BI GINGCC PHNG PHP PHN TCH SC KTS. Nguyn Ngc Vinh ngocvinhnguyen308@gmail.comSample Mobile phaset0t1t2t3t4t0t1t2t3t4DetectorsignalsTimeDetectorSample Mobile phaset0t1t2t3t4t0t1t2t3t4t0t1t2t3t4t0t1t2t3t4DetectorsignalsTimeDetectorKhoa Cng Ngh Ha Hc B mn Ha Phn TchH Cng Nghip TP.HCMTi liu tham kho1. C s l thuyt ca phng php sc k kh Phm Hng Vit NXB KH & KT2. Phng php phn tch sc k Nguyn Quang Khuyn NXBHQG TP.HCM3. Analytical gas chromatography (2nd) Walter Jennings Academic Press4. Chromatographyinthe petroleumindustry- E. R. Adlard Elsevier, 19955. Gaschromatography secondedition IanA. Fowlis JohnWiler & Sons, 1995.6. Highperformanceliquidchromatography SandyLindsay-John Wiler & Sons, 1987.7. Liquid chromatography mass spectrometry: an introduction R. E. Andrey John Wiley & Sons8. Gas chromatography and mass spectrometry, a practical guide F. G. Kitson Academic PressChng 1:CC KHI NIM C BN1. nh ngha, phn loi2. Nguyn l c bn3. Cc i lng o c bn1. nh ngha, phn loi Sc k (chromatography) c pht minh vo nm 1903 bi nh thcvt hc Nga Mikhail S. Tswetttrong qu trnh tch Chlorophills vXanthophylls bng CaCO3v alumina. Tuy nhin sau cc k thut ny b qun lng cho n cui nhngnm 1930 v u 1940 khi Martin v Synge gii thiu sc k lng lngdng tch cc acetyl amin acid vi pha tnh l silica. 1952 Archer J. P. Martin v Richard L. M. Synge c trao gii Nobel hahc cho cc pht minh v sc k phn b. Nguyn tc nn tng ca sck Tswett c p dng theo nhiu cch khc nhau xut hin nhiuloi sc k khc nhau, cho php phn tch cc phn t c cu trc gntng t nhau. Tn sc k vn c dng cho n ngy nay cho d cc phng phpsc k hin i khng cn lin quan n mu sc na.Ting Hy-lp:Chroma: muGraph:ghiSc k l g? Sc k l mt h cc k thut phn tch ha l dng tch, phn ly cccht trong mt hn hp da vo s phn b khc nhau ca chng giapha ng v pha tnh. Nguyn tc chung l cho mu cha cht cn phn tch trong pha ng(cht lng/kh) di chuyn qua pha tnh. Pha tnh tr hon s di chuyn cacc thnh phn trong mu. Cc thnh phn ny di chuyn qua h thngvi tc khc nhau chng s c tch khi nhau theo thi gian. Mithnh phn i qua h thng vi thi gian ring bit gi l thi gian lu (tR)S sc k kh mao qunCu trc tng qut ca mt thit b sc kS sc k lng hiu nng caoDegassePumpTim muColumnPhn loi sc k:K thut sc k c nhiu loi da theo trng thi ca mu v pha ng khitin hnh qu trnh sc k. l: Sc k kh (gas chromatography - GC) c pha ng (mobile phase - MP) lcht kh v mu a vo thit b phn tch dng kh. Trong trng hpny pha tnh (stationary phase - SP) c th l cht rn hoc cht lng Sc k lng (liquid chromatography - LC) c pha ng MP l cht lng vmu a vo thit b phn tch dng lng. Trong trng hp nySPcng c th l cht rn hay lngPha tnh trong 2 loi sc k ny c th l cht lng hoc rn nn ta c thm 2loi nh ca GC v LC nh trong bng 1.1 trnh by di y:Pha ng Pha tnhKhSc k kh - GCLng, Sc k kh lng, GLC (gas liquid chromatography)Rn, Sc k kh rn, GSC (gas solid chromatography)LngSc k lng - LCLng, sc k lng lng LLC (liquid liquid chromatography)Rn, sc k lng rn LSC (liquid solid chromatography)Sc k lng li c chia lm 2 nhm nh: Sc k lng p sut thng (sc k c in) Sc k lng hiu nng cao (High Performance Liquid Chromatography