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CHNG 3: TNG QUAN V SC K LNG


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3.1 PHNG PHP SC K LNG HIU NNG CAO (HPLC)
HPLC l mt phng php tch v phn tch cc hp cht c s dng rng ri
v ph bin nht hin nay v nhiu l do: c nhy tng i cao, c kh nng nh
lng tt, thch hp cho vic tch cc hp cht kh bay hi hoc d b phn hy nhit, c
phm vi ng dng tri rng trong nhiu lnh vc t nghin cu khoa hc trong cc phng
th nghim n cng nghip v mt s lnh vc khc.
Hp cht c th phn tch bng sc k lng nh acid amin, protein, acid nucleic,
hydrocacbon, carbohydrate, thuc khng sinh, thuc tr su, cc hp cht v c Da
vo s khc nhau v c ch chit tch s dng trong sc k lng hiu nng cao m ngi
ta c th phn chia n ra lm cc loi: sc k hp ph, sc k phn b, sc k ion, sc k
ry phn t trong sc k phn b c ng dng rng ri v ph bin. Ty theo
phn cc pha tnh v dung mi pha ng, ngi ta phn bit: sc k lng pha thng v
sc k lng pha o.
Sc k lng pha thng: pha tnh c phn cc cao hn phn cc ca dung
mi pha ng, dng tch v phn tch cc hp cht c phn cc cao vi phn t
lng khng ln lm.
Sc k lng pha o: ngc vi sc k pha thng, pha tnh c phn cc thp,
pha ng c phn cc cao hn. Phng php ny dng phn tch cc hp cht t
khng phn cc n phn cc va. Dung mi s dng l dung mi phn cc, trong
nc ng vai tr quan trng m li r tin, do sc k lng pha o c s dng
nhiu nht.
3.2 CC THNG S CA PHNG PHP SC K LNG
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3.2.1 H s phn b
Cn bng ca mt cu t trong h sc k c th c m t bng phng trnh n
gin sau:
A pha ng A pha tnh


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H s cn bng K cho cn bng ny c gi l t l phn b v c tnh nh sau:

Cs: nng cu t trong pha tnh
Cm: nng cu t trong pha ng
S: Pha tnh
M: Pha ng
K ty thuc bn cht pha tnh, pha ng v cht ha tan. K nh: cht di chuyn
nhanh; K ln: cht di chuyn chm.
Thng thng K > 1 cht c i lc vi pha tnh nu khng th n s di chuyn
nhanh qu lm cho kh tch. Hai cht mun tch ra khi nhau th phi c K khc nhau.
3.2.2 Thi gian lu t
R


L thi gian cht phn tch sau khi tim vo ct n u d c tnh theo cng
thc:
t
R
= t
R
+ t
M

t
M
: thi gian lu cht ca cu t khng b gi li trn ct, c tnh gn ng theo cng
thc:
2
M
0, 5 ( )
t
L dc
V

=
trong :
L: chiu di ct, cm
time
M
S
C
C
K =


15
dc: ng knh ct, cm
V: tc dng pha ng, mL/pht
C th nhn danh cht thng qua thi gian lu v trong iu kin th nghim trn
ct thit b sc k lng nht nh, thi gian lu ca cht l mt i lng xc nh.
Thi gian lu cng ln th h s phn b cng ln.
3.2.3 H s dung lng
m t tc lu ca cu t phn tch trong ct ngi ta s dng mt h s
quan trng gi l h s dung lng k.
Cs.Vs
'
C .V
M M M
Vs
k K
V
= =
Vs: th tch pha tnh
Vm: th tch pha ng
k' c th tnh theo cng thc
k phi > 1 peak ca cht tan tch khi peak ca dung mi nhng khng qu
ln v lc thi gian phn tch s ko di v mi b t do cht trong ct qu lu.
Khong k l tng l t 2 n 5 nhng khi phn tch mt hn hp phc tp chng ta c
th chp nhn k trong khong t 0,5 n 20.
k tng hoc gim ty thuc mnh ca dung mi pha ng. Trong sc k phn
b pha o ngc, mnh ca dung mi tng theo phn trm cht hu c.
Thng thng trong sc k phn b pha o ngc, khi gim dung mi hu c
trong pha ng 10% th k s tng ln gp 2 n 3 ln.
3.2.4 Hiu nng
Khi ni n hiu nng ca ct l ni n kh nng tch mi sc k ca mt cu t
trn ct. hiu nng N c biu din nh s a l thuyt ca ct. N cng ln hiu
nng tch ca ct cng ln.


