水產養殖用水中氨氮的流動螢光檢測

Abstract

水中氨氮對水生動物具有高度毒性，傳統檢測方法卻耗費人力、反應時間長，且反應試劑昂貴又含有酚或汞離子等毒物。本研究開發一高敏感性且具選擇性之螢光檢測方法，選用價格低且對環境友善的鄰苯二甲醛(OPA)與N-乙醯半胱氨酸(NAC)作為反應試劑，配合自動化軟體控制之序列注射系統與流動式螢光檢測儀搭配停流方法建立此快速且敏感的螢光檢測方法。 螢光檢測系統中，裝有5 mL注射筒的序列注射系統用於抽取水樣品或標準溶液，並與試劑混和，試劑為0.0160 M鄰苯二甲醛和0.0079M N-乙醯半胱氨酸分別溶於pH 6.8之0.1 M磷酸緩衝溶液，混和液由序列注射系統注入浸於63°C水浴槽之不鏽鋼反應管(內徑0.8 mm，長400 cm)，再進入流動式螢光檢測儀，以激發光/吸收光波長410/470 nm或380/470 nm檢測螢光訊號。本研究亦開發停流檢測方法，混和液流入反應管中即停止流動，等待加熱一分鐘後再檢測反應完成的衍生物來提高螢光強度。本螢光檢測系統中控制作動與螢光數據監測收集等功能皆於LabVIEW軟體開發之使用者介面上完成。 為了用於檢測水中氨氮濃度，在激發光/吸收光波長為410/470 nm下繪製了範圍為0.1至2.0 ppm的檢量線，相關係數達0.9987，相對標準偏差（n=3）小於5%，最小檢測極限至0.04 ppm，且試樣處理通量高於每小時15個。由於氨氮和一級胺的衍生物螢光光譜明顯不同，採用雙激發光波長檢測方法（激發光波長為410和380nm）可排除雜訊。以甘胺酸作為一級胺代表，氨氮和一級胺的計算結果誤差均小於10%。與現有檢測方法相比，本研究開發了快速、敏感性高且具選擇性的檢測方法，往後配合開發微型閥上檢測系統即可執行此安全、環保又便宜的螢光反應，有望應用於水產養殖產業、污水和畜禽養殖廢水等領域的水質檢測。

Ammonium is highly toxic for aquatic animals, but its conventional detection methods are labor-intensive, time-consuming and involved using toxic or expensive reagents. In this approach, the rapid and unstable reaction with the cheap and eco-friendly reagents, o-phthalaldehyde (OPA) and N-acetylcysteine (NAC), was conducted with a software-controlled lab-on-valve (LOV) system to achieve a highly sensitive and selective analytical performance by a stopped-flow strategy. A LOV system with a 5 mL syringe was used for sampling (pond water, the aqueous ammonium or glycine standard solutions for calibration) and mixing with the reagents (0.0160 M OPA and 0.0079 M NAC in 0.1 M phosphate buffer, pH 6.8); the mixture was then introduced through a stainless reaction coil (63°C, 0.8 mm i.d. × 400 cm) and then a flow-through fluorometer with the excitation/emission wavelengths at 410/470 or 380/470 nm. For the stopped-flow process, the flow was stopped and the reaction mixture was heated in the reaction coil for 1 minute, and the reacted mixture was propelled through the detector. The whole liquid-handling and fluorescence monitoring process were performed by user interfaces developed under LabVIEW platform. For ammonium determination, a calibration curve (410/470 nm) with the linear range from 0.1 to 2.0 ppm was obtained with the correlation coefficient of 0.9987; the relative standard deviation (n=3) for 1.0 ppm was less than 5%, and with limt of detection of 0.04 ppm. The sample throughput was higher than 15/hr. Since the fluorescence spectra of the OPA-adducts of ammonium and primary amines are distinctly different, two-wavelength (410/470 and 380/470) measurement was adopted when the sample solution contains residual primary amines such as amino acids. With glycine as the amine standard, both ammonium and primary amines can be calculated with the errors less than 10%. Compared with the prior arts, this quick, sensitive and selective approach used only a general-purpose mini-LOV system to execute a safe, green and economic reaction, which is promising for the water quality inspection of aquaculture, sewage and livestock wastewater.