EXPERMENT 9. To determination of Quinine by fluorescence spectroscopy. Introduction

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1 EXPERMENT 9 To determination of Quinine by fluorescence spectroscopy Introduction Many chemical compounds can be excited by electromagnetic radication from normally a singlet ground state S o to upper electronic excited states S 1 or S 2. In solution, S 2 state rapidly falls to its vibrational ground state and internally converts into S1 state, which also rapidly falls to its virational ground state subsequently, Such vibrational relaxation processes are so efficient that the average lifetime of vibrationally excited molecules is s or less. As shown in Figure 9.1, S 1 state, at its lowest vibrational level, can return to S o state by a combination of several mechanistic steps: radiative processes and radiationless processes. The favored route to the ground state is the one that minmizes the lifetime of the excited state. Figure 9.1 partial energy diagram for a photoluminescent system. Thus, if deactivation by fluorescence, with an average lifetime of about 10-8 s, is rapid with respect to the radiationless processes) such emission is observed. On the other hand, if a radiationless path has a more favorable rate constant, fluorescence is either absent or less intense. As shown in Fig. 1, S 1 state may undergo intersystem crossing to T 1 state, which is subject to subseguent deativation either by radiationless processes or by phosphorescence. 62

2 A T 1 S o transition is much less probable than a S 1 So transition, and the average lifetime of T 1 state with respect to emission ranges from 10-4 to several seconds. Therefore, intersystem crossing and external conversion compete so successfully with phosphorescence that the kind of emission is not frequently observed under ordinary circumstances. The various components of fluorescence instruments are similar to those found in ultraviolet-visible spectrophotometers Figure 9.2 shows a typical configuration for these components in a spectrofluorometer. Such instruments permit measurement of fluorescence and excitaton spectra. Figure 9.2 Components of a fluorometer or a spectrofluorometer The relationship of concentration and fluorescence may be derived from Beer's Law. The fraction of light transmitted is P/ P o = e-εbc (9-1) And the corresponding fraction of light absorbed is 1 - P/P o = 1-e-εbc (9-2) Rearranging, the amount of light absorbed is P o - P = P o ( 1-e -εbc ) (9-3) 63

3 the total fluorescence intensity is proportional to the number of light quanta absorbed and to fluorescence efficiency, which is the ratio of quanta absorbed to quanta emitted. F = (P o P ) Φ= P o Φ (1- e -εbc ) (9-4) For very dilute solutions in which not over 2% of the total excitation energy is absorbed and the term be is not greater than 0.05, equation (9-4) reduces to F = K P o Φεbc where the term K has been introduced to handle instrumental artifacts and the geometrical factor because fluorescnece is emitted in all directions but is viewed only through a limited aperture. In this experiment, students are required to perform the following procedure: (1) prepare several dilute standard quinine solutions. (2) measure their fluorescence at a fixed wavelength. (3) construct a calibration curve and determine the sensitivity limit. (4) Treat the unknown sample similarly. (5) Determine the concentration of the unknown sample. At first, students must measure the U.V. spectra of quinine in order to decide the wavelength for excitation. Molecular Fluorescence : The Determination of Quinine in Beverages Introduction Solutions of quinine fluoresce strongly when excited by radiation at 350 nm. The relative intensity of the fluorescent peak at 450 nm provides a sensitive method for the determination of quinine in beverages. Preliminary measurements are needed to define a concentration region in which fluorescent intensity is either linear or nearly so. The unknown is then diluted as necessary to produce readings within this range. Preparation of Reagent (a) Sulfuric acid, 0.05 M. 64