M
M R '
t
t t
k

=
2
2
1
R
2
R
w
t
5,55
w
t
16
H
L
N
|
|
.
|

\
|
=
|
.
|

\
|
= =


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Vi W: b rng mi sc k
W
1/2
: b rng ca mi phn na chiu cao
S a l thuyt cng ln, b rng W cng nh, mi cng nhn.
3.2.5 chn lc o


2 2 2
1 1 1
'

'
R M R
R M R
K t t t
K t t t

= = =


chn lc o l mt thng s rt quan trng, lin quan ti kh nng tch ca cc
cu t cn phn tch, n ty thuc bn cht ca pha tnh, pha ng.
Hai cht tch c khi o # 1 v o cng ln, kh nng tch cng cao.
C nhiu yu t nh hng n chn lc nh thnh phn dung mi pha ng,
pH ca mi trng, bn cht ca pha tnh, nhit . Trong s thay i pha ng t
dung mi ny sang dung mi khc s lm o thay i ng k.
3.2.6 phn gii
phn gii (R
s
) ty thuc vo iu kin pha ng, pha tnh v c tnh ca hp
cht cn phn tch m ta c c sc k vi nhng mi ph ln nhau hoc tch hn
nhau. phn gii R
s
l mt thng s dng nh gi mc tch ph, kh nng tch
ca cc mi sc k s c ngha quan trng trong vic phn tch mt cch nh lng.


Phng trnh trn thch hp cho vic tnh R
s
khi 2 mi tch hn nhau (R
s
> 1,5).
Trong trng hp R
s
nh hn, tc mi b trng lp th vic xc nh W
1
v W
2
s
kh khn hn l W
1/2
(1) v W
1/2
(2) lc ny R
s
s c tnh theo:
2 1
1/ 2(1) 1/ 2(2)
1,18
R R
t t
Rs
W W


'
A
'
B
A
B
K
K
K
K
= =
2
w w
t t
R
2 1
R R
S
1 2
+

=


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2
2
' 1
4 1 ' 1
s
k
R N
k
o
o
| |
| |
=
| |
+
\ .
\ .

Khi k
1
= k
2
, o1: ( )
1 '
1
4 ' 1
s
k
R N
k
o
| |
=
|
+
\ .

c th phn tch mt cch nh lng th 2 mi k nhau phi tch hn nhau, tc
R
s
> 1,3. Khi R
s
s 1 cn phi thay cc thng s thc nghim lm tng R
s
.
Tm li: khi tin hnh mt quy trnh phn tch bng sc k lng hiu nng cao, th
cn ch ti kh nng phn tch ca cc mi trn sc k . C nhiu yu t nh hng
ln php xc nh v ty theo yu cu cn phn tch nh tnh, nh lng hay bn nh
lng m chng ta s ti u ha cc thng s thc nghim ca pha tnh cng nh pha
ng cho hp cht cn nghin cu.
3.3 GII THIU CC B PHN CA THIT B SC K LNG HIU
NNG CAO
Thit b sc k lng hiu nng cao bao gm cc b phn chnh: bm, b phn tim
mu, ct sc k phn tch, u d, b phn iu khin v x l s liu.

Hnh 10: M hnh c bn ca HPLC.
Nguyn l hot ng: mu sau khi c tim vo ct s c pha ng li ko qua
ct. Da vo kh nng tng tc khc nhau gia cc cht c trong nn mu vi pha tnh
v pha ng m chng c tch ra khi nhau v sau khi ra khi ct s c ghi nhn bi
b d c th.


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Sc k lng c th ghp vi nhiu loi u d khc nhau nh u d ph t ngoi
kh kin UV-Vis, u d hunh quang FLD, u d ch s khc x RID, u d in ha
ECD, u d khi ph MS Hin nay u d khi ph c ng dng rng ri trong
phn tch vt v cc hp cht nhn danh chnh xc.
Ty theo tnh cht ca cht cn kho st m ta c th chn la pha tnh, pha ng
(bng 1).
Loi ct Pha tnh Dung mi Cht cn phn tch
C18 Octyldecyl ACN, MeOH, H
2
O Khng phn cc
C8 Octyl ACN, MeOH, H
2
O Khng phn cc
Phenyl Styryl ACN, MeOH, H
2
O Axit bo, cht c lin kt i
Cyano Cyanopropyl ACN,MeOH,H
2
O, THF Xeton, aldehyd
Amino Aminopropyl ACN, MeOH, H
2
O, THF,
CHCl
3
, CH
2
Cl
2

ng, anion
Diol Dihydroxyhexyl ACN, MeOH, H
2
O, THF Protein
SAX Aromatic Quaterany m Anion
Aromatic m SCX
Acid sulfonic ACN, MeOH, H
2
O
Cation
Alkyl ether m Diethyl
aminoethyl
DEAE
Diethylamine ACN, MeOH, H
2
O
Protein, anion
Alkyl ether m CM
Acid acetic ACN, MeOH, H
2
O
Protein, cation

Si Hexane Silica
Silanol chloroform
Hp cht hu c
phn cc, ng phn
Bng 1: Loi ct, pha tnh, pha ng v hp cht phn tch thng dng
13
.


