ai Cng Ve Sac Ky
(Chromatography)
PGS.TS. Nguyen c Tuan
Bo mon Hoa Phan Tch Kiem Nghiem
Khoa Dc ai hoc Y Dc TPHCM
Nguyen c Tuan ai hoc Y Dc TPHCM
ai Cng Ve Sac Ky
Nguyen c Tuan ai hoc Y Dc TPHCM
Muc tieu
- Trnh bay c nh ngha sac ky, cac giai oan cua qua trnh
sac ky, phan loai sac ky, hai yeu to chu yeu quyet nh qua trnh
tach sac ky, nguyen tac cua sac ky hap phu, phan bo, trao oi ion,
ray phan t, ai lc
- Giai thch c y ngha cua cac thong so sac ky
- ng dung c cach nh tnh, nh lng bang phng phap sac ky
ai Cng Ve Sac Ky
Nguyen c Tuan ai hoc Y Dc TPHCM
Ni dung
- Lch s - nh ngha
- Qua trnh sac ky
- Phan loai cac phng phap sac ky
- S tach sac ky va sac ky o
- Cac thong so ac trng cua sac ky
- ng dung cua sac ky
Lch s - nh ngha
Nguyen c Tuan ai hoc Y Dc TPHCM
Ether dau hoa
Sac to
TV
CaCO3
Chlorophyl
Xanthophyl
t0 t1
1906: Mikhail Tswett sac ky long hap phu tren cot
1938: Izmailov & Schraiber xay dng phng phap SKLM
1958: Stahl hoan thien phng phap SKLM
1941: Martin & Synge at nen tang cho sac ky phan bo: SKG va SKK
1946 - 1949: Cremer, Prior v Keulemans My SKK u tin
1952: Martin & James bai bao au tien ve SKK
Cuoi nam 1960: sac ky long cao ap ra i
1937 1972: 12 giai Nobel ve sac ky
2004: sac ky long sieu ap
2012: Sc k hi tu siu hiu nng
Nguyn c Tun i hc Y Dc TPHCM
Lch s pht trin ca sc k
Erika Cremer (1900 1996)
Fritz Prior (1921 1996)
My sc k kh u tin (1946 1949)
Kh nng ng dng ca UPCC
Nguyen c Tuan ai hoc Y Dc TPHCM
Lch s - nh ngha
Nguyen c Tuan ai hoc Y Dc TPHCM
Qui trnh trong o chat tan c tach rieng ra bi qua trnh dch chuyen
khac nhau ve ong lc hoc cua chung trong mot he thong hai hay nhieu
pha. Mot trong cac pha nay chuyen ong mot cach lien tuc theo mot hng
nhat nh va trong pha nay cac chat rieng biet the hien linh o khac nhau la
do co s khac nhau ve phan bo, hap phu, ien tch, kch thc phan t, o
hoa tan va ap suat hi
Sac ky oi hoi hai pha:
Pha ong: long, kh, long sieu ti han
Pha tnh: co nh trong cot hay tren be mat chat mang ran tr hoa hoc
Chat ran hay chat long c tam, hap phu hay lien ket hoa hoc
chat mang
Tng tac vi chat tan theo cac c che hap phu, phan bo, trao oi
ion, ai lc
Phng phap sac ky c ap dung trong cac nganh khoa hoc khac nhau
Nganh Dc: nghien cu, phan tch, kiem nghiem thuoc
Cht lng siu ti hn l g ?
