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BI GING

CC PHNG PHP PHN TCH SC K


TS. Nguyn Ngc Vinh ngocvinhnguyen308@gmail.com
Sample Mobile phase
t
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t
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D
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Time
Detector
Sample Mobile phase
t
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t
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t
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Time
Detector
Khoa Cng Ngh Ha Hc B mn Ha Phn Tch
H Cng Nghip TP.HCM
Ti liu tham kho
1. C s l thuyt ca phng php sc k kh Phm Hng Vit
NXB KH & KT
2. Phng php phn tch sc k Nguyn Quang Khuyn NXB
HQG TP.HCM
3. Analytical gas chromatography (2
nd
) Walter Jennings
Academic Press
4. Chromatography in the petroleum industry - E. R. Adlard
Elsevier, 1995
5. Gas chromatography second edition Ian A. Fowlis John
Wiler & Sons, 1995.
6. High performance liquid chromatography Sandy Lindsay -
John Wiler & Sons, 1987.
7. Liquid chromatography mass spectrometry: an introduction
R. E. Andrey John Wiley & Sons
8. Gas chromatography and mass spectrometry, a practical guide
F. G. Kitson Academic Press
Chng 1:
CC KHI NIM C BN
1. nh ngha, phn loi
2. Nguyn l c bn
3. Cc i lng o c bn
1. nh ngha, phn loi
Sc k (chromatography) c pht minh vo nm 1903 bi nh thc
vt hc Nga Mikhail S. Tswett trong qu trnh tch Chlorophills v
Xanthophylls bng CaCO
3
v alumina.
Tuy nhin sau cc k thut ny b qun lng cho n cui nhng
nm 1930 v u 1940 khi Martin v Synge gii thiu sc k lng lng
dng tch cc acetyl amin acid vi pha tnh l silica.
1952 Archer J. P. Martin v Richard L. M. Synge c trao gii Nobel ha
hc cho cc pht minh v sc k phn b. Nguyn tc nn tng ca sc
k Tswett c p dng theo nhiu cch khc nhau xut hin nhiu
loi sc k khc nhau, cho php phn tch cc phn t c cu trc gn
tng t nhau.
Tn sc k vn c dng cho n ngy nay cho d cc phng php
sc k hin i khng cn lin quan n mu sc na.
Ting Hy-lp: Chroma: mu
Graph: ghi