HPLC) hay sc k lng cao p (High Pressure Liquid Chromatography)Da vo bn cht ca qu trnh sc k, HPLC li c chia thnh nhiu loinh nh sau: Sc k phn b hay sc k chit nu pha tnh l cht lng Sc k pha thng (Normal phase chromatography) pha tnh l chthp ph Sc k pha o (Reversed phase chromatography) - pha tnh l cht hpph Sc k trao i ion (Ion exchange chromatography) pha tnh l chttrao i ion Sc k ghp cp ion (Ion pair chromatography) Sc k Gel hay ry phn t - pha tnh l gel.T=0T=10T=20Injector InjectorDetector DetectorMost Most Interaction with Stationary PhaseInteraction with Stationary Phase Least LeastFlow of Mobile Phase Flow of Mobile PhaseT=0T=10T=20Injector InjectorDetector DetectorMost Most Interaction with Stationary PhaseInteraction with Stationary Phase Least LeastFlow of Mobile Phase Flow of Mobile Phase2. Nguyn l c bnSc k l phng php tch cc cht da vo s phn b khc nhau ca chng giapha ng v pha tnh. Khi tip xc vi pha tnh, cc cu t ca hn hp s phn bgia pha ng v pha tnh tng ng vi tnh cht ca chng (tnh b hp ph, tnhtan, ).Trong h thng sc k ch c cc phn t pha ng mi chuyn ng dc theo hsc k. Cc cht khc nhau s c i lc khc nhau vi pha ng v pha tnh. Trongqu trnh chuyn ng dc theo h sc k ht lp pha tnh ny n lp pha tnh khc,s lp i lp li qu trnh hp ph, phn hp ph. H qu l cc cht c i lc ln vipha tnh s chuyn ng chm hn qua h thng sc k so vi cc cht tng tcyu hn pha ny. Nh c im ny m ngi ta c th tch cc cht qua qu trnhsc k.Gi s nu chng ta np mu phn tch gm hn hp cht phn tch A, B,C vo ct phn tch, kt qu cc cht A, B, C s c tch ra khi nhausau khi i qua ct tch. Quyt nh n hiu qu ca qu trnh tch yl tng hp ca cc tng tc c trnh by nh hnh v.Cht phn tch A, B, CPHA TNH PHA NGF1F3F2Tng ca ba tng tc ny s quyt nh cht no c ra gii ra khict tch trc tin khi lc gi trn ct l nh nht (F1) v ngc li.F tot = F1 + F2 + F3i vi mi cht, s lu gia c quyt nh bi ba lcF1, F2, F3.Trong , F1 v F2 gi vai tr quyt nh, cn F3 nh hng khng lnlm. y F1 l lc gi cht phn tch trn pha tnh trong ct tch. F2 llc ko ca pha ng i vi cht phn tch ra khi pha tnh.Nh vy vi cc cht khc nhau s c F1 v F2 khc nhau. Kt qu l cccht khc nhau s di chuyn trong ct vi tc khc nhau v tch rakhi nhau khi ra khi ct sc k nh hnh v di yPha ngABThi gianBAB ACCCSc k phn tch (Elution chromatography)Phn tch sc k: Cc cht tan b ra qua mt pha tnh nh s chuyn ng ca pha ng qua nMu Pha ngDetectort0t1t2t3t4t0t1t2t3t4Tn hiu detectorThi gianSc k (Chromatogrames)iu kin thu c sc k :- u d (Detector) c lp t im cui ca ct- u d tng thch vi cc cht cn pht hinSc k : Biu din s bin thin ca tnhiuratheo thi gian hoctheo th tchtiu hao ca pha ngCc peaks i xng (hoc khng i xng)Phn tch nh tnh (qualitative) v nh lng (quantitative)Sc k (Chromatogrames)S phn gii (resolution) Vn tc di chuyn tng i (relative migration rates) S gin peak (band broadening)3. Cc thng s c bn ca qu trnh sc k3.1. Cc qu trnh tch trong ct sc kS t- ng tc X-SP-MP trong ct tch + Nu gi: Cht PT: Xi, Pha tnh: SP, Pha ng: MP. Khichy sc k, qu trnh hp ph v gii hp lin tc xy ra, t lc np cht mu vo cho n lc cc cht i ra khi ct SK, trong ct tch lun lun c 3 t- ng tc:+ T- ng tc pha tnh (SP) vi cht phn tch (Xi), c lc F1.