5.4. Phng php sc k lp mng

Phng php sc k lp mng c dng nh tnh, th tinh khit v i khi bn nh lng hoc nh lng hot cht thuc.

Sc k lp mng l mt k thut tch cc cht c tin hnh khi cho pha ng di chuyn qua pha tnh trn chm hn hp cc cht cn tch. Pha tnh l cht hp ph c chn ph hp theo tng yu cu phn tch, c tri thnh lp mng ng nht v c c nh trn cc phin knh hoc phin kim loi. Pha ng l mt h dung mi n hoc a thnh phn c trn vi nhau theo t l quy nh trong tng chuyn lun. Trong qu trnh di chuyn qua lp hp ph, cc cu t trong hn hp mu th c di chuyn trn lp mng, theo hng pha ng, vi nhng tc khc nhau. Kt qu thu c l mt sc k trn lp mng. C ch ca s tch c th l c ch hp ph, phn b, trao i ion, sng lc phn t hay s phi hp ng thi ca nhiu c ch ty thuc vo tnh cht ca cht lm pha tnh v dung mi lm pha ng.

i lng c trng cho mc di chuyn ca cht phn tch l h s di chuyn Rf c tnh bng t l gia khong dch chuyn ca cht th v khong dch chuyn ca dung mi:

 

 

Trong : a l khong cch t im xut pht n tm ca vt mu th, tnh bng cm.

b l khong cch t im xut pht n mc dung mi o trn cng ng i ca vt, tnh bng cm.

Rf: Ch c gi tr t 0 n l.

Ngoi ra, khi sc k lin tc khng xc nh c tuyn dung mi, v tr vt cht th trn sc c th xc nh bng h s dch chuyn tng i Rr. H s dch chuyn tng i Rr c xc nh bng t s gia khong cch dch chuyn ca vt cht th v khong cch dch chuyn ca vt cht chun i chiu c sc k trong cng iu kin v trn cng bn mng vi mu th:

 

Trong : a l khong cch t im xut pht n tm ca vt mu th, tnh bng cm.

c l khong cch t im xut pht n tm ca vt cht chun, tnh bng cm.

Gi tr Rr c th ln hay nh hn l.

Cch tin hnh

Dng c

Bnh trin khai, thng bng thu tinh trong sut c kch thc ph hp vi cc phin knh cn dng v c np y kn.

n t ngoi, pht cc bc x c bc sng ngn 254 nm v bc sng di 365 nm.

Dng c phun thuc th.

T sy iu nhit hot ha v sy bn mng v sc k , hoc sy nng i vi mt s phn ng pht hin.

T ht hi c.

My sy dng sy kh sc k v cho php chm nhanh nhiu ln nhng dung dch pha long cht cn phn tch.

Mt my nh tt c th chp lu gi sc k nh sng ban ngy vi khong cch 30 - 50 cm.

T lnh bo qun nhng thuc th d hng.

Micropipet nhiu c t l, 2, 5, 10 n 20 ml, cc ng mao qun hoc dng c thch hp.

Bn mng trng sn cht hp ph c cht pht quang thch hp.

Trng hp phng th nghim khng c iu kin trang b cc loi bn mng trng sn th t chun b ly bn mng vi cc dng c sau y:

Cc tm knh phng c kch thc ph hp c x l trc bng ha cht ri ra sch bng nc v sy kh.

Thit b tri cht hp ph ln tm knh thnh mt lp mng ng u, c chiu dy thch hp.

Gi xp cc tm knh tri.

Chun b bn mng

Sp xp cc bn mng v chun b thit b: Cc phin knh phi c lau chi cn thn v ty sch hon ton cc cht bo bng cch ngm trong dung dch sulfocromic. Sau , ra di tia nc my ri trng nc ct v lm kh trn gi nhit thng hay trong t sy. t ln khung tri 5 phin knh 20 x 20 cm (hay phin knh 10 x 20 cm) dy bng nhau. iu chnh dy ca lp hp ph bng cc thng s trn thit b tri.

iu ch va ca cht hp ph: Cht hp ph c chn lc sao cho ph hp vi yu cu phn tch nh: Silicagel G, kieselguhr, cel1u1ose, nhm oxyd, trong s silicagel G c dng thng dng nht. Trn 25g silicagel G vi 50 ml nc ct v nho trong ci hoc lc mnh trong bnh nn c dung tch 200 - 250 ml, nt kn, trong 30 - 45 giy. Dch treo to c dng lng v ng nht. Rt ngay vo thit b tri iu chnh dy cho bn mng khong 0,25 mm (nu khng c ch dn trong chuyn lun ring). Sau khi rt dch treo vo thit b tri, lt tay n 180o v pha tri v ch cho n khi dch treo chy ra th bt u tri. Khi tri n phin cui cng lt tay n v pha phi gi cho dch treo khng chy xung.

ngyn cc phin knh ti ch khong 10 pht ti khi mt trn ht bng, hoc kh t nhin ti nhit phng.

