Sac Ky Lp Mong

(Thin Layer 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
Sac Ky Lp Mong (SKLM)
Nguyen c Tuan ai hoc Y Dc TPHCM
Muc tieu: Trnh bay c
C che chnh trong SKLM
Pha tnh va pha ong trong SKLM
Trang thiet b dung trong SKLM
Cac bc tien hanh trong SKLM
ng dung cua SKLM trong nganh Dc
Sac Ky Lp Mong (SKLM)
Nguyen c Tuan ai hoc Y Dc TPHCM
Dan bai
Gii thieu - nh ngha
Cac c che tach trong SKLM
Pha tnh
Pha ong
Trang thiet b
Cac bc tien hanh khi thc hien SKLM
Phat hien cac vet tren ban mong
Vai thong so ac trng cua SKLM
ng dung cua SKLM
anh gia SKLM
Gii thieu nh ngha
Nguyen c Tuan ai hoc Y Dc TPHCM
1938: Izmailov & Schraiber dung bot oxyd nhom e phan tch dch chiet alcaloid thc
vat
C che: hap phu chiem phan ln
Pha tnh: trng n theo ca chieu doc va chieu ngang
nh ngha: SKLM la mot phng phap sac ky dung chat hap phu lam pha tnh
trai thanh lp mong tren tam knh, nha hay kim loai. Qua trnh tach cac hp chat
xay ra khi cho pha ong la dung moi di chuyen qua pha tnh. Do o SKLM thuoc sac
ky long - ran.
SKLM co the dung e tm cac ieu kien toi u cho s tach bang sac ky long
tren cot.
Pha tnh
Nguyen c Tuan ai hoc Y Dc TPHCM
Silicagel (Kieselgel gel cua acid silicic)
S dung rong rai nhat
Kch thc hat 10 40 m, dien tch be mat 200 400 m
2
/g
Hoat tnh hap phu do OH tren be mat quyet nh
Ham am tang se lam giam hoat o
Hoat hoa 120
o
C/1 gi trc khi s dung
Si
HO OH
Si
HO OH
Si
HO OH
O O
Pha tnh
Nguyen c Tuan ai hoc Y Dc TPHCM
Nhom oxyd
Oxyd nhom trung tnh, acid, base
Hoat tnh tuy thuoc lng nc
Hoat hoa 120
o
C/1 gi trc khi s dung
Theo Brockman: 5 bac oxyd nhom
Bac hoat tnh I II III IV V
% nc 0 3 6 10 15

Pha tnh
Nguyen c Tuan ai hoc Y Dc TPHCM
Xac nh bac hoat tnh cua nhom oxyd: da vao R
f
cua mau chuan

