Chng1.

I CNG V SC K
I. LCH S PHT TRIN V PHNG PHP SC K:
Phng php sc k c pht trin vo nm 1903 do nh thc vt hoc ngi Nga Michael C.Txvet.
ng thc s l ngi u tin c cng tm ra phng php, gii quyt vn tch cc cht c tnh cht
ging nhau theo mt c ch c o, hon ton khc vi phng php tch tng c trc y, tuy nhin
pht hin ny b lng qun nhiu nm.
Nm 1941, Martin v Synge pht trin sc k phn b trn giy v a ra l thuyt a gii
thch cc qu trnh sc k, cc tc gi ng dng tch cc ancaloit t cc cy thuc phc v cho ch to
dc phm. Do c cng trong vic pht trin l thuyt ca phng php cho nn nm 1952 hai ng c
nhn gii thng Nobel v ho hc.
Cng t nm 1952 nhng my sc k mi ra i t ra c u th do c hiu qu tch rt cao. Ct mao
qun v cc detect sau ny c ci tin tng phn gii v nhy ca phng php ngi ta c th
phn tch c cc cht c hm lng nh c ppm v ppb. T y phng php c pht trin nhanh, ng
dng c nhiu trong thc t.
V sc k lng mt k thut mi c pht hin t nm 1970 lm tng hiu qu tch l sc k
lng hiu nng cao (high performance lipuid chromatography- HPLC): Cht nhi ct c ci tin, my tnh
v thit b vo b tr c a vo, tng cng kh nng ca phng php.
Ngy nay k thut ghp ni gia sc k v cc phng php khc c p dng. l k thut ghp
ni gia sc k v khi ph ( GC-MS, GC-MS) sc k v cng hng ht nhn, sc k a chiu, sc k in
mao qun (CEC)... Phng php c chnh xc v nhy rt cao, phn tch c nhiu i tng phc
tp hn.
II. C S L THUYT
1. NH NGHA - PHN LOI
1.1 nh ngha:
Sc k l qu trnh tch da trn s phn b lin tc cc cu t cht phn tch trn hai pha: mt pha
thng ng yn c kh nng hp th cht phn tch gi l pha tnh, mt pha di chuyn qua pha tnh gi l
pha ng; do cc cu t cht phn tch c i lc khc nhau vi pha tnh, chng di chuyn vi tc khc
nhau v tch ra khi nhau.
1.2 Phn loi cc phng php tch sc k
1.2.1 Phn loi theo h pha
Phng php u tin c nhiu ngi quan tm, y l phng php phn loi n gin theo h
pha tc l cht phn tch phn b gia hai pha l g?
h
pha
S phn loi cc phng php sc k

C
ch

1.2.2 Phn loi theo c ch tch

1.2.2.1 Sc k hp th:
Sc kh hp th l phng php da trn c s phn b cht phn tch gia pha tnh v pha ng nh
tng tc phn t thng qua cc trung tm hp th. Pha tnh l cc cht rn hoc lng c din tch b mt
ln, bn vng v mt ho hc. Chng hp th cht phn tch trn b mt ca chng cc mc khc nhau
khi cho pha ng cha cht phn tch tip xa vi chng. Tu thuc lc lin kt gia pha tnh v tng cu t
cht phn tch c trong pha ng, khi cho pha ng i qua pha tnh chng s di chuyn vi tc khc nhau.
1.2.2.2 Sc k phn b lng - lng
S phn bit gia sc k phn b lng-lng v s phn b thng thng l ch sc k phn b lng
- lng cn c gi l sc k chit, pha tnh l cht lng pha ng cng l cht lng, s phn b cht phn
tch gia hai pha lng ging nh qu trnh chit, cn s phn b ni chung l s phn chia cht phn tch
vo hai pha khng cn xt ti lng hay rn.
im khc nhau c bn gia sc k phn b lng - lng v sc k hp ph l ch: sc k phn b
lng- lng c ng ng nhit tuyn tnh khong nhit ln, phng php c nhy cao nhng c
nhc im l pha tnh khng c bn vng, hin tng tri mt pha tnh lm cho lp li b gim.
1.2.2.3 Sc k ion ( trao i ion)
Pha tnh thng l pha rn c kh nng trao i ion ca n vi cc cht phn tch trong pha
ng.Cht c kh nng trao i cation gi l cationit, cn cht c kh nng trao i anion gi l anionit. Lc
lin kt ch yu gia cht phn tch v pha tnh ch yu l lin kt tnh in , ph thuc nhiu vo in tch
ca ion cht phn tch, pH ca dung dch v bn knh hidrat ho ca cc ion cht phn tch. V d: phn ng
trao i ion gia cationit acid mnh v Ca2+ c th vit nh sau:
2R-SO3H + Ca2+ = (R-SO3)Ca + 2H+
Phn ng ca anionit baz mnh vi Cl- :
R-N(CH3)3OH + Cl- = R-N(CH3)3Cl + OH2

1.2.2.4 Sc k ry phn t (sc k loi c)

Pha tnh l cc cht rn c din tch b mt ln, xp, c nhng ng i trong lng cht rn, cn gi
l mao qun c kch thc c phn t. Cc phn t cht phn tch c th thm vo cht mc khc
nhau tu theo kch thc ca chng. Cc cht phn tch c kch thc ln khng th i vo su vo pha
tnh c, s b ra gii nhanh cn cc phn t cht phn tch c kch thc nh phn b su vo pha tnh s
b ra gii chm. Th t ra gii l cc cht c kich thc nh i ra sau v ngc li. Thi gian lu ca cc
cht t l nghch vi kch thc phn t ca chng. Tuy nhin cht phn tch c th c tng tc khc vi
pha tnh. Nh vy mt php sc k c th tch theo mt hay nhiu c ch khc nhau m chng ta khng xt
y.
1.2.3 Phn loi theo cch hnh thnh sc
1.2.3.1 Phn tch tin lu
Cc cu t A, B, C c i lc vi pha tnh l A < B< C. Cho hn hp cht phn tch lin tc i qua
ct, cht phn tch c i lc vi

pha tnh thp

Nng

nht i trc, tip theo l cht

c i lc yu

hn. Tuy nhin do chng ta cho

lin tc cht

phn tch vo nn i sau A l A

A+B+C. Ch c mt phn cht

tinh khit A

i trc

C
1.2.3.2

Phn

Trong sc k ny, chng

lng nh hn hp cht

tch th y
t. V

phn tch A,

Nng
Hnh 1: Sc phn tch tin lu

B, C vo ct sau cho cht

ta cng np
D l cht y

lin tc i qua ct.

i lc hp th l A

<B < C < D.

Do D y C, C li y B, B

li y A nn

sc c dng::
A

1.2.3.3 Phn
a mt lng nh
cc cht A, B, C vo ct sau

t, V
tch ra gii:

Hnh 2: Sc phn tch th y

(phn m l phn hai cu t xen k)

mu bao gm
dng cht E

c i lc hp th vi pha tnh yu nht ra gii lin tc. C A, B, C, E u tan tt trong pha ng . Th t

i lc hp th l E < A < B < C. Mt cu hi t ra lm sao E c i lc hp th yu nht vi pha tnh li c
th ra gii A, B, C ? tr li cu hi ny chng ta hnh dung l s tranh chp nhng trung tm hot ng
pha tnh khng phi ch c mt cu t cht phn tch tng tc vi cu t cht ra gii m y c th c
nhiu cu t ra gii tham gia (cu t c th l ion hoc phn t).

Gi s c cht tan C(phn t hoc ion) hp th vo pha tnh, phn b C tr li pha ng phi
cn ti nhiu cu t ra gii E. Quy lut u th s ng xem ra c tc dng. V vy khi ra gii ngi ta
thng cht ra gii c nng ln hn cht phn tch rt nhiu.
Tm

li

mt

nhn

theo

phn

loi

no

cng c th

lin

quan

n mt hay

nhiu cch

phn

loi

khc,

th

d sc k

lng-

rn

php sc k
cch

Chiu cao (h)

Din tch (S)

Thi gian (t)

ch n gin
l cht rn,
cht

lng

Hnh 3: Sc phn tch ra

gii

l pha tnh
pha ng l
theo

cch

phn loi theo lin kt pha nhng theo cch phn loi theo c ch th lng rn y c th l hp ph,trao
i ion. Tu theo pha tnh s dng, sc k lp mng c th l sc k trao i ion, hp ph, phn b, ry phn
t. Ngc li sc k hp ph xt v mt tng tc pha c th thuc loi sc k - rn hay lng- rn.
Sc k ra gii c ng dn Qu trnh tch sc k l mt chui cc phn ng phc tp t lc np mu
vo ct tch cho n khi cc cht phn tch trong mu ln lt i ra khi ct. hiu cc phanr ng xy ra
trong , trc ht ta xem th nghim ca Txvet, tin hnh nh sau:
Ha tan cc sc t thc vt trong t du ha, sau np mt lng nh vo ct nhi bt CaCO 3.
Tip theo, cho lin tc t du ha tinh khit i qua, sau mt thi gian nht nh s t thc vt tch thnh
cc gii ring bit. sau ny ngi ta bit c l cc -chlorophyl, -chlorophyl, -xantophyl, xantophyl, chng l cc cht mu nn khi tch chng to thnh cc di mu khc nhau nn ng gi l
Chromatography. Theo ting Hy Lp, Chroma l ,mu cn Graphy l ghi, danh t cn gi n
ngy nay.
Khi cc cht phn tch c np vo pha tnh, lc u cc cu t phn b pha tnh mt cch bt k
khng chn lc, chng c th phn b nhng im xen k, tuy nhin pha tnh c i lc lin kt vi tng
cu t khc nhau l khng ging nhau. Cc cht phn tch c i lc lin kt vi pha tnh ln hn s phn b
tt hn, cc cht c i lc vi pha tnh yu hn s phn b vo pha tnh t hn.
Khi cho dung mi tinh khit di qua ct, cc cht phn tch li c iu kin phn b li, cc cht
c phn b tt hn c thi gian pha tnh ln hn. Khi cht phn tch pha tnh chng khng di chuyn
cn khi pha ng chng di chuyn. V vy cc cht phn b tt hn, chng di chuyn chm hn, cc cht
phn b km hn c thi gian pha tnh t hn, kt qu l trong qu trnh tch sc k chng s di chuyn
nhanh hn. Ngha l sau mi ln hp th v gii hp cc cht phn tch c tnh cht ging nhau nn c mt
s chnh lch rt nh v thi gian nm pha tnh. Sau mi ln hp th v gii hp cc cht phn tch di
chuyn c nhng qung ng rt nh khc nhau.

Tip tc chop ha ng i qua ct, cht phn tch tip tc di chuyn, s kkhacs bit v qung ng di
chuyn s cng ln. tt nhin khi thi gian di chuyn cng ln, vunhf cht ca tng cu t cng rng do yu
t khuch tn dc theo ct, sau mt thi gian nht nh, cc cht phn tch tch khi nhau, l qu trnh
tch sc k.
g nhiu trong lnh vc phn tch do hiu qu tch cao

2. QU TRNH TCH SC K V CC LOI LC LIN KT TRONG H SC K

2.1 Qu trnh tch sc k
Qu trnh tch sc k l mt chui cc phn ng phc tp t lc np mu vo ct tch cho n khi cc cht
phn tch trong mu ln lt i ra khi ct. hiu cc phanr ng xy ra trong , trc ht ta xem th nghim ca
Txvet, tin hnh nh sau:
Ha tan cc sc t thc vt trong t du ha, sau np mt lng nh vo ct nhi bt CaCO 3. Tip theo,
cho lin tc t du ha tinh khit i qua, sau mt thi gian nht nh s t thc vt tch thnh cc gii ring bit.
sau ny ngi ta bit c l cc -chlorophyl, -chlorophyl, -xantophyl, -xantophyl, chng l cc cht mu
nn khi tch chng to thnh cc di mu khc nhau nn ng gi l Chromatography. Theo ting Hy Lp,
Chroma l ,mu cn Graphy l ghi, danh t cn gi n ngy nay.
Khi cc cht phn tch c np vo pha tnh, lc u cc cu t phn b pha tnh mt cch bt k khng
chn lc, chng c th phn b nhng im xen k, tuy nhin pha tnh c i lc lin kt vi tng cu t khc nhau l
khng ging nhau. Cc cht phn tch c i lc lin kt vi pha tnh ln hn s phn b tt hn, cc cht c i lc vi
pha tnh yu hn s phn b vo pha tnh t hn.
Khi cho dung mi tinh khit di qua ct, cc cht phn tch li c iu kin phn b li, cc cht c phn b
tt hn c thi gian pha tnh ln hn. Khi cht phn tch pha tnh chng khng di chuyn cn khi pha ng
chng di chuyn. V vy cc cht phn b tt hn, chng di chuyn chm hn, cc cht phn b km hn c thi gian
pha tnh t hn, kt qu l trong qu trnh tch sc k chng s di chuyn nhanh hn. Ngha l sau mi ln hp th v
gii hp cc cht phn tch c tnh cht ging nhau nn c mt s chnh lch rt nh v thi gian nm pha tnh. Sau
mi ln hp th v gii hp cc cht phn tch di chuyn c nhng qung ng rt nh khc nhau.

Hnh 4 :Qu trnh tch A v B trong ct tch sc k .

Tip tc chop ha ng i qua ct, cht phn tch tip tc di chuyn, s kkhacs bit v qung ng di chuyn
s cng ln. tt nhin khi thi gian di chuyn cng ln, vunhf cht ca tng cu t cng rng do yu t khuch tn dc
theo ct, sau mt thi gian nht nh, cc cht phn tch tch khi nhau, l qu trnh tch sc k

2.2 Cc loi lc lin kt trong h sc k

Trong h sc k c ba thnh phn ch yu tc quyt nh s tn ti ca h, cht phn tch, pha tnh,
pha ng to nn s phn b cn thit, gip cho s di chuyn cc cht phn tch dc theo chiu di ct vi
tc khc nhau. Chng ta thng ni cht phn tch phn b gia hai pha, tuy nhin y la cn bng ng
cht phn tch lun thay i pha lin kt (tr mt s trng hp c bit n ch mt pha trong sut qu
trnh phn tch) vn l ti sao cht phn tch c gi trong pha tnh? Hn na ba thnh phn ny tng
tc nh th no vi nhau, bngc lc lin kt no m kt qu cc cht phn tch li phn b vo pha tnh
mc khc nhau? Cu tr l do bn cht cc ca cc thnh phn trong h sc k. Cht phn tch c th l
ion, phn t trung ho hay cht phn cc, i tng lin kt ca n cng c th l ion, phn t, cht phn
cc. Nh vy tuy thuc vo ln ca in tch kich thc phn t, phn cc m chng tng tc vi
nhau theo cc lc lin kt khc nhau. Ngi ta chia cc lc lin kt thnh bn loi:
2.2.1 lc lin kt ion
Trn b mt pha tnh c cc trung tm hot ng l ion. l cc phn t c kh nng phn ly to
thnh cc ion, to nn trung tm hot ng ion chung tham gia lin kt vi cc ion cht phn tch trong dung
dch theo lc tng tc tnh in gi l lin kt ion. gi cc anion cht phn tch trong dung dch pha tnh
cn c cc trung tm cation trn b mt, v ngc li gi cc cation cht phn tch trong dung dch pha
tnh cn c cc ctrung tm anion cng tng t, pha ng cng c cc trung tm hot ng l cc ion, chng
tham gia tng tc vi cht phn tch v pha tnh. Chng c kh nng phn li trong dung dch to thnh cc
cation v anion.
Th d khi ra gii cc ion kim loi trn ct cationit, ta c th dng pha ng l dung dch HCl long.
Cc phng php sc k thng gp c s tham gia ca cc lin kt ion gi l sc k ion, sc k cp ion.
6

2.2.2 Lc phn cc
Trong phn t cht phn tch, cht ra gii trong pha ng cng nh cc phn t trn b mt pha tnh
c cc trung tm phn cc, chng c th l cc lng cc in c nh, cng c th l cc lng cc in do
cm ng. Cc trung tm phn cc ca cht ny lin kt vi trung tm phn cc ca cht khc bng lc lin
kt phn cc. S phn cc trong cc phn t i khi rt nh v s chnh lch v phn cc cng rt nh.
Tuy nhin, s chnh lch ny l yu t quan trng c bn lin kt khc nhau, l iu cn thit
phn chia chng trong cc php tch sc k.
Th d: cc Cresol (o-, m-,p- cresol) ch khc nhau v v tr nhm CH 3- trong phn t. Chnh s khc
nhau v v tr ny lm cho mt electron trong phn t cresol khc nhau to nn mt s khc bit nh v
phn cc, t lc lin kt vi pha tnh khc nhau. L nguyn nhn chnh lch thi gian lu trong qu
trnh sc k
2.2.3 Lc Van- de- Van (lc phn tn)
Lc ny c bn cht in, tuy nhin khng h c cc in trong mng li phn t, cng khng lin
quan ti phn t lng cc hay c nh.
Tng tc phn tn in hnh l mch hidrocacbon n l tng tc phn t .
Th d n-heptan l cht lng ch khng phi l cht kh s tng tc gia cc phn t n-heptan l lc
phn tn ln gi cht ny trng thi lng. tch cc hidrocacbon ngi ta dng hidrocacbon khc
lm pha tnh.
2.2.4 Lc tng tc c bit
Lc tng tc c bit l lc tng tc to thnh do cc lin kt hydro, lin kt cho nhn m c.S
hnh thnh lin kt gi nguyn t hidro v cc nguyn t c m in cao nh O, N, F... gi l lin kt
hidro. Khi tham gia lin kt, cc electron cn gi l m my electron chuyn dch sang nguyn t c m
in ln hn. Nh vy cc nguyn t c m in cao hn, lin kt hidro cng bn cht.
3. MT S I LNG DNG TRONG SC K
3.1 H s phn b KD v cch xc nh
Cu t A phn b gia pha ng v tnh khi cn bng c thit lp
As

Am

(1)

h s phn b c nh ngha l t s ca nng cht phn tch gia hai pha

KD =

[ As ]
(2)
[ Am ]

Trong : [Am ] l nng cht phn tch trong pha ng

[As ] l nng cht phn tch trong pha tnh
Cn bng phn b trong pha kh gi l cn bng ng l cn bng gia lng - lng, kh-lng, rnlng. Gi s cht phn tch c phn b phn no ca ct, a th n. Nhng do pha ng di chuyn,
cht phn tch li i xung a th (n+1) v y li thit lp cn bng mi ca c a th n v n+1.
cn bng thit lp nhanh mt yu cu rt cn thit l lp pha tnh phi mng, h s khuch tn ca
cht phn tch trong pha tnh ln hn h s khuch tn trong pha ng. H s phn b K D c th c xc
7

nh theo phng php tnh bng cch o nng cht phn tch trong pha ng v nng cht phn tch
trong pha tnh sau khi t cn bng. Mt cch gn ng KD c xc nh theo cng thc sau:
KD =

