Chng 1: K THUT PHN TCH

1.1. Phn tch sc k1.1.1. Nguyn l c bn- Sc k l k thut tch cc cu t ra khi hn hp da trn i lc khc nhau ca mi cu t i pha tnh v pha ng+ pha tnh: c tc dng gi cu t cn tch (lp cht c nh)+ pha ng: c tc dng ko cu t cn tch ra khi ct (cht lng, kh)

PPPT sc kc pht minh vo nm 1903 bi Tswett (1872-1919)u im:+ kh nng phn tch cao, c th tch nhng cht rt ging nhau v mt ha hc ln vt l+ l 1 trong nhng PPPT mnh nht

Ct sc k

Th t ra khi ctHp cht c i lc nhiu vi pha ng th c xu hng ra khi ct trcHp cht c i lc nhiu vi pha tnh th b gi li lu hn trong ct v ra sau

Sc k

Phn loi sc kPhn loi da trn trng thi ca pha ng:Sc k lng: + pha ng: cht lng+ pha tnh: rn, lngSc k kh+ pha ng: cht kh+ pha tnh: rn, lng

Phn loi sc kTheo c ch ca qu trnh tch:Sc k hp phSc k phn bSc k trao i ionSc k ry phn t

Cc i lng c bnt Mt RtRW

Thi gian lu tR , th tch lu VR- Sc : kt qu ca qu trnh tch sc k. Mi peak ca sc ng vi 1 hoc 1 nhm cu t ca hn hp cn tch.- Thi gian lu tR: thi gian t khi tim mu n khi ghi nhn c cc i ca mi sc k- Thi gian lu cht tM: thi gian 1 cht hon ton khng tng tc vi pha tnh hay cng l thi gian di chuyn ca pha ng t u ct n cui ct.

Thi gian lu hiu chnh tR , th tch lu hiu chnh VRThi gian lu hiu chnh tR: l thi gian cht b lu gi trong pha tnhtR = tR + tM = tM (1+K)VR = VM (1+K)- Thi gian lu v th tch lu hiu chnh ca mu:tR = tR - tMVR = VR - VM

H s phn b KCS : nng chung ca cht tan trong pha tnhCM : nng ca cht tan trong pha ngK cng ln ngha l cht b hp ph cng nhiu, cht chuyn ng cng chmK ch ph thuc vo bn cht ca cht tan, pha tnh v nhit m khng ph thuc vo kiu ct tch

H s cha (dung lng) KnS, nM : s mol ca cht tan trong pha tnh, pha ngb : t s phatR : thi gian lu hiu chnhtM : thi gian cht- K biu din mi tng quan gia thi gian ca cht tan lu li trong pha tnh so vi trong pha ng.- K ln: ct tch tt nhng thi gian phn tch ko di v vng b gin rng

S a l thuyt N trong ct sc kBit: d : lch chun ca peak - Trong TH l tng, peak c dng hnh Gauss, ta c rng ca peak:+ti im un: Wi = 2 d+ti mt na chiu cao: Wh = 2,355d+ ti y peak: Wb = 4 d- Ta c cc cng thc sau:

S a l thuyt N trong ct sc kN cng ln ngha l s lng cc cn bng tng th hiu qu phn chia trn ct sc k tng ln, b rng y cng nh, peak cng nhn, nh lng chng cng chnh xc.Sc k lng: N < 20.000Sc k kh: N > vi trm ngn? Sc k kh tch tt hn sc k lng ?- Chiu cao tng ng vi a l thuyt:H = L / NL: chiu di ct sc k

chn lc a chn lc a : dng nh gi 2 cht c tch c hay khng

a: ph thuc vo pha tnh, pha ng v bn cht ca cht tanPha tnh phi c cng bn cht vi cht tan gi chng li

H s phn gii RsH s phn gii Rs: dng nh gi 2 peak lin tip c th tch c hay khng (tt nht Rs = 1,5)

Kh nng tch cc cht Biu thc lin h gia 4 i lng trn:

ng dng ca PP sc knh tnhCu t c tnh theo gi tr tR. So snh tR ca mu v tR ca cu t chun trong cng k o trn my.nh lng: bng cch so snh vi dd chun:Chiu cao h ca mi (hp v i xng)Din tch mi S

Cc ng dng ca sc k

Phn tch da vo chiu cao peak

Phn tch da vo din tch peak

Xy dng ng chun (calibration with standards)

