TH NGHIM CHUYN

CNG NGH TCH CHIT HP CHT THIN NHIN

GV: ThS. Hong Minh Ho
KS. Nguyn Bnh Kha

TI LIU THAM KHO

[1]. Nguyn Kim Phi Phng (2007), Phng php c lp hp cht hu c, NXB H Quc Gia
TP. HCM.
[2]. Nguyn Khc Qunh C (1999), Bi ging chit xut dc liu. B mn dc liu.
H Y-Dc TP. H Ch Minh, 45-97.

BI 1: M U

Hp cht thin nhin (natural product), phn t sinh hc t nhin (biological molecule)
l cht bin dng th cp c to ra bi c th ca mt sinh vt, cht bin dng th
cp c th cn thit hoc nhiu khi khng cn thit cho c th sinh vt.

Cc cht bin dng th cp ca thc vt thng thuc cc nhm sau: alcaloid,
quinonoid, steroid, terpenoid, iridoid, flavonoid, glycosid.

Nu nhng cht i phn t c trng lng phn t ln, tnh cht ha hc tng i
ng nht, ngi ta c th xut c mt s qui trnh tng qut chit tch c lp
chng th cc hp cht thin nhin c trng lng phn t nh, tnh cht ha hc a
dng, khc bit nn khng c qui trnh tng qut no c th p dng chung cho tt c
cc nhm, m mi loi nhm phi c mt s phng php khc nhau.
Mc ch ca vic tch chit, c lp hp cht t nhin

Kho st thnh phn ha hc ca mt cy mi, trc cha ai nghin cu v xem
nhng cht ny c hot tnh sinh hc g? Mun bit c iu ny cn phi c lp hp
cht t tinh khit >95% mi c th kho st cu trc ha hc bng phng php
quang ph hin i.

Cn c thm lng mu mt hp cht bit cu trc ha hc, mun kho st thm v
hot tnh sinh hc ca cht . Nu vic th nghim cho kt qu hp dn th s xem xt
c th tng hp ha hc hp cht c s lng nhiu hn.

Tm hiu mt hp cht bit v xem cht ny c sn sinh ra t b phn no ca
sinh vt (s sinh tng hp).

Tm hiu s khc bit ca nhng cht bin dng th cp c sn sinh ra t cng mt
ngun t nhin nhng khng cng iu kin sinh thi: th d tm hiu xem hai thc vt
cng h (family), cng chi (genus), cng loi (species) nhng mc hai ni c diu
kin kh hu, t ai th nhng khc nhau c cha cng nhng hp cht t nhin v
nhng cht ny c cng hm lng hay khng?
 C nhiu phng php tch chit, c lp hp cht t nhin t cy c: Sc k ct, sc
k lp mng, sc k gel, sc k kh, sc k lng hiu nng cao.
 Trong hng hp cht thin nhin, vic c lp cht phc tp hn v khng bit c
cy ang kho st c cha cc hp cht vi cu trc ha hc nh th no. Bit rng cy
c cn kho st c cha nhiu loi hp cht hu c, t loi khng phn cc n loi rt
phn cc, v th nu mun c lp hp cht m p dng sc k ct trc tip ngay trn
cao th ban u s rt kh t c kt qu mong mun. V vy, ngi ta thng chun
b mt lot cc cao chit c tnh phn cc tng dn, nh th mi loi cao chit cha
tng i t hp cht, gip cho qu trnh c lp hp cht tinh cht d dng.
 Mun c cc loi cao c phn cc khc nhau, s dng cc dung mi chit c
phn cc khc nhau, da trn nguyn tc chung l cc cht ging nhau s ha tan

nhau: dung mi khng phn cc ha tan tt cc hp cht khng phn cc, dung mi c
tnh phn cc trung bnh s ha tan cc hp cht c tnh phn cc trung bnh v dung
mi phn cc mnh s ha tan tt cc hp cht phn cc.

