Published:Journal of Chromatographic Science,
ISSN 0021-9665Volume
39, Number 10, October 2001, pp.
Preparation and Characterization of 3-(Aza-18-Crown-6) Propylsilyl
Bonded Phase for Reversed-Phase Liquid Chromatography
Shi-Lu Da, Yu-Qi Feng, Hui-Ning Da, Yin-Han Gong, and
Yuan-Wei Zhang
39,
Number 5, May 2001, pp. 205-212
Liquid Chromatographic
Studies of the Effect of Phosphate on the Binding Properties of Silica-Immobilized
Bovine Serum Albumin W.A. Tao and R.K. Gilpin
Department of Chemistry, Wright State University, Dayton, OH 45435
High-performance
liquid chromatography has been used to examine how phosphate ions affect the
binding properties of bovine serum albumin (BSA) immobilized to porous silica.
In doing this, the time dependence of the protein to reach conformational equilibrium
is measured as a function of the concentration of phosphate in the eluent using
the D- and L-isomers of tryptophan and kynurenine as solutes. The overall binding
and chiral selectivity (aD,L) of the protein toward these solutes appear to
be related to two types of effects: one being those that are site-selective
and only influence the retention of the L-isomers and the other being those
that are nonselective and influence the retention of both enantiomers. An interesting
feature of the concentration-dependent data is a maximum in aD,L at intermediate
phosphate concentrations (i.e., 10 to 50mM phosphate) indicative of both cooperative
and antagonistic binding effects. Phosphate eluents within this concentration
range provide selectivity advantages, and those at higher concentrations decrease
the time required for the protein or column to reach equilibrium. A final set
of studies has also been carried out using four alternate buffer systems (i.e.,
borate, carbonate, acetate, and arsenate eluents). Although the borate eluents
affect the BSA’s binding properties and aD,L similar to the phosphate eluents,
the other buffers result in poor separations. Observations from this study are
useful in helping to optimize separations carried out on immobilized BSA as
well as addressing biological and mechanistic questions related to how anions
influence the native binding properties of serum albumins.
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