1

Chng 1: C S L THUYT CHUNG V TCH SC K 1.1. Gii thiu phng php

1.1.1 nh ngha sc k (Chromatography) nh ngha ca Mikhail S. Tsvett (1906): Sc k l mt phng php tch trong cc cu t ca mt hn hp c tch trn mt ct hp th t trong mt h thng ang chy. nh ngha ca IUPAC (1993): Sc k l mt phng php tch trong cc cu t c tch c phn b gia hai pha, mt trong hai pha l pha t nh ng yn cn pha kia chuyn ng theo mt hng xc nh. 1.1.2. Phn loi Ngi ta phn loi cc phng php sc k da vo c ch hot ng sc k: hp ph, phn b, trao i ion v vo tnh cht ca pha t nh cng nh phng php th hin sc k. V d: - Phng php sc k l ng rn trn ct, phng php sc k phn b kh l ng trn ct. - Phng php sc k phn b lng l ng trn bn phng hai chiu. - Phng php sc k phn b lng lng pha ngc p sut cao trn ct C ch sc k c nhiu nhng thc hin qu trnh sc k th ch c hai dng: dng ct v dng bn phng (bn knh, polime, kim loi, giy). Trong sc k ct, pha tnh c gi trong mt ct ngn v pha ng c cho chuyn ng qua ct bi p sut hoc do trng lc. Trong sc k bn mng, pha t nh c ph trn mt mt phng thy tinh hoc kim loi. Thng n gin ha, tuy khng chnh xc ngi ta gi tt cc phng php sc k: sc k kh, sc k l ng, sc k l ng cao p, sc k lp mng, sc k gel Trong s cc phng php sc k c bit, quan trng nht l sc k hp ph, sc k phn b v sc k trao i ion. Di y s gi i thiu ba phng php sc k ny nhm vo hai mc ch: chun b mu cho cht phn tch v phn tch mt hn hp cht.
Bng 1: Phn loi cc phng php sc k ct

Phn loi chung Sc k lng (LC) (Pha ng : lng)

Phng php c th - Lng- lng hoc phn b - Pha lng lin kt - Lng-rn hoc hp ph trao i ion

Pha tnh - Lng c ph trn mt cht rn - Cht hu c c gn trn mt b mt rn - Rn Nha trao i ion

Kiu cn bng - Phn b - Phn b gia cht l ng v b mt lin kt - Hp ph Trao i ion

C s phn tch sc k

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Sc k kh (GC) (pha ng: Kh)

- Kh-l ng - Kh - pha lin kt - Kh - rn Pha lng: cht lng siu ti hn (supercritical fluid)

Sc k lng siu ti hn

- Lng c ph trn mt cht rn - Cht hu c c lin kt trn mt b mt rn - Rn Cht hu c c lin kt trn mt b mt rn

- Phn b gia kh v l ng - Phn b gia l ng v b mt lin kt - Hp ph Phn b gia cht lng siu ti hn v b mt lin kt

Pha ng Mu Lp mng pha tnh Bn mng tr

im n dung mi Pha tnh Hng chy pha ng Cc cu t tch ra Nng ca B Hng chy pha ng Sc k Cc cu t gii tch ra Mu (A+B+C) Xut pht

Detector

Pha ng

Hnh 1.1: S nguyn tc a) sc k ct b) sc k bn mng

1.2. C s l thuyt chung ca phng php sc k

1.2.1 Qu trnh sc k Sc k l mt k thut tch trong cc cu t cn tch trong mt hn hp mu c vn chuyn bi pha ng i qua pha tnh. Mu i vo tng ng c mang theo dc h thng sc k (ct, bn phng) c cha pha tnh phn b u khp. Pha ng c th l pha l ng hoc kh, pha t nh c th l mt lp phim c ph trn b mt ca cht mang tr hoc mt b mt rn. S tng tc xy ra gia cc cu t vi pha tnh nh cc cu t s phn b gia pha ng v pha tnh.

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C s phn tch sc k

S i lc khc nhau ca cc cht tan trn pha t nh lm chng di chuyn vi nhng vn tc khc nhau trong pha ng ca h thng sc k. Kt qu l chng c tch thnh nhng di trong pha ng v vo lc cui ca qu trnh cc cu t ln lt hin ra theo trt t tng tc vi pha tnh. Cu t di chuyn chm (tng tc yu) ra trc, cu t b lu gi mnh hn ra sau d i dng cc nh (pic) tch ring r (hoc bc thang) ty thuc vo cch tin hnh sc k v c hin th di dng sc k . Hnh 1.1 minh ha mt qu trnh tch mt hn hp n gin gm hai cht A v B (lc tng tc vi pha tnh ca A < B) theo thi gian. 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 (ti th i im to). 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 t nh s xy ra (thi im t1). 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 b i pha t nh (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 kh i nhau. Nu t mt detect c kh nng pht hin c cc cht tan (cu t A v B) ti cui ct tch v tn hiu ca n c v li nh mt hm ca thi gian (hoc th tch c thm vo) th mt lot pic i xng s c ghi li v c gi l sc k . V tr ca cc pic theo thi gian c dng nhn din nh tnh v din tch ca pc c dng cho php phn tch nh lng ca cu t ang xt.

Bi Xun Vng

C s phn tch sc k

M u

Pha ng

Ct nhi

Detector

Sc k

Thi gian

Hnh 1.2: Qu trnh tch sc k trn ct ca hai cht A v B

1.2.2. Cc phng php tin hnh tch sc k thc hin tch sc k ngi ta c th s dng mt trong ba phng php sau: 1.2.2.1. Phng php ra gii K thut ny c s dng rng ri nht trong cc phng php sc k hin nay. Mt lng nh hn hp mu c gii thiu vo ct vi pha ng c i lc vi pha t nh b hn so vi bt k cu t cn tch c trong mu. V th cc cu t cn tch di chuyn
Bi Xun Vng C s phn tch sc k

vi tc chm hn so vi cht ra gii. Tc ny c xc nh bi i lc tng i ca m i cu t ln pha tnh so vi pha ng, chnh l h s phn b K = Csp/Cmp. y Csp, Cmp l nng ca cu t ang xt trn pha tnh v trong pha ng. Cc cu t c ra gii theo trt t i lc ca chng nhng tc di chuyn tng i ca chng ph thuc vo tng tc 3 thnh phn gia chng vi pha ng, vi pha tnh v gia pha ng vi pha t nh. Bi v cc cu t c tch kh i nhau vi mt vng pha ng gia chng nn phng php ny c s dng trong cc php tch vi mc ch phn tch. Pha ng c th khng thay i thnh phn dung mi trong sut qu trnh ra gii; c th thay i dung mi ra gii sau mt thi gian nh trc (ra gii theo tng giai on); cng c th khng thay i cc dung mi to nn pha ng nhng thay i nng ca cc thnh phn c trong pha ng sau mt thi gian nh trc (ra gii gradient). 1.2.2.2. Phng php tin lu Hn hp cn tch gm cc cht A, B v C c cho chy lin tc vo phn trn ca ct, trong A l cu t c i lc yu nht vi pha t nh. Do cc cu t A, B v C b lu gi trn ct, nn trc ht t ct chy ra ch c dung mi. A do c lc tng tc trn ct yu nht s di chuyn xung di cn cc cu t c i lc mnh hn A b pha tnh gi phn trn ca ct. Do dung dng c hn ca pha tnh nn khi vt dung lng ny th cu t A s di chuyn dc theo ct v ra khi ct dng nguyn cht sau l hn hp ca cc thnh phn tip theo A+B ri A+B+C. Phng php tin lu t c dng do khng thc hin c vic tch hon ton cc cu t, c bit khi s dng s tch sc k vo mc ch phn tch. 1.2.2.3. Phng php th y: Mu c cho vo ct, dng dung mi ra gii c i lc vi pha t nh mnh hn bt k mt cu t no ca hn hp tch y cc cu t cn tch thot ra khi ct. Cu t thot ra khi ct u tin l cu t tng tc yu nht vi pha t nh, sau n cc cu t khc c i lc vi pha tnh tng dn. Phng php ny to nn cc di ra gii khng hon ton c tch kh i nhau: c d i thu c cht nguyn cht nhng c di gia cc di nguyn cht th g m hn hp ca chng. Trong thc hnh phng th nghim tch cc hn hp phc tp ngi ta thng hay dng hn c l phng php ra gii.

Bi Xun Vng

C s phn tch sc k

Dung mi ra gii (E)

Pha tnh

Ra gii

Tin lu

Th y

n Detector

Tn hiu Detector

Th tch dung mi ra gii

Hnh 1.3: Cc phng php ra gii, tin lu, th y

1.2.3. c tnh sc k ca cht tan 1.2.3.1. Tnh cht lu gi Tnh cht lu gi phn nh s phn b ca cht tan gia pha t nh v pha ng v c biu th bng cc i lng th tch lu hay thi gian lu. Th tch lu VR l th tch pha ng cn thit vn chuyn cht tan i t thi im a mu vo, i qua ct v n detect (trn sc k l im cc i ca pic). Thi gian lu tRi l thi gian t lc cht tan i c np vo ct tch b phn tim mu cho n lc cht ra khi ct thi im c nng cc i.

Bi Xun Vng

C s phn tch sc k

Th tch lu c th nhn c trc tip t thi gian lu tR trn sc k bng cch nhn vi tc th tch dng Fc (th tch ca pha ng trn mt n v thi gian): VR = tR. Fc Tc dng c tnh theo cc thng s ca ct nh sau: Fc y: dc l ng knh ca ct, L l chiu di ct, t l rng ton phn ca cht nh i ct ( rng ca cht nh i ct biu th t s th tch k h ca cht nh i v th tch khi ton phn ca n: i vi cht nh i rn = 0.35 - 0.45, cht nh i xp = 0.70 - 0.90, ct mao qun = 1.0), Vc l th tch bn trong ct. L Tc tuyn tnh trung bnh u ca s dch chuyn cht tan l: u tR Tc tuyn tnh trung bnh u ca pha ng l:
u L tM

d c2 4

L tM

y tM hay to l thi gian khng b lu gi ca cht tan (thi gian cht tan lu trong pha ng). Trong sc k kh, tM c ly bng thi gian cn thit cho CH4 di chuyn i qua ct. Tng ng tM hay to c th tch VM (hoc Vo) biu th th tch trng ca ct (bao gm c th tch ca b phn tim mu (injector), th tch cc on ng n i, th tch rng ca chnh ct v th tch detect). Th tch lu hiu chnh VR hoc thi gian lu hiu chnh tR c cho bi: VR = VR VM hoc tR = tR tM (thi gian khng b lu gi ca cht tan gn ng c xem nh l thi gian lu ca pha ng).

Bi Xun Vng

C s phn tch sc k

Tim mu

Cng tn hiu Detector

Thi gian (th tch)

Hnh 1.4: Sc k minh ha cc thi gian lu t (hoc th tch lu) v rng c a y pic W ca cht tan khng b lu gi M v ca 2 cht b lu gi 1 v 2

1.2.3.2. H s phn b K (Partition coefficient) Mi cu t hoc cht tan c phn b gia 2 pha vi mt cn bng c thit lp v tt c cc qu trnh tch sc k c da trn s khc nhau v kh nng phn b ca cht tan gia pha ng v pha tnh. Khi cht tan i vo h thng sc k, n ngay lp tc phn b gia cc pha ng v pha tnh. Gi thit nu pha ng dng li vo bt k mt thi gian no, cht tan c mt s phn b cn bng gia hai pha, v nng ca cht tan trong mi pha c cho bi h s phn b nhit ng:
K Cs CM

y Cs v CM l nng cht tan trong pha tnh v pha ng tng ng. Trng hp khi K = 1 th cht tan c phn b bng nhau gia hai pha. H s phn b xc nh tc trung bnh ca m i vng cht tan (chnh xc l tm vng) do pha ng vn chuyn khi n i qua ct. i vi pic i xng, khi cc i pic xut hin li ra ca ct, mt na lng cht tan c ra khi ct trong th tch lu VR v mt na cn li c phn b gia th tch pha ng VM v th tch pha tnh Vs: VRCM = VMCM + VsCs hay hay VR = VM + KVs VR VM = KVs

Bi Xun Vng

C s phn tch sc k

Phng trnh ny ng vi ct phn b lng, cn i vi ct hp ph, Vs phi thay bng Ss l din tch b mt ca cht hp th. 1.2.3.3. H s dung lng k (The capacity factor) H s dung lng k l thng s thc nghim quan trng trong sc k ct c s dng rng ri m t cc tc dch chuyn ca cc cht tan trn ct. Cho mt cht tan, h s dung lng k c nh ngha nh l t s mol cht tan trong pha tnh v s mol cht tan trong pha ng: k =
CsVs C M VM K Vs VM

T s VM/Vs c gi l t s th tch pha H s dung lng cn c nh ngha l t s gia thi gian ca cht tan lu li trn pha tnh v thi gian cn cht tan di chuyn trong pha ng sut chiu di ca ct nu khng b lu gi tM. k'
' tR tM

tR tM

tM

VR VM VM

Khi k cho mt cht tan nh hn 1 nhiu, s ra gii xy ra rt nhanh nn vic xc nh thi gian lu l rt kh. Cn khi k khong 20 n 30 th thi gian ra gii s b ko di. V vy cc php tch nn c thc hin nhng iu kin m cc k ca cc cht tan nm trong khong gi tr t 1 n 5. Gi tr k trong sc k kh c th c thay i bi vic thay i nhit v cch nh i ct. Cn trong sc k l ng, k thng c iu khin cho php tch tt hn bi thay i thnh phn pha ng v pha tnh. 1.2.3.4. H s chn lc (The selectivity factor) Kh nng ca mt pha t nh tch 2 cu t A v B (B l cu t b lu gi mnh hn) c xc nh bi t s phn b tng i ca chng v cng l nhng h s lu gi ca chng i vi mt pha tnh cho. H s chn lc l mt hm ca s lu gi tng i ca m i cu t trn pha t nh:
' tR 2 ' t R1 ' kB ' kA

KB KA
C s phn tch sc k

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H s chn lc ca mt cp di (pic) gn nhau l mt hm ph thuc vo loi pha tnh c s dng, pha ng v nhit ca ct. cho mt s tch c tt th nn c ga tr ln hn 1. Hai cht cn tch cng tch ra xa kh i nhau khi cng ln nhng nu qu ln th thi gian phn tch cng ko di, nn trong khong t 1,05 n 2,0. 1.2.3.5. Nhng yu t nh hng n s lu gi Tc di chuyn ca mt di cht tan qua mt ct hay mt a sc k lp mng ph thuc vo s phn b ca cc phn t cht tan gia pha t nh v pha ng. Nhng nhn t nh hng n s phn b hay s lu gi l: Thnh phn v tnh cht ca pha ng Kiu v tnh cht ca pha tnh Cc lc tng tc cc phn t gia cc cu t ca pha ng v pha tnh Nhit Hai yu t gp phn tch tt cc hp cht bi sc k l: S khc nhau v thi gian phn b ca chng trong 2 pha: nu s khc nhau cng ln th s tch chng cng tt. dong rng ca cc pic nu cng rng th s tch chng cng km.

1. 3. S dong rng ca pic v hiu lc tch

1.3.1. M u Hiu lc ca mt ct tch chu tc ng bi ln ca s dong rng pic xy ra khi mt cht chuyn ng dc theo ct. Cht tan khi chy dc theo ct c khuynh hng phn b nh mt hnh phn b chun Gaussian vi lch chun (hnh 5). Nu cht tan tiu tn nhiu thi gian khi dch chuyn dc xung ct th pic cng b dong rng. dong rng ca mt pic thng c biu din thng qua cc gi tr: W1/2 l rng ca ca pic c o chiu cao ca pic, W l rng ca y pic c xc nh ti hai im ct ca hai ng tip tuyn ca hai bn thn pic vi ng nn.

Bi Xun Vng

C s phn tch sc k

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Tim mu

Chiu cao pic

Chiu cao pic

lch chun ca di

Trc thi gian

Hnh 1.5: S ca mt di cht tan v s nh gi ca di sc k cho hiu lc ct.

1.3.2. S khuch tn Nguyn nhn chnh dn n s dong rng ca pic l s khuch tn. H s khuch tn D o tc J ti mt cht di chuyn mt cch ngu nhin t mt vng c nng cao n vng c nng thp.

J(

mol ) m2 .s

dc dx

D l h s khuch tn c l nng x l di di chuyn S khuch tn trong cht lng thp hn trong cht kh khong 104 ln, cn cc phn t ln th khuch tn chm hn cc phn t nh t 10 n 100 ln. rng ca di sc k do khuch tn c biu din qua phng trnh sau:

c
y:

m e 4 Dt

x 2 /( 4 Dt )

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C s phn tch sc k

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c l nng (mol/cm3), t l thi gian, x l khong cch di chuyn c dc theo ct, D l h s khuch tn, m l s mol cht tan dch chuyn qua mt n v din tch tit din ngang ca ct. 2 Dt

Phng trnh trn cho bit lch chun ca di sc k: 1.3.3. Chiu cao a l thuyt H

Chiu cao a l thuyt H l mt hng s gia phng sai 2 ca di cht tan v khong cch x m n di chuyn. Tn g i ny l t l thuyt chng ct trong s tch c thc hin trong nhng giai on ri rc c gi l a tch. H = 2/L Vi L l chiu di ca ct nhi C th gi nh ct sc k c chia thnh N phn m ng hay N lp hay N a tch. m i a s phn b cht tan vo hai pha li t n m t trng thi cn bng mi (sc k l mt qu trnh ng, thc ra pha ng chy lin tc v thi gian khng thit lp trng thi cn bng). Chiu cao ca a tch cng c gi l chiu cao a l thuyt. Mc d trong ct sc k khng c cc a thc nhng ngi ta vn s dng khi nim ny ch m i quan h gia rng ca di sc k v khong cch dch chuyn dc theo ct sc k. Nu chiu cao a l thuyt cng nh (tc s a l thuyt cng nhiu) th rng hay s dong rng ca pic cng nh. Kh nng tch cc cu t ca mt hn hp ca mt ct c ci thin nu gim chiu cao mt a l thuyt. Cc cht tan khi dch chuyn qua cng mt ct tch c chiu cao a l thuyt khc nhau bi v chng c h s khuch tn khc nhau. Chiu cao mt a l thuyt khong t 0.1 n 1 mm trong sc k kh v khong 10 m trong sc k l ng hiu nng cao v nh hn 1m trong sc k in di mao qun. 1.3.4. S a l thuyt N N=

L H

L2
2

y L l chiu di ct, vi

L W nn: 4t R

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C s phn tch sc k

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2 16t R N= W 2
2 5.54 t R W 12 /2

Hay N = Vi:

tR l thi gian lu ca pic, W l rng ca pic y, W1/2 l chiu rng ca pic o na chiu cao t nh n y pic.

Cng thc ny c s dng xc nh thc nghim s a l thuyt ca mt ct tch. Hiu lc ca ct tch s tng ln khi s a l thuyt cng ln v khi chiu cao a cng nh.

1.4. Cc yu t nh hng n hiu lc tch ca ct

1.4.1. nh hng ca tc ca pha ng Mc nh hng ng hc ln hiu lc ca mt ct r rng ph thuc vo di th i gian ca pha ng tip xc vi pha tnh tc ph thuc vo tc pha ng. Nghin cu v hiu lc ct ni chung c thc hin bi vic xc nh chiu cao H nh l mt hm ph thuc vo tc pha ng. Kt qu cho thy c sc k kh ln sc k lng u c chiu cao a l thuyt t cc tiu trong vng tc dng thp ca pha ng v ni chung tc ca sc k l ng thp hn so vi sc k kh. 1.4.2. L thuyt v s dong rng pic Hn 40 nm qua, rt nhiu cng trnh nghin cu v thc nghim ln l thuyt v m i quan h nh lng gii thch nh hng ca cc thng s nh: vn tc tuyn tnh ca pha ng, h s khuch tn trong pha ng v trong pha t nh, t s phn b, ng knh ca ht nh i, b dy ca lp cht lng ph trn ht n chiu cao a l thuyt cho cc loi ct khc nhau. Nhng yu t nh hng ny c phn nh qua phng trnh ca Van Deemter nh sau:

H
y:

B u

Cs u CM u

H l chiu cao a l thuyt (cm); u l vn tc tuyn tnh ca pha ng (cm/s);

Bi Xun Vng

C s phn tch sc k

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A l h s m t nh hng n H ca s khuch tn xoy; B l h s khuch tn di; Cs v CM l cc h s chuyn kh i ca pha tnh v pha ng.

ng H = f(u) S hng B S hng Cs

S hng CM

S hng A

Vn tc tuyn tnh trung bnh u

Hnh 1.6: ng cong H-u (Van Deemter) cho mt ct sc k kh

1.4.2.1 S khuch tn xoy: Thnh phn A m t s khuch tn xoy ca cc cu t v cc ng i khc nhau ca pha ng chy xuyn qua pha tnh c nh i trong ct. V th cc cu t phi di chuy n vi nhng khong cch ng i khc nhau qua mt n v chiu di ca ct. S hng A c lp vi tc ca pha ng nhng ph thuc vo: ng knh ht nh i trong pha tnh dp, Cch chng c nhi trong ct hoc c ph trn bn mng c biu din qua l hm s ph thuc vo ng th ca cht nhi, dng hnh hc v kch thc ca ct: A = .dp Mt s phn t cht tan c ng i gn vi thnh ct s di chuyn nhanh hn so vi nhng phn t khc do mt nhi tng i thp, tc nhanh hn. Cc phn t i theo ng ngn hn c ra gii ra trc nhng phn t i theo nhng ng quanh co di hn dn n dong rng pic ca m i cht tan. Mt s tch v rng ca pic ti thiu s t c nu dng cc ht nhi c ng knh trung bnh nh v c nhi ng th vo trong ct. Tuy nhin, ht cng nh th p sut cn pha ng dch chuyn qua ct cng cao, c bit s gp kh khn nh i ct ng th. Trong sc k kh ct nhi ngi ta nhi ct vi cc ht c kch thc ti u

Bi Xun Vng

C s phn tch sc k

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khong 0.15 m. Trong sc k kh l ng khi s dng mng m ng trn thnh ct mao qun th s hng A = 0.
Ht pha tnh Tng tc cht tan vi ht pha tnh

Pha ng

Nng ca di

S khuch tn ra trc v sau ca di

Pha ng Di cht tan

Pha ng

Di chuyn vo pha tnh

Pha tnh

Cht tan b gi trong pha tnh Di chuyn khi pha tnh

Hnh 1.7: nh hng ca khuch tn xoy (a), khuch tn dc (b) s truyn khi (c) n phng trnh Van Deemter.