4 Add about 17 ml of 6 M Sulfuric acid to 2 L of distilled water. (b) Quinine sulfate standard, 1ppm. Weight g of quinine sulfate into a 1 L volumetric flask, and dilute to the mark with 0.05 M H 2 SO 4. Transfer ml of this solution to another 1 L volumetric flask, and again dilute to the mark with 0.05 M H 2 SO 4. This latter solution contains 1 ppm of quinine; it should be prepared daily and stored in the dark when not in use. Procedure Determination of a suitable concentration range To find a suitable working range, measure the relative fluorescent intensity of the 1 ppm standard at 450 nm. Use a graduated cylinder to dilute 10 ml of the 1 ppm solution with 10 ml of 0.05 M H 2 SO 4 ; again measure the relative fluorescence. Repeat this dilution and measurement process until the relative intensity approaches that of a blank consisting of 0.05 M H 2 SO 4. Make a plot of the data, and select a suitable range for the analysis (that is, a region within which the plot is linear). Preparation of a calibration curve Use volumetric glassware to prepare three or four standards that span the linear region; measure the fluorescence intensity for each. Plot the data. Analysis Obtain an unknown. Make suitable dilutions with 0.05 M H 2 SO 4 to bring its fluorescence intensity within the calibration range. Calculate the parts per million of quinine in the unknown. 65

5 實驗九 以螢光分光光譜儀測定 Quinine 之含量 一 目的 : 學習螢光分析法的原理及螢光分光光譜儀之使用, 並以檢量線法求得分析物的濃度 二 原理 : 螢光乃是一分子在吸收了電磁輻射後, 從基態 S 0 到達激發態 S 1 或 S 2, 接著以放射出電磁輻射的方式回到基態, 一般其所放射出的輻射能量比所吸收的輻射能量為低, 其產生的機構如下圖 9.1: S 0 表單重基態 ( 電子成對, 自旋方向相反 ) 圖 9.1 S 1 表單重第一激發態 ( 電子自旋方向相反, 其中一電子在較高能階 ) T 1 表三重第一激發態 ( 電子自旋方向相同, 其中一電子在較高能階 ) S 2 表表單重第二激發態 ( 電子自旋方向相反, 其中一電子在較高能階 ) 在激發態的電子不穩定 ( 半生期約 10-8 s ), 因分子間的碰撞以 vibrational relaxation 的方式放出熱能回到基態 : 或以放射出電磁輻射 ( S 1 S 0 + hν) 之螢光方 式回到基態 ; 不穩定的激發態 S 1 ) 亦可經過 intersystem crossing 到達三重激發態 (T 1 ) 66

6 後, 再以輻射的方式回到基態 (T 1 S 0 + hν), 此為磷光產生之原理 能夠產生螢光的物質, 其結構通常要具有一個以上芳香環或共軛結構者其螢光強度較強, 芳香環上之 -OH,-OCH 3 等推電子基具有增強螢光之作用 分子結構對磷光影響同螢光, 但分子必須在很低的溫度下, 才可能觀測到磷光的現象 螢光分析在定量上之分析基礎如下 : 螢光強度和待測物濃度關係式為 F = K P o bc F = K c F: 螢光強度 P o : 入射光強度 K: 測得之光子數 / 光子放射總數 : 光子產率 : 莫耳吸光係數 b: 光徑長 c: 濃度因此可藉螢光強度和濃度之線性關係製作檢量線以為定量分析之依據 此法可測得較吸收光譜法更低的濃度 ( 約 100 倍 ), 乃因當待測物濃度很低時, 可藉提高入射輻射之強度增強螢光強度 激發光譜儀之組件安排較紫外光可見光分光光譜儀略有不同, 其照射樣品之光束必須與欲偵測的放射光束成一角度, 通常為 90 度 一般組件安排如圖 9.2: 圖 9.2 激發光譜圖之測定 : 將 Filter 2 固定波長, 以 Filter 1 改變波長做波長掃描放射光譜圖之測定 : 將 Filter 1 固定波長, 以 Filter 2 改變波長做波長掃描 67

