Phuong Phap Tach - Chiet

7/29/2019 Phuong Phap Tach - Chiet

1/52

Cc Phng PhpTchChitPGS.TS. Nguyn c Tun

B mn Ha phn tch Kim nghim

Khoa Dc

i hc Y Dc TPHCM

Nguyen c Tuan ai hoc Y Dc TPHCM


2/52

Cac Phng Phap Tach - Chiet


Dan bai- M au

- Cac phng phap tach

- Phng phap loc

- Phng phap ly tam- Phng phap chia cat pha

- Phng phap tham thau va tham tch

- Chiet- Chiet pha ran


3/52

Cac Phng Phap Tach - Chiet


Muc tieu- Trnh bay c nguyen ly cua cac phng phap tach

- Phan biet c phng phap tham thau va tham tch

- Trnh bay c y ngha cua cac he so trong chiet long - long

- Trnh bay c c s ly thuyet cua phng phap sac ky

- Trnh bay c pham vi ap dung cua chiet long long

- Trnh bay c nguyen tac cua chiet pha ran


4/52

Chiet (Ly trch Extraction)


Chiet la mot phng phap dung dung moi (n hay hon hp) e tach lay mot chat hay mot

nhom cac chat t hon hp can nghien cu

Thng gap: chiet hoat chat t dung dch nc vao dung moi hu cMuc ch: nh tnh, nh lng, xac nh cau truc Chiet la mot phng phap tach bang chuyen pha da vao s phan bo cua chat tan trong hai pha

A va B

Phan loai:Chiet long long (liquid-liquid extraction, LLE), chit lng lng trn nn rn

(solid supported liquid liquid extraction, SLE)

Chiet long ran (liquid-solid extraction, LSE): ky thuat chiet pha ran (Solid phase extraction,

SPE)

Chiet co vai tro quan trong trong kiem nghiem


5/52

Chiet long - long


He so phan boA

B

C

C

K

CA, C

Blan lt la nong o S trong pha A va B

trang thai can bang

- Hang so mot nhiet o xac nh va trong nhng ieu kien ly tng- ac trng cho mot chat tan va mot cap dung moi xac nh A va B- Phu thuoc vao nhiet o, ap suat, tnh chat cua chat tan va dung moiK cang ln, qua trnh chiet cang hieu quaV du: Fe3+ Pha A Pha B K

Ether etylic Nc + HF 0,001

Ether etylic Nc + HCl 99,0


6/52


KS

1(pha 1) S

2(pha 2)

K: he so phan bo

1][ 2][

S

S

K

- Pha 1 (V 1) co m mol chat tan S, c chiet bang pha 2 (V

2)

- q1la % S con lai trong pha 1, nong o S trong pha 1:

1

1

V

qm

- (1-q1) la % S c chiet sang pha 2, nong o S trong pha 2:

2

)1

1(

V

mq

Chiet long - long

1

12

)1

1(

V

mq

V

mq

K

21

11 KVV

Vq


7/52


Tien hanh chiet lan 2:

2

)21

(1

121

12

KVV

Vq

KVV

Vq

Sau n lan chiet vi V2, S con lai trong pha 1:

n

KVV

V

nq

21

1

q luon luon nho hn 1, sau n lan chiet nao ay tc la qn se vo cung nho va co the coi nh bang 0

Chiet long - long

V du: Chat tan A trong nc - cloroform co K= 3, co nong o 0,01 M trong 100 ml dung dch nc

a) Chiet mot lan vi 500 ml cloroform

%2,6062,0

5003100

1001

q

b) Chiet 5 lan moi lan vi 100 ml dung moi

%1,0#00098,01003100

1005

5

q


8/52


Chiet long - long

He so phan bo bieu kien(He so phan chia D)

1

2

C

C

KD

C2, C1: tong nong o cac dan

khac nhau cua chat tan trong pha

va 2

KD: khong bat buoc la hang so

B la mot base hu c:

BH+ ch ton tai trong pha nc BH+ B + H+

Pha 1: pha nc

Pha 2: pha DMHC

11

2

][][

][

BHB

BD

Ta co: ===> [B]2= K[B]

1va

1

2

][

][

B

BK

1

1

][

][][

][

][][

BH

HB

BH

HBKa

][ HK

KKD

a

a He so phan chia phu thuoc vao pH


9/52


Chiet long - long

HA la mot acid: HA H+ + A-

A-

khong ton tai trong pha hu cPha 1: pha nc

Pha 2: pha DMHC

Ta co: ===> [HA]2= K[HA]

1va

He so phan chia phu thuoc vao pH

11

2

][][

][

AHA

HAD

1

2

][

][

HA

HAK

1][

][][

HA

HAKa

][

][

HK

HKD

a

He so phan bo bieu kien(He so phan chia D)

KD: khong bat buoc la hang so

1

2

C

C

KD

C2, C1: tong nong o cac dan

khac nhau cua chat tan trong pha

va 2


10/52


Chiet long - longV du: Dung dch nc cua mot amin 0,010 M co K = 3, K

b= 1 x 10-5, 50 ml dung dch tren c chiet bang

100 ml dung moi

a) pH = 10,00 73,2)100,1100,1(

)100,10,3(109

9

D q

5050 2 73 100

0 15 15%( , )

,

b) pH = 8,00 273,0)100,1100,1(

)100,10,3(89

9

D q

50

50 0 273 1000 65 65%

( , ),

So sanh:pH Nong o amin con lai

trong pha nc

10,00 0,0015 M

8,00 0,0065 M

0

-2

-4

-6

2 4 6 8 10 12

pH

Log DBH+ B

][

HK

KKD

a

a

n

DVV

V

nq

21

1


11/52


Chiet long - long

Hieu suat chiet (o chiet hay he so chiet):

AO

B

Q

QR

QAO

: lng chat tan S trong dung dch nc ban au

QB: toan bo lng QB cua S chiet c vao pha hu c


12/52


Cac phng phap chiet long - long Chiet n: hieu suat chiet thap

Hieu suat chiet: vi

Chiet lap: hieu suat chiet cao hn nhng ton nhieu thi gian, cong sc

Chiet n lan, VB ml dung moi/lan: vi

Chiet n lan, VB ml dung moi/n lan:

Phong th nghiem: chiet gian oan hay chiet lien tuc Chiet ngc dong: hieu suat chiet rat cao, tach c nhieu chat

'1

11

k

R

A

BD

V

VKk'

nk

R

)'1(

11

A

BD

V

VKk'

n

A

BD

V

V

n

KR

1

11


13/52


Cac phng phap chiet long - long

Ngam un hoi lu Soxhlet

Chiet bang may co bo phan

khuay va nghien

Bnh ngam kiet Ngam kiet lien tuc

Dung cu dungchiet gian oanva lien tuc


14/52

Chiet ngc dong


Nguyen tac: dung moi chiet va dung dch chiet di chuyn ngc chieuva tiep xuc vi nhau

Muc tieu: tach hai hay nhieu chat tan bang mot loat s phan chia giahai pha long long

Chiet gian oan qua nhieu bc

Chiet lien tuc qua nhieu bc


15/52



Gia s co hai chat tan A va B trong hon hp AB ang ton tai pha di L (lower

phase), c chiet bang pha tren U (upper phase)

Ban au:

[A] = 1 mM

[B] = 1 mM

DA

= [A]U/[A]

L= 4, D

B= [B]

U/[B]

L= 1. ieu kien can thiet cho s tach rieng la

hai chat phai co D hoan toan khac nhau


16/52

S o chiet gian oan qua nhieu bc


Bc 0

Bc 1

Bc 2

Bc 3

U0

L0

U0

L1mi

U1mi

L0

U2mi

L0

U1

L1

U0

L2mi

U3mi

L0

U2

L1

U0

L3mi

U1

L2


17/52



Pha A (mM) B (mM)