1.4.2.2. S khuch tn dc Khuch tn l qu trnh cht di chuyn t ni c nng cao n ni c nng thp hn vi tc t l vi s chnh lch nng v h s khuch tn ca cht ang xt. Trong sc k, khuch tn dc c dng m t s khuch tn din ra dc theo trc ca ct v song song vi ng i ca pha ng. N dn n s dch chuyn ca cht tan t trung tm nng ca mt di cht tan n nhng vng long hn v hng pha trc hoc sau ca dng chy. Khuch tn dc l mt ngun ca s dong rng ca pic trong sc k kh do tc khuch tn cao trong pha kh. Hin tng ny t hn trong sc k lng. ln ca B

Bi Xun Vng

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trong phng trnh Van Deemter c xc nh phn ln bi h s khuch tn DM ca cht phn tch trong pha ng v t l thun vi hng s ny: B = 2. DM y: l h s tr khng ca cht nh i ct n khuch tn dc, DM l h s khuch tn ca cht tan trong pha ng. Trong cc ct mao qun th c gi tr bng 1 cn trong ct nhi th khong 0.7. B nh hng ln trong phng trnh V-D khi tc pha ng thp nn trong sc k l ng nh hng ny t hn nhiu bi v pha ng c tc thp hn nhiu so vi ca sc k kh. 1.4.2.3. S truyn khi trong pha tnh Khi pha tnh l cht lng: h s truyn kh i Cs t l thun vi bnh phng chiu dy ca lp phim trn b mt cht mang v t l nghch vi h s khuch tn Ds ca cht tan trong lp phim. Gii thch: vi lp phim dy cc phn t cht tan phi di chuyn trung bnh xa hn tin n b mt, v vi h s khuch tn nh hn, chng s di chuyn n chm hn. Hu qu l tc truyn khi chm hn v chiu cao a l thuyt tng ln. Khi pha tnh l mt b mt rn: h s truyn khi pha tnh Cs t l thun vi thi gian c i h i cho nhng cu t b hp ph hoc gii hp, hay t l nghch hng s tc bc nht ca cc qu trnh ny. S chuyn ng ca phn t trong pha tnh tiu tn mt thi gian di hn, trong khi nhng phn t khc chuyn ng v pha trc cng vi pha ng, s dn n pic s dong rng. Pha ng i qua ct cng nhanh v tc truyn kh i trong pha t nh cng nh th pic cng dong rng hn. Khc phc: nhng cht lng t nht hn c th c chn lm pha tnh tng h s khuch tn. C th gim chiu dy ca pha t nh nhng dung lng ca ct s b gi m xung. 1.4.2.4. S truyn khi trong pha ng Cc qu trnh truyn kh i trong pha ng rt phc tp. S hiu bit tt v mt nh tnh ca nhng yu t t nguyn nhn ny tc ng n s dong rng ca pic dn n s ci thin ln lao trong tt c cc loi ct sc k. H s truyn khi trong pha ng CM: t l nghch vi h s khuch tn ca cht tan trong pha ng DM t l thun vi bnh phng ng knh ca ht nhi, t l thun vi bnh phng ca ng knh ct v tc chy ca pha ng.

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S dong rng ca di trong pha ng l do mt phn vo s ng cc ng i nh mt phn t hoc ion c th tm c cch ca n i qua ct nh i. Trn b mt ca pha tnh c th cha cc l khch thc khc nhau nn mt s cc phn t cht tan c th chuyn ng vo ra cc l ny dn n s b chm so vi nhng phn t khc. Cc dng cht lng ca pha ng gn cc ht chuyn ng chm hn dng cht lng gia khong cch cc ht. Nh vy thi gian lu trong ct cho cc phn t ca cng mt cht cng khc nhau. Cc phn t ny i n ct qua nhng khong thi gian khc nhau dn n s dong rng pic. nh hng ny thnh thong c g i l khuch tn xoy, c lp vi tc ca dung mi. 1.4.2.5. Cc phng php gim s dong rng pic Hai yu t quan trng nh hng n hiu qu ca ct l ng knh ca cc ht nhi v ng knh ca ct: Nu s dng cc ht nhi c ng knh cng nh th cng gim chiu cao a l thuyt. Trong khi li dng nh hng ca ng knh ct, cc ct ngy cng hp hn c s dng trong nhng nm gn y. Vi pha ng kh, tc ca khuch tn dc c th c gim mt cch ng k bi h thp nhit v nh vy lm gim h s khuch tn DM. iu ny dn n chiu cao a l thuyt s nh hn ti nhit thp. nh hng ny khng ng k trong trng hp sc k l ng bi v s khuch tn l chm nn s khuch tn dc c nh hng rt nh n chiu cao a l thuyt. i vi pha t nh lng, chiu dy ca lp cht lng hp th nn cng m ng cng tt bi v Cs trong phng trnh Van-Deemter t l thun vi bnh phng ca i lng ny.

1.5. phn gii ca ct (Column resolution)

1.5.1. nh ngha Trong sc k, phn gii Rs ca hai pic c xc nh nh sau (hnh 8): Rs =

tR Wav

VR Wav

t R 2 t R1 W2 W1 2

y : o tR v VR l hiu ca thi gian hoc th tch lu gi ca hai pic, o Wav l rng trung bnh ca hai pic.

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Tim mu

Thi gian

phn gii km, chn lc ti

phn gii km, chn lc tt nhng thi gian phn tch qu di, pic b dong rng

phn gii km, chn lc tt nhng thi gian phn tch di

phn gii km, chn lc tt v thi gian phn tch hp l

Hnh 1.8: S phn gii pic.

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phn gii ca mt ct tch cho bit kh nng tch hai cht phn tch bi ct. Nu ct tch c phn gii khong 1.5 th php tch A v B gn nh hon ton (s xen ph ch cn khong 0.3%), trong khi nu phn gii 0.75 th khng tch c cn phn gi i bng 1 th s xen ph ca hai pic cn khong 4%. phn gii cho mt pha tnh chn c th c ci thin bi tng chiu di ca ct tch (tc tng s a l thuyt N ca ct tch) nhng nh vy s lm ko di thi gian phn tch. 1.5.2. Cc yu t nh hng n phn gii Mi quan h gia s a l thuyt v phn gii l: T
k' tR tM
' k2 k1'

tM

2 16t R W 2

Rs =

tR 2 t R1 W2 W1 2

Rt ra: phn gii Rs =

N ( 4 1 )(
' k2 ) ' 1 k2

y: N l s a l thuyt, k2 l h s dung lng ca cu t th hai, =

' k2 k1'

Nu s a l thuyt ca 2 pic khng gi ng nhau th thay th N bng kav.

N1 N 2 v thay k2 bng

Ci thin Rs nh chiu cao a l thuyt

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Mt chi tit quan trng trong phng tr nh trn l phn gii t l thun vi

N . V

vy nu tng gp i chiu di ct th tng phn gii ln 2 ln. Tuy nhin khi tng s a l thuyt ln do tng chiu di ct th qu trnh sc k phi tiu tn nhiu thi gian hn tr khi s tng s a l do gim chiu cao mi a v khng tng chiu di ca ct, nhit ca ct (cho trng hp sc k kh) v chiu dy ca lp phim (cho sc k lng). Vic ti u ha tc pha ng cng c ch. Ci thin Rs nh h s chn lc Khi tng hay khi tng t s phn b th phn gii cng tng theo. Cch thay i l thay i pha t nh hoc thay i c pha t nh ln pha ng trong sc k l ng bi v thay i bn cht ca cc pha s dn n thay i . Khi t s phn b =2 th m bo cho s phn gii tt. Tuy nhin s ti u ny l cho mt cp cu t. Trong trng hp hn hp cha nhiu cu t th cn phi thay i k c s phn tch gia cc cu t. Trong sc k kh, gi tr ti u ha ca k c th t c bng chng trnh ha nhit cn trong sc k l ng th bng chng trnh ha thnh phn pha ng tc thay i t l gia cc dung mi ca pha ng. Ci thin Rs nh h s dung lng Thng s tch c th c ci thin mt cch ng k bng vic thay i h s dung lng k. Ni chung, s tng gi tr k lm tng phn gii. xc nh khong ti u ca gi tr k, phng trnh c vit di dng:

Rs

k' 1 k'

y Q cha phn cn li ca phng trnh. Thng gi tr ca k > 10 nn trnh v d t ng ln cht t phn gii nhng thi gian i h i cho s tch tng ln rt ng k. Gi tr ti u ca k trong khong t 1 n 5. Cch d nht ci thin phn gii l ti u ha k: i vi pha ng l kh, k thng c th c ci thin bi s thay i nhit . Cc phng php gim chiu cao ca a l thuyt nh c cp trong mc 4, bao gm gim kch thc ht nhi, ng knh ca ct

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Bi tp
Cu 1.1: Trnh by nh ngha ca cc khi nim sau: (2 im) a) b) c) d) e) f) g) h) S ra gii Pha ng Pha tnh H s phn b Thi gian lu H s dung lng chn lc Chiu cao a l thuyt

Cu 1.2: Trnh by cc yu t lm dong rng cc pic (1.5) Cu 1.3: M t phng php xc nh s a l thuyt ca mt ct sc k. Cu 1.4: Trnh by s khc nhau gia a) Sc k kh-lng v sc k kh-rn b) Sc k lng-lng v sc k l ng-rn Cu 1.5: Trnh by cc phng php ci thin phn gii ca hai cht trn mt ct sc k. Cu 1.6: Trong mt php phn tch sc k tinh du chanh, pic ca limonene c thi gian lu l 8.36 min vi rng y pic bng 0.96 pht. Terpinene ra gii 9.54 pht, vi rng y pic l 0.64 pht. Tnh phn gii ca 2 pic trn? (Ans: 1.48) Cu 1.7: a) Trong mt php phn tch sc k cc axit c trng lng phn t thp, butyric acid ra gi thi gian lu l 7.63 pht. Thi gian khng lu gi TM ca ct l 0.31 pht. Tnh h s dung lng ca butyric acid. (Ans: 23.6) b) Thi gian lu ca isobutyric acid l 5.98 pht. Hi chn lc ca isobutyric acid v butyric acid? (Ans: 1.29) Cu 1.8: Trong mt php phn tch sc k thuc tr su c clo Dieldrin cho mt pic vi thi gian lu l 8.68 pht v rng y pic l 0.29 pht. Hi c bao nhiu a l thuyt lin quan n php tch ny ? Cho chiu di ct l 2.0 met, Tnh chiu cao mt a l thuyt? (Ans:0.14 mm) Cu 1.9: Mt hn hp benzene, toluene, v methane c tim vo mt my sc k kh. Methane cho mt pic sc nhn 42s, trong khi benzene v toluene c thi gian lu 251 s v 333s. Tnh thi gian lu hiu chnh v h s dung lng cho m i cht tan. Tnh chn lc gia benzene v toluene. (Ans: = 1.39)

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Cu 1.10: Cc s liu sau y thu c t php tch 4 cu t trong ct mao qun di 20-m. Compound tr (min) w (min) A 8.04 0.15 B 8.26 0.15 C 8.43 0.16 (a) Tnh s a l thuyt ca m i hp cht v s a l thuyt trung bnh ca ct. (b) Tnh chiu cao trung bnh ca mt a l thuyt. (c) Gii thch ti sao m i hp cht c th c s a l thuyt khc nhau. Cu 1.11: Moody nghin cu hiu lc tch ca php tch sc k kh 2-butanone trn ct dinonyl phthalate . nh gi chiu cao a l thuyt nh mt hm ca tc dng pha ng theo phng trnh van Deemter vi A l 1.65 mm, B l 25.8 mm x mLx min1, and C l 0.0236 mm x min x mL1. (a) V th biu din H theo u (tc dng) trong khong 5120 mL/min. (b) Khong gi tr no ca tc dng pha ng cho m i gi tr ca A, B v C c nh hng ln nht. (c) Gi tr ti u ca tc dng v chiu cao ca a l thuyt ti gi tr (d) i vi ct mao qun m th gi tr A bng 0. Nu B v C vn khng thay i th tc dng ti u l bao nhiu v chiu cao a l thuyt gi tr ny?

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Chng 2: SC K KH 2.1. M u
2.1.1 Khi nim C s tch bng sc k kh l s phn b ca mu th gia hai pha: pha t nh c b mt tip xc ln, pha ng l kh thm qua ton b mt tnh . Nu pha tnh l rn th g i l sc k kh-rn. Cht rn nh i ct thng l silicagel, ry phn t hoc than hot tnh. Qu trnh ny ch yu l hp ph. Nu pha t nh l l ng, ta c sc k kh-l ng. Cht lng bao bc quanh b mt mt cht rn tr, gi l cht mang, to nn mt lp phim mng. C s cho s tch y chnh l s phn b ca mu trong v ngoi lp phim m ng ny. 2.1.2 Vi nt lch s 1905 Ramsey tch hn hp kh v hi da trn c s hp ph ca than hot. 1906 Tswett tch cc sc t thc vt trn ct v gi qu trnh tch l sc k 1941 Martin v Synge (gii thng Nobel) trnh by l thuyt a trong chng ct v chit sut ngc dng. Trn c s James v Martin xut ra sc k kh-l ng (1952) T n nay GC ngy cng hon thin. n nm 1968 c khong 18000 cng trnh v GC. Nhng nm gn y c s h tr ca cng ngh in t v tin hc, GC t c nhiu thnh tu hn na.

2.2.Thit b
2.2.1. Gii thiu Hin nay, hn 30 nh sn xut cung cp ra th trng khong 150 mu khc nhau ca thit b sc k kh. Trong thp nin 70, cc b tch phn k in t v x l s liu da trn my tnh tr nn thng dng. Thp nin 80 xut hin cc thit b sc k kh vi cc thng s nh nhit ca ct, tc dng, tim mu, c iu khin bng my tnh vi gi c va phi; v c l quan trng nht l s pht trin ca cc ct c kh nng tch nhng mu phc tp c cha nhiu cu t trong mt khong thi gian ngn. Nhng thnh phn chnh ca thit b sc k kh c m t trong hnh 2.1.

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Thit b o tc dng B iu dng B iu p B tim mu

Lp m cao su silicon

L Bnh kh pa ng

Ct tch

Hnh 2.1: S khi ca mt my sc k kh

Tim mu

Thi gian

Hnh 2.2: Mt sc k tiu biu tch cc este bo ha bi sc k khi-lng 1. methyl formate 2. methyl acetate 3. ethyl formate 4. ethyl acetate 5. n-propyl formate 6. iso-propyl acetate 7. n-butyl formate 8. sec-butyl acetate 9. iso-butyl acetate 10. n-butyl acetate

2.2.2 H thng cung cp kh mang Cc kh mang phi tr v mt ha hc nh He, Ar, N2, CO2 v H2 v vic chn la kh mang thng c quyt nh bi lo i detector s dng. H thng cung cp kh mang bao gm cc b iu chnh p sut (pressure regulators), cc thit b o p sut (gauges), v thit b o tc dng. H thng kh mang cn cha mt h thng lc phn t tch nc v cc cht nhi m bn khc. Tc dng c kim sot bi cc b iu chnh p sut hai giai on c lp vo cc bnh cha kh mang.
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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 nh i v khong t 1 n 25 ml/ph i vi ct mao qun. Ni chung, nu p sut i vo thit b khng i th tc dng s khng i. o tc dng kh ngi ta dng thit b o tc dng (flowmeter) vi bt x phng v ng h bm giy. Kh mang gim p sut t l vi nht kh mang nn cn chn kh mang c nht thp cho ct mao qun v ct nh i cht. Bng sau dn ra mt s c tnh c bn ca mt s kh mang Kh Argon Heli Nito Hydro dn in , 10-4 cal/cm. oK 0.52 4.08 0.37 5.47 50oC 242 208 188 94 nht 1 atm 100oC 200oC 271 321 229 270 208 246 103 121 300oC 367 307 139

Khi la chn cn ch n detector ang s dng nh sau: Detector o dn cn phi s dng kh mang c dn cao nh H2, He. Kh He c u im khng nguy him. Detector ion ha ngn la thng s dng kh mang N2 do r v khng nhuy him nhng trong trng hp ghp n i vi cc thit b khc, v d ghp ni vi kh i ph phi dng kh mang l heli. Detector cng kt ngn la thng dng kh mang l N2. 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 th i 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 y l dn nhit ca N2 rt gn vi dn ca nhiu kh v hi nhiu cht hu c nn c trng hp pic sc k c th b ngc.

2.2.3 H thng tim mu

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Cch thng dng nht a mu vo ct l s dng mt bm tim mu vi lng (microsyringe) tim mt mu lng hoc kh qua mt m cao su silicon (septum) chu nhit vo mt bung ha hi (injector). Bung ny c t nng vi nhit thch hp v c n i vi ct tch (hnh 3). i vi cc ct tch thng thng, c mu thng thay i t mt vi n 20l. Ct mao qun i hi lng mu a vo nh hn nn trong trng hp ny h thng chia dng mu c thit k trong b injector c s dng ch giao mt phn nh lng mu c tim i vo ct, phn cn li c thi ra ngoi. K thut s dng bm tim Ht dung dch mu vo bm tim v iu chnh dung dch n vch ri ko ngc pit tng ra sau lng mu chuyn ht vo thn bm tim (u kim rng). Sau khi xuyn kim qua lp m cao su silic ca injector yn khong 3 n 5 giy kim c cn bng nhit trong injector ri mi y pittong. Cch ny gip trnh mt s cu t kh bay hi cn ng li u kim gy sai s khi nh lng nhng cu t ny. Tim mu c chia dng (Split injection) Thch hp cho mu c cc cu t phn tch c nng ln hn 0.1% mu. Vi cc phn tch c phn gii cao, cc kt qu tt nht thu c cho lng nh nht ca mu ( 1l) tim vo c th c d tm khong 1ng cho m i cu t. Vic tim ton b mu s lm qu ti ct c ng knh 0.32 mm hoc nh hn. Trong khi tim mu c chia dng ch gi i thiu vo ct khong 0.2 n 2% lng mu. Mu c tim nhanh (< 1s) qua lp m cao su silic (septum) vo vng ha hi ca h thng tim mu (injector) c gi nhit cao (v d 350oC) qu trnh bay hi mu c din ra nhanh (hnh 4). Mt dng chy mnh ca kh mang li ko mu qua bung trn, c s ha hi hon ton v ha trn tt. Ti im chia dng, mt phn nh ca hi i vo trong ct sc k cn phn ln i qua van chia dng n l thng kh thi. T l ca mu khng c gii thiu vo ct c g i l t s chia dng (split ratio) c gi tr t 50:1 n 600:1. Sau khi mu b sc ra kh i bung tim mu (khong 30 s), van chia dng ng li v kh mang c gim tng ng. Mt l cht lng c tim vo s to khong 0.5 ml th tch kh v nhanh chng lm y bung tim mu. Vi kh c th thot ra tr li theo ng ca m cao su silicon. Cc cu t c nhit si thp s thot ra u tin ri n cc cu t c nhit si cao hn. Nhit ca bung tim mu nn ln gim thiu phn mt ny ca mu. Tuy nhin nu nhit qu cao, s phn hy mu c th xy ra. Trong sut qu trnh tim mu v sc k, c dng kh 1ml/ph lm sch lp m cao su silicon tch hi mu d v cc kh thot ra t lp m cao su ny (septum purge).

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Tim mu khng chia dng (splitless injection) Thch hp cho phn tch lng vt nhng cu t c nng nh hn 0.01% mu v cch tim ny c khong 80 % mu c gii thiu vo ct. .H thng tim mu tng t nh trng hp tim mu chia dng (hnh 2), nhng ng thy tinh trong bung tim mu l thng, trng v khng c bung trn mu. Mt th tch ln (khong 2 l) ca dung dch c pha long trong dung mi c nhit si thp c tim chm (khong 2s) vo trong ng thy tinh ca bung tim mu vi l thot chia dng b ng. Nhit ca bung tim mu cho ch khng chia dng gi thp hn (khong 220oC) so vi ch chia dng v mu b lu gi lu hn trong bung tim mu v trnh mu b phn hy nhit.

