I.Khi nim2II.Cu to21.Bnh cha dung mi v pha ng22.Pha tnh43.Bm54.B phn tim mu65.Ct sc k66.u d (detector) trong sc k lng hiu nng cao67.H thng thu nhn v x l s liu8III.Nguyn tc hot ng8IV.Phn loi101.Sc k phn b hiu nng cao102.Sc k hp ph hiu nng cao (sc k lng rn LSC)123.Sc k trao i ion hiu nng cao (IEC)134.Sc k cp ion (Ion pair chromatography- IPC)145.Sc k loi theo c (Size Exclusion Chromatography)17V.Mt s ai lng c ban trong phn tch sc k181.H s phn b182.Thi gian lu183.H s dung lng k194.Hiu nng195. chn lc206. phn gii20VI.ng dng20Ti liu tham kho22

I. Khi nimHPLC (High Performance Liquid Chromatography) l mt phng php chia tch trong pha ng l cht lng v pha tnh cha trong ct l cht rn c phn chia di dng tiu phn hoc mt cht lng ph ln mt cht mang rn, hay mt cht mang c bin bng lin kt ha hc vi cc nhm chc hu c. Phng php ny ngy cng c s dng rng ri v ph bin v nhiu l do: c nhy cao, kh nng nh lng tt, thch hp tch cc hp cht kh bay hi hoc d phn hy nhit.Bn cht chnh ca s tch trong loi sc k ny l da trn s hp ph b mt ca pha tnh. C ch tch l c ch hp ph. V th tt c cc tnh cht ng hc hp ph ca b mt pha tnh u c nh hng n kt qu ca s sc k mt hn hp cht mu.II. Cu toCc b phn c bn ca mt my HPLC c m t trong s nguyn tc chung:

Hnh 1. S my HPLC1. Bnh cha dung mi v pha ngBnh ng dung mi thng lm bng thy tinh th tch thay i t 100ml - 2l. Bnh lm bng thy tinh mu nu cha nhng cht nhy vi nh sng, bnh phi tht kn nu pha ng l nhng cht bay hi. V d nh aceton, methanol,Nu khng nng s b thay i.i khi phi c h thng kh kh v vi dung mi c th ha tan kh trong kh quyn nht l oxygen, ngi ta cng c th kh bng siu m hay bng cch sc kh Helium vo (He t tan trong dung mi nhng c th y cc kh khc ra khi dung mi).Pha ng l dung mi ra gii cht tan (cht phn tch) ra khi ct tch thc hin qu trnh sc k. Pha ng trong HPLC c th l dung mi hu c n nh methanol, acetonitrile, benzene, n-hexene, water, hay cng c th l hn hp ca hai hoc ba dung mi c trn vo nhau theo nhng t l ph hp. V d nh: hn hp methanol vi nc theo t l 70 methanol/ 30 nc, acetonitrile vi nc theo t l 80/20 N cng c th l dung mi nc c cha cc cht m pH, cht to phc, cht lm chm vi nng nht nh.Ni chung vi mi loi sc k s c cc h dung mi (pha ng) ra ring thch hp vi n, c th thu c kt qu tt nht. Sau pha tnh th pha ng l yu t th hai quyt nh hiu sut tch sc k ca mt hn hp mu. Ngha l trong mt h pha, th pha ng v pha tnh l hai yu t chnh ca qu trnh sc k. Hai yu t ny gi vai tr quyt nh thi gian lu gi ca cc cht mu v hiu qu s tch sc k.Ni chung, pha ng c th nh hng n nhng vn sau ca s tch sc k ca cc cht: chn lc ca h pha Thi gian lu gi ca cc cht tan Hiu lc ca ct tch phn gii ca cht trong pha tnh rng ca peak sc kDo , vi mt pha tnh chn ri, nu nh chng ta li chn c mt dung mi c thnh phn ph hp, th ta s c hiu tch sc k ca mt hn hp mu l tt nht i vi mt pha tnh chn.V vy, pha ng l mt yu t linh ng v phi tha mn mt s iu kin sau y: Pha ng phi tr i vi pha tnh, khng lm hng pha tnh theo bt k mt tnh cht no trong qu trnh sc k. Khng c s tng tc ph vi pha tnh sinh ra nhng sn phm khc lm phc tp cho qu trnh sc k. Pha ng phi ha tan c cht mu. C nh th mi lm cho cht mu c vn chuyn tt t u ct n cui ct tch khi thc hin qu trnh ra gii. Pha ng phi bn vng theo thi gian, ngha l khng b phn hy hay thay i khng theo mun khi chy sc k. Pha ng phi c tinh khit cao, m bo khng b nhim bn phn tch gy sai s cho kt qu phn tch. Ngha l cc cht pha ch pha ng phi c tinh khit cao. Phi nhanh t cn bng trong qu trnh sc k, nh cn bng hp ph, phn b, trao i ion, ty theo bn cht tng loi sc k. Phi ph hp vi loi detector dng pht hin cc cht phn tch. V d detector UV-Vis th pha ng phi trong sut vng ph , hay detector hunh quang th pha ng phi khng c tnh cht hunh quang. iu kin cui