Bc 0: Ong 0 U0 0,8 0,5L0 0,2 0,5

Bc 1: Ong 0 U1 (mi) 0,16 0,25L0 0,04 0,25

Ong 1 U0 0,64 0,25

L1 (mi) 0,16 0,25Bc 2: Pha U0 c chuyen vao ong coL2 mi

Pha U1 c chuyen vao ong coL1 cuPha U2 mi c olen pha L0 cu

Sau khi can bang moi ong s phan chia theo DA = 4, DB = 1


18/52



Soong (r)

0 1 2 3 4 5

Pha tren

(A)

0

Pha

tren

(B)

0

So

bc

(n)

Pha di

(A)

1

Pha

di

(B)

1

(A)

0,8

(B)

0,50

(A)

0,2

(B)

0,5

(A)

0,16

(B)

0,25

(A)

0,64

(B)

0,251

(A)0,04 (B)0,25 (A)0,16 (B)0,25

(A)

0,032

(B)

0,125

(A)

0,256

(B)

0,25

(A)

0,512

(B)

0,1252

(A)0,008

(B)0,125

(A)0,064

(B)0,25

(A)0,128

(B)0,125

(A)

0,0064

(B)

0,0625

(A)

0,0768

(B)

0,1875

(A)

0,3072

(B)

0,1875

(A)

0,4096

(B)

0,06253

(A)

0,0016

(B)

0,0625

(A)

0,0192

(B)

0,1875

(A)

0,0768

(B)

0,1875

(A)

0,1024

(B)

0,0625

(A)

0,00128

(B)

0,03125

(A)

0,02048

(B)

0,125

(A)

0,12288

(B)

0,1875

(A)

0,32768

(B)

0,125

(A)

0,32768

(B)

0,031254

(A)0,00032 (B)0,03125 (A)0,00512 (B)0,125 (A)0,03072 (B)0,1875 (A)0,08192 (B)0,125 (A)0,08192 (B)0,03125

(A)0,000256

(B)0,15625

(A)0,00512

(B)0,078125

(A)0,04096

(B)0,15625

(A)0,16384

(B)0,15625

(A)0,32768

(B)0,078125

(A)0,262144

(B)0,015625

5

(A)

0,000064

(B)

0,15625

(A)

0,00128

(B)

0,078125

(A)

0,01024

(B)

0,15625

(A)

0,04096

(B)

0,15625

(A)

0,08192

(B)

0,078125

(A)

0,065536

(B)

0,015625

S* (A)0,00032

(B)0,03125

(A)0,0064

(B)0,15625

(A)0,0512

(B)0,3125

(A)0,2048

(B)0,3125

(A)0,4096

(B)0,15625

(A)0,32768

(B)0,03125

Phan chia ngc dong cua A va BS: % A hoac B trong moi ong sau 5 bc


19/52



Sau 5 bc gop cac ong 0, 1 va 2 vao lo I va cac ong 4 va 5 vao

lo II. Ta co:

A (mM) Tyle B (mM) Tyle Mc otinh khiet*LoI 0,05792 0,5 50% B: 89%LoII 0,73728 73,7% 0,1875 A: 74%

tinh khitca A ctnh theo cng thc[A]/([A] + [B])

tinh khitca B: [B]/([A] + [B])

Nhan xet:A cang ngay cang cach xa B khi so bc cang tang


20/52



S o minh hoa chiet ngc dong lien tuc t s o chiet gian oan quanhieu bc

U9* U8* U7* U6 U5 U4 U3 U2 U1 U0 ===> MPSP L0 L1 L2 L3 L4 L5 L6 L7* L8* L9*

Dau * la cac pha mi

Vi:

Pha tren U di ong doc theo pha di L bat ong

Pha tren goi la pha ong MP (Mobile Phase)

Pha di goi la pha tnh SP (Stationary Phase)

Gia s A c chiet bang phan chia ngc dong theo s o tren. Phan oan

A pha tren la p% va pha di la q%. Ta co p + q = 1


21/52



Phan oan A moi ong sau moi bc di chuyen cua pha ong

Nhan xet: Nh thc (p + q)n khai trien

Soong rBcchiet n

0 1 2 3 4

1 q p

2 q2

2pq p2

3 q3 3pq2 3p2q p34 q4 4pq3 6p2q2 4p3q p4.... ..... ..... ..... ..... ....


22/52



Phan oan f (fraction) trong ong r sau bc chiet n:rnqrp

rrnnf !)!(!

Vi n ln, ong cha lng chat A cao nhat (rmax):

rmax # np

V du:A co p = 4/5 (DA = 4), sau 100 bc chiet ta co rmax # 100 x 4/5 # 80: ong 80B co p = (DB = 1), sau 100 bc chiet ta co rmax # 100 x 1/2 # 50: ong 50


23/52

o rong cua dai va nang suat phan giai


Tach A va B bang phan chia ngc dong qua 100 bc chiet

- Ong 70 - 90 hoan toan la A

- Ong 40 - 60 hoan toan la B

DA = 4 va DB = 7/3DA = 4 va DB = 1Ong59 - 72

Ong81-90

o tinh khiet

A 2,27% 38,90% 97,94%

B 66,06% 0,82% 96,68%

B A

Pnnhanomoon

Pnnhanomoon

B A

ng dung cua chiet long - long


24/52


Chiet bang dung moi hu c Nhieu phan t CHC va mot so t chat VC co o tan trong dung moi hu c ln gap nhieulan o tan trong nc

Cac yeu to anh hng:

Cau truc phan tThuoc th tao phc mang ien tch: Au, Fe trong HBr tao phc tan trong ether etylicpH: Xa phong/pH thap tao acid tan trong DMHC

Muoi alcaloid/pH cao chuyen alcaloid base tan trong DMHC



25/52


Chiet vi chelator kim loai Ion kim loai + chat phoi tr phc tan trong DMHC Chat phoi tr (ligand) hay dung:

NN

C SH

NN

H

N

OH

N

N

O

O

NH4

Dithizone 8-hydroxyquinolin Cupferron



26/52


Chiet vi chelator kim loaiHL H

+

+ L-

nL- + Mn+ MLn

MLn(Nc) ML

n(Hu c)

Trong o HL la ligand, Mn+ la ion kim loai.

He so phan chia D:

)(ML

)(MLD

n

n

ncPha

cHu

][

][

He so nay phu thuoc vao nong o cua ligand va pH cua dung dch



27/52


Chiet vi chelator kim loaiV du:nh lng Pb2+ trong nc bang phan ng tao phc vi dithizone

C6H5

N

N

C SH

NN

H

2C6H5

+ Pb2+Pb

NN

CS

C6H5

N N

C6H5

H

S

N

N

C

C6H5

NN

H

C6H5

+ 2H+

Dithizone (xanh) Khong mau o

Ong nghiem A (ml) B (ml) C (ml)Nc cat 10 0 0

Nc may 0 10 0

Dung dch Pb(NO3)2 2mM 0 0 10

Hexan 7 7 7Dithizone/CHCl3 0,01% 3 3 3

Lac yen ephan lp:

Mau cua lp hexan Xanh lam o o



28/52


Chiet cap ion Base (hay acid) + acid (hay base)/pha nc cap ion tan trong DMHC

A- + BH+ A-BH+

[A-BH+] (nc) [A-BH+] (hu c)

A-BH+: cap ion tao thanh

A- hoac BH+: tac nhan tao cap ion (ion pair agent, IPA)

C s cua phng phap chiet o quang, acid mau, acid mau-base hu c

ng dung trong kiem nghiem

Chiet va o quang: chlorpheniramin, loperamid, promethazine

Chuan o tao cap ion: dung dch chuan o la chat dien hoat anion dung nh lng alcaloid,ch th vang methyl, moi trng chloroform



29/52


Chiet cap ion Cac IPA thng dung

Cac acid manh: acid perchloric, acid sulfuric, acid phosphoric, acid hydrocloric,.

