Sürekli ve Kesikli Sistemlerde Borat Afinite Kromatografisi için Sabit Fazların Sentezi ve Ayırma Performanslarının Belirlenmesi

Abstract

Boronate affinity chromatography is being widely used in various applications in many different areas. In this study, monodisperse porous silica (SiO2) and magnetic silica (MagSiO2) microspheres were synthesized to utilize as a stationary phase in boronate affinity chromatography. The 3-aminophenylboronic acid (APBA) ligand was covalently attached to the microspheres, resulting in APBA-attached silica microspheres (SiO2APBA) and APBA-attached magnetic silica microspheres (MagSiO2APBA). The suitability of sorbents to use in boronate affinity chromatography was investigated through the adsorption desorption behaviors of two target biomolecules; ribonucleic acid (RNA) and β-nicotinamide adenine dinucleotide (β-NAD). Adsorption studies were carried out at pH 8.5 and room temperature, HEPES buffer was used as the adsorption buffer. The maximum equilibrium adsorption for RNA and β-NAD using SiO2APBA microspheres were found as 60 and 159 mg biomolecules / g sorbent, respectively, with quantitative desorption.

In the beginning, desorption studies run with pH 10.2 borate desorption buffer. Due to low desorption yields, the desorption studies of SiO2APBA were repeated with pH 7.0 phosphate buffer including different amounts of sorbitol as a competing diol. With the usage of the mild elution medium quantitative desorption efficiencies were obtained with SiO2APBA microspheres. MagSiO2APBA microspheres generally achieved quantative desorption yields with pH 10.2 borate buffer.

Microfluidic system studies were carried out with a 300 μm diameter microcolumn which is filled with SiO2APBA microspheres. Different feed concentrations were prepared for both RNA and β-NAD, where satisfactory isolation and desorption yields have been acquired. With the proposed microfluidic system, it is possible to recover valuable cis diol groups even if the sample volume is low.

Consequently, the adsorption desorption behaviors of the stationary phases synthesized were evaluated on RNA and β-NAD biomolecules for the boronate affinity chromatography. The results obtained at different biomolecule and sorbent concentrations indicated that the proposed system is very promising for the isolation of the cis-diol groups at low concentrations from very low volume of samples.