Lng/kh im ti hn Lng siu ti hn
Tng nhit vp sut
Tng nhit vp sut
Nguyn c Tun i hc Y Dc TPHCM
Mt s cht lng siu ti hn
Lng siu ti hn Nhit ti hn
(oC)
p sut ti hn
(psi)
N2O 36.5 1053.7
NH3 132.5 1653.3
n-butane 152.0 551.1
CO2 31.3 1071.3
Nguyn c Tun i hc Y Dc TPHCM
AB
Hp chat B
Hp chat A
SAC KY LONG HIEU NANG CAO
Qua trnh sac ky
Nguyen c Tuan ai hoc Y Dc TPHCM
a hon hp len pha tnh
Khai trien sac ky
Sac ky khai trien
Sac ky ra giai:
Qua trnh ra giai
Dung moi ra giai (eluent)
Dung dch ra giai (eluate)
Phat hien cac chat
Mau cua chat
en t ngoai
Phun thuoc th
au do (detector)
Mau
Trien khai sac ky
xxxO
X
A
F
Phan loai cac phng phap sac ky
Nguyen c Tuan ai hoc Y Dc TPHCM
Theo ban chat vat ly cac pha
Ten ky thuat Pha tnh Loai can bang
Sac ky long (liquid
chromatography): pha
ong la chat long
- Chat long c hap
phu tren 1 chat ran
(chat mang)
- Pha lien ket
- Chat ran
- Phan bo gia hai pha
long
- Phan bo gia pha long va
be mat pha lien ket
- Hap phu
Sac ky kh (gas
chromatography, GC):
pha ong la chat kh
- Chat long c hap
phu tren chat ran
- Pha lien ket
- Chat ran
- Phan bo gia pha kh va
pha long
- Phan bo gia pha kh va
pha lien ket
- Hap phu
Sac ky long sieu ti
han (supercritical fluid
chromatography, SFC):
pha ong la chat long
sieu ti han
- Pha lien ket - Phan bo gia pha long
sieu ti han va pha lien ket
Phan loai cac phng phap sac ky
Nguyen c Tuan ai hoc Y Dc TPHCM
Theo phng cach cho pha ong i qua pha tnh
Sac ky khai trien
Sac ky ra giai
Theo phng cach lu gi pha tnh
Sac ky cot
Sac ky lp mong
Sac ky giay
Phan loai cac phng phap sac ky
Nguyen c Tuan ai hoc Y Dc TPHCM
Theo ban chat cua qua trnh sac ky
Sac ky hap phu
Sac ky phan bo
Sac ky trao oi ion
Sac ky ray phan t
Sac ky ai lc
Sc k cp ion
Phan loai cac phng phap sac ky
Nguyen c Tuan ai hoc Y Dc TPHCM
Thong thng
Sac ky cot (Column Chromatography, CC)
Sac ky giay (Paper Chromatography, PC)
Sac ky lp mong (Thin Layer Chromatography, TLC)
Sac ky kh (Gas Chromatography, GC)
Sac ky long hieu nang cao (High Performance Liquid
Chromatography, HPLC)
Sac ky long sieu hieu nang (Ultra Performance Liquid
Chromatography, UPLC), UHPLC hoc UFLC (Ultra Fast
Liquid Chromatography)
Sc k hi t siu hiu nng (Ultra Performance
Convergence Chromatography, UPCC, UPC2)
C che tach Phan bo
Nguyen c Tuan ai hoc Y Dc TPHCM
Chat phan tch
hoa tan trong
pha tnh long
bao tren be
mat chat mang
ran tr
KS
1(pha 1) S
2(pha 2)
K: he so phan bo
1][2
][
S
SK
Chat
mang
ran
Pha
tnh
Chat
phan
tch
A
B
DC
CK KD: he so phan bo
bieu kien
C ch tch - Cp ion
Nguyen c Tuan ai hoc Y Dc TPHCM
C che tach Hap phu
Nguyen c Tuan ai hoc Y Dc TPHCM
n: so mol pha ong b thay the t be mat
S2
: phan t chat tan trong pha ong
S1
: phan t chat tan b hap phu
M2
: phan t pha ong
M1
: phan t pha ong b hap phu
: be mat hoat ong cua pha tnh
S2
+ nM1
S1
+ nM2
n
n
MS
MSK
]][[
]][[
12
21
- Pha ong luon chay theo mot chieu nhat nh nen luon xay ra qua trnh
hap phu va phan hap phu
- Cac chat tan se b keo theo pha ong va co ai lc khac nhau vi pha tnh
Chat tan hap
phu tren be
mat pha tnh
C che tach Trao oi ion
Nguyen c Tuan ai hoc Y Dc TPHCM
Pha tnh la cac chat trao oi ion. Gom 2 loai:
Nha trao oi cation (cationit):
Cationit acid manh -SO3
-H
+
Cationit acid yeu -COO-H
+
Nha trao oi anion (anionit):
Anionit base manh -N(CH3)3
+OH
-
Anionit base yeu -NH3
+OH
-
Tien trnh trao oi: (Mx+
va Ax-
: ion chat tan)
Cation: xRSO3
-H
++ M
x+(RSO
3
-)xM
x++ xH
+
Anion: xRN(CH3)3
+OH
-+ A
x-[RN(CH
3)3
+]xA
x-+ xOH
-
- Pha ong thng la cac dung dch em co pH va chat ien ly thch hp.
