2.4. Sc k gel [6, 9] Sc k gel c Mould D.L.

. pht trin t nm 1954 tch cc hp cht khng mang in tch theo th t trng lng phn t ca chng. 2.4.1. Nguyn tc c bn ca sc k lc gel Trong sc k gel, pha tnh l mng polymer c l rng v cc l rng ny c ph y dung mi dng lm pha ng. Cc l rng phi c kch thc ng nht v kch thc ca l ng vai tr quan trng trong qu trnh sc k. Mt hn hp gm nhiu hp cht c trng lng phn t khc nhau c th tch ring c hay khng l ty thuc vo kch thc l rng. Cc phn t c kch thc to hn l rng, khng th chui lt vo bn trong l rng, nhanh chng theo dng chy ca pha ng i ra khi ct. Cc phn t c trng lng phn t nh hn kch thc l rng, s ton phn hoc bn phn lt vo l rng, cui cng theo dng chy i ra khi ct.

Hnh 20: Nguyn tc ca sc k gel [6] Nh vy, cc thnh phn khc nhau ca hn hp mu khi i ngang qua ct sc k gel, s ra khi ct ln lt theo trng lng phn t, phn t ln i ra khi ct trc, phn t nh i ra khi ct sau. Lc hp thu trn b mt ht gel khng c k n, v th sc k gel c xem l sc k phn chia. 2.4.2. Cc loi ht gel ca sc k gel

Vic tch trong sc k gel ty vo c im ca l rng trong h khng gian ba chiu. Ht gel phi c kch c ging nhau, c tnh tr v mt ha hc, bn v mt c hc, cc l rng trong ht gel phi c hnh dng ng nht. Mun tch mt hn hp cha nhiu loi hp cht c trng lng phn t khc nhau th phi s dng nhiu loi gel khc nhau, mi loi s tch ring mt nhm hp cht c khong trng lng phn t xc nh. 2.4.2.1. Gel dextran ( tn thng mi l gel Sephadex) Gel dextran l polysaccharid thin nhin, mch thng, ngha l chui dy polyglucose ni vi nhau bi ni - 1,6. Nguyn liu ny khng ha tan trong nc. Mt chui dy polysaccharid ca dextran c to mng ngang, bi ni glyceryl, gia cc nhm - OH khi cho phn ng vi hp cht epicloro hydrin trong dung mi hu c iu kin kim. Sn phm to thnh l cht rn, dng hnh cu, khng tan trong nc, nhng c tnh i nc cao. Trong qu trnh thc hin phn ng, thay i t l gia dextran v epiclorohydrin, s lm thay i to mng ngang, t c th iu ch cc loi gel vi l rng kch c khc nhau. Gel Sephadex khng tan trong nc, bn trong cc dung dch nc mui, dung dch kim, c trong dung mi hu c. Ch khi tip xc lu vi acid v c mnh mi b phn hy. Gel n nh an ton trong khong pH = 2 - 12. Gel Sephadex thng mi thng c ghi km ch s ch lng nc m gel kh cn ht ly thnh gel trng n. Bng 9: Thi gian cn thit lm trng n gel Sephadex Loi gel Thi gian cn gel trng n ( gi) nhit phng G - 10, G - 15, G - 25, G - 50 G - 75 G - 100, G - 150, G - 200 3 24 72 trn bp cch thy 1000C 1 3 5

Bng 10: Mt s loi gel dextran Sephadex G v tnh cht ca gel

Loi gel Kch thc Lng nc Th tch gel Kh nng tch cc hp cht trong theo trng lng phn t Peptit, protein Cc hnh cu 0 - 700 0 - 1.500 0 - 1.500 phn nc ( ml/g) G - 10 G - 15 G - 25, th 40 - 120 40 - 120 100 - 300 1,0 0,1 1,5 0,1 2,5 0,2 23 2,5 - 3,5 46 Sephadex G ht gel kh gel c th ht n () ( ml/g)

on dextran 0 - 700 0 - 1.5700 100 - 1.500

G - 25, trung 50 - 150 bnh G - 25, mn LH - 20 G - 50, th 20 - 80 25 - 100 100 - 300 2,1 5,0 0,3 4,0 - 4,5 9 11 1- 30.000 500 - 10.000

