PHNG PHP SC K LP MNG Nguyn tc Cch tin hnh nh gi Nguyn tc: Phng php sc k lp mng c dng nh tnh, th tinh

h 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 t 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, ta thu c mt sc k trn lp mng. C ch ca s chia 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. 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 thch hp (vi ng knh Macro) 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 , c k bng bn chi di tia nc my ri trng nc ct v sy kh trn gi nhit thng hay trong t sy. 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, cellulose, 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, se li trong vi pht sau , v c bt b. 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). nguyn cc phin knh ti ch khong 10 pht ti khi mt trn ht bng, hoc kh t nhin qua m ti nhit phng. Hot ha: Cho cc bn mng kh mt vo t sy v sy 105 - 110oC 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 105-1100C trong 1 gi ri co b mt di mng 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 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 1 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 0,1 - 50 mg dng dung dch trong ether, c1oroform, nc hay dung mi thch hp khc. 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 sc k iu ch th lng cht c th ln ti 10 - 50 mg. 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 1,5cm - 2 cm v cch b mt dung mi t 0,8 - 1 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 gn nh thng ng vi bnh trin khai, cc vt chm phi trn b mt ca lp dung mi khai trin. y kn bnh v yn nhit khng i. 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. nh gi: 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. Vic sc k lp mng c tin hnh trong iu kin chun ho cho kt qu c tin cy cao hn. Hin nay ngi ta thng tin hnh sc k vi s gip ca h thng sc k lp mng hiu nng cao (Planar chromatography - HPTLC). Sc k lp mng (thin layer chromatography - TLC) l mt k thut sc k c dng tch cc cht trong hn hp[1]. Phng php sc k lp mng bao gm pha tnh l mt lp mng cc cht hp ph, thng l silica gel, aluminium oxide, hoc cellulose c ph trn mt mt phng cht tr. Pha ng bao gm dung dch cn phn tch c ha tan trong mt dung mi thch hp v c ht ln bn sc k bi mao dn, tch dung dch th nghim da trn tnh phn cc ca cc thnh phn trong dung dch. Sc k lp mng c s dng rng ri trong nhiu lnh vc:

xt nghim tinh khit ca cc ha cht phng x trong dc khoa xc nh cc sc t trong t bo thc vt pht hin thuc tr su, thuc dit cn trng trong thc n, hoc nhn bit nhng ha cht trong mt cht cho sn gim st cc phn ng hu c

Mt s ci tin c th kt hp phng php truyn thng t ng ha mt vi bc, lm tng dung gii ca sc k lp mng v cho s liu chnh xc hn. Phng php ny c gi l sc k lp mng hiu nng cao (high performance TLC - HPTLC). Chun b bn sc k Bn sc k c lm bng cch trn cht hp ph, nh silica gel, vi mt lng nh cht tr kt dnh, nh calcium sulfate (thch cao), v nc. Hn hp ny c tri ra nh mt lp va c trn mt b mt cht tr, nh thy tinh, nhm, hoc nha. Bn sc k ny s c kh v kch hot bng cch nung nng trong l trong 30 pht nhit 110C. dy ca lp hp ph thng l 0.1-0.25 mm cho ha hc phn tch, v khong 1-2mm cho sc k lp mng d b. Trong mi k thut sc k u bao gm 1 pha ng v 1 pha tnh. K thut Phng php tin hnh ging vi sc k giy vi li th l nhanh hn, tch hn hp hiu qu hn, v c s la chn gia cc "pha tnh" khc nhau. Bi tnh n gin v nhanh, sc k lp