7 激發光譜儀具有兩個單色光器, 以光徑而言, 一個安排在樣品槽前, 另一個則安排在樣品槽之後, 以利測量激發光譜和放射光譜 激發光譜之測量乃是將樣品槽後之單色光器固定適當波長, 並對樣品槽前之單色光器做波長掃描, 所得之光譜圖即為激發光譜 放射光譜操作則是將樣品槽前之單色光器固定適當波長, 並對樣品槽後之單色光器做波長掃描, 所得之光譜圖即為放射光譜 由激發光譜圖求得最大激發波長, 再由放射光譜來決定最大的放射波長, 由激發的 λ max 及螢光 λ max 來測量分析樣品的螢光強度 三 使用器材 20mL 量瓶 6 個比色槽架 1 個 螢光塑膠比色槽四面透光 9 個 藥品 1.0mg/L(ppm) Quinine 15 ml 0.05M H 2 SO 4 65 ml 四 實驗步驟 1. 以 1.0 mg/l 的 Quinine 溶液為 ( 溶液 1), 以 0.05 M H 2 SO 4 溶液為稀釋溶液亦為本實驗的 blank 2. 以 pipet 吸取 1.0 mg/l 的 Quinine 溶液 10.0 ml 到 20 ml 量瓶中 ( 溶液 2 ), 以 0.05 M H 2 SO 4 溶液稀釋到標線並混合均勻, 此為溶液 2 3. 取 ( 溶液 2 ) 10.0 ml 於 20 ml 量瓶中 ( 溶液 3 ), 以 0.05 M H 2 SO 4 溶液稀釋到標線並混合均勻, 此為溶液 3 4. 重覆步驟 3, 取前次較濃溶液依序以 0.05 M H 2 SO 4 溶液稀釋到標線並混合均勻, 直到配至溶液 7 5. 取待測溶液 1~7 到測光管中約八分滿測量吸收值 ( 放入測光管前, 請以 0.05 M 68

8 H 2 SO 4 徹底清洗測光管, 再用待測溶液潤洗測光管兩次, 測光管外壁以紙將水吸乾 ) 分別測量標準溶液之螢光值 6. 以螢光分光光譜儀測量 Quinine 之最大吸收波長及最大放射波長, 以溶液 1 測量 (1 ppm 標準溶液 ), 測量方法請參考儀器操做說明 a 儀器設定光譜掃描形式, 設定 Em = 450 nm, 掃描 Ex 範圍 300~600 nm, 讀取放射光譜圖 找到最大吸收波長即放射光之 max b 儀器設定光譜掃描形式, 設定 Ex = 上步驟所測得知最大放射波長 nm, 掃描 Em 範圍 300~600 nm, 讀取放激發光譜圖 找到最大吸收波長即激發光之 max 7. 以 Quinine 之吸收及放射之最大波長 依序放入 Blank 及溶液 1~7 的測光管於比色槽中, 重複測三次, 測量方法請參考儀器操作說明 8. 以標準溶液之螢光強度 v.s. 濃度作圖, 求出校正曲線 (Calibation Curve ) 9. 分析樣品 : 自行取一 unknown 樣品 5mL 於小燒杯中 測光管需先洗淨後再用 unknown 潤洗兩次, 放入測光管中約八分滿測量其吸收值, 並求出其濃度 若 over scale ( 超過儀器測量範圍 ), 則再重覆步驟 3 之稀釋步驟, 直到所測的螢光強度不會 over scale, 並且在校正曲線上 10. 求出樣品中 Quinine 的含量 (ppm) 69

9 五 實驗數據與結果使用儀器之廠牌型號 : Quinine 之最大激發 (EX) 波長 : Quinine 之最大放射 (EM) 波長 : A. 檢量線的製作 : Solution Blank 濃度 1 螢光值 2 3 平均值 Solution 分析樣品 濃度 1 螢光值 2 3 平均值 檢量線圖形以電腦作圖 檢量線方程式 : 分析樣品濃度值計算式 = 70

10 六 問題與討論問題 : 一 Quinine 之分子結構為何? 二 螢光分析法較分光光譜法之靈敏度為高,( 有較低之偵測極限 ), 為什麼? 三 螢光儀和紫外線可見光分光光譜儀有何相似及不同之處? 討論 : 請針對本次實驗的結果與收穫加以探討 71