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nh lng anthocyanin trong cc dch trch bng phng php HPLC ngi ta
cn kt hp u d UV/Vis vi u d ECD. Khng ging HPLC bn nh lng ch
lm sch, HPLC- UV/Vis- ECD dng phn tch. Cht phn tch c tim t ng v
cng vi pha ng ti ct. Cc hp phn khc tng tc khc nhau vi pha tnh v pha
ng. Cht phn tch sau khi c ra gii i n detector mt cch ring bit. Phng
php HPLC-UV/Vis-ECD c mt s c im sau:
Pha ng:
Pha ng trong HPLC c a lin tc vo h thng. Pha ng c th theo
chng trnh gradient dng hoc ng dng, nhng pha ng c nh thng mt thi
gian lu v kh tch cc mi. Hin nay thng dng nht l dng gradient. Khi ra gii
trong sc k pha o, lng dung mi hu c tng dn ln. Ti lc tim mu, mt lng
pha ng yu hn i vo h (t hp phn hu c hn). Sau li tng t l hu c trong
pha ng ln ra gii tt c nhng cht b gi li pha tnh.
Ct
10
:
C nhiu loi pha tnh khc nhau, dn n c ch ra gii cng khc nhau. Trong
nghin cu ny s dng ct pha o vi pha tnh khng phn cc v pha ng phn cc.
Pha tnh cha cc hp phn ging RMe
2
SiCl vi R l cc nhm alkyl (C
18
H
37
hoc
C
8
H
17
). Hp phn khng phn cc c i lc vi pha tnh nn thi gian lu di hn. Thi
gian lu c th iu chnh bng cch thay i phn cc ca pha ng. ng knh
trong ca ct ln to sc cha ln, c dng trong HPLC bn iu ch lm sch
anthocyanin. Ct phn tch dng trong HPLC-UV/Vis-ECD c ng knh trong ID nh
hn (C
18
250 x 4.6mm).
3.3.1. u d UV-Vis
u d c gn n UV (thng l n deuterim 190-360nm) v n Vis (n
tungsten, 360-800nm). V vy cc hp phn hp thu nh sng t khong 180- 800nm s
c u d pht hin.
Cht phn tch sau khi ra gii ra khi ct chuyn qua 1 t bo cm bin hnh tr.
nh sng trong vng UV/Vis truyn qua t bo ti mng quang in. Nu cht phn tch
hp thu c nh sng 1 bc sng no , mng quang in s pht hin ra s thay


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i. Tn hiu pht ra t mng quang in s chuyn ti b khuch i n b ghi o v h
thng thu d liu.
Cng dng sng truyn qua cell o (I) t l vi nng ca cht phn tch
theo nh lut Lamber- Beer (c- h s hp thu phn t, c- nng (mol/L), l- chiu di
ng truyn quang (l lun l 1 cm), I
o
l cng ca tia sng ti cell):
A= c l c
10
0
A log
tiem
I
I
DientichECD
C
F V n
DientichUV
C
l

|
o c
| |
=
|
\ .
=

=


Anthocyanin c 2 bc sng hp thu 530nm v 280nm, v vy c th s dng
u d UV/Vis. Trong HPLC nh lng, c th dng UV/Vis kt hp vi ECD hoc vi
MS.
3.3.2. u d ECD:
3.3.2.1 Tnh cht in ha ca anthocyanin:
Hot tnh chng oxi ha ca cc hp cht thin nhin ph thuc vo s lng cc
phn t hin din trong mu v cu trc ca chng, v d v tr nhm -OH hay -OCH
3
.
Anthocyanin c c tnh chng oxi ha do cc nhm -OH trn vng thm. Cht chng
oxi ha l mt cht kh khi tng tc vi cc gc t do, ngha l chng b oxi ha trn b
mt in cc. Cht chng oxi ha ngn s hnh thnh v hot ng ca oxi v nit.
Ta c th thy hnh 11, nhm catechol vng B v cc nhm resorcinol vng
A ca catechin c th b oxi ha. Vng A v B khng lin hp nhau nn s oxi ha cc
nhm -OH ca vng ny khng nh hng ti cc nhm -OH trn vng kia. Cu trc
catechin c cc nhm -OH gn vo vng thm c th b oxi ha. Vng B d b oxi ha
hn vng A, v vy khi p th 100 800mV th cc -OH trn vng B s b oxi ha, trn
1500mV vng A s b oxi ha.