( M M 1 ) .V
M 1.m

(3)

Trong : M l lng cht ban u tnh bng mg

M1 l lng cht phn tch cn li sau khi tip xc mg
V l th tch pha ng ml
m l lng cht hp th, gam
3.2 S lin h gia tc di chuyn ca cht phn tch v KD
Cht phn tch A mun di chuyn c phi pha ng. Gi f A l phn thi gian A pha ng:
fA =

[ A] m .Vm
[ A] m .Vm + [ A] s .[ V ] s

(4)

chia c t v mu cho [A]m . Vm ta c:

fA =

1
fA =
1+ k 'A

1
1+ KD

VS
Vm

(5)

Trong k A l h s dung tch.Tc tuyn tnh ca cu t A trong pha ng l:

u.fA = u / (1+kA)

uA =

(7)

U l tc tuyn tnh ca pha ng (cm/s). T biu thc (6) v (7) ta c nhn xt: Khi KD hoc fA cng ln ;
thi gian cht phn tch trong pha ng cng t, ni cch khc l cht phn tch di chuyn cng chm, ngc
li khi fA cng nh, cht phn tch di chuyn cng nhanh.
3.3 Thi gian lu tR, thi gian lu hiu chnh tR
Thi gian lu tR l thi gian lc bt u bm mu vo u ct ti khi pic t gi tr cc i.
tR =

(8)

L L(1 + k A )
=
uA
u

L/u l thi gian m cu t khng lu gi trn ct cn gi l thi gian cht

V
t R A = t m 1 + K D . s
Vm

(9)

= t m (1 + k ' A )

thi gian lu hiu chnh tR l thi gian lu khng tnh thi gian cht

t R' A = t R A tm = tm .k ' k ' =

t R'
V'
= R
tm Vm

(10)

3.4 Th tch lu Vm, th tch lu hiu chnh Vm

Th tch lu VR l th tch ca dung dch ra gii t khi bt u bm mu vo ct cho n khi pic t
cc i. Gi F l tc chy ca pha ng ml/pht,th tch lu V R c tnh theo cng thc:VRA = tRAF; Vm
= tmF
VRA = Vm(1 + k)

(11)
8

Th tch lu hiu chnh VR l th tch lu loi i th tch trng ca ct

VR = VR Vm = VmkA = KDAVS
Th tch lu ring Vg l th tch ring cho cu t, l th tch dung dch ra gii ra cht phn tch
trong mt n v trng lng hay th tch ca pha tnh
'
VRA
= K DA = Vg
VS

(12)

3.5 H s tch (cn gi l h s lu gi tng i)

H s tch l i lng nh gi kh nng tch hai cht bng phng php sc k. N ph thuc
vo h s phn b ca chng v t s ca h s phn b ca hai cht: mt cht phn tch A v mt cht khc
B hoc mt cht chun.
iu kin hai cht tch khi nhau l 1
=

k
K DA
= A' 1
K DB
kB

(13)

Ni mt cch khc h s phn b ca hai cht A v B gia pha tnh v pha ng phi khc nhau. Mt
khc t s th tch lu ca hai cht, tRA/ tRB cng chnh l t s ca h s phn b.
Vy c th tm tt l:

'
k
K DA
t'
VRA
= A' = RA
=
'
'
KDB
kB
t RB
VRB

(14)

3.6 a l thuyt v xc nh s a l thuyt bng thc nghim

3.6.1 Khi nim a l thuyt:
a l thuyt l phn no

ca ct

m cn bng c thit lp.

Theo quan

nim ca Martin v Synge, cn

bng c

thit lp nhanh chng, tc thi.

Tuy nhin

iu ny gp kh khn khi gii

thch hin

tng gin rng vng mu, hin

tng pic

sc

khng

cn

i.

Amundson v Lapidus v sau

Hnh 5:Xc nh s a l thuyt t thc nghim

Deemter cho rng a c cn

l
bng

Van
khi

t cn bng chuyn khi lng cht tan t pha ng vo pha tnh v ngc li v tc gi a ra thuyt
tc .
Nh vy c th ni rng phn nh ca ct gi l a l thuyt khi t c cn bng phn b cht
phn tch.
Gi n l s a l thuyt,

l lch chun; ~

. i vi ct sc k n cng ln pic cng hp,

thi gian lu t l vi s a l thuyt n, tR ~ n, nn ta c th vt:

2
tR
n
tR
=
hay n =

w = 4 hay =

Mt khc:

W
4

S a l thuyt c th vit li l:
(15)
3.6.2 a l thuyt c tnh thi gian khng lu gi, nef
nef

( t tm )
=16 R
w

nef = 16

2
16 ' 2
16
t
= 2 t m2 k '
2 ( R)
W
W

t R2
k2
k2
=
n
.
2
w2 ( 1 + k ) 2
(1+ k )

(16)

V t (8) ta c tm= tR/(1+k). Chiu cao a l thuyt hiu lc c tnh:

Hf =

L
n f(16)

T (15) v (16) ta c:
H ef

1 +k ' )
(
L
w2
=
=H
2
16 ( t R t m ) 2
k'

(17)

3.7 Sc .
Sc l s biu din s ph thuc din tch (S) hoc chiu cao (h) tn hiu o c c th l hp
th quang,cng c th l dn in hoc l tn s xung in ...lin quan cht ch ti nng cht phn
tch c trong mu v thi gian. Cc tn hiu ny c gi l pic sc k. tnh ton hm lng cht phn
tch, tnh din tch pic sc k l chnh xc nht.Ngy nay hu ht cc my hin i u c trang b cc
thit b tnh din tch ca pic sc k mt cch t ng

t RB .VRB

S, h

'
'
t RB
,VRB

t RA ,VRA
'
t ' RA , VRA

tm

t, V
t0
WB

WA

Hnh 6: Sc hai cu t A&B

3.8 phn gii v cch lm tng phn gii:
3.8.1 phn gii

10

 phn gii ni ln mc tch cu t khi nhau trong mt php tch sc k. Hai cu t A v B

c tch khi nhau cng trit , phn gii cng cao. phn gii c nh gi qua cc i lng c
trng l: thi gian lu, tRA, tRB v rng pic WA, WB.
phn gii R c tnh theo cng thc:

R=

2( t RB t RA )
W A + WB

(18)

Trong : tRB tRA= tR l s chnh lch thi gian lu(cng c th dng th tch lu V RA- VRB)ca hai cu t
A v B. i lng ny c so snh vi tng bn chiu rng pic, o bng giy hoc mililt, ni ln kh nng
tch ca ct.
Khi tng chiu di ct, s a l thuyt tng: tR ~ n, chiu rng pic t l vi
V vy

R~

n
n

hay

R~ L

3.8.2 Cch lm tng phn gii

Cch th nht: Da trn s thay i cu trc ct. Trong phng php ny c cc bin php: Tng
chiu di ct, s dng cht nhi c kch thc ht nh hn, ng u hn, thay i pha tnh v cui cng l
gim ng kinh ct
Cch th hai: Da trn cch vn hnh ct: l ti u ho tc pha ng, gim lng mu, gim
nhit ct (GC) v cui cng l thay i thnh phn pha ng (LC)
3.9 Thuyt tc , phng trnh Van Deemter
Thuyt c cc tc gi Lapidus v Amundsion a ra nm 1952, sau ny c Van Deemter pht
trin.
Theo thuyt ny cc yu t: ng knh ht nhi, k thut np ct v cc yu t tc ca pha
ng, h s khuch tn ca cht phn tch trong pha ng v pha tnh nh hng rt nhiu ti s gin rng
vng cht phn tch hay rng ca pic trong sc . i lng c trng cho gin rng ca vng cht
phn tch l chiu cao a l thuyt H, c hnh thnh do ba thnh phn H1,H2, H3.
H = H1 + H2 + H3

(19)

H1 l i lng khuch tn xoy, c trng cho s ng u ca ct nhi, ng knh ca ht nhi, kh

nng np ct
H1 = 2dp

(20)

Trong l h s np ct, nu np ct tt th = 1,5; dp l ng knh ht nhi

H2 l i lng khuch tn theo chiu dc. N c hnh thnh do s khuch tn ca cht phn tch v
hai u ca ct, ph thuc vo tc ca pha ng, h s khuch tn ca cht pht phn tch trong pha
ng, ng i ca cht phn tch, c biu din bng phng trnh:
H2 = B/u trong B= 2Dm

11

L i lng c trng cho h s khuch tn ca cht phn tch trong pha ng v h s ng i ca n.

Trong iu kin np ct tt, ng i ca cht phn tch khng qu khc khuu, = 1; Dm l h s khuch
tn ca cht phn tch trong pha ng, nh vy H2 c th vit li l:
H2 = 2Dm/u

(21)

H 3 l i lng c trng cho qu trnh chuyn khi lng l yu t ph thuc vo tc hp th v

gii hp. Qu trnh ny ph thuc vo c pha ng v pha tnh, v vy H3 c th vit: H3= (CS - Cm)u (22)
Trong Cs l h s dc trng cho qu trnh chuyn khi lng ca cht phn tch trong pha tnh, ph thuc
vo pha tnh, n c gi tr nh khi tc dng F1ml/pht, Cs 0

Cs= K.df2/Ds

(23)

Trong : K- h s
Ds- h s khuch tn cht phn tch trong pha tnh
df2- chiu dy pha tnh
Cm l hng s chuyn khi ca cht phn tch trong pha ng, ph thuc vo pha ng, c xc nh theo
biu thc:
Cm = dp2/Dm

(24)

l h s thc nghim c gi tr t 0,5- 1

Dm l h s khuch tn ca cht phn tch trong pha ng.
Tng hp li H c biu thc tng qut l:
H = 2dp + 2Dm/u + (Kdf2/Ds + df2/Dm)u

(25)

Phng trnh trn c vit tt l:

H = A+B/u +Cu

(26)

Trong thc t c th tnh

gn ng

H= 3-4dp

Cu

xt cc tiu chiu cao

thuyt ly o hm ca phng

ca a l
H min

Van Deemter ta c:

H = -B/u +C
Ti cc tiu H=0 => ut =

trnh

B
u

utu

B
C

u , cm / s

Hnh 7: ng cong Van Deemter

III. BI TP

Bi 1:
Trn sc k ngi ta thu c 3 pic 0,84 pht v 10,60 pht v 11,08 pht tng ng vi cc
hp cht A,B v C. Hp cht A khng lu gi bi pha tnh lng. Cc pic ca cc hp cht B v C c dng
ng Gauss c chiu rng tng ng 0,56 v 0,59 pht. Chiu cao ct sc k bng 28,3cm. .
1. Tnh s a l thuyt (n) ca ct sc k v chiu cao a l thuyt (H) ca pic B,C.
2. Tnh gi tr trung bnh T v ca ct.
3. Tnh ch s lu gi ca cht B,C.
12

4. Th tch ca cht lng c gi trn b mt ca cht mang ca ct bng 12,3ml. Th tch ca pha ng
bng 17,6ml. Hy tnh h s phn b B,C.
Gii:
1. Tnh n v H:
+ i vi pic B:
2

t
10, 60
nB = 16 RB = 16
= 5733
0,56
WB
+ i vi pic C
t
11, 08
nC = 16 RC = 16
= 5643
0,59
WC
Vy s a l thuyt ca ct tng ng 5688 a l thuyt.
+ Chiu cao a l thuyt:
H=

L 28,3
=
= 4,975.103 (cm)
n 5688

2. Tnh v T:
T n =

2
= n.H 2 = 5688.(4,975.103 ) 2 = 0,375(cm)
2
H

.tRB 10, 6.0,375

=
= 0,141( phut )
L
28,3

Ta c: TB =

.tRC 11, 08.0,375

=
= 0,147( phut )
L
28,3

TC =
T=

0,141 + 0,147
= 0,144( phut )
2

3. Ch s lu gi ca cht B,C:
Ta c:

RB =

tm 0,84
=
= 0, 079
t RB 10, 6

RC =

tm
0,84
=
= 0, 076
t RC 11, 08

4. H s phn b B,C:
Ta c: RB =

Vm
V (1 RB ) 17, 6(1 0, 079)
kB = m
=
= 16, 68
Vm + k B .VS
RB .VS
0, 079.12,3

RC =

Vm
V (1 RC ) 17, 6(1 0, 076)
kC = m
=
= 17, 40
Vm + kC .VS
RC .VS
0, 076.12,3

Bi 2
Tnh s a l thuyt hiu lc nef v Hef ca h sc k c tnh cht sau: t RA = 280 s; t RB = 300 s;
WA = 14s; WB = 15s. Thi gian cht l tm=10s, ct c chiu di l 3,2m.
13

Gii:
Theo l thuyt ta c s a l thuyt hiu lc nef l:
n=

k '2
; vi k l t s dung tch.
( k '+1) 2

t t
nef = 16 R M
W

280
nA = 16
= 6400
14
2

300
nB = 16
= 6400
15
T ta c:
t t 280 10
i vi pic A: k ' = R M =
= 27;
tM 10
2

nef A

27
= 6400
= 5951, 07
27 + 1

t t
i vi pic B: k ' = R M
tM

300 10
=
= 29;
10
2

29
nef B = 6400
= 5980, 4
29 + 1
Vy,

nef = 5065, 7 ; H ef =

300
= 0, 053 .
5965, 7

Bi 3
Pic sc k ca hp cht X c pht hin sau 15 pht sau khi a mu vo
(lc ca hp cht Y khng c gi bi vt liu ca ct xut hin qua 1,32 pht). Pic ca cht X c dng
ng phn b Gauss vi b rng ca y l 24,2s. di ca ct l 40,2cm.
1. Tnh s a l thuyt trong ct.
2. Tnh H ca ct.
3. Tnh T v ca ct.
4. Tnh ch s lu gi ca cht X.
5. T phng php chun b bit rng th tch ca cht lng c gi trn b mt ca cht mang ca ct
bng 9,9ml. Th tch ca pha ng bng 12,3ml. Hy tnh h s phn b X.
6. Tiu chun phn chia X v Y l bao nhiu nu yu t phn chia i vi X v Y bng 1,011.
Gii:
1. S a l thuyt (n):
2

t
15.60
n X = 16 RX = 16
= 22130
24,2
WX

14

2. Tnh H:
Ta c n =

L
L
40,2
H = =
= 1,82.10 3 (cm)
H
n 22130

3. Tnh v T:
T n =

2
= n.H 2 = 22130.(1,82.10 3 ) 2 = 0,27(cm)
H2
.tR

Ta c: T =

15.0,27
= 0,101( phut )
40,2

4. Ch s lu gi ca cht X:
Ta c:

R=

tm 1,32
=
= 0,088
tR
15

5. H s phn b X (kX):
Ta c: R =

Vm
V (1 R ) 12,3(1 0,088)
kX = m
=
= 12,9
Vm + k .VS
R.VS
0,088.9,9

6. Tiu chun phn chia (Rs):

Ta c: RS =
vi

n
1
(1 ) =
4

22130
1
(1
) = 0,4
4
1,011

l yu t phn chia.

Bi 4: Mt h thng sc k lng hot ng vi cc thng s sau y:

-

Chiu di ca phn ct c nhi pha tnh: 25,2 cm.

Tc dng: 0,312 ml/pht.

Th tch pha ng (Vm): 1,40 ml.

Th tch pha tnh (Vs): 0,168 ml.

Sau khi cho qua ct mt hn hp cha 4 cu t A,B,C v D thu c mt sc k cung cp cc d kin

sau:
Cu t
Khng lu gi bi ct
A
B
C
D
Hy tnh:

Thi gian lu gi,pht

1,2
4,7
8,8
12,5
13,8

Chiu rng chn pic,pht

0,48
0,92
1,34
1,44

1. S a l thuyt i vi mi pic,gi tr trung bnh, lch chun ca n v chiu cao a l thuyt ca

ct.
2. H s cha v h s phn b ca tng cu t.
3. phn gii RS, chn lc i vi 2 cu t C,D.
4. Chiu di ct nu mun t c phn gii 1,5 i vi 2 cu t C,D.
Gii
t 2
1. S dng phng trnh : n = 16( R ) tnh c s a l thuyt
w
15

A: n = 1534
B: n = 1463
C: n = 1392
D: n = 1469
ntb = 1500
H=

L 25, 2
=
= 0, 017cm
ntb 1500

2
= n.H 2 = 1500.(0, 017) 2 = 0, 66(cm)
2
H

n=

2. H s cha v h s phn b ca tng cu t.

p dng cng thc k ' X =

t R tm
ta tnh c h s cha ca tng cu t :
tm

kA = 2,9 ; kB = 6,3; kC = 9,4 kD = 10.

T cng thc k ' X =

K X VS
ta tnh c h s phn b ca tng cu t :
VM

KA = 24; KB = 53; KC = 78; KD = 87;

3. phn gii RS, chn lc i vi 2 cu t C,D.
Rs =

2t R
= 0,94
wC + wD

K D 87
=
= 1,12
K C 78

4.Chiu di ct phn gii gia cc cu t t yu cu ti thiu l 1,5 i vi 2 cu t C,D;k v

khng thay i khi n v L tng.
Gi n1,n2 l s a l thuyt ca ct c v ct mi,ta c:
Rs ,1
Rs ,2

n1
n2

0,94
1500
=
n2 = 3800
1,50
n2

Chiu di ct phn gii tng ln 1,5:

L2 = H.n2 = 0,017. 3800 = 65 cm.
Bi 5:
Cho h sc k c tnh cht sau: t RA = 280 s; t RB = 300 s; WA = 14s; WB = 15s. Ct c chiu di l
3,2m. Thay i chiu di t 320 cm ln 400 cm. Tnh phn gii?
Gii:
t RA = 280.

400
= 350( s )
320
2

t RB = 300.
2

t
280
nA = 16. RA = 16
= 6400
W
14

400
= 375( s)
320
2

t
300
nB = 16. RB = 16
= 6400
W
15

B
16

400
t
n = 16. R = 6400.
= 8000
320
W
2

16.t 2R
tR
Ta c: n = 16. W =
n
W
16.(350) 2
WA =
= 15, 65( s)
8000
R=

16.(375) 2
WB =
= 16, 77( s)
8000

2. ( 375 350 )
= 1,54
15, 65 + 16, 77

Bi 6
Trong sc k kh tc di chuyn ca pha ng c th o c trc tip, nu a vo mt lng no
ca cht tan tng t nh metan,n khng c gi bi pha tnh. Trong ct mao qun c chiu di 50m;
thi gian lu tr ca metan bng 71,5s;cn thi gian gi ca n-heptadecan l 12,6 pht. Hy cho bit:
a. Tc di chuyn ca pha ng bng bao nhiu?
b. Tc di chuyn ca di n-heptadecan bng bao nhiu?
Gii
a. Ta c L = 50 m = 5000 cm.
TR = 71,5 s
Vy tc di chuyn ca pha ng l:
F=

L 5000
=
= 69,93cm / s
t R 71,5

b. i vi n-heptadecan: tR= 12,6 pht = 756 s.

