開發用於分析生物微粒懸浮液及具揮發性化合物之複雜樣品的分離方法

Abstract

含有數百種化合物的複雜物質之化學分析是具有挑戰性的，所以在利用儀器進行偵測目標物質前減少樣品的複雜度是必須的。在此研究中，我們致力於開發兩種用於分析複雜樣品的方法。第一個方法用於生物粒子懸浮液的分析，而第二個方法用於具揮發性化合物之混合物的分析。

第一個分析方法利用了水動力層析法的優勢，藉由水動力層析法，在層流條件下產生拋物線流動分布使細胞和細胞聚集物分離。在目前的研究中，水動力分離法已與不同的線上及離線偵測器(光吸收/散射和顯微鏡)連接，並成功的應於於監測益生菌飲料中微生物的動態變化，優酪乳和克菲爾樣品在不同發酵時間下得到的層析圖譜，和顯微鏡圖片有良好的一致性。

第二個分析方法使用一定制開發的系統結合簡單微型蒸餾法與氣相層析法和電子游離質譜法。此系統以一儲存槽前後連接了蒸餾冷凝管與熔融二氧化矽毛細管之氣相層析管柱的進樣口。這樣的多維分離法(蒸餾-氣相層析-質譜法) 之開發始於一系列初步離線實驗；具有不同沸點的化合物分別被蒸餾出並以氣相層析質譜法進行即時分析，所得到的數據顯示蒸餾過程的動態變化。此蒸餾-氣相層析-質譜法最重要的優點是原始樣品 (如汽油) 可以直接被分析且無須去除可能汙染氣相層析儀進樣口及管柱的非揮發性物質。這兩個步驟是部分重疊的，此特性可以稍微增加整個分析的選擇性。實驗結果顯示簡單的儀器開發可以提升較少或是不需要前處理的複雜樣品之分析。

Chemical analysis of complex matrices – containing hundreds of compounds – is challenging. Reducing complexity of samples is often required before detection of the target analytes using standard instrumental platforms. In this work, we aimed to develop two analytical methods for analysis of complex samples. The first method enables analysis of bioparticle suspensions, while the second method enables analysis of mixtures of volatile compounds. The first method takes advantage of hydrodynamic chromatography (HDC). Separation of cells and cell aggregates in HDC arises from the parabolic flow profile under the laminar flow conditions. In the presented protocol, hydrodynamic separation is coupled with different on-line and off-line detectors (light absorption/scattering and microscopy). The method has successfully been applied in the monitoring of dynamic changes in the microbiome of probiotic drinks. Chromatographic profiles of yogurt and kefir samples obtained at different times during fermentation are in a good agreement with microscopic images. The second method uses a custom-developed system for coupling simple microscale distillation with gas chromatography (GC) and electron ionization mass spectrometry (MS). The developed system incorporates an interface between the distillation condenser and the injector of a fused silica capillary GC column. The development of this multidimensional separation (distillation-GC-MS) has been preceded by a series of preliminary off-line experiments. The components with different boiling points are fractionated and instantly analyzed by GC-MS. The obtained data sets illustrate the dynamics of the distillation process. An important advantage of the distillation-GC-MS technique is that raw samples (e.g. gasoline) can directly be analyzed without prior removal of the non-volatile matrix residues that could contaminate the GC injection port and the column. The two separation steps are partly orthogonal, what can slightly increase selectivity of the entire analysis. The experience gathered in this project shows that simple instrumental developments can upgrade standard analytical methodology, and make it suitable for analysis of complex samples following little or no sample preparation.