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Hnh 11: S oxi ha ca cc nhm catechol.
Mt s nghin cu cho thy cc nhm -OH v tr meta (trn vng A) kh b oxi
ha hn v tr para hoc ortho (trn vng B). Kt qu l, anthocyanin s b oxi ha
nhiu bc cc th p khc nhau, ty vo th t oxi ha ca cc nhm -OH khc nhau.
So vi u d UV/Vis, u d ECD nhy hn v chn lc hn i vi anthocyanin c
hm lng nh.
C ch ca qu trnh oxi ha xy ra theo th t sau:
1. S oxi ha cc nhm -OH trn vng B th dng b
2. S oxi ha cc nhm -OH trn vng A th cao hn
Th oxi ha ty thuc vo pH, khi pH tng ln s lm gim th oxi ha.
Ni chung, cc nhm -OH v tr ortho trn vng B b oxi ha 450mV. Nhm
methoxyl v ng lm tng kh nng chng oxi ha ca anthocyanin. Vng A v B
khng lin hp nhau nn s oxi ha ca cc nhm -OH trn vng A khng nh hng
ng k ti s oxi ha cc nhm -OH vng B v ngc li. V vy t phng php
quang ph c th kt lun rng vng A khng nh hng trn tnh cht ph ca cc gc
t do t vng B.
3.3.2.2 Nguyn l ca HPLC/ECD
6,14,15,16


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Thit k my HPLC - ECD v mt c bn khng khc g HPLC truyn thng,
ngoi tr u d quang hc c thay bng u d ECD. u d ECD c dng t
1974. Lc v c ch m t h thng HPLC - ECD hnh 14. Thit b gm bm
y dung mi ng bng p sut h thng. Ct phn tch cha vt liu nhi ct (thng l
lp nn tr c ph vi b mt k nc, chng hn cc dn xut ca hydrocacbon a
vng) phn tch cht da trn thi gian lu. u d in ha ni trc tip vi ct.
tng hiu sut v lp li, mu c tim bng h thng tim mu t ng, b to
gradient iu khin thnh phn pha ng, tt c cc h thng ny u phc v cho my
tnh v my tnh thu ph sc k . lm phong ph phn d liu, u d PDA ni trc
tip v t trc u d ECD. Thnh phn pha ng cha nng cht in ly cao
thun tin cho dng i qua u d ECD. Thng pha ng mi trng acid cha
Li
3
PO
4
hoc Na
3
PO
4
. Li
3
PO
4
d tan trong dung mi hu c (MeOH hoc ACN) v vi
khun khng pht trin khi c mt Li nn Li
3
PO
4
thng a dng trong dung mi ng.
Cn lm sch dung mi ng vi kh tr He loi oxi v oxi cn tr vic pht hin cht
phn tch siu nhy.
Khi cht phn tch i qua in cc lm vic (in hnh l in cc Cacbon nhng
thng l in cc kim loi), phn ng ha hc xy ra nh cht phn tch mt mt
(hoc nhiu) in t. Nhng in t ny c pht hin bng mt dng chy qua in
cc lm vic. V th, tn hiu ha hc chuyn thnh tn hiu in t m khng cn cht
mang t tnh trung gian. Dng chy qua in cc lm vic vo mt thi im no l
mt hm tuyn tnh vi s phn t b oxi ha ti b mt in cc. Th in ha ca in
cc lm vic c th bin i xc nh mt cch chn lc cht phn tch; bt k cht no
ra khi ct sc k m khng b oxi ha di th p vo th u d s khng pht hin
c. Do ECD c mt chc nng chn lc ni ti m cc h thng u d khc khng
c. Thm vo , ECD cc k nhy, gii hn pht hin nh gp 10 1000 ln nhy
ca u d UV/Vis v gp 10 ln u d hunh quang.


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Hnh 12: H thng LC-UV-ECD ti phng th nghim

Hnh 13: Nguyn l hot ng ca u d in ha.
A: ECD loi ampe gm u d gn vo thnh ca rnh hp, cht phn tch c chuyn
qua. Phn ng in ha chuyn cht X thnh sn phm b oxi ha Y.
B: ECD dng coulom gm mt in cc xp m cht phn tch s chuyn qua, phn ng
in ha hiu qu hn v ci thin c s truyn khi.
u d in ha hnh 13A l loi u d ampe. y l dng c bn nht ca
ECD. Khi cht phn tch chy qua in cc lm vic (thng l Cacbon nhng cng c