Phng php chun ni (internal-standard)

My sc k GC my sc k khHPLC my sc k lngsc k trao i ion

Sc k kh(Gas-Liquid Chromatography)Sc k kh Gas-Liquid Chromatography (GLC) hoc l Gas Chromatography (GC) Bc hi mu Tch cc cu t trong ct nh vo s phn b trong pha ng v pha tnh Pha ng: pha kh (N2, He, Ar) Pha tnh: pha rn hoc pha lng ph ln pha rn c gi trong ct Phng php cng c phn tch v xc nh cc hp cht ha hc

Sc k kh(Gas-Liquid Chromatography)Mu (sample) phn tch c: a vo b phn np mu (heated injector) Di chuyn qua mt ct phn tch (seperating column) nh mt dng kh mang tr (inert carrier gas) Pht hin v ghi li di dng cc peaks khi cc cu t i ra khi ct

Sc k kh(Gas-Liquid Chromatography)

Sc k kh(Gas-Liquid Chromatography)Ngun cung cp kh mang (Carrier Supply)

F = 25 150 ml.min-1: Ct nhi (Packed column)F = 1 25 ml.min-1: Ct mao qun (Open-tubular or Capillary column) N2: chi ph thp, an ton H2: chi ph thp, nguy c chy n He: thng thng, t Ar: Bnh cha p sut cao (pressurized tank) - Dng c iu chnh p sut (pressure regulator) - iu khin lu lng dng kh (Flow controller)Two stages pressure regulator

Sc k kh(Gas-Liquid Chromatography)Ngun cung cp kh mang (Carrier Supply)

Thit b tch N2 t khng kh nn (Pure Nitrogen Generator)- Thm thu chn lc N2- 0.5 ppm O2, > 0.5 ppm H2O, > 2.0 ppb halocarbons hoc CxHy. - Lu kng ti a 1 l/min. p sut 3,5 7 atm. Thit b cung cp kh H2 t nc ct (Hydrogen Generators)- Phng php in phn (Electrolysis)- Cht in ly: polymer rn (solid polymer electrolyte) - H2 99.999% - Kh nng lu tr H2: 4 litre- p sut: 1,4 7 atm. - Lu lng: 0 to 125 ml.min-1 v c th t n 1200 ml.min-1.

Sc k kh(Gas-Liquid Chromatography)H thng np mu (Sample Injection system)

Cc yu cu: - Lng mu thch hp - Tc np mu phi nhanh v mu np khi vo ct trng thi khGim s gin peak (band broadenning) v tng phn gii ca ct Microsyringe chun (calibrated) Septum: mng bng cao su silicone Gia nhit cho vng np mu: T > 50C ca cu t c nhit si cao nht Th tch np mu: 20 l i vi ct nhi (packed column) 0,2 l hoc nh hm i vi mao qun (open-tubular or capillary column)

B. Sc k kh (Gas-Liquid Chromatography)n nhit ct sc k (Column Thermostating)Mc ch: Bo m tnh lp li ca thi gian luL n nhit (thermostating oven)Topti.= f(Tsi), Topti Tsi vi RT= 2 30 phtNhit chng trnh ha (Temperature Programming)Isothermal: mu n ginMu phc tp: Tch cc cu t ca mu da vo s thay i ca T si

Sc k kh (Gas-Liquid Chromatography)So snh sc k hai ch : Isothermal v chng trnh ha nhit

Sc k kh (Gas-Liquid Chromatography)Cc vng c gia nhit ca h sc k kh (GC)

K thut sc k Mt trong cc thnh phn c bn nht: Detector (b d tn hiu). Chia 4 loi:Detetor ion ha mu FID (ion ha ngn la)Detetor da vo tnh cht vt l TCD (dn nhit).Detetor quang hc FPDDetetor in ha ECD (cng kt in t) Tn hiu ca Detetor ph thuc c tnh ca chng. Detector nhy cm nng tn hiu nhn c ph thuc tc dng kh i qua: TCD, ECD, FPD.

