TRNG I HC CNG NGHIP TP.

H CH MINH KHOA CNG NGH HA HC

PHNG PHP PHN TCH SC K

CHNG 4: PHNG PHP SC K KH

GVGD: T.S. NGUYN VN TRNG

Ni dung chng 4
4.1. Gii thiu chung v phng php sc k kh 4.2. Pha ng hay kh mang s dng trong sc k kh 4.3. Cc loi ct v pha tnh s dng trong sc k kh 4.4. Cc loi injector (bung tim) s dng trong sc k kh 4.4.1. Injector cho ct nhi 4.4.2. Injector cho ct mao qun 4.4.3. Van tim mu kh 4.5. Cc loi detector (u d) thng s dng trong sc k kh 4.5.1. Cc c tnh ca detector trong sc k kh 4.5.2. Cc detector thng s dng trong sc k kh 4.6. ng dng ca sc k kh

4.1. Gii thiu chung v phng php sc k kh

-

S lc v lch s sc k

- Cc khi nim v sc k kh - S sc k kh

- Qu trnh sc k

S lc v lch s sc k
Thp nin 1900, M. Tswett cng b cng trnh nghin cu da trn vic tch hn hp mu sc t l cy cha ca sc k (Father of chromatography). W. Ramsey cng b cng trnh tch hn hp kh da trn s hp th ca than Thp nin 1940, A. Martin v R. Synge l u tin cng b kh nng ca sc k kh trn tp ch Biochem. J., V35, 1358 (1941). Martin ginh gii thng Nobel cho cng trnh nghin cu sc k phn b.

Cc khi nim v sc k kh
- Sc

k kh l mt k thut sc k m c th c s dng

tch cc hp cht d bay hi

- Cc hp cht hu c c tch khi nhau nh s phn b khc nhau gia pha ng v pha tnh trong ct - Sc k kh l phng php sc k m pha ng dng kh lin tc chy qua pha tnh, cc cht tch ra do s tng tc khc nhau vi pha tnh.
+ Pha tnh: Rn hoc lng. + Pha ng: Kh.

QU TRNH SC K
(gas or liquid)

MOBILE PHASE

Mu vo

Mu ra

STATIONARY PHASE
(solid or heavy liquid coated onto a solid or support system)

S sc k kh
Mol-Sieve Traps Fixed Restrictors

Regulators Flow Controller

Injection Port

Detector

Electrometer

Recorder/ Integrator Column

Carrier Gas

Hydrogen

Air

Thit b trong sc k kh
Pha ng hay kh mang (carrier gas) B tim mu (injection port)

Pha tnh hay ct tch (mobile phase or

separation column)

u d (detectors)

4.2. Pha ng hay kh mang s dng trong sc k kh

Tnh cht ca kh mang Cc b phn ca kh mang Thit b by kh (lc kh) Phng trnh Van Deemter

Cc yu t nh hng n sc k kh

Tnh cht ca kh mang

Mc ch chnh: mang mu i qua ct Mc ch th 2: cung cp mt hn hp ph hp cho u d o cc cu t trong mu
Detector Thermal conductivity Flame ionization Electron capture Kh mang Helium Helium hoc nitrogen Very dry nitrogen hoc argon

Kh mang nn s dng loi tinh khit cao (minimum 99.995%)

Nu kh mang c nhim oxy v nc th chng s tn cng cc ha cht c trong ct v lm h hng ct Hm lng vt ca nc, s lm pha tnh mt tc dng dn n ng nn cao v xut hin cc peak ma (ghost peaks) Hm lng vt ca cc hydrocarbon c th l nguyn nhn gy ra ng nn cao khi s u d FID dn n nhy thp.

