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I /C s phng php sc k trao i ion ( Ion exchange

chromatography - IEC!
. "!#i$i thi%& ch&ng
- Sc ky la mt h cac k thut hoa hc phn tich dung tach cac cht trong mt
hn hop va uoc tin hanh ln u boi nha thuc vt hc nguoi Nga Mikhail
Tsvet (Mikhail Semyonovich Tsvet) vao nm 1903 khi ng ang nghin cuu
v chlorophyll.
- Sc ky trao i ion la mt phuong phap sc ky long rn ma pha tinh la hop cht
co kha nng trao i ion ( gi la cac ionit ).
- Sc ky trao i ion uoc phn loai dua trn co ch cua qua trinh tach : do ai luc
khac nhau cua cac ion trong dung dich ( pha ng) voi cac trung tm trao i ion
( nhom chua ion) trn cht rn ( pha tinh ).
- Dy la phuong phap uoc su dung nhiu trong cac phong thi nghim Hoa
sinh.Theo nhung thng k moi nht, IEC la k thut tach protein uoc su dung nhiu
nht trong cac quy trinh tinh ch (co th bao gm nhiu buoc va nhiu k thut khac
nhau):
IEC: 75,
sc ky ai luc (AC): 60,
sc ky lc gel (GF): 50 cac truong hop.
'!C ch( c)a phng php!
- Truoc tin, cht cn phn tich s gn thun nghich voi cac cht trao i bng t-
uong tac ion giua cac nhom mang in tich trai du.
- Sau o, cac ion trao i uoc chit rut ring bit bng phuong phap rua giai
hoc rua y.Co th tach chit bng cach thay i pH (it dung hon) khin cac
nhom tuong tac trn cht phn tich bi mt in tich .
Nguyn tc :
- Co so cua IEC la su canh tranh cac nhom tich in trai du trn cht trao i giua
cac ion cua cht cn tach voi cac ion khac.
- Khi nng ion canh tranh thp, cac ion cn tach s lin kt voi cht trao i,
Khi nng ion canh tranh cao, cac ion cn tach s roi ra.
Uu im :
- Co phn giai cao
- Da dang (cac loai nhua trao i khac nhau, nhiu cach kt hop giua m va pH).
Su phn tach cac ion tich in ( hinh vung) trong mi truong trao i anion ( hinh tron )
- Nguyn tc tach truc tip (cn cu trn khac bit in tich).
- D dang tin hanh.
*! Cc +o,i nh-a trao i ion
3.1. KN
Ionit la nhung ai phn tu acid hoc base khng tan trong nuoc hoc dung mi co
chua trn mang luoi nhung ion linh ng,co kha nng trao i theo uong luong va
thun nghich voi cac ion cung du trong dung dich cht in ly khi tip xuc.
3.2. Phn loi
Dua vao du in tich cua cac ion trao i, nguoi ta phn chia thanh 2 loai :
- Cationit ( RH, RNa, R
2
Mg, R
2
Ca. ): ion c inh cua mang luoi mang in tich
m, thuong la nhom acid. Ion linh ng mang in tich duong. Nhua co kha nng
trao i anion.
VD :
2R-Na CaCl
2
R
2
-Ca