cng l pha ng phi c tnh kinh t, khng him, khng qu t. l yu cu cn thit i vi mt h pha ng trong k thut tch HPLC cho phn tch hn hp mu nht nh.2. Pha tnhPha tnh trong HPLC chnh l cht nhi ct lm nhim v tch sc k mt hn hp cht phn tch.N l nhng cht rn xp v c kch thc ht nh. Song d dng no th pha tnh trong HPLC cng phi tha mn nhng yu cu c bn sau y:Phi tr v bn vng vi cc iu kin ca mi trng sc k, khng c phn ng ha hc ph vi dung mi ra gii hay vi cht phn tch, m bo c ch ca s tch l ng theo bn cht chnh ca pha tnh, hay khng mt cht phn tch cn tch.C kh nng tch chn lc mt hn hp cht tan nht nh trong iu kin sc k nht nh.Tnh cht b mt phi n nh, c bit l c c trng xp ca n v khng b thay i hay bin dng trong qu trnh sc k, khng b phn r di p sut cao ca qu trnh sc k.Cn bng ng ca qu trnh sc k phi xy ra nhanh v lp li tt. C nh th mi m bo thu c kt qu tch tt.C ht phi tng i ng nht, ngha l ng knh ca cc ht ln nht so vi cc ht nh nht khng chnh lch nhau qu 10% v trn 85% s ht phi nm trong vng c kch thc trung bnh. C nh th ct tch mi c hiu qu cao. l yu cu chung ca pha tnh trong k thut HPLC.Trong sc k hp ph pha o, pha tnh thng l cc silica alkyl ha, do n t phn cc hay khng phn cc. Pha ng l h dung mi phn cc.3. Bm Mc ch bm pha ng vo ct thc hin qu trnh chia tch sc k. Bm phi t c p sut cao khong 250 - 600bar v to dng lin tc. Lu lng bm t 0,1 n 10ml/pht.Gm cc b phn nh sau: u bm c hai van iu tit, mi van c mt l nh vi mt bi. Cc van ny m bo cho vic ht pha ng t thn bm vo ct sc k c an ton. Thn bm c mt pittng chuyn ng i li. Khi pittng tin ti, mt bi ng l pha ct li v bi khc nhc ra pha ng i vo than, khi pittng li li mt bi ng bnh ng dung mi v m l phi ct pha dao ng vo ct sc k.4. B phn tim mu a mu vo ct phn tch theo phng php khng ngng dng chy . Vi dung tch ca l bp l 5 - 100l .C 2 cch ly mu vo trong ct : Tim mu th cng (tim bng tay) Tim mu t ng (Autosample) .5. Ct sc kCt dng trong sc k lng thng l mt ng lm bng thp khng r hoc lm bng thy tinh c bit (ch dng khi p sut khong 60psi).Ct l tri tim ca mt qu trnh sc k, ct l mt yu t quan trng quyt nh n kt qu ca qu trnh sc k ca mt hn hp mu. Ct tch c nhiu kch thc khc nhau thng c chiu di t 10-25 cm, ng knh trong 2-5 mm.6. u d (detector) trong sc k lng hiu nng caoL b phn pht hin cc cht khi chng ra khi ct v cho cc tn hiu ghi trn sc k c th nh tnh v nh lng. Ty theo tnh cht ca cc cht phn tch m ngi ta la chn lai u d ph hp.Tn hiu u d thu c c th l: hp th quang, cng pht x, cng in th, dn in, dn nhit, chit sut,