Dng lm sch septum 2 mL/min Nhit bng nhit ban u ca l

Ct tch Van chia dng 1 mL/min

Tim mu chia dng

Tim mu khng chia dng

Tim mu vo ctj

Hnh 2.3: H thng tim mu c chia dng, khng chia dng v tim mu vo ct

2.3. Ct sc k
2.3.1. Gii thiu Trong thc t c nhiu dng ct tch khc nhau nhm tha mn cc mc ch nghin cu. Ni chung, ct tch sc k cn tha mn cc yu cu sau: m bo trao i cht tt gia pha ng v pha t nh 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. 2.3.2. Ct nhi

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Ct thng c lm bng thp khng r , nicken, thy tinh vi ng knh khong t 3 n 6 mm v chiu di khong t 1 n 5 m. Ct nh i cha cc ht cht mang rn c ph mt lp pha t nh lng hoc bn thn ht rn l pha tnh. Cht mang rn thng l diatomite c silan ha gim lin kt hydro vi cc cht phn cc:
OH OH ( CH 3 ) 3 SiO OSi(CH ) 3 3

- Si - O - Si - + (CH )3 SiNHSi(CH ) 3 3 3

- Si - O - Si - +

NH 3

Kch thc ht ng nht s lm gim chiu cao ct v tng phn gii. C ht nh s lm gim thi gian cn bng ha tan nh vy lm ci thin hiu qu ca ct. Tuy nhin, nu kch thc ht cng nh th cng t khng gian trng gia cc ht v p sut p pha ng qua ct phi cng cao. Kch thc ht c biu din theo micromet hoc c mesh, tc l c li m ht c th i qua hoc b gi li trn sng. V d cc ht c 80/100 mesh i qua c c sng 80 mesh (170m) nhng khng i qua c c 100 mesh (150 m). Yu cu ca cht mang rn l khng tham gia vo s tch v c kh nng gi c pha tnh (khng t hn 10 %). Mt s loi cht mang rn: C hai iu quan trng cn ch khi chn mt cht mang l: cu trc v c tnh b mt. Cu trc gp phn vo hiu qu ca cht mang, trong khi cc c trng b mt chi ph i s tham gia ca cht mang vo kt qu ca vic tch. Cc cht mang phi l cc cht tr v mt ha hc vi tt c cc loi mu. N nn c di n tch b mt ln pha l ng c th tri thnh mt lp phim m ng v cu trc b mt ph i thch hp lu gi lp phim mng . Tuy nhin, din tch b mt ln khng l iu m bo ct hiu qu. Hu ht cc cht mang trong sc k ct nhi c to t diatomite. Loi cht mang u tin dng trong sc k kh do hng Johns Manville ch to c tn thng mi l Chromosorb. Sau y l mt s loi cht mang thng dng. Chromosorb A dng qui m iu ch, c kh nng gi tt pha lng (ti a n 25%), c cu trc kh b ph v v c b mt kh tr. Thng c sn xut kch thc 10/20, 20/30 v 30/40 mesh. S dng cho nhng ct di, chnh lch p sut thp. Chromosorb G dng tch cc hp cht phn cc. Nh c din tch b mt thp, cng, t trng cao nn n c s dng cho cc trng hp tm pha lng hm lng thp. Tm 5% trn Chromosorb G tng ng vi tm 12 % trn Chromosorb W.

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Chromosorb P c ch to t gch chu la C-22 c mu hng (nn c k hiu P-pink) v tng i cng. B mt t hp th hn so vi cc loi Chromosorb khc, c dng cho nhng vic tch hydrocacbon. Chromosorb W c ch to t Celite 545, mu trng v d v hn so vi loi G. B mt tng i khng hp th v c dng tch cc hp cht hu c phn cc. C hai vn chung ca cc cht mang: Th nht, tng tc b mt cht mang (hp ph hoc xc tc) chnh l nguyn nhn gy ra nhng pic c ui hoc bin dng. Chnh nhng nhm -OH hay oxit trn b mt cht mang l nguyn nhn ca nhng tng tc . C th che cc nhm ny bng mt lng nh pha l ng phn cc. Nhng cch tt nht hin nay l silan ha vi cc thuc th DMCS (dimethyl dichorosilan) hoc HMDS (hexamethyl disilazane). Th hai, kch thc ht cng nh th s hng A trong phng trnh Van Deemter cng nh, nhng s chnh lch p sut trong ct s tng vt kh i gii hn lm vic thc t. Hiu sut ct ch c th ci thin nu dng cc ht c kch thc nh nhau. T l phn trm pha l ng Lng cht lng cn phi bao bc cc ht bng mt lp mng ng nht. Pha l ng qu nhiu s ng li nhng ch trng gia cc ht lm gim hiu sut ct. Trn cht mang Chromosrb khi t l pha l ng ln hn 30% hiu qu ca ct gim i rt nhiu. Trc y ngi ta hay tm 15-30% cht lng, nhng hin nay c khuynh hng ch t m 2-10%. V thi gian lu t l vi lng pha l ng trong ct nn php phn tch cng nhanh khi ct c lng pha l ng ph ln cng nh. Khi ti cht lng qu thp s xut hin nhng tm hp ph trn cht mang. Mun trnh iu ny cn phi dng nhng cht mang tr. bay hi ca mu th cng phi tnh n khi chn lng pha l ng. Cc cht kh bay hi nh steroit cn phi c phn tch trong nhng ct c ti thp (khong 3% hay thp hn). Cc cht rt d bay hi nh cc hydrocacbon nh th cn c ct ti cao (20-30%) v lng cht l ng cng nhiu thi gian cc cht c trong pha l ng cng lu, s phn b cng tt. Mt nhi Mt nh i c mt s nh hng r rt n s lu gi trn pha t nh. Pha tnh c ph trn mt cht mang trn c s phn trm trng lng, trong khi vt liu nhi c t trong ct trn c s th tch. Nu mt nh i ca cht mang tng ln, khi lng tng ca pha tnh trong ct tng ln. Chun b pha tnh v nh i ct:

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Tm pha t nh ln cht mang c nhiu cch, mt trong s l dng my ct quay chn khng. Ly mt lng pha tnh tnh ton sn ha tan vo trong mt dung mi thch hp trong mt bnh y trn, cho tip cht mang rn vi lng tnh ton thch hp vo. Lp bnh vo my ct quay. Quay bnh cho n khi dung mi bay ht. un nng bnh bng cch thy hoc bng n hng ngoi. Ly on ng thng c chiu di v ng knh mong mun, nt mt u bng bng th y tinh, u kia lp mt ci phu, y cht mang tm pha lng vo phu. Lc ct bng my lc hoc p nh vo ct n khi khng thy gim th tch cht mang trn phu na th ct np y. Nt u cn li bng bng thy tinh v cun ng li cho thch hp v i l n nhit ca my GC. Luyn ct: Ct cn phi t t nht 2 gi 25 oC trn nhit ti a m ct s s dng nhng khng c qu gii hn trn ca nhit bay hi ca pha lng. Mt lung kh mang nh (5-10 ml/ph) c cho qua ct. u ra ca ct h khng n i vi detector trnh lm bn n. 2.3.3. Ct mao qun a s cc php phn tch trong sc k kh s dng cc ct mao qun di t 15 n 100 m v ng knh trong rt nh t 0.10 n 0.53 mm. Cc ct ny c ch to t thy tinh oxit tinh khit nu chy c mc lin kt ngang cao hn nhiu so vi thy tinh thng nn bn v chu c nhit cao n 350oC. Lc cng cao ca ng thy tinh cho php ch to cc ct c thnh mng v d un. Cc ct mao qun m c lp phim m ng trng trn thnh ng cung cp phn gii cao hn, thi gian phn tch ngn hn v nhy cao hn ct nhi nhng chng c dung lng thp hn cho cc mu. Ct mao qun m hp cung cp phn gii cao hn ct mao qun m rng hn nhng chng i h i p sut cao hn hot ng v c dung lng cho mu nh hn. Lp phim m ng l ng pha tnh dy khong t 0.1 n 5 m trn b mt bn trong nh c minh ha trong hnh 2.5. Nu gim dy ca lp phim ny s lm tng phn gii, gim thi gian lu v gim dung lng cho mu. Mt loi khc l cc ct mao qun c cc ht rn cht mang ph mt lp pha t nh l ng c gn trn b mt bn trong ca ct. Bi v din tch b mt ca loi ny tng ln, ct ny c th x l nhng mu ln hn ct ph lp phim m ng trn thnh ct. Loi ct ny l trung gian gia ct mao qun ph phim m ng trn thnh v ct nhi.

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Hnh 2.4: Ct mao qun c lm bng polyamide v fused silica

Cht mang Pha lng

Ct nhi

Ct mao qun

Hnh 2.5: Minh ha cu trc bn trong ca cc ct nhi v ct mao qun

2.3.4. Nhng yu cu v pha tnh lng Mt pha t nh l ng l tng cho sc k kh l ng cn th hin chn lc v kh nng ha tan ca cc cu t c tch v c khong nhit lm vic rng. N cng phi bn ha hc v c p sut hi bo ha thp ti cc nhit cao.

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Nhit lm vic nh nht nn gn nhit phng, y cht lng pha t nh phi tn ti dng lng ch khng phi rn. Khi chn la pha t nh, mt s tha hip gia l thuyt v thc nghim phi c xem xt. V d theo phng trnh Van Deemter th hiu qu tch ca ct cao hn nu ct cha pha t nh lng t hn so vi ct nh vy cha pha tnh l ng nhiu hn. Nhng trong thc t pha tnh l ng ph trn cht mang m ng qu c th to nn mt s che ph khng y cc v tr hot ng trn b mt cht mang dn n s ko ui ca pic, khi cn tng phn trm ca pha t nh lng trn cht mang. Cc php tch trong sc k kh lng l kt qu ca nhng tng tc pha tnh ha tan ch n lc v s khc nhau ca hi bo ha ca cc cht tan. Lc tng tc chnh chu trch nhim trong tng tc ca cht ha tan vi pha tnh l phn b, cm ng, nh hng v tng tc cho nhn. Tng ca chng c o lng bi phn cc ca pha tnh i vi cht tan. Trong sc k kh l ng, kh nng chn lc ca m t ct chi ph i khong trng gia cc pic v ln cc i ca pic c tch. S khc nhau v chn lc l c ngha v chng cho php vic tch cc cht tan c cu trc gn ging nhau bi mt pha tnh chn lc. Cc thng s sau y 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. Mt vi loi pha t nh l ng thng dng c lit k trong bng v vic chn pha t nh l ng cho mt i tng mu c da trn qui tc cc cht cng ging nhau th cng tan tt vo nhau. Ct khng phn cc l tt nht cho mu phn tch khng phn cc. Cc ct c phn cc trung bnh th t t cho mu phn cc trung bnh v phn cc mnh th tt cho mu phn cc mnh. V d tch ancol ta phi chn pha lng phn cc nh cacbowax-20, ngc li tch hidrocacbon no mch thng phi s dng cc pha lng khng phn cc. Khi ct dng nhiu b lo ha th pha t nh 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 t nh b mt nhit cao, ngi ta gn chng vo b mt pha tnh silica bng cc lin kt cng ha tr. Ngi ta chia cc pha lng v cc i tng cn tch ra nhiu nhm theo tnh phn cc ca phn t nh sau:

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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. Cc loi pha tnh ph bin nht l Squalane, polysioxanes v polyethylene glycol. -Squalane

CH3

CH3

CH3

CH3

HC-(CH2)3-CH-(CH2)3-CH-(CH2)4-CH-(CH2)3-CH-(CH2)3-CH CH3 CH3 CH3 CH3

-Polysioxanes: l cc pha t nh c s dng rng ri nht cho cc ct sc k kh mao qun cng nh ct nh i. Bi v cu trc ca chng c th c gn thm mt s loi nhm chc a dng nn chng th hin tnh phn cc trong mt di rng. Cc loi silicon ny l dng gm nht nn c th ph tt trn silica v cng thch hp l tng cho ct sc k mao qun.

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Polyethylene glycol Cc pha t nh khng cha silicon c s dng rng ri nht l cc polyethylene glycol. Chng c sn trn th trng vi mt khong rng ca trng lng phn t nh Carbowax 20M v Superox-4. Cu trc chung ca mt polyethylene glycol c th c m t nh sau: HO-CH2-CH2-(-O-CH2-CH2-)n-O-CH2-CH2-OH Loi pha tnh lng ny c phn cc cao, nhng cng c mt vi hn ch. V d Carbowax 20M c nhit lm vic cc i kh thp 225oC v nhit lm vic t i thiu l 60oC. Hn na, nhng lng vt ca oxi v nc c nhng nh hng khng tt trn cc pha t nh lng nhng c bit khng tt cho carbowax 20M, lm tng nhanh qu trnh lo ha ca ct.

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2.3.5. Ch s lu Kovats Vic bo co cc s liu nh thi gian lu tuyt i tR l v ngha bi v hu nh m i thng s sc k v bt k dao ng thc nghim lin quan u tc ng n php o thi gian lu gi. Vi vic s dng s liu thi gian lu gi tng i ( = tR2/tR1) c mt vi ci thin, nhng vic thiu mt tiu chun thng nht thch hp cho mt khong nhit rng trn cc pha t nh c phn cc khc nhau lm gim nhng s tin ch ca n. Th tch lu tng i cng t c s dng v chng ph thuc vo rt nhiu thng s nh chiu di ct tch, lng pha tnh, lu lng dng Kovats v Ettre thng nht ngh chn dy ng ng n-ankan l cht chun nhm xc nh th tch lu tng i v: Cc n-ankan khng c d t v nhm chc nhm trnh cc hiu ng lng cc-lng cc, hiu ng cm ng gia phn t lng cc trung ha. Gia chng v pha t nh tng tc vi nhau ch theo lc khuch tn Van Der Waals nn th tch lu ca chng i vi bt c pha tnh no cng khng khc nhau nhiu hay c th xem l khng i. Trong s tip cn ca Kovat, ch s lu I ca mt n-ankan c cng nhn mt gi tr bng 100 ln s cacbon ca n. V d, cc gi tr I ca n-octan, n-decan, v n-dodecan l n lt bng 800, 1000 v 1200. Theo nh ngha I c th p dng trn bt k ct no, mao qun h hoc nh i, vi bt k pha lng v c lp vi mi iu kin sc k, bao gm c nhit ct. i vi tt c cc hp cht khc, cc iu kin sc k nh pha tnh, nng ca n, cht mang v nhit ct nhi phi c ch r. Bi v cc ch s lu cng l phng php thch hp cho vic bo co cc s liu lu gi vi cc ct mao qun nn pha t nh, dy lp phim v nhit ct cng phi c ch nh r cho nhng hp cht khc n-ankan; ngc li cc gi tr I l v ngha. Ch s lu I ca cu t khc c xc nh bng cch so snh thi gian lu ca n v i vi thi gian lu ca hai n-ankan, trong mt cu t c ra gii ra trc cn cu t kia c ra gii ra sau cht nghin cu trong nhng iu kin sc k c th. Ni cch khc, ch s lu ca mt cht phn tch ti nhit ct ng nhit c th c tnh ton t phng trnh sau:
I x 100Z 100
' (log t R ,x ' log t R ,Z 1 ' log tR ,Z ) ' log t R ,Z

y:

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tR,x l thi gian lu c hiu chnh ca cu t ang xt. tR,Z l thi gian lu c hiu chnh ca n-ankan ra gii trc cht nghin cu, tR,Z +1 l thi gian lu hiu chnh ca n-ankan r gii sau n, Z l s cacbon ca n-ankan c thi gian lu gi tR,Z . Vi nhng ln chy sc k theo chng trnh ha nhit , cc thi gian lu hiu chnh trong phng trnh trn c thay th bi cc nhit ra gii thch hp (K). Nu s khc bit v s cacbon gia hai n-ankan trn khng phi l 1 m l 2 hoc ln hn th phn s trong phng tr nh trn s c nhn vi 200 hoc mt bi s tng ng ca 100. V d: hy tnh ch s lu Kovats ca hp cht X c thi gian lu tR,x =15.7 pht Bit tR(n-octan) = 14.3 v tR,(n-nonan) = 18.5 pht v tR(metan) = 0.5 pht thu c trong cng iu kin sc k xc nh. Ta c thi gian lu hiu chnh ca cc cu t: tR,x =15.7 0.5 = 15.2 pht tR(n-octan) = 14.3 0.5 = 13.8 pht tR,(n-nonan) = 18.5 0.5 = 18.0 pht p dng phng trnh trn ta c:

Ix = 800 100(

lg15.2 lg13.8 ) = 836 lg18.0 lg12.8

Trong trng hp ny ta ni cu t X c s nguyn t cacbon biu kin l 8,36. V vy ta c th nh ngha ch s Kovats nh sau: ch s lu Kovats bng tch s ca 100 vi s nguyn t cacbon biu kin. Ch s lu lm chun ha cc bin thit b trong sc k kh cho php cc s liu lu gi c to trong nhng h thng sc k khc nhau c th so snh c. V d, isoamyl acetat vi ch s lu 1128 s b ra gii gia n-C11 v n-C12 di cng iu kin sc k. Nhng ch s lu gi cng rt hu ch trong vic so snh trt t ra gii ca mt lot cc cht tan trn mt ct c th ti mt nhit cho v trong vic so snh hai hoc nhiu ct. Mi lin h gia ch s lu v cu trc ca cc hp cht: Gia cu t cn xc nh v pha tnh c cc lc tng tc khc nhau: Tng tc lng cc-lng cc Lc lin kt hydro, kh nng to phc cho nhn

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Lc Van Der Waals Thng thng cc loi lc tng tc trn u xut hin ng thi, ty theo cu trc m lo i lc no s tri hn cc loi lc khc. Da vo ch s lu Kovats c th nghin cu m i quan h gia s lu gi v cu trc ha hc ca cc cu t nghin cu cng nh pha tnh. So snh ch s lu ca hai hp cht trong cng mt pha tnh Theo di ch s lu ca mt lot hp cht c cng s C nhng khc nhm chc rt ra nhng gia s ng vi thnh phn cu trc khc nhau. V d hai hp cht hexylxiclopropan v 1-octan. Cc ch s lu ca chng 80oC trn pha tnh squalan l 913.0 v 782.7. Cc hydrocacbon no mch thng tng ng l n-nonan (I80 = 900) v n-octan (I80 = 800). Nh vy cc gia s ng vi cc nhm chc trn l +13 v -17.3. D on ch s lu ca hp cht 5-hexenylxiclopropan l = 900 + 13 17.3 = 895.7 Gi tr thc nghim o c ca hp cht ny I80 ca hp cht ny ng bng 895.7. L do ca s trng hp ny l hai nhm chc xa nhau nn khng c s tng tc qua li. So snh ch s lu ca mt hp cht trn hai pha tnh khc nhau minh ha ta xt mt lot gi tr ch s lu ca cc hp cht c 6C nh trong bng sau: Cu trc CH2=CH-(CH2)3CH3 Tn cht Hexen-1 IS 584.5 IDC200 591.2 IPPG 610.5 I 26.0

Xiclohexan CH3-(CH2)4-CH2-Cl 1-clohexan

674.8 829.1

672.3 851.6

699.2 922.6

24.4 93.5

Benzene CH3-(CH2)4-CHO CH3-(CH2)3CH2-CO-CH3

O

651.0

692.3

767.9

116.9

Hexanal Hexanon-2 Xiclohexanon Hexanol-2

741.5 726.0 831.1 798.5

790.2 781.2 893.9 891.3

899.0 891.0 1038.2 1103.8

157.5 165.0 207.1 305.3

CH3-(CH2)CH2-OH

IS = ch s lu vi ct Squalan (100m x 0.25 mm), T = 100oC

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IDC200 = ch s lu ct DC 200 (100m x 0.25 mm) , T = 100oC IPPG = ch s lu vi ct Polypropylenglycol (100mx0.25 mm), T = 100oC I = IPPG - IS Trong ba pha t nh s dng y squalan c tnh khng phn cc; popropylen c tnh phn cc mnh; cn du silicon c tnh phn cc trung bnh. Theo nh ngha ca Kovats th n-hexan c ch s lu l 600. Cc gi tr trong bng c th c gii thch nh sau: n-hexanol c I l ln nht v chng c tng tc lng cc-lng cc c bit mnh v i s tham gia cu n i hydro gia cc nhm hydroxyl vi nguyn t oxi ca ete ca polypropylenglycol. Cc amin bc mt v bc hai tng ng cn c tng tc mnh hn na. Hexen-1 v xiclohexan c cc gi tr I nh nht. Cc olefin c tng tc mnh hn do c cha cc in t pi cho tng tc lng cc cm ng. Gi tr I ca benzene ln hn hn so vi ca 1- hexen do c cu trc h pi lin hp. Phn t 1-clohexan c cha lng cc C-Cl nn phn t ny c phn cc mnh tng ng vi phn cc ca 3 lin kt i gp li. C ba hp cht cha nhm cacbonyl u cho cc gi tr I tng ng nhau. Tuy nhin xiclohexan ln hn r rt so vi hexanon-2 do cu trc mch vng ng mt vai tr ng k. Qua v d trn ta thy c mt mi tng quan cht ch gia cu trc ha hc ca cc cht vi cc ch s lu ca chng. 2.3.5. S phn loi cc pha tnh ca Mc Reynolds. 2.3.5.1 Cch tip cn ca Mc Reynolds H thng c s dng rng ri nht trong phn loi cc pha t nh l h thng Mc Reynolds. Mc Reynolds chn 10 cht ha tan thm d c nhng chc khc nhau, m i chc c nh r o mt tng tc c th vi mt pha lng. ng o cc gi tr I ca chng trn khong 200 pha khc nhau, bao gm Squalane, c phc v nh mt pha lng tham kho di cng mt iu kin sc k. Mc Renolds tnh ton cho mi cht gi tr I: I = I liquiphase- I squalane Khi s khc nhau trong ch s lu cho mt cht d trn mt pha lng cho v squalane tng ln, mc tng tc c th lin quan n cht d tng ln. 2.3.5.2. Bng cc cht d c s dng trong s phn loi cc pha tnh lng