+ T- ng tc pha ng (MP) vi cht phn tch (Xi), c lc F2.+ T- ng tc pha ng (MP) vi pha tnh (SP), c lc F3.+ Chng ta c th m phng nh-trong hnh sau:+ Trong 3 t- ng tc ny: +F3 l khng i (trong 1 h pha). +F1 v F2 ng- c chiu nhau.+F1 gi cht phn tch Xi li trn pha tnh (SP). +F2 ko cht phn tchXi vo pha ng (MP). Nh-vy lc l- u gi ca cc cht trong qu trnh sc k l tng i s ca 3 lc t- ng tc , ngha l ta c : Ftot= ( F1 +F2+F3 ) Do trong mt hn hp mu, cht phn tch no:+ C lc Ftot nh nht s - c ra khi ct tch tr- c tin. + Clc Ftot ln nht s ra sau cng. V th m to ra qu trnh sc k tch cc cht. +NhngchtcFtotbngnhau,haygnnhauthkhngtch- c khi nhau, chng trng nhau, hay chen ln nhau (Hnh 5a). X iSPMPF1F2F33.2. Thi gian l- u ca cht (Rettention Time)+ Trong mt h pha, thi gian l- u tRca cht trong ct tch.+ L 1 i l- ng c tr- ng cho mi cht PT,+T lc np cht vo n lc n ra v tr c nng cc i.+ Thi gian l- u c (hnh 7): + Thi gian l- u tng cng (hay ton phn):tR+ Thi gian khng l- u gi:t0(hay tM),+ Thi gian l- u hiu lc, tRv : tR=( tR- t0)(1)Pc sc k v thi gian l- u+ Thi gian l- u tRica cht ph thuc:+ Pha tnh (Loi, c ht, xp, loi xp, iu kin ch to), H8.+ Pha ng (Loi, thnh phn, tc ,..).+ Bn cht ca cht Phn tch X.+ pH ca pha ng (ch h IE-, IP).+ Nhit tch, v.v.Cc v d - c ch ra trong mc ni v nh h- ng ca SP v MPThi gian l-u, tRi(pht) ?Thi gian l- u t Riph thuc vo lai SP3.3. H s tch ca 2 chtTrong 1 h pha sc k HPLC, h s tch :-- c biu th: o=( tR(A) / tR(B) ) (2)Trong : tR(A) v tR(B) l thi gian l- u ca cht A v cht B.-Nh-vy:+ Nuo=1 khng c s tch sc k 2 cht A v B.+ Nuo =1 s c s tch sc k .Mun c s tch tt, th gi trophi khc 1 nhiu. Cho v d v gii thch.+ Tch hn hp PAHs (hnh 9a) + Tch cc axit HC (hnh 9b).V d tch ca 5 cht trong 5 iu kin MP khc nhauCh iu kin MP 4 v 5 c s tch ttV d tch 10 Hydro cacbon thm Ch cht 3 v 4 ch- a tch - c hon ton3.4. H s phn b Kpb(Partition Coefficience)Trong mt h pha sc k: Trong qu trnh sc k, s phn b ca cht Xivo SP v MP l cc cn bng nhit ng, cc cn bng ny - c c tr- ng bi h s phn b Kpb. V h s ny :-H s phn b Kpbl mt hng s nhit ng.-N c tr- ng cho s phn b ca cht gia 2 pha (SP & MP).-V cho ta bit cht i phn b vo mi pha th no.-- c tnh:Kpb= Ci(SP)/Ci(MP)(3)Trong : Ci(SP)v Ci(MP): Nng cht i trong pha SP v MP. Kpb ny ph thuc: + Bn cht ca cht PT, cu trc phn t, nhm th,.. + Bn cht v cu trc ca pha SP, + Bn cht v cu trc ca pha MP v thnh phn, + Gi tr pH ca MP (trong IEX-, IP-HPLC), + Nhit chy sc k. Kpb theo quy lut phn b Lng-Mua.C 3 dng: (1): Phn b Tuyn tnh: (2): Phn b li: (3): Phn b lm (Xem hnh 5 trn). +Cn bng phn b ny c 3 dng: 1. Dng tuyn tnh, khi nng cht nhDng ny cho: + Pic sc k cn i, khng dong chn. + Hiu qu tch cao (tc l H nh).2. Hai dng khng tuyn tnh, khi nng cht ln (hnh 5).Hai dng ny: + Pic sc k khng cn i, pic SK dong chn. + Hiu qu tch nh (tc l H ln).Tr- ng hp khng tuyn tnh ny c 2 kiu:a)- Phn b li, chn pic SK dong pha tr- c (hnh 5). b)- Phn b lm, chn pic sc k dong pha sau (hnh 5).Quy lut phn bca cht v pic sc k3.5. Tc pha nga). Tc th tch, F+ Biu th bng s mL MP chy qua ct tch trong 1 pht.