BI 2: SC L LP MNG V NH TNH S HIN DIN CA

MT S NHM HU C
I. Mc ch

Trang b nhng k nng, thao tc khi kho st cc hp cht t cy c bng k thut sc
k lp mng.

Sinh vin c th nh tnh s hin din ca mt s nhm hp cht hu c bng phng
php vt l v ha hc.
II. C s l thuyt
Sc k lp mng (SKLM) (Thin Layer Chromatography (TLC)) cn gi l sc k phng
(planar chromatography), da ch yu vo hin tng hp thu, trong pha ng l mt dung
mi hay mt hn hp dung mi, di chuyn qua mt pha tnh l mt cht hp thu tr. Th d:
silica gel hay oxit alumin. Pha tnh ny c trng thnh mt lp mng, u, ph ln mt nn
phng nh tm king, tm nhm hoc tm plastic. Do cht hp thu c trng thnh mt lp
mng nn phng php ny gi l sc k lp mng.
 Bnh sc k: Mt chu, h, lbng thy tinh, c np y.
 Pha tnh: Mt lp mng khong 0,25 mm ca mt loi cht hp thu th d nh silica
gel, alumin.
 Mu phn tch: Mu cht cn phn tch thng l hn hp gm nhiu hp cht vi
phn cc khc nhau.
 Pha ng: Dung mi hoc hn hp hai dung mi, di chuyn chm chm dc theo tm
lp mng v li ko mu cht i theo n.
III. Cch tin hnh
1. Cc bc chun b trc khi SKLM
1.1 Chun b vi qun
Dng c







ng vi qun
n cn
Kp
L thy tinh
Giy thm
Aceton

Cch tin hnh

Dng 2 tay cm 2 u ng vi qun, h nng on gia ca vi qun n khi mm do.
em vi qun trnh khi ngn la ri ko t t hai u ng ra xa. Gi yn cho n khi thy
tinh c cng li.
Tin hnh ra vi qun ko bng cch chm vo l thy tinh c cha aceton, ly vi qun
ra v chm vo giy thm rt b aceton. Lm li vi ln vi qun c sch.
1.2 . Chm mu ln tm bn mng
1.2.1. SKLM bn nh
Dng c:








Tm bn mng thng mi 20x20cm

Ko ct
Thc
Bt ch
Vi qun ko
Mu chm bn mng
My sy

Ha cht: eter du ha, cloroform, etyl acetat, metanol, aceton, nc ct

Cch tin hnh
Mu l cht lng, ly mu v chm trc tip trn bn mng. Mu l cht rn th phi ha
tan mu trong dung mi hu c ph hp, nng 2-5%, dung mi ha tan mu khng nht
thit l dung mi gii ly.
Ct tm bn mng nh (5x2cm), dng vit ch k mt ng thng pha di bng cao
1cm lm mc xut pht. Mt ng thng pha trn bn mng cao 0.5cm lm tin tuyn dung
mi.
Chm vi qun nhng vo dung dch mu, chm nh u vi qun vo tm bn mng ti
vch xut pht. Nhanh chng nhc vi qun ri khi tm bn mng vt chm ch lan rng ra
thnh vt trn c ng knh 2-5mm.
Sau khi chm xong, sy nh dung mi bay ra khi vt chm ri nhng vo dung dch
gii ly.
Nu cn kho st mt lt nhiu mu khc nhau, chun b v chm mi mu mt vt trn
bn mng. Vt ny cch vt kia 1cm. Hai vt ngoi ba phi cch b cnh 1,5cm.
1.2.2. Sc k lp mng iu ch (20x20cm)
Vi SKLM iu ch, dung dch mu cht s c chm ln thnh mt ng di, u, dc
theo vch xut pht bng cch dng vi qun ko mt ng di dc theo vch xut pht.
1.3. Gii ly bn mng
1.3.1. Chun b bnh gii ly bn mng