Chat mau
Bac hoat tnh cua oxyd nhom theo Brockman
II III IV V
Gia tr R
f

Aminoazobenzen
Sudan o
Sudan vang
0,59
0,00
0,01
0,74
0,10
0,25
0,85
0,33
0,57
0,95
0,56
0,78
Pha tnh
Nguyen c Tuan ai hoc Y Dc TPHCM
Kieselguhr
Bot nhe, vang xam cha 70 95% SiO
2
Chat hap phu yeu
Tach cac chat phan cc: cetoacid, lacton,
Chat mang pha tnh trong sac ky phan bo
Kieselguhr silicagel hay kieselguhr thach cao (1:1)
Thach cao
Cellulose: khong can chat ket dnh, tach cac chat than nc
Polyamid: tach cac phenol
Nha trao oi ion: silicagel sulfon hoa
Pha tnh
Nguyen c Tuan ai hoc Y Dc TPHCM
Chat hap phu a c tieu chuan hoa
Chat hap phu co chat ket dnh: silicagel G (Gypseum), kieselguhr G, oxyd nhom G tron san 5%
thach cao
Chat hap phu khong ket dnh: silicagel H, oxyd nhom H
Chat hap phu co them chat ch th huynh quang: silicagel GF
254
, silicagel HF
254
,
Ban sac ky trang san vi gia : thuy tinh, giay nhom, polymer
u iem ban mong trang san
o on nh c hoc cao, thao tac n gian va de dang hn
Tnh ong nhat cao: thch hp e ban nh lng, nh lng
Hieu lc tach cao hn ban mong trang thu cong
Trang thiet b Pha tnh
Nguyen c Tuan ai hoc Y Dc TPHCM
Tnh chat Cac chat c
Pha tnh o acid Hoat tnh Tac dung
phan chia
dung e phan tach
Silicagel Acid Co hoat tnh Hap phu Hau het tat ca
Dan chat siloxan Phan bo Cac chat t phan cc
Oxyd nhom Base Co hoat tnh Hap phu Hydrocarbon, alcaloid, chat mau thc pham, lipid
Cat bien Phan bo ng, acid beo
Kieselguhr Trung tnh Khong co hoat tnh Phan bo ng, cac triglycerid, cac acid beo bac cao
Magie diphosphat Hoat tnh yeu Hap phu Carotinoic, tocopherol
Magie trisilicat Hoat tnh yeu Hap phu Carotinoic, tocopherol
Calci hydroxid Base Hoat tnh yeu Hap phu Carotinoic, tocopherol
Calci phosphat Base yeu Hoat tnh yeu Hap phu Carotinoic, tocopherol
Calci sulfat Hoat tnh yeu Hap phu Carotinoic, tocopherol, cac acid beo glycerid k nc
Silicagel + oxyd nhom (1:1) Acid + base Co hoat tnh Hap phu Cac chat mau, cac acid barbituric
Sat hydroxid Co hoat tnh Hap phu Cac chat phan cc
Than Co hoat tnh Hap phu Cac chat khong phan cc
Cellulose Thc te trung
tnh
Khong co hoat tnh Phan bo Acid amin, carbohydrat, nucleotid
Polyamid Trung tnh Khong co hoat tnh (Trao oi ?) Cac flavon
Polyacrylonitril Trung tnh Khong co hoat tnh (Trao oi, phan bo ?) Cac antoxyan
Nha trao oi ion Acid + base Khong co hoat tnh Trao oi Acid nucleic, nucleotid, ion kim loai, halogenic
Gel sephadex Ray phan t Polymer, protein, phc kim loai
Pha ong
Nguyen c Tuan ai hoc Y Dc TPHCM
Thay oi tuy theo c che tach
Tang cng sc ra giai: thng ket hp 2 dung moi
Khong co qui tac cu the e la chon dung moi
Sac ky s bo e tham do he dung moi
Nen chon he dung moi n gian va dung loai tinh khiet
Hng dan la chon pha tnh va pha ong:
Tnh chat cua mau phan tch: o hoa tan, o phan cc, so lng va ac tnh cac nhom chc
C che sac ky cua pha tnh
Dung moi: o bay hi, o nht, o tinh khiet, s phan lp
Chat phan tch ion hay phan cc: dung moi phan cc nh butanol nc, them acid acetic hoac amoniac
Pha tnh silicagel hoac cac chat hap phu phan cc khac: o phan cc pha ong quyet nh toc o di chuyen
va R
f
ieu chnh sc ra giai cua pha ong e R
f
: 0,2 0,8 at o phan giai cc tr
Pha ong
Nguyen c Tuan ai hoc Y Dc TPHCM
Mot so nhom chc co kha nang hap phu tang dan:
CH=CH < OCH
3
< COOR < C=O < CHO < SH < NH
2
< OH < COOH
Dung moi vi sc ra giai (sc dung moi) tang dan:
Dung moi c
0
Dung moi c
0
Dung moi c
0

Ether dau hoa 0,01 Ether etyl ic 0,38 Acetonitril 0,65
Hexan 0,01 Cloroform 0,40 Pyridin 0,71
Heptan 0,01 Methylen clorid 0,42 Dimethyl sulfoxid 0,76
Cyclohexan 0,04 Dicloroetan 0,49 Isopropanol 0,82
Carbon tetraclorid 0,18 Aceton 0,56 Ethanol 0,88
Ether isopropyl ic 0,28 Dioxan 0,56 Methanol 0,95
Toluen 0,29 Butanol 0,56 Nc 1,00
Benzen 0,32 Ethyl acetat 0,58

Trang thiet b Ban mong
Nguyen c Tuan ai hoc Y Dc TPHCM
Ban mong dnh chac: chat hap phu c tron them 5 15% chat ket
dnh (thach cao, tinh bot, dextrin)
Ban mong khong dnh chac
5 x 20 10 x 20 20 x 20 (cm)
Trang thiet b Bnh sac ky
Nguyen c Tuan ai hoc Y Dc TPHCM
Bnh ay tron, vuong hoac ch nhat
Cac loai bnh sac ky
Cac bc tien hanh khi thc hien SKLM
Nguyen c Tuan ai hoc Y Dc TPHCM
Chuan b ban mong, bnh sac ky thch hp, pha ong
Cham dung dch len ban mong: 1-10g
Khai trien sac ky
Phat hien cac vet tren ban mong
Mau sac t nhien cua vet
Phun thuoc th hien mau
Soi di en UV 254 hoac 365 nm: them chat phat huynh quang khong
tan vao pha tnh
Dung may o mat o quang (densitometer): o cng o tia phan xa
t be mat ban mong khi soi di en UV. Chat phan tch hap thu
bc xa c ghi lai thanh pic sac ky

