Macrocyclic Polyamine Bonded Phase and Gold Nanoparticles Self-assembled Column for Bioanalytical Applications
Date Issued
2007
Date
2007
Author(s)
Chen, Tse-Hsien
DOI
zh-TW
Abstract
Two macrocyclic polyamines, [32]ane-N8 and [28]ane-N6O2 were prepared and employed as a selective modifier for the capillary electrochromatographic separation. Under the same coating process, it has been observed that [28]ane-N6O2 coated column showed higher electroosmotic flow (e.g. μeo = -9.67 × 10-4 cm2V-1s-1) than that of [32]ane-N8 (μeo = -6.19 × 10-4 cm2V-1s-1) at pH 4. It is noteworthy to mention that μeo values increase if pyridine is added into the solution of coating process. As for example, μeo increases from -1.31 × 10-4 to -4.43 × 10-4 cm2V-1s-1 at pH 6, indicating that a greater amount of the bonded ligands were found with the addition of pyridine. With [32]ane-N8 coated column, the optimum conditions for the separation of aminothiols were at -20 kV using an acetate buffer (30 mM, pH 4.0), hydrodynamic injection and detection wavelength at 214 nm. A basic mixture (pH 7.76) of sulfur-containing biomolecules was baseline separated in less than 15 min. In the similar experimental conditions, the [28]ane-N6O2 coated capillary column showed only five peaks and no characteristic splitting for reduced and oxidized form of glutathione. With pH gradient mode, the baseline separation of the acidic mixture (pH 2.73) was demonstrated using an acetate buffer (30 mM) with inlet buffer pH 4 and outlet buffer pH 3 (pH 4-3). But ethanol was needed in the mobile phase for the separation of acidic mixture (pH 1.89).
A mixture of angiotensin (I、II and ST)、β-casomorphin (bovine and human)、oxytocin acetate、tocinoic acid、vasopressin and FMRF amide could be separated using phosphate buffer (pH 7, 30 mM). Column efficiency was found with the average theoretical plate numbers of 70000 m-1 and relative standard deviation (RSD) of < 1 % (n = 6) The prepared column can be used with injections more than 600 with the EOF increase only 5.91 %. [Met5]-enkephalin and [Leu5]-enkephalin could also be separated with the bonded phase in 30 mM acetate using pH gradient method (pH 4-3). The [32]ane-N8 coated column was also applied to separate of mono- and disaccharides. The reductive aminated product was carried out with p-aminobenzoic acid. A synergic effect was observed for the separation of borate complexes with the bonded phase. A complete separation of all compounds as well as the excess derivatizing agent by using borate buffer (pH 9.0) in a mode of concentration gradient (60 mM inlet side and 70 mM outlet side) could be achieved. The RSD of the migration time measured for each sample was less than 4 % in six continuous runs, suggesting that the bonded phase along with the gradient formed inside the column was quite stable. The results indicated that with the mixing modes of anion coordination, anion exchange, and shape discrimination, the interaction adequately accomplishes the separation of carbohydrates which are epimers or have different glycosidic linkage, although the electrophoretic migration is also involved in the separation mechanism.
The gold nanoparticles(Au NPs) was immobilized in fused-silica capillary column wall by (3-mercaptopropyl)trimethoxysilane for the separation of sulfur-containing biomolecules. We used high concentration buffer (80 ~ 120 mM) to avoid bubble formation. Baseline separation of basic mixture was obtained at 20 kV, using an acetate buffer (80 mM, pH 4). The results suggested that the interaction between analytes and the bonded groups on the wall is ligand exchange. The Au NPs solutions were added into both 32- and 28-membered macrocyclic polyamines for realizing the status of NPs in the presence of these ligands. Spectral changes and TEM pictures demonstrate that the macrocyclic polyamines, which possess positive charges, trigger aggregation of negatively charged citrate-capped nanoparticles. Then the Au NPs self-assembled in a [32]ane-N8 coated column wall. The good separation efficiency was procured not only sulfur-containing biomolecules but also proteins include six ovalbumin peaks are observed.
Subjects
巨環多胺化合物
金奈米粒子
毛細管電層析
macrocyclic polyamine
gold nanoparticles
capillary electrochromatography
Type
thesis