- Pha ong co them mot so dung moi hu c nh MeOH, EtOH,e lam
tang o hoa tan hay o chon loc cho chat tan cac ion chat tan canh
tranh tren mang ion
Ion
phan
tch
Ion trai
dau
Ionit
ien tch
co nh
Qua trnh trao oi ion
C che tach Trao oi ion
Nguyen c Tuan ai hoc Y Dc TPHCM
S dng nha trao i ion kh khong nc
C che tach Ray phan t
Nguyen c Tuan ai hoc Y Dc TPHCM
- trang thai can bang: Cs C
m
Cs,
Cm:
nong o chat tan trong pha tnh va pha ong
He so K c tnh theo phng trnh:
sVmVR
V
mCsCK
Trong o:
Vm:
tong the tch cua pha ong nam trong cac lo xop
cua cac hat pha tnh
VS: the tch dung moi gia cac lo
VR:
the tch b lu gi cua chat tan
- Sac ky ray phan t = Sac ky loc qua gel = Sac ky tham thau gel.
- Dung trong sinh hoa e tach cac protein, cac carbohydrat,
C che tach Ai lc
Nguyen c Tuan ai hoc Y Dc TPHCM
- Mi va co tnh chon loc cao
- Da vao tng tac ac hieu gia mot
loai phan t chat tan vi mot phan t th
hai lien ket cong hoa tr vi pha tnh
V du: phan t lien ket cong hoa tr vi
pha tnh la mot khang the cua mot
protein. Khi cho hon hp gom hang ngan
protein qua cot, ch co mot pretein phan
ng vi khang the c gi lai tren cot,
cac protein con lai c ra khoi cot.
Protein b gi lai tren cot se c tach ra
khoi khang the bang cach thay oi pH
hay nong o ion trong pha ong.
Sac ky ai lc
Mot phan t
tng tac vi
phan t lien ket
cong hoa tr vi
pha tnh
Cac phan t
khac c ra
khoi cot
Qua trnh tach sac ky
Nguyen c Tuan ai hoc Y Dc TPHCM
Hai cach
tach hai chat
xen phu
S tach sac ky va
sac ky o
Nong o
chat tan tai
cac dai A va
B tren cotA B
A B
t1 t2
Cac thong so ac trng trong sac ky
Nguyen c Tuan ai hoc Y Dc TPHCM
e tach sac ky c tot:
Toc o di chuyen cua cac chat tan
Hien tng gian pic
Pic hep va can oi
Nguyen c Tuan ai hoc Y Dc TPHCM
Sc k giy v sc k lp mng
V tr cac vet tren sac ky o co the xac nh nh ch so Rf(Retardation factor)
Rd
d
v
Vf
R
M
0
===> 0 Rf 1
x x
dM
dR
1 2
ng
dung
moi
- Rf= 0: chat tan hoan toan khong di chuyen, nam
tai iem xuat phat
- Rf= 1: chat tan di chuyen cung vi van toc cua
dung moi
- dR: khoang di chuyen c cua chat can tach
- dM: khoang di chuyen c cua dung moi
- v: van toc di chuyen cua chat can tach
- V0: van toc di chuyen cua dung moi
Toc o di chuyen cua cac chat
Nguyen c Tuan ai hoc Y Dc TPHCM
Sac ky kh va sac ky long hieu nang cao
Thi gian lu tR
(phut): thi gian can thiet e mot chat di chuyen t ni
tiem mau qua cot sac ky, ti detector va cho pic tren sac ky o (tnh t
luc tiem en luc xuat hien nh cua pic).
tM
(hoac to): thi gian lu cua
mot chat khong b lu gi, con
goi la thi gian chet cua cot.
tR
cang ln, chat tan b lu gi
cang manh va toc o di chuyen
cua no cang nho.