Vy tc di chuyn ca di l:
F=

5000
= 6, 61cm / s .
756

Bi 7
Mt ct sc k mao qun di 30m, chy mt cht chun hai tc pha ng khc nhau, cho cc s
liu sau:
Tc (cm/s)

S a l thuyt

0,5

150 000
120 000

Xc nh vn tc ti u,chiu cao a l thuyt cc tiu v s a l thuyt vn tc ti u ,cho

A=0.
Gii
TN1

H1 = 3000/150000 = 0,02 cm

TN2

H2 = 3000/120000 = 0,025 cm.

Ta c phng trnh:
17

B
+ 0,5C
0,5
B
0, 025 = + C
1
0, 02 =

Gii phng trnh ta c : C= 0,02 s ; B = 0,005 cm2/s.

U ti u = (B/C)1/2 = 0,5 cm/s
Hmin = 2(B.C)1/2 = 0,02 cm.
S a l thuyt vn tc ti u l:
n=

3000
= 150000 .
0, 02

Bi 8 Mt ct di 360 cm dng tch propan v butan bng phng php sc k kh. Dng thc ngi
ta o c khong cch t lc bm mu n nh ca pic sc k ln lt l 152,5 mm v 415,5 mm,b rng
chn cc pic ln lt l 13,5 mm v 33,8 mm. Tnh s a l thuyt v chiu cao a l thuyt ng vi mi
cht.
Gii
t 2
S dng phng trnh : n = 16( R ) tnh c s a l thuyt
w
n(propan) = 2042
n(butan) = 2416
Chiu cao a l thuyt H =

L
ta c
n

H(propan) = 0,176 cm
H(butan) = 0,149 cm.
Bi 9 Mt hn hp cha metyl xiclohexan,metyl xiclohexen v toluen c tch bng phng php sc k
lng kh trn ct nhi di 42 cm. Sc k cung cp thi gian lu ca chng ln lt l 10,1 ; 11,0 ; 13,5
v chiu rng pic ln lt l 0,75 ; 0,80 ; 1,05 pht. Hy tnh:
1. S a l thuyt i vi mi pic, gi tr trung bnh, lch chun ca n v chiu cao a l thuyt ca
ct.
2. phn gii gia metyl xiclohexan v metyl xiclohexen,gia metyl xiclohexen v toluen. Cho bit
kh c s dng l khng kh vi tR(KK) = 1,2 pht (khng kh c xem l cu t khng b lu gi
bi ct).
3. Hy tnh chiu di ct phn gii gia cc cu t t yu cu ti thiu l 1,5.
Gii
K hiu A : metyl xiclohexan
B: metyl xiclohexen
C: toluen.
t 2
1. S dng phng trnh : n = 16( R ) tnh c s a l thuyt
w
18

A: n = 2902
B: n = 3025
C: n =2645
ntb = 2900
H=

L
420
=
= 0, 015cm
ntb 2900

2
= n.H 2 = 2900.(0, 015) 2 = 0,81(cm)
H2

n=

2. phn gii gia metyl xiclohexan v metyl xiclohexen,gia metyl xiclohexen v toluen
Rs , A B =

2t R
= 1,16
wA + wB

Rs , B C =

2t R
= 2, 70
wA + wB

3.Chiu di ct phn gii gia cc cu t t yu cu ti thiu l 1,5.

V Rs,A-B = 2,70>1,5 nn ch cn tnh chiu di phn gii gia A-B t yu cu 1,5.
Gi n1,n2 l s a l thuyt ca ct c v ct mi,ta c:
Rs ,1
Rs ,2

n1
n2

1,16
2900
=
n2 = 4800
1,50
n2

Chiu di ct phn gii tng ln 1,5:

L2 = H.n2 = 0,015. 4800 = 73 cm.
Bi 10:
Mt php sc k kh s dng ct di 36 m, chy cht chun 3 th nghim vi tc pha ng khc
nhau cho cc pic c thi gian lu v rng pic nh sau:
Th nghim
u (cm/s)
tR (s)
w (s)
1
10
300
20
2
20
250
16
3
30
150
10
Xc nh vn tc ti u, s a l thuyt cho ct vn hnh vn tc ti u
Gii :
Theo phng trnh Van-Deemter, mun xc nh c vn tc ti u, Hmin v s a l thuyt ta cn
xc nh cc h s A, B, C. Mun vy ta xc nh s a l thuyt v chiu cao a l thuyt cho tng th
nghim :
Ta c : L=36 m = 3600 cm
2

TN 1:

300
n1 = 16
= 3600
20

TN 2:

250
n2 = 16
= 3906
16

H1 =

3600
= 1,00(cm)
3600

H2 =

3600
= 0,92(cm)
3906

19

150
n3 = 16
= 3600
10

TN 3:

H3 =

3600
= 1,00(cm)
3600

Thay cc s liu vo phng trnh trn ta c h phng trnh sau:

1,00 = A +

B
+ 10C (1)
10

0,92 = A +

B
+ 20C (2)
20

1,00 = A +

B
+ 30C (3)
30

Gii h gm 3 phng trnh (1), (2) v (3) ta c:

A= 0,36 (cm), B = 4,8 (cm2/s) v C = 0,016 (s)
Vn tc ti u, Hmin v s a l thuyt c tnh l :
utoiuu =

B
=
C

4,8
= 17,32(cm / s )
0,016

H min = A + 2 B.C = 0,36 + 2 4,8.0,016 = 0,914(cm)

n=

L
3600
=
= 3939
H
0,914

TI LIU THAM KHO.

1. T Vng Nghi , Nguyn Vn Ri, Nguyn Xun Trung, Trn T Hiu Ho hc phn tch
Tp 2 NXB Khoa hc k thut.
2.TS-Nguyn nh Lm- C s l thuyt v ng dng ca sc k-Bi ging.
3.TS- Nguyn B Hoi Anh-i cng v sc k Bi ging.
4.KS-Phan Hin Lng- Phng php sc k lng HPLC.

Chng 2. SC K LNG HIU NNG CAO

1.1. KHI NIM V PHNG PHP SC K

20

Sc k l qu trnh tch da trn s phn b lin tc cc cu t cht phn tch ln 2 pha: mt pha thng ng
yn, c kh nng hp ph cht phn tch gi l pha tnh, mt pha di chuyn qua pha tnh gi l pha ng; do cc cu t
cht phn tch c i lc khc nhau vi pha tnh, chng di chuyn vi tc khc nhau v tch ra khi nhau.
Qu trnh sc k khc c bn vi qu trnh tch khc 2 im:
- S phn b cht phn tch ln 2 pha lp i lp li nhiu ln (hng ngn ln, thm ch hng triu ln).
- Pha ng i qua pha tnh.
1.2. VI NT V LCH S PHT TRIN CA PHNG PHP HPLC
Phng php sc k lng hiu nng cao (HPLC High Performance Liquid Chromatography), trc kia gi
l phng php sc k lng cao p (High Pressure Liquid Chromatography).
Phng php ny ra i t nm 1967-1968 trn c s pht trin v ci tin t phng php sc k ct c in.
Ban u, phng php c thc hin p sut thng v cht nhi ct c kch thc ln. C ch tch l sc k hp
ph, sc k phn b nhng do tc pht trin nhanh ca phng php, cc cht nhi ct khc nhau ngy cng c
ci tin, hiu qu tch ca ct ngy cng c nng cao.
Sc k lng hiu nng cao bao gm nhiu phng php c c th ring, l sc k lng pha lin kt, sc k lng
lng, sc k trao i ion lng rn, sc k hp ph lng rn.
Hin nay phng php HPLC ngy cng pht trin v hin i ho cao nh s pht trin nhanh chng ca
ngnh ch to my phn tch v c s dng nhiu trong lnh vc kim nghim, c bit l trong kim nghim thuc
v hin l cng c c lc trong phn tch cc thuc a thnh phn (cho php nh tnh v nh lng).
1.3. NGUYN TC CA PHNG PHP HPLC
Nguyn tc ca phng php HPLC l qu trnh tch cc cht trng thi lng da trn s phn b lin tc
cc cht ln 2 pha: Mt pha ng yn c kh nng hp ph cht phn tch gi l pha tnh; mt pha di chuyn qua pha
tnh mang cht phn tch ra khi ct tch gi l pha ng. Do cc cht phn tch c i lc khc nhau vi pha tnh
nn chng di chuyn vi tc khc nhau v c tch ra khi nhau.

Hnh: Minh ho qu trnh tch cc cht A v B trong ct tch sc k.

Nh vy c th ni HPLC l mt sc k c in i km vi mt detect nhy pht hin cht phn tch sau
khi tch ra khi ct trong qu trnh chy sc k.
Vi nhng tin b k thut v ct tch v detect chuyn phng php phn tch ny c mt tc nhanh
v chnh xc nh h thng bm cao p khong 300 atm nhm to dng chy vi lu lng vi ml/pht. V vy ngi
ta gi l sc k lng hiu nng cao.

21

u im ca phng php ny l lng mu phn tch rt t ch cn khong 20L/1 ln phn tch v mu ch

cn ho tan m khng cn lm bay hi, do HPLC c th phn tch c cc cht m khng s gy ra s phn hy
do nhit trong qu trnh phn tch.
1.4. CC GIAI ON CHY SC K HPLC
* Giai on tch:
- Thc hin php tch cc cht trng thi lng trong ct sc k di p sut cao (200 500 atm). V vy
phi chuyn ton b cht phn tch vo trong dung dch (thng l ha tan trong dung mi lm pha ng). Phng
php ny thch hp cho tch cc cht c nhit si cao cng nh nhit si thp (tr nhng cht l th kh iu
kin thng).
- Dng thit b bm mu bm cht phn tch vo u ct tch cht phn tch c hp ph trn b mt
pha tnh.
- Dng bm cao p bm dung mi ra gii qua ct (c th l dung mi n hoc hn hp cc dung mi)
ra gii cht phn tch ra khi ct (do lc lin kt gia cc cu t cht phn tch vi pha tnh khc nhau m chng tch
ra khi nhau).
- K thut chy sc k:
+ K thut c nh pha ng (gi thnh phn v vn tc pha ng khng i).
+ K thut graient pha ng (bin i pha ng lin tc). Graient c th l tng bc hoc lin tc ty thuc vo mu
phn tch v thnh phn pha ng sao cho qu trnh tch sc k xy ra hon ton.
* Giai on pht hin v x l kt qu phn tch:
- Cc cht phn tch sau khi tch ra khi nhau c pht hin nh 1 b d gi l detect.
- Vic ghi nhn v x k kt qu c thc hin nh my tnh chuyn dng, kt qu cho mt sc k trong
cha cc thng tin cn thit nh : thi gian lu, din tch v chiu cao pic, h s phn gii, h s i xng
+ Vi 1 cht s c 1 thi gian lu c trng cho cht nn ta c th cn c vo tnh cht ny phn tch nh tnh.
+ ln pic c c trng bng din tch hay chiu cao, 2 i lng ny t l vi nng cht phn tch trong 1
khong xc nh no , c s dng nh lng cht phn tch.
1.5. CT TCH TRONG HPLC
Ct tch l b phn quan trng nht ca h thng sc k, bao gm cc vn : cc loi ct tch, cht liu lm
ct, kch thc ct, kch thc ht nhi, cch np ct,
Hn hp cc cht tan c th c tch hon ton ra khi nhau hay khng c xc nh bi hiu qu ca ct
tch v bi chn lc ca h pha. Do vy iu quan trng l phi ch to c cc ct tch c hiu qu.
* Ct tch trong HPLC c 3 loi:
+ Ct thng.
+ Ct mao qun.
+ Ct kch thc nh (ct micro).
* Cht liu lm ct: l thp c bit, nu lm bng thy tinh th phi bc thp ngoi v php sc k ny c
thc hin p sut rt cao.
* Kch thc ct:
Loi ct tch
Ct thng

ng knh

Chiu di

Tc chy

(mm)

(cm)

(mL/pht)

4,0 4,6

15 30

12

22

S a l thuyt
40.000 60.000

Ct mao qun
4,0 4,6
4 13
24
40.000 60.000
Ct micro
1,0 2,0
15 100
0,03 1
1.000.000
- Trong ct c ng knh 4.6mm c dng nhiu nht, chim 44,5%;ct ng knh 4,0 mm l 16,7%
- Ct mao qun c thi gian phn tch rt ngn, d thay i tc chy t ct quy c sang ct mao qun.
Tuy nhin c nhng bt tin l phi trang b th tch ct ph, th tch cuvet ca detector phi nh, vic nhn v x l
tn hiu phi nhanh.
- Ct micro tit kim dung mi pha ng.
- Kch thc ca ct ph thuc vo cht nhi: ht nhi nh th kch thc ct nh, ht nhi ln th kch thc
ct ln sao cho s a l thuyt thc hin php tch hon ton.

Hnh: Ct sc k.
* Kch thc ht nhi:
- Pha tnh trong sc k lng hiu nng cao c th l cht rn hoc cht lng:
+ Pha tnh rn c nhi vo ct to thnh cht nhi hay ct sc k (cc ht cht nhi trong pha tnh rn c d = 3 10
m, ph bin nht hin nay l 3 7 m).
+ Pha tnh lng th c gi trong ct sc k nh vo ht cht mang tr. Mng mng thng dng l cc loi du nh
silicon c b dy d = 13 m, loi pha tnh ny c dung tch nh hn pha tnh rn.
Nh vy ty thuc vo pha tnh m ta c phng php sc k lng - lng (pha tnh lng) hoc lng - rn (pha
tnh rn).
- Ht cht nhi c ng knh 30 m thch hp cho ct tch c ng knh trong 2 mm. Khi s dng cht
nhi c ng knh nh hn 10 m, ng knh trong ct tch khong 3 4 mm s cho hiu sut tch v lp li
tt. Nh vy kch thc ht dng trong HPLC nh hn so vi sc k kh.
- Nhm thu c cc ct tch c hiu qu cc cht nhi c a vo trong ct nh l lp ng nht v cht
nhm thu c s n nh. y mt s yu cu t ra cho cc cht nhi l phn b kch thc ht cht nhi cn phi
hp v cc ht cc nh hoc cc ln phi khng c, v cc ht c kch thc ln gy nhiu lon ng k s ng nht
qu trnh nhi, trong khi cc ht qu nh li lm gim s thm t...

23

Hnh: Ht nhi trong HPLC.

* Cch nhi ct:
- Trc khi nhi, ct tch lm bng thp khng g c lm sch bng HNO 3 50%, ra bng nc ct nhiu
ln, trng bng axeton, clorofom ri li bng axeton v cui cng lm kh bng kh N 2 hoc khng kh nng.
- Cc phn g lp u v cui ct tch vi cc b phn khc cng rt quan trng v lm th tch cht t nht.
cc u ct tch phi s dng cc mng lc bng thp khng g, thy tinh xp p, teflon xp. Bng thy tinh thng
c dng ngn cc ht nh. L xp ca vt liu lm ngn phi nh hn kch thc cc ht nhi t nht l 5 ln.
- Trong thc t c 2 cch nhi: nhi kh v nhi t (huyn ph). Vic la chn cch nhi ct tch ph thuc
vo kch thc ca cht nhi:
+ Cc cht nhi c ng knh ln hn 20m thng c nhi kh, tng phn nh cht nhi c thm vo ct
(khong 0,5 ml hoc t hn), va rt va g hoc rung. Trong mt s trng hp c th ni vi bm sc k v luyn
ct tc dng cao v p sut cao trong 10 pht. Ct nhi c coi l tt (ng knh ht cht nhi 3040 m)
tc ra gii 3 cm/s v chiu cao a l thuyt ca ct tch khong 1 mm.
+ Vi kch thc ht cht nhi khong 10m hoc 5m ngi ta hay s dng phng php nhi t. Trong qu trnh
nhi ct, huyn ph phi bn vng, khng c s sa lng. Huyn ph bn vng khi :
Gia cc tiu phn v cht lng huyn ph c t trng nh nhau (phng php cn bng t trng).
nht ca mi trng huyn ph ln n mc tc sa lng ca cc tiu phn l cc tiu (phng php nht
cao).
Cc cht lng lm huyn ph thng c dng l metanol, axeton, dn xut halogen, ioxan... hay hn hp
ca chng, cc cht lng c nht cao nh parafin, xiclohexano.
* Ngoi ra, ngi ta cn s dng ct bo v t trc ct phn tch gim thiu s hp ph bt thun nghch
ca cht tan i vi pha tnh v s tc nghn ct chnh. Ct bo v ni chung ging ct phn tch nhng c ch to
t nhng vt liu r hn vi ngha l ct chp nhn hi sinh bo v tui th ca ct phn tch.
1.6. CC LOI PHA TNH THNG DNG TRONG HPLC
1.6.1. Nguyn tc chn pha tnh trong HPLC
Chn pha tnh trong HPLC sao cho:
- Cn bng chuyn khi lng cht tan t pha ng vo pha tnh v ngc li phi c thit lp nhanh.
- Pha tnh c dung lng ln hp ph mt lng ln cht phn tch.
- Khong p sut lm vic tng i rng.

24

1.6.2. Cc loi pha tnh trong HPLC

* Pha tnh hp ph pha thng :
- Loi pha tnh ny c ch to t: silicagen, polime (polistiren).
- Silicagen c cu to c bit, xp, b mt g gh, c nhng mao qun cho nn din tch b mt ca silicagen
ln, trn b mt ca n cn c cc nhm silanol cng c th tham gia cc lin kt vi cht phn tch. Silicagen c tnh
axit, pH = 3 5 nn hp ph tt cc cht c tnh baz hn l cc hp cht c tnh axit. V nhng c tnh trn m
Silicagel thng c s dng tch, phn li cc hp cht thuc h ru, phenol, andehit, axit hu c, lipit, amin,
.). Trn b mt ca n c cha nhm OH l nhm phn cc. Cc nhm OH c th tng tc rt mnh vi cc
phn t c cha nhm chc. ln ca s tng tc ph thuc vo kiu v s cc nhm chc trong cc phn t cht
tan.