G - 50, trung 50 - 150 bnh G - 50, mn G - 75 G - 100 G - 150 G - 200 20 80 40 - 120 40 - 120 40 - 120 40 - 120 7,5 0,5 10 1 15 1,5 20 2 12 15 15 20 20 30 30 40 3 70.000 4 - 150.000 5 - 400.000 5 - 800.000 1 - 50.000 1 - 100.000 1 - 150.000 1 - 200.000

2.4.2.2. Gel polyacrylamid ( gel Bio - gel loi P) L loi polymer tng hp di dng ht hnh cu, ch to t acrylamid ( CH 2 = CH - CONH2) vi N, N - metylenbisacrylamid. Thay i t l ca hai cu phn monomer nn trn s lm thay i kch thc ca l rng. Ht gel c kch thc trung bnh 45 - 180 m. Bio - gel tng i tr, nhng km bn hn Sephadex. Gel n nh bn mi trng pH = 2 - 11. Trong mi trng kim mnh, cc ni amid b thy gii thnh acid carboxylic, iu ny khin cho gel c tnh cht gn ging nha trao i ion, v th khng nn s dng Bio - gel cho trng hp sc k thc hin thi gian di, trong iu kin pH < 9.

Gel polyacrylamid c tnh cht i nc mnh nh cc nhm - NH2. Mun gel trng n tt, cn ht gel tip xc vi nc trong thi gian nht nh ty thuc vo loi gel. 2.4.2.3. Gel stryragel Gel ny l polyme c tng hp t styren v divinylbenzen. Ht gel c c tnh khng trng n. Ht gel hnh cu, kch thc 40 - 80 m. Vi loi gel ny, nn gii ly ct vi cc dung mi tetrahydrofuran, o - diclorobenzen, toluen, ...v khng nn s dng dung mi phn cc nh nc, metanol, aceton hoc acid formic. 2.4.3. Cc nguyn tc c bn v thc hnh sc k gel 2.4.3.1. Nhi gel vo ct Ct s dng cho sc k gel cng ging nh ct sc k c in, l ct bng thy tinh. Cc ht gel c lm trng n cho vo ct theo k thut nhi ct st. Sau khi rt huyn ph gel vo ct, cn bng ct bng cch cho dung mi gii ly ( loi khi u cho vic gii ly) chy ngang qua ct, lng dung mi c th tch bng 2 - 3 ln th tch ct. Kim tra s cht ch ca ct bng cch cho mt lng mu th vo u ct. Mu th thng c chn l dextran blue 2000, c mu xanh dng, c th quan st bng mt thng s di chuyn ca mu trong ct. Th tch dung dch mu < 10% th tch ct. Cho dung mi gii ly ui hon ton lng mu th ny ra khi ct. Trong sut qu trnh np gel cng nh trin khai sc k, khng c kh lp mt gel trn u ct. 2.4.3.2. t mu cn sc k ln u ct gel: Dung dch mu cn tch phi c lc b nhng hp cht cn dng rn, cn. Dung dch mu c ha tan vo dung dch m, t ln u ct ging nh trong sc k ct c in. 2.4.3.3. Trin khai sc k gel: Ch s dng k thut dung mi n nng . Gii ly c th bng trng lc nhng tt nht l s dng bm y t u ct. Hng dung mi gii ly trong nhng l nh, mi l c mt th tch nht nh. Cc cht tan s ra khi ct theo th t trng lng phn t gim dn. 2.4.3.4. V d c th v sc k gel Sephadex LH - 20

