PHNG PHP SC K LP MNG Nguyn tc: Phng php sc k lp mng c dng nh tnh, th tinh khit v i khi bn nh lng hoc

oc 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. HPLC Nguyn tc chung v phng php HPLC Sc k lng l qu trnh tch xy ra trn ct tch vi pha tnh l cht rn v pha ng l cht lng (sc k lng - rn). Mu phn tch c chuyn ln ct tchdi dng dung dch. Khi tin hnh chy sc k, cc cht phn tch c phn blin tc gia pha ng v pha tnh. Trong hn hp cc cht phn tch, do cu trc phn t v tnh cht l ho ca cc cht khc nhau, nn kh nng tng tc cachng vi pha tnh v pha ng khc nhau. Do vy, chng di chuyn vi tc khc nhau v tch ra khi nhau Sc k lng hiu nng cao bao gm nhiu phng php c tnh c th ring, l sc k lng pha lin kt, sc k phn b lng lng v sc k trao i lng rn, sc k hp ph lng rn KHI PH L phng php nghin cu cc cht bng cch o, phn tch chnh xc khi lng phn t ca cht da trn s chuyn ng ca cc ht mang in hay ion trong mt in trng hoc t trng nht nh K thut ny c nhiu ng dng v c kt hp vi nhiu phng php: Khi ph kt hp vi sc k kh.

 Khi ph kt hp vi sc k lng. Khi ph kt hp vi in di.

SC K TRAO I ION
Nguyn tc: Trc tin, protein s gn thun nghch vi cc cht trao i bng tng tc ion gia cc nhm mang in tch tri du. Sau , protein gn c chit rt ring bit, thng l nh vo vic tng dn lc ion (khin cho tng tc ion b b gy hon ton). Hay dng nht l gradient NACl ca m. C th thay i bng cch thay i PH khin cc nhm tng tc trn prtein b mt in tch. Trong phng php ny, cc phn t protein c phn lp da trn in tch ion ha b mt ca chng bng vic s dng cc vt liu lm ct l cc ht mang cc nhm chc tch in m hoc dng (y cn c gi l pha tnh). Cc phn t protein tng tc yu vi cc ht (chng hn nh mt phn t protein tch in dng c cho chy qua ct mang cc ht tch in m) s c hi lu khi s dng mt dung dch mui long chy qua ct sau (dung dch chy mu c gi l pha ng). Cc phn t tng tc vi pha ng cng mnh, cng cn dung dch hm lng mui cao hi lu mu (bi mui lm "trung ha" cc vng mang in tch v v vy cho php cc phn t protein c gii phng khi ct. Bng vic tng dn nng mui trong cc dung dch m thu hi mu, cc phn t protein khc nhau, k c cc phn t c c tnh tch in gn ging nhau cng c phn tch thnh cc phn on khc nhau khi chng c hi lu t ct.

SC K LC GEL
L k thut chun tch v xc nh cc i phn t c kch thc khc nhau, vd. cc protein. Dung dch gm hn hp cc i phn t c a vo nh ct v chy qua ct nh trng trng. Cc phn t nh hn b cn tr li khi dch chuyn xung pha di ct v chng c kh nng xm nhp qua cc l thu hp gia cc phn t gel tt hn. Cc phn t qu ln khng th xm nhp qua l c v do vy chng qua ct mt cch nhanh chng. Bng vic phn tch cht lng nh

xung y ct (dch chit hp) sau nhng khong thi gian v so snh vi chun (thu c bng cch cho chy qua ct cc i phn t bit r), ta c c thng tin v kch thc v khi lng phn t ca cc thnh phn ca hn hp. Loi gel thng phm thng dng nht l Sephadex. SC K KH Sc k kh l mt phng php phn tch c nguyn l d a trn hai qu trnh hp th (hoc hp ph ) v gii hp lin tc gia pha tnh l cht rn hoc lng v pha ng l cht kh. Do pha ng hon ton khng c tng tc vi cht cn phn tch nn thi gian lu gi ca cc cht trong ct phn tch ch ph thuc vo bn cht ca tng tc gia cht phn tch v i pha tnh. Da vo s khc nhau v i lc gia cc cht cn phn tch v i pha tnh v nhi t si m cc cht cn phn tch trong mu c tch ra khi nhau nh s chuyn dch lin tc ca pha ng dc lp pha tnh. Khi pha tnh l mt cht hp ph rn th k thut phn tch c gi l sc k khrn (GSC). Khi pha tnh l cht lng c gn ln b mt ca cht mang tr hoc c ph di dng mt lp phim mng ln thnh ct mao qun th k thut ny c gi l sc k kh-lng (GLC).

IN DI in di l hin tng dch chuyn ca cc vt th mang in tch di tc ng ca in trng. S dch chuyn ny do thnh phn lc in trong lc Lorentz. Nguyn tc in di: in di l k thut c s dng trong th nghim phn tch cc i phn t tch in. Trong phng th nghim sinh hc phn t ngi ta thng s dng phng php in di tch ly, pht hin phn t DNA nguyn vn, DNA b ct hn ch v DNA ca sn phm PCR. Da vo c tnh cu to ca cc axit nucleic, cc axit nucleic l cc i phn t tch in m ng u trn khp b mt nn khi chu tc ng ca in trng chng s di chuyn t cc m sang cc dng. Tnh linh ng ca phn t ph thuc vo hai ch tiu l khi lng phn t tc l s lng nucleotic hay cp nucleotic v nng cc cht cu thnh gel. Hai loi gel c s dng trong axit nucleic l gel polyacrylamide v gel agaroza. Vic chn loi gel cng nh nng cc cht to thnh gel ph thuc kch thc

trung bnh ca cc on axit nucleic. Trn cng mt bng gel, c cng mt dng in nhng phn t DNA khc nhau v trng lng nn khc nhau v in tch v chy c nhng qung ng khc nhau sau mt gian nh nhau. Sau khi phn tch bng in di, pht hin phn t DNA, ngi ta dng phng php lm hin hnh. i vi gel agarose, ngi ta nhum bng ethidi bromua (C21H20BrN3). Cht ny s gn xen cc baz ca phn t DNA v pht quang di tia t ngoi. Nh vy d dng cho php pht hin v tr cc on DNA trn gel v c th phn bit c phn t DNA trn cng mt bng gel. i vi gel polyacrylamid, cc phn t thng c nh du bng ng v phng x v v tr ca chng s c pht hin bng k thut phng x t ghi. Trong in di ngi ta s dng thang DNA chun, thng l DNA ngi, khi in di cho chy cng vi mu nghin cu, qua c th so snh vi DNA mu vi DNA chun bit trng lng phn t hoc trch ly c DNA khc nhau.