Dng c
 Bnh gii ly (bnh phi c np y)
 Giy lc (ct sao cho b va vo bnh gii ly)
 ng ong 10ml, 100ml
 a thy tinh
Ha cht: Eter du ha, cloroform, etyl acetat, metanol, aceton, nc ct
1.3.2. Gii ly bn mng
Cch tin hnh
Pha dung mi (h dung mi) ph hp cho vo bnh gii ly c t sn mt tm giy thm
(giy lc), nghing o nh dung mi thm t t giy lc (lm cho dung mi trong bnh
c bo ha).
t tm bn mng vo bnh gii ly, cnh y ca bn mng chm vo y ca bnh v
ngp vo dung mi. Cc vt chm mu khng c ngp vo dung mi.
Khi dung mi n mc tin tuyn (vch pha trn) bn mng th ngng qu trnh gii ly.
Ly bn mng ra, sy kh dung mi. Tin hnh hin hnh mu th bng cc thuc th c
trng.
2. Hin hnh cc vt sau khi gii ly
Sau khi gii ly xong, cc hp cht c mu s c nhn bng mt thng, nhng phn ln
cc cht hu c khng c mu nn mun nhn thy cc vt cn s dng cc phng php ha
hc hoc vt l.
2.1. Phng php vt l
Pht hin bng tia t ngoi (UV)
Dng c:
 n chiu UV
 Vit ch
Cch tin hnh: Bn mng sau khi gii ly xong, sy kh, t bn mng vo n UV, quan st
mu, nu quan tm th dung vit ch khoanh li.
2.2. Phng php ha hc:
Pht hin bng cc thuc th c trng
Dng c:
 Kp
 My sy
 Cun bng keo
 Ko
Ha cht
 I2
 Dd FeCl3
 Dd H2SO4 50%

Thuc th

Mu ca vt

Hp cht

Vng hoc nu

Hp cht hu c ni chung

H2SO4

Vng m n da cam

Flavon, Flavonol

H2SO4

Mu hoc xanh dng

Chalcon, auron

H2SO4

Mu cam n

FeCl3

Xanh lc n xanh en

H2SO4

Mu sm (max: 545nm)
Mu
Mu tmxanh dng en (max: 595nm)

Hi I2

Flavonoid
Sesquiterpen
Triterpen

Cch tin hnh: Bn mng sau khi gii ly xong, sy kh. Nu hin hnh bng hi I2 th t
bn mng trong bnh cha I2. Nu hin hnh bng dung dch FeCl3, hay H2SO4 th nhng tm
bn mng vo l cha dung dch , ly ra v lc nh cho thuc th chy xung ht. Tin hnh
nng bn mng bng my sy hay bp in, cc vt mu s hin ln, dng bng keo dn tm
bn mng li tm bn mng khng b v tin cho quan st ln sau. Trn tm bn mng
phi ghi r dung mi hoc h dung mi gii ly.

BI 3: CHIT XUT TINH DU

I. Mc ch:
Gip sinh vin nm c phng php tch chit tinh du bng phng php li cun hi
nc.
II. C s l thuyt
Mun kho st tinh du trong cy nn tin hnh vic li cun hi nc trn mu cy ti,
trnh tht thot tinh du v tinh du c tnh bay hi cao.
Nc khi b un nng s bc thnh hi bay ln, hi nc bay ln mang theo tinh du.
Dng c, ha cht v cch tin hnh
Dng c
 Bnh cu, erlen
 Bp un, ng ngng hi
 Cc ng ni bng thy tinh
Ha cht
 Nc
 V bi, v chanh
Cch tin hnh
Mu c ct nhuyn, c t vo bnh cu, cho nc ct vo bnh sao cho phn th tch
ca c mu v nc ch chim ti a hai phn ba th tch bnh cu. Lp h thng v cm bp
in un nng.