OH

Si

OH

Si

OH

OH
Si

Si

Khi gp nhng hn hp phc tp th ta hot ha nhm OH bng mt s nhm phn cc: -C 3H6CN, -(CH3)2O-CH2-CH(OH)-CH2(OH), -C3H6NH2 Nhm ny l nhng nhm a nc v d to cu lin kt hiro vi cc phn
t nc nn lm gim hot tnh sc k pha tnh.
- S lu gi ca cc cht tan trong cc h xy ra l do tng tc gia cc phn t cht tan v cc v tr hot
ng trn b mt ca cht rn. V cc phn t dung mi c th tng tc vi cc v tr ny, s hp ph c th coi nh
l qu trnh cnh tranh gia cc phn t cht tan v cc phn t dung mi trong s tn cng cc v tr hp ph trn b
mt ca cht rn. iu c ngha l cc cht tan c mt trong
pha lng thay th mt hoc nhiu cc phn t dung mi t b mt m chnh
n b hp ph.
- Nh vy loi pha tnh ny thch hp vi vic tch cc cht tan vi cc kiu nhm chc khc nhau hoc s
cc nhm chc khc nhau v i vi vic tch cc ng phn, min l cc cht tan khng phn cc hoc phn cc km
bi chng phi ho tan trong pha ng t phn cc.
* Pha tnh hp ph pha ngc :
- Pha tnh l nhng silicagen trung tnh c ankyl ha cc nhm -OH trn b mt bng cc gc ankyl (C 3,
C5, C8, C18 ..., thng dng nht l C18).

V vy m cht nhi ny c b mt khng phn cc, n thuc loi cht nhi k nc tc l nc khng lm
nh hng n b mt ca n.
- Loi pha tnh ny c s dng tch cc cht t khng phn cc n phn cc. V vy phm vi s dng
a dng, phong ph hn.
* Pha tnh hp ph trao i ion:

25

- Pha tnh l ionit. Ionit l nhng i phn t axit hoc baz khng tan trong nc v cc loi dung mi, cha
bn trong mng li nhng ion linh ng c kh nng trao i theo ng lng v thun nghch vi cc ion cng du
trong dung dch cht in ly khi tip xc. Ionit c th dng rn hay dng lng.
+ Khi ion trao i mang in tch dng (ion c nh ca mng li khng gian mang in tch m) th ionit gi l
cationit. V d:

R-SO3H + M+ RSO3M + H+

+ Khi ion trao i mang in tch m (ion c nh ca mng li khng gian mang in tch dng) th ionit gi l
anionit.
+ Cc ionit c kh nng trao i c cation v anion cng mt lc th c gi l ionit lng tnh. V d:
R-N(CH3)3OH + X- R-N(CH3)3X + OH- Ionit th hin tnh cht nh mt axit mnh (cationit axit mnh) hoc baz mnh (anionit baz mnh) c kh
nng trao i mi gi tr pH v dung lng
hp th t thay i theo pH.
- Ionit th hin tnh cht nh mt axit yu (cationit axit yu) c i lc vi H + - l ion c dng trao i
ca ionit ln hn so vi cc ion khc trong dung dch phn tch nn cationit axit yu khng c kh nng trao i
cation trong mi trng axit (tc ch lm vic mt gi tr pH xc nh) v dung lng hp th thay i nhiu theo
pH.
- Ionit th hin tnh cht nh mt baz yu (anionit baz yu) c i lc vi OH - - l ion c dng trao i
ca ionit ln hn so vi cc ion khc trong dung dch phn tch nn anionit axit yu khng c kh nng trao i
anion trong mi trng baz (tc ch lm vic mt gi tr pH xc nh) v dung lng hp th thay i nhiu theo
pH.
- Cationit axit v anionit baz c kh nng trao i trung bnh gia hai loi mnh v yu. Cationit axit trao i
trong mi trng t axit yu n baz, anionit baz trao i trong mi trng t baz yu n axit.
* Pha tnh ry phn t:
Pha tnh c ch to t cc vt liu bn vng, b mt xp, c cha cc mao qun kch thc c phn t kch thc mao qun phi c la da vo kch thc ca cc phn t cht phn tch nh: polime tng hp, oxit
nhm, silicagen.
1.7. PHA NG TRONG HPLC
1.7.1. Nguyn tc chung chn pha ng
- Phi tr vi pha tnh khng lm hng cht nhi.
- Ha tan c mu phn tch.
- Bn vng, n nh trong thi gian chy sc k.
- C tinh khit cao.
- Ph hp vi detecto.
1.7.2. Chn pha ng ph hp vi pha tnh
Pha ng trong sc k lng hiu nng cao l mt dung mi n hoc hn hp cc dung mi c trn theo
nhng t l xc nh. Tuy nhin ngi ta hay dng hn hp hai hay ba dung mi c mt pha ng c phn cc
ph hp vi php phn tch. Tch cc hn hp mu phc tp ngi ta phi dng pha ng c thnh phn l hn hp
cc dung mi.
* Pha ng khi s dng ct sc k hp ph pha thng:
- V pha tnh c cha nhm OH d to cu lin kt H vi cc phn t phn cc nn pha ng cho loi pha

26

tnh ny l nhng dung mi khng phn cc hoc t phn cc nh C6H6, C6H5CH3, nhexan, CCl4. Nu dng dung mi
phn cc s lm hng pha tnh.
* Pha ng khi s dng ct sc k hp ph pha ngc
- Pha tnh khng phn cc, v vy pha ng l dung mi phn cc hoc khng phn cc, thng thng ngi
ta ly hn hp nc vi cc dung mi hu c tng i phn cc lm pha ng, thng dng nht l: CH 3OH H2O
v axetonitril H2O vi t l thch hp.
Bng: Ch s phn cc ca mt s dung mi hu c thng dng
Stt
Pha ng
Ch s phn cc (P)
Cc i hp th (nm)
1
Xyclohexan
0,04
210
2
n-hexan
0,1
210
3
Cacbon tetraclorua
1,6
265
4
i-propyl ete
2,4
220
5
Toluen
2,4
286
6
ietylete
2,8
218
7
Tetrahydrofuran
4,0
220
8
Etanol
4,3
210
8
Etyl axetat
4,4
255
9
ioxan
4,8
215
10
Metanol
5,1
210
11
Axetonitrin
5,8
190
12
Nc
10,2
---- c pha ng vi ch s phn cc trung gian ch cn trn hai hay nhiu dung mi trn vi t l thch hp
tnh theo cng thc sau :
P(AB) = P(A).V(A) + P(B).V(B)
Trong :
P(A), P(B) ln lt l ch s phn cc ca A, B.
V(A), V(B) ln lt l th tch ca A, B.
P(AB) l ch s phn cc ca hn hp gm A, B theo ng t l trn.
- thay i h s chn lc ca mt cp cht tan ta thay i mt hoc nhiu dung mi lm pha ng. Cc
dung mi thng c s dng cho mc ch ny l axetonitrin, metanol, tetrahydrofuran (THF). la chn h dung
mi tt nht ta dng s tam gic dung mi.

Hnh: Tam gic dung mi thay i chn lc ca mt cp cht tan.

S tch c ti u ha trc ht vic thay i t l axetonitrin trong nc n khi t c s phn tch tt
nht trong thi gian phn tch cn thit (metanol, THF cng c th c chn u tin). Sau , cc h dung mi ny

27

c iu chnh (nu cn thit) thnh lp im ba ca tam gic dung mi. S kt hp ca 4 dung mi ny s to ra

7 h dung mi nh trn s . T y cho php d on nhng thay i trong s hnh thnh pha ng tc ng n
s tch trong qu trnh sc k.
Bng: S to thnh cc pha ng c hiu nng phn tch tng ng.

* Pha ng khi s dng ct sc k hp ph trao i ion:

- Pha ng l dung mi nc c cha cc cht m pH, cht to phc, cht lm chm lm n nh nng
ion H+.
- tch cation kim loi, dng dung mi c pH thp v khi nng cation H + cao nn cation H+ cnh
tranh mnh v y cation kim loi ra.
- tch anion, dng dung mi c pH cao v khi nng anion OH- cao nn anion OH- cnh tranh
mnh v y anion gc axit ra.
* Pha ng khi s dng ct sc k ry phn t:
- Dung mi lm pha ng l dung mi khng phn cc, khng cha cation v anion.
1.8 CC K THUT TCH
Sc k lng hiu nng cao l phng php c dng kh ph bin trong phn tch hn hp cc cht hu c,
qu trnh phn tch gm 2 giai on : giai on tch hn hp cc cht v giai on pht hin.
*Giai on tch : hn hp cht phn tch c bm qua ct sc k, c lu gi trong ct v ln lt ra gii
khi ct nh mt pha di ng chy qua ct di 1 p lc ln.
*Giai on pht hin v x l kt qu phn tch :
+ Cc cht phn tch sau khi tch ra khi nhau c pht hin nh 1 b d gi l detect.
+ Vic ghi nhn v x k kt qu c thc hin nh my tnh chuyn dng, kt qu cho mt sc k
trong cha cc thng tin cn thit nh : thi gian lu, din tch v chiu cao pic, h s phn gii, h s i xng.
+ Vi 1 cht s c 1 thi gian xc nh c trng cho cht nn ta c th cn c vo tch cht ny
phn tch nh tnh.
+ ln pic c c trng bng din tch hay chiu cao, 2 i lng ny t l vi nng cht phn
tch trong 1 khong xc nh no , c s dng phn tch nh lng theo phng php lp ng chun hay so
snh vi cht bit chnh xc hm lng.
HPLC l qu trnh tch hn hp cc cht ct tch sc k v trng thi lng. V vy m cc cht phn
tch mun tch c theo phng php ny th trc ht phi ha tan trong mt cht lng no m thng l ha tan
trong dung mi lm pha ng. Phng php ny thch hp cho tch cc cht c nhit si cao cng nh nhit si
thp (tr nhng cht l th kh iu kin thng).

28

- Pha tnh trong sc k lng hiu nng cao c th l cht rn hoc cht lng. Pha tnh rn c nhi vo
ct to thnh cht nhi hay ct sc k, cn pha tnh lng th c gi trong ct sc k nh vo ht cht mang tr.
Mng mng thng dng l cc loi du nh silicon c b dy d = 1-3 m , loi pha tnh ny c dung tch nh hn
pha tnh rn. Nh vy ty thuc vo pha tnh m ta c phng php sc k lng - lng hoc lng - rn.
- Pha ng trong sc k lng hiu nng cao l mt dung mi n hoc hn hp cc dung mi c trn
theo nhng t l xc nh. Vic thc hin tch sc k c th theo 2 k thut khc nhau :
+ K thut gi thnh phn v vn tc pha ng khng i.
+ K thut gradient pha ng (bin i pha ng lin tc). Gradient c th l tng bc hoc lin tc l ty
thuc vo mu phn tch v thnh phn pha ng sao cho qu trnh tch sc k xy ra hon ton.
1.9. SC K LNG PHA LIN KT (BPC)
Trong BPC ( bonded-phase chromatography), cht phn tch c lin kt vi pha tnh c gi l pha lin kt.
1.9.1 Pha tnh
- loi pha tnh ph bin nht c ch to t silicagel. N c cu to c bit, xp, b mt g gh, c nhiu
mao qun nn din tch b mt ln. Trn b mt n cn c cc nhm silanol (OH) c th lin kt vi cht phn tch.
Khi c ankylsilan kt hp vi silicagel, cc nhm ankyl khc c th gn vo b mt ca n.
-Si- OH+ Si(CH3)2RCl -Si-O-(CH3)2R + HCl
R l cc gc khc nhau v s lng cacbon v s nhm chc. V vy, pha tnh c th phn cc (c cha nhm
nh -NH2; -CN...); t phn cc hoc khng phn cc (phn t ch cha hidro cacbon).
Cht nhi pha lin kt thng c phn cc khc nhau, c chia thnh hai loi
- Sc k pha thng
- Sc k pha o
1.9.1.1 Sc k pha thng
- Trn b mt ca n c cha cc nhm -OH l nhm phn cc. Khi gp nhng hn hp phc tp th ta hot
ha nhm -OH bng mt s nhm phn cc nh: -C3H6CN; -CH2-CH(OH)-CH2-OH; -C3H6NH2... Cc nhm ny l
nhm a nc v d to cu lin kt hidro vi cc phn t nc, v vy n lm gim hot tnh sc k pha tnh. Loi
pha tnh ny c s dng tch cc cht khng phn cc hoc t phn cc.
1.9.1.2 Sc k pha ngc- L nhng silica trung tnh c ankyl ha cc nhm -OH trn b mt bng cc gc
ankyl (C3, C5, C8, C18..., thng dng nht l C18)
-Si-OH + Si(CH3)2RCl -Si-O-Si(CH3)2R + HCl
- V vy m cc cht nhi ny c b mt khng phn cc, n thuc cht nhi k nc ngha l nc khng
lm nh hng n b mt ca n.
- Loi pha tnh ny dng tch cc cht c phn cc rt a dng, t khng phn cc n phn cc
1.9.2 Pha ng
* Nguyn tc chung chn pha ng
Pha ng trong HPLC c chn ty thuc vo mu phn tch, n c th l dung mi n hay hn hp 2,3
dung mi c trn theo nhng t l nht nh. Khi chn pha ng ta phi ch n nhng yu cu sau:
- Phi tr (khng tc dng) vi pha tnh khng lm hng cht nhi.
- Phi ha tan c mu phn tch ( mang c cht phn tch ra)
- Bn vng, n nh trong thi gian chy sc k.

29

- C tinh khit cao

- ph hp vi detector
* Chn pha ng ph hp vi pha tnh
1.9.2.1 Pha ng khi s dng ct sc k hp th pha thng
- V pha tnh c cha nhm -OH phn cc d to cu lin kt hidro vi cc phn t phn cc, nn pha ng cho loi
pha tnh ny l nhng dung mi khng phn cc hay t phn cc. Nu dng dung mi phn cc s lm hng pha tnh.
- Ni chung l dung mi hu c khng ha tan trong nc nh benzen; metylbenzen; n-hexan,
tetraclormetan... h dung mi hu c ny dng tch cc hidrocacbon hoc cc hp cht hu c t phn cc.
1.9.2.2 Pha ng khi s dng ct sc k hp th pha ngc
-V pha tnh khng hoc t phn cc nn pha ng phi l dung mi phn cc hoc hn hp cc dung mi hu
c phn cc vi nc. Thng dng nht l metanol- nc v axetonitril- nc.
-H sc k ny c th dng tch nhiu hn hp t phn cc n khng phn cc. V vy phm vi s dng
a dng, phong ph hn.

stt

Pha ng

Ch s phn cc (P')

Cc i hp th (nm)

Ciclohexan

0,04

210

n-hexan

0,1

210

Carbon tetraclorur

1,6

265

Nc

10,2

---

Toluen

2,4

286

Dietyleter

2,8

218

Tetrahydrofuran

4,0

220

Etanol

4,3

210

Etylacetat

4,4

255

10

Dioxan

4,8

215

11

Metanol

5,1

210

12

Acetonitril

5,8

190

c pha ng vi ch s phn cc trung gian ch cn trn hai hay nhiu dung mi trn vi t l
thch hp c tnh theo cng thc sau:

P' (AB)= P'(A). VA+ P'(B). VB

Vi P' (A); P'(B) l ch s phn cc ca A, B; VA; VB l th tch pha trn ca A v B; P'(AB) l ch s

phn cc ca hn hp gm A, B theo ng t l trn.
- S tch c ti u ha trc ht vic thay i t l acetonitrl trong nc n khi t c s phn
tch tt nht trong thi gian phn tch cn thit (metanol, THF cng c th c chn u tin). Sau , cc
30

h dung mi ny c iu chnh (nu cn thit) thnh lp im ba cu tam gic dung mi. S kt hp

ca 4 dung mi ny s to ra 7 h dung mi nh trn s . T y cho php ta d on nhng thay i
trong s hnh thnh pha ng tc ng n qu trnh tch sc k.

Loi

Pha tnh

Pha ng

Cht phn tch

Pha thng

B mt phn cc (nhm

Khng phn cc hoc t

Tch cc cht khng

-OH,...)

phn cc

phn cc hoc t phn

cc nh hidrocacbon;
ete; aldehid; ceton...

Pha o

B mt khng phn cc

Phn cc hoc hn hp

Tch cc cht t khng

( ankyl ha)

dung mi hu c phn

phn cc n phn cc

cc vi nc
1.10 SC K TRAO I ION, SC K CP ION, SC K LNG LNG
1.10.1 Sc k trao i ion
V c bn sc k trao i ion v sc k trao i ion ging nhau, c ch tch tng t nhau, cng da trn c
s ca phn ng trao i ion. Cng thc c bn ca sc k trao i ion l:
H dn dung dch ra gii + ct tch trao i ion+ ct loi tr + detetor dn
Phn ng loi tr l phn ng loi b yu t cn tr thu c tn hiu ca ion cn tch.
V d: Khi tch cc halogenua, s dng NaX ra gii (X: OH -, Br-, F-). Nu cho NaX i vo detetor th
tn hiu rt ln, ln t c tn hiu ca cc halogenua cn tch, v vy cn loi tr tn hiu nn i. S dng anionit dng
R-HCO3 cc phn ng nh sau:
ct tch:

R-HCO3 + X- + Na+

ct loi tr: R-H + Na+ +HCO3-

RX + Na+ + HCO3 RNa + H2CO3

Nh vy c Na+ v X- khng qua detetor, ch c cc ion halogenua cn tch i qua v tn hiu o c l ca

chng.