Thi gian lu hieu chnh t'R
c
tnh theo cong thc:
t'R
= tR
- tM
(hay t'R
= tR
- to)
Toc o di chuyen cua cac chat
Th tch cht ca ct (mL)
vM 0,5 x 10-3 x L x d2
L: chiu di ct (mm)
d: ng knh trong ca ct (mm)
May HPLC
Nguyen c Tuan ai hoc Y Dc TPHCM
1
2 34
5
6
7
8
910
11
12
131415
16
17
detector
18
1. Nguon heli
2. Bnh dung moi
3. au phun
4. au loc
5. Van ty le dung moi
6. Bm
7. Van kiem tra vao
8. Van kiem tra ra
9. Kh xung
10. au thai
11. Van xa
12. Moi bm
13. Loc dung moi
14. ieu hoa ap suat
ngc
15. Chuyen oi ap suat
16. Van tiem mau
17. Cot bao ve + cot
sac ky
18. Ti detector.
Toc o di chuyen cua cac chat
Nguyen c Tuan ai hoc Y Dc TPHCM
Thi gian lu hiu chnh
tR
= tR
tM
He so dung lng k
tR
= tM
(1 + k)
k: ban chat cac pha, chat tan, nhiet o, ac iem cot
2 < k < 10 (co the chap nhan en 1 < k < 20)
He so chon loc =
B lu gi manh hn A nen > 1
1,05 < < 2,00
K
K
k
k
t
t
B
A
B
A
R B
R A
'
'
'
'
,
,
Din tch pic, chiu cao pic
Hnh dang pic S bat oi cua pic
Nguyen c Tuan ai hoc Y Dc TPHCM
ng cong phan bo
chuan Gaussian
AC
BCAF He so oi xng (0,8 < AF < 1,5)
BC: na chieu rong pha sau cua pic c o
1/10 chieu cao cua pic.
AC: na chieu rong pha trc cua pic c o
1/10 chieu cao cua pic.
He so keo uoi (1/20 chieu cao)
AC
BCACAs
2
Hnh dang pic S bat oi cua pic
Nguyen c Tuan ai hoc Y Dc TPHCM
AC
BCAF He so oi xng (0,8 < AF < 1,5)
Hieu lc cot va so a ly thuyet biu kin
Nguyen c Tuan ai hoc Y Dc TPHCM
2
2/1
2
54,516
W
t
W
t
H
LN RRbk
Nbk
: so a ly thuyet biu kin
H: chieu cao a ly thuyet
W: chieu rong cua pic ay pic
W1/2
: chieu rong cua pic o na chieu cao
Nbk
tot khi gan bang Nmax
pd
LN 4000max
L: chieu dai cot (cm)
dp: ng knh hat pha tnh (m)
Hieu lc cot va so a ly thuyet biu kin
Nguyen c Tuan ai hoc Y Dc TPHCM
Sac ky o minh hoa cach tnh N
So a ly thuyet
Nguyen c Tuan ai hoc Y Dc TPHCM
L
NN bk100
N: so a ly thuyet
Nbk
: s a l thuyt biu kin
L: chiu di ct (cm)
S gian pic (peak spreading) m rong dai (band broadening)
Nguyen c Tuan ai hoc Y Dc TPHCM
Hien tng ac biet trong sac ky
Nghien cu qua thuyet ve a va thuyet ong hoc
La ket qua di chuyen nhanh cham khac nhau cua cac phan t cung mot
chat khi qua cot
Pic ra muon tu hn pic ra sm
Cot sac ky tot (hieu lc cao) se cho pic nhon (t b gian)
S gian pic va ly thuyet ong hoc
Nguyen c Tuan ai hoc Y Dc TPHCM
Phng trnh Van Deemter
Cuu
BAH
a
b
c e
d
Khuech tan xoay A
Khuech tan doc B
Chuyen khoi C
Moi lien quan cua H vi cac yeu to ong hoc
trong qua trnh ra giai:
Khuech tan chat tan khi di chuyen trong cot
Thiet lap can bang theo tng tac: chat tan pha ong pha tnh
Toc o cua cac qua trnh tng tac dien ra trong cot
Khc phc A
- Ht pha tnh nh