Sc k l g?
Sc k l mt h cc k thut phn tch ha l dng tch, phn ly cc
cht trong mt hn hp da vo s phn b khc nhau ca chng gia
pha ng v pha tnh.
Nguyn tc chung l cho mu cha cht cn phn tch trong pha ng
(cht lng/kh) di chuyn qua pha tnh. Pha tnh tr hon s di chuyn ca
cc thnh phn trong mu. Cc thnh phn ny di chuyn qua h thng
vi tc khc nhau chng s c tch khi nhau theo thi gian. Mi
thnh phn i qua h thng vi thi gian ring bit gi l thi gian lu (t
R
)
S sc k kh mao qun
Cu trc tng qut ca mt thit b sc k
S sc k lng hiu nng cao
Degasse
Pump
Tim
mu
Column
Phn loi sc k:
K thut sc k c nhiu loi da theo trng thi ca mu v pha ng khi
tin hnh qu trnh sc k. l:
Sc k kh (gas chromatography - GC) c pha ng (mobile phase - MP) l
cht kh v mu a vo thit b phn tch dng kh. Trong trng hp
ny pha tnh (stationary phase - SP) c th l cht rn hoc cht lng
Sc k lng (liquid chromatography - LC) c pha ng MP l cht lng v
mu a vo thit b phn tch dng lng. Trong trng hp ny SP
cng c th l cht rn hay lng
Pha tnh trong 2 loi sc k ny c th l cht lng hoc rn nn ta c thm 2
loi nh ca GC v LC nh trong bng 1.1 trnh by di y:
Pha ng Pha tnh
Kh
Sc k kh - GC
Lng, Sc k kh lng, GLC (gas liquid chromatography)
Rn, Sc k kh rn, GSC (gas solid chromatography)
Lng
Sc k lng - LC
Lng, sc k lng lng LLC (liquid liquid chromatography)
Rn, sc k lng rn LSC (liquid solid chromatography)
Sc k lng li c chia lm 2 nhm nh:
Sc k lng p sut thng (sc k c in)
Sc k lng hiu nng cao (High Performance Liquid Chromatography
HPLC) hay sc k lng cao p (High Pressure Liquid Chromatography)
Da vo bn cht ca qu trnh sc k, HPLC li c chia thnh nhiu loi
nh nh sau:
Sc k phn b hay sc k chit nu pha tnh l cht lng
Sc k pha thng (Normal phase chromatography) pha tnh l cht
hp ph
Sc k pha o (Reversed phase chromatography) - pha tnh l cht hp
ph
Sc k trao i ion (Ion exchange chromatography) pha tnh l cht
trao i ion
Sc k ghp cp ion (Ion pair chromatography)
Sc k Gel hay ry phn t - pha tnh l gel.
T=0
T=10
T=20
Injector Injector
Detector Detector
Most Most Interaction with Stationary Phase Interaction with Stationary Phase Least Least
Flow of Mobile Phase Flow of Mobile Phase
T=0
T=10
T=20
Injector Injector
Detector Detector
Most Most Interaction with Stationary Phase Interaction with Stationary Phase Least Least
Flow of Mobile Phase Flow of Mobile Phase
2. Nguyn l c bn
Sc k l phng php tch cc cht da vo s phn b khc nhau ca chng gia
pha ng v pha tnh. Khi tip xc vi pha tnh, cc cu t ca hn hp s phn b
gia pha ng v pha tnh tng ng vi tnh cht ca chng (tnh b hp ph, tnh
tan, ).
Trong h thng sc k ch c cc phn t pha ng mi chuyn ng dc theo h
sc k. Cc cht khc nhau s c i lc khc nhau vi pha ng v pha tnh. Trong
qu trnh chuyn ng dc theo h sc k ht lp pha tnh ny n lp pha tnh khc,
s lp i lp li qu trnh hp ph, phn hp ph. H qu l cc cht c i lc ln vi
pha tnh s chuyn ng chm hn qua h thng sc k so vi cc cht tng tc
yu hn pha ny. Nh c im ny m ngi ta c th tch cc cht qua qu trnh
sc k.
Gi s nu chng ta np mu phn tch gm hn hp cht phn tch A, B,
C vo ct phn tch, kt qu cc cht A, B, C s c tch ra khi nhau
sau khi i qua ct tch. Quyt nh n hiu qu ca qu trnh tch y
l tng hp ca cc tng tc c trnh by nh hnh v.
Cht phn tch A, B, C
PHA TNH PHA NG
F1
F3
F2
Tng ca ba tng tc ny s quyt nh cht no c ra gii ra khi
ct tch trc tin khi lc gi trn ct l nh nht (F1) v ngc li.
F
tot
= F
1
+ F
2
+ F
3
i vi mi cht, s lu gia c quyt nh bi ba lc F1, F2, F3.
Trong , F1 v F2 gi vai tr quyt nh, cn F3 nh hng khng ln
lm. y F1 l lc gi cht phn tch trn pha tnh trong ct tch. F2 l
lc ko ca pha ng i vi cht phn tch ra khi pha tnh.
Nh vy vi cc cht khc nhau s c F1 v F2 khc nhau. Kt qu l cc
cht khc nhau s di chuyn trong ct vi tc khc nhau v tch ra
khi nhau khi ra khi ct sc k nh hnh v di y
Pha ng
A
B
Thi gian
B
A
B A
C
C
C
Sc k phn tch
(Elution chromatography)
Phn tch sc k: Cc cht tan b ra qua mt pha tnh nh s chuyn ng
ca pha ng qua n
Mu Pha ng
Detector
t
0
t
1
t
2
t
3
t
4
t
0
t
1
t
2
t
3
t
4
T

n

h
i

u

d
e
t
e
c
t
o
r
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,

Hiu qu tch cao.


+ Biu th theo cng thc:
-
Chiu cao L thuyt, H:
H = L/N = (L/16).(W
i
/t
i
)
2
(13)
-
Chiu cao Hiu lc, H
ef
:
H
ef
= (L/16). { W/( t
i
-t
o
) }
2
(14)
b). S a N ca ct tch
L mt i l- ng:
+ Biu th tng s lp cht di chuyn trong ct tch.
+ S N ln Hiu qu tch cao. Ti thiu: N
ef
>= 4500.
+ - c biu th theo cng thc:
- S a L thuyt, N:
N = 16.(t
i
/W
i
)
2
(15)
- S a Hiu lc, N
ef
:
N
ef
= 16.{ ( t
i
-t
o
)/W }
2
(16)
+ Hay tnh gn ng:
N
ef
= [3500.L(cm)] / d
p
(m) 17)

c). Ph- ng trnh Van Deemter
Ph- ng trnh thc nghim, Do Van Deemter xy dng
Ph- ng trnh ny:
+ Biu th mi quan h gia H v u, tc l H =f(u).
+ Ph- ng trnh:
H
tot
= ( A + B/u + C1.u + C2.u ) (18)
Const.
H
tot
= ( H1 + H2 + H3 + H4 )
Trong : A = 2.d
p
; B = 2.D
MP
;
C1= .(d
p
)/D
MP
; v C2=k./D
SP


Trong 4 phn ca H
tot
:
+ H1 : L d ht pha tnh quyt nh, phn chnh quyt nh H
t
.
+ H2 : S khuch tn ca cht trong pha ng,
+ H3 : S khuch tn ca cht vo pha tnh,
+ H4 : S khuch tn ca cht trong pha tnh.

- ngha u:
- Ti u
o
: C H nh nht,nn N ln nhtHiu qu tch tt nht.
- Cc gi tr u < u
o
: khi u gim, H tng theo. Nn N gim.
- Cc gi tr u > u
o
: khi u tng, H tng theo. Nn N gim.
- dc ca 2 nhnh: Gim, khi d
p
nh dn (hnh 15).

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