Gel Sephadex LH - 20 l gel Sephadex G - 25 c hydroxylpropyl ha ngha l bin i mt s nhm - OH trong gel dextran thnh nhm - O - CH2 - CH2 CH2 - OH. Vic iu ch dn xut ny gip cho gel c thm tnh thn du bn cnh tnh i nc sn c. Bn cnh vic gel trng n trong nc, gel c th trng n trong dung mi hu c c tnh phn cc nh dimetylsulfoxid, pyridin, metanol, tetrahydrofuran,... Nh kh nng gel c th trng n trong dung mi hu c phn cc, gel Sephadex LH - 20 c dng trong vic tch cc hp cht thin nhin, loi tan trong dung mi hu c. Gel Sephadex LH - 20 tch hp cht theo hai k thut sc k khc nhau ty vo dung mi gii ly. Khi gii ly ct vi n dung mi, gel Sephadex LH - 20 tch hp cht bng k thut sc k gel nh vo s khc bit v trng lng phn t. Cc hp cht c trng lng phn t khong 4000 dalton thng khng b gi li trong gel Sephadex LH 20 nn c gii ly ra khi ct ngay trong th tch trng ca ct. Kh nng gel tch cc hp cht theo trng lng phn t khong 100 - 4000. Khi gii ly ct gel vi hn hp dung mi, gel Sephadex LH - 20 tch hp cht bng k thut phn chia, cc hp cht c tch ring ra ty vo i lc ca hp cht i vi mt trong hai pha lng trong ct. Khi s dng hn hp hai dung mi (tt nht l mt dung mi phn cc v mt dung mi c tnh km phn cc), ge l Sephadex LH - 20 c i lc vi dung mi c tnh phn cc mnh hn, a n h qu to thnh hai pha, pha ng lng v pha lng. Kch c ct: Thng chn ct bng thy tinh, c chiu di khong 40 - 100 cm, ng knh ct 1,5 - 2,5 cm. Kch thc ct c chn ty theo th tch gel s dng. Lng mu phn tch v lng gel: Trong thc nghim, sc k t kt qu tt khi s dng 1 mg mu cht cn 1 ml gel trng n. Gel Sphadex LH - 20 c th trng n trong nhiu loi dung mi hu c. Nh kh nng ny, gel Sephadex LH - 20 thng c dng trong vic tch cc hp cht thin nhin, loi tan c trong dung mi hu c.

Bng 11: Lng dung mi hu c gel Sephadex LH - 20 c th hp thu Dung mi Lng dung mi hp thu ( ml/g gel kh) Dimetylformamid Nc Metanol Etanol Clorofom ( 1% metanol) Clorofom n- Butanol Tetrahydrofuran Aceton Etyl acetat Toluen 2,2 2,1 1,9 1,8 1,8 1,6 1,6 1,4 0,8 0,4 0,2 Th tch ht gel ( ml/g) 4,0 - 4,5 4,0 - 4,5 4,0 - 4,5 3,5 - 4,5 3,5 - 4,5 3,0 - 3,5 3,0 - 3,5 3,0 - 3,5 -

Np gel vo ct: Ct c bt thng ng trn gi. M van kha bn di, m mt phn, rt dung dch huyn ph gel vo ct. ng thi bn di c dung mi chy ra. Rt dung dch gel vo ct thnh dng nh u, rt mt ln y ct. Trnh rt gel thnh nhiu t, s lm lp gel nn khng u trong ct. Cn bng gel trong ct: Sau khi gel c np hon tt vo ct, tip tc cho dung mi chy ngang qua ct vi vn tc 1 - 5 ml/ pht trong vi gi ct c nn u. Lu l phi lun c dung mi trn u ct, khng c gel trn mt ct kh. Lng dung mi cn ha tan mu: Mu c ha tan trong dung mi ph hp. Nu mc ch l loi b mui, lng dung dch mu np trn u ct c th chim khong 30% th tch ct gel. Nu mc ch sc k gel l tch ring cc hp cht, lng dung mi dng ha tan mu phi vo khong 1 - 5% th tch ct gel. Np mu cn sc k ln u ct: - M kha cho dung mi chy ra khi ct, h mc dung mi trong ct xung st mt thong ca cht hp thu trong ct.

- ng kha li, np dung dch mu vo u ct. Dng pipet ht dch mu, cho dung dch mu chy dc thnh trong ca ct, chm xung b mt ca cht hp thu. - M kha bn di cho dung mi chy ra khi ct, lm cho dung dch mu thm ht vo cht hp thu trn u ct. - Cho mt lp bng, giy lc, ...ph ln b mt ca cht hp thu bo v mt ct. - Cho dung mi vo y ct bt u qu trnh gii ly. Gii ly ct: s dng k thut gii ly vi dung mi n nng . Gii ly nh vo trng lc vi vn tc dng chy 2 - 4 ml/ pht ( cho ct gel 200 ml). C th s dng bm nh. Thu dung dch gii ly trong cc l nh 2.4.4. Cc ng dng ca sc k gel xc nh trng lng phn t ca mt hp cht i phn t tch mt hn hp nhiu cht thnh cc nhm hp cht ring r loi b mui ra khi mt hp cht hoc dung dch loi b cc cht mu tp bn ra khi hp cht ang kho st. Amino acid homopolymes occurring in natural