1

CC QU TRNH TCH
TRONG S C K L NG
TS NGUYN B HOI ANH
2
Qu trnh tch l qu trnh quan trng nht
trong phng php sc k ni chung v sc k
lng ni ring.
Hiu qu ca qu trnh ny ph thuc rt nhiu
vo tng tc gia cc cht trong pha tnh v
pha ng.
Mc ch chnh ca sc k l tch v nh tnh
cc cht trong hn hp cht phc tp
Tuy nhin trong thc t khng phi lc no vic
tch cc cht cng xy ra han ho do cc
tng tc l rt phc tp v tng tc ca mi
cht khi nm ring r v nm trong hn hp li
phn no khc nhau
3
Mt s lai sc k
High Performance Liquid chromatography
(HPLC) : sc k lng hiu nng cao
Gas Chromatography (GC) : sc k kh
Thin-Layer Chromatography (TLC) : sc k
lp mng
Capillary Electrophoresis(CE) : sc k in di
mao qun
4
u im ca phng php sc k
C th phn tch ng thi nhiu hp
cht
phn gii cao nh qu trnh tch trn
ct
nhy cao (ppm-ppb) nh u d
Th tch mu phn tch nh (1-100uL)
5
HPLC
High performance liquid chromatography
High pressure liquid chromatography
High price liquid chromatography
High problem liquid chromatography
High pleasure liquid chromatography
6
Mt s dng HPLC
Pha thng (thun) : Normal phase
Pha o : Reversed phase
Ghp cp ion : Reversed phase ion pairing
Sc k ion : Ion exchange (IC)
Sc k gel : SEC (GPC/GFC)
Sc k tch ng phn quang hc : Chiral separation
Cch phn chia ny ph thuc vo ct tch
Phn chia HPLC theo cu to thit b
H ng dng (ng thnh phn pha ng) :
Isocratic elution
H thay i thnh phn pha ng : Gradient elution
7
Hn hp cht tch khi nhau th
no ?
Pha tnh
Hng dng chy
8
Ti sao li c s khc nhau nh vy?
Do lc tng tc khc nhau
Mnh
Yu
9
Qu
Qu

tr
tr

nh t
nh t

ch di
ch di

n ra th
n ra th

n
n

o
o
Qu trnh tch din ra trong ct sc k.
Vt liu nhi ct, 3-5um
column
10
Tng tc gia cht phn tch
v vt liu nhi
Do s tng tc khc nhau gia cc cht
phn tch v vt liu nhi ct m qu trnh
tch xy ra.
packing
material
sample A
sample B
11
c
o
l
u
m
n
mixed sample
Qu
Qu

tr
tr

nh t
nh t

ch
ch
Mobile phase
12
u im ca HPLC
iu kin phn tch kh d dng :
- Khng cn bay hi mu nh GC, do phn tch c
cc cht km bn nhit.
D dng thu hi cht phn tch vi tinh khit
cao nu gn b thu hi phn on (fraction
collector) , thng dng trong iu ch, tch
tinh du, dc phm
lp li cao
Thng khng phn hy mu
13
Sc k pha thun (thng)
Normal Phase Mode
M.
M.
Tswett
Tswett
:
:
L ngi u tin pht trin phng
php sc k, s dng tch
Chlorophylls
Ber. Deut. Botan. Ges., 24, 384 (1906)
Adsorptionsanalyse und
chromatographische
chromatographische
Methode. Anwendung auf die Chemie des
Chlorophylls.
14
Normal Phase
Petroleum ether
Petroleum ether
CaCO
CaCO
3 3
Chlorophyll's
Chlorophyll's
Chromato
Chromatograph
Color
Color
15
Normal Phase
Trong h thng sc k u tin ngi
ta s dng
CaCO
3
lm cht nhi ct (pha tnh)
Petroleum ether lm dung mi pha ng
V t pha thun c nh ngha
nh sau :
Normal Phase
Normal Phase
C
C