mng thng c dng gim st cc phn ng ha hc v phn tch cht lng sn phm ca phn ng. Mt vt nh dung dch cha mu th c thm ln bn sc k, khong 1 cm t di ln. Bn sc k sau c nhng vo mt dung mi thch hp, nh ethanol hoc nc, v c t vo trong mt vt cha c np. Dung mi di chuyn ln bn sc k bi mao dn, gp phi mu th v dch chuyn mu th ln bn sc k. Cc hp cht khc nhau trong hn hp mu th dch chuyn vi tc khc nhau do chng c sc ht khc nhau i vi pha tnh, v tan khc nhau trong dung mi. Cc hp cht c tch ra da trn s cnh tranh ca cht tan v pha ng c c ch lin kt vi pha tnh. Th d, nu silica gel c dng nh pha tnh, n c xem l phn cc. Cho trc 2 hp cht c tnh phn cc khc nhau, cht no c tnh phn cc ln hn s c s lin kt vi silica gel ln hn v v th s c kh nng y pha ng ra khi cc ch lin kt. Do , hp cht c tnh phn cc nh hn s di chuyn ln cao hn trn bn sc k (kt qu l h s lu Rf s ln hn). Nu pha ng c thay bng mt dung mi phn cc hn hoc l mt hn hp cc dung mi, n s c kh nng y cc cht tan ra khi ch lin kt vi silica gel, v tt c cc hp cht trn bn sc k s dch chuyn ln cao hn. Trn thc t, nu chng ta dng mt hn hp ethyl acetate v heptane nh l pha ng, tng thm ethyl acetate s cho h s lu Rf cao hn cho tt c cc hp cht trn bn sc k. Thay i phn cc ca pha ng s khng lm cc hp cht c th t di chuyn ngc li trn bn sc k. Nu mun c mt th t ngc li trn bn sc k, mt pha tnh khng phn cc s c s dng, nh l C18-chc nng ha silica.

Qu trnh sc k lp mng: mt hn hp ca mt ho85p cht v mt hp cht lam c tch bit trong qu trnh sc k (dung mi mu xanh nht di chuyn ln trn bn sc k. Dung mi thch hp dng trong sc k lp mng s l mt dung mi c tnh phn cc khc vi pha tnh. Nu mt dung mi phn cc c dng ha tan mu th trn mt pha tnh phn cc, vt nh mu th s lan trn do mao dn, v cc vt khc nhau c th trn ln vo nhau. Do , hn ch s lan trn ca cc vt mu, dung mi c s dng ha tan mu th phi khng phn cc, hoc phn cc mt phn, nu pha tnh phn cc, v ngc li. Phn tch Do mt s ha cht khi c tch ra s tr nn khng mu, mt vi phng php c s dng quan st nhng vt ny:

Thng thng, mt lng nh cht hunh quang, thng l maganese-activated zinc silicate, c cho thm vo cht hp ph c th quan st c nhng vt ny di nh sng en (tia cc tm UV254). Lp hp ph v th s t pht ra nh sng lc, nhng cc vt mu s lm tt nh sng ny. Hi Iodine cng l mt loi thuc th cho mu ging nhau. Mt s thuc th cho mu ring bit c dng nhng bn sc k vo, hoc phun ln bn sc k.

Trong trng hp ca cht bo, sc ph c th s c chuyn qua mt mng polyvinylidene fluoride (PVDF) v sau s c phn tch su hn, chng hn nh khi ph.

Mt khi tr nn quan st c, h s lu Rf ca mi vt mu s c xc nh bng cch chia khong cch di chuyn c ca hp cht cho khong cch di chuyn c ca dung mi. Nhng s liu ny ph thuc vo cc loi dung mi c s dng v cc loi bn sc k, v khng phi l hng s. [sa] ng dng Trong ha hc hu c, phn ng c gim st cht lng bi sc k lp mng. Cc mu th c thm ln bn sc k bng mt ng mao dn: mt vt nh cht ban u, mt vt nh t hn hp phn ng, v mt vt nh gm c 2 cht. Mt bn sc k nh (3 cm x 7 cm) s mt khong khong hai, ba pht vn hnh. Qu trnh ny phn tch cht lng v s ch ra nu cht ban u bin mt, sn phm c to thnh, v bao nhiu sn phm c to thnh. ng tic rng sc k lp mng i vi cc phn ng xy ra nhit thp c th cho ra kt qu khng ng, bi v cc mu th s c lm m ln trong mao dn. Mt trong nhng phn ng nh vy l phn ng kh ester bi DIBALH thnh aldehyde. Mt v d l sc k c p dng cho mt phn l xanh ( y l rau chn vt) qua 7 bc. Carotene tch ra nhanh chng v ch quan st c cho n bc 2. Chlorophyll A v B hin r gia bn sc k trong bc cui cng v lutein l hp cht u tin nhum mu vng ln bn sc k.

Trong mt nghin cu, sc k lp mng c s dng lc phn ng ha hc hu c[2], nh tng hp BINAP t 2-naphtol. Trong phng php ny, cn v dung dch xc tc (chng hn nh iron(III) chloride c t ring r trn ng gc, sau phn ng vi nhau v c phn tch ngay lp tc.