Mau
Trien khai sac ky
x x x
O
X
A
F
Thuoc th hien mau
Nguyen c Tuan ai hoc Y Dc TPHCM
Thuoc th Mau cua vet ng dung phan tch
Thuoc th chung
Acid phosphomolypdic Xanh toi Nhieu chat hu c
Acid sulfuric ac Nau en Tat ca cac chat hu c
Hi iod Nau Nhieu chat hu c
Thuoc th chon loc
Ninhydrin Hong tm Acid amin, cac nhom chc amin
2,4-dinitrophenylhydrazon Da cam, o Cac hp chat carbonyl
Xanh hoac luc bromocresol Vang Cac acid hu c
2,7 fluorescein Vang luc Nhieu chat hu c
Vanilin/acid sulfuric Xanh luc hong Alcol, ceton
Rhodamin B Huynh quang o Cac chat lipid
Anisaldehyd/SbCl
3
Nhieu mau Cac chat steroid
Diphenylamin/Zn
2+
Nhieu mau Cac chat bao ve thc vat
Thong so ac trng cua SKLM: He so lu gi R
f
(Retardation factor)
Nguyen c Tuan ai hoc Y Dc TPHCM
0
V
V
d
d
R
M
R
f
= =
===> 0 s R
f
s 1
x x
d
M

d
R

1 2
ng
dung moi
- R
f
= 0: chat tan hoan toan khong di chuyen, nam tai iem xuat
phat
- R
f
= 1: chat tan di chuyen cung vi van toc cua dung moi
- d
R
: khoang di chuyen c cua chat can tach
- d
M
: khoang di chuyen c cua dung moi
- v: van toc di chuyen cua chat can tach
- V
0
: van toc di chuyen cua dung moi
Thong so ac trng cua SKLM: He so lu gi R
f
(Retardation factor)
Nguyen c Tuan ai hoc Y Dc TPHCM
- Cac yeu to anh hng en R
f
Chat lng va hoat tnh cua chat hap phu
Chieu day cua lp mong, quang ng sac ky, lng chat
cham
V tr va so lng chat can tach tren ban mong
Thanh phan va o tinh khiet cua pha ong
Phng phap khai trien sac ky
o bao hoa cua dung moi trong bnh sac ky (hien tng b)
Anh hng cua cac cau t khac co trong thanh phan hon hp
can tach
o am, nhiet o va pH...
Hien tng b
Thong so ac trng cua SKLM: He so lu gi R
f
(Retardation factor)
Nguyen c Tuan ai hoc Y Dc TPHCM
- S dung chat oi chieu

R X
R X
R A
OX
OF
OA
OF
OX
OA
s
f
f
( )
( )
( )
= = =
* Hai chat giong nhau lan lt phai co R
f
va R
s
bang nhau.
* Hai chat phai co cung lng cham nh nhau va c trien khai trong
cung ieu kien sac ky
x x x
O
X
A
F
Sac ky o sac ky
oi chng
Thong so ac trng cua SKLM
Nguyen c Tuan ai hoc Y Dc TPHCM
f
f
R
R M
R
R
d
d d
k

=

=
1
'
2
16
|
.
|

\
|
=
W
d
N
R
x x
d
M

d
R

1 2
ng
dung moi
W
B
d
A
d
B
W
A
W

B A
A f B f
s
W W
R R
R
+

=
] [ 2
) ( ) (
H s dung lng
S a l thuyt biu kin
phn gii
( )
2 / 1
) (
) (
) (
1 1 N
R
R
R R
B f
A f
A f S
|
|
.
|

\
|
=
Cac ky thuat khai trien SKLM
Nguyen c Tuan ai hoc Y Dc TPHCM
Sac ky mot chieu
Sac ky hai chieu
Mau phan tch nhieu chat
Sac ky mot chieu khong u o tin cay