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Trong bng ny, 5 hng s u tin ca McRenolds thng c xut hin v c biu din bi cc k hiu X, Y, Z, U v S. Mt s tip cn tng t c thc hin trc bi Rohrscheiner vi 5 cht ha tan. Mi cht d c n nh gi tr zero vi squalane nh l pha l ng tham kho. K hiu X Cht d ca McReynolds Benzen Cht d ca Rohrschneider Benzen Tng tc c o Mt electron cho cc hydrocacbon thm v olefin. Nng lc cho nhn proton (ancohol,axit, nitrils). Tng tc nhn electron (ketone, ete, andehit, este). Cc tng tc lng cc Tng tc nhn electron mnh Tng tc ancol c th tng t nh n-butanol. Cc tng tc ankan phn cc Tng tc hydrocacbon khng bo ha ging nh benzene Tng tc nhn proton Tng tc phn tn

n-Butanol

Etanol

2-Pentanone

2-butanone

U S H

Nitropropane Pyridine 2-methyl 2-metanol

Nitrometan Pyridine -

J K L M

Iodobutan 2-octyne 1,4 dioxane Cis-hydrindane

ng dng: Mt trong nhng ng dng quan trng nht ca cc hng s McRenolds l nhn bit c nhng pha tnh ging nhau hoc tng t nhau nhm trnh c vic s dng trng lp. Mt ng dng khc l s dng la chn cc ct tch thch hp. Ni chung gi tr hng s McRenolds cng ln th pha tnh c kh nng gi c cu t cn tch lu hn. V d pha t nh cabowax 20M tch ancol thch hp hn OV-17 hoc OV-101 (gi tr tng ng ca cc pha l 536, 158, 57). tch cc hp cht mang nhm chc khc nhau th d ancol khi xeton, este th gi tr X, Y, Z cn phi khc nhau xa. Da vo hng s McRenolds ta c th d on c th t ra gii ca cc cu t. Cc hng s McRenolds ca cc pha tnh ph bin cho ct nhi GC c lit trong bng sau:

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Bng cc hng s McRenolds v cc pha tnh c s dng thng thng

Pha Squalane DC200 DC700 SE-30 SE-54 OV-1 OV-3 OV-7 OV-11 OV-17 OV-22 OV-25 OV-61 OV-73 OV-101 OV-105 OV-202 OV-210 OV-215 OV-225 OV-275 OV-330 OV-351 OV-1701 SP 2100 SP2250 SP2300 SP2310 SP2330 SP2340 SP2401 T(oC) min/max 20/100 0/200 5/250 50/300 50/300 100/350 0/350 0/350 0/350 0/375 0/350 0/350 0/350 0/325 0/350 0/275 0/275 0/275 0/275 0/265 25/275 0/250 50/270 0/250 0/350 0/375 20/275 25/275 25/275 25/275 0/275 Bn cht ha hc Paraphin Dimetyl silicon Phenylmetyl silicon Dimetyl 5%phenyl 1% vinyl Dimetyl 10%phenyl-phenyl metyl dimetyl 20%phenyl-phenyl metyl dimetyl 35%phenyl-phenyl metyl dimetyl 50% phenyl 50% metyl 65%phenyl-phenyl metyl dimetyl 75%phenyl-phenyl metyl dimetyl 33%phenyl-diphenyl dimetyl 5.5%phenyl-diphenyl dimetyl Dimetyl (lng) Cyanopropylmetyl-dimetyl Trifluoropropyl-metyl(lng) Trifluoropropyl-metyl(lng) Trifluoropropyl-metyl(gm) Cyanopropylmetylphenylmetyl Dicyanoallyl Phenylsilicon-cabowax copolimer Cabowax nitroterephtalic acid polimer 14%cyanopropylphenyl Metyl 50%phenyl 50%cyanopropyl 55%cyanopropyl 90%cyanopropyl 100%cyanopropyl trluoropropyl X 0 16 107 15 33 16 44 69 102 119 160 178 101 40 17 36 146 146 149 225 629 222 335 67 17 119 316 440 490 529 146 Y 0 57 149 53 72 55 86 113 142 158 188 204 143 86 57 108 238 238 240 369 872 391 552 170 57 156 495 637 725 757 238 Z 0 45 153 44 66 44 81 111 145 162 191 208 142 76 45 95 358 358 363 338 763 273 382 153 45 162 446 605 630 659 358 U 0 66 228 64 99 65 124 171 219 243 283 305 213 114 67 139 468 468 478 492 1106 417 583 228 67 243 637 840 913 942 468 Z 0 43 190 41 67 42 88 128 178 202 253 280 174 85 43 86 310 310 315 386 849 368 540 171 43 202 530 670 778 800 310 SP-2100,SE30,OV-1 DC-710 SP2250 Cc pha c cu trc tng t SP-2100,SE30,OV-101,OV-1 SP-2100, OV-101, OV-1 SP-2100

SP-2401 SP2300 SP2340

SP-1000

SE-30,OV101,OV-1 OV-17 OV-225

OV210

2.4. Detector

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2.4.1. Detector o dn nhit (Thermal conductivity detector) 2.4.1.1. Gii thiu Trc y, detector o dn nhit l loi thng dng nht trong sc k kh bi v n n gin v p ng hu ht cc cht phn tch. Khng may, dn nhit khng nhy d tm nhng lng nh ca cht phn tch tch t ct mao qun m c ng knh ct nh hn 0.53 mm. Cc detector o dn nhit vn cn s dng cho ct mao qun ng knh 0.53 mm v cho cc ct nh i.
Bng dn nhit ti 273 K v 1 atm

Kh H2 He NH3 N2 C2H4 O2 Ar C3H8 CO2 Cl2 2.4.1.2. Nguyn tc

dn nhit J/(K.m.s) 0.170 0.141 0.0215 0.0243 0.0170 0.0246 0.0162 0.0151 0.0144 0.0076

Phn t lng 2 4 17 28 28 32 40 44 44 71

dn nhit o kh nng ca mt cht vn chuyn nhit t mt vng nng sang vng lnh (bng). Nhit mt i khi cc phn t kh p vo si dy t nng ri bt ra vi mt ng nng ln hn. S va chm phn t vi si t trong mt n v thi gian cng ln th tc mt nhit cng ln. S khc nhau v tnh dn nhit ca cc kh l do tnh linh ng, ngha l tc kh khuch tn ti v i khi si t. Tc ca phn t li l hm ca trng lng phn t, do 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. N cng l kh c dn nhit cao th hai (sau H2), nn bt k cht phn tch no trn vi dng kh He cng lm gim dn nhit ca dng kh. Trong detector (hnh 5), cht ra gii t ct sc k chy qua si dy tc nung nng Tungsten-Rhenium. Khi cht phn tch xut hin t ct, dn nhit ca dng kh b gim xung, si dy tc tr nn nng hn nn in tr ca n tng ln v th p vo si dy ny thay i. Detector s o s thay i th ny theo cu Wheatstone nh sau

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Hnh 2.6: Detector o dn nhit - Cu ni Wheatstone

Mt dng nh kh mang lm kh so snh i qua R1 v R2. Kh mang v mu sau khi qua ct c dn qua S1 v S2. Khi c 4 si t S1, S2, R1, R2 cng mt nhit , chng s c cng gi tr in tr th cu cn bng, tn hiu ra bng khng. Nu in tr S1 v S2 thay i do kh mang cun theo cng vi cc cu t cht phn tch th cu mt cn bng, tn hiu ra xut hin. nhy ca detector o dn nhit t l nghch vi tc dng kh. N nhy hn v i tc dng thp hn. nhy tng theo khi s khc nhau v nhit gia si dy t vi vng kh i xung quanh tng ln. nhy cng tng theo bnh phng ca cng dng ca dy t. 2.4.2. Detector ion ha ngn la 2.4.2.1 Nguyn tc hot ng Da trn s bin i dn in ca ngn la c to bi hn hp kh H2 v O2 trong khng kh (ngun ion ha) v c t trong mt in trng (bung in cc). Kh mang sau khi ri kh i ct i qua ngun ion ha vo bung in cc. chng b bn ph bi cc chm ht electron to thnh cc ht tch in (ion dng, ion m, electron). Kt qu to thnh dng in gia hai in cc. Dng in ny i qua in tr o R. S ri th Eo qua in tr R ny s c khuch i ri a ra my ghi. Nu ch c kh mang i qua 2 bn ca in cc, dng in sinh ra gi l dng nn, gim thiu dng ny ngi ta dng mt dng in b chnh. Khi kh mang em theo cc phn t mu, cc phn t ny b ion ha v dng in qua in cc tng ln. Cc tn hiu c ghi li di dng cc pic.

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2.4.2.2. ng dng: nhy ca detector FID cao hn rt nhiu so vi nhy ca detector TCD khong 100 - 1000 ln. Tuy nhin n ch s dng thch hp nht vi cc hp cht hu c cha cacbon. C mt s hp cht khng th pht hin bng detector ny 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. 2.4.3. Detector cng kt in t (ECD) 2.4.3.1. Nguyn tc hot ng B phn chnh ca detector ECD l bung ion ha, ni din ra cc qu trnh ion ha, bt gi electron v ti lin hp. Qu trnh ion ha: t mt ngun tia phng x 63 Ni c lp sn trong detector, pht ra mt chm tia vi tc khong 108, 109 ht /giy. Cc ht ny s ion ha phn t kh mang (N2 hoc 5% metan trong Ar) to ra cc ion dng ca phn t kh mang v in t t do s cp. So vi cc in t ca chm tia , cc in t t do ny chm hn. Chng c gia tc nh mt in trng v chuyn dch v pha anot. Ti y chng b ly mt in tch v qua to ra dng nn ca detector. Qu trnh cng kt in t din ra khi cc phn t cc cht phn tch vi i lc electron cao i vo detector. Chng bt mt s electron dn n detector thay i hiu th gia anot v catot duy tr dng nn khng i. S thay i ny c ghi nhn di dng pic sc k trn giy ghi hoc bng computer. 2.4.3.2. ng dng Detector loi ny c bit rt nhy vi nhng phn t hp cht c kh nng cng kt v i cc in t t do trong pha kh nh cc phn t cha halogen, cacbonyl lin hp, nitril, hp cht nitro, v hp cht c kim. N tng i km nhy vi cc phn t hp cht hydrocacbon, cc ru v xeton. Nh vy nhy ca ECD ph thuc vo: ln ca dng nn. Kh nng i in t ca cht cn phn tch. Bn cht ca kh mang v hiu th t vo 2 bn in cc. 2.4.4. Detector khi ph 2.4.4.1. Gii thiu

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My kh i ph l mt detector vn nng cho phn tch nh tnh v nh lng cc cht hu c c sc k kh ln sc k l ng. Trong phng php kh i ph, cc phn t kh b ion ha (thng to cation) c tng tc trong mt trng in t v sau c tch theo khi lng ca n. Qu trnh ion ha thng cung cp nng lng n phn t ph v n thnh nhng mnh khc nhau. Mt ph khi l mt biu cho thy lng tng i ca m i mnh v to ra t detector khi ph. Trong sc k kh m i pic ca sc k tng ng vi mt cht phn tch c tch ra c nhn din bi mt ph kh i. Hnh l mt ph kh i ca hp cht c nhn din khi so snh vi th vin ph ca nhng hp cht bit. Trc honh ca ph khi l m/z, khi lng ca ion chia cho in tch ca ion. Phn ln cc ion u mang in tch +1 nn m/z thng chnh l kh i lng ca ion (.v.C). Trc tung ch thnh phn % tng i ca cc ion so vi ion c lng ln nht c qui c 100%. Trong ph khi c mt pic quan trng ca ion phn t khng b ph v thnh cc mnh. Da vo t l ca cc ng v ca mt nguyn t trong t nhin, ta c th nhn thy t l ny xut hin trong ph khi qua t l ca cc ion phn t ca cc ng v. V d ta c th hiu iu ny trong ph khi ca 1-brombutan. V t l ca nguyn t brom trong t nhin l 50.52% 79Br v 49.48% 81Br, pic ti m/z=136 ca ion phn t C4H979Br c cng ln hn mt cht so vi pic ca ion phn t ti m/z=138 ca phn t C4H981Br.

Hnh 2.7: Sc k v ph MS tng ng ca 1-Butanol v 1-Bromobutane.

2.4.4.2.My khi ph nam chm hnh qut Nguyn tc hot ng vi ngun ion ha bng chm electron

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Cc phn t kh i vo ngun ion ha bng va chm electron v b bn ph bi cc electron c nng lng ng hc khong 70 eV. Nng lng ny ln ion ha cc phn t (M + e M+ + 2e) v ph v chng thnh nhng mnh v nh hn. Mt th hiu dng nh a y (repeller plate) y nhng ion hng v ng phn tch v mt th nh cc a h i t ion (ion focus plates) gia tc ion gip cc ion c vn tc cao b tng kh i phn y ca b phn ny v i vo ng phn tch. ng phn tch (Analyser tube) c duy tr p sut chn khng cao (khong 10-5 Pa) cc ion chuyn ng qua ng khng b lch hng bi nhng va chm vi cc phn t kh pha ng. Cc cation di chuyn qua ng phn tch chu tc ng ca t trng t vung gc v i hng di chuyn ca chng. Trng ny s hng cc cation v detector t cui ng phn tch. Mt khe detector ch cho nhng ion trong mt khong gi tr khi lng nh xc nh tip cn detector. Cc phn t c khi lng khng ph hp va phi cc thnh ng hoc b loi b bi khe thot ion nn khng vn ti c detector. Ti b phn thu ion (ion collector), mi cation n b trung ha bi mt electron. Dng in i hi trung ha chm ion t l vi s lng cc ion n b phn thu ion ny. Ph kh i l mt th cho thy dng in nh l mt hm ca s khi c chn bi t trng. Phng php ion ha bi tng tc electron (electron impact ionization) cho ph kh i c nhiu mnh v. Ion phn t c th c hm lng thp thm ch b bin mt. Nu phn t ln th vic c qu nhiu mnh v s lm cho vic gii thch ph khi ca n gp nhiu kh khn. Trong trng hp ny cc chng tr nh my tnh c th c s dng lm khp ph ca cht cha bit ny vi mt ph khi bit trong th vin ph lu trong my tnh. Nguyn tc hot ng vi ngun ion ha ha hc: Mt k thut tinh t sn sinh ra t mnh v hn l s ion ha ha hc (chemical ionization). Trong trng hp ny, ngun ion ha c y metan p sut khong 100 Pa. Cc electron c cung cp nng lng chuyn CH4 n nhng sn phm hot ng khc: CH4 + e CH4+ + 2e CH4+ +CH4 CH5+ + CH3 CH5+ l cht nhng photon khi tng tc vi cht phn tch cho MH+, thng l ion c hm lng ln nht trong ph kh i ion ha ha hc metan (methane chemical ionization mass spectrum).

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CH5+ + M CH4 + MH+ 2.4.4.3.My khi ph t cc My kh i ph nam chm hnh qut thng t c chn ghp n i vi sc k bi v t trng khng th thay i nhanh ghi ph ca nhng di sc k hp. Loi c s dng ph bin hin nay kt ni vi sc k l my kh i ph t cc (quadrupole mass spectrometer). Khi m i cht ha tan trong ct sc k c tch c th hin di dng pic. Cht c dn n bung ion ha tng tc electron c p sut chn khng, ti y cc phn t cht phn tch b cc electron c gia tc bn ph to cc ion mnh v. Cc ion ny li tip tc c gia tc bi mt th khong 5-15 V trc khi i vo b tch theo khi lng t cc (quadupole mass seperator). B tch gm 4 thanh kim loi song song vi nhau, trong mt cp s c p vo mt th khng i v cp kia mt th hiu dao ng tn s radio. T trng c to ra lm lch cc hnh trnh phc tp khi chng di chuyn t bung ion ha hng n detector v ch cc ion no c t s khi lng vi in tch (m/z) chn trc mi tip cn v i detector. Cc ion khc va p vo cc thanh kim loi v b mt trc khi vn n detector. Bng vic thay i nhanh chng th p vo, cc ion c khi lng khc nhau c chn lc c tip cn vi detector. Cc b tch khi ph t cc c qut nhanh ghi hai n tm ph trong mt giy vi khong 800 n v kh i.

2.5. Pht trin mt phng php phn tch bng sc k kh

Do c nhiu thng s nh hng n qu trnh sc k nn c th lm b i ri cho nhng ngi m i bt u lm quen vi vic chn mt th tc cho vic phn tch sc k mt i tng c th no . Ni chung, c nhiu gii php khc nhau c th tha mn. Trong phn ny s tho lun cch chn mt phng php s dng. Trt t cn xem xt theo th t sau: mc tiu phn tch, chun b mu, detector, ct v bm mu. 2.5.1. Mc tiu ca php phn tch Mc tiu ca php phn tch l phn tch nh tnh hay nh lng? C cn phi thc hin mt php tch phn gii cao mt cu t hoc ch cn mt php tch c phn gii tt cho mt phn no thi trong sc k ? C cn hy sinh phn gii rt ngn thi gian phn tch? Cn phn tch nh lng ch mt cu t hoc nhiu cu t trong mu v c cn chnh xc cao khng? Cht phn tch hin din trong mu nng thch hp hoc cn c nhng k thut c bit (c mu v mt detector rt nhy) cho phn tch siu vt.

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Mi yu t cp dn n mt vi s tha hip trong vic chn cc k thut thch hp. 2.5.2. Chun b mu Cc mu trong thc t rt phong ph v thng c thnh phn phc tp: C mu c hm lng tng t nhau nhng cng c mu bn cnh cu t chnh mu cn cha mt s cu t vi hm lng vt. C mu c cc cu t vi nhit si khc nhau trong mt khong rng. Mu c th dng dung dch nhng cng c dng kh hoc dng rn. Mu cha nhiu cu t phn cc hoc cha nhiu cu t t hoc km phn cc. Cha kha c mt qu trnh sc k thnh cng cho mt mu phc tp l cn phi lm sch n trc khi gii thiu n vo ct. Cc phng php nh vi chit pha rn, chit l ng - l ng, chit rn - l ng, s gii hp nhit cc cc cu t d bay hi v nhiu phng php mi khc gip c lp nhng cu t cn phn tch trong mu phc tp t nhng cht cn tr. Cc phng php ny gip c lp v lm giu cc cu t cn phn tch n mc c th d tm c. Nu ta khng lm sch mu th sc k thu c s cha mt rng cc pic km phn gii v cc cu t khng bay hi s cn gi trong ct v ph hy cc ct t tin. 2.5.3. Chn detector Giai on tip theo l chn detector cho qu trnh sc k. Vn t ra l cn bit m i thng tin trong mu hay ch cn mt detector chuyn bit d tm mt hoc mt nhm cc cu t c bit no trong mu. Detector c s dng rng ri nht l detector khi ph nhng t tin. Detector ion ha ngn la cng l loi c dng ph bin nht nhng n ch p ng tt cho hp cht cha mch cacbon di hoc cc hydrocacbon. Loi ny khng nhy nh detector bt electron hoc detector hunh quang. Detector ion ha ngn la i h i mu phi cha ln hn 10 ppm cht cn phn tch cho phn tch tim mu chia dng. Detector dn nhit l cch ph bin nht d tm cc cht nhng n khng nhy cho nhng ct mao qun m c phn gii cao. Cc detector nhy cho phn tch siu vt ch p ng vi mt loi hp cht no thi. V d detector bt electron l loi c bit cho cc hp cht cha halogen, cacbonyl lin hp, nitril, v hp cht nitro. Nu ch cn bit nhng thng tin nh tnh nhn din cc cht ra gii, cc detector kh i ph v hng ngoi l nhng chn la tt. Detector hng ngoi ging detector o dn nhit thng khng nhy cho nhng ct mao qun m.

