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 Hi I2 H2SO4 H2SO4 H2SO4 FeCl3

Mu ca vt Vng hoc nu Vng m n da cam Mu hoc xanh dng Mu cam n Xanh lc n xanh en Mu sm (max: 545nm) Mu xanh dng en (max: 595nm) Mu tm

Hp cht Hp cht hu c ni chung Flavon, Flavonol Chalcon, auron Flavonoid Sesquiterpen Triterpen

H2SO4

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 <100 mg 0,5-1,0 g 1,0-10,0 g 10,0-20,0 g ng knh trong ca phu (cm) 0,5-1,0 2,0-3,0 5 10 Chiu cao lp hp thu 4 4 5 5 Phn on dung dch gii ly (ml) 10-15 20-30 30-50 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.