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)

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

Nguyen c Tuan

ai hoc Y Dc TPHCM

Sac Ky Lp Mong (SKLM)

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

Nguyen c Tuan

ai hoc Y Dc TPHCM

Gii thieu nh ngha

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.
Nguyen c Tuan ai hoc Y Dc TPHCM

Pha tnh
Silicagel (Kieselgel gel cua acid silicic) S dung rong rai nhat Kch thc hat 10 40 m, dien tch be mat 200 400 m2/g Hoat tnh hap phu do OH tren be mat quyet nh Ham am tang se lam giam hoat o Hoat hoa 120oC/1 gi trc khi s dung

HO Si

OH HO Si O

OH HO Si O

OH

Nguyen c Tuan

ai hoc Y Dc TPHCM

Pha tnh
Nhom oxyd Oxyd nhom trung tnh, acid, base Hoat tnh tuy thuoc lng nc Hoat hoa 120oC/1 gi trc khi s dung Theo Brockman: 5 bac oxyd nhom

Bac hoat tnh % nc

I 0

II 3

III 6

IV 10

V 15

Nguyen c Tuan

ai hoc Y Dc TPHCM

Pha tnh
Xac nh bac hoat tnh cua nhom oxyd: da vao Rf cua mau chuan
Bac hoat tnh cua oxyd nhom theo Brockman Chat mau Aminoazobenzen Sudan o Sudan vang II 0,59 0,00 0,01 III 0,74 0,10 0,25 IV Gia tr Rf 0,85 0,33 0,57 0,95 0,56 0,78 V

Nguyen c Tuan

ai hoc Y Dc TPHCM

Pha tnh
Kieselguhr Bot nhe, vang xam cha 70 95% SiO2 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
Nguyen c Tuan ai hoc Y Dc TPHCM

Pha tnh
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 GF254, silicagel HF254, 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
Nguyen c Tuan ai hoc Y Dc TPHCM

Trang thiet b Pha tnh

Tnh chat Pha tnh Silicagel Dan chat siloxan Oxyd nhom Cat bien Kieselguhr Magie diphosphat Magie trisilicat Calci hydroxid Calci phosphat Calci sulfat Silicagel + oxyd nhom (1:1) Sat hydroxid Than Cellulose Polyamid Polyacrylonitril Nha trao oi ion Gel sephadex Thc te trung tnh Trung tnh Trung tnh Acid + base Acid + base Base Base yeu Trung tnh Khong co hoat tnh Hoat tnh yeu Hoat tnh yeu Hoat tnh yeu Hoat tnh yeu Hoat tnh yeu Co hoat tnh Co hoat tnh Co hoat tnh Khong co hoat tnh Khong co hoat tnh Khong co hoat tnh Khong co hoat tnh Base Co hoat tnh o acid Acid Hoat tnh Co hoat tnh Tac dung phan chia Hap phu Phan bo Hap phu Phan bo Phan bo Hap phu Hap phu Hap phu Hap phu Hap phu Hap phu Hap phu Hap phu Phan bo (Trao oi ?) (Trao oi, phan bo ?) Trao oi Ray phan t Cac chat c dung e phan tach Hau het tat ca Cac chat t phan cc Hydrocarbon, alcaloid, chat mau thc pham, lipid ng, acid beo ng, cac triglycerid, cac acid beo bac cao Carotinoic, tocopherol Carotinoic, tocopherol Carotinoic, tocopherol Carotinoic, tocopherol Carotinoic, tocopherol, cac acid beo glycerid k nc Cac chat mau, cac acid barbituric Cac chat phan cc Cac chat khong phan cc Acid amin, carbohydrat, nucleotid Cac flavon Cac antoxyan Acid nucleic, nucleotid, ion kim loai, halogenic Polymer, protein, phc kim loai

Nguyen c Tuan

ai hoc Y Dc TPHCM

Pha ong
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 Rf ieu chnh sc ra giai cua pha ong e Rf: 0,2 0,8 at o phan giai cc tr
Nguyen c Tuan ai hoc Y Dc TPHCM

Pha ong
Mot so nhom chc co kha nang hap phu tang dan:
CH=CH < OCH3 < COOR < C=O < CHO < SH < NH2 < OH < COOH

Dung moi vi sc ra giai (sc dung moi) tang dan:

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

Nguyen c Tuan

ai hoc Y Dc TPHCM

Trang thiet b Ban mong

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
Nguyen c Tuan

10 x 20

20 x 20

(cm)
ai hoc Y Dc TPHCM

Trang thiet b Bnh sac ky

Bnh ay tron, vuong hoac ch nhat

Cac loai bnh sac ky

Nguyen c Tuan ai hoc Y Dc TPHCM

Cac bc tien hanh khi thc hien SKLM

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
Nguyen c Tuan Mau F X A

Trien khai sac ky

ai hoc Y Dc TPHCM

Thuoc th hien mau

Thuoc th Thuoc th chung Acid phosphomolypdic Acid sulfuric ac Hi iod Thuoc th chon loc Ninhydrin 2,4-dinitrophenylhydrazon Xanh hoac luc bromocresol 2,7 fluorescein Vanilin/acid sulfuric Rhodamin B Anisaldehyd/SbCl3 Diphenylamin/Zn2+
Nguyen c Tuan

Mau cua vet Xanh toi Nau en Nau Hong tm Da cam, o Vang Vang luc Xanh luc hong Huynh quang o Nhieu mau Nhieu mau

ng dung phan tch Nhieu chat hu c Tat ca cac chat hu c Nhieu chat hu c Acid amin, cac nhom chc amin Cac hp chat carbonyl Cac acid hu c Nhieu chat hu c Alcol, ceton Cac chat lipid Cac chat steroid Cac chat bao ve thc vat
ai hoc Y Dc TPHCM

Thong so ac trng cua SKLM: He so lu gi Rf (Retardation factor)

dR V Rf = = d M V0
nam tai iem xuat phat dung moi

===>

0 Rf 1

ng dung moi

- Rf = 0: chat tan hoan toan khong di chuyen, - Rf = 1: chat tan di chuyen cung vi van toc cua - dR: khoang di chuyen c cua chat can tach dM

- 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

dR x x 1 2

Nguyen c Tuan

ai hoc Y Dc TPHCM

Thong so ac trng cua SKLM: He so lu gi Rf (Retardation factor)

- Cac yeu to anh hng en Rf

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

Nguyen c Tuan

ai hoc Y Dc TPHCM

Thong so ac trng cua SKLM: He so lu gi Rf (Retardation factor)

- S dung chat oi chieu

OX R f ( X ) OF OX Rs ( X ) = = = OA OA R f ( A) OF
* Hai chat giong nhau lan lt phai co Rf va Rs bang nhau. * Hai chat phai co cung lng cham nh nhau va c trien khai trong cung ieu kien sac ky

F X A

Sac ky o sac ky oi chng

Nguyen c Tuan

ai hoc Y Dc TPHCM

Thong so ac trng cua SKLM

H s dung lng

dM dR 1 Rf = k'= dR Rf

S a l thuyt

dR N = 16 W
Rs =

ng dung moi
dB dA

phn gii

2[ R f ( B ) R f ( A) ] W A + WB

dM

WB

dR

WA

R f ( A) RS = (1 R f ( A) )1 R f (B)
Nguyen c Tuan

1/ 2 N

x x 1 2

ai hoc Y Dc TPHCM

Cac ky thuat khai trien SKLM

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

Nguyen c Tuan ai hoc Y Dc TPHCM

Cac ky thuat khai trien SKLM

ng dung moi ng cham mau

Dung moi B

Dung moi A 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. Nguyen c Tuan Sac ky o cac dan chat xanthin tren ban mong pha ao C-18. Pha ong: metanol/K2HPO4 0,1 M (55:45 tt/tt). Phat hien: hi iod. Thi gian khai trien: 1 gi. Cac gia tr Rf: theobromin 0,68; theophylin 0,56; cafein: 0,44; 3-isobutylmetyl xanthin 0,21. ai hoc Y Dc TPHCM

Cac ky thuat khai trien SKLM

Sac ky che hoa
Tach cac chat vi lng nho trong trng hp khong the tach bang cac phng phap khac Dung moi Dung moi

Ky thuat sac ky che hoa (anh dau va cao rieng tng vung)

CHP cha A

CHP cha B

phan hap phu

Nguyen c Tuan

ai hoc Y Dc TPHCM

ng dung cua SKLM

nh tnh va th tinh khiet
Sac ky so sanh chat th X vi chat oi chieu A tren cung mot sac o ieu kien X la A: Rf(X) = Rf(A) vi cac he dung moi khac nhau Chat th c coi la tinh khiet khi tren sac ky o khong co vet la Da vao Rf

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
Nguyen c Tuan ai hoc Y Dc TPHCM

SKLM hieu nang cao (High Performance Thin Layer Chromatography, HPTLC) 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

Nguyen c Tuan

ai hoc Y Dc TPHCM

anh gia SKLM: u iem

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
Nguyen c Tuan ai hoc Y Dc TPHCM

anh gia SKLM: Nhc iem

Tr so Rf 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

Nguyen c Tuan

ai hoc Y Dc TPHCM