- Ct mao qun c bao pha tnh b mt bn trong
S gian pic va ly thuyet ong hoc
Nguyen c Tuan ai hoc Y Dc TPHCM
Phng trnh Van Deemter
Cuu
BAH
Khuch tn xoy A
S gian pic va ly thuyet ong hoc
Nguyen c Tuan ai hoc Y Dc TPHCM
Phng trnh Van Deemter
Cuu
BAH
Khuch tn dc B
Khc phc B
- S dng ct sc k c ng knh nh
(Ct mao qun)
S gian pic va ly thuyet ong hoc
Nguyen c Tuan ai hoc Y Dc TPHCM
Phng trnh Van Deemter Cuu
BAH Chuyn khi C
Khc phc C
- Tng nhit ct gim nht pha ng v
gim qu trnh chuyn khi
- Ct mao qun bao mt lp pha tnh mng v u
- Cht mang bao mt lp pha tnh mng v u
Pha tnh
Cht mang
S m rong dai do cac nguyen nhan khac
Nguyen c Tuan ai hoc Y Dc TPHCM
Dong chay tang
Hr u
Dex
M
2
24
Hex
: yeu to anh hng en H do nguyen
nhan khac
r (cm): ban knh cac ong noi cac bo phan
cua may HPLC
u: toc o dong cua pha ong
DM: he so khuech tan trong pha ong
Anh hng cua toc o dong len hieu lc cot
Nguyen c Tuan ai hoc Y Dc TPHCM
(a) sac ky long
Toc o dong (cm/s)
H
(mm)
(b) Sac ky kh - long
H
(mm)
Toc o dong (cm/s)
S k vng t UPCC
Nguyen c Tuan ai hoc Y Dc TPHCM
Anh hng cua kch thc hat nhoi cot
Nguyen c Tuan ai hoc Y Dc TPHCM
C uf d p d c u
DuM
M
( , , )2 2
Toc o dong (cm/giay)
Chieu cao
a ly
thuyet H
(mm)
Anh hng cua c mau len hieu lc cot
Nguyen c Tuan ai hoc Y Dc TPHCM
HETP (mm)
g mau/g chat nhoi cot
Cot Spherisorb ODS
Phan bo k = 4,3
Phan bo k = 2,3
Hap phu
Pha ao k = 4,3
Pha ao k = 2,2
Pha ao k = 1,2
Phan bo k = 1,0
o phan giai
Nguyen c Tuan ai hoc Y Dc TPHCM
R
t t
W Ws
R B R A
A B
2 ( ) ( )
Rs
= 0,75: hai pic khong tach tot, con xen
phu nhau nhieu.
Rs
= 1,0 : hai pic tach kha tot, con xen
phu nhau 4%.
Rs
= 1,5 : hai pic tach hoan toan (ch
xen phu 0,3%).
Moi lien quan gia Rs
tR
H va k
Nguyen c Tuan ai hoc Y Dc TPHCM
Rs l hm
s ca
k(1+k)
Nguyen c Tuan ai hoc Y Dc TPHCM
Anh hng cua hieu lc cot len o phan giai
t
t
a)
b)
t
t
a)
b)
Gia tang hieu lc cot
Anh hng cua hieu lc cot
len o phan giai trong trng
hp he so chon loc khong oi
(a) Hieu lc cot thap
(b) Hieu lc cot cao
Hieu lc cot cao va he so chon loc giam
(a) Hieu lc cot toi a vi he so chon loc
thap
(b) Gia tang he so chon loc va van gi
hieu lc cot toi a
Nguyen c Tuan ai hoc Y Dc TPHCM
o phan giai
RN k
ks
B
B
4 1
'
'Tang R
s:
- Tang N: bang cach dung cot dai hn (ap suat, thi gian va o rong cua
pic cung tang theo) hoac pha tnh co kch thc hat nho hn. Cung co
the giam toc o dong pha ong.
- Tang k'B: bang cach thay oi thanh phan pha ong (sac ky long), nhiet
o (sac ky kh). kB
tang se tang thi gian phan tch. e dung hoa Rs
va
thi gian ra giai : 2 < kB
< 5.
- Tang bang cach thay oi loai pha tnh hoac thanh phan pha ong, ke
ca pH. Viec tang co anh hng manh en Rs.