Hnh 5: S h sc k trao i ion

1.Bnh ng dung dch ra gii ; 2: Bm cao p; 3: Van bm mu;
4: Ct tch ;

5: Ct loi tr;

1.10.2 Sc k cp ion

31

6: Detector ; 7: My ghi

Sc k cp ion (SKCI) c pht trin vo nm 1970 v ngy nay vn c p dng do c nhng u im

v kh nng thch ng vi mu phn tch. Khi nim cp v cht phn tch v ion i to thnh cp ion tham gia vo
qu trnh tch sc k. Ion i c a vo pha ng to cp vi cht phn tch, lm cho n c nhng tnh cht cn
thit ph hp vi pha tnh cng nh phng php tch.
C hai loi : SKCI pha thng v SKCI pha o
+ SKCI pha thng: l phng php s dng cht hp thu a nc nh silicagen, Al 2O3 thm ch silicagen
bin tnh nhng vn a nc lm pha tnh. y l nhng cht hp thu c din tch b mt ln, c cc mao qun
kch thc c phn t. Ngi ta cn a thm vo b mt ca chng cc axit v c. Pha ng l cc dung mi khng
phn cc cha cc cht phn tch.
+ SKCI pha o: s dng silicagen bin tnh lm pha tnh, v d ODS hay C18, pha ng l cc dung mi phn
cc nh nc hoc hn hp cc dung mi phn cc hay t phn cc
Vai tr ca ion i rt quan trng, chng lin kt vi ion cn tch trc khi phn b vo pha tnh. Chnh nh
ion i, cc cht phn tch cng vi ion i th hin r bn cht ca n v kh nng lin kt, v iu dn n tnh
cht ca cp ion by gi khc nhau so vi ion n gin ban u. y l iu cn thit ca php tch sc k, v c nh
vy th h s phn b ca chng mi khc nhau v chng tch khi nhau.
V d: amin R-NH2 c th tch theo c hai phng php pha o v pha thng
tch pha thng, pha tnh l lp nc trn silicagel cha HClO 4 cn cc amin trong dung mi t phn cc,
phn ng cp ion xy ra nh sau:

H + + ClO 4 + R NH 2

R NH 3+ ClO 4

Chnh cp R-NH3+ClO4- tham gia lin kt vi pha tnh khi cho pha ng t phn cc tip xc v pha tnh. Cc
amin c cc gc R khc nhau lin kt vi pha tnh khc nhau l c s tch khi nhau. tch pha o, pha tnh by
gi l cht khng phn cc, th d ODS, pha ng l dung mi phn cc c cha HclO 4 0,1M / nc/ ACN. Cc cp
ion amin lin kt vi pha tnh khng phn cc theo cc cc lin kt khc nhau l c s tch sc k.
1.10.3 Sc k lng- lng
S phn b ca cht tan trong h lng-lng c c trng bng nhit ng hc bi phng trnh:

a is
K= m
ai
Trong : K: h s phn b nhit ng hc ca cht i

a i s : hot ca cht tan i trong cht lng s;

a i m : hot ca cht tan i trong cht lng m

s pha long v cng ca cht tan, h s phn b c th c biu th bng cc i lng phn s mol v
cc h s hot ca cht tan i trong pha lng ng vi:

x is fis
K xi = m = m
xi
fi
Trong : Kxi : h s phn b ca cht tan i s pha long v cng

x i s , x i m : cc phn s mol ca cht tan i trong pha s v m

fi s ,f i m : h s hot ca cht tan i trong pha s v m
Sc k lng lng gm hai kiu pha thng v pha ngc, ph thuc vo pha tnh c s dng l phn cc
hay khng phn cc. Nu pha tnh phn cc (LLC pha thng) th b mt cht mang rn cn phi phn cc v i vi

32

pha tnh khng phn cc (LLC pha ngc) th b mt cht mang rn phi khng phn cc. Pha tnh c tri cng trn
b mt cht mang rn nh l lp cht lng c b dy ng nht iu ny c tc dng lm tng s vn chuyn khi ca
hai pha lng. Ngha l cht mang rn cn phi c nhng tnh cht hp ph ln cho pha tnh c th gi cht n di
dng chy, ni mt cch khc cht mang rn cn phi c lm t d dng hn bi pha tnh so vi pha ng.
*K thut kt ta: Dung dch ca pha tnh trong dung mi hu c c bm qua ct tch c nhi trc,
ct ny c cn bng trc vi cng dung mi hu c. Khi ct c bm y dung dch pha tnh, dung mi
khc ( trn ln hon ton vi cht pha long nhng khng trn ln vi pha tnh) c bm qua ct tch, pha tnh c
kt ta v lp y cc l ca cht mang. Sau ct tch c cn bng vi pha ng bo ho pha tnh. i vi cc h
lng-lng trong pha tnh biu th i lc ln ng k i vi cht mang v ho tan chng mc no trong pha ng
th k thut tm ng lc c th c ng dng c kt qu.
* K thut tm ng lc: Pha ng c bo ho vi pha tnh c bm lin tc qua ct tch c nhi
trc. V i lc ln vi cht mang, pha tnh c ko ra khi pha ng v hp ph u tin ln cht mang.
* K thut bay hi dung mi: u tin cht tan c tm vi pha tnh v sau c nhi kh vo trong ct
tch. Mt lng chnh xc pha tnh c ho tan vo trong cht lng d bay hi v trn cn thn vi cht mang kh.
Sau dung mi c lm bay hi u t hn hp trong my bay hi quay chn khng, pha tnh c nm li trong
cc l ca cht mang.
* Din tch b mt c trng ca cht mang: cho phng php sc k lng lng thun tu, cht mang rn
cn phi tr i vi cc cht tan c cc tnh cht hp ph km theo i vi pha tnh, ngi ta thy rng trong thc t
c th gp s hp ph cnh tranh gia cht tan v pha tnh trn cht mang. Mt khc, s hp ph c th gy ra hiu
ng dng hoc m i vi chn lc v iu lm gim u im ca lp li nhit ng. Khuynh hng hp
ph cht tan trn cht rn ph thuc vo bn cht v din tch b mt ca cht mang v vo lng tm ca pha tnh.
* La chn cc h lng lng: V mt l thuyt, tt c cc t hp cht lng, tch thnh hai pha c th c p
dng nh l h pha trong LLC. Tuy nhin, trong thc t s cc cht lng l t hn do cc l do thc tin, chng hn
nhng cht c nht cao d pht hin hoc c hot tnh ho hc l khng chp nhn c. Ngoi cc l do hin
nhin ny, s phn cc khc nhau gia hai pha lng l iu rt quan trng, c tnh cht quyt nh c c cc ct
LLC chn lc v bn vng.
V d: Nu phn cc khc nhau rt ln th cc cht tan hoc s khng b gi hoc s b gi rt mnh trn
ct. Mt khc, chn lc ni chung s cao.

-S khc nhau v phn cc khng thch hp vi tan ca cc cht tan cng lm tng tnh khng
n nh ca cc h sc k. Khng nhng th, trong trng hp ny chn lc s rt thp. Do , trong thc
t cn phi c mt s khc nhau xc nh v phn cc gia hai pha thu c mt s lu gi va phi
v cc ct LLC bn vng
* ng dng:

- Sc k lng-

lng l phng php ph bin c dng tch nhiu cht ha hc

- Sc k lng-lng ng ng dng trong vic tch cc kiu cht tan khc nhau nh cc cht dit c cha clor,
cc esterphosphate, cc phenol, cc hp cht thm a vng c hidroxyl ha, cc cht dit c urea v cc dn xut
2,4-dinitrophenylhydrazon ca ceton v cc aldehid
1.11. DETECTOR TRONG HPLC

33

Detector l mt b phn quan trng quyt nh nhy ca phng php, n bao gm hai loi:
- Loi theo di tnh cht ca pha ng: L theo di tnh cht vt l hay ho hc ca pha ng c lin quan n
cht phn tch. S lin quan ny c nh gi bng s tng hay gim tnh cht ca pha ng t l vi s c mt ca
cht tan trong dung dch nn c th nh lng c. l cc detector ch s khc x (RI), detector hng s in mi
v detector dn
- Loi theo di tnh cht ca cht phn tch thng qua cc phn ng c trng, detector ny ch nhy i vi
mt s tnh cht ho hc hoc vt l ca cht tan, chng khng ph thuc vo t l thnh phn cht ra gii. l cc
my quang ph hp th t ngoi kh kin (UV-Vis), my ph hunh quang, cc detector in ho v khi ph.
1. Detector ch s khc x
Khc x l tnh cht ca tt c cc loi phn t. Tuy nhin khong gi tr ch s khc x ca hu ht cc cht
hu c v v c l kh nh v n ph thuc ng k vo nhit . Do phng php o ch s khc x l phng
php vn nng nhng khng phi l phng php nhy v chn lc, gii hn pht hin nm trong khong 1ng-1pg.
Cc detector RI ch yu c dng pht hin lng c g ca mu cht khng hp th trong vng t ngoi kh
kin, khng nhy hunh quang, khng c hot tnh in ho v khng d dng b dn xut ho. Ni mt cch khc loi
detector ny ngy cng khng c gi tr.
2. Detector hp th t ngoi- kh kin UV-Vis
y l loi detector rt thng dng, hu ht cc thit b sc k lng c trang b loi detector ny. N c cu
to gn ging vi my trc quang bnh thng, im khc nhau bung mu, phn cuvet tnh c thay bng
Flowcell. Nng cht phn tch ti thiu c th pht hin c thng qua cng thc:

A = log

I0
= lC
I

Trong : A l mt quang; I0 v I l cng ca tia ti v tia truyn qua;

l h s tt phn t; l chiu

dy cuvet (cm);C nng cht tan cn xc nh (mol/L)

Gii hn pht hin ca detector UV-vis l 10-6(mol/L), ngy nay cc my c phn gii cao s cho nhy
cao hn.

Hnh 2:
1: Ngun sng ;
5: flowcell ;

2: Gng ; 3,7: Cc bn gng ;

6,10: T bo quang in ;

Detectror UV- VIS

4,8: Cc b n sc

9: B phn so snh ; 11: X l s liu

3. Detector hunh quang

Detector hunh quang c u im l nhy cao v bc x pht ra c o i vi nn ti, nhng c s
dng t hn detector UV-vis v cht pht hunh quang khng nhiu. chn lc ca vic pht hin da trn tnh cht
hunh quang c t ra do phi dng n hai bc sng trong qu trnh o: bc sng kch thch v bc sng pht

34

x v c bit l cc hp cht phi c cu trc nht nh c kh nng pht hunh quang. Detector s dng cho cc
cht c kh nng pht hunh quang bao gm cc cht bn thn chng pht hunh quang v trong c trng hp cht
phn tch phn ng vi thuc th to thnh hp cht c kh nng pht hunh quang. Ngun kch thch c bc
sng ngn, thng dng n dteri 190-400 nm.

Hnh 3: Detector hunh quang

1: Ngun sng kch thch ; 2,5: Cc b n sc ; 3: Bn gng ; 4: Flowcell
6,7: Cc t bo quang in ; 8: B phn x l tn hiu
Khi cht phn tch i vo flowcell nh c nh sng kch thch chng pht ra nh sng th cp, qua b n sc
5 v ti t bo quang in 6 bin i thnh dng in. Tn hiu ny c khuch i v so snh vi tn hiu t t
bo quang in 7, ghi li pic nh b x l 8.
4. Detector dn
Detector dn ph hp vi cc cht c hot tnh in ho, v d mt s ion kim loi chuyn tip nh Cu 2+,
Ag+, Ni2+Flowcell ca detector c cu to c bit gm mt h in cc l in cc lm vic v in cc so snh,
i khi ngi ta dng thm in cc ph tr.

Hnh 4:

Detector dn

1: Flowcell c gn cc in cc
2: in tr ; 3: B phn x l s liu ; 4: Bin tr
m bo tn hiu ca detector n nh, phn nh ng nng cht phn tch, dng chy qua flowcell
phi khng thay i vn tc tuyn tnh, iu c ngha ng knh mao qun qua flowcell cng u cng tt. Khi
cht phn tch i qua lm thay i dn din ca dung dch l c s pht hin v nh lng chng. loi
tr nh hng ca tn hiu nn, thng thng ngi ta s dng cc cht ra gii c dn in km nh axit benzoic,
axit salisilic, axit phtalic Trong iu kin nh vy, dung dch i qua detector c dn in nh.

35

B phn o v x l tn hiu s cn bng dng in gia hai nhnh I v II. Khi c cht phn tch i qua
flowcell thng l cc ion c dn in tt lm thay i dng I v II. B phn ghi o s ghi li tn hiu trn di
dng pic.
.5. Detector bc sng n
Cc detector bc sng n dng n thu ngn p sut thp c hiu sut nng lng cao ti 254 nm. Cc
detector ny n gin, chc chn, r v lm vic vi nhiu thp. Hn ch chung ca detector ny l cht tan cn
nghin cu phi hp th 254 nm m khng b cc phn t ca dung mi hoc cc cht khc cn tr. Ngy nay cn c
detector a bc sng kiu ny (dng n thu ngn khc cng vi b lc; 365nm; c thm n cadimi v km).
6. Detector dng dy diot quang tuyn tnh
Ngy nay detector dy diot quang tuyn tnh l loi detector a bc sng c s dng nhiu nht. B dit
bao trm mt cch c trng khong 190-600 nm vi phn gii 2-5 nm. Ton b quang ph c th nhn c ch
trong khong 10 ms v c sau mi 10 ms th qu trnh c lp li. C th tin hnh o ghi ng thi bng cch
dng ti 8 bc sng chn lc, cc tn hiu ca chng c th c biu din thnh th bng cc cch khc nhau, c
th thu c cc ph hon chnh ti nh pic v ti cc im un. C th biu th cc d kin di dng th ba chiu
hoc hai chiu. Detector dng dy diot quang tuyn tnh c dng gii quyt cc vn sau: nh gi pic, xc
nh pic cha bit v kim tra pic. So vi cc detector hp th t ngoi- kh kin UV-Vis loi detector ny c nhy
thp hn.
7. Detector ph kh
Vic kt hp LC v ph khi a n mt k thut tch c nhy chn lc cao. Trong mi nm gn
y ngi ta s dng phng php ny nhn bit v xc nh cc hp cht khi chng rt kh tch bng phng
php GC-MS. Chng hn nh cc hp cht c trng lng phn t tng i cao, cc hp cht khng bn vi nhit v
cc hp cht phn cc, chng thng l cc hp cht t nhin, cc cht cn b mi trng, cc loi thuc v cc sn
phm phn hu ca chng.
C hai phng php sau y c dng thit k cc h LC-MS
+ Phng php vn chuyn: Phn cht i ra khi h LC c tm vo mt dy curoa hoc dy kim loi v
dung mi c loi b trc khi n c a n my khi ph. K thut a trc tip cho php a trc tip mu
cht i ra khi h LC i vo my ph khi nh l mt vi phun qua mt mao qun dng nht hoc qua mt l hp
1-5 m .K thut ny rt thch hp cho phng php LC ct hp, cho php a ton b lng cht ra khi h LC v
cho php s dng phng php ion ho ho hc ( LC-MS tinh vi v LC-MS phun nhit)

+ Phng php a thng cht lng vo: N bao gm vic lm bay hi nhanh trong mao qun kim
loi khi c mt ca mui nh amoni axetat. Phng php ny c nhiu trin vng v h ra hu c - nc c
th a vi tc 1-2 ml/pht
Hin nay c mt s kiu ghp LC-MS mi kiu c mt gi tr ring ca n. Cc yu t cn n
khi phi la chn mt kiu l: gii hn pht hin cn thit, tnh b hay khng b ca cht phn tch, tc
dng chy ra trong h LCphng php ny rt thch hp cho phng php nhn bit.
1.12 TI U HA MT PHP PHN TCH SC K LNG HIU NNG CAO
Ti u ho mt php sc k l nghin cu cc iu kin ti u php tch sc k c thc hin
hon ton.
1.12.1 Chun b mu :
36

Mu a vo ct tch ca HPLC phi trng thi lng do nu mu l cht kh hay l cht rn th phi ho
tan bng mt dung mi thch hp chuyn cht phn tch v trng thi lng (x l mu). Dung mi ho tan cht phn
tch thng l dung mi c chn lm pha ng. Tu thuc vo bn cht ca cht phn tch v mu cha cht phn
tch m chn phng php x l mu ph hp.
-

Cc phng php x l mu nh: K thut v c ho t, k thut v c ho kh, k thut kh t kt

hp, cc k thut chit (chit thng thng, chit pha rn, chit pha kh, chit siu m), cc k thut sc k, cc k
thut chng ct, k thut in phn
- V d: Chun b mu xc nh hm lng vitamin A trong mu rau qu.
Ly 10 gam mu nghin mn cho vo bnh nn c nt nhm, thm 20 gam Na 2SO4khan, 1 gam MgCO3 khan, 40
mL dung mi n hexan, lc chit 5 pht, lng, gn ly dung dch. Thm 40 mL dung mi n hexan vo b v lc
chit tip. Lc ly dung dch v gp c hai ln dung dch n hexan vo bnh ct quay chn khng. Ct quay t t cho
o

dung mi n hexan bay hi (gi nc lm lnh 4 C ) n cn 2 mL, ngui, ho tan b snh cn li trong 5 mL
MeOH hay n hexan, ly tm ly dung dch mu xc nh cc cht vitamin A bng phng php HPLC. Th tich`
tim c th l: 20 L,50 L,100 L
1.12.2. Chn cc iu kin ti u
* Chn ct sc k:
Chn pha tnh ph hp vi phng php sc k:
+ Sc k hp ph pha thng
+ Sc k hp ph pha ngc
+ Sc k phn b lng - lng
+ Sc k trao i ion rn - lng
+ Sc k hp ph rn - lng
+ Kch thc ht ti u, xp.
+ Chiu di ct c nh.
* Chn pha ng:
Pha ng c chn ph thuc vo pha tnh. C th l dung mi n hay l hn hp 2,3 dung mi trn ln theo t l th
tch nht nh cho s tch c hiu qu.
+ Chn pha ng phi ch cc iu kin:
+ Tr, khng tc dng vi pha tnh (trnh lm hng cht nhi).
+ Bn v n nh trong qu trnh chy sc k.
+ Ho tan tt cht phn tch mang cht phn tch ra khi ct.
+ Ph hp vi loi detect la chn.
+ Tc ca pha ng.
+ Cht m pH, cht to phc
* Chn detector v cc thng s ca n php phn tch c nhy cao nht:
Tu thuc vo bn cht ca cht phn tch la chn detector ph hp: Cht c kh nng pht hunh quang hoc to vi
thuc th to thnh hp cht c kh nng pht hunh quang th chn detector hunh quang, nhng cht c hot tnh in ho
th ph hp vi detector dn, nu cht phn tch c kh nng hp th trong vng t ngoi - kh kin th dng detector hp th
t ngoi - kh kin UV Vis. Nu khng dng c cc loi detector trn th dng loi detector ch s khc x RI.