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2.5.4.Chn ct Nhng chn la chnh l pha t nh, ng knh ct v chiu di ct. Chn pha tnh theo nguyn t c cc cht c cu to cng ging nhau th tng tc tt vi nhau. Cc pha t nh khng phn cc c s dng nhiu. Pha t nh c phn cc trung bnh c s dng hu ht nhng php tch m pha t nh khng phn cc khng th. i vi nhng hp cht phn cc cao th mt ct phn cc mnh l cn thit. Cc ng phn quang hc v nhng hp cht c cu trc hnh hc gn ging nhau i h i nhng pha tnh c bit cho php tch ny. Gia ng knh ct v b dy ca lp phim c m i quan h trong vic nh hng n phn gii. phn gii cao nht c th t c bi nhng ct hp nht vi pha tnh mng nht. S kt hp ny lm gim thiu tr khng i vi s chuyn kh i (s hn C trong phng trnh Van Deemter) trong c hai pha t nh v pha ng nh vy lm gim chiu cao a l thuyt. Cc ct hp v lp phim mng c bit thch hp cho vic tch cc hn hp ca cc cht c nhit si cao nn b lu gi qu mnh trn cc ct c lp phim m ng. Cc thi gian lu ngn cung cp nhng php phn tch nhanh. Tuy nhin, cc ct hp c lp phim mng c dung lng mu rt thp, i h i nhng detector c nhy cao (detecter ion ha ngn la c th khng tng thch), khng lu gi tt nhng hp cht d bay hi v c th chu nhng nh hng do nhng v tr hot ng trn b mt c th b l ra. Cc ct c hp c lp phim dy cung cp mt tha hip tt gia phn gii v dung lng mu. Chng c th c s dng vi nhiu detector (nhng thng khng phi cc detector o dn in hoc hng ngoi) v vi nhng hp cht d bay hi. Cc thi gian lu gi di hn so vi ct c lp phim mng. Cc ct c rng v c lp phim dy c i h i cho vic s dng cc detector o dn in v hng ngoi. Chng c dung lng mu cao v c th x l cc hp cht d bay hi nhng cho phn gii thp v thi gian lu gi lu. Nu mt ct chuyn bit p ng vi hu ht nhng yu cu t ra nhng khng cung cp phn gii th ct di hn cng loi c th c s dng. Chiu di gp i ca ct lm tng gp i s a l thuyt v theo phng trnh tnh phn gii th lm tng phn gii ln 2 ln. iu ny khng phi l cch tt nht tng phn gii bi v n lm tng gp i thi gian lu.

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Vic s dng mt ct hp hn hoc lp pha tnh mng hn s lm tng phn gii m khng ko di thi gian lu gi. Vic thay i pha t nh lm thay i hon ton s lu gi tng i ca nhng hp cht khc nhau ( trong phng trnh) v c th phn gii cc cu t ang quan tm. 5.5. Chn phng php tim mu Quyt nh chnh cu i cng l lm th no tim mu. Ch tim mu chia dng: Tt nht cho nhng mu phn tch c nng cao hoc phn tch kh, nhng phn tch nh lng c th c cc cu t kh bay hi b mt trong sut qu trnh tim mu. Tim mu chia dng cung cp phn gii cao v c th x l nhng mu bn nu nh i vo ng trong b tim mu mt cht hp ph thch hp. Cc hp cht khng bn nhit c th phn hy trong sut qu trnh tim mu nhit cao. Ch tim mu khng chia dng: c yu cu cho nhng dung dch rt long. N cung cp phn gii tt nhng trong phn tch nh lng c th c cc hp cht km bay hi b mt trong qu trnh tim. N tt hn ch tim mu chia dng i vi nhng hp cht c bn nhit trung bnh bi v nhit tim mu h thp hn. Tim mu khng chia dng gii thiu mu vo ct chm v vy by dung mi v by lnh c i hi. Cc mu cha m i cht phn tch t hn 100 ppm c th c phn tch trong nhng ct c chiu dy lp phim < 1m vi ch tim mu khng chia dng. Nhng mu c m i cht phn tch t 100-1000 ppm i h i lp phim dy hn 1m. Tim mu trn ct (on-column injection): Tt nht cho phn tch nh lng v cho nhng hp cht km bn nhit. N l mt k thut c phn gii thp v khng th s dng cho nhng ct c ng knh nh hn 0.25 mm. N c th x l nhng dung dch c cng nh long. 2.5.6. Chng trnh ha nhit v p sut 2.5.6.1.Nhng thun li ca chng trnh ha nhit ct Ct sc k hot ng nhit khng i c mt s im bt li:

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Nu nhit c chn qu thp, nhng pic ha tan u tin s c khong cch rt gn nhau, trong khi cc cu t b lu gi mnh s c cc pic b dong rng v nm thp. Ngoi ra nu nhit ct hi thp th thi gian phn tch s tng theo cp s nhn. Khi phn tch hn hp g m cc cu t c nhit si khc nhau nm trong mt khong hp th vic chn nhit cho ct ch khng i khng thc s kh khn. Tuy nhin, i vi nhng mu phc tp nhiu cu t c khong nhit si rng th khng th dng ch nhit ct khng i. Nu chn nhit thch hp cho cc cu t c im si thp th cc cu t c im si cao s b lu gi lu trong ct tch dn n pic ca chng b dong rng. Ngc li nu thc hin vic tch nhit cao th cc cu t c im si cao s ra gii nhanh v c phn gii tt, nhng cc cu t c im si thp th b tch chm v c phn gi i km, thm ch nhiu pic b chp ln nhau. Nhng cu t lu gi mnh c th c nhanh chng ra gii nu chn nhit cao hn, v cng ci thin kh nng d tm chng. Tuy nhin, nu lm nh vy th cc cu t cng b ra gii tng ln dn n cc pic cng st gn nhau v s lm mt phn gii cho cc pic u sc k . Tr ngi ny c th c gii quyt trong GC bng chng trnh ha nhit , y nhit ca ct trong l c tng dn ti mt tc tuyn tnh sut qu trnh phn tch. Chng trnh ha nhit c mt vi u im: N lm gim thi gian tin hnh phn tch v ci thin kh nng d tm ca cc cu t (pic s nhn hn v c cc di cch u nhau trong c sc k ). Trong trng hp cc mu khng bit thnh phn hoc mu phc tp, nhng cu t c nhit si cao nu iu kin ng nhit c th khng ra gii c hoc khng th d tm, th c th th hin mt thi gian lu gi thch hp hn. Chng trnh ha nhit cng c th gip ra sch mt ct b nhim bn cc cu t c nhit si cao trong nhng ln tim trc. Chng trnh ha nhit thng thit lp nhit cho ct tch tng ng vi nhit ca cu t c nhit si thp nht, thch hp l di 90oC. Sau nhit c nng ln theo tc chn trc cho n nhit cui cng tng ng vi nhit ca cu t c im si cao nht. Khi nhit ca ct tng ln th cc cu t s ln lt tch kh i ct vo thi im nhit ca ct t n nhit si ca tng cu t. C ba kiu chng tr nh ha nhit c s dng trong sc k kh: kiu ng n (ballistic), kiu tuyn tnh v kiu a tuyn tnh.

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T c t A B C

Thi gian
Chng trnh kiu n o din ra khi mt l (oven) c duy tr ti mt nhit nhanh chng thay i n mt nhit cao hn (ng A), v thnh thong c s dng cho vic iu kin ha ct sc k kh rn sau khi n c s dng trong mt thi gian di. Tuy nhin, mt ct sc k c duy tr ti nhit cao sau nhanh chng nng nhit ln cao th lp phim pha tnh c th b ph hy do b sc nhit. Chng trnh ha nhit c s dng rng ri nht l kiu tuyn tnh (ng B). Bt u t nhit thp v c duy tr ng nhit trong mt khong thi gian no , sau nhit trong l cha ct c tng vi mt tc tuyn tnh n mt nhit cui cng c chn sn. Nhit cui cng ny cng c duy tr trong mt khong thi gian c th no . Nhit ban u v tc tng tuyn tnh sau c xc nh t mt php phn tch thm d trc vi ghi ch nhng nhit ra gii. S chn la nhng iu kin ban u thch hp s cho php vic tch nhng cht c nhit si thp, trong khi nhit cui cng c chn nn ln ra gii c nhiu hn nhng cu t b lu gi mnh (lu gii hn nhit cao nht ca pha tnh). Kiu a tuyn tnh c thc hin trong mt vi trng hp d tm hoc tng cng phn gii trong mt php tch, nhng c s dng thng dng hn trong vic kt hp vi tim mu trn ct. Trong chng trnh ha nhit , nhit ca mt ct c tng sut qu trnh tch tng p sut hi ca cht ha tan v nh gim thi gian lu ca nhng cu t b ra gii chm. Mt v d c trnh by trong hnh 6. Ti nhit hng nh 150 oC, cc hp cht d bay hi hn xut hin gn nhau v cc hp cht t bay hi c th thm ch khng

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b ra gii t ct. Nu nhit c cho tng t 50 oC n 250 oC vi tc tng 8oC/pht, tt c cc cu t c ra gii v cc pic xut hin kh u. Cn trnh tng nhit ln qu cao ngn s phn hy nhit ca cht phn tch v pha t nh. Hin nay, cc my sc k phn ln u c trang b cc b in t kim sot p sut c th chng trnh ha ca cc kh mang i vo ct. S tng p sut vo ct lm tng tc pha ng v nh vy lm gim thi gian lu.

Hnh 2.7: (a) tch ng nhit ca cc ankan tng t 150oC. Ct 3% Apiezon-L on 100120 mesh VarAport 30. Kh mang helium 10 ml min1. (b) Mu v ct nh (a) nhng nhit tch c chng trnh ha cho thy phn gii cao hn v thi gian ngn hn.

2.6. Phn tch nh tnh trong sc k kh

Trong phn tch nh tnh, hai detector c th nhn din cc hp cht l detector khi ph v detector hng ngoi chuyn ha Fourier. Mt pic c th nhn din bng cch so snh ph ca chng vi mt th vin ph c lu gi trong my tnh.

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Mt phng php km tinh t hn l nhn din thi gian lu ca mt cht vi thi gian lu ca cht trong mt mu bit trc (mu chun) trn cc ct c phn cc khc nhau (hnh 7). Cch ng tin cy nht l so snh cc thi gian lu trong cng mt sc k thu c b i mu bit trc (mu chun) c thm mu cn d tm vo trong cng mt iu kin sc k. Nu nh cht cn d tm trng vi cht c trong mu chun th pic ca cht trong mu thm s c din tch hay chiu cao tng ln so vi khi cha thm cht cha bit vo. S nhn din ch mc thm d khi thc hin trn mt ct, nhng khng nh hn khi thc hin trn mt vi ct trn nhng loi pha tnh khc nhau. Trong hnh 17 cho thy c th nhn din c cc nh 2,3,4,7 v 9 l metyl, etyl, npropyl, n-butyl, v n-amyl alcol. Cn ch rng c th c nhng cht khc nhau nhng c thi gian lu ging hoc rt gn nhau. V vy cn xc nh trn nhiu pha tnh khc nhau hoc s dng cc detector khi ph hoc hng ngoi xc nh.

Hnh 2.8: Phn tch nh tnh bng cch so snh thi gian lu vi cht chun

2.7. Phn tch nh lng

2.7.1 Mt s nguyn nhn gy ra sai s

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Chun b mu: cng nh mi phng php khc, mu em phn tch c ly mu sao i din ng cho c l nguyn liu hay sn phm. Cc qui tc ly mu cn phi tun th cho tng loi mu. Mu cn c lm sch trc khi tim mu vo GC. Vic ny nu lm khng tt c th gy nn mt cu t cn xc nh. Tim mu: khi cht lng tim vo bung tim mu th nhit thit lp nu qu cao c th gy nn s phn hy mu, hoc mu c tham d vo mt phn ng no . K thut tim mu cng c th gy sai s. Mu b phn hy hoc b hp ph: c nhiu trng hp c s phn hy hoc hp ph trong bung tim mu, trong ct, trong detector c th lm cho cc pic khng i din cho lng ca chng c trong mu. khc phc iu ny ta nn dng phng php lp ng chun bit din tch hay chiu cao ca pic c t l tuyn tnh vi lng mu a vo hay khng. p ng ca detector: mi detector p ng khc nhau vi cc hp cht khc nhau. V vy cn bit r cc h s p ng ny. Hn na khi iu kin lm vic thay i th p ng ca detector cng thay i. Trong GC c th s dng phng php chun ni khc phc iu ny. K thut ly tch phn: trong GC c nhiu cch thit lp quan h gia thng tin nhn c t pic sc k vi hm lng ca cu t: o chiu cao pic, dng my ghi v tch phn, ct v cn giy. Cc cch ny c th c nhng sai s ring trong qu trnh x l. Ngy nay vi s ghp n i my tnh v cc phn mm h tr vic tch phn ha din tch cc pic tr nn d dng v thng dng. Kt qu c bo co y cc thng tin ca pic nh chiu cao pic, din tch pic, phn trm trong mu 2.7.2 Cc phng php tnh ton nh lng 2.7.2.1. Phng php chun ha din tch y l phng php tnh thnh phn phn trm ca mu bng cch o din tch tng pic trn sc k . Theo cch ny em din tch pic ca cht quan tm A cho tng din tch ca cc pic: %A = (din tch pic A/tng din tch cc pic)x100 % Khi phn tch thnh phn c im si st nhau ca mt dy ng ng, phng php ny c th dng tnh t l phn trm khi lng. Phng php ny ch ng nu tt c cc cu t u c ra gii v p ng ca detector vi m i cu t l gi ng nhau. Nu nhng iu kin ny tha mn th y l phng php nhanh v hiu qu. 2.7.2.2. Phng php tnh theo h s hiu chnh Nh bit detector p ng khc nhau i vi cc cht khc nhau. V vy cn phi tnh h s hiu chnh. Nh h s ny c th tnh c thnh phn phn trm ca cc cu t trong mu.

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%A

dien tich cua A / F ( A)

i

(dien tich cua Ai / F ( Ai ))

Cch xc nh h s hiu chnh: Tim dung dch chun bit nng cc cu t A, B, C vo GC Sc k thu c c cc pic phn gii hon ton v din tch thu c tng ng SA, SB, SC tng ng vi cc khi lng trong mu mA, mB, mC Chn mt pic lm chun v d A c t l SA/mA c gn gi tr FA = 1 T t l SB/mB, SC/mC suy ra FB, FC

2.7.2.3. Phng php lp ng chun: Lp cc ng chun ring r i vi tng cu t trong hn hp bng cch tim nhng th tch bng nhau ca mt lot dung dch hn hp cht chun c nng khc nhau. Nh vy mt lot cc nng ca cc cht chun c phn tch v din tch ca chng c xc nh. Mt ng chun c dng cho m i cu t vi mt trc nng v trc kia l din tch tng ng kim tra s tuyn tnh ca p ng ca detector. Tim cng th tch ca mu c cc cu t cn phn tch v chy sc k trong cng iu kin nh khi chy chun. T cc din tch thu c ca cc cu t cn phn tch v ng chun va thit lp suy ra c nng ca chng. 2.7.2.4. Phng php dng chun ni Phng php ny cn c gi l phng php chun ha tng i hay gin tip. nh lng mt cu t X ta cn phi chn mt cht chun S sao cho: Nu trn ln X vi S ta phi thu c 2 nh ring bit trn sc k . Pic ca X v S phi kh gn nhau. Sau ta phi pha cc hn hp c t l trng lng ca X v S bit trc, chy sc k, o din tch cc pic, lp t s din tch tng ng, cui cng lp ng chun tng i.

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Sc/Ss

Wc/Ws
y :

Chun

Cu t

Sc/Ss l t l din tch ca cc cp cu t cn xc nh X v cht chun n i Wc/Ws l t l trng lng ca cc cp cu t cn xc nh X v cht chun n i Khi phn tch mu tht, ta cho mt lng bit trc cht chun n i S vo mu ri tin hnh sc k hn hp. T t l din tch o c, bng ng chun tng i va dng ta c t l trng lng. Vi trng lng chun S thm vo bit ta tnh c trng lng ca cht X. Phng php ny c cc u im: Khng cn bit n p ng ca detector Khng cn duy tr nghim ngt cc iu kin tin hnh sc k v nhng thay i c lo i tr theo cch tnh t s.

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BI TP
Cu 2.1: a) Trnh by s khc nhau gia sc k kh lng v sc k kh rn. b) Cho bit nhng loi cu t no c th tch c bng phng php sc k kh rn v sc k kh l ng c) Ti sao sc kh kh rn hu nh khng c s dng rng ri nh sc kh kh lng Cu 2.2: Nu cc nguyn tc hot ng ca cc loi detector c s dng trong sc k kh: a) b) c) d) Detector o dn nhit Detector ion ha ngn la Detector bt electron Detector khi ph Nhng u im v nhc im ca cc loi detector trn

Cu 2.3: Trnh by nh hng ca dy lp phim pha tnh ln sc k trong sc k kh Cu 2.4: Gii thch ti sao ct mao qun m cung cp phn gii tt hn so vi ct nh i trong sc k kh? Cu 2.5: Khi no cn s dng ch tim chia dng, khng chia dng v tim thng vo ct trong sc k kh? Cu 2.6: Lm th no ci thin phn gii ca 2 pic qu st nhau trong sc k kh? Cu 2.7: Trnh cc yu t nh hng n s dong rng ca pic sc k v nhng kt lun cn rt ra gim s dong rng pic khi tin hnh sc k kh l ng. Cu 2.8: Trnh by cc phng php xc nh nh lng c s dng trong sc k kh: a) Phng php chun ha din tch b) Phng php tnh theo h s hiu chnh c) Phng php lp ng chun d) Phng php dng chun n i Cu 2.9: Cho bit trt t ra gii ca hexan, heptan, octan, butanol v 2-pentanone ra kh i ct c pha tnh (diphenyl)0.35 (dimetyl)0.65 polysioxane. Bit ch s lu Kovats ca butanol v 2pentanone trn ct ny ln lt l 717 v 777. Cu 2.10: Cho bit trt t ra gii ca hexan, heptan, octan, nonane, benzene, nitropropane v pyridine ra khi ct c pha tnh (diphenyl)0.05 (dimetyl)0.95 polysioxane. Bit ch s lu Kovats ca benzene, nitropropane v pyridine trn ct ny ln lt l 672, 745 v 761.

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Cu 2.11: Mt hn hp cc methyl esters ca cc axit bo c sc k trn ct Carbowax 20 M cho cc din tch pic v cc h s p ng detector nh sau: Ester Din tch pic, cm2 H s p ng methyl-n-butyrate 2.95 0.81 methyl-iso valerate 0.86 0.88 methyl-n-octanoate 1.66 0.98 methyl-n-decanoate 4.52 1.00 Tnh thnh phn % hn hp. Cu 2.12: Trong mt php tch mt hn hp cc hydrocacbon, cc thi gian lu hiu chnh c o nh sau: propane 2.23 min isobutane 5.71 min butane 6.67 min Tnh ch s lu Kovat ca cc hydrocacbon ny? Cu 2.13: Mt hn hp n-heptane, tetrahydrofuran, 2-butanone, and n-propanol ra gii theo trt t ny khi s dng ct c pha tnh phn cc nh Carbowax. Trt t ra gii s o ngc li chnh xc theo th t trn khi s dng pha t nh khng phn cc nh polydimethyl siloxane. Gii thch trt t ra gii trong mi trng hp.

Cu 2.14: Cc s liu sau y l cc thi gian lu hiu chnh ca cc n-ankan s dng pha tnh Carbowax 20M. tr Alkane (min) Pentane 0.79 hexane 1.99 heptane 4.47 octane 14.12 nonane 33.11 Hi ch s lu Kovat c thi gian lu hiu chnh l 9.36 min? Cu 2.15: (7) Cc s liu sau y thu c khi phn tch sc k kh p-xylene v methyl isobutylketone (MIBK) trn ct mao qun. Injection Mode split tr Peak Peak Width Compound (min) Area (min) MIBK 1.878 54285 0.028 p-xylene 5.234 123483 0.044 splitless MIBK 3.420 2493005 1.057 p-xylene 5.795 3396656 1.051 Gii thch s khc nhau gia thi gian lu, din tch pic, v rng pick khi chuyn t tim mu chia dng sang tim mu khng chia dng.

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Chng 3: SC K LNG
(LIQUID CHROMATOGRAPHY)

3.1.

M u
Sc k l ng ct l mt trong nhng nhnh ca phn tch cng c pht trin nhanh nht, c p dng cho nhiu lo i mu hn bt k k thut tch no v c kh nng phn tch nhng hn hp mu cc k phc tp trong ha hc v trong sinh hc. Nghin cu k thut tch bng LC l cn thit v n s tip tc ng mt vai tr quan trng trong tt c nhng lnh vc ca phn tch ha hc, mc d c nhng tin b hn na trong cc thuc th chn lc v nhng ci tin trong cc k thut vt l o lng. Vi s pht trin trong sut nhng thp nin qua v thit b v s nh i ct, sc k lng hiu nng cao (HPLC) ni ln nh mt phng php c a thch cho k thut tch v phn tch nh lng ca rt nhiu loi mu. Phn tch HPLC nhanh, hiu qu v c th d tm lng mu nh n 200 pg. Nhng c h i p dng HPLC hu nh khng gii hn do HPLC tr thnh mt cng c khng th thiu trong khoa hc v cng nghip. Sc k l ng da trn hin tng: di cng mt iu kin, m i cu t trong mt hn hp tng tc vi mi trng xung quanh khc vi tt c cc cu t khc. Khi c s dng nh mt cng c phn tch, LC c th xc nh cc cu t trong mt hn hp v lng ca chng trong mu. Khi kt hp vi nhng k thut phn tch khc th c th nhn din nh tnh cc cu t trong mu. Php tch c i h i khi: Mt hn hp qu phc tp cho vic phn tch trc tip v d o ph. Cc cht phn tch rt ging nhau v d cc ng phn. S cn thit phi iu ch mt cht c tinh khit cao. Xc nh lng ca ca mt cht no . Sc k ct m, sc k bn mng c s dng trong nhng trng hp tch tng i n gin, cn HPLC l k thut tch nhng hn hp phc tp v c kh nng tch vi phn gii cao gii quyt nhng vn nhanh hn v tt hn. Trong HPLC nhiu thng s phi c xc nh v thit lp trc khi mu c cho vo trong my. V th thnh cng vi HPLC, cn thi gian thit lp ch v iu kin hot ng thch hp cho my trong mi php phn tch. iu ny c th mt hng gi hoc ngy ty theo kinh nghim v kin thc ca ngi phn tch.

