propyl chains anchoredvia siloxane bridges to the silica surface proper and of polar moieties attached to the hydrocarbonaceous functions. The polar
top layer contains weak cationic and/or hydrophobic binding sites, is strongly hydrated in contact with aqueous eluents and
bars the access by large biopolymers to the hydrocarbonaceous sublayer. In reversed-phase chromatography of small non polar
molecules with hydro-organic eluents, however, this layer is accessible and engenders a retentive behavior typical for weak
hydro-carbonaceous bonded phases. As a result the stationary phases, depending on the nature of the sample and the mobile
phase, exhibit the properties of "soft" phases for the chromatography of biopolymers under mild elution conditions and those
of "hard" phases for the separation of small non-polar molecules under conditions generally employed in reversed-phase chromatography.
The retention of nucleic acid constituents on most of the stationary phases investigated subject to a dual mechanism as a
result of the interplay of electrostatic and hydrophobic interactions between the eluites and the binding sites on the stationary
phase surface. Siliceous stationary phases having surface morphology described above are suitable for the separation of nucleic
acid constituents having widely ranging molecular weights up to 3 × 106 Daltons provided the support has appropriate pore dimensions. This is demonstrated by the separation of mixtures arising
from digesting t-RNApha or polyadenylic acids as well as those of ribosomal RNA’s and different forms of the plasmid pBR322 DNA.
Presented at the 15th International Symposium on Chromatography, Nürnberg, October 1984 |
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Keywords: | Column liquid chromatography Novel stationary phases Nucleic acids constituents |
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