Hot ha: Cho cc bn mng kh mt vo t sy v sy l05 - l10oC trong 30 pht (nu khng c ch dn chuyn lun ring). ngui ri bo qun trong bnh ht m. Khi dng, nu cn th hot ha li bng cch sy l05 - l100C trong l gi ri co b mt di cht hp ph dc hai bn cnh ca tm knh.

Chun b bnh khai trin: Cc bnh khai trin thng l bnh thy tinh, hnh hp hay hnh tr, c np y kn, kch thc thay i ty theo yu cu ca cc bn mng s dng. Bo ha hi dung mi vo kh quyn trong bnh bng cch lt giy lc xung quanh thnh trong ca bnh, ri rt mt lng va dung mi vo bnh, lc ri giy lc thm u dung mi. Lng dung mi s dng sao cho sau khi thm u giy lc cn li mt lp dy khong 5 mm n 10 mm y bnh. y kn np bnh v yn l gi nhit 20 - 25oC. Mun thu c nhng kt qu lp li, ta ch nn dng nhng dung mi tht tinh khit, loi dng cho sc k. Nhng dung mi d bn i v ha hc ch nn pha trc khi dng. Nu s dng nhng h pha ng phc tp phi ch n nhng thnh phn d bay hi lm thay i thnh phn ca h pha ng dn n hin tng khng lp li ca tr s Rf.

Chm cht phn tch ln bn mng: Lng cht hoc hn hp cht a ln bn mng c ngha quan trng i vi hiu qu tch sc k, c bt nh hng rt ln n tr s Rf. Lng cht qu ln lm cho vt sc k ln v ko di, khi , vt ca cc cht c tr s Rf gn nhau s b chng lp. Lng cht nh qu c th khng pht hin c do nhy ca thuc th khng (thng thng nhy ca cc thuc th trn 0,005 mg). Lng mu thng thng cn a ln bn mng l 0,l - 50 mg dng dung dch trong dung mi thch hp. Th tch dung dch t 0,001 ml n 0,005 ml i vi trng hp a mu ln bn mng di dng im v t 0,l - 0,2 ml khi a mu ln bn mng di dng vch nh trong trng hp sc k iu ch. i vi cc dung dch c nng rt long th c th lm giu trc tip trn bn mng bng cch chm nhiu ln cng mt v tr v sy kh sau mi ln chm.

ng xut pht phi cch mp di ca bn mng l,5cm - 2 cm v cch b mt dung mi t 0,8 - l cm. Cc vt chm phi nh, c ng knh 2 - 6 mm v cch nhau 15 mm. Cc vt ba phi cch b bn ca bn mng t nht 1 cm trnh hiu ng b. Khi lm sc k lp mng bn nh lng, chnh xc ca kt qu phn tch ph thuc rt nhiu vo chnh xc ca lng cht th a ln bn mng, tc l th tch dung dch chm ln bn mng. Do , vi nhng trng hp phn tch bn nh lng phi dng cc mao qun nh mc chnh xc. Khi khng cn nh lng dng micropipet hoc ng mo qun thng.

Trin khai sc k: t bn mng vo bnh trin khai. Ch lng dung mi dng cc vt chm phi trn b mt ca lp dung mi. y kn bnh v trin khai nhit t 20 - 25o C v trnh nh mt tri. Khi dung mi trin khai trn bn mng c mt on theo quy nh trong chuyn lun, ly bn mng ra khi bnh, nh du mc dung mi, lm bay hi dung mi cn ng li trn bn mng ri hin vt theo ch dn trong chuyn lun ring. Quan st cc vt xut hin, tnh gi tr Rf hoc Rr v tin hnh nh tnh, pht hin tp cht hoc nh lng nh quy nh trong chuyn lun ring.

Ch :

Khi phn tch bng sc k lp mng lun phi chm cht i chiu bn cnh vt cht th vi lng tng t nh d on c trong vt cht th so snh kt qu v mu sc, kch thc v gi tr Rf hoc Rr trong cng iu kin.

Hiu nng ca bn mng c kim tra nh sau: Chm mt th tch thch hp (10 ml ) cho lp mng thng thng v 1 ml n 2 ml cho loi bn mng c kch thc ht mn) dung dch th hiu nng lp mng gm lc bromocresol, da cam methyl, methyl v sudan G (thuc th). Tin hnh sc k vi h dung mi methanol toluen ( 20 : 80). Kt qu trn sc k phi c 04 vt tch r rng c Rf nh sau: Lc bromocresol : < 0,15; da cam methyl: 0,1 - 0,25, methyl: 0,35 - 0, 55 v sudan G : 0, 75 - 0, 98.