Tnh cht ca kh mang

Cc kh thng dng nht: nito (r v c sn), heli (hiu qu tch cao).
Cc cht khng c php c trong kh mang: oxy, kh cacbonic, hi nc v cc halogen. Yu cu ca kh mang: - Tr vi cu t kho st. - C t khi nh ( nht thp) lm tng vn tc kh mang. - Tn ti dng tinh khit hay d lm cho tinh khit. - Khi xt quan h gia chiu cao a l thuyt H v vn tc tuyn tnh ca dng kh

mang, loi kh mang no cho cc tiu cng tri rng cng tt.
Lu : - Chn cht kh lm kh mang phi ph hp vi detector s s dng. - iu chnh vn tc v p sut kh mang qua ct phi ht sc cht ch v lu lng kh mang nh hng n qu trnh tch.

La chn kh mang
Detector o dn nhit (TCD) H2, He. Kh He c u im khng nguy him. Detector ion ha ngn la (FID) N2 do r v khng nhuy him nhng trong trng hp ghp ni vi khi ph phi dng kh mang l heli. p sut ca kh vo thit b nm trong khong t 10 n 50 psi c tc dng t khong 30 n 150 ml/ph i vi ct nhi v khong t 1 n 25 ml/ph i vi ct mao qun

Sau y l c im ca mt s kh mang thng dng

Kh H2 khi s dng lm kh mang cn dng kh nit lm kh bo v thi qua ct trc. Trong cc phng th nghim ngi ta dng ph bin my sn xut kh hydro vi cng sut t 125 ml/ph n 225 ml/ph. Khi dng H2 trong phng th nghim phi c my d ch h H2 v cm la. Kh He, argon l kh tr ha hc rt thch hp cho sc k nhit cao. Kh nit do khng nguy him, gi r v d dng lm tinh khit nn N2 c dng nhiu cho sc k kh. Cn ch l dn nhit ca N2 rt gn vi dn ca nhiu kh v nhiu hp cht hydrocarbon nn c trng hp peak sc k c th b ngc.

My to kh H2 (hydrogen gas generators suppliers)

Cc b phn ca kh mang

Cc b phn kh mang

Two-stage gas regulator

THIT B BY KH

Traps shown: A. Moisture Trap: B. Hydrocarbon Trap: C. Oxygen Trap:

Traps

Molecular Sieve Trap (moisture trap)

Hydrocarbon Trap

Oxygen Trap

18

PHNG TRNH VANDEEMTER

B H H A H B H C A Cu u
Trong :
- HA: Chiu cao ring phn th hin cht lng ca ct nhi gy nh hng n tc chuyn dch khc nhau ca cc phn t trong ct

nhi.
- HB: Chiu cao ring phn biu din s phn tn ca cu t kho st trong pha ng. - HC: Chiu cao ring phn biu din s hp ph v gii hp ph ca cu t trn pha tnh v s phn tn ca cu t trong hai pha.

Van Deemter Plot

Plate Height, H

A + B/u + Cu
Mass Transfer (both), Cu Multipath Term, A

Longitudinal diffusion, B/u

Linear Velocity, u

NH HNG CA KH MANG N HIU NNG TCH CA CT

4.3. Cc loi ct v pha tnh s dng trong sc k kh

Mt s yu cu v ct
Phn loi ct tch trong sc k kh Thnh phn v tnh cht pha tnh trong sc k kh Cc yu t nh hng n qu trnh tch ca ct sc k

Mt s yu cu v ct
m bo trao i cht tt gia pha ng v pha tnh nh vic ti u ha cc thng s ca phng trnh Van Deemter. thm cao tc c gim p sut nh vi mt tc kh mang nht nh. Kh nng ti trng cao ca ct. C khong nhit s dng rng v chu c nhit cao.