t
t
:
:
mang t
mang t

nh phn c
nh phn c

c
c
Pha
Pha

ng :
ng :
mang t
mang t

nh khng phn c
nh khng phn c

c
c
16
Cc loi ct HPLC pha thun
Ct Silica gel : dng a mc ch (general
use)
Ct Cyano : dng a mc ch (general
use)
Ct Amino : phn tch ng
Ct Diol : phn tch protein
Silica gel
Si
Si
-Si-CH
2
CH
2
CH
2
CN
-Si-CH
2
CH
2
CH
2
NH
2
-Si-CH
2
CH
2
CH
2
OCH(OH)-CH
2
(OH)
Sillica gel bin tnh (b tr)
17
Cc lin kt xy ra th no?
Silica gel (polar)
Hydrogen bonding
Hydrogen bonding
Non-polar
Hydrogen bonding : Lin kt
hydrogen
Nu cht phn tch c
-COOH : Nhm Carboxyl
-NH
2
: Nhm Amino
-OH : Nhm Hydroxyl
Lin k
Lin k

t Hydrogen
t Hydrogen
s
s

m
m

nh.
nh.
Nu mu c nhm tert-butyl hoc cc nhm
khng phn cc ln
Do ch
Do ch

ng ng
ng ng

i l
i l

p th
p th

Lin k
Lin k

t Hydrogen
t Hydrogen
s
s

y
y

u
u
19
Retention Time v lin kt
Hydrogen
OH
HO
SiOH
SiOH
M
M

nh
nh
Y
Y

u
u
or
R
R

t y
t y

u
u
1
1
2
2
20
Dung mi pha ng s dng cho
sc k lng pha thun
Dung mi ch yu (khng phn cc) :
Hydrocarbons (Pentane, Hexane, Heptane, Octane)
Aromatic Hydrocarbons (Benzene, Toluene, Xylene)
Methylene chloride
Chloroform
Carbon tetrachloride
Dung mi ph (phn cc hoc hi phn cc) :
Methyl-t-butyl ether (MTBE), Diethyl ether, Tetrahydrofuran (THF),
Dioxane, Pyridine, Ethyl acetate, Acetonitrile, Acetone, 2-propaol,
ethanol, methanol
Dung mi ch yu c s dng chnh lm pha ng, dung
mi ph thng c thm vo vi t l nht nh thay
i thi gian lu.
Cc dung mi thng khng hp thu trong vng UV d
dng cho vic xc nh.
21
nh hng ca phn cc
1 : Dioctyl phthalate
2 : Dibutyl phthalate
3 : Diethyl phthalate
4 : Dimethyl phthalate
Pha ng : Hexane
0 % 2 % 5% /
0 % 2 % 5% /
MeOH
MeOH
MeOH
MeOH
MeOH
MeOH
22
Ct : Mang tnh khng phn cc
Pha ng : Mang tnh phn cc
Normal phase
Normal phase
C
C

t
t
:
:
mang t
mang t

nh phn c
nh phn c

c
c
Pha
Pha

ng :
ng :
mang t
mang t

nh khng phn c
nh khng phn c

c
c
Pha
Pha

o : Reversed
o : Reversed
Phase
Phase
23
Cc lai ct pha o
Ct C18 (ODS)
Ct C8 (octyl)
Ct C4 (butyl)
Ct Phenyl
Ct TMS
-Si-C
18
H
37
Si
Non
Non
-
-
polar property
polar property
24
Cc lin kt xy ra th no?
Lin k
Lin k

t k
t k

n
n

c
c
Khng phn cc
Dung mi phn cc
25
Tnh k nc
Nu mu c nhiu
CH
3
CH
2
CH
2
--- : dy Carbon
: nhm thm (Aromatic)
Nu mu c nhiu :
-COOH : nhm Carboxyl
-NH
2
: Nhm Amino
-OH : Nhm Hydroxyl
T
T

