熱透鏡吸收光譜法在毛細管電泳上的應用

Applications of Thermal Lens Spectrometry (TLS) on Capillary Electrophoresis

陳男政(Nan-Zheng Chen)

指導教授 ： 林震煌

國立臺灣師範大學/理學院/化學系/碩士(2006年)

摘要

熱透鏡吸收法是利用分析物吸收雷射光而產生折射率上的改變，做為偵測分析物的方法，是一種具有極高靈敏度的分析方法。本研究利用此原理，開發毛細管電泳/熱透鏡吸收偵測法，針對非螢光性奈米粒子(奈米金、奈米鑽石)及非螢光性物質(孔雀石綠、甲醛)進行研究。
奈米鑽石的生物相容性非常好，表面積大而且可吸附許多藥物分子或生物分子，如蛋白質分子等。奈米鑽石的研究，將有可能取代現行在生物醫學實驗上使用螢光染料分子標記蛋白質的方法。但是，目前沒有理想的方法可以用來分離與分析奈米鑽石與其標識物。本研究首先使用不同粒徑的奈米金，進行毛細管電泳/熱透鏡吸收儀器系統的測試。利用奈米金表面微帶負電的特性，順利的分離了平均粒徑為15及70 nm的奈米金粒子。隨後以此系統，亦成功的分離了非螢光性奈米鑽石(平均粒徑35 nm)與吸附了胺基酸(L-Lysine、Glycine、L-(+)-Cysteine)的奈米鑽石。其中，胺基酸與奈米鑽石分別以濃度比10，100，500，900的比例混合後進行電泳分離，實驗發現奈米鑽石可以吸附平均重量比為500倍的胺基酸。
孔雀石綠是一種疑似具有致癌性的三苯甲烷類非螢光性有機染料，經常被拿來做為魚貨類的消毒劑或殺菌劑。一般對於孔雀石綠的檢驗方式以液相層析/紫外光吸收法最為普遍。但是紫外光吸收法的偵測靈敏度較差，不足以應付例行性/低濃度孔雀石綠的檢驗工作。本研究則嘗試選用熱透鏡吸收法進行研究，這是因為該方法的偵測靈敏度可隨著雷射功率的增加而提高。實驗發現，配合毛細管電泳/線上濃縮技術的使用，在最佳化條件之下，偵測下限可達~12 ppb。再者，低濃度的甲醇或甲醛難以使用氣相層析質譜法加以偵測。目前僅能以呈色法做為檢驗甲醇的簡易方法。這是將含有甲醇的溶液(例如脫色後的假酒)，加入透明無色的晶紅酸試劑(又名希夫試劑)。經過加熱處理後，可以看見顏色上的變化(若有甲醇會呈紫色)。但是這樣的方法，過於粗糙，誤判機率高。僅能篩選具有高濃度甲醇的假酒，不足以用來做為檢驗日常食品中，是否含有低濃度甲醇或甲醛的方法。本研究則同樣利用毛細管電泳/熱透鏡吸收法，以綠光雷射為光源，針對紫色產物進行分析與研究。實驗結果發現，傳統上的呈色法，其紫色產物並非僅為單一物質。實驗並發現晶紅酸試劑與甲醛的反應複雜，且其產物組成伴隨著反應時間改變而有所不同。此現象可由本研究的電泳圖譜上觀察到多數譜峰而被證明。

關鍵字

熱透鏡吸收光譜法 ； 毛細管電泳

並列摘要

Thermal lens spectrometry is a highly sensitive detection method, which makes use of analytes to absorb laser light and produce the change of refractive index. The study based on this principle, developing a capillary electrophoresis/thermal lens absorbance method to detect non-fluorescence materials, including nanoparticles (nanogold and nanodiamond) and organic materials (malachite green and formaldehyde).
The biological compatibility of nanodiamond is very good, the surface accumulates greatly and can adsorb many medicinal molecules or living creature molecules, as proteins etc... The research of nanodiamond will replace the current method that using fluorescent dye molecules to label proteins in the biomedical science. Nonetheless at present there is no ideal method to separate nanodiamond and its labeling materials. This study first used nanogold in different size to test the capillary electrophoresis/thermal lens absorbance instrumental system. Nanogold particles with average diameters of 15 and 70 nm were usefully separated by the character of its slightly negative surface. Later on with this system, non-fluorescence nanodiamond (average diameters of 35 nm) and nanodiamond adsorbing amino acids (L – lysine、glycine and L – (+) – cysteine) were also successfully separated . Among them, amino acids were individually mixed with nanodiamond by the concentration ratio 10, 100, 500, 900 to proceed CE analysis. Experimental results showed nanodiamond could adsorb amino acids that were 500 times with the gravimetric ratio.
Malachite green, a non-fluorescent organic dye, that is potentially dangerous to human health, has been used illegally in the treatment of certain fish diseases, mainly, against parasites in fishwater and marine fishes. Generally the widespread examination method for malachite green is liquid chromatography/ultraviolet absorbance. Nevertheless the detection sensitivity of ultraviolet absorbance is poor and is not enough to cope with routine and low concentration examination task for malachite green. This study use thermal lens absorbance method to undergo research because the detection sensitivity can be improved with the advancement of laser power. By the use of capillary electrophoresis/on-line sample concentration, the limits of detection could be improved to 12 ppb at optimal condition. Furthermore, low concentration methanol or formaldehyde is hard to be detected by gas chromatography. The chromogenic method is the only simple examination method for methanol at present, which is that the solution containing methanol (such as color-rejected fake wines ) adding to colorless Schiff , s reagent. After heating the reactant, the color changes (The solution will be purple if methanol exists ). Yet such method is crude and highly miss-judged. It can only sift fake wines containing high concentration methanol, and isn't enough to examine whether low concentration methanol or formaldehyde exists in daily foodstuffs. This study also used capillary electrophoresis/thermal lens absorbance method and made use of green laser as light source to analyze the purple product. Experiment results showed the purple product was not unique by the traditional chromogenic method. It also found that the reaction between Schiff , s reagent and formaldehyde was complicated, and its product components varied with reaction time. This phenomenon could be proof by a lot of peaks observed from electropherograms.

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