The Ionization Constant of An Indicator

一、目的
To combine acid-base equilibrium and pH measurement with
spectrophotometer and illustrate the “isosbestic point”.

二、原理
A series of curves of absorbance vs. wavelength for an indicator which
is in the different pH conditions is obtains which ideally should all
intersect at a point called the isosbestic point. For a family of curves
like,this shows an isosbestic point, two conditions are necessary:
1. The total indicator concentration must be the same in every case.
2. The indicator must exist in only two forms, whose proportion change
as the condition are changed (in this case, as the pH is changed). A
good isosbestic point shows good experimental technique.

三、儀器設備及藥品
1. pH meter
2. “JASCO”Model 7800 Spectrophotometer
3. 0.1M of Acetic acid
4. 0.1M of NaOH
5. A solution of bromocresol green made by dissolving 100 mg of
solid indicator in about 3 mL of 0.1M NaOH, adding water, and
marking up to 100 mL. The concentration of the indicator should
be about 0.1% but need not be accurately known.

四、實驗步驟
1. Prepare five solutions:
(1) 10mL of 0.1M NaOH
(2) 10mL of 0.1M NaOH + 10mL of 0.1M Acetic acid
(3) 10mL of 0.1M NaOH + 20mL of 0.1M Acetic acid
(4) 10mL of 0.1M NaOH + 25mL of 0.1M Acetic acid
(5) 10mL of 0.1M Acetic acid. This amount does not be measured
exactly.
2. To each of five solutions, use pipet to add exactly the same volume of
the 0.1% indicator solution; 0.50mL is a suitable volume, but
whatever volume is chosen, it must the same for all.
3. Transfer each solution to 100mL flask and make up to the mark with
 water.
4. Measure represent absorbance of these five solutions from wavelength
400 to 600nm at intervals of 20nm, and plot the values on the same
graph paper.
5. Measure the pH of each of the solutions (2),(3),and (4).

五、儀器操作：
A. UV-Vis 光譜儀
1. 打開主機電源開關和 plotter 電源開關，熱機 15min.

2. 在主機上，按 一下“Method”鍵，接著續按”▼”鍵，直到顯示
螢幕上出現“31.Output”，然後按“Enter” 輸入以選擇 “＃31
plotter (2)on”。(見下圖)

3. baseline 校正：
a) 清空樣品槽(兩樣品槽中不放任何東西)，接著按”Method “
鍵，續按”▼” 鍵，直到顯示螢幕上出現 “23:correction”。
b) 按“Enter” 鍵，出現 “1. baseline”，按“Enter” 鍵。
 c) 利用”◣(向右鍵)”選擇”＃23. baseline (3) measure”，然後
按“Enter” 鍵。
d) 下方”measure” 鍵上亮紅燈，待紅燈熄滅時，表示 baseline
已校正完畢。(見下圖)

4. 測量參數設定：
a) 按一下”Method”鍵，然後續按”▼”，直到顯示螢幕上出現
”4. spectrum”， 接著按“Enter” 鍵。
b) 再按”▼”及”Enter”鍵，顯示螢幕上出現”4.start WL “，
將起始測量波長改為”620nm” ，再按“Enter”鍵。
c) 再按”▼”及”Enter”，將”4. end WL “之最終測量波長改
為”400nm”， 再按“Enter”鍵。
d) 再按”▼”及”Enter”鍵，將”WL scale “用”◣(向右鍵)改
為 “(4) 50 nm “，再按“Enter”鍵。
5. 樣品之測量(雙光束分析法)：
a) 將參考溶液( solvent)裝入 cell 中，以透明面朝光行徑方向方
式放入參考溶液槽中(見上 3 之圖)。
b) 將待測溶液裝入 cell 中，以透明面朝光行徑方向方式放入待
測溶液槽中並蓋上蓋子。
c) 按”measure”，出現紅燈。
d) 待積分器列出 UV 光譜後，更換待測溶液，依次測完所有樣品
之吸收度。
e) 所有待測溶液依下一步驟測量 pH 值。
6. pH 值之測量
a) 按”ON/OFF”鍵，然後再按”CLEAR”，此時狀態指示燈
AUTOLOCK 指示燈亮且 STAND 燈在閃動。(注意：若燈顯示
不對，按 MODE 直到燈顯示與上同)。
 b) 將 pH 電極浸入 7.00 之緩衝溶液中，此時 STAND 燈在閃動，
按”STAND”鍵，待 WAIT 燈消失，而 SLOPE 燈閃動時，表
示 pH 7.00 已校正完畢。
c) 取出電極，用去離子水洗淨並擦乾，放入 4.01 之緩衝溶液中，
按 “SLOPE” 鍵，待 WAIT 燈消失，表示 4.01 已校正完畢。
7. 取出電極，用去離子水洗淨並擦乾，放入待測溶液中。此時本
機、STAND、SLOPE、ATC、AUTOLOCK 燈均亮，按
“MEASURE”鍵，待 WAIT 燈消失，所顯示數字即為 pH 值。
8. 使用完畢，請將電極以去離子水洗淨後，裝回裝有蒸餾水的套
子中。

六、數據處理
1. Calculate the ionization constant of bromocresol green from these pH
measurement and the absorbance curves :
Read the absorbances at two wavelengths.One is the wavelength of
maximum absorbance of acid form of indicator (W1). Another is
wavelength of the maximum of the absorbance of the base form (W2).
Let the absorbances at W1 be P for acid form, absorbance at W2 be R for
the base form and W for the buffers. The concentration of the base and
acid forms in the buffers are in the ratio (P-Q) to (Q-R).
Thus the ionization constant is therefore:

Ka= [H+][In-] = [H+](A2-A4)•A1

[HIn] (A1-A3)•A2

The three ionization constants of the indicator that has the different Q
in the three buffers (2),(3) and (4).