HPLC Primer

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Text of HPLC Primer

  • Bi ng Thanh -1- K thut sc k lng

    Chng 1 - NHNG KHI NIM M U V SC K LNG CAO P

    1.1. Phn loi k thut sc k

    Ngun gc ca sc k lng bt u vo u nhng nm 1900 c trong cc nghin cu ca nh thc vt hc ngi Nga, Mikhail S. Tswett. Nhng nghin cu ni ting ca ng tp trung vo tch cc cht sc t trong l cy, nhng sc t ny c chit t thc vt nh dung mi.

    ng y mt ct thu tinh vi ht nhi. ng tm thy hai loi vt liu c trng c ch l phn (canxi cacbonat) v alumina. ng rt mu ca mnh (dung mi chit mu thc vt qua ng th ho) vo trong ct. Khi mu c a xung dc theo ct nh trng lc c th thy xut hin nhng bng mu khc nhau c tch ra do c mt s di chuyn nhanh hn s cn li. ng m t mi lin quan ca nhng tch bit ny, nhng bng mu khc nhau vi cc cht khc nhau. ng to ra mt php tch sc k nhng hp cht ny da trn lc ht ho hc ca mt s hp cht vi ht (pha tnh). Mt s hp cht gn vo ht khi ang chy xung, trong khi nhng cht khc gn vo dung mi (pha ng) s di chuyn nhanh hn. Qu trnh ny c th c m t nh sau: Cc hp cht mu phn b hay phn chia khc nhau gia pha ng ang di chuyn v pha tnh to ra hin tng tch cc cht. Tswett t tn qu trnh l chromatography [t Hy Lp chroma l mu sc v graphy l vit ra, ngha l color writing m t thc nghim colorful]. Ngy nay, sc k lng trong nhiu dng khc nhau tr thnh mt trong nhng cng c mnh m nht trong ho hc phn tch.

    Cui nhng nm 1930 v u nhng nm 1940 Martin v Syng a ra mt dng sc k lng-lng bng cch dng silica gel lm vt liu pha tnh trong mi trng nc, v di dng lp ht nhi v n c s dng tch mt s dng acetyl amino acid. H cng b nghin cu ca mnh vo nm 1941 v trong c ngh thay pha ng cht lng bng mt loi kh ph hp lm tng tc dch chuyn gia hai pha v to ra hiu qu tch cao hn, t y khi nim sc k kh ra i. Trong mt cng b tng t vo nm 1941, Martin v Synge ngh s dng ht nh v p lc cao trong sc k lng ci thin kh nng tch, m ng cho s pht trin sc k lng hiu qu cao.

    Do , s t c H.E.T.P nh nht (hiu qu cao nht) nh s dng nhng ht rt nh v p lc cao khc nhau qua ct.

    Cng b ca Martin a ra vo nm 1941 c tt c cc iu kin thu c c hiu qu cao ln phn gii cao trn cc ct LC hin i. Tuy nhin, bt chp nhng ngh ca ng, mi gn n nhng nm 1950, cc khi nim ca ng mi c s m hoa kt tri. Hot ng ca lnh vc sc k lng b lu m trong nhng nm 1950 do s ra i ca sc k kh v do hng lot n lc ci thin ci thin k thut LC khng thc hin c cho n khi s pht trin ca sc k kh gn nh hon tt vo gia nhng nm 1960. Tr ngi chnh i vi s pht trin LC l thiu cc detector nhy cao, v n vn l tr ngi cho n khi A. Tiselius v D. Claesson pht trin detector ch s khc x vo nm 1942, gi m kh nng pht trin k thut LC hiu qu cao.

    Sc k lng c th c thc hin theo 3 cch c bn. Trong tt c cc trng hp th mu ho tan vo trong cht lng c chuyn ln trn hoc vo trong thit b, dng c sc k nh dung mi. Cch th nht. Mu c t ln trn v sau chy qua mt lp mng cc ht sc k c c nh trn b mt a thu tinh. Mu xut hin vi mu en, nhng hin ti thnh mu vng, v xanh. Cnh y ca a thu tinh c t trong mt dung mi. Dng chy to ra bi dung mi khuych tn qua lp ht kh (nh lc mao qun) di chuyn ln trn a thu tinh. Cch ny c gi l sc k lp mng

    Thin Layer Chromatography (TLC).

  • Bi ng Thanh -2- K thut sc k lng

    Cch th 2. Mu c im ln trn giy, dung mi c thm vo to ra dng chy. Cch ny c gi l sc k giy Paper Chromatography.