Nc trong bnh cu khi b un nng s bc thnh hi bay ln, hi nc bay ln mang

tinh du, hi ny b ng ngng hi lm lnh, ngng t tr thnh th lng, ri xung ng gn.
Trong ng gn, dung dch tch thnh hai lp gm lp nc v lp tinh du. Ty theo tinh
du nh hn nc hay nh hn nc, khi rp h thng s la chn ng gn cho ph hp. Vn
tip tc un nng h thng, lp nc trong ng gn cng lc cng nhiu, s c tr v bnh
cu nh ng nhnh ngang thng nhau. Nh ng ny m khng cn phi tip thm nc cho
h thng trong qu trnh chng ct. Lc ny ch cn m kha ng gn l c th thu ly tinh
du.

BI 4: SC K CT
I. Mc ch
y l mt trong nhng k thut nhm phn on cao th ban u (cao etanol, metanol,
butanol) ra thnh cc phn on cao nh hn, c phn cc khc nhau, hoc c lp ra hp
cht tinh khit t mt phn on cao nh.
II. C s l thuyt
S sc k l mt phng php vt l tch mt hn hp gm nhiu loi hp cht ra
ring thnh tng loi n cht, da vo tnh i lc khc nhau ca nhng loi hp cht i
vi mt h thng (h thng gm hai pha: mt pha ng v mt pha tnh).
Vic tch hai hp cht no ra ring c t c kt qu tt hay khng l ty vo h
s phn chia. Bt k mt hp cht no khi t vo mt h thng gm hai pha (hai pha: lnglng hoc rn-lng), lc t n trng thi cn bng, hp cht s phn b vo mi pha vi
mt t l nng c nh, t l ny thay i ty vo cc tnh cht ng hc ca hp cht v
ca c hai pha.
Cng tng t, mt hn hp gm nhiu loi hp cht khc nhau khi c t vo h
thng gm hai pha, v th s c tng tc mnh/yu khc nhau i vi pha tnh. H qu l mi
loi hp cht s di chuyn ngang qua pha tnh vi mt vn tc khc nhau, nh vy, k thut
sc k c th tch ring cc loi hp cht.
Sc k ct h c tin hnh iu kin p sut kh quyn. Pha tnh thng nhng ht
c kch thc tng i ln (50-150m), c np trong mt ct bng thy tinh. Mu cht cn
phn tch c t pha trn u pha tnh, c mt lp bng thy tinh t ln trn b mt
khng b xo trn lp mt. Dung mi gii ly c a ra v hng trong nhng l nh pha
di ct, ri em i c quay ui dung mi, dng sc k lp mng theo di qu trnh gii
ly.
Trong loi sc k ct vi pha tnh l silica gel loi thng, hp cht khng phn cc
c gii ly ra khi ct trc, hp cht phn cc c gii ly ra sau:
Bng 1: Cc loi hp cht c tnh phn cc tng dn
Loi hp cht
Hydrocarbon
Alken
Eter
Hydrocarbon R-H
Hp cht thm

Th t gii ly ra khi ct

Ceton
Aldehyde
Ester
Alcol
Amin
Acid carboxylic
Cc hp cht kim mnh

Chm nht, cn dung mi phn cc

III. Dng c, ha cht

Sc k nhanh-ct kh (Dry-column flash chromatography)
Dng c
 Phu lc xp bng thy tinh vi xp ca l thuc loi 2 hoc loi 3 dng chy ca
dung mi c thun li, ng knh trung bnh ca l xp 41-100m.
 Chn s v chy nh
 Bnh tam gic
 H thng to chn khng nh bng vi nc (my bm).
 2 ci mung
 Bng gn, ko
 5 l thy tinh (100ml)
Ha cht
 Silica gel kh (c ht 15-40 m)
 Eter du ha, cloroform, etyl acetat, metanol, nc ct, aceton.
Sc k ct h (open-column chromatography)
Dng c








Ct thy tinh (ng knh 1.5cm, cao 60cm)

2 ci kp
1 gi (gi ct)
1 becher 100ml, a thy tinh
ng ong 100ml
Bng gn thm nc
Phu thy tinh nh, cc l thy tinh (100ml, 50ml)

