Photo-response assisted enantiomer separations on an azobenzene-modified gamma-cyclodextrin stationary phase in micro-HPLC.
The photo-responses of the retention and enantioseparation of several optical isomers were evaluated using an azobenzene-modified gamma-cyclodextrin stationary phase (Az gamma-CDSP) in micro-HPLC. UV light irradiation induced a decrease in the retention and the chiral selectivity for N-(3,5-dinitrobenzoyl)-1-phenylethylamine (DNBPEA) and N-(3,5-dinitrobenzoyl)-1-(1-naphtylethyl)amine (DNBNEA), while an increase was induced for dansylphenylalanine (DnsPhe) using a mixture of methanol and aqueous phosphate buffer as the mobile phase. No changes in the retention and the enantiomer separation of benzoin were observed with UV light irradiation. The retention behaviors were recovered by visible-light irradiation. It was speculated that the main factor of the change in the retention behavior was a change in the pi-pi interaction due to the azobenzene moiety of the stationary phase with photo-irradiation. Comparing the retention behavior before and after UV light irradiation, a suitable condition for obtaining a better resolution and enantiomer separation would be chosen using Az gamma-CDSP.
Separation of proteins by polymeric adsorbents containing azobenzene moiety as a ligand
Polymeric adsorbents containing an azobenzene moiety as a ligand were prepared, and the photoinduced adsorption behavior of proteins to the adsorbent was studied. This method regulates by light the adsorption/desorption of proteins in hydrophobic chromatography. In the dark, the amount of protein adsorbed onto the adsorbent increased with increasing hydrophobicity of either adsorbent or protein. On irradiation with UV light, the amount of protein adsorbed decreased. Such a photoinduced change of the adsorption of protein was considered to be caused by the change of the hydrophobic interaction between the adsorbent and the protein due to the photoisomerization of the azobenzene moiety accompanying the polarity change of the adsorbent. It is also found that the desorption of protein was dependent on the balance of the hydrophobicity between the adsorbents and proteins. When column chromatography was carried out, the proteins were adsorbed in the dark and could be eluted after photoirradiation, with water as the single solvent. Furthermore, mixture of proteins could be separated by using a hydrophobic gradient column which was constituted by two polymeric adsorbents having different hydrophobicity.
Temperature-controlled high-performance liquid chromatography using a uniformly sized temperature-responsive polymer-based packing material.
Uniformly sized polymer-based packing materials modified with a temperature-responsive polymeric selector, poly(N-isopropylacrylamide), on either the internal or the external surface were prepared by an in situ surface-selective modification method. These packing materials were utilized as stationary phases in reversed-phase high-performance liquid chromatography to evaluate their ability to thermally control chromatographic separation selectivity. A packing material having a modified internal surface showed temperature-dependent separation selectivity in drug separation. Drugs that could not be separated below the phase transition temperature of the poly(N-isopropylacrylamide) selector were separated faster and with much better resolution above the phase transition temperature, while the unmodified base packing material afforded worse resolution of the drugs above the phase transition temperature due to the peak overlapping resulting from faster elution of all the solutes employed. In addition, a packing material modified on the external surface showed a temperature-responsive shift of the elution region for a polypeptide such as BSA, which was thought to be useful in direct injection of a serum sample.