Khi cho cht tan i ngang qua t bo o, u d s to nn nhng tn hiu in. Mi s thay i v ln vt l ca cht tan s c chuyn i thnh xung in v c th c c trn my ghi.S gii p ca u d t l vi lng cht tan hay nng cht tan trong t bo, lp li v c lp vi cht ra gii (tr trng hp ra gii vi gradient i vi vi loi u d). u d tt l phi cho kt qu bn, nhanh, lp li. N cng phi c nhy tt vi cng nng v khng bin i cht lng tch.Da vo tnh cht ca mu cht cn phn tch m ta c cc loi detector sau: Detector UV-Vis Detector hunh quang Detector in ha: o dng, cc ph, dn. Detector khi ph Detector diode pht quang v diode mng Detector UV-Vis:y l loi ph bin nht trong HPLC. Nguyn tc ca n l cho pha ng t ct c chy qua mt ng o (flowcell) nh c mt chm bc x n sc UV/VIS chiu qua. S hp thu bc x ca cht tan l mt hm ph thuc tuyn tnh vo nng C ca n theo nh lut Lambert - Beer:D = lC y:D l i lng mt quang;l l chiu di ca cell; l h s hp thu phn t v l hng s cho mt cht tan v bc sng.n v ca ph thuc vo th nguyn ca l v C. Trong h SI th nguyn ca C l mol m-3, l l mt nhng trong thc t C c o vi mol dm-3 cn l l cm nn l dm3 mol-1 cm-1.nh lut Lambert Beer ch ng khi chm sng b hp thu l n sc, nhng h thng detector thng khng cung cp chm tia n sc m ch mt di hp cc chm tia quanh gi tr bc sng c chn. Mt ng o (flow cell) trong UV detector c ng knh trong khong 1 mm v chiu di nh sng i qua khong 10 mm, nh vy th tch cha khong di 8 l.7. H thng thu nhn v x l s liu l cc trm d liu hin i tip nhn v lu gi cc tn hiu pht ra t cc detector v in cc sc k hon chnh vi chiu cao, din tch cc nh, cc thng s nh tnh mu v cc bin s ca phng php. Chng cn c s dng lp chng trnh sc k, kim sot hu ht cc bin s v theo di thng xuyn trong thi gian vn hnh di khng c ngi trng coi.Vi tin b ca cng ngh thng tin, hin nay b phn thu nhn v x l s liu ca cc h thng HPLC thng l cc my vi tnh hin i c kh nng ghi nhn, lu gi, bin tp, x l thng tin ht sc hiu qu.Nh cc phn mm ci t tng thch vi cc chng trnh ca cc detector khc nhau, cc my vi tnh h tr rt hiu qu cho vic nh tnh, nh lng cc cht phn tch da vo cc c s d liu c ci sn.Nguyn tc hot ng ca h thng bm cao p bm lin tc qua ct vi tc dng c iu chnh. Khi mu c tim vo loop v theo dng dung mi i vo ct. Qu trnh tch sc k xy ra trong ct. Sau khi mu cht c tch ra khi ct th c u d ghi nhn tn hiu v chuyn thnh tn hiu in th hin tng peak ln sc k .III. Nguyn tc hot ngMu cht lng c ha tan trong mt dung mi thch hp, a vo bung bm mu sau c bm t ng vo ct tch. Qu trnh tch xy ra y.Do cc cu t cht phn tch c i lc khc nhau vi pha tnh, chng di chuyn dc trn ct vi tc khc nhau. Sau khi cc cu t tch ra khi nhau v thot ra khi ct s ln lt i vo detector, y tn hiu c ghi li, chuyn thnh tn hiu in. Tn hiu ny c khuych i ri chuyn sang b phn ghi. Cc tn hiu c x l v chuyn ra ngoi mt sc k .Trn sc k nhn c, s c cc tn hiu ng vi cc cu t c tch gi l peak. Qu trnh tch trong sc k: L s vn chuyn v phn b lin tc ca cht phn tch t u ct n cui ct. Qu trnh tch da vo tnh cht ha hc, vt l v ha l ca cc cht. Da trn 2 qu trnh l hp ph v gii hp ph. Xy ra lin tc gia 2 pha: pha tnh l cht lng hoc cht rn c nhim v gi cht phn tch, pha ng l cht lng (1 cht hoc hn hp nhiu cht) c nhim v ha tan v di chuyn cht phn tch. Cht phn tch lun phn b gia 2 pha, trong pha ng lun chy qua ct tch vi mt tc nht nh. Hiu qu ca qu trnh tch ph thuc rt nhiu vo tng tc gia cc cht trong pha tnh v pha ng.V d: Tch mt hn hp gm hai cht A v B:Mu cha A v B c tim vo ct. Khi cho mt cht ra gii bt u chy qua ct, phn ca mu c ha tan trong pha ng c di chuyn ti phn u ca ct. y cc cu t A v B t phn b gia hai pha. Tip tc cho pha ng i qua ct th n s y phn ha tan ny chy xung di v mt s phn b mi gia pha ng v pha tnh s xy ra. ng thi s phn b gia dung mi mi v pha tnh cng din ra ti v tr ca mu lc u. Vic thm tip dung mi s mang cc phn t ha tan chy xung ct trong mt lot lin tip cc chuyn bin gia hai pha. Bi v s di chuyn ca cht tan ch xy ra trong pha ng, nn tc trung bnh ca s di chuyn cht tan ph thuc vo phn thi gian cht tan y nm trong pha . Phn thi gian ny l nh i vi cht tan b lu gi mnh bi pha tnh (cu t B trong v d trn) v ln i vi cht tan (cu t A) c s lu gi trong pha ng mnh hn. Sau mt thi gian cc phn t cht A v B dn dn c tch khi nhau.