Ha cht
 Silica gel (50-150m)
 Eter du ha, chloroform, etyl acetat, metanol, acetone, nc.
IV. Cch tin hnh
Sc k nhanh-ct kh (Dry-column flash chromatography)
a) Np cht hp thu vo ct
Rp h thng gm phu lc xp gn trn bnh tam gic, bnh ny ni vi mt my bm
ht to chn khng, cho my bm hot ng. Mc tng lng nh silica gel kh cho vo
phu, dng mung nn nh silica gel xung y phu, mi ln mi t, tng lp, to thnh
mt khi rn, ng nht, cht ch, c b mt bng phng. Chiu dy lp silica gel khong
5cm. Nu cn tch mt lng mu cht nhiu hn th s dng phu to hn, c ng knh
ln hn.
Bng 2: Mi lin quan gia lng mu v kch thc phu trong sc k-nhanh ct kh.
Lng mu

ng knh trong
ca phu (cm)

Chiu cao lp hp
thu

Phn on dung
dch gii ly (ml)

<100 mg

0,5-1,0

10-15

0,5-1,0 g

2,0-3,0

20-30

1,0-10,0 g

30-50

10,0-20,0 g

10

50

b) Cn bng ct sc k
Sau khi np pha tnh xong, chnh lp b mt pha tnh cho bng phng, t mt lp bng
gn dy ln b mt ct bo v lp mt.
Cn bng ct bng loi dung mi t phn cc nht, loi dung mi dng gii ly u
tin. Cho mt lng ln dung mi ln u ct, m my bm, dung mi s c ht xung
bnh tam gic. Thc hin vi ln, n khi cht hp thu ng nht trong ct th dng, ct
c cn bng.
c) Np mu ln u ct v gii ly ct
Np mu th: Dng mung mc tng lng nh silica gel vo cao th ban u, nghin
thnh bt, sau khi nghin thnh bt mn xong, ly lp bng gn ra khi ct, dng mung
mc bt nghin vo u ct, dng mung tri u phn bt ny c mt lp mng, khng
c nn lp bt ny.
Ly lp bng gn lc ny ph tr li ln mt ct, b mt ny s khng b xo trn khi
gii ly.

Cho my bm hot ng, rt mt lng va phi dung mi ln b mt phu, khi my

bm rt ht phn dung mi, ngt p sut, rt phn dung dch va gii ly ang cha trong
bnh tam gic v cc l c nh s th t. Gn phu vo h thng, rt dung mi, cho my
bm ht hot ng, ht phu kh. Mi ln phi ht kh phu ri mi cho dung mi mi
vo.
Ghi ch: i khi mi bt u gii ly, ct b nght, dung mi khng ht qua c l do cc
hp cht phn cc no kt tinh li khi gp dung mi t phn cc. Gii quyt bng
cch dng spatule khuy xi trn lp b mt ca ct ln khong 0,5cm, ri tip tc gii ly
bnh thng.
Sc k ct h (open-column chromatography)
a) Chn cht hp thu nhi ct
Silica gel pha thng (50-150m)
b) Chn dung mi cho qu trnh sc k ct
Trc khi trin khai sc k ct, nht thit phi s dng sc k lp mng d tm h
dung mi gii ly cho ph hp. Cc bc tun t nh sau:
+ Mu cht cn sc k c ha tan trong dung mi ph hp, vi nng 10mg/ml. Gi l
dd mu (A).
+ Chun b 5-6 tm bn mng (2x5cm), chm ln nhng tm bng ny, mi chm khong
2-5l dung dch mu (A).
+ Mi bng c gii ly vi cc dung mi hoc h dung mi c phn cc khc nhau,
tin hnh hin hnh cc vt mi bng bng UV hay thuc th.
+ i vi mu cao kh chit xut t cy c (cao cha cc hp cht t t phn cc n phn
cc), chn dung mi gii ly u tin l dung mi c th y vt t phn cc nht ca cao
chit ln v tr bng vi Rf = 0,5 v chn dung mi chm dt sc k ct l dung mi c
th y vt phn cc nht ca cao chit vi Rf = 0,2.
c) T l gia lng mu cht cn tch i vi kch thc ct
- T l gia lng mu cn tch v lng cht hp thu s dng: Trng lng cht hp thu
phi ln hn 25-50 ln trng lng ca mu.
- T l gia chiu cao cht hp thu trong ct v ng knh trong ct sc k: Chiu cao
cht hp thu: ng knh trong ca ca ct = 10:1.
d) Np cht hp thu dng st vo ct
- Gi cho ct thng ng trn gi, u ra ca ct dng ming thy tinh xp hoc ming
bng gn chn.
- Trong mt becher c sn dung mi (dung mi u tin cho qu trnh gii ly ct), cho
cht hp thu vo becher, u n mi ln mt lng nh, va rt, va khuy nh u.
Lng dung mi s dng phi va hn hp khng c qu st, khin cho bt kh s
b bt gi trong ct v cng khng c qu lng.