nh k
nh k

n
n

c m
c m

nh
nh
hn
hn
.
.
T
T

nh k
nh k

n
n

c
c
s
s

y
y

u hn
u hn
.
.
26
Thi gian lu v Tnh k nc
OH
OH
C18 (ODS)
Mnh
Yu
1
1
2
2
27
Cc dung mi s dng trong
HPLC pha o
Dung dch m + dung mi hu c
Khi s dng dung dch m, nng m v pH l
nhng thng s rt quan trng.
Methanol (MeOH), acetonitrile (ACN) hay THF l cc
dung mi thng s dng nht cho sc k pha o.
Vic ti u ha t l ca dung dch m v
dung mi hu c cng rt quan trng.
Vi cng mt lai vt liu nhi ct, ty theo
kch c ht nhi ct, hng ch to ct thnh
phn pha ng cng c th phi thay i cho
ph hp
28
nh hng khi tng phn cc
20% H
20% H
2 2
O 30% H
O 30% H
2 2
O 40% H
O 40% H
2 2
O
O
1 : p-Hydoxymethylbenzoate
2 : p-Hydoxyethylbenzoate
3 : p-Hydoxypropylbenzoate
4 : p-Hydoxybutylbenzoate
Pha ng : MeOH
29
nh hng ca cc pha tnh
C18 (ODS)
Mnh
C8
sample
sample
sample
C4
Trung bnh
Yu
30
nh hng ca cc pha tnh
Analytical Conditions
Column : Shim-pack CLC-ODS
Mobile phase : MeOH : H2O = 7 :3
Flow rate : 1.0 mL/min
Temperature : 40 C
Injection volume : 10 uL
Detection : UV-254 nm
Peaks
1. Methyl benzoate
2. Ethyl benzoate
3. n-Propyl benzoate
4. n- Butyl benzoate
ODS C8 TMS
ODS C8 TMS
Thng s Normal Phase Reverse Phase
phn cc ca ct Cao Thp
phn cc ca dung
mi
Thp Cao
Th t ra gii Cht km phn cc
ra trc
Cht phn cc ra
trc
Tng phn cc dung
mi
Ra gii nhanh
hn
Ra gii chm hn
So snh Normal phase v Reversed phase
32
Normal Phase
Tch tt cc ng phn lp th, v
d nh Vitamin E etc
Thi gian lu km n nh
Reversed Phase
lp li thi gian lu tt .
Pha tnh khng ng u nhau.
So snh Normal phase v Reversed phase
33
Sc k cp ion pha o
Reversed Phase Ion-Pair Chromatography
Cht ghp cp ion
34
Cc cht ghp cp
Ghp cp vi cc Anion
Tetra-n-butylammonium hydroxide (TBA)
Ghp cp vi cc Cation
Butanesulfonic acid sodium salt (C4)
Pentanesulfonic acid sodium salt (C5)
Hexanesulfonic acid sodium salt (C6)
Heptanesulfonic acid sodium salt (C7)
Octanesulfonic acid sodium salt (C8)
Decanesulfonic acid sodium salt (C10)
35
Tch cc Carboxylic Acid bng
ghp cp ion
Column:
bonded C18
Mobile phase:
H
2
O/MeOH1:1
tetrabutyl ammonium hydroxide
36
Tch cc hp cht Amino bng
cch ghp cp
Analytical Conditions