Nguyen c Tuan ai hoc Y Dc TPHCM
nh tnh va th tinh khiet
SKG & SKLM
V tr cac vet tren sac ky o co the xac nh nh ch so Rf(Retardation factor)
Rd
d
v
Vf
R
M
0
===> 0 Rf 1
x x
dM
dR
1 2
ng
dung
moi
- Rf
= 0: chat tan hoan toan khong di chuyen,
nam tai iem xuat phat
- Rf= 1: chat tan di chuyen cung vi van toc cua
dung moi
- dR: khoang di chuyen c cua chat can tach
- dM: khoang di chuyen c cua dung moi
- v: van toc di chuyen cua chat can tach
- V0: van toc di chuyen cua dung moi
ng dung cua sac ky
Nguyen c Tuan ai hoc Y Dc TPHCM
- S dung chat oi chieu
R XR X
R A
OX
OFOA
OF
OX
OAs
f
f
( )( )
( )
* Hai chat giong nhau lan lt phai co Rfva
Rs
bang nhau.
* Hai chat phai co cung lng cham nh nhau
va c trien khai trong cung ieu kien sac ky
xxxO
X
A
F
Sac ky o sac
ky oi chng
ng dung cua sac ky
SKG & SKLM
Nguyen c Tuan ai hoc Y Dc TPHCM
Sac ky kh va sac ky long hieu nang cao
Thi gian lu tR
(phut): thi gian can thiet e mot chat di chuyen t ni
tiem mau qua cot sac ky, ti detector va cho peak tren sac ky o (tnh t
luc tiem en luc xuat hien nh cua peak).
tM
(hoac to): thi gian lu cua
mot chat khong b lu gi, con
goi la thi gian chet.
tR
cang ln, chat tan b lu gi
cang manh va toc o di chuyen
cua no cang nho.
Thi gian lu hieu chnh t'R
c
tnh theo cong thc:
t'R
= tR
- tM
(hay t'R
= tR
- to)
ng dung cua sac ky
Nguyen c Tuan ai hoc Y Dc TPHCM
* Hai chat giong nhau lan lt phai co tR
va tR
bang nhau
* Hai chat phai co cung the tch tiem mau nh nhau va
c trien khai trong cung ieu kien sac ky
Neu tren truc hoanh cua sac ky o s dung n v o la the
tch dung moi:
- the tch lu VR
- the tch lu hieu chnh V'R
- the tch chet cua cot VM
(the tch rong Vo
):the tch dung
moi t ni tiem trong cot en detector
ng dung cua sac ky
Sac ky kh va sac ky long hieu nang cao
Nguyen c Tuan ai hoc Y Dc TPHCM
ng dung cua sac ky
nh lng Nguyen tac chung: da tren s so sanh chieu cao hoac dien tch cua
pic trong mau th vi mot hay nhieu mau chuan.
Pic hep va oi xng: o chieu cao cua pic
Pic tu hay lech phng: o dien tch cua pic
Phng phap nh lng
C chun
Xay dng ng chuan (chuan ngoai nhieu iem)
So sanh trc tiep (chuan ngoai 1 iem)
Them chuan
Noi chuan
Khng c chun
Chuan hoa dien tch
Nguyen c Tuan ai hoc Y Dc TPHCM
ng dung cua sac ky
Dung ng chuan (chuan ngoai nhieu iem)
Pha cac dung dch chuan co nong o khac nhau
Tien hanh sac ky cac mau chuan
Ve ng chuan o bieu dien s phu thuoc chieu cao hay dien tch pic
theo nong o hoc thit lp phng trnh hi quy
Tien hanh sac ky mau th. Da vao ng chuan hoc phng trnh
hi quy suy ra nong o mau th
Nguyen c Tuan ai hoc Y Dc TPHCM
ng dung cua sac ky
So sanh trc tiep (chuan ngoai 1 iem)
Phan tch bang chieu cao cua pic:
ho: Chieu cao pic chuan, h
x: Chieu cao pic th
Phan tch bang dien tch pic:
So: Dien tch pic cua chuan, S
x: Dien tch pic cua th
C Ch
hx o
x
o
C CS
Sx o
x
o
C0