3.2.

Cc b phn c bn ca my HPLC
C s phn tch sc k

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Hn hp cn phn tch c ha tan trong mt dung mi thch hp, tim mt th tch chnh xc vo b phn tim mu v c mang vo ct bi mt dng chy lin tc ca cng dung mi (pha ng) trong mu c ha tan. S tch din ra trong ct c cha nhng ht xp c din tch b mt ln (pha t nh). Cc cu t trong mu lin tc tng tc vi pha t nh. Pha ng (cn g i l cht ra gii) c bm qua ct c nh i cht cc ht sc k. Vi vic chn pha ng v vt liu nh i ct thch hp, cc cu t trong mu s di chuy n dc trn ct vi nhng tc khc nhau. Khi nhng cu t ln lt thot ra khi ct v i vo detector thch hp, y tn hiu c ghi li, x l v chuyn ra ngoi mt sc k , cho bit s hin din ca m i cu t di dng mt pic. Khi lng cu t c trong mu c tnh ton da vo chiu cao hoc din tch ca pic ca n. Cc b phn c bn ca mt my HPLC c m t trong s nguyn tc chung (hnh 3.1):

Bnh cha dung mi

Ct tch

Van tim mu Computor Sc k

L n nhit

Hnh 3.1: S my HPLC

3.2.1 Bnh cha dung mi Bnh cha dung mi l bnh thy tinh 1 dm3 c np c khoan l dn ng nha ( 1/8 inche) dn dung mi t bnh n bm. Dung mi bm vo phi khng c cha bi bn hoc nhng vt l khc v iu ny c th lm nh hng n tui th ca bm hoc lm kt cc ct tch.

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Cn lc dung mi qua giy lc 0.45m trc khi s dng v an ton hn u ca ng nha trong bnh cha dung mi cn phi c u lc. Cn loi b hoc ui cc khng kh ha tan hoc cc bt khng kh trong dung mi v kh ha tan c th lm bin dng cc pic v sinh bt kh lm nhiu ng nn. Loi b bng cch chy siu m, hoc sc kh tr nh heli Trong phng php ra gii thng ch cn mt bnh dung mi. Trong phng php ra gii gradient thng dng 2, 3 ho c 4 bnh dung mi khc nhau v h dung mi ra gii l hn hp ca cc loi dung mi trn c trn vi t l bin i theo chng trnh lp trc.

3.2.2. p sut trong ct tch p sut trong ct tch c th ln hn hai trm ln p sut kh quyn. Theo h thng SI th p sut c tnh bng n v Pascal (Pa). p sut ca kh quyn khong 105 Pa. V s thun l i trong vic th hin p sut bi nhng s nh hn nn nhiu ngi lm thc nghim v cc nh sn sut cng s dng nhiu n v khc nh bar, psi (pounds per square inche) hoc thnh thong kg cm-2. 1bar = 10 5 Pa nn 1 bar tng ng bng p sut kh quyn (760 mm Hg hoc 1 atm). 1 psi = mt lc ca 0.4536 x 9.81 N tc ng ln mt din tch 0.02542 m2 1 psi = 6897 Pa 1 bar = 14.5 psi Ct tch c th chu n 200 bar nhng hu ht nhng th nghim trong HPLC ch s dng p sut trong ct khong 20 n 100 bar. p sut trong ct ph thuc vo chiu di ct, kch thc ht ca pha tnh, nht v tc dng ca pha ng. 3.2.3. H thng bm Chc nng chnh ca bm l vn chuyn pha ng i qua ct tch vi mt tc xc nh. Bm cn p ng mt s yu cu kh cao v tnh nng nh sau: o Pha trong bm khng b tn cng bi bt k loi dung mi no c s dng. o Mt khong rng ca tc dng c th chn la v d thay i tc dng. o D thay i t vic s dng dung mi ny n loi dung mi khc. o D bo dng v sa cha. Mt s loi bm thng dng nh sau: Bm tc dng hng nh (constant flow pump) y l bm kiu syringe y dung mi lin tc, khi ht dung mi trong ng bm (khong 250 ml) th dng li np li.

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Bm kiu piston th y ht lun phin. Loi ny c dng ph bin v c nhiu u im. 3.2.4. H thng tim mu Phng php tim mu u tin mn k thut tim mu trong GC, trong dng syringe tim mu qua mt m cao su t hn kn c t ti u ca b tim mu u ct tch. Mt phng php na (stopped flow) l ngi ta dng bm v dng syringe tim mu vo ct tch thng qua mt ci kha. Phng php ph bin hin nay l dng van tim mu c mt vng mu (sample loop) c dung tch khc nhau t 5 n 500 l. Phng php ny rt d s dng, d t ng ha v cho chnh xc rt cao, mc du tng i t tin. Vi phng php ny mu u tin p sut kh quyn c tim bng xilanh vo vng mu. Sau chuyn van t v tr np mu (load ) n v tr tim mu (inject ) ni vng mu vo v tr ca dng chy ca pha ng, y lng mu chnh xc trong vng mu s c vn chuyn vo ct tch. 3.2.5. Ct sc k lng hiu nng cao Cc ct s dng trong HPLC c lm bng vt liu thp khng r (thp Cr-Ni-Mo) vi mt trong ca ct c lm nhn. Kch thc ph bin ca mt ct l ng knh ngoi bng 6.35 mm (1/4 in.), 4.6 mm ng knh trong, v chiu di nhiu c n 25 cm. Ct c nh i cc ht c c ng knh 3, 4, 5, 10 m. Cht nh i trong ct thng l silicagen hoc silicagen c bc hoc gn ha hc mt mng m ng cht hu c. Bn cnh silicagen ngi ta cn dng Al2O3, ht polime xp, ht cht trao i ion. B lc tin ct / ct bo v Sau b phn tim mu v trc ct tch cn phi lp ct bo v (hay b lc tin ct) gip bo v nhng ct t tin kh i s xm nhp ca ca cc ht bn nh c th c trong dung mi v trong mu tim vo. Ct bo v khng lm thay i hiu qu tch ca ct. N c thit k vi mt th tch tng cng tng i nh v th tch cht nh, nh vy khng lm dong pic. Ct bo v c y cc ht nh i trng phim m ng hoc c pha lin kt nh trong ct phn tch. Ct bo v c mt dung lng xc nh cho cht nhim bn. S ln tim mu, s suy gim ca hnh dng pic hoc phn gii l du hiu cn phi thay ct bo v. 3.2.6. Detector

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Chc nng ca detector trong HPLC l gim st pha ng i ra t ct phn tch. Tn hiu ra ca detector l tn hiu in t l thun vi vi mt vi tnh cht ca pha ng hoc ca cht tan trong mu. C kh nhiu thit b c ng dng lm detector trong HPLC. Nhng c trng quan trng ca detector trong HPLC l: nhy tuyn tnh p ng (respond) chn lc hoc thng dng p ng khng b tc ng bi nhng thay i ca cc iu kin th tch cht thp khng d b ph hy r tin, ng tin cy v d s dng. Khng c detector no c th s hu tt c nhng c trng trn v chng khng hon ton c lp vi nhau nn ci thin mt c tnh ca n c th lm ti thm ci kia. Bt k mt detector no cng chu ting n ca thit b, ch yu l phn in, l n ca n xc nh lng cht ha tan nh nht c th c d tm. Bi v khi ta gi m lng cht phn tch n mt gi tr no th tn hiu t detector s qu nh c th phn bit n vi ting n ca detector. nhy ca detector thng c xem nh nng to ra tn hiu c nh tn hiu to ra bi ting n. Mt detector tuyn tnh c tn hiu t l thun vi lng hoc nng ca cht tan. Khong tuyn tnh ca detector chnh l khong nng m s tuyn tnh ny tun theo. Gii hn pht hin cho bit lng ti thiu ca cu t c th c thy mt cch ng tin cy trn sc k . V mt thc nghim gii hn d tm ca mt cht l lng cht cho mt pic gp vi ln ting n ca nn (thng 3 ln). Mt detector vn nng th nhy vi m i th c trong mu v d detector khi ph, cn mt detector chn lc th ch nhy vi mt vi cu t no nh detector ECD nhy vi hp cht cha halogen. Kiu detector UV-VIS Hunh quang o dn o ch s khc x p ng Chn lc Chn lc Chn lc Ph thng Mc n 10-4 a.u 10-7 a.u 10-2 S/cm 10-7 r.i.u Gii hn pht hin (g/cm3) 10-8 10-12 10-7 10-6 Th tch flowcell (l) 1-8 8-25 1-5 5-15

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3.2.6.1. 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.

3.2.6.2. Detector chui photodiot (Photodiode Aray Detector) Trong detector UV-VIS chm sng a sc c cho i qua mu sau hi t vo khe vo ca b n sc ha (monochromator) tch chm tia n sc cn o hng vo b d tm. Mt quang ca mt mu c tm thy nh so snh gia cng ca chm tia ti b d tm khi khng c mu (cn gi l mu trng) vi chm tia i qua mu. o mt quang ti nhng bc sng khc nhau, bc sng c thay i nh quay chm lng knh hoc b cch t (grating) trong b n sc ha. S hin din ca b phn quay trong thit b gii thiu thm sai s (v d do s git li c hc) v vic quay chm c th gy tn thi gian. Trong detector chui photodiot, chm tia a sc sau khi i qua mu c tn x bi b cch t c c nh ri ri vo mt chui cc photodiot. Mi photodiot o mt di hp cc bc sng trong quang ph, nh vy n l b thu nh n s liu song song, ngha l tt c cc im c o ng thi. H thng ny c mt s thun li nh sau:

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Bi v l b thu nhn s liu song song nn vic x l v lu tr quang ph c th c thc hin trong khong 0.5 s so vi vi pht trong detector UV. V khng c b phn di chuyn c th b lm mi mn nn nhng sai s khi thit lp li mt bc sng cn chn c gim xung v thit b cng t i h i bo dng nh thit b khc. Nh kh nng thc hin nhng php o nhiu bc sng cng mt lc v tc thu nhn s liu nn nhng k thut ly trung bnh s liu khc nhau c th c s dng lm gim ting n v tng nhy. Khi c s dng nh mt detector trong HPLC, mc d t tin hn so vi loi UV truyn thng nhng n cng c nhng u im ng k: Quang ph ca m i pic trong sc k c th c lu gi v ln lt so snh vi ph chun gip tin li trong vic nhn din cc pic. Bc sng ti u cho vic d tm cng d dng c tm thy v nhng thay i ca bc sng trong qu trnh d tm cng c th c chng tr nh ha xy ra t i nhng im khc nhau trong sc k . N cng cung cp nhy cc i cho cc pic. 3.2.6.3. Detector hunh quang (fluorescence detector) Nhiu hp cht c kh nng hp th chm tia UV ri sau ngay lp tc pht ra bc x c bc sng di hn (hunh quang) hoc sau mt s tr thi gian (ln quang phosphorescence). Thng phn nng lng b hp th sau pht x tr li l tng i thp nhng c mt vi hp cht, phn pht x tr li c th t 0.1-1 nn thch hp cho vic d tm hunh quang. Nhng hp cht nh vy thng c vng lin hp nh cc hp cht vng thm c nhiu nhn. Nhiu hp cht khng c ph hunh quang c th chuyn sang cc dn xut c ph ny nh x l vi nhng thuc th thch hp. 3.2.6.4. Detector o ch s khc x (Refrative index detector) Detector (RI) ny nhy vi s khc nhau gia ch s khc x ca cc cht ra gii t ct vi dng chy ca pha ng nguyn cht. y l loi detector thng dng nht v bt k cht ha tan no cng c th c d tm min l n c ch s khc x khc vi dng chy ca pha ng nguyn cht.Tuy nhin, loi detector ny km nhy khong 1000 ln so vi detector UV. Mc ting n tt nht c th thu c vo khong 10-7 n v ch s khc x, iu ny tng ng vi nng tng ng ting n khong 10-6 g.cm-3 cho hu ht cc cht tan. C nhiu mu thit k cho detector o ch s khc x, nguyn tc c bn hot ng: Cuvet c hai bnh tam gic c th tch 5 n 10 microlit trong m i bnh c dung mi nguyn cht v dung dch cht tan chy vo.
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Chm sng kh kin lc cc bc x hng ngoi (c th lm nng mu) c chiu qua cuvet c dung mi nguyn cht chy qua 2 bnh tam gic v c hng vo ng nhn quang in nh mt a deflection. Khi cht tan vi ch s khc x khc i vo cuvet, chm sng b lch lm tn hiu ca ng nhn quang in thay i. Detector RI khng dng trong sc k ra gii bin i nng u (gradient) v n khng th d tm chnh xc khi thnh phn pha ng b thay i. RI nhy vi s thay i nh ca nhit v p sut (0.01oC). V c nhy thp nn n khng c dng trong phn tch lng vt. RI cng c khong nng tuyn tnh hp.

3.3. Cc phng php sc k lng hiu nng cao

3.3.1. Sc k phn b hiu nng cao Gm hai loi phng php sc k l ng-lng v sc k pha lin kt 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. Sc k pha lin kt (bonded phase chromatography) Pha t nh c gn ha hc (lin kt) vi cht mang (ht silicagen) to nn hp cht csiloxan (hnh 3.2). Nu R l mt nhm t phn cc nh octyl (C8), octadecyl (C18) hay phenyl v pha ng 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: o Cc phn t khng phn cc, t phn cc, ion, hoc c th ion ha c th c tch mt cch hiu qu. o Pha tnh c mt khong phn cc rt rng c th chn la. o N cho php chn mt khong rng cht ra gii. o Cc cht dung mi ra gii rt thng dng v khng t (nc, methanol v acetonitril). o K thut ra gii gradient c th c s dng m khng s hng pha tnh.
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o Khng c hn ch v p sut pha ng i vo ct. o N cho kt qu tch tt vi rt nhiu i tng tch.

Hnh 3.2: S tng hp cc pha tnh pha lin kt ha hc

Chn cc pha: thng chn pha tnh c tnh phn cc gi ng 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 l n lt ra sau. ng dng: sc k l ng 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

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3.3.2.

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 mu

Sc k hp ph hiu nng cao (sc k lng rn) 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 nhng bt mn, silicagen v nhm oxit l nhng cht c a dng. y cc cht cng b phn cc cng b lu gi mnh v ra chm khi ra gii. Pha ng: 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. 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. Dung mi Ete du ha Hexan Heptan Xiclohecxan Tetraclocacbon Ete propylic Toluene Benzene Ete etylic Clorofom Clometylen Dicloetan Aceton dioxan Butanol Etyl acetat Acetonitril Pyridin Dimetylsunfoxit Ru isopropylic Ru etylic o 0.01 0.01 0.01 0.04 0.18 0.28 0.29 0.32 0.38 0.40 0.42 0.49 0.56 0.56 0.56 0.58 0.65 0.71 0.75 0.82 0.88

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Ru metylic

0.95

3.3.3. Sc k trao i ion hiu nng cao 3.3.3.1.Nha trao i ion (ionit) L phng php sc k trong cc nha trao i ion c dng lm pha tnh di dng bt mn. Nha trao i ion (ionit) l nhng hp cht cao phn t c cha nhm chc cha ion c kh nng trao i. Nha trao i cation (cationit) c hai loi: cationit axit mnh c cha nhm SO3-H+ c ng dng rng ri, cationit axit yu c nhm COOH. Nha trao i anion (anionit) cng c hai loi: anionit baz mnh c nhm amoni bc 4 N(CH3)3+OH- v anionit yu c nhm trao i l cc amin bc 2 hay 3. Cc nha trao i ion c ch to bi s ng trng hp gia styrene v divinyl benzene vi divinyl benzene c hm lng thay i t 1 dn 16 % tng mc lin kt ngang ca polime, dn n ch trng n nhng khng ha tan trong nc. Cc nhn benzene sau c gn vo cc nhm SO3H to nha cationit hoc cc nhm amin NR3+ to nha anionit. Nu axit metaacrylic c dng thay th cho styrene th nha cationit axit yu COOH c to thnh.

Lin kt ngang Nhm chc trao i ion

Hnh 3.3: S tng hp nha ionit

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Cc ionit khng tan trong nc, khi tip xc vi dung dch trao i ion th c s trao i. Vi cationit c s trao i cation, v d s trao i cation Ca2+ vi nha cationit axit mnh: 2RSO3-H+(rn) + Ca2+ (dd) (RSO3)2Ca(r) + 2H+(dd) Vi anionit ta c v d: 2R(CH3)3+OH- (r) + SO42-(dd) [R(CH3)3]2SO42-(r ) + 2OH-(dd) 3.3.3.2. Cn bng trao i ion Qu trnh trao i ion gia nha cationit axit mnh vi ion Ca2+ trong dung dch c th vit: 2H+(rn) + Ca2+(dd) Ca2+(r) + 2H+(dd) p dng nh lut khi lng ta c: K' [Ca 2 ]r [ H ]2 d 2 [Ca ]dd [ H ]2 r

y pha rn l nha ionit. Biu thc trn c th biu din li nh sau:

K [Ca 2 ]r [Ca 2 ]dd K' [ H ]r [ H ]2 dd
2

K l h s phn b ca Ca2+ gia dung dch v nha ionit. Ion no c K ln s b lu gi mnh trn ionit, K nh s b lu gi yu. K ph thuc vo in tch v kch thc ca ion b hydrat ha. Ni chung, nha trao i ion u tin trao i vi ion c in tch cao hn, bn knh hydrat ha nh hn. Mt th t u tin v s chn la cho cc cation c th c biu din nh sau: La3+ > Ce3+ >Eu3+ >Y3+ > Sc3+ > Al3+ > Ba2+ > Pb2+ > Sr2+ > Ca2+ > Ni2+ > Cd2+ > Cu2+ > Co2+ > Zn2+ > Mg2+ > Ag+ > Rb+ > K+ > NH4+ > Na+ > H+ > Li+ 3.3.3.3. Pha ng trong sc k trao i ion: Ra gii gradient vi s tng lc ion hoc thay i pH c gi tr rt ln trong sc k trao i ion. Gi s xt mt ct m anion A- b lu gi mnh hn anion B-. Ta tch A- khi B- bng cch ra gii vi ion C- c i lc vi ct yu hn c A- v B- . Khi nng C- tng ln, C- dn dn b thay th v di chuyn dc theo ct v ra kh i ct. Khi nng ca C- tip tc tng ln th A- s b ra gii. Trong hnh 10 cho thy mt s tng dn nng H+ c s dng tch trao i ion. Trt t ra gii gi ng vi trt t ca cc h s chn lc ca cc cation. H s chn lc cng ln th nng H+ i hi cng cao ra gii cation ra khi ct.

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Hnh 3.4: Ra gii La(III) t ct nha trao i cation s dng gradient nng H+ tch cch cation lu gi mnh hn. La(III) c d tm bi phn ng vi thuc th to mu sau khi ra gii.

Khi tm gi tr thch hp pH cho mt php tch c th, iu cn bit l gi tr pKA ca cc cu t c trong mu. ng dng : Phng 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 kh i ct l Mg2+ , Zn2+ , Ca2+ v Ba2+ . C th pht hin cc ion c ra gii ra kh i ct nh detector o dn in. 3.3.4. 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 loc km. Trong ICP ta c th s dng c sc k pha thun ln sc k pha o, m i loi c mt thun li ring ca n. Pha t nh ca sc k pha o c th gm c c pha tnh silic c silan ha c s dng 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 t nh lin kt) v mt ion i c in tch ngc du vi ion phn t mu.