Cac hp chat sulfonic:

Heptansulfonat natri, laurylsulfat natri

Helianthin, tropeolin,

Xanh bromothymol, xanh bromophenol, xanh bromocresol,

Dan chat sulfonic cua naphthalen

Dan chat sulfonic cua fluorescein

Cac ammonium: tetrabutylammonium (C4H

9)4N+

Chit lng lng


30/52

Nguyn c Tun i hc Y Dc TPHCM

Chit lng lng

X l nh trong qu trnh chit

Chiet long ran


31/52


Pha ran (nhom oxid, than hoat, nhom silicat) chiet cac chat t pha long: hap phu

Pha long (dung moi hay he dung moi) chiet cac chat t mau phan tch ran (bot

dc lieu)

Ky thuat chiet pha ran (Solid Phase Extraction, SPE)


32/52


Tach chat phan tch t mau bang chat ran

Ra giai bang dung moi thch hp

Tinh che dch chiet trong can bang chiet long long

Khong nhng la ky thuat tach chiet oc lap, ma con c cai at vao GC-MS

hoac HPLC-MS

Chiet pha ran Thc hanh


33/52


Lam sach mau Keo dai tuoi tho cot sac ky

Co ac mau tang o nhay

u iem hn chiet long long:

Mau sach hn

Hieu suat chiet (ty le hoi phuc) cao hn

Dung moi chiet t hn

t ton thi gian hn

T ong hoa



34/52


Trang thiet b Ong hnh tru bang thuy tinh hay polypropylen: 1, 3, 6 ml

Chat hap phu: hat silica 40 m, ng knh lo xop 60 A, be matco nhom silanol Si-OH, ham lng 100, 200, 500, 1000 mg

Phan cc: cyano, diol, amino, silica

Khong phan cc: C1, C8, C18, phenyl

Trao oi ion: benzensulfonyl propyl, amin bac 4

Mang loc polyethylen, teflon, thep khong r

He thong tao chan khong

Bo phan noi cac ong, bnh cha dung moi

Thc hanh chiet pha ran


35/52

Nguyen c Tuan

Tien hanh Can bang (on nh) cot SPE

a mau len cot

Ra cot

Ra giai chat phan tch

ai hoc Y Dc TPHCM



36/52

Nguyen c Tuan

La chon chat hap phu

o phan cc cua chat hap phu tng ng vi chat phan tch o phan cc cua dung moi va chat hap phu phai khac nhau

Tuy thuoc chat phan tch e chon lng pha ran va the tch ra giai khac nhau

Lng chat phan tch Lng pha ran The tch nen

500 mg < 25 mg 1 ml

1000 mg < 50 mg 2 ml

5 g < 0,25 g 10 ml

ai hoc Y Dc TPHCM



37/52


Quy trnh chiet

X ly cot bang dung moi hoac dung dch em thch hp e chuyen pha ran sang trang thai co

the lu gi chat phan tch trong mau

Tach chat phan tch: mau c hoa tan trong dung moi va cho qua cot. Pha ran se lu gi chat

phan tch va mot so tap chat

Loai tap: dung dung moi hoac dung dch em cho qua cot e loai tap a c gi lai tren pha

ran va lam giau mau phan tch. Hoac co the ra giai chat can phan tch ra trc va gi lai tap

tren cot

Ra giai: dung dung moi thch hp ay chat phan tch khoi pha ran. Dch chiet thu c se

c tiep tuc phan tch bang cac phng phap thch hp



38/52




39/52




40/52


Dung moiChat hap phu phan cc Sc dung moi Chat hap phu khong phan ccHexan YEU NcIsooctan Methanol

