Hydroxyapatite (HAp) is a natural calcium-phosphate (Ca10(PO4)6(OH)2) ceramic, commonly used in protein separations. HAp microspheres are extensively employed in a variety of biomedical and chromatographic applications. Conventional methods for HAp preparation often yield polydisperse particles through time-consuming and resource-intensive processes. This manuscript highlights an innovative approach, focusing on direct and rapid conversion of calcium carbonate (calcite) particles into HAp microspheres using an economical dissolution precipitation technique. The investigation systematically examines the impact of crucial reaction parameters, including reaction temperature, reaction time, and mechanical stirring rates, on the conversion process. By optimizing these parameters, the study identifies conditions that promote complete HAp conversion, as evidenced by X-ray diffraction (XRD) studies. Higher reaction temperatures and prolonged reaction times facilitate complete conversion to HAp, while lower values result in residual calcite. Scanning electron microscopy (SEM) images elucidate how these reaction parameters influence the surface microstructure of the generated microspheres, notably observing disintegration of HAp microspheres at higher stirring rates.
The optimized parameters from this investigation were utilized to produce eggshell-derived HAp (ECHAp), aiming to address the cost concerns associated with HAp matrices. The study presents a distinctive process, wherein eggshell calcite is transformed into vaterite microspheres, which are subsequently transformed into HAp microspheres. Comparison with synthetic-source calcium carbonate-derived HAp microspheres (CHAp) and a commercial HAp matrix (CHT) validates the promising properties of ECHAp, showcasing larger surface area (33.8 m2 g−1) and equivalent protein binding capacity. Importantly, ECHAp excels in the separation of proteins in bovine serum albumin (BSA) and lysozyme mixtures, surpassing both CHAp and CHT matrices. Furthermore, the ECHAp matrix proved highly effective in binding and purifying IgY under the chromatographic conditions specified in this thesis. Moreover, ECHAp demonstrates exceptional stability over multiple purification cycles. This study underscores the potential of utilizing eggshell waste as a novel and economical route for HAp matrix preparation, offering ease of synthesis and economic viability for chromatographic purification of biomolecules.
In addition, this research aims to modify the chromatographic properties of HAp by incorporating metal ions into its structure, allowing it to function as an immobilized metal affinity chromatography matrix. The unique crystal structure of HAp allows for various substitutions, providing an opportunity to modify its properties. In this particular study, the spray-drying method was utilized to prepare nickel-ion substituted HAp (NiSHAp). The material was extensively characterized using Scanning Electron Microscopy (SEM), X-ray diffraction (XRD), and Fourier Transform Infrared spectroscopy (FTIR). The results confirmed the purity of the NiSHAp without any impurity phases, and the particle size analysis revealed micron-sized particles with a narrow distribution. To evaluate its chromatographic performance, experiments were conducted to purify recombinant L-asparaginase. The nickel-ion substitution demonstrated a significant impact on the separation of L-asparaginase protein, surpassing the capabilities of conventional HAp that failed to effectively separate the protein from impurities. These findings highlight the potential of spray-dried nickel-ion substituted hydroxyapatite as a viable alternative to commercial metal-affinity matrices for chromatographic purification of recombinant proteins.