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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 b: Bng sau tm tt vi h thng ICP c bo co trong cc ti liu vi m i ng dng ch r pha ng, pha tnh, ion i. Bng: Cc h thng sc k cp ion pha thun
pH ca nc pH 9 0.1MHClO4 Pha ng Cyclohexane/CHCl3/1-pentanol Cc hn hp ca tributyl phosphate, ethyl acetate, butanol, CH2Cl2,v/hoc hexane Butanol/CH2Cl2/Hexane CH2Cl2/ CHCl3/ butanol v /hoc Pentanol/CH2Cl2 v/hoc CHCl3 Butanol/heptane Butanol/CH2Cl2/Hexane Butanol/CH2Cl2/Hexane Ion i N,N-dimethylprotrityline ClO4(Butyl)4N+ ClO4Picrate (Butyl)4N+ ClO4CH3SO3(Butyl)4N+ Loi mu Cc axit cacboxylic

Cc amin

m HPO42- /PO430.1MHClO4 pH 5-6 pH 6-8.5 0.2-0.25M HClO4 0.1M methanesunfonic acid pH 8.3

Cc axit cacboxylic Cc amin Cc amin Cc sunfoamit Amin v cc amin bc bn Cc amin Cc axit cacboxylic

Butanol/CH2Cl2/Hexane Bng : cc h thng sc k cp ion pha o

Nhng 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. o S tng nng ca ion i dn n s tng h s dung lng k trong ICP pha o. 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). o Phn t ion i cng ln th gi tr kcng ln trong sc k pha o v cng nh trong sc k pha thun.

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phn cc ca dung mi: o Lc dung mi trong ICP pha thun v pha o c th c thay i bi thay i phn cc ca pha ng. o Trong cc h thng pha o, hn hp nc vi methanol hoc acetonitril ni chung 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 o 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). o 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 m i hp cht s thay i theo pH. o Ni chung pH ca pha ng c duy tr nh th no cho cc hp cht mu c ion ha hon ton, cn s to thnh ion i th t cc i. o V d khi tch cc axit nu pH ca pha ng thp th anion mu X- s nm d i dng HX dn n s lng mu nm dng ion i trn pha tnh s thp. ng dng: ICP 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. 3.3.5. 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 nh i 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 nh i 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 lo i 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.

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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 nh i ct, bn hn, cho php s dng mt khong rng cc dung mi bao g m 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 lo i 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 d b mt ca mt cht nhi a nc c cu trc: OH OH SiCH2CH2CH2CH2OCH2CHCH2 Phng trnh ra gii: Trong sc k theo gel th tch ca pha ng Vm thng c g i l th tch rng (Void volume) Vo. i lng Kav c nh ngha l: Kav = (Vr Vo)/(Vt - Vo) y o Vr l th tch lu ca mt cht tan, o Vt l th tch tng ca ct = r2 vi r l bn knh). o i vi mt phn t ln v khng xm nhp vo gel th Vr = Vo v Kav = 0. o i vi cc phn t nh xm nhp t do vo gel th Vr Vt v Kav 1. o Cc phn t c kch thc trung bnh xm nhp mt phn vo cc l th c Kav nm trong khong t 0 n 1. Thc t do lun lun c s hp ph nn Kav lun lun ln hn 1. Th tch rng c o bi cho mt loi phn t tr c kch thc ln i qua ct. Th tch ra gii ca n chnh l Vo.

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Hnh 3.4: S cu trc mt poliacrylamide

3.4. Pha ng
3.4.1. S quan trng ca s phn cc trong pha ng HPLC S phn b tng i gia 2 pha c xc nh bi s tng tc ca cu t cht tan gia hai pha vi cng c xc nh bi bi s phn cc ca cc phn t mu, ca pha ng v ca pha tnh. Cc lc tng tc gia cc phn t c th gy ra bi: o Mt phn t cht ha tan c momen nh n c th tng tc mt cch chn lc vi nhng lng cc khc. o Nu phn t l cht cho hoc nhn proton tt th n c th tng tc tt vi nhng phn t nh vy nh lin kt hydro. o Mt phn t cng c th tng tc lng cc cm ng nh b phn cc bi cc phn t khc.

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phn cc l mt i lng c s dng trong sc k nh l mt ch s o kh nng ca cc hp cht tng tc vi cht khc theo nhng cch khc nhau trn: mt phn t cng phn cc th cng tng tc mnh vi nhng phn t khc. Nu phn cc ca pha t nh v pha ng gi ng nhau th tng tc ca cht ha tan v i hai pha ny l nh nhau dn n s tch cc cht ha tan l km. Nh vy vi pha t nh khng phn cc kiu hydrocacbon ta cn mt pha ng phn cc mnh, trong khi pha tnh l silica phn cc mnh th ta cn pha ng phn cc tng i thp. Nu ta quan tm tch cc hp cht tan gn gi ng nhau v mt ha hc th ta nn chn pha tnh gn gi ng v mt ha hc nh cc cht tan cn tch m ta quan tm. S lu gi ca cc cht tan thng thay i khi ta thay i phn cc ca pha ng. 3.4.2. o phn cc D thy rng nc l dung mi phn cc hn heptan. Nc c mt lng cc nn l cht cho nhn proton v d ha tan cc cht ion. Tng t, metanol v acetonitril u phn cc hn heptan, nhng khng d dng xc lp phn cc tng i cho metanol v acetonitril. tin li c th biu din phn cc ca mt dung mi hoc hn hp dung mi di dng mt con s. iu ny c thc hin bi nhiu cch v khng c cch no hon ton tha mn. Tuy nhin, chng cho php ta sp xp cc dung mi theo trt t tng dn ca phn cc v gip ta d on c phn cc ca hn hp cc dung mi. Bng sau cho gi tr phn cc ca cc dung mi c sp xp theo trt t tng dn ca thng s sc ra gii .

Dung mi Hexan Metyl benzene Tetrahydrofuran Triclometan Butanon Etyl axetat Diclo metan 1,2 dicloetan Propanon 1,4 dioxan 2-metoxyl etanol Axetonitril Etanol Methanol Nc

14.9 18.2 18.6 19.0 19.0 19.6 19.8 20.0 20.2 20.4 23.3 23.9 25.9 29.4 47.8

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Thng s sc ra gii ca mt hn hp cc dung mi c th c d on s dng qui tc n gin sau:

m i i

y i l t l th tch ca dung mi i v i l thng s sc ra gii ca dung mi i. Nh vy ta c th to mt dung dch hn hp c gi tr phn cc cn c bng cch s dng mt hoc nhiu dung mi c phn cc cao hn trn vi mt hoc vi dung mi c phn cc thp hn phn cc cn i h i. V d i vi mt dung dch hn hp gm hai dung mi methanol (M) v nc (w)
m w w M M w M M w M

(1
w

) (

Trong sc k pha o th cc hn hp cha cc dung mi khc nhau c cng phn cc ton phn c gi l cc dung dch hn hp cng lc ra gii (isoeluotropic). Trong thc t vic thay th pha ng bng mt hn hp isoeluotropic s to thi gian phn tch mu gn bng nh trc nhng cc gi tr k ca cc cu t trong mu s thay i do nhng hiu ng nh cp tc l phn gii ca chng s b thay i. Mt cch phn loi khc theo sc ra gii ca dung mi trn ct pha tnh nhm oxit v i nc c chn l dung mi c sc ra gii ln nht bng 1. Bng sau 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 xp theo kh nng y cc cht ra kh i nhm oxit, nhng vi silicagen ta cng c dy tng t. Mun c dung mi pha ng vi tr trung gian khng c trong bng, ta dng hn hp dung mi theo t l thch hp. Dung mi Ete du ha He xan Heptan Cyclohe xan Tetraclorua cacbon Ete propylic Toluen Ben zen E te etylic Cloroform Clorua methylen Dicloroetan Aceton Dioxan 0.01 0.01 0.01 0.04 0.18 0.28 0.29 0.32 0.38 0.40 0.42 0.49 0.56 0.56

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Butanol Etyl acetat Acetonitril Pyridin Dimetyl sunfua Ru Izopropylic Ru etylic Ru mtylic

0.56 0.58 0.65 0.71 0.75 0.82 0.88 0.95

3.4.3. Ti u ha pha ng Khi pht trin mt phng php HPLC, ta mun t c mt mc tch c th chp nhn c tt c cc cu t ta quan tm trong mu vi mt thi gian va phi v vi mt phn gii va xc nh. Mt s mu ta ch cn phn tch mt hoc vi cu t c trong mu. mt mc no , s tch c th chu nh hng ca cc thng s nh nhit , ct tch, tc dng ca pha ng, nhng yu t quan trng nht kim sot vic tch vn l thnh phn ca pha ng. Nu php tch khng tha mn bi vic s dng pha ng l hn hp 2 dung mi th c th s dng 3 hoc thm ch trong mt s trng hp n 4 dung mi cho thnh phn ca pha ng. Vn l t l thnh phn pha ng ca hn hp ny l bao nhiu? S ti u ha pha ng c nhiu nhm nghin cu trong thi gian qua v sau y l mt s chin lc ti u ha pha ng c c kt. 3.4.3.1. Tm mt pha ng thch hp cho pha o Khi s dng mt ct pha lin kt theo kiu pha o, vic tm thnh phn pha ng tt nht cho mt php tch theo mt cch tip cn chung: u tin nhn din mt dung mi mnh (v d metanol, acetonitril hoc tetrahydro furan) m mu c th ho tan. Bc tip theo l chn mt dung mi yu m mu ha tan mt cch hn ch. Phn ln thng chn nc. Mt khi mt dung mi mnh v yu c nhn din, vic tim mu vo ct c thc hin s dng dung mi mnh va chn lm pha ng. Trong trng hp ny tt c cc cu t u ha tan to mt pic ti Vo. Nu c s lu gi khi s dng 100% dung mi mnh lm pha ng th mt dung mi mnh hn cn c tm thay th. V d, nu s lu gi xy ra vi 100% metanol th th 100% acetonitril lm dung mi mnh cho pha ng. Sau khi xc nh khng c s lu gi ca cc cu t trong pha ng 100% dung mi mnh, pha ng nn c lm cho yu hn nh trn vi dung mi yu vi nhng t l khc nhau.
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Mu sau c tim vo mi pha ng mi ny v s lu gi c ghi chp li cho n khi s lu gi xy ra. T l dung mi mnh v yu nn c pha trn theo nhng bc logic (v d 100%, 80%, 60%, 40%, 20%). Nh vy s lu gi ca cc pic c th c so snh tm thnh phn pha ng thch hp m c th t c phn gi i mong i. Cn ch n tc pha ng trong qu trnh kho st trn. Thng mt tc pha ng nhanh (trong vng gii hn p sut ca h thng sc k) c s dng hn ch vic tn thi gian cho vic pht trin phng php. 3.4.3.2. Pht trin mt pha ng ci thin chn lc Khi pht trin mt php tch, mc tiu u tin l tm cho c mt cp dung mi tt nht. Nhng thnh thong vic trn hai dung mi nh cch chn trn khng to pha ng c th tch tt. V th cn phi th mt dung dch hn hp mi, v d nu hn hp metanol/nc khng cho php tch tt nh mong i th c th ngh n hn hp acetonitril/nc, hoc mt hn hp dung mi hu c khc/nc. Ni chung nu metanol khng thch hp th chn la tip theo l acetonitril. Cc pha ng c lc ra gii ging nhau c g i l cc isoeluotropic. Khi s dung cc pha ng isoeluotropic thay th th c th tng cng chn lc ca nhng hp cht c cng gi tr k khi pha ng l dung dch hn hp c chn lc ban u, ngha l c th tch c chng. i khi mt hn hp hai dung mi khng th gip php tch t c. Trong trng hp ny hn hp 3 dung mi l hu ch. C nhiu ti liu v vic s dng nhiu hn mt dung mi hu c cho pha ng t c chn lc. Cng c nhiu phn mm tr gip pht trin cc pha ng s dng ti 4 dung mi trn ln (v d metanol/acetonitril/ tetrahydrofuran/nc). Tuy nhin, cc pha ng s dng ba dung mi khng phi xy ra ph bin, thc t ch cn n chng cho nhng trng hp rt kh khn trong vic tch v thng i h i s tr gip ca nhng chng trnh my tnh.

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Hnh 3.5: Ti u ha php tch HPLC s dng cc pha ng c thnh phn thay i. Cc pic: (1) paracetamol, (2) caffein, (3) aspirin, (4) phenacetin, s dng ct C18, 5m, detection 235 nm.

3.4.3.3. S dng cc pha ng khng nc Bi v cc php tch pha o c da trn kh nng ha tan ca mu trong cc pha ng v pha tnh nn khng cn thit s dng nc nu kh nng ha tan b hn ch.

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Thng hai dung mi hu c (mt dung mi hu c mnh v mt yu) c th v nn c s dng hn l mt dung mi hu c mnh v nc. Chng hn nu mu hon ton khng ha tan trong nc, dung mi yu phi l mt dung mi khc nc. V d trong trng hp tch triglycerit (cc cht bo), nc c xc nh l dung mi qu yu v cc cht bo thm ch khng ha tan mt phn trong nc. Trong php tch pha o, mt dung mi mnh l THF v dung mi yu kia l acetonitril. Nh rng vic pht trin mt php tch pha o khng i hi vic s dng mt dung mi l nc mc d nhiu ngi khng ng nh vy. 3.4.3.4. Ti u ha vi hai hoc ba dung mi cho sc k pha o Hnh 3.6 m t mt qu trnh thc hin mt cch c h thng pht trin mt php tch vi s kt hp ca cc dung mi.

Hnh 3.6: Pht trin phng php HPLC sc k pha o theo kiu tam gic u. THF l tetrahydrofuran. Hnh trnh by th tc c p dng cho mt php tch sc k thc t.

Cc bc cn thc hin t c mt php tch thch hp p ng nhng tiu ch t ra (c th khng cn qua tt c cc bc): o Bc 1: Ti u ha php tch vi acetonitril/dung dch m thu c sc k A. o Bc 2: Ti u ha php tch vi metanol/dung dch m thu c sc k B. o Bc 3: Ti u ha php tch vi tetrahydrofuran/dung d ch m thu c sc k C. o Bc 4: Trn cc dung mi s dng A, B, C theo tng cp, vi m i cp c t l 1:1 to cc sc k D, E v F. o Bc 5: Xy dng mt hn hp 1:1:1 ca cc dung mi A,B,C to sc k G. o Bc 6: Nu cc kt qu t A n G l tt th chn 2 im tt nht v trn dung mi thu c cc im gia hai im ny.

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Acetonitril/H2 O

Metanol/H2 O

Tetrahydrofuran/H2 O

Hnh 3.7: Gin cho thy phn trm th tch ca dung mi c cng lc ra gii. ng thng ng giao vi giao vi mi ng dung mi to nhng im c cng lc ra gii. V d 30% th tch acetonitril/70% nc c cng lc ra gii nh 40% th tch methanol hoc 22%V ca tetrahydrofuran.

3.4.4. Tch bng pha ng c thnh phn bin i u (Gradient separations) Nhiu trng hp mu cha nhiu cu t c phn cc khc nhau trong khong rng nn mt php tch vi pha ng khng thay i thnh phn c th dn n : - Mt s cu t c h s dung lng k nh cn mt s th rt ln nn khi lc ra gii ca pha ng thp phn gii cc pic ra u tin th s ra gii cc pic sau cng rt chm lm ko di thi gian phn tch v lng ph dung mi. - Mt s pic c th b ko ui. t c phn gii nhng gim thi gian phn tch, mt s bin i nng thnh phn nn c chn. Pht trin php tch gradient. Bc u tin nn kho st trong mt khong rng ca lc ra gii theo ch khng thay i thnh phn dung mi (isocratic elution), t 10 n 100% nng ca dung mi hu c mnh v d acetonitril trong nc. o iu ny giup ta xc nh c cc gi tr k ca cc cu t c trong mu cho m i trng hp. o Nu cc gi tr k ca cc cu t kh gn nhau th trong sc k pha o ta c th gi m nh thnh phn dung mi hu c xung s gip ci thin phn gii. Nu khng th thay i dung mi khc hoc thay i pha tnh. o Nu cc gi tr k ca cc (hoc mt s) cu t c k khc nhau kh ln v vt ngoi khong 5-10 th nn s dng ch ra gii c thnh phn pha ng bin i u (gradient). Bc tip theo l tri cc pic rng ra bng cch chn khong bin thin gradient hp hn:

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o Biu gradient biu din s ph thuc ca thi gian t lc tim mu vi % ca dung mi acetonitril ca sc (a) trong hnh 3.8 tng u t 10 dn 90 % trong 40 pht (vi thi gian ch dung mi l gn 5 pht). Pic 1 c ra gii thi im pht 14 khi thnh phn dung mi l 28% v pic 8 c ra gii gn 35.5 pht tng ng thnh phn % dung mi l 71%. Nh vy s bin thin thnh phn dung mi t 10 n 28% v t 71 n 90 % l khng cn thit. o V th trong ln chy sc k th hai c th thay i thnh phn pha ng tuyn tnh acetonitril t 28 n 71% trong thi gian 40 pht. Trong sc (b) iu kin c chn hi khc mt t l t 30 n 82% trong 40 pht. Kt qu cc pic c tri rng hn v thi gian cc pic tch kh i nhau cn 32 pht. Trong sc k (c), ta mun xem th liu c th s dng khong gradient hp hn gim thi gian chy sc k. Thnh phn dung mi pha ng trong ln ny bin i nh trong (b) nhng trong khong thi gian 20 pht. Kt qu sc (c) cho thy cc pic 6 v 7 khng c phn gii hon ton nn sc k (b) hp l cho php tch gradient. Nu php tch trong sc k (b) khng th chp nhn th ta c th th ci thin n bng cch gim tc dng pha ng hoc tin hnh gradient phn on. L l ca gradient phn on l s dng thnh phn dung mi tt cho m i vng ca sc k v sau tng % ca pha ng cho vng tip theo. iu ny tin hnh d dng. Vi tt c cc cng c trn, ta c th tm ra cch cc cu t ca mt hn hp nu n khng cha qu nhiu cu t. Nu sc k pha o tht bi cn chuyn sang mt phng php khc nh sc k pha thun.

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Hnh 3.8: Cc php tch gradient tuyn tnh ca cng mt mu c s dng trn trong cng mt ct tch v h thng dung mi tc dng 1.0 ml/phut. Thi gian ch dung mi l 5 pht.

3.5. Pht trin mt php tch

Khi pht trin mt php tch, mt chin lc ca vic chn pha ng c cp. Tuy nhin khi phn t c phn phn cc v mt phn khng phn cc th nhng mc tiu h tr chin lc pht trin phng php hi khc vi nhng tho lun trn. minh ha cho vic tip cn pht trin phng php cho loi mu ny, ba loi estrogen trong mt mu dc phm s c tch. Cu trc ca ba loi ny trong hnh 3.9.

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Hnh 3.9: Cu trc ca 3 estrogen lin hp

Hnh 3.10: Kt qu tch cui cng ca 3 estrogen (a) Mu chun, (b) Mu vin thuc. Pha ng s dng 35/65 MeOH/H2O(v/v) + m KH2PO4 0.001M

Trong v d ny trong khi tt c cc hp cht c cu to tng t nhau v mt cu trc, chng ch khc v mc bo ha trong vng th hai, iu ny ng v s khc nhau ca phn khng phn cc trong cc phn t ny. Cc phn t ny l nhng mu i natri v c in tch -1 khi ha tan trong nc.

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S dng ct trao i ion c l khng l mt tng tt v tt c cc hp cht ny u c cng in tch v khng may pKa ca chng khng c bit. Bi v c s khc bit nhau v cu trc phn khng phn cc trong phn t, ta nn ch n pha tnh m c th phn bit i x gia s khc nhau ca nhng phn khng phn cc ny (nu pKa ca chng khc nhau ng k th c th tip cn phng php sc k trao i ion). Mt pha lin kt khng phn cc nh C18 l mt la chn thch hp. Trong nhiu trng hp, vic c sn nhiu loi ct khng phn cc s quyt nh loi ct no c chn cho php tch. Tuy nhin, nh c cp trc y, bi v ct C18 cho s lu gi tt vi mt khong rng ca lc ra gii ca pha ng, n l ct dng cho nhng mc ch ph bin tt nht cho pha o. Sau khi ct C18 c chn, nhng dung mi mnh v yu phi c chn. Bi v ct khng phn cc c chn nn nc c chn nh l dung mi yu cn metanol nh l dung mi mnh. Tuy nhin, acetonitril hoc THF c l cng c th c chn nh l mt dung mi mnh. Lc dung mi theo trt t sau : metanol < acetonitril < THF. V th, chn metanol trc s cho php uyn chuyn trong vic chn hai dung mi sau nu metalnol khng cho kt qu tch tt. Ngoi ra metanol c xem l dung mi t c hn so vi hai dung mi kia. Bi v im bt u l mt ct pha lin kt C18 10 m v mt pha ng gm metanol/nc, cn chun b mt mu cha 3 cht chun trong mt hn hp 50:50 metanol/nc. y l mt s la chn ty v thnh phn pha ng cha c xc nh. Dung dch mu cng c ha tan trong cng mt dung mi nh trng hp dung dch cht chun, c trn trong thi gian ngn v c lc tch nhng cht khng ha tan trong vin thuc. Bc tip theo l bt u chy sc k vi 100 % metanol nh l pha ng vi tc pha ng thch hp. Metanol c kim tra nh l mt dung mi mnh bi vic quan st sau khi tim mu, tt c u c ha tan ti th tch cht v th khng c s tch y. Nh vy metanol l dung mi qu mnh lm cho cc cu t cn tch trong mu u ha tan vo trong pha ng v khng b lu gi trn pha tnh. Cht ra gii ny phi c lm yu hn nh thm vo n phn trm bit ca nc v mu c tim vo. Qa trnh ny c tip tc cho n khi s lu gi bt u r rng. Trong v d ny l trng hp 50:50 metanol/nc v kt qu tch tt nht ti 35:65 metanol/nc. Bc tip theo l tim cc chun ca estrogen v xc nh li s lu gi ca chng.