    Cch th ba: Theo cch hiu lc nht ny th mu c a qua ct hoc thit b c np ht ph hp. Nhng ht ny c gi l vt liu nhi sc k, pha tnh hay cn gi l cht hp ph. Dung mi chy lin tc qua ct. Ti mt im no trong mt thi im no , mt php tim dung dch mu vo dng dung mi c thc hin, dng dung mi ny mang mu chy qua ct. Trong thc nghim ca Tswett, cc hp cht trong trong mu c th c tch nh di chuyn nhng tc ring khc nhau qua thit b.

    Khi dng ct hoc cartridge c s dng, c vi cch to ra dng chy. Trng lc hoc chn

    khng c th c s dng i vi dng ct, dng khng c thit k lm vic vi p lc. in hnh th ht dng cho dng ct c kch thc ln hn (>50 micron) v v th d to ra dng chy hn i vi ct thu tinh (thc nghim ca Tswett). Thm vo cc ct nha di hnh dng tang xylanh c th c np y ht vt liu nhi v c dng thc hin cng vic tch mu. Kiu ny c gi l chit pha rn Solid Phase Extraction . y dng c sc k cn c gi l mt Cartridge c dng vi chn khng lm sch mu rt phc tp trc khi n c a i phn tch thm.

    Ch i vi loi giy c bit ny kh nng tch l khc nhau. Mu c mu sc ho trn, mt s c tch v mt s khc khng c tch. Theo m hnh c bn th mu mu en c cha mu vng, v xanh c p ln giy. Chng ta nhn thy mt vng trong mu xanh l cy v mt vng trn mu . Giy khng tch c mu vng v mu xanh nc bin. Trong v d di cng mu ch c mu vng v mu xanh nc bin. Ch giy khng tch hai mu ny. V d gia l mu cha mu v mu xanh nc bin v chng c tch tt. C nhiu chn la cho cc nh sc k

    la chn p ng kh nng tch mong i

    ci thin nng lc tch, nhng ht c kch thc nh hn (

  • Bi ng Thanh -3- K thut sc k lng

    - Tnh cht hp ph, - Tnh cht trao i ion, to cp ion, - S ry phn t theo kch thc ca chng, - S to phc v lin hp phn t - S phn b ca cc cht gia hai pha khng tan vo nhau,.

    K thut sc k c hai loi c tn gi da theo trng thi cht mu khi tin hnh sc k. l loi sc k kh v sc k lng. Trong k thut sc k lng li c chia thnh cc nhm sau:

    - Sc k lng p sut thng - Sc k lng p sut cao (HPLC) hay sc k ct p sut cao - Sc k lng siu cao p, hoc siu hiu qu (UPLC)

    V tn gi cho cc k thut sc k lng, ngi ta cng thng gi HPLC l sc k lng hiu qu cao v UPLC l sc k lng siu hiu qu.

    Nguyn bn th tn HPLC da trn mt thc t l cn p lc cao to ra dng chy cho sc k lng trong ct nhi. Thi k u cc cu kin thit b ch c kh nng to ra p lc 500 psi (35 bar). V v th n c gi l sc k lng cao p High Pressure Liquid Chromatography (HPLC). u nhng nm 1970 c bc nhy to ln v cng ngh. Cc thit b HPLC mi ny c th t n p lc 6000 psi (400 bar). Vi nhng t ph lin tc v hiu qu, tn gi c i thnh sc k lng hiu qu cao High Performance Liquid Chromatography (HPLC). By gi sc k lng hiu qu cao l mt trong nhng cng c mnh m nht trong phn tch ho hc, vi kh nng tch, nhn din v nh lng cc hp cht c mt trong bt k mu no c th c ho tan trong cht lng. Ngy nay k thut sc k lng c th d dng t n kh nng tch v pht hin cc cht dng vt thp n c phn nghn t parts per trillion (ppt). HPLC c dng trn bt k loi mu no nh mu dc phm, thc phm, nutraceutical, m phm, mi trng, y php v cc cht ho hc cng nghip.

    Bt u t nm 2004, nhng t ph hn na trong cng ngh thit b v ct tch gia tng ng k phn gii (Resolution), tc (Speed) v nhy (Sensitivity) trong sc k lng. Ct vi kch thc ht nh (1.7 micron) v trang thit b c thit k mang n 15000 psi (1000 bar) cng vi nhng kh nng c bit t c mt mc hiu qu mi.

    Cng ngh mi ny c gi l sc k lng siu hiu qu ULTRA PERFORMANCE LIQUID CHROMATOGRAPHY (cng ngh UPLC).

    Vi thnh cng ny bt ny ngh vo mt thi im no trong tng lai nhng nghin cu c bn ngy nay ang c cc nh khoa hc tin hnh s t n ct c kch thc ht ch l 1 micron v thit b c kh nng lm vic p lc 100000 psi (6800 bar).