va Cx
cang gan nhau ket qua cang chnh xac
Phng php ni chun
Kt qu phn tch khng lp li khi p dng phng php ngoi chun (x
l mu qua nhiu giai on, hm lng cht phn tch thp hay th tch tim
mu khng lp li)
Nguyn tc
Thm cng hm lng mt cht chun khc (chun ni) vi cht cn
nh lng vo mu th v chun
Mu phn tch gm nhiu cht: chn mt trong cc cht lm chun ni
T s din tch pic ca cht phn tch v chun ni l thng s c s
dng xy dng ng chun (S/SIS, C)
Yu cu ca chun ni
Pic ca chun ni phi tch khi pic ca cc thnh phn khc
tR ca chun ni phi gn vi tR ca cht phn tch
Nng chun ni thm vo gn bng nng cht phn tch
Nguyn c Tun i hc Y Dc TPHCM
Phng php nh lng bng HPLC
Phng php ni chun
nhy pht hin ca chun ni v cht phn tch thng khc nhau
Xc nh yu t hiu chnh Fs ca chun ni v cht phn tch
S dng dung dch chun tinh khit
FS C
S Cs
i s
s i
Nng Cx ca mu th c din tch nh Sx
CS
SC Fx
x
i
i s
- Cs: nng ca dung dch chun ngoi
- Ci: nng ca dung dch chun ni
- Ss: din tch nh ca chun ngoi
- Si: din tch nh ca chun ni
Nguyn c Tun i hc Y Dc TPHCM
Phng php nh lng bng HPLC
Phng php thm chun
Thm mt lng xc nh cht chun vo dung dch mu th
Nng Cx ca mu th c din tch pic l Sx
Thm C vo lm tng S
p dng khi c nh hng ca cht ph
C SC
Sx x
Nguyn c Tun i hc Y Dc TPHCM
Phng php nh lng bng HPLC
Phng php qui v 100% din tch pic
Hay p dng xc nh tinh khit ca hot cht
Yu cu
Mi cu t trong hn hp cn phn tch u phi c ra gii, pht
hin v tch hon ton
S p ng ca u d trn cc cu t l nh nhau
zyx
x
AAA
AX
100%
- % X: gi tr % ca cu t X trong hn hp X, Y, Z
- Ax, Ay, Az: din tch pic ca cu t X, Y, Z
Nguyn c Tun i hc Y Dc TPHCM
Phng php nh lng bng HPLC
Nguyn c Tun i hc Y Dc TPHCM
ng dng HPLC trong KN dc liu v HCTN
nh lng dc liu v HCTN Khng c cht chun
p dng phng php qui v 100% din tch (xc nh tinh khit sc k)
Yu cu: tt c cc cht trong mu th u phi c tch v pht hin
Gi nh: mi cht trong hn hp tim vo my HPLC cho 1 pic duy nht
Khi lng ca cht trong mi pic c tm thy
[S1/(S1 + S2 + + Sn)] x M
M: tng khi lng cht tan trong mu tim
ng dng rt hn ch khi nh lng a thnh phn
C th p dng nh lng gn ng cc HCTN c tinh khit cao
Pic dung mi v pic chnh tng ng vi cht phn tch
T l % ca din tch pic cho bit hm lng tng i ca cht
Nn s dng pha ng lm dung mi pha mu th
Nguyn c Tun i hc Y Dc TPHCM
ng dng HPLC trong KN dc liu v HCTN
Kim tra tinh khit ginsenosid-Rg1 trc v sau khi tinh ch
Ct Supelcosil LC-18 (250 x 4,6 mm; 5 m); Pha ng: MeOH H2O (60:40);F = 0,9 ml/pht; u d PDA: 203 nm
Xc nh tinh khit
nh hng ca t l dung mi ln phn gii
30%
MeOH
40%
MeOH
50%
MeOH
60% MeOH70% MeOH
Nguyn c Tun i hc Y Dc TPHCM
nh hng ca dung mi ln phn gii
50% MeOH
50% H2O
32% THF
68% H2O
25% THF
17% MeOH
58% H2O
Nguyn c Tun i hc Y Dc TPHCM
nh hng ca pha tnh ln phn gii
Ct C18
Ct cyano
Ct phenyl
A. cortison; B. dexamethason; C. corticosteron; D. orthonitrophenol;
E. flourobenzen. Bc sng pht hin: 254 nm
Nguyn c Tun i hc Y Dc TPHCM