37

* Chn khong nng :

Tng lin tc nng ca cht bin tnh trong pha ng cc h pha thng v pha ngc hay thay i pH hoc nng
i ion trong cc h trao i ion: tch cc hn hp phc tp cc cht tan c s khc nhau ln v phn cc trong mt ln
chy sc k v trong mt thi gian ngn.
* Tin hnh sc k vi nhiu ct tch:
Nhm lm gim thi gian tch hn hp cha nhiu nhm cht tan khc nhau rt ln v phn cc v cn ti u chn
lc bng cch s dng cc ct tch vi cc pha ng khc nhau.
* Chn nhit thch hp.
1.12.3. Bm mu vo ct tch
-Bm lng mu vo sao cho nhy ca php phn tch (chn lng mu khong bao nhiu L bm vo ct sc
k ).
- Chng trnh gradien pha ng.
1.12.4. Chn phng php phn tch
Da vo c im ca cht phn tch trong hn hp cn tch (phn t lng, kh nng ho tan trong nc, tnh cht phn
cc ) m la chn phng php phn tch ph hp.
V d:
- Nu nhng cht c phn t lng ln (M >2000):
+ C kh nng tan trong nc: s dng phng php sc k gel, dung mi nc.
+ Khng tan trong nc: s dng phng php sc k gel, dung mi hu c.
- Nhng cht c phn t lng nh (M < 2000):
+ Khng tan trong nc:
Cc cht thuc ng ng: S dng phng php sc k phn b.
Cc cht l ng phn: S dng phng php sc k hp ph.
Cc cht c kch thc khc nhau: S dng phng php sc k gel.
+ Tan trong nc:
Khng phi l hp cht ion: S dng phng php sc k phn b pha o hoc sc k gel, dung mi nc.
Hp cht ion: Nu l baz th sc k trao i cation, cn nu l axit th sc k trao i anion.
C hp cht ion v hp cht khng ion: Sc k pha o to cp ion.
1.13. MY SC K LNG HIU NNG CAO V S GHP NI CA HPLC VI KHI PH
1.13.1. S my HPLC

38

Hnh 1: Gii thiu h thng HPLC

Hnh 2: S chc nng thit b HPLC

Trong * Dung mi ra gii (hay pha ng bi v n di chuyn)
Trong sc k pha thng, pha ng l cc dung mi khng phn cc nh benzen, n-hexan, toluen
Trong sc k pha o, pha ng l cc dung mi phn cc c th l nc, metanol, axetonitril
Tuy nhin, ngi ta thng dng hn hp hai hay ba dung mi c mt pha ng c phn cc ph hp vi
php phn tch.
* Bm cao p (h thng qun l dung mi)
Bm cao p l dng c bm pha ng qua ct tch vi 1 tc dng xc nh, ph hp vi qu trnh ra gii
cc cht ra khi ct tch sc k, thng vi tc mL/pht.

39

C nhiu loi bm cao p nhng hai loi hay c s dng l bm mng tc l nn dung dch qua mng v bm
pittong l nn trc tip bng pittong .
Bm nn qua mng c u im l ngn pittong v ngn dung mi ring nn du khng nh hng n dung
mi. to c dng dung dch mt chiu, ngi ta dng cc vin bi.

Hnh 3: Bm cao p s dng mng (bm mng)

Bm pittong ch c mt ngn, c ngha l dung mi tip xc vi pittong v xilanh, nh vy vt liu lm bm

phi chu mi mn v ha cht khng lm bn dung mi. to c dng dung dch mt chiu, ngi ta dng cc
vin bi.

Hnh 4: Bm cao p kiu pittong

* Van tim mu (van bm mu)
Van bm mu l thit b rt quan trng trong h sc k. N c chc nng np tng lng chnh xc dung dch
cht phn tch vo u ct, ng thi chu c p sut cao v tr vi ha cht.
Van bm mu thng dng c 6 ca, c vng mu dung tch khc nhau t 5 L 50 L.

40

(a) Van bm mu v tr np mu

b) Van bm mu v tr phun mu
Hnh 5. Van bm mu
* Ct tch
Ct tch c th c lm bng thp hay thy tinh thch anh c v bc bng thp chu c p sut cao trong
vng t 200 500 atm.
Kch thc ca ct ph thuc vo c ht cht nhi c trong ct, c ht cht nhi ln th kch thc ct ln, c ht
cht nhi nh th kch thc ht nh, sao cho m bo s a l thuyt thc hin php tch t hiu sut cao.
Cht nhi c kch thc 3 10 m th ct c chiu di 3 6 cm.
Cht nhi trong ct tch c gi l pha tnh.
* Detector
Detector l b phn thu nhn tn hiu cht phn tch theo 1 tnh cht ha hc, vt l, ha l no .
Mt s detector c s dng nh: detector UV-VIS, detector hunh quang, detector bt in t, detector o
dn in (Xem r hn mc detect).
* B phn ghi nhn kt qu
B phn ghi nhn kt qu l mt my tnh chuyn dng, t v th, x l s liu, tnh ton kt qu
* Bnh thi
Pha ng sau khi thot ra khi detector, s c chuyn ti bnh thi.
1.13.2.Ghp ni HPLC vi khi ph(LC-MS)
S ghp ni HPLC vi khi ph cho php ngi ta nh dng v xc nh mt s hp cht m vi phng php
sc k khi ph khng thc hin c, v d nh xc nh hp cht c khi lng phn t tng i cao, cc hp
cht khng bn nhit, cc hp cht phn cc cao, mt s dc phm,
1.14. PHN TCH NH TNH BNG PHNG PHP HPLC
Mi cht phn tch trong nhng iu kin tng ng c la chn c mt thi gian lu xc nh. V vy thi
gian lu l tiu chun phn tch nh tnh cht phn tch bng phng php sc k lng cao p.

Nguyn tc:
Chy sc k mu chun theo nhng iu kin ti u la chn xc nh thi gian lu ca tng cht trong
mu . Sau chy mu phn tch cng trong nhng iu kin nh chy mu chun v so snh thi gian lu kt
lun s c mt ca tng cht.

41

Hnh 6: S phn tch nh tnh

Qua s trn ta thy mu phn tch c B v C.
1.15. PHN TCH NH LNG BNG PHNG PHP HPLC
Tn hiu o l din tch ca pic hay chiu cao ca pic. tin hnh nh lng cht phn tch, ngi ta s dng cc cht
chun l cht phn tch c tinh khit cao. Tin hnh kho st cht chun tm iu kin o ca cht chun, mu gi v cui
cng mi o trn mu thc t.
Mi cht phn tch trong nhng iu kin ti u la chn th din tch (chiu cao) ca pic t l vi nng ca cht phn
tch:

S = K.C (S: din tch ca pic)

h = K.C (h: chiu cao ca pic)
Cc phng trnh ny dng nh lng cht phn tch bng HPLC, thng ngi ta dng din tch ca pic chnh

xc cao hn.
C nhiu phng php nh lng cht phn tch, c th:
1.15.1. Phng php ngoi chun
Tu thuc vo tnh cht ca mu: hm lng, yu t cn tr, tnh cht phc tp ca nn cng nh chnh xc yu cu m
ngi phn tch c th p dng cc cch nh lng khc nhau: Chun ho mt im v chun ho nhiu im.
Chun ho mt im

42

- Nguyn tc:
Chun ho mt im l s dng mt php o ca cht chun bit trc nng , o c lp v song song vi php o
cht cn phn tch.
- Phng php ny thng p dng:
+ i vi cht phn tch c hm lng ln hoc lm giu n mc cn thit.
+ nh gi khi khng yu cu chnh xc cao v y b qua cc yu t cn, cc sai s ngu nhin c th
lm gim tnh chnh xc ca php o.
+ Nng ca cht phn tch Cx phi nm trong khong tuyn tnh.
- Phng php:
S dng hai php o c lp:
+ Php th nht: Chy sc k cht chun trong iu kin tng t cht phn tch (nn v cc thng s khc ging
nhau).
+ Php th hai: Chy sc k cht phn tch.
- Kt qu tnh nh sau:

Hnh 7: Chun ho mt im

C
C
C x Cc
=
C x = c h x hay: C x = c Sx
h
hc
hc
Sc
Ta c: x
Chun ho nhiu im (phng php ng chun)
- Chun b mt dy dung dch chun c nng khc nhau trong iu kin tng t mu phn tch. Tin hnh php tch
sc k v tnh din tch ca pic i vi dy cht cht chun. Sau dng ng chun hay ng hi quy tuyn tnh biu din s
ph thuc chiu cao (din tch) ca pic vo nng cht phn tch. xc nh nng cht phn tch Cx ta cng thc hin php
o i vi cht phn tch, c Sx (hx) nht nh, i chiu vi ng chun ta c nng cht phn tch trong mu.

43

Hnh 8: ng chun nhiu im xc nh cht phn tch

Phng php ny c u im l sau khi dng ng chun ri th c th s dng phn tch hng lot trong cng iu
kin c kho st. Nu hm lng cht phn tch qu b nm di gii hn ca ng chun th phi s dng phng php lm
giu tng nng ca cht phn tch n nm trong vng tuyn tnh. Trong nhiu trng hp mu phn tch c thnh phn
phc tp, chng ta cha bit chnh xc nn khng th chun b dy mu u tho mn nhng quy nh cho phng php ny do
khng xc nh chnh xc v tr ca ng chun, v vy kt qu phn tch mc sai s ln. Trng hp ny khng nn s dng
phng php ng chun m tt nht l s dng phng php thm chun xc nh nng ca cht phn tch trong mu.
1.15.2. Phng php thm chun
- Nguyn tc ca phng php ny l dng ngay mu phn tch lm nn chun b dy mu u.
- Chun b mu: Ly mt lng mu phn tch nht nh (Cx) ri thm vo nhng lng chnh xc cht cn xc nh
vo C1, C2, C3, C4, C5 ta c dy mu u c hm lng cht xc nh l:

Co = C x
CI = C x + C1
CII = C x + C2
C III = C x + C3
C IV = C x + C4
C V = C x + C5
- Tin hnh php tch sc k v tnh din tch (chiu cao) ca pic i vi dy mu u. Sau dng ng chun hay
ng hi quy tuyn tnh biu din s ph thuc chiu cao (din tch) ca pic vo nng cht phn tch.
h,S

Ao
Cx

0 C1 CxC2 C3 C4

44

C5

Hnh 9: ng chun ca phng php thm chun

Xc nh Cx bng phng php ngoi suy: Ko di ng chun v pha tri, ng ny ct trc honh ti im Cx, on
OCx chnh bng gi tr nng Cx cn tm. Cng c th xc nh Cx bng cch t gc to O k mt ng thng song song vi
ng chun, t im Ao k mt ng thng song song vi trc honh, hai ng ny ct nhau ti im M, t M h ng
vung gc xung trc honh. im ct ny l Cx. on OCx chnh bng gi tr nng Cx cn tm.
Phng php ny c u im l qu trnh chun b mu d dng, khng cn c nhiu ho cht c tinh khit cao
chun b dy mu u. Mt khc li loi tr hon ton nh hng v thnh phn ca mu cng nh cu trc vt l ca mu, nhng
sai s ngu nhin do thao tc Phng php ny xc nh c hm lng cht phn tch nng b m phng php ng
chun khng phn tch c v cho chnh xc cao, c nhiu ngi s dng. Nhng phi ch rng, nng thm ca cht
chun phi theo tng bc v khong cch ca cc bc phi xp x bng Cx th phn ni suy tuyn tnh mi chnh xc.
1.15.3. Phng php ni chun
Trong mu, cc cht phn tch c th b nh hng ca nhiu yu t: nh hng nn, cc iu kin khc gy sai s h
thng. loi tr nh hng ny ngi ta thm vo mu cht ni chun c tnh cht gn ging tnh cht ca cht phn tch, c
nng cng gn bng nng ca cht phn tch (Cx).
Tin hnh 3 php o sc k: mt php cho cht chun ni, mt php cho cht chun c nng xc nh (t cc cht
chun), t rt ra c h s nh hng Fx, php sc k th 3 cho cht phn tch cha bit v t tnh ra nng ca n. H
s Fx chnh l i lng a vo loi tr nh hng ca nn cng nh cc iu kin o khc.

Hnh 10: Phng php chun ni xc nh cht phn tch

C c Ci
C .h
: = F x F x= c i
h c hi
h c .Ci .

C x Cc
C
=
C x = i .h x .Fx
h x hc
hi

1.15.4. Phng php vi sai

- Phng php vi sai nng ln:
Chun b mu:
Dung dch chun 1: C1
Dung dch chun 2: C2 (C2 > C1)
Dung dch phn tch: Cx (Cx > C1)

45

Tin hnh php tch sc k dung dch chun 2 v dung dch phn tch trn nn dung dch chun 1 v tnh din tch (chiu
cao) ca pic:

S21 = S2 S1 , Sx1 = Sx S1 .
Ta c:

C x C1 Sx1
S
=
C x = x1 ( C2 C1 ) + C1
C 2 C1 S21
S21
.
- Phng php vi sai nng b:
Chun b mu:
Dung dch chun 1: C1
Dung dch chun 2: C2 (C2 < C1)
Dung dch phn tch: Cx (Cx < C1)
Tin hnh php tch sc k dung dch chun 1 trn nn dung dch chun 2 v v dung dch phn tch. Tnh din tch (chiu
cao) ca pic:

S12 = S1 S2 , S1x = S1 Sx .
Ta c:

C1 C x S1x
S
=
C x = 12 ( C1 C 2 ) + C1
C1 C 2 S12
S1x
.
Phng php ny s dng xc nh cc cht c nng qu ln hoc qu b.

Phn 2
BI TP V SC K LNG HIU NNG CAO (HPLC)
Bi 1: Hm lng fluoranthene (PAHs - mt loi hirocacbon a vng thm) trong du c th c xc nh bng
phng php HPLC vi detector UV/Vis sau khi c chit bng metylenclorua.
Ly 2,03g du th x l bng 20,00ml metylenclorua, lc b cht rn khng tan; ly 1ml dung dch sau khi lc pha
long thnh 10ml vi axetonitrin. Ly5 l dung dch thu c phn tch bng HPLC th PAHs cho tn hiu pic 0,2170

46

(din tch pic hay chiu cao pic). 5 l dung dch chun cha fluoranthene 20.0 ppm c phn tch trong cng iu
kin, du hiu pic tng ng l 0,2580. Xc nh hm lng PAHs trong du theo ppm.

Fluoranthene(PAHs)
Bi gii:
- Ta bit, din tch pic S (hay chiu cao H tng ng) t l thun tuyn tnh vi nng cht phn tch theo phng
trnh: S = kC
+ Vi dung dch chun ta c: S1 = kC1
+ Vi mu phn tch : Sx = kCx (phn tch cng iu kin nn xem nh cng gi tr
- Suy ra:

S1 C1
S
0, 217
= 16,8 ppm
=
C x = C1 2 = 20.
S1
0, 258
S2 Cx

- Khi lng PAHs trong mu: 16,8 ( g/ml).10.20 = 3360 g

- Nng PAHs trong mu:

3360
= 1670 ppm
2, 013

Bi 2: Thnh phn ca a vitamin c xc nh hiu qu bng phng php HPLC vi detector UV Vis. 5l mu
chun c cha 170 ppm vitaminC, 130 ppm niacin, 120ppm niacinamide, 150ppm pyridoxine, 60ppmm thiamine, 15
ppm axit folic v 10ppm riboflavin phn tch bng HPLC, cc tn hiu ln lt l 0,22, 1,35, 0,90, 1,37, 0,82, 0,36 v
0,29. Chun b mu vitamin, nghin thnh bt v chuyn vo bnh dung tch 125ml c cha 10ml dung dch NH 3 1%
(v/v) trong dimethy sulfoxide. Sau khi khoy trong 2 pht, thm vo 90ml CH 3COOH 2%, khoy hn hp trong mt
1 pht, lm nng 400C trong 5 pht, lc bng phu. Ly 5l mu o bng HPLC thu c cc tn hiu ph 0,87 ng
vi vitaminC, 0,00 ng vi niacin, 1,40 ng vi niaciamide, 0,22 ng vi pyridoxine, 0,19 ng vi thiamine, 0,11 ng
vi axit folic v 0,44 ng vi riboflavin. Tnh s miligam ng vi mi loi vitamin c trong mu phn tch.
Bi gii:
- p dng cng thc:

S = kC cho tng vitamin ta c:

S0 = kC0
S
0,87
C x = C0 x = 170.
= 672, 27 ppm
S
=
kC
S
0,
22
x
x
0

+ VitaminC :

Vy khi lng vitaminC trong mu : 672,27( g/ml).100(ml) = 67227 g hay 67,227mg.

+ Niacin:

S0 = kC0
S
0
Cx = C0 x = 130
=0

S0
1,35
S x = kCx

Vy khi lng Niacin trong mu bng 0

+ Niacinamide :

S0 = kC0
S
1, 4
Cx = C0 x = 120.
= 186, 667 ppm

S0
0,9
S x = kCx

Vy khi lng niacinamide trong mu : 186,667.100 = 18666,7 g hay 18,6667mg

+ Tng t ta c khi lng cc vitamin cn li trong mu:
mpyridoxide = 2,4mg

47

mthiamine = 1,390mg
maxit folic = 0,0463mg
mriboflavin = 1,517mg
Bi 3: Hm lng cafein trong thuc ng c xc nh bng phng php HPLC, s dng phng php ng
chun. Chun b dung dch chun v phn tch 10l, kt qu thu c:
Nng (ppm)
max (nm)
50,00
8354
100,00
16925
150,00
25218
200,00
33584
250,00
42002
Chun b mu bng cch cho mt vin thuc ng vo mt bese, thm vo 10ml methanol. Sau
L;. khi thuc tan, chuyn vo bnh c dung tch 25ml; tip tc pha long n th tch bng metanol; lc ly
dung dch. Ly 1ml dung dch trn pha long thnh 10ml bng metanol, tin hnh phn tch bng phng php sc k
lng hiu nng cao HPLC, du hiu ca cht cafein c pht hin bc sng 21469nm. Xc nh s miligam
cafein trong vin thuc.
Bi gii:
- X l s liu trn ta c phng trnh ng thng:

max = max (C ) = 167,91Cx + 30,1

(*)

- Hm lng cafein trong thuc:

Thay max = 21469 vo (*) ta c: Cx = 127,68 ppm.
- Khi lng cafein:

127,68( g/ml).10ml.25ml = 31920 g hay 31,920 mg

- Kt lun: mcafein = 31,920 mg

Bi 4: Kagel v Farwell nh lng nng ca axit acetylsalicylic(ASA) v caffein (CAF) trong thuc ng bng
phng php sc k lng pha ngc, dng axit salisylic (SA) lm cht nn. Dy mu chun c chun b bng cch
cho ASA v CAF vo bnh dung tch 250ml, thm tip vo 100ml metanol, thm tip 10ml axit salisylic vo mi bnh.
Kt qu thu c tng ng t cc mu chun nh sau:
Mu

ASA
CAF
T s chiu cao pic
T s chiu cao pic
(miligam)
(miligam)
ASA/SA
CAF/SA
01
200,00
20,00
20,5
10,6
02
250,00
40,00
25,1
23,0
03
300,0
60,00
30,9
36,8
Ha tan vin thuc ng bng 100ml metanol, thm tip vo dung dch 10ml dung dch nn, lc ly dung
dch. Phn tch mu thu c t s chiu cao ca pic nh sau: ASA/SA = 23,2 v CAF/SA = 17,9.
Xc nh s miligam ASA v CAF trong vin thuc.
Bi gii:
- Dng phng trnh H(S) = kC

H1 20,5
=
= 0,1025
C1 200
H 2 25,1
=
= 0,1004
H2 = k2C2 k2
C2 250
H1 = k1C1 k1 =

48

H 3 30,9
=
= 0,103
C3 300
k +k +k
0,1025 + 0,1004 + 0,103
k= 1 2 3 =
= 0,1019
3
3
H= k C

H3 = k3C3 k3 =

- Hm lng ASA : thay H = 23,2 vo phng trnh trn ta c:

C ASA =

H
23, 2
=
= 227, 6742 miligam
k 0,1019

- Tng t, hm lng CAF trong thuc ng l :

CCAF =

31,312 miligam

Bi 5: Bohman dng sc k lng cao p HPLC pha ngc phn tch nh lng vitamin A trong thc phm bng
phng php thm chun. Ly 10,067g mu ng cc cho vo bnh dung tch 250ml, thm vo 1g natri ascorbate, 40
ml etanol v 10ml KOH 50%. Sau khi khuy 30 pht, tip tc thm vo 60ml etanol. Vitamin A sau c chit ba
ln bng100ml hexan, sau lm bay hi, phn cn li cha vitamin A c chuyn vo bnh 5ml v pha long bng
metanol. Dung dch thm chun pha t 10,093g ng cc x l cng iu kin trn v thm vo 0,02mg vitamin A.
o mu v dung dch chun bng HPLC thu c din tch pic tng ng l 6,77.10 3 v 1,32.104. Xc nh s mg
vitamin A trong 100g ng cc.
Bi gii:
- Ta c : S = k.C
Cx l hm lng vitamin A trong 10,067g mu, suy ra vi 10,093g mu hm lng vitamin A s l:

Cx

10, 093
= 1, 00258C x = C
10, 067

- Vi dung dch phn tch: Sx = kCx

- Vi dung dch chun: S = k(C + 0,02)

S
Cx
0, 02 S x
Sx
Cx
x =
Cx =
=
S ' 1, 00258C x + 0, 02
S ' 1, 00258S x
S ' C '+ 0, 02

Cx =

0, 02.6, 77.103
= 0, 0211 mg
(1,32.104 1, 00258.6, 77.103 )

- Hm lng vitamin A trong 100g thc phm l: 0, 0211.

