界面活性劑在逆流層析分離的應用暨在逆流層析術中溶質分散之研究

Abstract

逆流層析常用的動靜相系統為有機-水相系統，本論文使用界面活性劑分子修飾於動相或是靜相溶劑，應用於逆流層析之分離。本論文的第一部分，是利用含有界面活性劑sodium 1-heptanesulfonate (SHS)的正己烷-水相系統，分離類固醇，以正己烷為動相沖提，SHS的濃度尚未到達臨界微胞濃度以前，類固醇的滯留時間僅微幅增加，反之，在高於臨界微胞濃度之後，類固醇的滯留時間會大幅增加，此外，微胞靜相系統對於極性太低的芳香族化合物無滯留能力，對於酯類也無明顯的滯留效果，微胞對於分離疏水性化合物提供了一種溶劑系統的選擇。 本論文的第二部分，利用界面活性劑bis(2-ethylhexyl) sodium sulfosuccinate形成反微胞，將含有反微胞的正己烷為逆流層析靜相，分離肌紅蛋白、細胞色素C以及溶菌酶，藉由動相中的酸鹼值以及離子強度的梯度變化，能調整蛋白質在水相-微胞相中的分佈，增加分離的解析度，細胞色素C的製備回收率約90%，溶菌酶的回收率約為82%，利用反微胞靜相製備分離蛋白質，對於蛋白質還有濃縮的效果，期望可應用於大量的蛋白質製備分離。 本論文的第三部分，是於逆流層析儀中，使用連續萃取法分離銀奈米粒子，銀奈米粒子因表面修飾負電荷，在陽離子界面活性劑(tetraoctylammonium bromide, TOAB)的幫助下，由離子對萃取方式，從水中萃取至甲苯/正己烷(1:1)混合溶液，較小的奈米粒子較容易萃取到有機溶液中，我們發現以0.02 mM TOAB做為動相萃取，其分離效果最好，原本粒徑分佈為15.8 ± 5.3 nm的奈米粒子，經過分離後，可得奈米粒子為13.7 ± 1.9 nm、14.1 ± 3.5 nm、19.2 ± 4.3 nm、22.2 ± 4.9 nm分佈的四支收集管，比起批式萃取法，逆流層析連續萃取法可提供較好的分離解析度。 本論文的最後一個部分，則是探討逆流層析儀，轉動螺旋管柱中的流動訊號，藉由觀察一系列分子的流動訊號，包含銀奈米粒子、牛血清蛋白、溶菌酶、抗壞血酸、染料分子、碘化鉀金屬鹽，分析其在轉動螺旋管柱中的運動行為，若樣品的擴散較差，會產生不對稱的波形，稱為convection peak，再將CCC轉速提高，原本不對稱的波形會變成對稱的類高斯波形。我們進一步歸納出，逆流層析轉動之下理論板數的計算方式，可為逆流層析的基本原理以及訊號波形等，提供更詳細的研究資訊。

The most commonly used stationary/mobile phase solvent systems in counter-current chromatography (CCC) are organic-water systems. However, we employed surfactant-modified solvent systems in CCC separations. In the first part of this thesis, an n-hexane/surfactant -containing water solvent system was developed to separate a steroid sample. Retention times of steroids increased slightly by increasing the sodium 1-heptanesulfonate (SHS) concentration below the critical micellar concentration. However, the retention times increased drastically while the SHS concentrations were above the CMC. Aromatic hydrocarbons were not retained by the stationary phase no matter what the surfactant concentrations were. The retention times of esters, however, were only slightly affected by the surfactant addition even above the CMC. The micellar solvent systems provide an alternative way for hydrophobic sample separations in CCC In the second part, a protein mixture consisting of myoglobin, cytochrome c, and lysozyme was separated by CCC using a two-phase aqueous/AOT reverse micelle-containing organic solvent system. Separations were manipulated mainly by pH gradients and incorporating an ionic strength gradient along with the pH gradient. The recovery of cytochrome c and lysozyme reached 90% and 82%. Furthermore, concentration or enrichment of these two proteins was achieved from a large-volume sample load. This technique can potentially be employed in the recovery and enrichment of proteins from large-volume aqueous solutions. In the third part, size separation of silver nanoparticles was investigated in CCC based on a continuous extraction process. The aqueous nanoparticles were readily transferred to the organic phase (toluene/hexane = 1:1, v/v) together with the phase transfer catalyst, tetraoctylammonium bromide (TOAB), owing to the ion-pair adduct formation between silver nanoparticle anions and tetraoctylammonium cations. Smaller nanoparticles were found to be more readily transferred to the organic phase compared to larger nanoparticles. It appeared that a concentration of 0.02 mM TOAB was adequate to achieve optimum separation and recovery for the aqueous Ag nanoparticle sample. Samples of 15.8 ± 5.3 nm were separated; the distributions of four fractions collected were 13.7 ± 1.9, 14.1 ± 3.5, 19.2 ± 4.3, and 22.2 ± 4.9 nm. Compared with the stepwise extraction performed in this study, the step-gradient extractions using CCC provided much better size discrimination. In the last part of this thesis, the dispersion behavior of solutes was investigated in a rotating flowing coiled tube. Silver nanoparticles, bovine serum albumin, lysozyme, ascorbic acid, tartrazine, and potassium iodide samples were eluted in a coiled tube of counter-current chromatography (CCC) apparatus with a single phase. Apparent convection peaks of low-diffusivity solutes appeared in the static CCC tube, while Gaussian-like peaks showed up for the high-diffusivity solutes. When the rotation speed of the CCC apparatus was elevated, all solute peak widths became smaller, and the convection peaks of silver nanoparticles and BSA were minimized and formed Gaussian-like peaks. We studied the effect of rotating to the theoretical plate of the signals. The same reasoning could also be used to rationalize other special band shapes encountered in two-phase CCC separations