    Trong sc k ct rn - lng, k thut sc k lng p sut thng ra i v c ng dng t lu (trn 40 nm). Nhng hiu sut tch khng cao, nhy thp v tn nhiu thi gian cng nh dung mi. Ngc li k thut HPLC tuy ra i mun hn nhng hin nay v ang pht trin rt nhanh chng, ng dng rt rng ri trong nhiu lnh vc. V k thut ny c nhy cao, hiu sut tch cao v tn t thi gian cng nh lng mu. Nhn chung trong thi gian va qua, k thut phn tch HPLC (v thi gian gn y c thm s xut hin ca k thut UPLC) chim n 70% trong tng s cc cng trnh nghin cu v ng dng v sc k. c bit vi s pht trin nh v bo ca k thut phn tch ph khi, cc k thut v phng php LC/MS ang c ng dng rt nhiu trong lnh vc khoa hc i sng (nghin cu tm kim thuc, nghin cu ch th sinh hc, gii trnh t v nhn din protein,.)

    K thut HPLC li bao gm 2 nhm: - Sc k lp mng p sut cao (HPTLC), - Sc k lng p sut cao (HPLC),

    Trong nhm HPLC, tu theo bn cht qu trnh sc k ca pha tnh trong ct tch ngi ta li chia thnh:

  • Bi ng Thanh -4- K thut sc k lng

    1. Sc k phn b (PC) ca cht tan gia 2 pha khng trn ln. 2. Sc k hp ph pha thng (NP-HPLC), 3. Sc k hp ph pha ngc (RP-HPLC), 4. Sc k trao i ion (IE-HPLC) v cp ion (IP-HPLC), 5. Sc k ry phn t (FG-HPLC).

    Trong 5 loi ny th loi th 5 ch dnh cho cc cht c phn t lng ln hn 2000 n v oxy. Mt cch tng qut chng ta c minh ho trong s hnh 1.1.

    Hnh 1.1: S phn loi ca HPLC

    S phn chia trn y l da theo tnh cht ca qu trnh sc k, tt nhin y cng ch l s phn chia tng i. V trong nhiu trng hp qu trnh sc k xy ra khng phi ch theo mt tnh cht (mt c ch), m ng thi theo nhiu c ch. V d trong sc k cp ion dng cht hp ph pha ngc Hypersil ODS tch cc t him, vi pha ng l methanol v nc th s tch li xy ra theo c c ch trao i ion v c ch hp ph pha ngc.

    Mt khc khi xem xt v trng thi ca pha tnh ngi ta li chia thnh:

    - Sc k lng - lng (LLC).

    - Sc k lng - rn (LSC).

    y trong h LLC th pha tnh l cht lng, n c gi trong ct tch nh mt cht mang tr. Cht mang tr ny thng l cc silicagel tr, v d nh Kiselgure ca hng Merck. Cn trong h

    LSC th pha tnh l mt cht rn, xp v c c ht ph bin t 3 n 10 m (ng knh). Ngy nay vi s pht trin mnh m ca nghnh khoa hc ch to vt liu pha tnh tch sc k ni chung v

    sc k lng ni ring, c ht c th h xung 1,7m vi hiu qu tch sc k lng cao hn nhiu

    ln so vi kch thc ht ph bin t 3,5m tr ln (c v nhy, tc v phn gii).

    Cng vi pha tnh, trong k thut HPLC, cc cht lng hay hn hp cc cht lng dng ra gii cc cht phn tch trong qu trnh sc k c gi l pha ng (MP). Pha ng c th ch l mt dung mi hu c nh methanol, acetonitril, butanol hay cng c th l hn hp ca 2 dung mi nh methanol v nc. Hoc cng c th l dung mi c thm cc cht m, cht dn in, cht to phc,vv

    Song song vi k thut HPLC, k thut sc k kh cng c pht trin v ng dng trong nhiu lnh vc. c bit l k thut sc k kh mao qun v sc k kh phn gii cao ghp ni khi ph, ph hng ngoi bin i fourier. Cc k thut ny c gii thiu trong cc gio trnh v SC K KH ca cc gio s Nguyn Xun Dng v Phm Hng Vit (trng i hc Khoa hc T nhin - i hc Quc gia H ni - Vit Nam).

    1.2. Nguyn tc cu to ca h thng my HPLC

    thc hin vic tch bng k thut HPLC, chng ta cn c cc h thng trang b v k thut ny. Mt h thng trang b HPLC c bn v n gin lm vic c theo k thut HPLC cn c

    K thut HPLC K thut UPLC

    HPT