- Nh mt phu lc c ui di t trn ct, rt hn hp st vo ct, va m nh kha

bn di ct cho dung mi chy ra, hng vo mt becher trng bn di ct, dung
mi ny c s dng li rt tr li ln u ct.
- Tip tc rt cht st vo ct cho n khi ht, va rt va dng mt thanh cao su g nh
bn ngoi thnh ct cht hp thu nn u trong ct.
- Sau khi np xong, cho dung mi chy ra v rt tr li u ct vi ba ln vic np ct
c cht ch, cho n khi thy cht hp thu trong ct c dng ng nht.
Lu trong qu trnh np ct, dung mi vn lin tc chy nh u ra khi ct, hng lng
dung mi ny c s dng li rt tr li ln u ct, khng cho u ct b kh,
ngha l phi lun c mt lng dung mi trn u ct.
- Sau khi np xong, mt thong cht hp thu u ct phi nm ngang. Nu mt thong
khng nm ngang, phi cho them dung mi them ln trn u ct, dng a thy tinh
khuy o nh phn dung mi gn st mt thong, lm xo mt phn cht hp thu trn
u ct, yn, cht hp thu lng xung t t to nn mt mt thong bng phng.
e) t mu cht cn tch ln u ct sc k
- t mu cao trong mt chn s, cho mt t silica gel vo, ly chy nghin mn, dng phu
thy tinh t mu vo ct. Khi t mu bt kh ln u ct xong, dng mt t dung mi
(dung mi u tin chy ct) thm t phn bt (mu cht v silica gel) ng thi
ra phn cn dnh trn ct.
- Cho dung mi vo y ct bt u qu trnh gii ly.
f) Theo di qu trnh gii ly ct.
- Hng dung dch gii ly trong nhng l thy tinh c nh s th t, hng mi l 50ml.
- S dng sc k lp mng gom cc l thnh cc phn on c kt qu SKLM ging
nhau.

BI 5: TCH CHIT CC HP CHT HU C BNG PHNG PHP

SC K CT
I. Mc ch
Sinh vin s c lp mt vi hp cht tinh khit t hn hp cc cht ban u bng
phng php sc k ct kt hp vi SKBM.
II. C s l thuyt
Xem lai bi sc k ct v sc k bn mng
III. Dng c v ha cht
Dng c
 Ct thy tinh (s c la chn sau)
 2 ci kp (gi ct)
 1 gi (gi ct)
 2 becher, a thy tinh
 ng ong 100ml
 Bng gn
 Phu thy tinh nh, cc l thy tinh (cc l bi)
 Ko, vi qun ko, bn mng (20x20cm)
 1 ci kp bn mng
 My sy hoc bp in nng bn
 Bnh gii ly bn
 n soi UV
 H2SO4 50%
Ha cht: eter du ha, cloroform, etyl acetat, metanol, aceton, nc ct
IV. Cch tin hnh
Xem li phn sc k ct h ca bi sc k ct.