Chromatographic Characterization of Phosphonate Analog EDTA-Modified Zirconia Support for Biochromatographic Applications

Abstract

Zirconium dioxide (zirconia) has a great affinity for inorganic and organic phosphate. Previous work from this laboratory demonstrated the utility of phosphate-modified microparticulate zirconia as a support for protein separations. We have extended this investigation to include the study of ethylenediamine-N,N'-tetramethylphosphonic acid (EDTPA), a phosphonate analog of EDTA, as a surface modifier for zirconia. Our work explores the use of EDTPA-modified zirconia (PEZ) for its potential use as a high-performance inorganic cation-exchange support for the separation of proteins. The phosphate groups in EDTPA very effectively block the sites responsible for strong interactions of hard Lewis bases with zirconia's surface. Modification of zirconia with EDTPA provides a "biocompatible" stationary phase, resulting in high mass recoveries of proteins. We compare PEZ with inorganic phosphate-modified zirconia to show increased efficiency, as well as unique selectivities for chromatography of proteins on the chelator-modified surface. Finally, the selectivity, efficiency, and separation mechanism are reported. The studies show that PEZ is a useful high-performance ion-exchange support for the separation of cationic proteins and for modulating the sites responsible for the high affinity of zirconia toward certain classes of anions.

Preparation and Characterization of Alkylphosphonate-Modified Magnesia-Zirconia Composite for Reversed-Phase Liquid Chromatography

“…It was found to be 6.1 µmol m -2 , which is similar to phosphate coverage, 17 but greater than EDTPA coverage on zirconia. 18 The larger coverage of the alkylphosphonate might be ascribed to its linear structure (Fig. 1) and greater concentration of Lewis acid sites of the magnesia-zirconia composite.…”

“…5,7 Carr and co-workers have extensively studied carbon [8][9][10] and polymers 11-15 coatings on zirconia as surface modifiers of zirconia to create anion-exchange, hydrophobic and hydrophilictype phases with varying degrees of success. Since bare zirconia has a great affinity for Lewis bases, Carr and coworkers have studied modification of zirconia with Lewis bases such as fluoride, 16 phosphate 17 and ethylenediamine-N,N′-tetramethylphosphonic acid (EDTPA), 18 and their application in separation of proteins. We have studied preparation of spherical zirconia by sol-gel process 19 and modification of zirconia with stearic acid as reversed-phase for HPLC.…”

The use of confocal laser scanning microscopy to study the transport of biomacromolecules in a macroporous support

“…(C) Plate heights for lysozyme on EDTPA modified zirconia beads, 25 _m in diameter and pore size of 22 nm. Data adapted from (20). the solute within the bead, one should visualize fluorescent stain within the bead radius.…”

Cations on Ion Chromatography by Phosphate-Coated Zirconia Stationary Phase Column

“…Therefore, many researchers have attempted to use supports such as graphite carbon [1,2], apatite [3], and titania [4,5] as alternatives to silica gel or polymers. Use of a graphite carbon stationary phase, which has excellent chemical and physical resistance [6], has been considered effective because it can fix ion-exchangers such as ionized surfactants [2] and can use a buffer solution over a wide range of pH (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14). The ion-exchangers are primarily fixed by hydrophobic interaction with the graphite carbon stationary phase, which itself shows no ligand-exchange behavior.…”

“…To enhance analyte selectivity, Carr et al [7][8][9][10][11] and other researchers [12][13][14] modified various inorganic and organic species possessing ionic functional groups and applied them to liquid chromatographic columns. However, approaches to ion chromatographic columns are discussed in fewer reports.…”