IV. Phn loiDa vo s khc nhau v c ch tch chit s dng trong HPLC, ngi ta chia HPLC thnh 4 loi: Sc k phn b hiu nng cao (Partition chromatography). Sc k hp ph hiu nng cao (Sc k lng - rn LSC). Sc k trao i ion hiu nng cao (IEC). Sc k ry phn t (size exclusion/gel permeation chromatography). Sc k loi theo c (Size Exclusion Chromatography)1. Sc k phn b hiu nng caoGm hai loi phng php sc k lng lng v sc k pha lin kt.a) Sc k lng lng (LLC)Pha tnh l cht lng c ph trn b mt ca cc ht cht mang (support). Pha tnh thng b dung mi ha tan v mt dn lm ct dn mt hiu lc nn phng php ny ngy cng t c s dng.b) Sc k pha lin kt (Bonded phase Chromatography) Pha tnh c gn ha hc (lin kt) vi cht mang (ht silicagen) to nn hp cht c siloxan.Nu R l mt nhm t phn cc nh octyl (C8), octadecyl (C18) hay phenyl v phang l dung mi phn cc nh H2O, methanol, acetonitril th c sc k pha o (hay sc k pha ngc). y pha tnh t phn cc hn pha ng, tri vi truyn thng ca sc k trc y l pha tnh phn cc hn pha ng. Nu R l nhm kh phn cc nh alkylamin (CH2)nNH2 hay alkylnitril (CH2)n-CN v pha ng l dung mi t phn cc nh hexan th ta c sc k pha thun. Hin nay sc k pha o c dng rng ri v nhng l do sau: Cc phn t khng phn cc, t phn cc, ion, hoc c th ion ha c th c tch mt cch hiu qu. Pha tnh c mt khong phn cc rt rng c th chn la. N cho php chn mt khong rng cht ra gii. Cc cht dung mi ra gii rt thng dng v khng t (nc, methanol v acetonitril). K thut ra gii gradient c th c s dng m khng s hng pha tnh. Khng c hn ch v p sut pha ng i vo ct. N cho kt qu tch tt vi rt nhiu i tng tch. Chn cc pha: thng chn pha tnh c tnh phn cc ging vi cc cht cn tch v khc vi pha ng. Cc cht hu c c phn cc tng theo th t sau: Hydrocacbon no, lefin, hydrocacbon thm, halogenua, sunfua, te, dn xut nitro, este = andehit = xeton, ancol = amin, sunfon, sunfoxit, amit, axit cacboxilic, nc. Th t ra gii trong sc k pha thun l cc cht khng phn cc ra trc ri n cc cht phn cc mnh hn. Trong sc k pha o cc cht phn cc ra trc ri n cc cht t v khng phn cc ln lt ra sau. ng dngSc k lng phn b hiu nng cao c dng trong vic tch cc cht thuc nhiu lnh vc nh: Thuc khng sinh, an thn, gim au, steroid Axit amin, protein, hydrat cacbon, lipid ng nhn to, cht chng oxi ha, cht ph gia, aflatoxin Cht thm, cht in hot, cht mu, ha cht cng nghip Thuc tr su, dit c, phenol v cc dn xut phenol Thuc, cht c, cn trong mu2. Sc k hp ph hiu nng cao (sc k lng rn LSC)L phng php pht trin sm nht v c dng rt ph bin. Trong phng php ny, cht tan b gi trn b mt pha tnh tc l cht hp ph v b dung mi y ra (phn hp ph) Pha tnh: pha tnh l cht rn m trn b mt c cha cc nhm hidroxil phn cc nh nhng bt mn, silicagen v nhm oxit. y cc cht cng b phn cc cng b lu gi mnh v ra chm khi ra gii. Pha ng: l mt dung mi khng phn cc bng di cho ta mt dy cc dung mi vi sc ra gii tng dn t trn xung (thng s sc dung mi tng dn). Dy ny c xp theo kh nng y cc cht ra khi nhm oxit. Nhng vi silicagen ta cng c dy tng t. Mun c dung mi c v tr trung gian khng c trong bng th ta dng hn hp dung mi theo t l thch hp.Dung mi