100
= 0, 209mg
10, 067

- Kt lun: mvitamin A = 0,209 mg.

Bi 6: Hai cht A, B c thi gian lu tng ng l 16,40 v 17,63 pht. Cht khng lu gi ct mt 1,30 pht. B
rng chn pic ca cht A l 1,11; cht B l 1,21 pht. Tnh:
1. phn gii ct
2.S a l thuyt trung bnh ca ct
3. Chiu cao a, bit ct di 30,0cm
4. Chiu di ca ct t phn gii 1,5
5. Thi gian cn ra gii cht B ct di hn.
Bi gii:
1. phn gii ct:

49

Ta c : R =

2(t RB t RA ) 2(17, 63 16, 40)

=
= 1, 060
wA + wB
1,11 + 1, 21

2. S a l thuyt trung bnh

2

16, 4
t
N A = 16 RA = 16
= 3493 a l thuyt
w
1,11
2

17, 63
N B = 16
= 3397 a l thuyt
1, 21
N=

3493 + 3397
= 3445 a l thuyt
2

3. Chiu cao ca a:

HA =

L
30
=
= 0, 00858cm
N A 3493

HB =

L
30
=
= 0, 00883cm
N B 3397

H = 8, 7.103 cm
4. Chiu di ct:
Tac:

N =

2 R (t RA + tRB ) 2.1,5(17, 63 + 16, 40)

=
= 83 N = 6889
t RA t RB
17, 63 16, 40

L = H .N = 8, 7.103.6889 = 60cm .
5. Thi gian ra gii B lu hn: 1,3 + 16,40 +17,63 = 35,33 pht.
Bi 7: Trong qu trnh tin hnh sc k mt hn hp cht phn tch gm 3 cht X, Y, Z trong ct sc k c chiu di
28,3 cm. Ngi ta thu c trn sc 3 pic tng ng vi thi gian lu: 0,84 pht, 10,60 pht, v 11,08 pht tng
ng vi 3 cu t X, Y, Z. Cc pic ny u c dng ng Gauss, rng pic ca Y v Z ln lt l W Y = 0,56 pht v
WZ = 0,59 pht, cn cht X khng c lu gi bi vt liu ct.
1. Tnh s a l thuyt trung bnh v chiu cao a theo pic Y v Z.
2. Tnh s a l thuyt hiu lc trung bnh ca ct sc k.
3. Tnh h s lu gi tng i gia Y v Z.
4. Th tch ca cht lng c gi trn b mt cht mang ca ct l 12,3 ml. Th tch pha ng bng 17,6 ml. Tnh h
s phn b ca Y v Z.
Bi gii:
1. Tnh s a l thuyt trung bnh ( n ) v chiu cao mi a (H) theo pic Y v Z.
t
* S a l thuyt: n = 16 R
W

(1)
2

10,6
= 5733
0,56

- i vi cu t Y: nY = 16

11,08
= 5643
0,59

- i vi cu t Z: nZ = 16

50

 S a l thuyt trung bnh : n =

* Chiu cao a trung bnh: H =

nY + n Z 5733
=
= 5688
2
5643

L
28,3
=
= 4,975.10 3 (cm)
n 5688
2

t t
n f = 16. R m
W

2. S a l thuyt hiu lc ca ct sc k:
2

- Cu t Y: n fy

10,60 0,84
= 16
= 4860
0,56

11,08 0,84
= 4820
0,59

- Cu t Z: n fz = 16

S a l thuyt hiu lc trung bnh: n f =

4860 + 4820
= 4840
2

3. Tnh h s lu gi tng i gia Y v Z:

t RY t RY t m 10,06 0,84
=
=
= 0,900
t RZ t RZ t m 11,08 0,84

4. Ta c :
2

t t
n f = 16. R m
W

(2)

V
t R = t m (1 + K D . s )
Vm

tm =

tR
1+ KD.

Vs
Vm

(3)

Thay (3) vo (2) ta c:

tR

tR
V

1+ KD. s

Vm
n f = 16.
W

KD. s
2

V
tR
m

= 16.
W

1 + K . Vs

Vm

Thay (1) vo (4) ta c:

KD. s
Vm
n f = n.

Vs
1+ KD.
Vm

- Xt cu t Y:

- Xt cu t Z:

n f Vm
1
KD = n V
nf
s

K DY =

K DZ =

4860 17,6

5733 12,3

4820 17,6

5643 12,3

1
4860
5733

1
1

4820
5643

= 16,62

= 17,45

51

(4)

Bi 8: (Trang 67, schBi tp v s tay phn tch nh lng, Nguyn Th Thu Vn)
Mt h thng sc k lng hot ng vi cc thng s sau:
- Chiu di ca phn ct c nhi pha tnh (L): 25,2 cm.
- Tc dng ca pha ng (F): 0,312 ml/pht.
- Th tch pha ng (Vm): 1,40 ml.
- Th tch pha tnh (Vs): 0,168 ml.
Sau khi cho qua ct mt hn hp cha 4 cu t A, B, C, D thu c mt sc k cung cp cc d kin sau:
Cu t
Khng lu gi bi ct
A
B
C
D
Hy tnh:

Thi gian lu gi tR (pht)

1,2
4,7
8,8
12,5
13,8

Chiu rng chn pic W (pht)

0,48
0,92
1,34
1,44

1. S a l thuyt i vi mi pic, s a l thuyt trung bnh ca ct, lch chun ca s a l thuyt v chiu cao
a l thuyt ca ct.
2. H s dung tch v h s phn b ca tng cu t.
3. phn gii R, h s tch i vi 2 cu t C v D.
4. Chiu di ct nu mun t phn gii 1,5 i vi 2 cu t C,D.
Bi gii:
1.
- Tnh s a l thuyt (n) i vi mi pic:
t
p dng phng trnh: n = 16 R
W

( vi tR l thi gian lu, W l chiu rng ca pic)

4,7
=1534
i vi pic A: nA = 16
0,48
2

8,8
= 1464
0,92

i vi pic B: nB = 16

12,5
=1392
i vi pic C: nC = 16
1,34
2

13,8
=1469
1,44

i vi pic D: nD = 16

- S a l thuyt trung bnh ca ct: ntb = 1465

- Chiu cao a l thuyt ca ct: H =

L
25,2
=
0,017(cm)
ntb 1465

- lch chun ca s a l thuyt ca ct:

T n =

2
= n.H 2 = 1465.0,017 2 = 0,651(cm)
2
H

2.
- H s dung tch ca tng cu t (kX): k X =

tR tm
tm

52

i vi cu t A: k A =

4,7 1,2
= 2,92
1,2

i vi cu t B: k B =

8,8 1,2
= 6,33
1,2

i vi cu t C: k C =

12,5 1,2
= 9,42
1,2

i vi cu t D: k D =

13,8 1,2
=10,5
1,2

- H s phn b ca tng cu t (KD):

T cng thc:

k X =

K D VS
Vm

KD =

i vi cu t A: K DA =

2,92 1,40
= 24,33
0,168

i vi cu t B: K DB =

6,33 1,40
= 52,75
0,168

i vi cu t C: K DC =

9,42 1,40
= 78,5
0,168

i vi cu t D: K DD =

10,5 1,40
= 87,5
0,168

k X Vm
Vs

3.
- phn gii R i vi 2 cu t C v D: RC D =
- H s tch i vi 2 cu t C v D: =

2.(t RD t RC ) 2.(13,8 12,5)

=
= 0,94
WC + WD
1,34 + 1,44

K D 87,5
=
= 1,115
KC
78,5

4. . Chiu di ct phn gii gia cc cu t t yu cu ti thiu l 1,5

- Gi L1, L2 ln lt l chiu di ca ct c v ct mi.

R
Ta c: 2 =
R1

L2
. T suy ra:
L1

L2 =

L1 .R22 25,2 1,5 2

=
= 64,17(cm)
R12
0,94 2

Bi 9: Trn ct sc k cho, hai cht c thi gian lu hiu chnh ln lt l 15 v 20 pht. Trn mt ct khc vi th
tch pha tnh ln hn, cu t th nht c thi gian lu hiu chnh l 25 pht. Hi thi gian lu hiu chnh ca cu t
th 2 trn ct ny bng bao nhiu?
Bi gii:
- Gi 2 cht cho l A v B:

= t RA tm = tm K DA
- Ta c : t RA

Vs
Vm

= t RB t m = t m K DB
t RB

Vs
Vm

- Cht A:
+ Vi ct sc k th nht:

)1 = t m K DA
(t RA

(Vs )1
Vm

+ Vi ct sc k th hai:

) 2 = t m K DA
(tRA

(Vs ) 2
Vm

53

- Tng t vi cht B.
- Lp t l ta c:
(t RB ) 2 =

(t RA )1 (VS )1 (t RB )1 15
=
=
=
(t RA ) 2 (VS ) 2 (t RB ) 2 25

25 (t RB )1 25 20
=
= 33,33( pht )
15
15

Vy thi gian lu hiu chnh ca cu t th 2 trn ct sc k th 2 l: 33,33 pht.

Bi 10: Hiu qu ca ct sc k cho l 4200 a l thuyt, vi thi gian lu gi octadecan v 2-metylheptadecan
ln lt l 15,05 v 14,82 pht. phn gii ca cc hp cht ny trn ct l bao nhiu? Cn bao nhiu a l thuyt
vi nhng thi gian lu gi trn th phn gii bng 1,5?
Bi gii
- K hiu A: octadecan
B: 2-metylheptadecan
- Ta c:
n = 4200
tRA = 14,82 pht = 889,2s
tRB = 15,05 pht = 903s
tR
W

- S a l thuyt n = 16

w = 16

(vi W l rng ca pic)

t R2
n

wA =

16

889,2 2
= 54,883 ( s )
4200

wB =

16

903 2
= 55,734 ( s )
4200

- phn gii R

R =

2( t RB t RA )
wA + wB

R =

2( 903 889,2 )
= 0,249
54,883 + 55,734

Vy R = 0,249
- Khi R = 1,5 th:
1,5(wA + wB) = 2(tRB - tRA)

54

2
2
16 t RA
16 t RB
4
+
= ( t RB t RA )
n
n
3
4t RA
4t RB
4
( t RB t RA )
+
=
3
n
n
2

3(t + t RB )

n = RA
( t RB t RA )

3( 889,2 + 903)

n =
903 889,2

= 151795

Vy cn 151795 a l thuyt c phn gii R =1,5

Bi 11: Ct sc k lng c chiu di 2m c hiu qu 2450 a l thuyt tc ca dng 15ml/pht v hiu qu 2200
a l thuyt tc dng 40ml/pht. Vy nng ti u ca dng phi bng bao nhiu v hiu qu tc ca
dng s gn bng bao nhiu ?
Bi gii :
Ta c :

H=

B
+ C u
u

Vi u1 = 15 ml/pht

H1 =

L
200
=
= 0,0816
N1 2450

cm

Vi u2 = 40 ml/pht

H2 =

L
200
=
= 0,0909
N 2 2200

cm

Ta c h :

Ut =
Hmin =

B
+ C 15
15
B
0,0909 =
+ C 40
40
B
0,829
=
= 21,74 (ml/pht)
C
1,754 10 3
0,0816 =

B = 0,829

C = 1,754.10-3

2 B C = 2 0,829 1,754 10 3 = 0,076

(cm)

Bi 12: Ngi ta th nghim ct sc k kh - lng c chiu di 2m bao tc khc nhau ca dng, mt khc,
lm pha di ng ngi ta dng hli. Kt qu th nghim tm thy rng ct c cc c trng sau :
Mtan(pha di ng)

n-octaecan

tR

tR

18.2 giy

2020 giy

223 giy

8.0 giy

888 giy

99 giy

55

5.0 giy

558 giy

68 giy

a> Hy xc nh tc di chuyn ca pha ng i vi mi dng.

b> Hy xc nh s a l thuyt v gi tr H i vi mi dng.
c> Bng cch gii ng thi cc phng trnh cn thit hy tm cc gi tr ca cc hng s trong phng trnh sau
:

B
H = A + + C u
u
d> Tc ti u ca s di chuyn ca pha di ng bng bao nhiu ?
Bi gii :

a> Tc di chuyn ca pha di ng

u1 =

200
= 11 cm/s
18,2

; u2 =

200
200
= 25 cm/s ; u3 =
= 40 cm/s
8
5

b> S a l thuyt v gi tr H :
2

tR
2020
N1 = 16 1 = 16
= 1310
W
223

1
2

2 100
= 0,1526 (cm)
1310

tR
888
N2 = 16 2 = 16
= 1287
W
99

2
2

H1 =

H2 =

tR
558
N3 = 16 3 = 16
= 1076
W
68

H3 =

2 100
= 0,1554 (cm)
1287

2 100
= 0,1859
1067

(cm)

c> Xc nh A, B, C :
Ta c h :

B
+ C 11
11
B
0,1554 = A +
+ C 25
25
B
0,1859 = A +
+ C 40
40
0,1526 = A +

A = 0,059
B = 0,695
C = 2,729 103

d> Tc ti u :
Ut =

B
0,695
=
= 15,96
C
2,729 103

cm/s

Bi 13: Tin hnh sc k hn hp 2 cht A v B trn ct sc k c chiu di L=4m c s a l thuyt n = 800 a.

56

Tc tuyn tnh ca 2 cu t A v B trong pha ng ln lt l 2 cm/s ; 1,6 cm/s, t m = 10s.

a> Tnh tRA v tRB
b> C th tch A v B ra khi nhau c khng ?
c> Tnh phn gii ca php sc k.
Bi gii:
a> Ta c :

tR =

L
U

Vi : L - chiu cao ct sc k

U - Tc tuyn tnh ca cu t
- Cu t A :

- Cu t B:

t RA =
t RB =

L
400
=
= 200s
UA
2

L 400
=
= 250s
U B 1, 6

b> Tnh h s tch :

t R' A
t R' B

t RA t m
t RB tm

200 10
= 0,8 1
250 10

Vy c th tch 2 cht A v B ra khi nhau.

Bi 14: Cc thi gian gi ca - cholestan v - cholestan trong h cht lng pha rn trn ct sc k c chiu di
1m v vi hiu qu 104 a l thuyt tng ng bng 4025 v 4100 giy. Nu 2 hp cht ny cn c phn chia vi
phn gii bng mt th cn bao nhiu a l thuyt t c mc ch ny ? Chiu di no ca dng ny cn
nhn c phn gii ch ra, nu H= 0,1mm?
Bi gii :
Ta c :

tR t RB
R = 2 A
=1
WA + WB

WA + WB = 150 s (1)

Vi t R=A 4025 s
R

t =B4100 s
Mt khc :

t RA
WA

t RB
WB

t RB WA t RA WB = 0

T (1) v (2) ta c : WA = 74,31 giy

(2)

N=46942 a

WB = 75,69 giy
Vi H = 0,1 mm L = 4,6942 m
Bi 15: Pic sc k ca hp cht c pht hin sau 15 pht khi a mu vo (lc pic ca hp cht Y khng c

57

gi bi vt liu ca ct xut hin qua 1,32 pht). Pc ca cht X c dng ng phn b Gauss vi b rng ca y
l 24,2s. di ca ct l 40,2 cm.
a> Tnh s a l thuyt trong ct.
b> Tnh H ca ct
c> Tnh T v ca ct.
d> Tnh ch s lu gi ca X .
e> T phng php chun b bit rng th tch ca cht lng gi trn b mt
ca cht mang ca ct bng 9,9. Th tch ca pha ng bng 12,3 ml. Tnh hng s phn b K D.
Bi gii :
a> Tnh N :
2

15.60
t
N = 16 R = 16

W
24,2

=22130 a

b> Tnh H :

H=

L
40, 2
=
= 0,0181 mm
N 22130

c> Tnh T v :

T=

W 24,2
=
= 6,05 giy
4
4

2
N= 2
H

d> Tnh R :

R=

e> Tnh KD :

= H N = 1,81.103. 22130 = 0,27

cm

1,32
= 0,088
15
R=

1
V
1 + KD S
VM

1
=

9,9
1 + KD
12,3

58

= 0,088 KD = 12,9

Chng 3. SC K KH
1. Nguyn tc v mt s yu cu ca phng php sc k kh
1.1. Nguyn tc
Da vo tng tc khc nhau ca cc cht i vi pha tnh. Vi pha tnh rn (GSC) ph thuc vo lc hp
ph, vi pha tnh lng (GLC) ph thuc vo s phn b ca cu t. Trong khi , pha ng cha cht kh mang tr
chuyn ng lin tc qua ct trong qu trnh tch. y, do s khc nhau v i lc lin kt vi pha tnh, nn sau
mi ln hp th v gii hp cc cht phn tch di chuyn c cc qung ng rt nh khc nhau. Pha ng di
chuyn lin tc qua ct, ko theo cc cu t khc nhau di chuyn c nhng qung ng khc xa nhau. Sau
mt thi gian nht nh, cc cu t s tch ra khi nhau.
1.2. Mt s yu cu ca phng php phn tch sc kh
Mt s yu cu php phn tch sc k kh c hiu qu: Ct tch c pha tnh ph hp vi mu phn tch. Detecto
ph hp vi phng php cng nh tnh cht v hm lng mu.Kh mang ph hp vi cht phn tch v
nguyn l lm vic ca Detecto. Kh ph tr c thnh phn v tnh cht ph hp.
a ra quy trnh vn hnh, bao gm: nhit ct tch, nhit Detecto, nhit
ha mu, cch bm mu, tc dng kh mang, kh ph tr,
1.3. S my v qu trnh phn tch
php phn tch sc k kh c hiu qu, cn c vo mu phn tch, ngi phn tch bng nhng nguyn tc c bn
v bng kinh nghim thc t a ra mt cu hnh my v iu kin vn hnh ph hp bao gm:
Ct tch c pha tnh ph hp vi mu phn tch
Detetor ph hp vi phng php cng nh tnh cht v hm lng mu

59

Kh mang ph hp vi cht phn tch v nguyn l lm vic ca detetor

a ra quy trnh vn hnh bao gm nhit ca ct tch, nhit detetor, nhit b phn bm mu, cch
bm mu, tc dng kh mang, kh ph trNhng thng s ny c t thng qua my tnh iu khin ca thit
b.

