Dai cuong ve sac ky

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


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


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


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


Lch s - nh ngha


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


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


AB

Hp chat B

Hp chat A

SAC KY LONG HIEU NANG CAO

Qua trnh sac ky


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


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



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



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



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


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


C che tach Hap phu


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


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


S dng nha trao i ion kh khong nc

C che tach Ray phan t


- 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


- 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


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


e tach sac ky c tot:

Toc o di chuyen cua cac chat tan

Hien tng gian pic

Pic hep va can oi


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


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)


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


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.



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


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


AC

BCAF He so oi xng (0,8 < AF < 1,5)

Hieu lc cot va so a ly thuyet biu kin


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


Sac ky o minh hoa cach tnh N

So a ly thuyet


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)


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


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




Cuu

BAH

Khuch tn xoy A




Cuu

BAH

Khuch tn dc B

Khc phc B

- S dng ct sc k c ng knh nh

(Ct mao qun)



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


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


(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


Anh hng cua kch thc hat nhoi cot


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


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


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


Rs l hm

s ca

k(1+k)


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


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.


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


- 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



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


* 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



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


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


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


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



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



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




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


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


nh hng ca dung mi ln phn gii

50% MeOH

50% H2O

32% THF

68% H2O

25% THF

17% MeOH

58% H2O


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


Dai cuong ve sac ky

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