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研究生:陳哲明
研究生(外文):Chen, Jei-ming
論文名稱:毛細管液相層析兩點線上偵測系統對蛋白質分離過程的探討
論文名稱(外文):Direct Investigations of the Separation Process of the Protein in Reversed-Phase Liquid Chromatography Using Dual on-column Detections
指導教授:陳淑慧陳淑慧引用關係
指導教授(外文):Chen Shu-Hui
學位類別:碩士
校院名稱:國立成功大學
系所名稱:化學系
學門:自然科學學門
學類:化學學類
論文種類:學術論文
論文出版年:1998
畢業學年度:86
語文別:中文
論文頁數:90
中文關鍵詞:毛細管液相層析構形
外文關鍵詞:Capillary ChromatographyConformation
相關次數:
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蛋白質因具有複雜的三度空間結構,在分離過程中其分子構形易在複
雜的層析環境中隨著動相溶劑組成、壓力、以及與靜相接觸時間等的因素
變動而改變。而此現象卻不易在一般管柱後的偵測被觀察到。利用毛細管
液相層析兩點線上螢光偵測的方法,分離過程可以直接被觀察。本實驗是
用約80cm長度200μm內徑的fused silica毛細管柱
填充5μm內徑的碳十八(C18)粒子,兩偵測點以串聯方式分置於管
柱前後端而距離可隨需要而調整。兩螢光偵測儀皆具有光譜掃瞄之功能。
整體系統經注射標準樣品評估測試後即被應用於對lysozyme分離
過程的探討,探重點主要分為等位沖提(第二章)、梯度沖提(第三章)
、以及壓力影響(第四章)。 等位沖提部份,以不同比率以及不同的
沖提溶劑(氰甲烷、甲醇)沖提lysozyme,層析圖譜顯示有兩根
主要波峰。由容量因子對有機溶劑比率的作圖顯示,較早沖提出來的波峰
與靜相無作用而被直接沖提出來;較晚沖提出來的波峰呈現吸附與瞬間脫
附的現象。氰甲烷呈現較強的脫附能力。二根波峰的分佈比率與lyso
zyme在注射溶劑與動相沖提溶劑中的溶劑化形態差異有關。此外由U
V光譜顯示,氰甲烷會促成靜相對lysozyme的三級構形改變而甲
醇則無此現象。由觀察結果可以導出lysozyme在C18液相層析
的沖提機制。 梯度沖提部份,以相同梯度速率但不同沖提溶劑比率起
點沖提,結果顯示最先沖提出來的波峰面積取決於lysozyme溶劑
化的程度。利用線性溶劑強度(LSS,Linear solvent
 strength)理論所得到梯度沖提的Z number與等位沖
提的不同,這可能是脫附速度比層析速度慢造成梯度下來不及平衡所致。
另外以氰甲烷的差異較甲醇大,這可能是由於氰甲烷沖提過程中lyso
zyme三級構形的改變較大所致。 除了沖提溶劑外本實驗也針對以
不同壓力在等位沖提下,對lysozyme沖提行為做探討。實驗結果
發現以氰甲烷沖提,lysozyme在動相中的莫耳體積比在其靜相中
大84.5 cm /mole;而甲醇則為39.9 cm /mole
,這顯示壓力對lysozyme之溶劑化形態及其在管柱中的沖提行為
具有顯著的影響。
The 3-dimensional folding struc
ture of proteins could be contin
uously altered during the separa
tion process by chromatographic 
parameters such as       the com
position of the mobile phase, pr
essure, and the contact time wit
h the stationary phase. However,
 these phenomena cannot be direc
tly observed by off-columndetect
i on. By the use of dual on-colu
mn fluorescence detections,the c
hro matographic elution of the 
protein could be observed in-sit
u along the separation column. I
n this study, a 80-cm, 200-μm i.
d. fused silica capillary column
 was packed with 5-μm i.d.C18 pa
rticles and the two fluorescence
 detectors were placed in series
 along thecolumn at variable pos
itions. Two fluorescence detecto
rs are capable of on-the-fly spe
ctra scanning. Column performanc
e was confirmed by the chromatog
ram obtained from the injectiono
f a standard test mixture before
 use. In this work,chromatograph
ic conditions are systematically
 varied to conduct studieson iso
cratic elution (chapter 2), grad
ient elution (chapter 3),and pre
ssure effect (chapter 4). In is
ocratic elution, two peaks of ly
sozyme were observed to occur co
nsistently upon the injection of
 lysozyme in buffer solution wit
h variousacetonitrile or methano
l compositions in the mobile pha
se. Fromthe relationship between
 capacity factor and the composi
tion of organic solvent,the earl
y eluted peak appears to have no
 retention throughout the examin
edrange of organic solvent compo
sition and the later eluted peak
 appearsto be eluted instantly u
pon the organic solvent composit
ion exceedsa critical value. The
 critical organic solvent compos
itions are foundto be 29% and 70
% for acetonitrile and methanol 
respectively, indicatingthat ace
tonitrile is a stronger desorpti
on agent. Fromthe chromatograms 
of lysozyme in various injection
 solvents, the non-retentive spe
cies is found to experience a mo
bile phase-inducedconformational
 exposure upon the injection and
 the retentive speciesappears to
 have no such exposure. Moreover
, UV spectra obtained from on-co
lumn detectionsindicate that ace
tonitrile tends to promote the r
etentive species to undergoa sta
tionary phase-induced conformati
onal change along the columnwher
eas methanol does not appear to 
promote the conformational chang
e of lysozyme. Based on the obse
rvation, we could derive the elu
tionmechanism of lysozyme in rev
ersed-phase liquid chromatograph
y. In gradient elution, lysozym
e is eluted with a linear gradie
nt from 29%-38% and 55%-65% for 
acetonitrile and methanol respec
tively. The contact area between
 the protein and the desorption 
organic solvent can be represent
ed by a Z number which can be de
duced from eitherisocratic or gr
adient elution. The obtained Z v
alue from the gradient elution i
s found to be different from tha
t obtained from the isocratic el
ution and can be attributed to t
he slower desorption rate of the
 proteinfrom the C18 surface com
pared to the chromatographic pro
grammingrate. Moreover, the Z nu
mber between two detectors is fo
und tovary to a greater extent u
nder acetonitrile elution than m
ethanol.This observation is cons
istent with a greater conformati
onal change of lysozymeunder ace
tonitrile elution than methanol 
elution as described above. In 
addition to elution solvents, we
 have also investigated pressure
 effecton the elution behaviour 
of lysozyme under isocratic elut
ion.From the plot of ln k' versu
s pressure, the change of molar 
volume of lysozymefrom the stati
onary to the mobile phase can be
 estimated. The experimental dat
a reveal that the molar volume o
f lysozyme inthe mobile phase is
 larger than that in the station
ary phase byabout 84.5 cm  and 3
9.9 cm  under acetonitrile and m
ethanol elution,respectively. Th
e results can also be attributed
 to the greater conformationalch
ange of lysozyme under acetonitr
ile elution than methanol elutio
n.Apparently, the pressure has a
 pronounced influence on thesolv
ation type of lysozyme and its e
lution behaviour. 

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