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khi l kim loi), phn ng ha hc xy ra lc ny cht phn tch mt i mt hoc nhiu
in t (tc l cht phn tch b oxi ha). Nhng in t ny c pht hin di hnh
thc dng in chy qua in cc lm vic. Do thng tin ha hc v cht phn tch
c chuyn thnh tn hiu in m khng cn cht mang t tnh hoc quang hc trung
gian. Dng chy qua pin in ha bt k thi im no u tuyn tnh vi s mol c
oxi ha trn b mt pin ti thi im . Th in ha ca in cc lm vic c th bin
i cho php xc nh cht phn tch chn lc hn da trn th oxi ha; bt k hp cht
no i ra khi ct m khng oxi ha di th p vo cell o th u d s khng nhn ra.
Do vy u d ECD c mt chc nng chn lc ni ti m cc u d quang hc khng
c. Tuy nhin, loi u d ny t nhiu khng hiu qu v ch c 5 - 10% cht phn tch
chuyn qua in cc lm vic s khuch tn n b mt in cc v thc hin qu trnh
in ha. Do dng hnh hc ca u d nn ch cho php s oxi ha ti lp phn cch
(boundary), ch mt phn cht phn tch truyn qua u d c oxi ha. Loi u d
coulom tt hn (hnh 13B), loi ny c matrix xp, thng l Cacbon. in cc so snh
v in cc b tr (counter) dng kim loi (thng l Paladium). i vi dung dch
long, u d coulom gn nh y ht cht phn tch chuyn ha trn in cc di th
p ca u d. Dng c hng trc tip vo u d ch khng phi truyn qua. Din
tch b mt in cc tng ng k, dng chuyn ng mnh nn pha trn lp dung dch
phn bin gip cc iu kin c tha mn. V vy, u d nhy hn 10 - 20 ln u
d ampe v ci thin c s truyn khi. Trong thc hnh u d ampe v u d
coulom khng khc nhau nhiu v nhy nhng nhn chung u d culong c a
chung hn.
3.3.2.3 C ch
Cc cht c th kh hoc oxi ha tri qua phn ng oxi ha trn b mt in cc,
to s thay i dng v c u d ECD ghi nhn. Hnh 14 m t c ch ca pin in
ha: 1 cp in cc c t vo trong pin v p th dc theo in cc. Khi th ln,
phn ng din ra v xut hin dng in.
Cng dng I c ghi o theo thi gian, din tch peak ng vi tng in tch
Q trao i gia in cc v cht c hot tnh in ha, c tnh theo cng thc sau:
Din tch peak = Q = }i
.
dt


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nh lut in phn Faraday lin kt Q vi nng cht hot ng in cc qua
phng trnh:
Q = n F n
tim

Vi n
tim
= [C] V
tim

Q = n [C] V
tim
F
Trong my Dionex chng ti biu din theo hiu in th V thay v cng I,
cng dng ny c bin i thnh hiu th bng cch nhn vi in tr R
Din tch peak = R i
.
dt = | n [C] V
tim
F
Vi:
R: l in tr ca thit b (c th tnh ton hoc bit c t nh sn xut), cn gi
l h s |
i: dng o c bi u d in ha.
n: s electron trao i lin quan ti qu trnh in cc.
C: nng ca cht hot ng in cc.
V
tim
: th tch tim (L)
F: hng s Faraday = 96500 (C/mol).
n
tim
l s mol cht phn tch c tim vo my.
Nu bit c s electron trao i n trong hp cht c tnh in ha, s bit c
nng cht phn tch t din tch peak sc k HPLC- ECD.



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Hnh 14: S ca h HPLC- ECD. ng mi tn m l pha ng. ng t nt ch
mi lin h gia cc thit b.

Hnh 15: (a) m phng phn ng trn pin in ha (2 in cc t trong dung dch cha
hp cht c hot tnh in ha); (b) pin in ha tht- in cc b tr (A) p th c nh bi
Amp.1, th c chn lc t my in th (P) kt ni vi ngun in. Dng chy qua in cc
lm vic c iu khin bng b ampe th 2 (Amp.2) v d liu i ra hng vo u c hoc
h thng qun l d liu; (c) h in cc culong s dng in cc carbon graphite l xp, trn
mi n v in cc c 1 in cc carbon l xp, trn c 2 mt c gn in cc so snh v in
cc b tr. Khi p sut dc theo in cc kh nh, cc n v in cc c th kt ni li thnh
chui, to mng array.
Mt pin in ha c bn cn 3 in cc: in cc lm vic ( y s din ra qu
trnh oxi ha hoc kh), in cc b tr v in cc so snh (dng in chy qua rt b,
dung ha bt k s thay i trong dn nn ca pha ng). Pin in ha c s
dng c nhy cao, b mt rng m bo 100% cht phn tch hot tnh in cc s b
oxi ha hoc kh. Dng o u ra c n v mV (U = I R, U c n v l mV, I c
n v l mA, R c n v l O).
Vic trao i in t xy ra ti ranh gii ca in cc lm vic v cht hot ng
trong dung dch in dn. T l trao i in t, tc ln ca dng chuyn qua, chnh l
tc phn ng ti th p. T l ny cng ty thuc vo tc ca cht kho st n b
mt in cc bng khuch tn v i lu.
S electron trao i n ca cc cht in hot c th c xc nh bng phng
php vol-ampe. Nguyn tc ca phng php c m t nh sau:


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Cch xc nh s electron trao i bng vol-ampe
19,20,21
:
Cc phng php in ha s dng trong h sinh hc m phng bn cht chng
oxi ha da trn mi lin h cht ch gia kh nng oxi ha kh ca ha cht trong dung
dch v hot tnh in ha ti b mt in cc, cng ca cht chng oxi ha trong
mi trng v th p chng b kh trn b mt in cc (cht kh cng mnh th p
cng t dng). Thc t mt cht chng oxi ha l cht kh trong tng tc vi gc t do,
lc ny chng s b oxi ha, tuy nhin khng phi lc no cng vy. Nhng cht chng
oxi ha khc nhau phn ng vi cc gc t do v cc cht oxi ha khc nhau nhiu mc
, cng nh chng s hot ng trn cc b mt in cc khc nhau vi mc khc
nhau. D bin i nh vy nhng chng cng cho php nh danh v nh lng. Mc
khc, cht chng oxi ha ngn s hnh thnh v kh nng hot ng ca nitrogen v
oxygen t nhng dng hot ng khc.
Trong phng php vol-ampe, th p ca in cc lm vic thay i tuyn tnh vi
thi gian, bt u t th E m khng xy ra phn ng in ha n gi tr E xy ra
s kh/oxi ha cht nghin cu. Sau khi i ngang qua vng th c mt hoc hai phn ng
in ha xy ra, hng qut tuyn tnh o li, cc sn phm cng nh sn phm trung
gian to ra t ln qut u s thc hin phn ng in ha v c ghi nhn. Khong thi
gian th nghim (chnh l vn tc qut v th E nghin cu) c th bin i 10
2
-10
-3
s mc
d cc phn ng nh lng thng gii hn 10-10
-3
s. Cht in ly b tr c mt km
hm s di chuyn ca cc in tch.
Cc th nghim c thc hin trn cell 3 in cc kn kh ni vi dng argon,
dung dch c sc kh argon 5-10 pht ui oxi trc mi ln o, th tch cell l
10mL. Qut th t -200 ti 1200mV v -200 ti 600mV. in cc so snh l calomen
bo ha cch bit vi dung dch bi cu ni, bn trong cu ni c cng dung mi v cht
in ly b tr. in cc b tr c dng xon din tch b mt 1cm
2
, l 1 si platium di
khong 5cm ng knh 1mm. in cc lm vic gm nhng a thu c t mt ct
ngang ca si dy bng vng c cc ng knh khc nhau t bn trong vt liu thy
tinh. Gia cc ln chy, in cc lm vic c nh bng bng bt alumina trong 3 pht
loi b cc sn phm oxi ha hp th mnh trn b mt in cc, nhng sn phm ny
s cn tr b mt in cc, nh hng ti ln th th 2 d l pH no. in cc lm vic


28
cng c th lm bng Cacbon ng knh 3mm hoc c dng a quay ng knh 2mm
nm gia li Teflon, hot ng vn tc gc e = 105rad/s, qut th 20mV/s m bo
trng thi n nh.
S electron trong phn ng in ha c th c xc nh bng nhiu cch.
Phng php ph bin nht dng php o in lng coulometry, xc nh s lng ca
cht in phn gii phng trong thi gian in phn bng cch o s culong s dng.
Tuy nhin khi s dng gi tr n t phng php culong gii thch ph CV thng gp
tr ngi, v khong thi gian v cc iu kin phn ng tng i khc nhau. Trong php
o in lng coulometry thi gian l 1 gi v lp phn ng l dung dch gc ca cell o,
cn trong phng php CV thi gian ngn hn 1 giy v lp phn ng khong mt vi
mm tnh t b mt in cc.
Cc bi bo trc y thng dng mt phng php rt ph bin xc nh n
trong CV l: cht chun, thng l ferrocene (n = 1), c thm vo cng vi mu trong
cell o CV, ri so snh trc tip cng peak ca chun v cht nghin cu. Tuy nhin
vic so snh ny da trn gi thit h s khuch tn ca chun v mu l nh nhau
(thng h s khuch tn rt kh bit trc). Ngoi ra cng phi tnh n cc phn ng
ha hc ng th v d th c th nh hng ti hnh dng v chiu cao ca sng in
th. Do phng php ny lun thiu chnh xc. Tuy nhin, nu o lng t k thut
tc thi:
I
transient
= n D
1/2
F A c
o
(t T
c
)
-1/2

i
transient
t l vi n D
1/2

Vi T
c
: thi gian kho st ring.
A: din tch b mt in cc.
F: hng s Faraday = 96500.
D: h s khuch tn b mt.
c
o
: nng ca dung dch.
Kt hp vi php o in lng voltametry trng thi tnh (o dng Faraday i
qua dung dch in phn khi p th thch hp vo in cc lm vic)


29
i
steady state
= n D 4F c
o
r
o

i
steady state
t l vi n D.
D: h s khuch tn cu.
r
o
: bn knh.

