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n
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Thi gian
Sc k
(Chromatogrames)
iu kin thu c sc k :
- u d (Detector) c lp t im cui ca ct
- u d tng thch vi cc cht cn pht hin
Sc k : Biu din s bin thin ca tn
hiu ra theo thi gian hoc theo th tch
tiu hao ca pha ng
Cc peaks i xng (hoc khng i xng)
Phn tch nh tnh (qualitative) v nh lng (quantitative)
Sc k
(Chromatogrames)
S phn gii (resolution)
Vn tc di chuyn tng i (relative migration rates)
S gin peak (band broadening)
3. Cc thng s c bn ca qu trnh sc k
3.1. Cc qu trnh tch trong ct sc k
S t- ng tc X-SP-MP trong ct tch
+ Nu gi: Cht PT: Xi, Pha tnh: SP, Pha ng: MP. Khi
chy sc k, qu trnh hp ph v gii hp lin tc xy ra,
t lc np cht mu vo cho n lc cc cht i ra khi ct
SK, trong ct tch lun lun c 3 t- ng tc:
+ T- ng tc pha tnh (SP) vi cht phn tch (Xi), c lc
F1.
+ T- ng tc pha ng (MP) vi cht phn tch (Xi), c lc
F2.
+ T- ng tc pha ng (MP) vi pha tnh (SP), c lc F3.
+ Chng ta c th m phng nh- trong hnh sau:
+ Trong 3 t- ng tc ny:
+ F3 l khng i (trong 1 h pha).
+ F1 v F2 ng- c chiu nhau.
+ F1 gi cht phn tch X
i
li trn pha tnh (SP).
+ F2 ko cht phn tch Xi vo pha ng (MP).
Nh- vy lc l- u gi ca cc cht trong qu trnh sc k l tng i s
ca 3 lc t- ng tc , ngha l ta c :
F
tot
= ( F
1
+ F
2
+ F
3
)
Do trong mt hn hp mu, cht phn tch no:
+ C lc F
tot
nh nht s - c ra khi ct tch tr- c tin.
+ C lc F
tot
ln nht s ra sau cng.
V th m to ra qu trnh sc k tch cc cht.
+ Nhng cht c F
tot
bng nhau, hay gn nhau th khng tch - c
khi nhau, chng trng nhau, hay chen ln nhau (Hnh 5a).
X i
SP
MP
F1
F2
F3
3.2. Thi gian l- u ca cht (Rettention Time)
+ Trong mt h pha, thi gian l- u t
R
ca cht trong ct tch.
+ L 1 i l- ng c tr- ng cho mi cht PT,
+T lc np cht vo n lc n ra v tr c nng cc i.
+ Thi gian l- u c (hnh 7):
+ Thi gian l- u tng cng (hay ton phn): t
R
+ Thi gian khng l- u gi: t
0
(hay t
M
),
+ Thi gian l- u hiu lc, t
R
v :
t
R
= ( t
R
- t
0
) (1)
Pc sc k v thi gian l- u
+ Thi gian l- u t
Ri
ca cht ph thuc:
+ Pha tnh (Loi, c ht, xp, loi xp, iu kin ch to), H8.
+ Pha ng (Loi, thnh phn, tc ,..).
+ Bn cht ca cht Phn tch X.
+ pH ca pha ng (ch h IE-, IP).
+ Nhit tch, v.v.
Cc v d - c ch ra trong mc ni v nh h- ng ca SP v MP
Thi gian l-u, t
Ri
(pht) ?
Thi gian l- u t Ri
ph thuc vo lai SP
3.3. H s tch ca 2 cht
Trong 1 h pha sc k HPLC, h s tch :
-
- c biu th:
o
= ( t
R(A)
/ t
R(B)
) (2)
Trong : t
R(A)
v t
R(B)
l thi gian l- u ca cht A v cht B.
-
Nh- vy:
+ Nu
o
= 1 khng c s tch sc k 2 cht A v B.
+ Nu
o =
1 s c s tch sc k .
Mun c s tch tt, th gi tr
o
phi khc 1 nhiu.
Cho v d v gii thch.
+ Tch hn hp PAHs (hnh 9a)
+ Tch cc axit HC (hnh 9b).
V d tch ca 5 cht trong 5 iu kin MP khc nhau
Ch iu kin MP 4 v 5 c s tch tt
V d tch 10 Hydro cacbon thm
Ch cht 3 v 4 ch- a tch - c hon ton
3.4. H s phn b K
pb
(Partition Coefficience)
Trong mt h pha sc k:
Trong qu trnh sc k, s phn b ca cht X
i
vo SP v MP l cc
cn bng nhit ng, cc cn bng ny - c c tr- ng bi h s
phn b K
pb
. V h s ny :
-
H s phn b K
pb
l mt hng s nhit ng.
-
N c tr- ng cho s phn b ca cht gia 2 pha (SP & MP).
-
V cho ta bit cht i phn b vo mi pha th no.
-
- c tnh:
K
pb
= Ci
(SP)
/Ci
(MP)
(3)
Trong : Ci
(SP)
v Ci
(MP)
: Nng cht i trong pha SP v MP.