3.6. Sc k lng trn ct m

3.6.1 Gii thiu Trong phng php ny ngi ta s dng ct m hay ct c nh i vi pha ng ch y qua. Phng php ny c phn bit vi phng php sc k l ng hiu nng cao (HPLC) ch dung mi pha ng chy qua ct do trng lc ca n.

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K thut ny c s dng vi tt c cc qu trnh: hp th, phn b, trao i ion, v sc k qua gel. Thit b v thit lp thc nghim c trnh by trong hnh 3.11:

Hnh 3.11: Cu to v nguyn tc hot ng ca ct sc k ct m

Hin nay nhiu ng dng phn tch nh tnh ca k thut ny c thay th bi sc k bn m ng v HPLC. S ph bin ca HPLC cng lm gim nhiu ng dng khc ca k thut ny. Tuy nhin, n vn c s dng nhiu trong nhng php tch vi lng ln (> 10 g) ca nhng hn hp phn ng trong tng hp hu c, nhng cng vic c lp v chit tch cc hp cht hu c t nhin. 3.6.2. Cc vn thc nghim 3.6.2.1. Ct v cc th tc nhi ct Sc k ct m c thc hin ch yu trong cc ct thy tinh. Kch thc ca ct ph thuc vo s lng ca cht cn tch. Ct nh nht c ng knh ch vi milimet v chiu di vi cm, trong khi ct ln nht c th vi cm ng knh v chiu di tng ng di hn. thu c hiu qu ln nht ct phi c nhi thch hp. nh hng ca kch thc ht v th tc nh i ct c tho lun trong chng trc. Do phng php s dng trng lc ca dung mi chy xuyn qua ct nn kch thc ht nhi phi > 150 micromet thu c nhng tc dng chy c th chp nhn c. Ct phi c nhi ng nht lm gim thiu n mc nh nht s bin dng ca sc k. Mt vn na cn ch y l bt khng kh trong qu trnh nh i ct. ngn nh hng ny n mc c th, cht nhi ct nn c trn vi dung mi vi t l 1:3 v rt thnh
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dng mng vo vo trong ct. Nu kch thc ht l ng nht th ct d hn nh i. Khi nh i ct hoc trong qu trnh tch khng c cho ct kh bt k v tr no v nh vy dn n khng kh xm nhp vo cc v tr . 3.6.2.2. Np mu Mu nn c a vo u ct khi chun b xong ct v c pha vi mt lng dung mi dng ra gii thch hp. Phn trn ca ct c th c bo v bi mt lp bng th y tinh, giy lc Khi tt c mu c hp ph ht th rt dung mi ra gii vo v tin hnh sc k. Dung mi c th c np lin tc theo yu cu ca php tch. 3.6.2.3. Cc th tc ra gii C ba th tc ra gii chnh: ra gii ng h (isocratic), ra gii phn on fractional (stepwise) v ra gii bin thin gradient: Isocratic l th tc ra gii vi hn hp dung mi c thnh phn khng thay i trong qu trnh chy sc k cho n khi hon thnh s tch. Factional l th tc ra gii s dng mt dung mi hoc hn hp dung mi thch hp c lp v tch mt hoc mt nhm cu t cn tch trong mu. Thnh thong c th s dng nhiu dung mi khc nhau c lc ra gii tng dn tch cc cu t hoc nhm cu t khc nhau. Gradient l th tc s dng mi trng ra gii c thay i lin tc, ngha l nng ca cc dung mi trong hn hp c thay i bin thin u theo thi gian. nh hng ca th tc ny l c th ra gii ln lt cc cht hp ph mnh dn v gim hin tng ko ui. iu ny c ngha l cc di sc k s c khuynh hng m dc hn trong ct v chim trn ct t hn. 3.6.3. Sc k hp ph 3.6.3.1. Dung mi Cht ra gii s dng trong sc k hp ph c da trn cc dung mi khng phn cc nh hexan hoc cha lng nh nhm phn cc nh 2-propanol. Khi mu c a vo ct th cc phn t mu c cha cc nhm chc phn cc s lin kt vi cc v tr hot ha trn cht nh i. Chng s b thay th ln lt bi cc phn t c cha nhm phn cc ca cht ra gi i khi khai trin sc k v s di chuyn xung cc v tr mi pha di ca ct. Vic thay th ny d hay khng ph thuc vo phn cc tng i ca chng. Cc phn t cng phn cc th s cng hp ph mnh v v th s di chuyn chm trn ct.

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Cc dung mi nh cp ng vai tr cnh tranh vi cc phn t trong mu cc v tr hot ng trn cht hp ph. Nh vy, cc phn t dung mi lin kt cng mnh v i pha t nh th cc cht trong mu nm trong pha ng lu hn v nh th chng cng c ra gii nhanh hn. V th s lu gi khng b nh hng qu nhiu bi kh nng ha tan ca mu trong cht ra gii m bi kh nng hp ph ca dung mi. Dung mi ban u nn chn c nhng lc ra gii trung bnh nhng dung mi mnh hn tip theo c th c th. Ni chung, vi cc cht hp ph phn cc nh oxit nhm hoc silicagen, kh nng hp ph tng ln khi cht b hp ph c tnh phn cc cng tng. i vi cacbon th trt t ngc li. Trappe tm thy lc ra gii ca mt lot dung mi cho nhng cht hp ph trn ct cha silicagen gim theo trt t nh sau: Nc nguyn cht > methanol> etanol > propanol > aceton > etyl axetat > dietyl te > clorofom > diclometan > benzene > toluene > tricloetylen > cacbon tetraclorua > cyclohexan > hexan. Thc t, dung mi l yu t kim sot trong sc k hp ph. Vi dy trn ta c th chn mt dung mi hoc hn hp dung mi vi lc ra gii thch hp. 3.6.3.2. Cht hp ph pha tnh Trn b mt mng li ca cht hp ph xp nh Al2O3, silicagen c cha cc nhm phn cc hydroxyl OH, v nhng nhm ny s tng tc b mt vi cc phn t cht tan (Al2O3 c nhng chi tit cu trc thm s c tho lun phn sau) Nhiu cht rn c s dng lm cht hp ph c lit k trong bng cng vi nhng hp cht cn tch ch yu l nhng cht hu c (cc cht v c cn tch th cn s dng phng php sc k ion).

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Cc cht hp ph c s dng lm pha tnh c tnh phn cc mnh nh Al2O3, silicagen, vi th c kh nng hp ph mnh cc hp cht c cc nhm cng phn cc theo trt t nh sau: -CH=CH- < -OCH3 < -COOR < =C=O < -CHO < -SH < -NH2 < -OH < -COOH Trt t ny o ngc i vi cacbon. 3.6.3.3. Chun b cht hp ph Silicagen Silica hay silica gel hay axit silixic l tn chung cho vt liu c iu ch bi cho natri silicat tc dng vi axit sunfuric to gel, ra v sy kh. pH cui cng ca dung dch cha gel s xc nh din tch b mt c th. V d, pH 3.72 din tch b mt l 830 m2g-1, cn pH 5.72 l 348 m2g-1. Khi iu ch nhiu ln th cn gi gi tr pH khng i nh dung dch m axetic axit/axetat natri. Cc v tr hot ng trn b mt g m cc nhm silanol Si-OH vi khong cch khong 5Ao. Cc th tc b sung tnh hot ng b mt ny ph thuc vo vic tch hoc thm nc. B mt hp ph nc s che cc v tr hot ng c th tch mt cch n gin b i gia nhit. Tuy nhin khi un nng nhit cao c 400oC dn n s mt hon ton tnh hot ng ca b mt do s tch loi phn t nc t hai nhm silanol k nhau v to thnh m i lin kt ete ca hai nhm ny (khng c ho t tnh sc k). Alumina B mt ca oxit nhm c kh nng th hin cc kiu tng tc cht tan-cht hp th khc nhau. iu ny l do trc ht c trng dng in rt mnh xung quanh Al3+, cho php tng tc vi cc cht d b phn cc ha. V vy n thch hp hn silicagen trong vic phn gii cc hp cht thm t olefin. Th hai, cc v tr baz (c l l O2- ) cho php tng tc vi cc cht nhng proton . S chun b v hot ha cc alumina khc vi silicagen ch hot tnh ca n tng ln khi tng nhit hot ha. Alumina hot tnh cao c th c to bi un nng nhit 400oC qua m. B mt c th b mt hot tnh bi nc hydrat tr li. Alumina t c s dng rng ri hn silicagen do chng c khuynh hng lm xc tc cho nhiu phn ng. 3.6.4. Sc k phn b Trong sc k phn b cht hp ph rn c thay th bi nh i vo ct cht mang rn trn c ph pha t nh l ng. Pha tnh lng khng c ha tan v khng trn ln vi pha ng. Sc k phn b l k thut li dng kh nng ca mt cht tan phn b ca n gia hai pha.

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C s ca phng php ny l do s khc nhau trong cc h s phn b ca cc cu t khc nhau, khi cho pha ng thch hp i qua th hn hp s b phn gii. Cht mang rn Cc cht mang ni chung c s dng l silicagel, diatomit, v xenlulo.

Cht mang s dng trong pha t nh l cht tr i vi cc cht cn tch. Cht rn c ph pha lng c nhi vo ct nh trong sc k hp ph. Ct cng tng t. Cht mang ph i hp ph v lu gi pha t nh l ng v phi c din tch b mt rng. N phi bn c v d dng nh i vo ct khi c ph pha tnh lng v khng cn tr dng chy pha ng.

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Kh khn ln nht khng th trnh kh i l s tn cng ca hiu ng hp ph. Thm ch nu b mt ca cht mang c ph kn vi pha lng tnh, hiu ng hp ph cng hy cn. Khi s ph kn ca b mt khng d t c th s hp ph c th l nh hng chnh trong s tch.

3.7. Sc k lng trn mt phng

Sc k giy v sc k lp mng c xy dng, pht trin v ng dng t lu. Tuy nhin hin nay sc k giy t c s dng. Trong sc k mt phng, qu trnh sc k c tin hnh trn mt t giy hay mt lp bt ri u v c gi trn mt mt tm knh, cht do hay kim loi, v d nhm. Bn thn lp mng c th l pha tnh hoc ch l cht mang gi pha tnh trn . Pha ng l cht lng s thm v chy trn lp m ng do tc dng ca lc mao dn v i khi c trng lc v in th. Ph bin nht hin nay l sc k lp m ng. nc ta phng php sc k lp mng c s dng rng ri, c nhiu ti liu, chuyn gia c kinh nghim v phng php ny. 3.7.1. Vai tr ca phng php SKLM V mt l thuyt cng nh v chng loi cc pha tnh v ng, v mt ng dng, SKLM v sc k l ng trn ct kh gi ng nhau. V th SKLM c th dng tm cc iu kin t i u cho s tch bng sc k lng trn ct. u im ca SKLM l c th tin hnh nhanh chng v t t n km cc th nghim thm d cho sc k ct. SKLM c dng th tinh khit ca sn phm dng nhiu trong phng th nghim, trong nghin cu sinh hc, ha sinh 3.7.2 Nguyn tc ca SKLM SKLM thng c tin hnh trn mt bn thy tinh c ri mt lp m ng dnh chc ca cc ht bt mn. Lp mng ny thng c dng lm pha t nh. Cc pha tnh v ng trong SKLM cng tng t cc pha trong sc k l ng hiu nng cao (sc k hp ph, sc k phn b pha thun v pha ngc, sc k trao i ion, sc k trn gel). Chun b cc lp mng Bt dng ch to lp m ng ( v d bt silicagen) thng c thm nc thnh bt nho v ri ln b mt sach ca mt tm knh hay cht do (hoc phin knh dng soi trn knh hin vi) thnh mt lp mng. Ngi ta thng cho thm mt cht kt dnh (nh bt b) v bt nho gi cc ht dnh vi nhau v dnh trn mt knh. Tm knh c yn cho lp mng nh hnh v dnh vo mt knh sau c em sy vi gi trong t sy.
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Mt s hng ha cht ch to sn nhiu loi bn mng nh bn silicagen ch th hunh quang di tia t ngoi bc sng 254 nm. Khai trin sc k L qu trnh cho pha ng chy xuyn qua pha t nh v ko mu phn tch di chuyn trn pha tnh. Thng ngi ta chm mt git mu phn tch gn mt mp ca bn mng. Dng bt ch nh du ni chm mu. Sau khi git mu kh, t bn m ng vo bnh sc k bo ha hi dung mi ca pha ng, mp pha chm mu c nhng vo dung mi pha ng nhng khng c cho im chm mu chm trc tip vo dung mi ng. Sau khi dung mi chy c na hay hai phn ba bn m ng th ly ra sy kh.

C hai cch trin khai: o Sc k ln o Sc k ngang Trn hnh l sc hai chiu mt s axit amin. Mu th c chm mt gc ca lp mng (ni nh du x) cho dung mi A chy, bay hi dung mi , quay 90o cho dung mi B chy, bay hi dung mi, phun nihidrin l thuc th hin mu ca cc axit amin. Xc nh cc cht bng cch so snh v tr vt ca mu vi cc vt ca chun.

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Hnh 33: Khai trin sc k theo hai chiu

Pht hin cc vt trn bn mng C nhiu phng php pht hin cc vt: Hai cch ph bin c th dng pht hin hu ht cc cht hu c l phun dung dch iot hay axit sunfuric: s xut hin cc vt mu ti. C th phun cc thuc th c hiu (nh ninhydrin). Cng c th thm cht hunh quang vo pha t nh: c bn m ng s sng (hunh quang) khi soi n t ngoi (v d n c bc x 254 nm) ni c cht phn tch th s ti hoc c mu hunh quang khc vi mu nn sng ca bn mng. C th tm tt cc phng php pht hin nh sau: Nu hp cht c mu th ch cn nh sng ban ngy. Dng tia t ngoi UV cho nhng hp cht pht c hunh quang (c cc h ni i lin hp) Phun hi iot cho nhng hp cht cha t nht mt ni i. Dng thuc th Dragendorff d tm cc alkalo id. Dng thuc th FeCl3 cho cc hp cht h pheno l. Dng aniline phtalat pht hin cc loi ng. Dng Ninhydrin pht hin cc hp cht nitro nh amin, amino axit. Phn tch bn nh lng C th c lng (bn nh lng) lng cht trn sc bng cch so snh din tch vt ca mu th vi din tch ca vt chun. C th thu c kt qu tt hn nu co ly bt cha vt, chit ly cht phn tch v em nh lng bng mt phng php l ha thch hp.

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Bi tp
Cu 3.1: Trnh by s khc nhau c bn gia sc k hp ph, sc k phn b, sc k trao i ion v sc k ry phn t. Cu 3.2: Gii thch ti sao lc ra gii tng ln khi dung mi pha ng tr nn t phn cc hn trong sc k pha o, trong khi lc ra gi tng ln khi dung mi pha ng tr nn phn cc hn trong sc k pha thun. Cu 3.3: Ti sao p sut cao cn thit trong sc k l ng hiu nng cao? Pha lin kt trong sc k lng l g? Cho bit mt s pha lng lin kt khng phn cc , phn cc trung bnh v phn cc mnh. Cu 3.4: Trnh by nguyn tc hot ng ca cc loi detector c s dng trong HPLC: a) b) c) d) Detector UV-VIS Detector Photo diodArray Detector o ch s khc x Detector pht hunh quang

Cu 3.5: Cho bit cc loi cu t no c th tch c bng sc k lng hiu nng cao nhng khng th tch c bng sc k kh. Cu 3.6: Cho bit cc bc c bn trong pht trin php tch isocratic trong sc k pha o s dng mt dung mi hu c v hai dung mi hu c Cu 3.7: D on trt t ra gii ca cc cht sau: a) n-hexan, n-hexanol, benzene b) ethyl axetate, diethyl ether, nitrobutane Khi thc hin php tch l sc k pha thng v sc k pha o. Cu 3.8: Cc cht phn cc c tch ra bng HPLC c ct tch cha pha tnh phn cc polyetylenglycol. Hi thi gian lu ca chng thay i nh th no nu pha ng gm CH3CN/H2O c nng acetonitril thay i t 40 ln 60%. Cu 3.9: Khi bn c gng tch mt hn hp cc cht cha bit bng sc k pha o vi pha ng l 50% acetonil/50% nc th thu c sc k c cc pic nm qu gn nhau v c ra gii trong khong k=2-6. Vy trong ln chy tip theo bn nn tng hay gim thnh phn acetonitril trong pha ng ci thin phn gii. Gii thch ?

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Cu 3.10: Mt hn hp n-heptane, tetrahydrofuran, 2-butanone, v n-propanol ra gii theo th t ny ra kh i ct pha tnh phn cc nh Carbowax. Trt t ra gii s o ngc theo ng th t trn nu s dng ct pha o khng phn cc nh C18. Hy gii thch trt t ra gi trong m i trng hp. Cu 3.11: Haddad v cng s bo co cc h s dung lng sau ca mt php tch pha o ca salicylamide (k sal) v caffeine (k caff). %v/v methanol 30% 35% 40% 45% 50% 55% ksal 2.4 1.6 1.6 1.0 0.7 0.7 kcaff 4.3 2.8 2.3 1.4 1.1 0.9 Gii thch s thay i s thay i cc gi tr k khi tng nng methanol trong pha ng. Explain the changes in capacity factor. C thun li g khi s dng pha ng c % methanol thp ? c bt li g khng? Cu 3.12: Gi s bn cn tch mt hn hp benzoic acid, aspartame, v caffeine trong mt nc gii kht soda. Cc thng tin thu c nh sau:

Hp cht

tr khi pha ng c m pH bng 3.0 3.5 4.0

4.5

benzoic acid 7.4 7.0 6.9 4.4 aspartame 5.9 6.0 7.1 8.1 caffeine 3.6 3.7 4.1 4.4 (a) Gii thch s thay i gi tr thi gian lu cho m i hp cht khi thay i pH pha ng. (b) Gii thch thi gian lu ca 3 hp cht khi pha ng c cng pH. Cu 3.13: 2) Nng ca PAHs trong t c th c xc nh bng cch chit PAHs bng methylene chloride. Dch chit sau c pha long, nu cn thit, v cc PAHs c tch bng HPLC s dng detector UV/Vis hoc detector pht hunh quang. Trong mt php phn tch tiu biu, cn 2.013-g mu t kh ri chit bng 20.00 mL methylene chloride. Sau khi lc t, ly 1-mL ca phn dch chit pha long n 10 mL bng acetonitrile. Tim 5 L ca dch chit pha long ny vo my HPLC cho tn hiu 0.217 (th nguyn ty chn) cho trng hp fluoranthene. Khi tim 5 L dung dch chun 20.0-ppm fluoranthene vo my HPLC trong cng iu kin th tn hiu thu c l 0.258. Tinhd nng ppm ca fluoranthene trong mu t trn. Cu 3.14: Thnh phn ca mt vin thuc multivitamin c xc nh bng HPLC vi diode array UV/Vis detector. Tim 5- L mu chun c cha 170 ppm vitamin C, 130 ppm niacin, 120 ppm niacinamide, 150 ppm pyridoxine, 60 ppm thiamine, 15 ppm folic acid, and 10 ppm riboflavin vo my HPLC, cho cc tn hiu ln lt thoe th t cc cht trn (th nguyn ty chn), 0.22, 1.35, 0.90, 1.37, 0.82, 0.36, v 0.29. Vin thuc c chun b cho phn tch bng cch ngin mn thnh bt ri chuyn vo bnh tam gic c 125-mL cha 10 mL of 1% v/v NH3 trong dimethyl sulfoxide. Sau khi siu m trong 2 min, thm vo 90 mL of 2% acetic acid, v hn h dung dch c khuy trong and 1 min v siu m 40 C trong 5 min. Sau dch c em lc vi giy lc c 0.45- m. Tim 5- L

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mu va chun b trong my HPLC trong cng iu kin cho cc tn hiu ln lt l 0.87 cho vitamin C, 0.00 cho niacin, 1.40 cho niacinamide, 0.22 cho pyridoxine, 0.19 cho thiamine, 0.11 cho folic acid, v 0.44 cho riboflavin. Tnh s milligrams ca m i cht c trong vin vitamin. Cu 3.15: . Lng caffeine trong mt vin thuc giamr au c xc nh bng HPLC s dng phng php lp ng chun. Cc dung dch chun caffeine c chun b v phn tch s dng vng tim mu c th tch 10- L. Kt qu o cc chun nh sau Nng cc chun Tn hiu (ppm) (th nguyn ty ch n) 50.0 8354 100.0 16925 150.0 25218 200.0 33584 250.0 42002

Mu c chun b bng cch cho mt vin thuc trong mt cc nh v thm vo 10 mL methanol. Sau khi mu ha tan chuyn ton b phn dch c phn khng tan trong cc vo bnh nh mc 25-mL v thm methanol n vch. Mu sau c em lc, ly 1.00-mL phn lc cho vo bnh nh mc 10-mL ri thm methanol n vch. Khi phn tch bng HPLC, tn hiu thu c l 21469. Tnh s milligrams caffeine trong vin thuc gim au.

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