Column : Shim-pack CLC-ODS
Mobile phase : [A] : [B] = 9 : 1
[A]
100 mM phosphate buffer (pH=2.1)
0.8 mM sodium octane sulfonate
[B] acetonitrile
Flow rate : 1.5 mL/min
Temperature : 40 C
Injection volume : 10 uL
Peaks
1. Nicotinic acid 6. Thiamine
2. Nicotinamide 7. Caffeine
3. Pantothenate 8. Folic acid
4. Pyridoxine 9. Biotin
5. Riboflavin 10. Riboflavin
Phosphate
37
Nhng lu quan trng trong
sc k ghp cp ion
Loi cht ghp cp
Nng cht ghp cp
pH ca pha ng
R-COOH RCOO
-
+ H
+
(pKa=4.5)
R-NH
2
+ H
+
R-NH
3
+
(pKa=6.0)
38
nh hng ca cht ghp cp
Mobile Phase: H
2
O/MeOH
1:1,with 0.005M ion pairing
reagent and 1% HOAc
1 Maleic Acid
2 Phenylephrine
3 Phenylpropanolamine
4 Naphazoline
5 Phenacetin
6 Pyrilamine
Hexane Sulfonate Pentane Sulfonate
39
nh hng ca nng cht
ghp cp
40
Ra ct khi s dng TBA
Nu s dng cht ghp cp l amine t cp hoc TBA,
ct phn tch cn phi ra sau khi kt thc phn tch.
Dung dch ra hiu qu nht thng l mui sodium
perchlorate, bi v sodium perchlorate c i lc rt mnh
i vi cc hp cht amine.
Dung dch vi thnh phn nh sau thng c s dng
ra ct :
Dung dch 0.1% phosphoric acid c cha 100 mM
sodium perchlorate : methanol = 1 : 1
41
Sc k ion : Ion Exchange
N
+
R
R
R
Sample
SO
3
-
Sample
+
+
+
+ +
+
+
+
+
+
+ +
+
Lc ion
42
Ion Exchange
S dng trong lnh vc sinh hc
(phn tch protein, peptide, amino acid)
Sc k Ion : phn tch cc hp cht ion
Bao gm 2 loi ct:
Ct trao i Cation
Strong Cation Exchange (SCX) (R-SO
3
-
)
Weak Cation Exchange (WCX) (R-COO
-
)
Ct trao i Anion
Strong Anion Exchange (SAX) (R
4
N
+
)
Weak Anion Exchange (WAX) (DEAE diethyl
aminoethyl)
43
Phn tch Protein s dng ct WCX
Analytical Conditions
Column : Shim-pack WCX-1
Mobile phase :
[A] 20 mM phosphate buffer (pH=6.0)
[B] A+0.25M sodium sulfate
[A] - [B] 30 min linear gradient
Flow rate : 1.0 mL/min
Temperature : ambient
Detector : UV-280 nm
Injection volume : 10 uL
Peaks
1. albumin
2. myoglobin
3. -chymotrypsinogen A
4. liponuclease A
5. lisozyme
44
Nhng im quan trng cn lu
trong sc k ion
pH ca dung dch m
Nng ca dung dch m
Phng php ra gii
ng dng : Isocratic
Gradient pH
Gradient lc ion
45
Sc k gel SEC