Sc k kh (Gas-Liquid Chromatography)u d (Detectors)Mt s yu cu: Tn hu thu c tuyn tnh hoc gn tuyn tnh vi lng mu Thi gian tr li nhanh Pht hin a dng (universal detection) Tn hiu ra khng ph thuc v nhit Lm vic n nh t nhit thng n 400C (i vi GC) Thermal Conductivity Detector (TCD) Flame Ionization Detector (FID) Electron Capture Detector (ECD) Nitrogen-Phosphorous Detector (NPD) Flame Photometric Detector (FPD): FID tweaked for S compounds Photoionization Detector (PID)

Detector dn nhit (TCD)Detector TCD da vo tnh cht in dn.Nguyn tc: o lin tc dn nhit ca kh mang (tinh khit hoc cha cu t tch) gia bung o v bung so snh c lp cc dy in tr Vonfram hoc Platin.c ng dng rng ri c th x hu ht cc cht tch t ct sc k.Nhy cm nng , gii hn pht hin 10-9g/ml. dn in ca He, H2 ln hn 6 10 ln so vi cc hp cht hu c.

Sc k kh (Gas-Liquid Chromatography)Thermal Conductivity Detector (TCD)S nguyn lMeasures heat loss from a hot filament nearly universal Filament heated to const T When only carrier gas flows heat loss to metal block is constant, filament T remains constant When an analyte species flows past the filament generally thermal conductivity goes down, T of filament will rise. (resistance of the filament will rise).

Sc k kh (Gas-Liquid Chromatography)Thermal Conductivity Detector (TCD)

Detector ion ha ngn la FIDNguyn tc: da trn s bin i dn in ca ngn la H2 t trong mt in trng khi c cht hu c chuyn qua.Cc cht hu c b oxy ha nh oxy khng kh to cc ion tri du tng ng, chng chuyn v cc bn in cc nm 2 pha ngn la.CH* + O* CHO+e-- S lng cc ion ph thuc H2/khng kh- Detector nhy cm khi lng, khng nhy vi kh v c N2, N2O, NO, CO2, CS2, COS- nhy l 10-12g/s, thch hp phn tch cc hydrocacbon

Sc k kh (Gas-Liquid Chromatography)Flame Ionization Detector (FID)Sensitive towards organics

Analyte is burned in H2/air, which produces CH and CHO+ radicals CHO+ radicals are reduced at a cathode which produces a current proportional to the radical quantity 10-12 A Specific for organic carbon, insensitive to inorganics, CO2, SO2 etc. Generally DL 100x less than TCD about pg/s (flow rate dependent) Response to specific organic depends on the number of organic carbons.

Detector cng kt in t ECDDetetor nhy cm nng Xc nh cc hp cht cha nhm chc hoc a lin kt Da trn kh nng cng kt cc in t t do trong pha kh ca dng kh mang c ion ha bng tia pht ra t ngun phng x (63Ni) to ra ngun e- v cc ion phn t kh mang M+M + M+ + e- nhy: kh rng 1 106Thng s dng phn tch thuc tr su, dit c c cha vng benzen clo ha

Electron Capture Detector (ECD)Sensitive to electron withdrawing groups especially towards organics containing F, -Cl, -Br, -I also, -CN, NO2

Nickel-63 source emits energetic electrons collides with N2 (introduced as make-up gas or can be used as carrier gas) producing more electrons:Ni-63 e-, e- + N2 2e- + N2+ The result is a constant current that is detected by the electron collector (anode). As an analyte flows through past the Ni-63 source, electron capture is possible by electron-withdrawing species: A + e- A- Current decreases as a result of e- capture by analyte. This is one of the few instances in which a signal is produced by a decrease in detectable phenomenon. Very low DL for detected species 10-15g/ml for many halogenated substances

Sc k kh (Gas-Liquid Chromatography)Electron Capture Detector (ECD) Radioactive Ni-63 source Easily contaminated with O2, H2O, sample overloading. High maintenance device. Highly variable response to halogenated substances Sometimes complementary information from FID helps.The bad

Sc k kh (Gas-Liquid Chromatography)Thu thp v x l s liu (Data Acquisition and Processing )

B. Sc k kh (Gas-Liquid Chromatography)Mt s ng dng ca GC (GLC) p dng i vi cc mu bc hi v n nh nhit n vi trm C C kh nng pht hin v phn tch rt nhiu cht v hn hp c ng dng rng ri tch v xc nh cc cu t trong cc mu t nhiu chng loi khc nhauMt vi v d: Ketones: polydimthyl siloxane Alkalodes: 5% phenyl polydimethyl siloxane Sterods: 50% phenyl polydimethyl siloxane Chlorinated Aromatics: 50% Trifluoropropyl polydimethyl siloxane Alcohols: Polyethylenglycol Esters: 50% Cyanopropyl polydimethyl siloxane Sc k kh kt hp khi ph