Phn loi ct tch trong sc k kh

Ct nhi
Pha tnh: rn
Gas Solid Chromatography

Ct mao qun
Golay pht minh nm 1957 Capillary column hay open tubular column K hiu: OT

K hiu: GSC Pha tnh: rn c ph lp mng lng

Gas Liquid Chromatography

K hiu: GLC

Phn loi ct tch trong sc k kh

CT NHI (packed column)

GLC: Gas Liquid Chromatography GSC: Gas Solid Chromatography

CT MAO QUN (capillary column)

Ct mao qun

c tnh ca ct
Cc loi ct
FSOT Length (m) ID (mm) Efficiency (Plate/m) Sample size (ng) Relative pressure Relative speed Flexible Chemical inertness 10-100 0.1-0.3, 0.53* 2000-4000 10-75 Low Fast Yes Best WCOT 10-100 0.25-0.75 1000-4000 10-100 Low Fast No SCOT 10-100 0.5 600-1200 10-1000 Low Fast No Packed 1-6 2-4 500-1000 10-106 High Slow No Poor
31

FSOT: Fused-silica open tubular column

Kch thc ca ct mao qun (Column Dimensions)

Chiu di ct: 10 60m (thng thng 30m)

ng knh trong ca ct: 0.10 0.53 mm

B dy lp phim ca pha tnh: 0.10 0.25 mm
32

Ct sc k kh lm vic nh th no?

Thnh phn v tnh cht trong pha tnh

Pha rn (Solid phase)
Ch yu s dng tch cc hp cht c khi lng phn t nh v cc cht kh. Ph bin nh: silica, alumina, ry phn t nh zeolites, cabosieves, carbon blacks

Pha lng (Liquid phase)

Trn 300 loi pha tnh lng khc nhau c s dng rng ri. Cc nhm pha lng thng dng
Non-polar, polar, intermediate and special phases

Pha lng polymer

34

Pha tnh Polymers (stationary phase polymer)

Siloxane

Arylene

Polyethylene glycol

35

Pha lng
Pha khng phn cc
S tch dc vo bay hi ca cc cht S gii hp cc cht ra khi ct ph thuc vo nhit si ca mi cht Pha ph bin nht: dimethylpolysiloxane, dimethylphenylpolysiloxane

Pha phn cc
Cha cc nhm chc phn cc S tch ph thuc vo nhit bay hi ca mi cht v ph thuc vo tng tc polar polar Pha ph bin: polyethyleneglycol

Pha trung bnh

36

Common stationary phase coating for capillary column

Composition 100% dimethyl polysiloxane (Gum) 100% dimethyl polysiloxane (Fluid) 5% diphenyl 95% dimethyl polysiloxane 14% cyanopropyl phenyl polysiloxane 50% phenyl, 50% methyl polysiloxane 50% cyanopropylmethyl, 50% phenylmethyl polysiloxane 50% trifluoropropyl polysiloxane Polyethylene glycol TPA modified Polyethylene glycol Polarity Nonpolar Nonpolar Nonpolar Immediate Immediate Immediate Immediate Polar Polar Applicaitons Phenols, Hydrocarbons, Amines, Sulfur compounds, Pesticides, PCBs Amino acid derivatives, Essential oils Fatty acids, Methyl esters, Alkaloids, Drugs, Halogenated compounds Drugs, Steroids, Pesticides Drugs, Steroids, Pesticides, Glycols Fatty acids, Methyl esters, Alditol acetates Halogenated compounds, +Aromatics Acids, Alcohols, Aldehydes acrylates, Nitriles, Ketones Free acids, Alcohols, Ethers, Essential oils, Glycols, Solvents Temp limits -60oC to 325oC 0oC to 280oC -60oC to 325oC -20oC to 280oC 60oC to 240oC 60oC to 240oC 45oC to 240oC 60oC to 240oC 60oC to 220oC
37

38

Pha tnh Polysiloxane

Pha tnh polysiloxane l pha tnh khng p

Thay mt s nhm methyl bng mt s nhm phn cc hn nh (phenyl, vinyl, cyanopropyl, Trifluoropropyl, aryl) phn cc ca pha tnh tng

Poly(dimethyldiphenyl) siloxane x = 5%n, y = 95%n : low polarity

x = 14%n, y = 86%n : low polarity

x = 50%n, y = 50%n : medium polarity

Poly(cyanopropyldimethyl) solixane x = 6%n, y = 94%n : low polarity x = 14%n, y = 86%n : medium polarity x = 35%n, y = 65%n : medium polarity x = 50%n, y = 50%n : high polarity

Poly(cyanopropyldimethyl) siloxane
x y

x = 33%n, y = 67%n : high polarity

Poly(cyanopropylmethylmethylphenyl) sioloxane

Poly(methylphenyl-dimethylphenylene) siloxane

Cc yu t nh hng n chn lc
Bn cht ca pha tnh Nng ca pha tnh Nhit ct Vic chn la v x l s b cht mang rn

hoc cht hp ph.

