本研究是利用[C4mim][PF6]當作萃取劑，藉由調控水溶液的 pH 值，蛋白質能在水溶液與乙酸乙酯和[C4mim][PF6]的混合物間進行正 向及反向萃取，並發現珠狀蛋白在低pH 值且有金屬鹽類存在所呈現 的熔珠態會明顯地影響蛋白質在低pH 值時的萃取;不過如果無金屬 鹽類存在，則不會在低pH 值時有影響萃取的現象發生。
另外，本實驗測試了水溶液中 KCl 對於蛋白質萃取的影響，由結 果可知，水溶液中的KCl 對細胞色素 c 及核糖核酸酶 A 存在遮蔽效 應而影響蛋白質的萃取，但是對於較疏水性的肌紅蛋白則無干擾的情 形產生。此外，嘗試在正丁醇裡加入有機鹽類TBAB 以模擬 IL 的性 質，當TBAB 濃度越高，肌紅蛋白萃取率越大，顯示 IL 能萃取蛋白 質或許是由於其近似醇類的極性及鹽類的特性。
至於萃取機制主要是蛋白質與 IL 之間的靜電作用力所致，但是 其他如蛋白質的疏水性、蛋白質與IL 間的其他作用力等等，可能也 是影響IL 萃取蛋白質的因素。此液相／液相萃取技術或許可使用於 蛋白質的純化及濃縮，並且開拓ILs 新的應用領域。
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表一、陽離子對稱性對於Ionic Liquids 熔點的影響5
表二、陰離子團大小對於Ionic Liquids 熔點的影響5
資料來源: http://www.mariecurie.org/annals/volume3/berthod.pdf
20°C的物理化學性質
[TBAB] added Extraction efficiency increased (%)
N N CH 3
C 4 H 9 Cl
N N
CH 3
C 4 H 9 Cl
-+
K + PF 6 - H 2 O
phase separation
N N
CH 3
C 4 H 9
+ PF 6
-圖四、1-butyl-3-methyl imidazolium hexafluorophosphate, ([C4mim][PF6]) 合成示意圖
HE 3.73 singlet 圖五、 [C4mim][PF6] NMR圖譜
HA 0.72 triplet HB 1.15 sextet
HC 1.68 quintet
HD 4.05 triplet HG 7.22 singlet
HH 7.30 singlet HF 8.26 singlet
HE 3.73 singlet
Cytochrome c
Cytochrome c
Cytochrome c
Mixture of Ethyl Acetate and [C4mim][PF6]
Mixture of Ethyl Acetate and [C4mim][PF6] Mixture of Ethyl Acetate
and [C4mim][PF6]
0 1 2 3 4 5 6 7 8 9 10 11 12 0
20 40 60 80 100
Extr action ( % )
pH
圖七、pH 值對蛋白質細胞色素 c 正向萃取率的效應 (■) 以手搖晃混合 10 秒 (●) 以磁石攪拌 1 小時
300 350 400 450
Wavelength (nm) J. Am. Chem. Soc. 124 (2002) 14840.
4
450 500 550 600 650 700 750
0.0 0.1 0.2
Absorbance (AU)
Wavelength (nm)
(a)
(b) (d) (c)
圖九、蛋白質細胞色素c 在不同 pH 值水溶液的吸收光譜( 450-750 nm )。
Molten Globule pH 2
pH 2
pH 7
Wavelength (nm) J. Am. Chem. Soc. 124 (2002) 14840.
250 300 350 400 450 500 550 600
300 350 400 450 500 0
1 2 3 4 5 6
Fluor escence I n tensity
Wavelength (nm)
(a)
(b) (c) (d)
圖十二、蛋白質細胞色素c 在不同 pH 值水溶液的螢光光譜圖 (a) pH 1 (b) pH 2.7 (c) pH 7 (原始態) (d) pH 11
激發波長: 280 nm
200 220 240
0 1 2 3 4 5 6 7 8 9 10 0
20 40 60 80 100
Extraction (%)
pH
200 400 600
0 1 2 3
Absorbance (AU)
Wavelength (nm) (d)
(a) (b) (c)
圖十四、pH 值對蛋白質肌紅蛋白正向萃取率的效應
圖十五、蛋白質肌紅蛋白在不同pH 值水溶液的吸收光譜圖
300 400 500 600 700 0
1 2 3
Absorbance (AU)
Wavelength (nm) Myoglobin in Aqueous ( pH = 5 ) Myoglobin in Mixture of IL and EA (forward extraction at pH 5)
5 6 7 8 9 10 11 12 13
0 10 20 30 40 50
Back-extraction (%)
pH
圖十六、蛋白質肌紅蛋白在不同相中的吸收光譜圖
300 400 500 7 (native state)
13
1
222 nm 208 nm
圖十八、蛋白質肌紅蛋白在不同pH 值水溶液的螢光光譜圖，激發波長: 280 nm
1 2 3 4 5 0
10 20 30 40 50 60
Extraction (%)
pH
2 3 4 5 6 7 8 9 10 11 12 13
0 10 20 30 40 50 60
Back-Extraction (%)
pH
圖二十、pH 值對蛋白質核糖核酸酶 A 正向萃取率的效應
300 350 400 450
0.0 0.2 0.4 0.6 0.8 1.0
200 300 400 500 600 700
0.0
0.0 0.2 0.4 0.6 0.8 1.0 0
20 40 60 80 100
Extraction (%)
KCl (M)
0.0 0.2 0.4 0.6 0.8 1.0
0 20 40 60 80 100
Extraction (%)
KCl (M)
圖二十六、pH 值及 KCl 濃度對蛋白質肌紅蛋白的萃取效應 (■) pH 5 (●) pH 1
圖二十八、不同pH 值時，胺基酸進入[C6mim][BF4]的分佈係數 與胺基酸疏水性的關係。。
圖二十九、胺基酸進入[C6mim][BF4]的分佈係數與 pH 值的關係
資料來源: Green. Chem. 7 (2005) 196.