Ete du ha0,01

Hexan0,01

Heptan0,01

Xiclohexan0,04

Tetraclocacbon0,18

Ete propylic0,28

Toluene0,29

Benzene0,32

Ete etylic0,38

Clorofom0,40

Clometylen0,42

Dicloetan0,49

Aceton0,56

Dioxan0,56

Butanol0,56

Etyl acetat0,58

Acetonitril0,65

Pyridin0,71

Dimetylsunfoxit0,75

Ru isopropylic0,82

Ru etylic0,88

ng dng: Sc k hp th c dng nhiu tch cc cht tng i t phn cc, cc cht hu c khng tan trong nc c phn t lng nh hn khong 5000. Phng php ny mnh hn hn cc phng php khc trong vic tch ng phn.3. Sc k trao i ion hiu nng cao (IEC)Pha tnh cha cc nha trao i ln di dng bt mn, pha ng thng l dung dch nc.Pha tnh l nhng ht mang sn mt in tch nht nh, nhng ht ny s tng tc vi cc phn t (protein) mang in tch tri du vi chng. C th, nu ht mang in m tin trnh c gi l sc k trao i ion dng, th s tng tc vi nhng phn t mang in tch dng. Ngc li, nu ht mang in tch dng gi l sc k trao i ion m, th tng tc vi phn t mang in tch m. V th, nhng protein cng du vi ct s chy ra khi ct trong khi nhng protein tri du b gi li ct. phng thch nhng protein ny, ta tng nng ion ca pha ng, nhng ion ny s th phn t protein tng tc vi cc ht mang in tch. V d, trong sc k trao i ion dng, ta thm mui natri clorua hay mui khc trong dung dch tch gii bi v ion natri s tranh bm vo ct vi cc protein c in tch dng, do , nhng protein mang in tch dng c phng thch ra ngoi ct ln lt theo ln v in tch. ng dngPhng php ny c dng tch cc ion v c v hu c. V d khi cho dung dch hn hp cc ion Ba2+, Ca2+, Zn2+, Mg2+ i qua ct cationit axit mnh th cc ion ny b lu gi trn ct . Khi dng mt dung dch axit long ra gii th ln lt cc ion i ra khi ct l Mg2+ , Zn2+ , Ca2+ v Ba2+ . C th pht hin cc ion c ra gii ra khi ct nh detector o dn in.4. Sc k cp ion (Ion pair chromatography- IPC)Phn ln sc k cp ion (IPC) ny sinh do nhng hn ch ca sc k trao i ion nh: Cc ct c khuynh hng t hiu qu hn cc ct LC khc. Cc ct thng khng cho kt qu lp li mt mc no . Cc ct thng t bn. chn lc km.Trong ICP ta c th s dng c sc k pha thun ln sc k pha o, mi loi c mt thun li ring ca n. Pha tnh ca sc k pha o c th gm c c pha tnh silic c silan ha c s d ng bnh thng v d pha lin kt C18 hoc C8.Pha ng trong ICP gm mt dung dch m trong nc (cng vi mt dung mi hu c nh methanol hoc acetonitril cho php tch trong pha tnh lin kt) v mt ion i c in tch ngc du vi ion phn t mu.V d, gi s cn tch mt s cc axit hu c, pha ng c m pH =7 tt c cc hp cht trong mu nm di dng RCOO-. Ion i trong trng hp ny c th dng ion tetrabutyl amoni (TBA+). Ct dng trong ICP c chun b ging nh trong sc k pha lin kt v pha thun. Pha phn bBng sau tm tt vi h thng ICP c bo co trong cc ti liu vi mi ng dng ch r pha ng, pha tnh, ion i. pH ca ncPha ngIon iLoi mu

pH 9Cyclohexan/CHCl3N,N-dimethyl protrilylineCc axit cacboxylic

0,1 MHClO4Cc hn hp ca tributyl phosphate, ethyl acetate, butanol, CH2Cl2, v/hoc hexanClO4-Cc amin