Cch chn pha tnh

Ct khng phn cc l tt nht cho mu phn tch khng phn cc. Cc ct c phn cc trung bnh th tt cho mu phn cc trung bnh v phn cc mnh th tt cho mu phn cc mnh. Khi ct dng nhiu b lo ha th pha tnh b t bay mt nn cc nhm silan (Si-OH) b l ra v cc pic b ko di ui tng ln. Nu trong ct c oxi nhit cao cng dn n s thoi ha ct v pic b ko ui. gim khuynh hng pha tnh b mt nhit cao, ngi ta gn chng vo b mt pha tnh silica bng cc lin kt cng ha tr.

 phn cc ca i tng phn tch

Nhm rt phn cc: H2O, glycol, glyxerin, poliphenol, axit dicacboxylic. Nhm phn cc: ancol mch thng, axit bo hu c, phenol, amin bc 1,2, hp cht nitro v nitril c cha hydro . Nhm phn cc trung bnh: ete, ankanal, ankanon, este, amin bc 3, hp cht nitro v nitril khng cha hidro . Nhm t phn cc: hidrocacbon thm, anken, hidrocacbon b halogen ha mt phn. Nhm khng phn cc: ankan, thioankanol, sunfua, ankan b halogen ha.

Cch lp ct mao qun?

Chng trnh nhit trong GC

S tch cc hp cht bng GC Chng trnh ng nhit Chng trnh nhit Cch set up 1 chng trnh nhit

S tch cc hp cht bng GC

Trong GC, cc cht phn tch tch c da vo:
Nhit si S phn cc Cc tnh cht khc

Cc cht c nhit si thp s ra trc cc cht c nhit si cao hn Tng tc ca cc cht da vo phn cc, nu s dng ct khng phn cc, th cc cht phn cc mnh s ra nhanh hn, trong khi cc cht phn cc km hn s b lu gi lu hn

Chng trnh ng nhit (isothermal temperature)

Nhit trong sut c qu trnh l khng i Cc hp cht b gii hp nhanh c th b chng lp mi sc k, cc cht kh gii hp s c thi gian lu rt di v hnh dng ca mi sc k s rng Chng trnh ng nhit khng phi l mt s la chn tt cho mu c cha cc cht phn tch c im si khc nhau

Chng trnh nhit (temperature programming)

Bng cch gia tng nhit nh l mt hm theo thi gian, n c th tch cc cht c nhit si khc nhau v vn gi c mt sc k tt. Mt khc, tng thi gian phn tch c rt ngn

Isothermal GC temperature/ Temperature programming

Isotherm column temperature

Temperature programming

www.chem.agilent.com

Set up chng trnh nhit

S dng k thut th ng sai (trial and error technique) C gng tm nhng bi bo c cng b v phn tch cc cht quan tm, thng thng chng trnh nhit c bo co. S dng cc trang web chuyn v sc k nh: http://chromatographyonline.findpharma.com/lc gc/ http://spectroscopymag.findpharma.com/spectro scopy

 Lun s dng dung dch chun setting up mt chng trnh nhit Nhit ca injector lun c ti u ha trc khi bt u. Nu nhit injector qu thp th cc cht khng bay hi ht, ngc li th c th mt cht phn tch, v th cn phi tra nhit si ca cht cn phn tch. Cn thn khi s dng ct, cn kim tra li nhit si ca dung mi Nhit u c xc nh da trn kh nng tch tt ca mt vi cu t u. Kinh nghim thay i chng trnh nhit sao cho gia nhit u v cui c tch tt nht v ngn nht