+ K hiu: F (mL/ph.): + Lin quan n tit din ngang ca ct tch.+ Tnh theo:F = Vi/ti(6)b). Tc tuyn tnh-Biu th bng s mm a sc k di chuyn - c trong ct tchtrong 1 n v thi gian.-K hiu:u (mm/pht).-Theo theo: u= L/ti(7)hay l: u= F. [ 1/ (Ecl.r2.t) ] (8)Trong :- Ecl:H s c tr- ng xp.- r : Bn knh ct tch.- L : Chiu di ct tch. c). nh h- ng tc MP n kt qu tch Th hin :+Loi dung mi lm MP (hnh 12a).+Thnh phn MP (hnh 12b).+Tc MP (hnh 12c). (b) (a)V nh h- ng ca dung mi lm MP(a)- MeOH/H2O : 70/30. (b)- THF/H2O : 70/30nh h- ng ca thnh phn MPCt SP: ShimPack ODS,7 m, 200x4 mm. Detector: UV = 214 nm. MP: MeOH/H2O, vi: 90/10; 80/20; 60/40; 50/50; F =1,00 mL/pht.Cht PT: Metyl (1), Etyl (2), n-Propyl (3), v n-Butyl-Paraben (4). Thi gian l-u, tRi (pht) Hnh 12c. V nh h- ng ca tc MP (a)- F= 0,8 mL/pht v(b)- F= 0,16 mL/pht. Mu: Nucleotides: AMP(1), ADP(2), ATP(3), GMP(4), GDP(5), GTP(6), MP: Dung dch: [(KH2PO4:0,015-0,25 M)/KCl 0,22 M]. 3.6. phn gii, RL 1 i l- ng:+ Cho bit kh nng tch ca ct sc k.+ iu kin 2 cht tch khi nhau. + RSti thiu c s tch: Theo quy tc Rayley.+ - c tnh theo: RS=(N/4)1/2.{ 1 - [(1+k1')/(1+k2')]} (9)Hay : RS= (1/4).[( o -1 )/o ].(Nef)1/2(10)Hnh sau y biu th quy tc Rayley.L iu kin ti thiu 2 cht st nhau tch thnh 2 pic.(a)(b) (c)tRi ? tRi ? tRi ?Quy tc Rayley v s tch(a)- Khng tch - c. (b)-Va tch. (c)- Tch xa nhau3.7. Nng cht ca pic sc k+Khi ra gii (chy sc k), cc cht PT s ra:+ Lc u Nng nh, ri tng dn.+ t cc i (nh pic),+ Sau li gim dn.+Nng cht nh pic sc k:Cmax.= Qi/ ( oi. ) 2 (t) (11)Trong :Qi = Vi.Ci: Th tch v nng mu - c np vo ct tch.oi: L lch chun ca pic sc k chti.+Biu v d (hnh v). HpicNng cht trongpic SK khi ra gii V nng cht CXti mt thi gian bt k tXng vi th tch ra gii VX mL ca pic sc k l - c tnh theo cng thc sau: (VMaxVX)2CX = CMax. exp { (- Nef/2). ----------------- }(12)(VMax.VX)2Cng thc ny cho php chng ta tnh - c nng ca cht X ti bt k lc no trn pic sc k.3.8. Chiu cao a H v s a N ca ct tch a). Chiu cao a, H+ L b dy ca lp cht trong ct tch.+ Trong ct tch, Gi tr H nh, Hiu qu tch cao.+ Biu th theo cng thc:-Chiu cao L thuyt, H:H = L/N=(L/16).(Wi/ti)2(13)-Chiu cao Hiu lc, Hef :Hef=(L/16). { W/( ti-to) }2(14)b). S a N ca ct tch L mt i l- ng: + Biu th tng s lp cht di chuyn trong ct tch. + S Nln Hiu qu tch cao. Ti thiu: Nef >= 4500. + - c biu th theo cng thc: - S a L thuyt, N:N =16.(ti/Wi)2 (15) - S a Hiu lc, Nef:Nef = 16.{ ( ti-to)/W }2 (16) + Hay tnh gn ng: Nef =[3500.L(cm)] / dp(m)17) c). Ph- ng trnh Van Deemter Ph- ng trnh thc nghim, Do Van Deemter xy dng Ph- ng trnh ny: + Biu th mi quan h giaHvu, tc l H =f(u). + Ph- ng trnh: Htot = ( A +B/u+C1.u +C2.u )(18) Const.Htot=( H1+H2+ H3+ H4 )Trong :A = 2.dp ;B = 2.DMP ; C1= .(dp)/DMP ; vC2=k./DSP Trong 4 phn ca Htot: + H1 : L d ht pha tnh quyt nh, phn chnh quyt nh Ht.+ H2 : S khuch tn ca cht trong pha ng, + H3 : S khuch tn ca cht vo pha tnh, + H4 : S khuch tn ca cht trong pha tnh. - ngha u:- Ti uo: C H nh nht,nn N ln nhtHiu qu tch tt nht. - Cc gi tru < uo: khi u gim, H tng theo. Nn N gim. - Cc gi tru > uo : khi u tng, H tng theo. Nn N gim. - dc ca 2 nhnh: Gim, khi dp nh dn (hnh 15).