Toluen Isopropanol

Cloroform Acetonitril

Methylen chlorid Aceton

Tetrahydrofuran Ethyl acetat

Ethyl ether Ethyl ether

Ethyl acetat Tetrahydrofuran

Aceton Methylen chlorid

Acetonitril Cloroform

Isopropanol Toluen

Methanol Isooctan

Nc MANH Hexan



41/52


La chon dung moi: tuy theo tng giai oan tien hanhOn nh cot: s dung 2 loai dung moi

Dung moi 1: manh hn dung moi ra giai loai bo tap chat

Dung moi 2: khong manh hn dung moi ra giai tranh hieu suat chiet giam

The tch dung moi: 1 2 ml/100 mg chat hap phu

a mau len cot: Dung moi hoa tan mau phai yeu so vi chat hap phu s dung (trong trng hp chat phan tch b hap phu

tren cot)

Ra cot: Dung moi tng ng hoac hi manh hn dung moi hoa tan mau

The tch: 0,5 0,8 ml/100 mg chat hap phu

Ra giai: The tch: 0,5 0,8 ml/100 mg chat hap phu

Chon dung moi da tren mau chuan a biet nong o khi cho qua cot SPE



42/52


Trien khai phng phap V du: s dung cot SPE (500 mg chat hap phu khong phan cc) e lam sach mau cha thuoc tr sau phosphor

Can bang cot: methanol, nc

a mau len cot: 3 ml dung dch chuan (dung moi nc)

Ra cot/ra giai:

3 ml nc

3 ml MeOH 10%

3 ml MeOH 20%

3 ml MeOH 30%

..

3 ml MeOH 100%

3 ml aceton

Xac nh hieu suat chiet (ty le hoi phuc)

sau khi tm c ieu kien toi u



43/52


V du: quy trnh chiet vitamin B12 t nc tieu

X ly cot C18 (1 g)

2 ml MeOH, 2 ml nc

NaH2PO4 0,05M

Cho qua cot 10 ml nc tieu

Ra cot bang nc

Ra giai vitamin B12

2 ml hon hp EtOH - NaH2PO4 0,05M (1:1)

3 ml nc

ng dung cua thc hanh chiet pha ran


44/52


ng dung Thuoc diet co Triazin



45/52


ng dung: Thuoc tr sau chlor



46/52


ng dung: Caffein trong huyet thanh



47/52


ng dung: Caffein trong huyet thanh



48/52


ng dung: Barbiturat trong huyet thanh



49/52


ng dung: Barbiturat trong huyet thanh



50/52


ng dung: Benzodiazepin trong huyet thanh



51/52


ng dung: Benzodiazepin trong huyet thanh

So sanh chiet long long va chiet long - ran i Kh t i


52/52


uim Khuytim

Chitlnglng ngin,dthchin. Dng nhiu dung mi, nhhngnsckhengi phn tch v gy

nhim mi trng. Kh khn trong vic kt ni viGC, HPLC, ... gy cn tr cho qutrnh tng ha vic phn tch. i khi tonhtng lm sai lchktqu.

Chit pha rn Lng dung mi dng t hnnhiu so vichitlnglng. c mtsthitbktnichitpha rnvi GC hoc HPLC, d dngtng ha phn tch mu. C nhiu la chn pha rn dng

cho SPE, nn c c ch chit adng, ph hp hn vi cht phntch, tnh chnlctthn.

Kh lugicht phn ccmnh. Tnh chn lc ch da vo tngtc phn cc, tng tc k nc,cha da vo c im ca chtphn tch.

Lng dung mi dng gim

nhiunhng hy cn ln.

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Phuong Phap Tach - Chiet