2NaCl
- Anionit ( ROH, RCl, R
2
CO
3
, R
2
SO
4 .) :
ion c inh la nhom base mang in tich
duong. Ion linh ng tich in m. Nhua co kha nng trao i cathion.
VD :
2RCl NaSO
4
R
2
SO
4
2NaCl
- Ionit lung tinh: co th trao i ca ion mang in tich duong ln ion mang in tich
m.
Dua vao cac nhom co cung
ban cht :
- Ionit v co thin nhin va
tng hop.
- Ionit huu co thin nhin va
tng hop.
- Ionit c bit : co nhom tao
phuc , ionit lung tinh , ionit
redox, ionit long, mang trao i
ion.
Mt s nhm trao i ion trn
nha.
.!Chi(t tch cc ion c)a ch/t c0n ph1n t2ch kh3i c4t sc k!
Phuong phap uoc su dung nhiu nht la phuong phap rua giai :
- Cho V(ml) dung dich chua hn hop cac cu tu (vi du, hn hop hai cu tu A va B,
trong o A co ai luc voi ct nho hon B) chay qua ct. Cac cu tu A, B chua trong
Vml truoc ht s bi giu lai o phn trn cua ct.
- Sau o cho dung dich rua (thuong la dung mi hoa tan cac cu tu) chay qua ct.
Luc o cac cu tu bi giu o phn trn cua ct s bi dung mi 'rua va ua dn xung
phia duoi. Cu tu A co ai luc voi ct nho hon B nn chuyn ng xung phia duoi
nhanh hon B. Nu ct u dai va ch chay cua dung dich rua thich hop thi sau mt
thoi gian cho chay dung dich rua, cac cu tu tach ra thanh tung vung. Cac vung nay
s tun tu thoat ra khoi ct, mi vung lai
uoc cach nhau bng mt phn dung mi.
Trong phuong phap rua giai, nguoi ta
cung hay dung nhung dung dich chua mt
cu tu co ai luc voi ct nhung phai nho
hon ai luc cua cac cu tu cn tach voi ct.
Su dung mt dung mi on gian co
thanh phn khng i: isocratic
Su dung hai hay nhiu hon cac h
dung mi co phn cuc (polarity)
khac nhau nhiu: gradient elution=>
tng cht luong cua qua trinh tach
(improve seperation eIIiciency)
Hnh bn di biu din ng cong
thot c!a "u trnh r#a gi$i.
Hiu qu tch bng phng php
gradient elution
5. Cch ch&5n 67 89 ti sinh c4t
- Cn rua, lam truong (mt s nhua uoc ban o dang uoc truong sn co th dung
ngay), va xu ly nhua thanh dang cn thit truoc khi dung. Cac IEC co th tai su dung
nhiu ln, sau mi ln su dung nht thit phai tai sinh (hi nguyn) nhua.
- Rua va lam truong trn nhua voi khoang 10 th tich m, lng, gan bo cac hat lo
lung( am bao tc chay n inh).
II/ Cch ti(n h9nh ph1n t2ch h9m +:ng ;itrate 89 ;itrite 6<ng
phng php sc k trao i ion
1. Ch&5n 67 m=&!
- Ly 10g mu nghin nho, thm 50ml nuoc 60
o
C .
- Trn u va lng trong 10 phut.
- Chuyn sang binh inh muc 200ml. Thm 50ml
acetonitrile va ao nhe.
- D o nhit phong. Thm nuoc tinh khit toi muc,
lc u.
- Lc qua giy lc. Sau o cho dung dich hoa tan qua
mt mang lc, kich thuoc l khoang 0.45m .
'!>i?& ki%n ti(n h9nh @
Ct sc ky : Waters IC-PacTM Anion HC 150 x 4.6 mm column
Pha ng : m Lithium borate gluconate , gradient tach (rua giai ) :
acetonitrile/ater (pH6.5) , mu rau phn tich.
Tc dong : 1.0 mL/min.
Detector quang : V
Acetonitrile
Buoc song : 205 nm
Dung tich mu : 40 L
*!A% thBng thi(t 67@
- Binh chua dung mi pha ng
- B phn khu khi
- Bom cao ap
-B phn tim mu ( bng tay hay Autosample)
-Ct sc ky ( Pha tinh ) ( ngoai mi truong hay trong b iu nhit )
- Detector ( nhn tin hiu )
-H thng may tinh gn phn mm nhn,tin hiu va su ly du liu va iu khin h
thng HPLC.
- In du liu .
.! C(t D&E @
% Dua vao thoi gian luu cua Nitrate va Nitrite o dung dich chun th hin o pic sc
ky u tin, ta co th xac inh uoc nh Nitrate/Nitrite trong mu cn phn tich
trong tp hop cac cht ma sc ky trao i ion tach uoc o pic 2 va 3.
- A : D hp thu ( Absorbance nit).
F! C(t +&Gn phng php @
Hi&u "u$' ch(n )(c cao' *ng d+ng rng r,i
-h tich m.u nh/
0 th 1h2ng 3h h!4 m.u 5nond6structi76 o8 sam3)69
:;nh )<ng d d=ng
>3 d+ng <c 7i cc m.u 1h2ng ba4 h?i 7= 1h2ng b@n nhi&t
>3 d+ng <c cho cc ion 72 c?
Aii hBn 3hCn tDch c!a 3h?ng 3h3 1h2ng cao 5chE Fc ;nh <c G'H mg
IJK%L )it 9.

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