4.4. Cc loi injector (bung tim) s dng trong sc k kh

Injector cho ct nhi Injector cho ct mao qun Van tim mu kh

Injector cho ct nhi v ct mao qun

H thng bm mu
Gm hai b phn chnh
Micro xilanh (Micro-syringes) B tim mu (Injector ports)

55

B tim mu
Ln hn 500C so vi nhit si ca cu t Kch thc mu
Mu lng: 0.1 - 10mL Mu kh: 0.5 - 5mL

Carrier gas

Septum Septum purge

Heated injection port

Vent Inlet liner

Split mode (1:100) Split/splitless mode Autosampler

Column
56

Bung ha hi trong injector

Thit k c bn
Mt ng thy tinh thng chu nhit Thn injector c lm bng thp khng g

Mu c a vo
Bay hi nhanh nhit cao c di chuyn vo ct bng kh mang

57

Microsyringes/ Autosampler

Gas-tight syringe

Microsyringe

Autosampler

59

Inlet Liners

60

Accessories: Vial, Septa and Caps

61

Cc k thut bm mu
Bm mu bng tay (manual) Bm mu t ng (auto sampler) K thut bm mu headspace K thut bm mu chia dng v khng chia dng (split/splitless)

Bm mu bng tay (manual)

Bm mu t ng (auto sampler) (xem phim)

K thut bm mu headspace

K thut bm mu chia dng v khng chia dng (split/splitless)

Mt s lu khi bm chia dng

Thch hp vi mu c hm lng cht phn tch cao T l chia thng dng 1:1 500:1 Nhit ca inlet khong 2500C, nhit u ct thng nh hn 100C so vi nhit si ca cht phn tch Chia dng trnh c hin tng qu ti ca ct

V d v bm chia dng

4.5. Cc loi detector (u d) thng s dng trong sc k kh

Cc c tnh ca detector trong sc k kh Cc detector thng s dng trong sc k kh
Detector ion ho ngn la (Flame ionization detector FID) Detector dn nhit (Thermal conductivity detector TCD) Detector cng kt in t (Electron capture detector ECD) Detector quang ho ngn la (Flame photometric detector FPD) Detector ion ho pht x ngn la (Flame thermionic detector FTD hay NPD) Detector khi ph (Mass spectrocopic)

c tnh ca detectors
Detector nh th no c gi l l tng? Phn loi detector
Phn loi theo s phn hy v khng phan hy mu Phn loi theo tnh chn lc v tnh a nng Tnh nng ca cc u d

Detector nh th no c gi l l tng?
nhy phi t 10-8 10-15 g cht tan

n nh v lp li tt
p ng theo ng thng i vi cht phn tch Khong nhit t nhit phng ti t nht l 400oC Thi gian p ng ngn tin cy cao v d s dng

S gng nhau v mt p ng
n ca ng nn thp

Phn loi detector theo s phn hy v khng phan hy mu

Detector phn hy mu Mass Spectral (CI/EI)

Detector khng phn hy mu

Thermal Conductivity (TCD)

Flame Ionization (FID)

Nitrogen-Phosphorus (NPD) Flame Photometric (FPD) Electrolytic Conductivity

Electron Capture (ECD)

Photo Ionization (PID)

Phn loi theo nhy v tnh a nng

Detectors a nng: TCD
Xc nh tt c cc cht
Thun li trong vic nh tnh

Detectors chn lc: ECD,NPD,FPD

p ng tng loi c tnh ca hp cht Tng nhy khi phn tch hm lng vt
75

Cc detector thng s dng trong sc k kh

GC Detector
Type
Flame ionization (FID)

Applicable samples
Hydrocarbons

Typical detection limit

0.2 pg/s

Thermal conductivity (TCD)

Electron Capture (ECD) Mass Spectometer (MSD) Nitrogen-phosphorous (NPD)

Universal detector
Halogenated compounds Tunable for any species N, P compounds

500 pg/mL
5 fg/s 0.25-100 pg 0.1 pg/s (P) 1 pg/s (N) 0.5 pg Cl/s

Electrolytic conductivity (ELCD)