B. Sc k lng hiu qu cao (Hight-Performance Liquid Chromatography - HPLC)Phn loi HPLC da bn cht tng tc Sc k phn b (partition chromatography) Sc k hp ph hoc lng-rn (adsorption or liquid-solid chromatography) Sc k trao i ion (ion exchange chromatography) Sc k loi tr kch thc (size exclusion chromatography)

B. Sc k lng hiu qu cao (Hight-Performance Liquid Chromatography - HPLC)Phn loi HPLC da vo vt liu nhi Pha thng thng (Normal phase): vt liu nhi l silica n gin Trao i ion: silica bin tnh (mdified silica) Pha o (reverse-phase): silica bin tnhPhn ln cc HPLC l pha o Cht phn tch c gi trn pha tnh phn cc nh hn cho n khi b ra tri bi pha ng phn cc ln Thao tc n gin Hiu qu cao Ct lm vic n nhC th phn tch cho c hai loi cu t c c tnh tng t hoc khc xa nhau

B. Sc k lng hiu qu cao (Hight-Performance Liquid Chromatography - HPLC)Mc du c nhiu l thuyt nghin cu v vic s dng pha o nhng phn ln cc chng trnh HPLC pha o u thu c t phng php th v sai (by trial and error).

B. Sc k lng hiu qu cao (Hight-Performance Liquid Chromatography - HPLC) Gc R l C8 (n-octyl), C12 (n-octyl) hoc C18 (n-octyldecyl). Pha ng l H2O + dung mi ha tan (acetonitrile, methanol, ethanol, isopropanol). Cc cu t phn cc s b ra ra nhanh nht, tng phn cc ca pha ng s lm tng thi gian chy muPha tnh-Pha o(Stationary Phases for Reversed-Phase LC)Pha tnh bnh thng ca LC(Stationary Phases for Normal LC) Pha ng tng i khng phn cc: Hexane, Isopropyl eter, toluene Cc cu t khng phn cc s b ra ra nhanh nht, tng phn cc ca pha ng s gim thi gian chy mu

B. Sc k lng hiu qu cao (Hight-Performance Liquid Chromatography - HPLC)nh hng ca bn cht pha tnh n cht lng tch

B. Sc k lng hiu qu cao (Hight-Performance Liquid Chromatography - HPLC)S nguyn l ca HPLC

B. Sc k lng hiu qu cao (Hight-Performance Liquid Chromatography - HPLC)Cc yu cu i vi dung mi p sut bm: vi trm atm (6000psi), lu lng 0,1 10 ml.min-1 vi E

B. Sc k lng hiu qu cao (Hight-Performance Liquid Chromatography - HPLC)

B. Sc k lng hiu qu cao (Hight-Performance Liquid Chromatography - HPLC)Polar Solvents

Water > Methanol > Acetonitrile > Ethanol > Oxydipropionitrile

Non-polar Solvents

N-Decane > N-Hexane > N-Pentane > Cyclohexane phn cc ca mt s dung mi s dng trong HPLCLa chn pha ng v pha tnhCh yu da vo s phn cc ca cu t phn tch, pha ng, pha tnhQuy tc chung: phn cc (polarity) ca cu t cn phn tch v pha tnh l tng ng cn pha ng c phn cc sai bitKhi phn cc ca cu t v pha tnh qu ging nhau: thng tc mnh gia cu t cn phn tch v pha tnh thi gian phn tch ko di

B. Sc k lng hiu qu cao (Hight-Performance Liquid Chromatography - HPLC)

B. Sc k lng hiu qu cao (Hight-Performance Liquid Chromatography - HPLC)H thng np mu (Sample Injection Systems) S dng valve 6 cngNp mu qua vng ly mu (sampling loops) Sc k lng hin iC th thay th sampling loops t 5 l n 500 lSai s ca lng mu np di 1%

