Co(II)-Luminol-NH2OH之化學發光及應用

Abstract

本研究利用流動注入分析 (Flow injection analysis) 系統探討Co(II)和hydroxylamine (NH2OH) 增強luminol的化學發光之研究，發現在此系統中不需要額外加入氧化劑，推測是在強鹼的環境中經由Co(II)的催化下，運用還原劑NH2OH與水中溶氧作用產生O2˙-，生成的O2˙-再與luminol作用，使luminol被氧化因而產生明顯的化學發光訊號。Co(II)的催化對於此系統的化學發光增強非常的明顯，在pH 13下，加入10-5M Co(II)與沒添加Co(II)做比較約可增強93倍。並且對組裝的管路配置、流速、pH值、反應物 (Co(II)、luminol、NH2OH) 之濃度找出最佳化條件，發現此系統在雙管路配置，流速5 mL/min，pH 13，2*10-4 M NH2OH，10-4 M Co(II)與3 μM luminol作用有最強的化學發光訊號。 在放光光譜中，很明顯可以看到在425 nm附近有最大的放光強度，可以確認本系統的放光是由luminol 所造成的。此外從除氧測試中得知，除氧後化學發光訊號抑制66.65%，推測是產生的O2˙-減少所導致，而在自由基消滅劑的測試當中，在本系統除了O2˙-之外，也會產生部分的1O2和OH˙使化學發光增強，因此加入這些有專一性的自由基消滅劑會使化學發光變弱，對於其他金屬離子如Cr(III)、Ni(II)、Mn(II)、Fe(II)與Co(II)在化學發光的比較上，發現Co(II)的化學發光訊號最強，因此Co(II)在本系統是個相當良好的催化劑。 之後運用Co(II)-luminol-NH2OH之化學發光系統可以用來檢測不同的抗氧化劑物質，在此對於每一種分析物做一系列的抑制最佳化，改變luminol、NH2OH、Co(II)的濃度，pH值，使得各分析物有更好的靈敏度，能夠檢測更低的濃度，如多酚類化合物dopamine (偵測範圍1~5 μM，偵測極限為0.18 μM)、hydroquinone (偵測範圍0.05~0.25 μM，偵測極限為0.013 μM)、catechol (偵測範圍1~5 μM，偵測極限為0.11 μM)、resorcinol (偵測範圍1~6 μM，偵測極限為0.20 μM)，以及ascorbic acid (偵測範圍0.5~4 μM，偵測極限為0.14 μM)、tannic acid (偵測範圍0.2~1.0 μM，偵測極限為0.042 μM)，還有對一些胺基酸，如methionine (偵測範圍5~40 μM，偵測極限為1.64 μM)、tyrosine (偵測範圍5~25 μM，偵測極限為1.82 μM) 進行檢測，而這些抗氧化劑會與水中溶氧和一些活性含氧物質等自由基作用，造成luminol的化學發光產生抑制，因而進行檢測。此外還有對一些金屬離子造成本系統的干擾進行檢測，發現添加10-5 M Cr(III)，10-4 M Ni(II)，10-4 M Mn(II)都會對於本系統造成干擾，應該要避免此些離子的存在。

In this study, a chemiluminescence (CL) method based on the Co(II)-luminol-NH2OH system has been developed. The flow-injection analysis (FIA) system was used to measure the CL produced from the oxidation of luminol. The reducing agent, hydroxylamine, interacted with dissolved oxygen in water to produce superoxide ion (O2˙-), which caused the CL with luminol in the presence of cobalt(II) as a catalyst in an alkaline solution. For the Co(II)-luminol-NH2OH CL, a 93-fold increase in CL intensity was observed upon addition of 10-5 M Co(II) at pH 13. The effects of pH, flow rate, concentration of reagents (cobalt(II), luminol, hydroxylamine), and modes of reagent mixing on CL emission were also investigated and optimized. We found flow rate: 5 mL/min, pH 13, 2*10-4 M NH2OH, 10-4 M Co(II) with 3 μM luminol for optimum CL. In emission spectrum, the CL maximum signal occurred at 425 nm. So the CL is caused by luminol in Co(II)-luminol-NH2OH system. When the dissolved oxygen was removed from the solution by purging with nitrogen, the CL intensity decreased by 66.65%, this might indicate that superoxide ion played a major role in this CL reaction. The scavengers of reactive oxygen species, such as superoxide dismutase, ascorbic acid, DMSO, and 1,4-diazabicyclo[2,2,2]octane were added into the reaction system. The CL intensity decreased greatly in the presence of these scavengers of radical. These results showed that in addition to O2˙-, 1O2 and OH˙ also participated in the CL reaction. The CL system has been applied to the determination of antioxidants such as dopamine (dynamic range: 1~5 μM, LOD: 0.18 μM), hydroquinone (dynamic range: 0.05~0.25 μM, LOD: 0.013 μM), catechol (dynamic range: 1~5 μM, LOD: 0.11 μM), resorcinol (dynamic range: 1~6 μM, LOD: 0.20 μM), ascorbic acid (dynamic range: 0.5~4 μM, LOD: 0.14 μM), tannic acid (dynamic range: 0.2~1.0 μM, LOD: 0.042 μM), and some amino acids such as methionine (dynamic range: 5~40 μM, LOD: 1.64 μM), and tyrosine (dynamic range: 5~25 μM, LOD: 1.82 μM). The antioxidants destroyed the radicals involved in the CL reaction, causing a decrease in CL emission. The dynamic ranges varied for different antioxidants with different antioxidative power. Some interferences such as 10-5 M Cr(III), 10-4 M Ni(II), and 10-4 M Mn(II) should be avoided.