利用膠體滲透層析儀配合黏度計與光散射儀量測微脂體顆粒粒徑與樹枝狀高分子分子量的探討

Determinations Of Liposome Size And Dendrimer Molecular Weight By Using Gel-Permeation Chromatography Coupled With Differential Viscometer And Light Scattering Detector

鄭涵玲(Han-Ling Cheng)

指導教授 ： 王少君

國立中正大學/理學院/化學暨生物化學系/碩士(2012年)

摘要

膠體滲透層析儀(Gel-Permeation Chromatography, GPC)為一種常用於測量高分子聚合物之分子量分佈的技術，其利用尺寸大小排除法的原理搭配具有低角度(7°)(low-angle laser light scattering；簡稱LALLS)與直角度(90°)(right-angle laser light scattering；簡稱RALLS)光散射儀、微差黏度計(differential viscometer；簡稱DV)以及折射率儀(refractive index；簡稱RI)的多功能偵測器，可在溶液中直接測得不同大分子的粒徑大小，並在不需使用高分子標準品即可計算出絕對分子量。
首先使用一系列pullulan標準品(5k, 10k, 20k, 50k, 100k, 200k, 400k, 800k)利用泛用分子量校正法(Universal Calibration；簡稱UC)來建立GPC分析管柱(Viscogel column)的關係曲線，由實驗上所獲得的分子量對滯留體積(retention volume)的線性關係，確定此管柱適當的流析條件。
此外，利用LALLS、RALLS及前兩者結合Viscometer等三種不同偵測方法，配合RI偵測器得到的層析圖譜，來比較使用不同類型管柱(Viscogel分析管柱和保護管柱guard column)量測推估pullulan分子量與真實分子量的相對誤差。亦可由RI、LALLS、RALLS與Viscometer四種偵測器訊號推得水力半徑(Radius of hydrodynamic, Rh)以及迴轉半徑(Radius of gyration, Rg)。
三種偵測方法("RI+RALLS+DV"、"RI+RALLS"、"RI+LALLS")，測得P400k分子量相對誤差不大(~3%)，最小的量測誤差出現在"RI+RALLS+DV"方法，推測原因為在高角度光散射下量測訊號靈敏度較高再加上搭配黏度計的修正，將以此法作為主要量測方法。
在微脂粒顆粒量測方面，使用超音波震盪法來合成微脂體(Liposome)，藉由所建立之GPC靜態光散射法來量測微脂粒的顆粒大小，其旋轉半徑(Rg)值約44~48nm，並以動態光散射分析方法(水力半徑值約55 nm)及穿透式電子顯微鏡(約47 nm)來驗證之。
最後在聚乙二胺樹枝狀高分子量測方面，雖然在缺乏適當儀器常數校正下，無法準確計算出樹枝狀高分子真實分子量。但利用參考文獻資料(粒徑、分子量)加上GPC所得數據(IV、RALLS積分面積、濃度)建立以RALLS積分面積除以濃度值和分子量的尺度關係圖，可得到呈高度相關的R2值。另外並以愛因斯坦黏度方程式來確認極限黏度和代數之間的關係。
期望日後由樹枝狀聚合物數據製成之上述尺度關係圖可進一步探討不同代數間的反應動力學研究。

關鍵字

樹枝狀分子 ； 微脂體 ； 膠體滲透層析儀

並列摘要

Gel-Permeation Chromatography (GPC) is the technique which can measure the molecular weight (M.W.) and polydispersity index (PDI) of polymer using size exclusion mechanism. GPC method with multi-functional detectors including low-angle laser light scattering (LALLS), right-angle laser light scattering (RALLS), differential viscometer (DV) and refractive index (RI) enables to determine the particle size of different macro-molecules in solution and to calculate the absolute molecular weight without using any polymer standards.
First, we use pullulan standards (5k, 10k, 20k, 50k, 100k, 200k, 400k, and 800k) to carry out universal calibration (UC) method to establish the relation curve using GPC (Viscogel) column, which shows the linear relation of the molecular weight versus retention volume to confirm the size ranges of proper exclusion.
Next, the chromatograms acquired with the detection combinations of LALLS, RALLS, and DV along with RI detector are able to compare the determination bias of pullulan M.W. between using Viscogel column and guard column. One also can employ the above four-detector methods to estimate the hydrodynamic radius (Rh) and radius of gyration (Rg) of macro-molecules.
The relative error of pullulan 400k M.W. determination differences between using three detection methods ("RI+RALLS+DV", "RI+RALLS", "RI+LALLS") are small (~3%). The least determination measurement error is found using "RI+RALLS+DV" detection method because of better signal sensitivity of light scattering collected at the right angle and angular correction with viscometry measurement.
We can also use the GPC method with two static light scattering detectors collecting at right-angle and low-angle, respectively to measure liposome particle size. The Rg of liposome is determined as 44-48 nm using GPC with light scattering detectors which is verified by comparing the Rh of liposome measured as 55 nm with dynamic light scattering method and the particle size of 47 nm imaged with transmission electron microscope.
Finally, we use characterized particle size and M.W. data of polyamidoamine dendrimer standards to confirm the scaling relation of RALLS intensity/ concentration versus M.W. Although dendrimer M.W. cannot be determined because of lacking proper instrument constant calibration. We also use Einstein viscosity equation to confirm the relation of intrinsic viscosity versus dendrimer generations.
Further study of dendrimer reaction kinetics in different generations can be investigated by using the above scaling relations.

並列關鍵字

Liposome ； Gel-Permeation Chromatography ； Dendrimer

參考文獻

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Springer-Verlag: Berlin, 1999.

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3. Striegel, A. M.; Yau, W. W.; Kirkland, J. J.; Bly, D. D. Modern Size-Exclusion Liquid Chromatography, 2nd ed.; Wiley: New York, 2009.

4. Grubisic, Z.; Rempp, P.; Benoit, H. J. Polym. Sci. B Polym. Phys.1996, 34, 1707-1713.