B. Sc k lng hiu qu cao (Hight-Performance Liquid Chromatography - HPLC)Ct sc k HPLC Thng thng:L = 10 30 cm v c th ni tip 2 ct hoc nhiu hnID = 4 10 mm, kch thc ht nhi: 3, 5 v 10m40.000 60.000 a/m ct Ct tc cao v hiu qu hn L = 3 - 7 cm v c th ni tip 2 ct hoc nhiu hnID = 1 4,6 mm, kch thc ht nhi: 3 hoc 5 m100.000 a/m ctCt bo v (Guard Column)c lp t trc ct phn tch ko di tui th ca ct Thnh phn = thnh phn ca ct phn tch nhng c ht ln hn gim tn tht p sut

B. Sc k lng hiu qu cao (Hight-Performance Liquid Chromatography - HPLC)n nh nhit ca ct (Column Thermostats) Phn ln ng dng cua HPLC c thc hin nhit phng Tuy vy cht lng ca sc k s tt hn nu duy tr nhit ca ct khng thay i (sai s < 0,05C) Thit b HPLC hin i c trang b thm l gia nhit cho ct (Column heater) n nh nhit gn 150C vi sai s < 0,05C Trang b h thng phun nc lm lnh (water jackets fed) t b n nhit khng ch chnh xc nhit

B. Sc k lng hiu qu cao (Hight-Performance Liquid Chromatography - HPLC)u d (Detector) dng cho HPLCKhng nhy v c kh nng phn tch a dng nh detector ca GCThng gp nht l Detector UV-Vis

B. Sc k lng hiu qu cao (Hight-Performance Liquid Chromatography - HPLC)UV-Vis and Fluorescence Detector = 200-400nm s dng 254 nm

B. Sc k lng hiu qu cao (Hight-Performance Liquid Chromatography - HPLC)Cc phng php nng cao phn gii trong HPLCTng chiu di ca ct (Increase column length)Gim ng knh ca ct (Decrease column diameter)Gim lu lng pha ng (Decrease flow-rate)Pha tnh (vt liu nhi ct) ng nht (Uniform stationary phase (packing))Gim th tch bm mu (Decrease sample size)La chn pha tnh sch hn (Select proper stationary phase)La chon pha ng tinh khit hn (Select proper mobile phase)S dng p sut n nh hn (Use proper pressure)Thnh phn ca pha ng thay i hp l (Use gradient elution)

So snh HPLC v GC (Comparison of HPLC and GLC)Cc c im chung: Hiu qu, chn lc cao, ng dng rng ri Th tich mu nh C th khng ph hy mu (nondestructive of sample) nh lng d dngu im ca HPLC p dng c vi cc mu khng bay hi v khng bn nhitp dng c cho cc ion v cu im ca GC Thit b n gin v r Nhanh chng D dng kt ni vi ph khi

p dngS liu ban u: (tR)A=16.4 pht, (tR)B=17.63 pht, (tR)M=1.3 pht, chiu di ct: L=30 cm rng ca peak ti ng nn: WA=1.11 pht v WB=1.21 phtTnh ton: Rs, N, H, Chiu di ca ct bo m Rs=1.5 v (tR)B tng ng.Rs= 2(17.63 16.4)/(1.11+1.21) = 1.06Gii:N = 16(16.4/1.11)2 = 3493 v N = 16(17.63/1.21)2 = 3397 N = (3493+3397)/2= 3445H = L/N = 30/3445 = 8.710-3 cmDo kB v khng thay i khi tng chiu cao ca ct, ta c:

Bi tp1) Cho 2 cht c thi gian lu tR1=1,43, tR2= 4,5 v b rng y Wb1=0,1, Wb2 = 0,3. Tnh s a l thuyt ng vi mi cht2) Mi cp peak c chn lc a = 1,1 v h s phn gii Rs=1, h s cha K2=0,5. Tnh s a l thuyt N?

Bi tp3) Mt hn hp gm 2 cht c h s cha K1= 2,58 v K2=2,64. Tnh h s chn lc a?

Bi tp3) Mt h thng sc k lng hot ng vi cc thng s sau:Chiu di phn ct c nhi pha tnh: 25,2 cm,Tc dng: 0,312ml/ph, Th tch pha ng: VM=1,4ml, Th tch pha tnh: Vs=0,168mlSau khi cho qua ct cha 2 cu t AB ta c sc k vi d liu sau: tM = 1,2ph; thi gian lu ca A v B l 12,5 v 13,8ph, chiu rng chn peak A v B l 1,34 v 1,44ph. Hy tnh: N, H, K, K, Rs, a