m HPO42-/PO43-Butanol/ CH2Cl2/Hexan(Butyl)4N+Cc axit cacboxylic

pH 5-6Pentanol/ CH2Cl2 v/hoc CHCl3PicrateCc amin

pH 6-8,5Butanol / Heptane(Butyl)4N+Cc sunfoamit

0,2-0,25M HClO4Butanol/ CH2Cl2/HexaneClO4-Amin v cc amin bc 4

0,1M methane- sunfonic acidButanol/ CH2Cl2/HexaneCH3SO3-Cc amin

pH 8,3Butanol/ CH2Cl2/Hexane(Butyl)4N+Cc axit cacboxylic

Bng. Cc h thng sc k cp ion pha thunNhng nguyn tc chung trong vic kim sot s tch trong ICP tng t nh trong sc k pha o v pha thun. Vai tr ca ion i: Trong sc k pha o, lc dung mi c thay i bi thay i ion i hoc thay i nng ca n. S tng nng ca ion i dn n s tng h s dung lng k trong ICP pha o.Trong sc k ICP pha thun v pha o th k c th c thay i bi thay i loi ion i trong (v d thay th pentasulfonat bng heptansunfonat chng hn). S cng thm mt nhm CH2- dn n k tng ln khong 2.5 ln (nh hng s ln hn nu nng ca ion i thp). Phn t ion i cng ln th gi tr kcng ln trong sc k pha o v cng nh trong sc k pha thun. phn cc ca dung mi Lc dung mi trong ICP pha thun v pha o c th c thay i bi thay i phn cc ca pha ng. Trong cc h thng pha o, hn hp nc vi methanol hoc acetonitril nichung c s dng l pha ng. Khi phn trm ca nc gim xung th dung mi tr nn mnh hn v k ca mu gim xung. o Cc hn hp gia butanol v pentanol vi CH2Cl2, CHCl3 c hoc khng thm hexan l nhng pha ng thng dng trong sc k pha thun ICP. nh hng ca pH Gi tr k ca mu s chu tc ng nhiu trong ICP bi thay i pH hoc thay i dung mi ny bng dung mi khc (v d t acetat etyl thay bng butanol). Nu chng ta bit gi tr pKA ca cc axit hoc pKB ca cc baz ang tch th ta c th d on c s lu gi tng i ca mi hp cht s thay i theo pH. Ni chung pH ca pha ng c duy tr nh th no cho cc hp cht muc ion ha hon ton, cn s to thnh ion i th t cc i. V d khi tch cc axit nu pH ca pha ng thp th anion mu X- s nm di dng HX dn n s lng mu nm dng ion i trn pha tnh s thp. ng dngICP c ng dng trong nhng mu phn cc nh cc baz, cc ion, v cc hp cht c cc nhm ion ha. Cc mu nc, bao gm cc dch sinh l, cng nhn c s quan tm ca ICP.5. Sc k loi theo c (Size Exclusion Chromatography) Sc k loi theo c hay cn gi l sc k trn gel l mt cng c mnh cho phn tch cc cht c khi lng phn t ln. Cht nhi ct cho sc k ny l cc ht nh (c 10 micromet) polime hoc silica cha mng li cc l ng nht trong cc cht tan v dung mi c th khuch tn vo. Trong cc l cc phn t b by mt cch hiu qu v b tch t dng chy ca pha ng. Thi gian c ng trung bnh trong cc l ph thuc vo kch thc hiu dng ca cc phn t. Cc phn t c kch thc ln hn so vi kch thc trung bnh ca cc l ca cht nhi th khng b lu gi. Cn cc phn t c ng knh nh hn ng k kch thc l th thm nhp cng su vo trong su l v nh vy cn nhiu thi gian hn ra khi l, dn n b ra gii ra sau cng. Nm gia hai loi ny l cc phn t c kch thc trung bnh s xm nhp vo trong cc l nhiu hay t ty thuc ung knh ca chng. Ch y rng trong sc k loi theo c th khc vi cc trng hp xt trc y, y khng c s tng tc ha hc hoc vt l gia cc phn t cht tan v pha tnh. Cht nhi ct C hai loi: Cc ht polime v silica c ng knh t 5 n 10 micromet. Loi sau cng hn nn d nhi ct, bn hn, cho php s dng mt khong rng cc dung mi bao gm c nc, cn bng thit lp nhanh hn trong nhng dung mi mi, v bn nhit cao hn. Nhng nhc im ca ht silica l c khuynh hng lu gi cht tan bi s hp ph v c th c tim nng xc tc cho cc phn ng phn hy cc phn t cht tan. Hu ht sc k loi theo c u tin c thc hin trn cht nha ng trng hp gia divinyl benzene vi styrene. Kch thc l ca loi polime ny c kim sot bi s m rng lin kt ngang v v th lin quan n phn trm divinylbenzen s dng sn xut nha. V vy c nhiu loi gel vi kch c l khc nhau.Lc u, cc nha ng trng hp ny l k nc v ch c s dng vi pha ng l cc dung mi khng nc. Hin nay, cc gel a nc l c sn (v d poliacrylamide) dn dn c th s dng dung mi nc cho cc php tch cc phn t cht tan trong nc v d cc loi ng. Cc loi silica v ht thy tinh c kch thc l trung bnh ca khong 40 n 2500 Ao hin c sn trn th trng. gim s hp ph, b mt ca nhng cht ny thng c chuyn ha bi nhng cht th hu c. V. Mt s ai lng c ban trong phn tch sc k1. H s phn bCn bng ca mt cu t X trong h sc k c th c m t bng phng trnh nh sau:

Hng s cn bng K cho cn bng ny c gi l t l phn b hay hng s phn b (partition coefficient) v c tnh nh sau:Vi CS : nng cu t trong pha tnh.CM : nng cu t trong pha ng.H s K ty thuc vo bn cht pha tnh, pha ng v cht phn tch.2. Thi gian lutR : thi gian lu ca mt cu t t khi vo ct n khi tch ra ngi ct.tO : thi gian cho cht no khng c i lc vi pha tnh i qua ct; cng l thi gian pha ng i t u ct n cui ct v cn gi l thi gian lu cht.tR' : thi gian lu tht ca mt cu t.tR=tR-to

Hnh 2. Thi gian lu ca cu t phn tch3. H s dung lng kk c nh ngha theo cng thc sau:

Vi VS :th tch pha tnhVM :th tch pha ngNu k~ 0, tR~ tO: cht ra rt nhanh, ct khng c kh nng gi cht li.Nu k cng ln (tRcng ln): cht trong ct cng lu, thi gian phn tch cng lu, mi c kh nng b t.Khong k l tng l 2 - 5, nhng khi phn tch mt hn hp phc tp, k c th chp nhn trong khong rng 1 - 20.4. Hiu nngHiu nng hay s a l thuyt N ca ct c trng cho kh nng tch mi sc k ca cc cu t trn ct. N cng ln, hiu nng tch cng cao.S a l thuyt c th o trn sc k . Ngi ta chng minh c:

Hay

Vi W1/2 l chiu rng mi sc k v tr chiu cao mi (pht)W l chiu rng mi sc k v tr y mi (pht)5. chn lcc trng cho kh nng tch hai cht ca ct.

6. phn giiy l i lng biu th r c ba kh nng ca ct sc k: s gii hp, s chn lc v hiu qu tch. N c xc nh qua phng trnh sau:

Hay

VI. ng dngHPLC c xem l phng tin nh tnh, nh lng c nhy cao, chnh xc, gim thi gian sc k, t tn mu, c th p dng cho hu ht cc cht nh: cc cht d b phn hy bi nhit , cc acid amin, protein, acid nucleic, cacbohydrat, khng sinh, thuc tr su, cc cht v c,Thit b HPLC cng ngy cng pht trin v hin i hn nh s pht trin nhanh chng ca ngnh ch to v sn xut thit b phn tch. m bo cht lng kt qu phn tch nhiu loi ch tiu trn cc nn mu khc nhau, p ng c nhu cu phn tch a dng ca khch hng, CASE cng u t cc h thng HPLC hin i ca cc hng ni ting trn th gii trong sn xut thit b phn tch nh Shimadzu (model 10A, 20A), Agilent (LC 1200), Dionex (UltiMate 3000), Thermo, Mehtromvi hu ht cc u d nh quang ph t ngoi kh kin (UV-VIS), hunh quang (RF), khc x (RI), o dn (sc k ion -IC), khi ph (MS), Cc h thng HPLC ny u c gn cc b chch mu t ng nhm nng cao tnh chnh xc v c th phn tch ng thi nhiu mu.H thng HPLC 10A :

H thng HPLC 20A

Ti liu tham kho1. Bi ging Phn tch thc phm2. https://en.wikipedia.org/wiki/High-performance_liquid_chromatography3. http://elchem.kaist.ac.kr/vt/chem-ed/sep/lc/hplc.htm4. http://case.vn/vi-VN/34/96/118/details.case

11