磁性膨脹床吸附介質其物理化學性質探討

Physical and biochemical characterization of magnetic adsorbent applied in expanded bed chromatography

雷嘉雯(Chia-Wen Lei)

指導教授 ： 藍祺偉

元智大學/工程學院/化學工程與材料科學學系/碩士(2012年)

摘要

膨脹床吸附（Expanded bed adsorption, EBA）技術為一種應用於生化回收與程序整合的新型態生物分離技術。相較於傳統層析吸附，EBA集固液分離、濃縮及初步純化等單元操作結合成單一步驟，可以從發酵液中直接回收生物分子。
本研究以纖維素為基本原料，並且利用不鏽鋼粉末以及四氧化三鐵奈米級磁性粉末當作增重劑，採用反相懸浮熱再生技術製備磁性吸附劑。並分析其物理性質，包括濕真密度、含水率、孔度、孔容、比表面積、平均孔徑等。結果指出濕真密度為1.12 g mL-1到1.31 g mL-1，粒徑大小為150~300 μm，含水率從74%到57%，比表面積1.4~2.3 m2 mL-1。流體化性質方面以滯留時間分佈法進行量測，其關鍵因子包括理論塔板數、等板高度、Bodenstein number以及軸向擴散係數等。實驗結果發現自製磁性吸附劑M10在外加磁場時擁有較佳的流體混和性質，且操作流速可達500 cm h-1，是未加磁場M10和STREAMLINE Base的兩倍。
利用光學顯微鏡、掃描式電子顯微鏡與傅立葉轉換紅外線光譜儀對自製磁性吸附劑的外觀、表面結構與官能基進行觀察。發現自製吸附劑擁有完整的球形形狀，磁性粉末亦均勻分布於微球中。表面上有豐富的網狀結構，而紅外線光譜顯示出自製磁性吸附劑的吸收峰(–OH、C=O、C–O和Fe–O鍵)。從以上基本性質測試與表面型態分析結果顯示自製磁性吸附劑M10具有較高的比表面積以及理想的膨脹床介質條件。
接著以染劑固定化方法將介質表面接上親和性官能基之吸附劑，以等溫吸附實驗探討新型磁性吸附劑的吸附效果，以及探討熱力學和動力學參數，與STREAMLINE Base做比較。吸附結果發現，M10在溫度為308 K時有最佳吸附效果，吸附BSA蛋白和Lysozyme的最大吸附量分別為18.04 mg g-1和17.28 mg g-1。以熱力學分析表明，本研究的吸附系統為自發性吸熱反應。最後將此親和性磁性吸附劑填入管柱中，實際進行膨脹床程序吸附BSA蛋白，以相同膨脹度不同流速為基準，探討不同類型吸附劑的吸附結果。結果指出，外加磁場M10吸附BSA蛋白，其產率可高於其他兩者10%，回收率則是高於其他兩者5%。因此，本研究能夠成功製備出應用於膨脹床層析技術的磁性吸附介質，於膨脹床外添加磁場後，可操作流速可加快2倍，並且吸附效果能夠提高，與市售吸附劑STREAMLINE Base具有競爭力。

關鍵字

磁性顆粒 ； Fe3O4 ； STREAMLINE Base ； 膨脹床

並列摘要

Compared with the conventional adsorption chromatography, the expanded bed adsorption (EBA) technology offers the advantages of combining the unit operations of particulate removal, product concentration and product capture into one single step in protein recovery. Biomolecules can be recovered directly from fermentation broth.
The cellulose, stainless steel powder and Fe3O4 nano magnetic powder were employed for custom-make adsorbent through the water-in-oil suspension thermal regeneration method in the present study. The results of physical characterizations of the magnetic particles showed the wet densities in the values between 1.12 to 1.31 g mL-1 and the size distribution is in the range of 150 to 300 μm. The water content is from 57 % to 74 %, and the specific surface area is around 1.4~2.3 m2 mL-1. The liquid mixing performances in the expanded bed were studied through residence time distribution (RTD) test, which showed that flow velocity was an important parameter effecting on the stability of expanded bed. Using RTD analysis indicate the best characterizations of fludisation is M10 under the magnetism. However, the opration flow velocity (500 cm h-1) is 2-folds higher than STREAMLINE Base and M10 without magnetism (250 cm h-1) at identical expansion ratio.
The structure and morphology of magnetic particles were characterized using optical photomicrographs, scanning electron microscope (SEM), and Fourier transform infrared spectroscopy (FT-IR). The optical photomicrographs indicated that the spherical magnetic particles were dispersed uniformly. The SEM micrograph shows that the magnetic particles have a porous surface structure. Infrared spectroscopy depicted characteristics of absorption bands for magnetic particles (O–H, C=C, C–O–C and Fe–O bonds).
The STREAMLINE Base and M10 were subsequently immobilized with dye-ligand and played as affinity adsorbent to bind bovine serum albumin (BSA) and lysozyme in isothermal adsorption and different chromatography mode. In addition, thermodynamics and adsorption kinetics were determined. From the result when raised the temperature (298 K to 308 K) that increased the effect of adsorption capacity. The dynamic adsorption of BSA protein in EBA with Streanline Base and M10 were investigated at identical expansion ratio.
The maximum adsorption capacity and optimal adsorption temperature of BSA protein for STREAMLINE Base and M10 were 35.48 mg g-1 at 313 K and 18.04 mg g-1 at 308 K, respectively. The maximum adsorption capacity and optimal temperature for adsorption of lysozyme on M10 resin was 17.28 mg g-1 at 308 K. The Kd values decreased when adsorbed BSA/lysozyme increased for either STREAMLINE Base or M10 resins between 298K and 318K. The negative values of Gibbs free energy were observed which demonstrated BSA protein and lysozyme adsorption system for STREAMLINE Base and M10 were spontaneous reaction.
Finally, the dynamic adsorption capacity of BSA using M10 adsorbent at EBA with external magnetism has higher 10% yield and 5% recovery than STREAMLINE Base and M10 without magnetism. The results demonstrated that M10 is a competitive adsorbent for recovery of biomolecules under expanded bed operation.

並列關鍵字

magnetic particle ； Fe3O4 ； STREAMLINE Base ； expanded bed

參考文獻

Al-Marzouqi, I., Levy, M. S., & Lye, G. J. (2005) Hydrodynamics of PEG-phosphate aqueous two-phase systems in a J-type multilayer countercurrent chromatograph. Journal of Liquid Chromatography and Related Technologies, 28, 1311-1332.

Altintas, E. B. & Denizli, A. (2006) Efficient removal of albumin from human serum by monosize dye-affinity beads. Journal of Chromatography B: Analytical Technologies in the Biomedical and Life Sciences, 832, 216-223.

Amersham Pharmacia Biotech (1997) Ion exchange chromatography principles and methods.

Amersham Pharmacia Biotech (2001) Protein Purification Handbook.

Amigo-Benavent, M., Athanasopoulos, V. I., & del Castillo, M. D. (2010) Ion exchange chromatographic conditions for obtaining individual subunits of soybean b-conglycinin. Journal of Chromatography B, 878, 2453-2456.