K
pb
ny ph thuc:
+ Bn cht ca cht PT, cu trc phn t, nhm th,..
+ Bn cht v cu trc ca pha SP,
+ Bn cht v cu trc ca pha MP v thnh phn,
+ Gi tr pH ca MP (trong IE
X
-, IP-HPLC),
+ Nhit chy sc k.
K
pb
theo quy lut phn b Lng-Mua. C 3 dng:
(1): Phn b Tuyn tnh:
(2): Phn b li:
(3): Phn b lm (Xem hnh 5 trn).
+
Cn bng phn b ny c 3 dng:
1. Dng tuyn tnh, khi nng cht nh
Dng ny cho:
+ Pic sc k cn i, khng dong chn.
+ Hiu qu tch cao (tc l H nh).
2. Hai dng khng tuyn tnh, khi nng cht ln (hnh 5).
Hai dng ny:
+ Pic sc k khng cn i, pic SK dong chn.
+ Hiu qu tch nh (tc l H ln).
Tr- ng hp khng tuyn tnh ny c 2 kiu:
a)- Phn b li, chn pic SK dong pha tr- c (hnh 5).
b)- Phn b lm, chn pic sc k dong pha sau (hnh 5).
Quy lut phn b
ca cht v pic sc k
3.5. Tc pha ng
a). Tc th tch, F
+ Biu th bng s mL MP chy qua ct tch trong 1 pht.
+ K hiu: F (mL/ph.):
+ Lin quan n tit din ngang ca ct tch.
+ Tnh theo:
F = V
i
/t
i
(6)
b). Tc tuyn tnh
-
Biu th bng s mm a sc k di chuyn - c trong ct tch
trong 1 n v thi gian.
-
K hiu: u (mm/pht).
-
Theo theo:
u = L/t
i
(7)
hay l: u = F. [ 1/ (E
cl
.r
2
.
t
) ] (8)
Trong :
- E
cl
: H s c tr- ng xp.
- r : Bn knh ct tch.
- L : Chiu di ct tch.
c). nh h- ng tc MP n kt qu tch
Th hin :
+ Loi dung mi lm MP (hnh 12a).
+ Thnh phn MP (hnh 12b).
+ Tc MP (hnh 12c).
(b) (a)
V nh h- ng ca dung mi lm MP
(a)- MeOH/H
2
O : 70/30. (b)- THF/H
2
O : 70/30
nh h- ng ca thnh phn MP
Ct SP: ShimPack ODS,7 m, 200x4 mm. Detector: UV = 214 nm.
MP: MeOH/H
2
O, vi: 90/10; 80/20; 60/40; 50/50; F =1,00 mL/pht.
Cht PT: Metyl (1), Etyl (2), n-Propyl (3), v n-Butyl-Paraben (4).
Thi gian l-u, t
Ri
(pht)
Hnh 12c. V nh h- ng ca tc MP
(a)- F= 0,8 mL/pht v (b)- F= 0,16 mL/pht.
Mu: Nucleotides: AMP(1), ADP(2), ATP(3), GMP(4), GDP(5), GTP(6),
MP: Dung dch: [(KH
2
PO
4
:0,015-0,25 M)/KCl 0,22 M].
3.6. phn gii, R
L 1 i l- ng:
+ Cho bit kh nng tch ca ct sc k.
+ iu kin 2 cht tch khi nhau.
+ R
S
ti thiu c s tch: Theo quy tc Rayley.
+ - c tnh theo:
R
S
= (N/4)
1/2
.{ 1 - [(1+k
1
')/(1+k
2
')]} (9)
Hay : R
S
= (1/4).[( o -1 )/o ].(N
ef
)
1/2
(10)
Hnh sau y biu th quy tc Rayley.
L iu kin ti thiu 2 cht st nhau tch thnh 2 pic.
(a) (b) (c)
t
Ri
? t
Ri
? t
Ri
?
Quy tc Rayley v s tch
(a)- Khng tch - c. (b)-Va tch. (c)- Tch xa nhau
3.7. Nng cht ca pic sc k
+
Khi ra gii (chy sc k), cc cht PT s ra:
+ Lc u Nng nh, ri tng dn.
+ t cc i (nh pic),
+ Sau li gim dn.
+
Nng cht nh pic sc k:
C
max.
= Qi/ (
o
i. ) 2 ( t
) (11)
Trong :
Qi = V
i
.C
i
: Th tch v nng mu - c np vo ct tch.
o
i : L lch chun ca pic sc k cht i.
+
Biu v d (hnh v).
H
pic
Nng cht trong pic SK khi ra gii
V nng cht C
X
ti mt thi gian bt k t
X
ng vi th
tch ra gii V
X
mL ca pic sc k l - c tnh theo cng thc
sau:
(V
Max
V
X
)
2
C
X
= C
Max
. exp { (- N
ef
/2). ----------------- } (12)
(V
Max
.V
X
)
2
Cng thc ny cho php chng ta tnh - c nng ca cht
X ti bt k lc no trn pic sc k.
3.8. Chiu cao a H v s a N ca ct tch
a). Chiu cao a, H
+ L b dy ca lp cht trong ct tch.
+ Trong ct tch, Gi tr H nh,