S
S

c gel (s
c gel (s

c k size phn t
c k size phn t

) (S
) (S
EC)
EC)
th
th

ng
ng

c bi
c bi

t d
t d

i tn :
i tn :
GPC (Gel Permeation
chromatography) : sc k thm thu gel,
thng s dng trong lnh vc polymer.
GFC (Gel Filtration Chromatography) :
sc k tinh lc gel, thng s dng
trong lnh vc sinh ha.
46
Nguyn l ca SEC

Khng s
Khng s

d
d

ng hi
ng hi

u
u

ng tng t
ng tng t

c.
c.

T
T

ch d
ch d

a trn s
a trn s

kh
kh

c nhau v
c nhau v

th
th

i
i
gian di chuy
gian di chuy

n c
n c

a ch
a ch

t.
t.
47
Th t ra gii
SEC
Column
48
Mc ch ca GPC / GFC
GPC

S
S

d
d

ng
ng

x
x

c
c

nh tr
nh tr

ng l
ng l

ng
ng
phn t
phn t

c
c

a polymer.
a polymer.
GFC

T
T

ch c
ch c

c protein
c protein
49
Mi tng quan gia trng lng
phn t (MW) v thi gian lu RT
M
o
l
e
c
u
l
a
r

W
e
i
g
h
t

(
L
o
g
M
W
)
Gii hn loi tr
Gii hn thm thu
Time
50
Lp ng chun
Tim tng dung dch chun polymer c
phn t lng khc nhau bit c mi
quan h gia trng lng phn t v thi
gian lu .
No. time(min) mol. wet.
1 22.0 5500000
2 22.6 1800000
3 23.4 860000
4 25.0 400000
5 27.4 160000
6 31.0 50000
7 33.8 20000
8 38.4 4000
9 39.8 2000
10 42.8 600
11 46.8 80
51
Tim mu tht
tnh ton MW, cn phi c phn mm
GPC
52
Tch Protein s dng ct GFC
Analytical Conditions
Column : Asahipak GFA-50
Mobile phase :
0.1 M sodium phosphate
0.1 M NaCl (pH=7.0)
Flow rate : 0.5 mL/min
Temperature : ambient
Detector : UV-280 nm
Injection volume : 10 uL
Peaks
1. glutamate dehydrogenase
2. lactate dehydrogenase
3. enolase
4. adenylate kinase
5. cytochrome C
53
Sc k tch ng phn quang hc
C*
H
COOH
COOH
NH
2
R
R
HOOC
HOOC
*C
NH
2
R
R
H
M
i
r
r
o
r
(L) form (R) form
Tnh cht vt l ca cc cht ny ging ht
nhau, ngoi tr s quay cc.
*Chiral Center
54
Tch cc ng phn quang hc
cch 1
Cho 2 ng phn to dn xut vi cht
hu trin to cht lng hnh
(L) + (R) (L)-(R)
(R) + (R) (R)-(R)
Cht i hnh Cht lng hnh
Cc sn phm to thnh c th tch bng
Sc k pha thun hoc pha o
55
Tch trc tip s dng ct Chiral
- Pha tnh c gn mt ng phn quang
hc bit.
(R)
(R)
(L)
(R)
Tng tc mnh
Tng tc yu
Tch cc ng phn quang hc
cch 2
56
Cch la chn loi sc k
Loi sc k Dung mi s dng Cht phn tch
Reverse Phase H
2
O/Buffer, ACN, Cc cht trung ha hoc
MeOH khng ion ha, tan trong
hn hp nc/dung mi hu c
Ion-Pair RP Ging RP nhng Cc cht dng ion hoc c th
thm cht to cp ion ion ha
Normal Phase Hexane, CH
2
Cl
2
Hn hp cc cht khng tan
trong hn hp dung mi hu
c nc.
Ion Exchange H
2
O/m Cc ion v c, protein, axit
nucleic
57
Cch la chn loi sc k
Lai sc k Dung mi s dng Cht phn tch
GPC/GFC H
2
O, THF, DMF, Cc cht c phn t lng
. ln
Chiral Hn hp dung mi Cc ng phn quang hc
hu c nc
58
Cc cch ra gii trong HPLC
Ra gii vi ch Isocratic
Thnh phn pha ng khng thay i
trong sut qu trnh ra gii.
Ra gii vi ch Gradient
Pha ng vi thnh phn thay i trong
qu trnh chy.
59
Gradient
Time
B
c
o
n
c
e
n
t
r
a
t
i
o
n
Time
B
c
o
n
c
e
n
t
r
a
t
i
o
n
Isocratic
Cc cch ra gii trong HPLC
60
Isocratic
Th
Th

i gian phn t
i gian phn t

ch d
ch d

i
i
Kh
Kh

nng t
nng t

ch k
ch k

m
m
MeOH / H
2
O = 6 / 4
MeOH / H
2
O = 8 / 2
( ct C18 )
61
Gradient
95%
30%
%

M
e
O
H
62
u v nhc im ca ch Isocratic
u:
Thnh phn pha ng chnh xc.
H thng phn tch n gin, r tin.
ng nn n nh do thnh phn khng i
nn s dng tt cho cc u d thnh phn
nn nh hng nhiu n tn hiu nh dn
(CDD), o ch s khc x (RI)
Thnh phn pha ng c th trn trc bng
tay hoc trn lin tc (nu c b gradient)
63
u v nhc im ca ch Isocratic
Nhc:
Kh nng tch km, c bit trong cc mu
phc tp.
Vic kho st thay i thnh phn pha ng
cho ph hp thnh phn mu khng th thc
hin nhanh.
Thi gian phn tch di nu mun tch tt.
Nu trn trc (khi khng c b gradient p
sut thp) thnh phn pha ng c th thay
i theo thi gian khi lu
64
u v nhc im ca ch Gradient
u:
Kh nng tch tt.
Thnh phn pha ng kh chnh xc,
c bit vi h gradient p sut cao.
C th thay i rt nhiu thnh phn
trong pha ng. chnh xc ca thnh
phn theo thi gian gn nh khng i
Thi gian phn tch kh ngn
65
u v nhc im ca ch Gradient
Nhc:
Thit b t tin hn, c bit l h
gradient p sut cao.
chnh xc ca thnh phn pha ng
km hn h isocratic.
Khi mt thnh phn c t l nh, thnh
phn pha ng s km chnh xc v
khng n nh.