transient
steadystate
i
n
i
~ , lc ny h s khuch tn D c gin lc.
Nu s dng cc in cc khc nhau cho php o tc thi v trng thi tnh, cn
iu chnh dng o c v c hiu ng din tch in cc. Tuy vy chng ta c th trnh
vic chun ha ny nu cc php o ging nhau thc hin trn cng in cc v cng mt
cht chun bit n v D ging nh ferrocene.
3.3.3 Gii thiu v LC/MS/MS
12
Sc k lng ghp khi ph l mt thit b l tng cho cc phng th nghim.
LC/MS l phng php c dng trong phn tch vt v cc hp cht cn nhn danh
chnh xc v trong nhng iu kin vn hnh nht nh ngoi thi gian lu c trng, ha
cht cn c nhn danh bng khi ph ca n. nhy LC/MS ty thuc vo hp cht
cn phn tch v giao din s dng. Trong sc k lng ghp khi ph, hn hp cht phn
tch sau khi tch ra khi ct s i qua mt ng truyn n u d khi ph. Ct sc k
lng cho php tch cht cn quan tm, khi ph cho php tch ion cn quan tm v cho
bit phn t lng ca cht phn tch. Mt h thng LC/MS/MS s phn mnh ion m
thnh cc ion con v tch cc ion con nh danh v nh lng.
Mt h LC/MS c bn gm h thng bm sc k lng, b phn tim mu, ct sc
k, u d khi ph.
Nhim v ca u d khi ph:
Nhn danh ha cht:
Mt mi trn sc k vi thi gian lu xc nh c trng cho ha cht, t
R
ny
chnh l thi gian t lc ha cht bt u vo ct v ra khi ct vo u d khi ph.


30
Mi ny biu din tng cng cc ion sinh ra t phn t ha cht thng qua k
thut ion ha xc nh. Tng cc mi ny trn sc hp thnh sc tng ion (total ion
chromatogram).
Mi ny cng c th biu din cng ca mt loi ion sinh ra t s phn mnh
ca phn t ha cht hoc t s phn mnh ca mt loi ion ca ha cht.
Khi ph tng ng ca ha cht hoc khi ph biu din cng cc ion sinh
ra t mt ion nht nh. Nh th vic nhn danh thng qua va thi gian lu va khi
ph s chnh xc hn l ch da vo thi gian lu.
nh lng nng ca ha cht trong mu.
Cc b phn chnh ca khi ph gm: b to ion, b tch ion, b d ion.
So snh 2 k thut ion ha APCI v ESI:
Ging nhau:
- u thuc loi ion ha mm, t to s phn mnh, trong nhiu trng hp cn thy
c phn t ban u.
- Nu c s phn mnh ion th cch phn mnh cng n gin, gip on nhn cu
trc cht ban u d dng hn.
- C hai k thut u c th to ion m hoc dng ty theo cu trc hp cht kho
st.
31
Khc nhau:
K thut phun ion (ESI: electrospray
ionization)

- Ion to trong pha dung dch
- Thch hp cho cht khng bn nhit
- C th to s phn mnh t hn APCI
- Thch hp cho cht kh phn cc
- Thch hp cho cht c phn t khi
ln
K thut ion ha ha hc p sut thng
(APCI: atmospheric pressure chemical
ionization)
- Ion to trong pha hi
- Thch hp cho cht khng bn nhit
- C th to s phn mnh nhiu hn ESI
- Thch hp cho cht t phn cc
- Thch hp cho cht c phn t khi nh


32

K thut APCI: khi phn t cht phn tch v dung mi i qua mt l sy s ha hi,
cc phn t hi c kh N
2
phun ra v i vo vng phng in (corona discharge). Hin
tng ion ha xy ra:
C ch to ion dng:
N
2
+ e
-
N
2
+ 2e
-

H
2
O + e
-
H
3
O
+
+2e
-

H
2
O + H
3
O
+
H
3
O
+
+ OH


M + H
3
O
+
MH
+
+ H
2
O
C ch to ion m: vi nhng phn t c H linh ng, trong vng phng
in, c s ion ha ca phn t bng cch mt i H
+
, phn cn li l mt anion:
OH

+ M (M-H)
-
+ H
2
O
K thut phun ion ESI:
- Cc phn t ha cht v dung mi ra khi ct sc k c a vo mt ng mao
qun bng kim loi cao th v sau c phun mn bng kh N
2
thot ra chung quanh ng
mao qun.
- Di tc dng ca in th cao, c s to thnh nhng ht nhuyn mang in tch
thot ra t u ng mao qun.
- Sau cc phn t dung mi t t bc hi. Cc ht mang in c th tch nh dn
v do s y nhau gia cc in tch cng du, chng s b ra thnh nhng ht nh mang
in tch.
- Sau cc ion dng hoc m to thnh c a vo b phn tch ion qua mt
ca rt nh. Dung mi v kh N
2
b bm ht ra ngoi.