Ky thuat sac ky hai chieu
Cac ky thuat khai trien SKLM
Nguyen c Tuan ai hoc Y Dc TPHCM
Sac ky o SKLM hai chieu cua mot vai acid amin.
Dung moi A: toluen/2-chloroetanol/pyridine.
Dung moi B: Chloroform/benzyl alcohol/acid acetic.
1. acid aspartic, 2. acid glutamic, 3. serine, 4. |-alanine, 5. glycine, 6. alanine, 7.
methionine, 8. valine, 9. isoleucine, 10. cysteine.
Dung moi A
Dung moi B
Sac ky o cac dan chat xanthin tren ban mong pha ao
C-18.
Pha ong: metanol/K
2
HPO
4
0,1 M (55:45 tt/tt). Phat hien: hi
iod. Thi gian khai trien: 1 gi. Cac gia tr R
f
: theobromin
0,68; theophylin 0,56; cafein: 0,44; 3-isobutyl-metyl xanthin 0,21.
ng dung moi ng cham mau
Cac ky thuat khai trien SKLM
Nguyen c Tuan ai hoc Y Dc TPHCM
Sac ky che hoa
Tach cac chat vi lng nho trong trng hp khong the tach bang cac phng phap khac
Ky thuat sac ky
che hoa (anh dau
va cao rieng tng
vung)
Dung moi Dung moi
CHP cha
A
CHP cha B
phan hap phu
ng dung cua SKLM
Nguyen c Tuan ai hoc Y Dc TPHCM
nh tnh va th tinh khiet
Da vao R
f
Sac ky so sanh chat th X vi chat oi chieu A tren cung mot sac o
ieu kien X la A: R
f
(X) = R
f
(A) vi cac he dung moi khac nhau
Chat th c coi la tinh khiet khi tren sac ky o khong co vet la
Ban nh lng nh lng
Tach chiet chat phan tch trong vet sac ky bang dung moi thch hp, lam sach dch chiet, nh lng bang ky
thuat thch hp: t dung, ton thi gian
nh lng trc tiep tren ban mong: o dien tch hay cng o mau vet sac ky
Dung may o mat o quang (Densitometer): chieu chum tia vao vet sac ky va o cng o hap thu
hoac huynh quang
X ly anh vi camera ky thuat so o phan giai cao: quet ban mong vi he thong phan tch thu nhan
hnh anh cua vet sac ky. X ly d lieu anh bang may tnh
SKLM hieu nang cao
(High Performance Thin Layer Chromatography, HPTLC)
Nguyen c Tuan ai hoc Y Dc TPHCM

S dung ban mong trang san co be day 0,1 mm
Pha tnh silica va pha lien ket siloxan 5 m
Tang o tin cay cua ket qua phan tch
Tang o phan giai va o nhay:
Vet sac ky nho hn
Thi gian sac ky ngan hn
Lng dung moi dung t hn
Sai so cua phng phap: 5 10% chu yeu do a mau len ban mong
SKLM hieu nang cao
(High Performance Thin Layer Chromatography, HPTLC)
Nguyen c Tuan ai hoc Y Dc TPHCM

SKLM hieu nang cao
(High Performance Thin Layer Chromatography, HPTLC)
Nguyen c Tuan ai hoc Y Dc TPHCM

SKLM hieu nang cao
(High Performance Thin Layer Chromatography, HPTLC)
Nguyen c Tuan ai hoc Y Dc TPHCM

anh gia SKLM: u iem
Nguyen c Tuan ai hoc Y Dc TPHCM

Phng phap n gian: th tinh khiet, xac nh gii han tap chat, phat hien cac chat la, nh tnh, ban nh
lng va nh lng
S dung pho bien e nghien cu, sang loc
Ky thuat va thiet b n gian
S dung linh hoat: de thay oi chat hap phu, dung moi khai trien cung nh thuoc th phat hien, ke ca cac loai
thuoc th an mon ma sac ky giay khong dung c
Thi gian tien hanh nhanh (15 - 30 phut, toi a vai gi) va o nhay cao hn sac ky giay
La mot phng phap vi phan tch
Co the dung e tm cac ieu kien toi u cho s tach bang sac ky long tren cot
Pham vi ng dung rong rai: kiem tra hieu qua chiet tach cua cac phng phap khac nh sac ky cot, chiet xuat,
chng cat, tai ket tinh,
Phat hien tat ca cac chat ke ca cac chat khong di chuyen theo pha ong
Thc hien tach de dang cac mau co nhieu thanh phan
anh gia SKLM: Nhc iem
Nguyen c Tuan ai hoc Y Dc TPHCM

Tr so R
f
co o lap lai thap do thanh phan pha ong thay oi trong qua trnh
khai trien sac ky
Tang gian rong vt do khuech tan v toc o dong pha ong thap