Compounds containing halogens, S, or N

2 pg S/s 4 pg N/s

Photoionization (PID)
Flame Photometric (FPD) Fourier Transform IR (FTIR)

Compounds ionized by UV
S, P compounds Organic compounds

2 pg/C/s
10-100 pg (S) 1-10 pg (P) 0.2-40 ng
77

Flame Ionization Detector (FID) (u d ion ha ngn la)

Chn lc:
Cc hp cht c lin kt C-H

p ng km cho cht hu c khng c cha H (e.g. hexachlorobenzene)

Km nhy i vi cc cht kh khng chy nh H2O, CO2, SO2 and NOx

78

FID Assembly

80

Detector FID
Nguyn tc Mu b bn ph bi cc chm electron to thnh cc ht in tchdng in Nu ch c kh mang, dng in i qua gi l dng nn gim thiu ng nn ngi ta dng dng in b chnh ng dng
C nhy cao hn TCD 1000 ln Ch thch hp cho cc hp cht hu c cha cacbon Mt s hp cht khng th pht hin bng FID nh: CO, CO2, axit formic, formaldehit, NOx, NH3, hp cht halogen, CS2, H2S, H2O v bn thn cc kh cn thit dng hot ng detector ny nh H2, N2, He.

Detector TCD (u d o dn nhit)

Nguyn tc
dn nhit o kh nng ca mt cht vn chuyn nhit t mt vng nng sang vng lnh S va chm phn t vi si t trong mt n v thi gian cng ln th tc mt nhit cng ln. Phn t cng nh tc chuyn ng cng cao, dn nhit cng tt. Heli l kh mang thng dng c s dng cho detector o dn nhit. Detector s o s thay i th ny theo cu Wheatstone Ph hp xc nh cc cht kh nh: O2 , H2, CO, N2, Sox, Trnh s dng phn tch cc hp cht c tnh axit hoc halogen v chng s lm hng filament v lm gim in tr ca h gim nhy

Bng dn nhit ti 273 K v 1 atm

Cu to u d TCD

ECD detector (u d bt gi in t)

Gii thiu v GC-MS

Gas Chromatography
E A

D B

Gas Chromatograph
C

Sample: mixture of volatile liquids (~1mL)

Gas Chromatogram B A C E

Abundance

D
0 5 10 Time (minutes) 15 20

Gas Chromatograph

Injection Port

Detector Capillary Column

Data System or Recorder

Carrier Gas Supply

Oven

Mass Spectrometry
O H3C C N C O N H C C C H3 N C H N Mass Spectrometer

Typical sample: isolated compound (~1 nanogram)

Mass Spectrum

194

67

109

55 82 42

94 40 60 80 100 120

136 140

165 160 180 200

Mass (amu)

Mass Spectrometer

Sample Introduction

Data Output

Inlet

Data System

Ion Source

Mass Analyzer

Ion Detector

Vacuum Pumps

Electron Impact Ionization Source

~70 Volts Electron Collector (Trap)

Positive Ions Repeller Neutral Molecules

Inlet + + +

_ _
+ + + to Analyzer

e-

e_

eElectrons Extraction Plate

Filament

Magnetic Sector Mass Analyzer

ion trajectory in register ion trajectory not in register (too light)

S Ion Source N Electromagnet Detector ion trajectory not in register (too heavy)

Quadrupole Ion Filter

resonant ion non-resonant ion

_ + + _

Detector

Ion Source

DC and AC Voltages

ng dng ca phn tch sc k kh

Methyl tert-butyl ether (MTBE) MSD, Extended 8020 Analysis

Environmental-Hydrocarbons Unleaded Gasoline

Diesel Fuel Column: DB-5ms

Environmental-Pesticides and Herbicides

Environmental-Pesticides and Herbicides

Organophosphorous Pesticides

Environmental-Semivolatiles

Environmental-Semivolatiles

118 compounds in EPA Method 525.2 GCMS

Food and Flavors by GCMS

Perfume