還原劑及Co(II) 誘導Luminol 之化學發光及其分析上的應用

Chemiluminescence of Luminol Induced by Reducing Agents and Co(II) and Its Analytical Applications

方珣(Shiun Fang)

指導教授 ： 林萬寅

國立臺灣大學/理學院/化學研究所/碩士(2009年)

https://doi.org/10.6342/NTU.2009.02863

摘要

化學發光 (chemiluminescence) 為經由化學反應，產生光的現象。本研究利用流動注入分析系統 (flow injection analysis) 以及停止流光譜儀 (stopped-flow spectrometer) 探討luminol與還原劑的化學發光現象。Luminol在強鹼環境中化學發光極微弱，通常會使用催化劑與增強劑以增加化學發光訊號。本研究中，以金屬離子催化並使用還原劑誘導luminol的化學發光反應，在流動注入分析系統中，NH2OH可使luminol-Co(II)在鹼性環境產生化學發光，而N2H4則可增強NH2OH-luminol-Co(II) 化學發光並使訊號較為穩定。利用停止流光譜儀分析，Co(II)的催化可使 NH2OH-luminol在鹼性環境中的反應時間縮短且增強化學發光訊號。
流動注入分析系統中，探討 pH 值、反應物（luminol、Co(II)、NH2OH、N2H4）濃度與混合的方式等因素影響化學發光強度，並找出系統最佳化條件。本系統使用單管流路配置（流速設定在5 ml/min)，當pH = 13.0、[luminol] = 30 μM、[Co(II)] = 100 μM、[NH2OH] = 200 μM與[N2H4] = 500 μM 的條件下有最強的化學發光訊號且再現性佳 (n = 21， RSD = 1.1 %)。本系統中，將試劑除氧後化學發光訊號減弱20 %，表示此化學發光與溶氧有關。加入專一性消滅．O2-、1O2 和．OH的試劑都會使化學發光訊號降低，推測這些活性含氧物質與 luminol 作用導致化學發光的增強。此化學發光系統可應用於偵測能與活性含氧物質作用而抑制化學發光的物質，並針對每一種分析物進行抑制最佳化，可獲得更高的靈敏度。例如應用於偵測酚類化合物 hydroquinone、catechol 及 resorcinol，其偵測範圍分別為 2 ~ 400 nM、3 ~ 500 nM 及 0.3 ~ 10 μM，偵測極限分別為 1.05 nM、1.35 nM 及 0.10 μM。此方法具有簡便、快速以及靈敏度佳等優點。
利用停止流光譜儀偵測 luminol-NH2OH-Co(II)-OH- 系統的化學發光現象，並探討pH值、反應物（luminol、Co(II)、NH2OH）濃度對化學發光強度及時間的影響。在Co(II) 與 luminol 存在下，改變NH2OH濃度，當NH2OH濃度慢慢增加( 0 ~ 50 μM )化學發光強度增加且時間縮短，將濃度提高( 100~200 μM )可觀察到兩根peak 出現，若NH2OH濃度高於250 μM則只見短時間的peak，不同時間產生的化學發光現象主要與 NH2OH 濃度有關。在200 μM NH2OH條件下，改變Co(II) 濃度，當Co(II) 濃度增加，化學發光強度增加且時間縮短，其中又以時間長的peak 2 改變較為顯著，時間短的peak 1變化不明顯，添加EDTA 則結果相反，且當EDTA 濃度略高於Co(II) 時，化學發光消失。改變 luminol 濃度，當濃度增加( 0~30 μM ) 整體化學發光強度增加且peak 2 時間略為增加，若luminol 濃度高於35μM，化學發光強度則隨luminol 濃度提高而降低，且反應時間拉長，此時 peak 只剩下一根。改變pH 值，化學發光的強度隨pH 值增加而增強，且反應時間縮短。將試劑除氧後化學發光訊號減弱，其中peak 2 減弱程度明顯，表示此化學發光與溶氧有關。在luminol化學發光系統中，也測試金屬離子：Co(II)、Hg(II)、Zn(II)、Cr(III)、Fe(III)、Mg(II)、Cu(II)、Ni(II)、Mn(II) 對化學發光的影響，發現Co(II) 反應時間較短且化學發光效率較高，便於偵測，因此催化劑以Co(II) 為最佳選擇。加入專一性消滅．O2-、1O2 和OH．的試劑，其中，消滅．O2-和．OH的試劑會使化學發光訊號降低，對此系統影響較大，而消滅1O2 的試劑則使化學發光增強，．O2-和．OH可能與luminol作用導致化學發光的增強。
在luminol-NH2OH-Co(II)-OH- 系統中，推測peak 2的化學發光是因luminol、Co(II)、NH2OH等試劑形成錯合物而造成的，若改變Co(II) 在溶液中的量，則直接影響化學發光強度及時間。另外，推測 peak 1的化學發光主要是溶氧與試劑產生的活性含氧物質所引起的。此系統應用於檢測酚類化合物，在不同實驗條件下，當溶液中存在 hydroquinone以及catechol時，不同的抗氧化劑分子具有不同的反應活性，可提升對酚類化合物的選擇性及靈敏度，增加定性及定量上的應用。