33
- in th dng u kim to ion dng, in th m th to ion m v c quyt
nh ty theo cht kho st.
- Cc cht c tnh acid to ion m pH cao, cn cht c tnh baz to ion dng pH
thp.
3.3.4 Gii thiu thit b khi ph FINNIGAN LCQ deca
17
(hng Thermo Quest
Finnigan).
Thit b HPLC (TPS Spectra system) bao gm b ly mu t ng (spectra system
AS3000), bm P4000 gradient pump (spectra system P4000) v b loi kh chn khng,
u d UV 6000 (spectra system SN4000) LP flow cell 5cm ni vi u d MS (LCQ
Deca). Th tch anthocyanin khong 10L tim vo ct Phenomenex (Torrance, California,
USA) Luna C18 (150mm 2.0mm, kch thc ht 3m), chng trnh ra gii bng 3.

Hnh 16: H thng LC-UV- MS ti phng th nghim
u d MS l mt by ion LCQ-Deca (ThermoFinnigan) vi giao din ion ha phun
in t chy dng ion dng. p th dng +3 +4kV vo kim silica, dung mi to
git bay ln, kh Helium y cc ht kh v gip phn nh, cc ht kh ny va chm nhau,
dng in 80mA, th mao qun 23mV, nhit mao qun 225
o
C, thu knh (tube lens) ci


34
t hiu th -5V, sheath gas (He) 1.2 L/pht v kh b tr (He) 6 L/pht. Ph hp thu
DAD t bc sng 200 ti 800nm. By ion chy dng qut scan MS/MS ty thuc d
liu, ln qut u ci t khong m/z= 200-1200. Ln qut th hai khai bo ion c cng
cao nht t ln qut u v thu ph MS ln hai, tng t ln qut 3 MS
3
khai bo ion
cng cao ln 2. Nng lng va chm 28%, thi gian kch hot 30ms. Sau khi c
tch nh, ion con c in tch ln hn in tch ca phn t m, ESI l k thut ion ha
mm, ch cn vi giy thc hin s phn mnh.

Hnh 17: S ca giao din ESI
14
Nhn chung, 1 cht i vo my MS tri qua 3 bc:
- u tin, phn t c bn ph bi dng electron nng lng cao, chuyn phn t
thnh ion. Ion phn t tng tc n trng tnh in.
- Tip theo, trong trng tnh in hay nam chm in, cc ion gia tc c phn tch
ty theo t l khi lng/in tch.
- Cui cng, cc ion c t l khi lng/in tch c trng c pht hin. Dng ra
ca u d c khuch i v ghi o.
Cht phn tch t sc k lng hoc bm xy lanh chuyn qua kim phun, to cc git
tch in cui u kim. Dng He khng tch in trung ha cht lng v gip dung mi
bay hi thnh git. Khi dung mi bay hi, cc phn t cht phn tch b y gn nhau hn,


35
chng y ln nhau v cc git b v ra. Qu trnh ny do lc khng culong gia cc ion
gy ra. Qu trnh ny s dng li khi cc phn t khng cn lin kt vi dung mi v tr
thnh cc ion ring r. Sau chng c pht hin. Anthocyanin dng ion dng, v
vy chng khng cn ion ha m trc tip i vo my MS sau khi bay hi dung mi. Qu
trnh nh sau:

Th nghim khi tandem mass chnh l cc bc la chn khi ph vi cc dng v
xut hin gia cc qu trnh. Trong th nghim tandem mass, thng tin v cu trc c tm
thy qua cc mnh v ln 2 v 3 (MS
2
, MS
3
). Vi anthocyanin, khi cc nhm ng mt
i, ty vo khi lng phn t b mt, c th xc nh l ng hexose hay pentose. Sau
khi mt gc ng, s nhn bit nhm aglycone. Loi aglycone c nhn danh khi so
snh vi nhng nhm bit.

Hnh 18: M t phng php tandem mass to ra cc mnh v ion t MS
2
trong thit b
triple quadropole.
Trong ion trap, cc ion b loi ra khi by ngoi tr ion cn c bn ph ln 2.
Thit b p mt tn s sng radio svi tn s cng hng bng vi vn tc gc e ca ion
trong by. Nh tn s sng ny, nng lng ng lc c chuyn vo ion v ion bt u
lun chuyn trong ion trap mt bn knh ln hn.
E
kinetic
= m r e
2



36
Ion c ngun nng lng ng hc ny s va chm vi mt trong cc nguyn t He
vn sn trong ion trap, lc ny qu trnh bn ph xut hin. Nng lng ng hc ny ty
vo khuch i ca tn hiu sng radio v c hiu chnh bi s % do b vn hnh MS,
trong my LC-UV-MS ti trng Roskidle khong 25-50%.

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