關鍵字

化學發光 ； 還原劑 ； 鈷離子 ； 酚類化合物 ； luminol

並列摘要

Chemiluminescence (CL) is the emission of light from an electronic excited state of a species produced in a chemical reaction. In this study, the CL reaction of luminol with reducing agents and metal ions was studied by flow injection analysis and stopped-flow spectrometry. Upon addition of hydroxylamine to the luminol-Co(II) system, the CL intensity increased significantly. The use of hydrazine could further increase the CL intensity by five folds. The presence of Co(II) increased the CL intensity and reduced the CL duration.
The effects of pH, concentration of reagents (luminol, Co(II), NH2OH, N2H4) and modes of reagent mixing on CL intensity were also investigated and optimized. The optimal conditions for maximum CL intensity are: flow rate = 5 mL/min, pH = 13.0, [luminol] = 30 μM, [Co(II)] = 100 μM, [NH2OH] = 200 μM and [N2H4] = 500 μM. The CL signal showed good reproducibility (RSD = 1.1% for n = 21). Deoxydenation of sample solutions by purging with nitrogen reduced the CL intensity by 20%, indicating that oxygen is involved in this CL reaction. Specific scavengers for ．O2- , 1O2 and ．OH decreased the CL intensity greatly, suggesting that these reactive oxygen species (ROS) played significant roles in the CL reaction. The CL detection system can be used to determine substance such as phenolic compounds that can effectively destruct ROS. For the determination of hydroquinone, catechol, and resorcinol, the dynamic ranges are 2~400 nM, 3 ~ 500 nM, and 0.3 ~ 10 μM, respectively, and the limits of diction are 1.05 nM, 1.35 nM and 0.10 μM, respectively. The proposed CL method is simple, rapid, convenient and sensitive.
In this study, we have used the stopped-flow technique to study the effect of pH, reagent concentration, ROS scavengers on the CL intensity and duration. When increasing amount of NH2OH (0-50 μM) was added to the Co(II)-luminol solutions at pH 13.0, progressive increase in CL intensity and decrease in CL duration were observed. Interestingly, two distinct CL peaks appeared at higher concentrations of NH2OH (100~200 μM) and then a single peak was present at 250 μM NH2OH. The presence of increasing amount of Co(II) in luminol-NH2OH solutions enhanced the CL intensity and reduced the CL duration progressively, especially for the second peak. The CL intensity increased rapidly as the concentration of luminol increased, reached a maximum at 30 μM, and then dropped considerably at higher concentrations. Changing the pH has a profound effect on the CL emission. Strong and shortened CL emission was observed at high pH. These results indicate that the peak shape and duration of CL emission can be controlled by appropriate adjustments of reagent concentrations. It is found that CL peaks obtained at different conditions responded differently to the analytes in terms of selectivity and sensitivity.
To gain a deeper insight into the mechanism of CL emission, we have investigated the effect of EDTA, ROS-scavenging, and deoxygenation on the CL emission. The addition of EDTA reduced the CL intensity and prolonged the CL emission dramatically. No CL emission was observed when the added EDTA exceeds the amount of Co(II), suggesting that free Co(II) is essential for CL. Deoxygenation of sample solutions reduced the CL intensity considerably. The extent of reduction in CL intensity is greater for the second peak than for the first peak. Thus oxygen is important in CL enhancement. ROS-scavenging studies revealed that ．O2- and ．OH are critical for the CL enhancement since the presence of scavengers for these two radicals decreased the CL intensity dramatically. It is postulated that complexation of Co(II) with suitable ligands such as NH2OH, OH-, O2, or luminol anion may lead to the generation of ROS (e.g., ．O2-, ．OH) or other reactive intermediate. Subsequent reactions of these reactive species with luminol results in the strong enhancement in CL emission.

並列關鍵字

Chemiluminescence ； reducing agent ； Co(II) ； phenolic compound ； luminol

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