1.3.1. S thit b sc k kh

4
6

5
2

9
8

Phn mm vi s l

H i u k h i n v x l s li u
10
Hnh 1.1. S my
S sc
kt hkh
i t b s c k k h
1.

kh mang

Ngun

6.Detetor

2. iu chnh p sut

7. Khuch i

3. Lc kh

8. My ghi

4. B phn bm mu

9. My tch phn

5. Ct tch

10. Phn mm v my tnh

Trung tm ca php tch sc k l ct tch 5, n l b phn quan trng nht, quyt nh s thnh cng ca php tch.
Tip theo l detetor 6, y l b phn nh hng ln ti tc nhy ca phng php. Cc b phn tip theo l ngun
kh mang, kh ph tr v phn h iu khin v x l tn hiu.
Sau khi mu c a vo b phn bm mu 4 (injector), n c ha hi do y c t mt nhit
ha hi ph hp. Kh mang ngun 1 c iu chnh p sut 2 v lm sch b phn lc kh 3, s y mu i dc
theo ct. Nhit ct c khng ch cht ch bng h rle v cp nhit in. Cc cht phn tch sau khi c tch
trn ct ln lt i vo detetor. Ti y mi cht phn tch cho mt tn hiu nht nh, cc tn hiu ny c khuch
i, x l v ghi li nh cc b phn 7, 8, 9.
Ngy nay, hu ht cc my sc k u c b phn my tnh c ci t cc chng trnh hot ng cho my
bao gm tc dng kh mang, nhit ca cc b phn bm mu, ct tch, detetor v x l tn hiu sau ct 10.

60

Ngoi ra, b phn t ng bm mu v cc ch lm vic khc ph hp vi tng i tng phn tch v detetor
cng c p dng.
1.3.2. Cc bc tin hnh ca php phn tch sc k kh
- Chun b h sc k
Kim tra: mc r r ca kh mang, s lin thng gia thit b dng dng tch cc cu t trong hn hp
cn phn tch ra khi nhau, thit b nhn bit cht tan ra khi ct v th hin kt qu vi thit b iu khin khc.
Nu l ct mi th phi luyn ct trc khi s dng bng cch cho kh mang chy qua ct t nht 30 pht
o
nhit khong 200 250OC ty theo Tmax ca ct trc khi tin hnh bm mu.

- t ch chy sc k
t ch cho tc pha ng, iu kin (ch ) nhit , thi gian chy sc k, chn detetor v iu kin
lm vic ca detetor; cc iu kin phn dng t bung bm mu.
- Tin hnh chy sc k
- Bm mu vo ct
- Chy sc k
- Phn tch d liu thu c t sc : nh tnh, nh lng
Tm li yu cu tng qut ca qu trnh vn hnh: lp li ca th nghim phi cao, hn ch ti a sai s ngu
nhin.

2. Ct tch sc k kh
Ct thng lm bng thy tinh hoc bng thp khng g c cha pha tnh. C hai loi ct: ct nhi v ct mao
qun. Ct nhi thng di t 1 5m, ng knh trong 5mm. Ct nhi c nhi y pha tnh hoc c ph bn
trong bng pha tnh. Ct mao qun di t 10 n 100m, ng knh trong 250 m . Pha tnh c ph trn b mt
pha trong ca ct. Ct mao qun c kh nng tch cao hn ct nhi nhng c c pic li d b rong khi bm qu nhiu
mu.

Hnh 2.1. Ct nhi (Brain M. Tissue, 17/9/2003)

61

Hnh 2.2.Ct mao qun (Brain M. Tissue, 17/9/2003)

Ct tch l trung tm ca php tch sc k. Ngy nay, hu ht cc my sc k u s dng ct tch mao
qun, c pha tnh l cc hp cht cao phn t gm 4 loi: cc ancol, cc ete v este, cc hp cht amin, cc silicon.
2.1. Cc loi ct tch.
2.1.1 . Ct nhi.
Ct nhi l loi ct m pha tnh hoc cht mang pha tnh c nhi vo y ct.

Ct

Cht nhi
ct
Hnh 2.3. Hnh nh m phng cht nhi v ct nhi
- i vi Sc k kh-rn (GSC)
Cht nhi ng thi l pha tnh thng dng l cc polyme vi xp, silicagel; alumina hoc l mt loi ry
phn t no .
i vi loi ny ngoi lc hp ph l chnh thng km theo lc phn b v lm cho sc bt i xng (c dng
ui).
Do pha tnh thng l cc cht phn cc v c tnh hp ph mnh nn khi tch cc cht phn tch c phn
cc mnh th thi gian lu ca chng cng s ln hn nhiu so vi GLC v th ch thun li khi tch cc hn hp c
phn cc thp.
i vi cc cu t c phn cc mnh nh nc, cc ancol do c i lc rt ln i vi pha tnh loi ny
nn khng th tch c chng ra khi nhau trong hn hp ng ng ca chng. iu ny phi c ch khi ch
bin v x l mu phn tch.
Lu : Trong GSC, cht nhi cng c th ng vai tr nh mt cht xc tc, lm xy ra cc phn ng ha hc trn ct
m cht phn tch cng tham gia (nh silicagel; alumina)
Cc loi polyme vi xp thng c to ra t phn ng trng hp styren v ivinylbennzen, cho nn ty
thuc vo hm lng ivinylbenzen m c cc kch thc l xp khc nhau. Loi ny hp ph c c nhng cht
phn cc rt yu k c nc.
- i vi Sc k kh lng(GLC)
Cht mang thng dng l diatomit. Diatomit l loi vt liu silicat, n l cu t chnh trong gch chu la.
Diatomit c u im l tr v phng din ha hc, c kh nng thm pha tnh l lng tt, c b mt ring ln, chu
c nhit cao. Nhng cng c nhc im l d v nn kch thc v hnh dng bn ngoi khng ng nht, d
c ngun gc t nhin nn n c cha nhiu tp cht.
Ch to: s ch t diatomit sau trn u vi xa t l thch hp, nung hn hp 900 oC ri nghin thnh bt.
Cu trc b mt silica:

62

OH

OH
Si

Si

Trong thc t, nng cao cht lng ca cht mang s dng vo mc ch c th no , ngi ta tin hnh hot
ha chng bng cc cht ph thch hp. Th d, sau khi ch to ngi ta ph b mt sn phm lun gi 1% (v khi
lng) pha tnh c tnh cht chn lc c bit no , khi to c cc ct c tnh nng s dng tng ng nhau
(tng t nh hng ch to ra nhiu ct ging nhau).
Lu : Do yu cu b dy ca mng pha tnh trn ton ct phi ng nht nn thng ha tan pha tnh vo dung mi
c nhit si thp, sau khi thm u trn ct, rung, lc, cho chy dung dch d, sau tin hnh ui dung mi. Ct
sau khi nhi mi c un cong cho ph hp kch thc l.
Pha tnh l tng phi khng phn ng vi cht phn tch, c KD xc nh c r rng i vi cc cht phn tch, gi
tr ny khng c qu ln hoc qu nh - thch hp ng thi c nhit si cao (thch hp vi h sc k khng
hn ch kh nng lm vic ca ct)
- u im v nhc im ca ct nhi.
Tr v mt ha hc, c kh nng thm c pha tnh tt, c b mt ring ln v chiu c nhit cao. Nhng cha
nhiu tp cht v d v.
2.1.2.Ct mao qun
Loi ct ny, ngi ta ph thnh ct bng mt lp pha tnh. Do gim p sut trong ct thp nn cc ct ny thng
rt di, c khong hn 105 l thuyt th ct phi di khong 10 -100m trong khi cc ct nhi ch di khong 1 - 5m.
C 3 loi ct mao qun h:
- SCOT: support coated open tubular columns (ct mao qun h ph pha tnh)
- PLOT: porous layer open tubular columns (ct mao qun h ph lp xp)
- WCOT: wall coated open tubular columns (ct mao qun h ph thnh hoc phim). Loi ct ny thng c
ch to t ng SiO2 ng knh trong khong 0,25 0,50 mm, c gm (n mn khng nhn nh cc ng thy
tinh) v ph mt mng tnh (phim), thng c b dy l 0,5 0,25 m .
Do ng knh mao qun nh nn cos a l thuyt ln
lng mu a vo ct phi nh < 100ng
cn
lng mu nh (cn c s tng quan vi kh nng tng thch ca detetor). Ct ny c th thc hin cc cn bng
chit trong khong 1000 a ch cn n khng qu 1 giy (s).
Cc hnh di y th hin mt s thng s ca cc loi ct mao qun

Pha
tnh

20
SCOT

20
PLOT

0,5 0,25

63

WCO

Hnh 2.4. M hnh m phng mt ct ngang ca cc loi ct mao qun

u im:

+ Cn bng thit lp nhanh;

+ phn gii tt (do dng lng mu nh)

+ ng nht cao (i vi loi ct n mn bng phng php ha hc)
+ Gii hn pht hin nh
Nhc im ca ct mao qun:+ Di, nh
kh ch to
+ Lng mu nh
cn c detetor c kh nng tng thch cao.
2.2. Cch nhi pha tnh ct tch
Ngi ta ph pha tnh ln cht mang (pha tnh + dung mi + cht mang) sau t nng lm bay hi dung
mi. Khi cht mang c tm pha tnh tip tc c sy trong t chn khng nhit thch hp, ngui ri ray
trn ray c c s thch hp sau nhi vo ct. Cht nhi c a vo ct bng: mt u ct bng mt phu u
ct kia c gn vi mt h thng chn khng ( nh vo bm chn khng). Ton b ct c c nm ngang trn mt
b rung c th iu khin c tc .
Bng 2.1. Cu to v cc thng s v cc ct tch

Kiu ct tch
c trng
ng knh trong
Chiu di
Hiu qu Ct

Ct mao qun phim

mng WCOT

Ct mao qun
lp mng
SCCT

Ct mao

Ct nhi

qun nhi

thng thng

0,,25 0,50 mm
10 100 m

0,50 mm
10 100 m

1 mm
16m

2 4 mm
14m

1000 3000 a/m

600 1200 a/m

1000 3000 a/m

500 1000 a/m

20 30 cm/s

20 30 cm/s

8 15 cm/s

4 6 cm/s

Tc dng

hydro; heli

hydro; heli

hydro; heli

hydro; heli

(ti u)

10 15 cm/s

10 15 cm/s

3 10 cm/s

2 5 cm/s

nit; argon
1 5 ml/pht

nit; argon
2 8 ml/pht

nit; argon
2 6 ml/pht

nit; argon
2060 ml/pht

Tc th tch

hydro; heli

hydro; heli

hydro; heli

hydro; heli

dng (ti u)

0,5 4 ml/pht

1 4 ml/pht

1 3 ml/pht

15 50 ml/pht

nit; argon

nit; argon

nit; argon

nit; argon

10 100 ng

10ng 1g

10ng 10g

10ng 1mg

Thp

Thp
i hi

tch

Lng mu
p sut i hi
Kh b tr
Cho detetor
Tc phn tch
Tr ha hc
Tnh thm

i hi thng thng
Nhanh
Tt
Cao

thng

Rt cao
i hi

thng
Nhanh

thng
Trung bnh

Cao

Thp

thng

Cao
i hi thng thng
Chm
Km nht
Thp

Hin nay Vit Nam ct nhi thng thng vn l loi ct sc k ph bin nht, trong khi a s cc phng
th nghim, k c trong k ngh, nhiu nc khoa hc tin tin trn th gii hu nh b hn ct nhi m ch cn
s dng ct mao qun. Tuy nhin so vi phng php sc k kh ct nhi, sc k kh s dng ct mao qun i hi

64

nhng yu cu ht sc c bit i vi qu trnh bm mu cng nh i vi qu trnh pht hin cc cu t (v d i

vi cc ct mao qun phim mng WCOT lng mu c a vo ct khong 10 -7g.
2.3. Cc loi pha tnh lng
Pha tnh loi ny c nh ga da vo

tsoi

(tc l khong nhit lm vic ca ct) v phn cc

s dng hiu qu ca pha tnh, ngi ta a ra mt s nguyn tc

Dng cc pha tnh phn cc tch cc mu gm cc cu t phn cc
- Cu t c p sut hi thp cn phi c tch nhit kh cao nn cn pha tnh lng l cht chu c nhit
cao
- Cng cn lu rng khi tch nhit thp th phn gii ca GC s tt hn
Th d mt s pha tnh thng gp
Bng 2.2. Mt s pha tnh thng s dng

Thnh phn ha hc

t soi max
( o Cphn
) cc

ng dng chnh (i tng phn

Squalane (C30H62)

150

NP

Cc hydrocacbon

Polymethyl siloxane

350

NP

Cc hp cht khng phn

Polycarboran siloxane

400

NP

Cc hp cht c p sut hi thp

200

Cc ester, acide bo, pesticides

250

Cc ru, cc cht thm, cc ester

250

270

Diethyleneglycol
adipate
Polyethylene glycol
Polytrifluopropylmeth
yl siloxance
Polycyanomethyl
siloxance

tch)

Cc amino acide, steorit, cc hp

cht cha N
Cc ancaloit, dc phm, cc hp
cht halogeno

NP: khng phn cc; I: t phn cc; P: phn cc

3. Cc loi kh mang (pha ng) c s dng trong sc k kh.

C th s dng kh n hoc hn hp 2 hoc 3 kh theo t l thch hp. Kh mang c vai tr a cht phn
tch i dc theo ct, ra gii v lm sch h thng sc k. Vic chn kh no lm kh mang ph thuc vo cht
phn tch, ph thuc vo Detecto, v k thut sc k. Thng s dng cc kh nh: He, H2, Ar, N2, Kr, c
tinh khit cao khong 99,995% n 99,9999%. Tc trung bnh ca kh mang: 100 250 ml/pht.
'
'
'
T mi lin h gia t R vi F (lu lng dng pha ng) qua phng trnh VR = t R .F m F v tc chuyn

di u ca pha ng ph thuc vo p sut ca n trn ct thng qua h s Martin J (J c gi l hiu s hiu chnh). J

l hm s, ph thuc vo p sut u v p sut cui ct qua phng trnh:

65

J=

3.( PPdc ) 2 1
2.( PPdc )3 1

Cn phi lu rng do tnh cht d b nn ca cc cht kh nn tc chuyn di ca kh mang khng t l thun vi

p sut kh mang.
ng thi cng cn thit phi nhc li l khi p sut hi bo ha ca cu t tng (cht d bay hi), thi gian lu ca
cu t s gim khi tng nhit ca ct sc k.
3.1. Yu cu i vi kh mang:
Do gim p sut qua ct tch t l vi nht ca kh mang theo phng trnh:

P =

.L.u
Bo

Trong : P = P - Pc
`

: nht ca kh mang
u : tc dng trung bnh
Bo : thm thu ring

Do yu cu ca i tng cn tch v kh nng tng thch ca detetor nn kh mang cn c cc yu cu chnh sau:

i vi ct nhi cn kh mang c nht thp. Ct mao qun c th s dng c cc kh mang c nht cao.
Kh mang c dng phi khng thay i trng thi l, ha hc khi i qua my sc k kh
Phi tng thch vi detetor. Chng hn i vi detetor dn in (TCD) cn s dng kh mang c dn
cao nh H2, He; trong mt s trng hp c th dng N 2, nhng cn phi ch n khong tuyn tnh ca detetor. i
vi detetor ion ha ngn la th c th s dng tt c cc kh mang tr O 2.
Ngoi ra i vi kh mn cn lu cc im:
an ton khi s dng
Hiu qu kinh t, tinh khit cao
3.2. c im ca mt s kh mang thng c s dng
Kh hydro: kh hydro thng mi thng t tiu chun cho sc k kh. Trong phn tch lng vt, khi s
dng pha tnh d b hng v trong sc k iu ch cn phi lm sch v kh kh. Khi s dng kh hydro lm kh mang
cn dng nit lm kh bo v thi qua ct trc. Cc ng dn kh hydro phi dy tt nht l dng ng kim loi nh
va kn va tit kim kh. Hydro dng cho cc ct tch lm vic di 200 oC vn t ra tr. Trong cc phng th nghim
hin i dng kh ph bin my sn xut kh hydro vi cng sut t 125ml/pht n 225ml/pht. Khi dng my ny,
lc u phi ch cho my t p sut nht nh mi c a kh vo ct tch. Trong phng sc k s dng kh hydro
phi c my d h hydro v cm la.
Kh hli: l kh tr ha hc rt thch hp cho sc k kh nhit cao. Khi s dng detetor ion ha bng tia
phng x phi s dng hli tinh khit.
Kh argon: cng nh cc kh tr khc, argon trn c c khng c hot tnh ha hc c dng cho sc k kh
nhit cao. Do nht ca argon cao, yu cu v dy dn khi s dng n khng gp kh khn lm. Kh argon
ngy cng c s dng nhiu lm kh mang.
Kh nit: do khng nguy him, gi r v d dng lm tinh khit nn nit c s dng rt nhiu trong sc k
kh. Nhng cn ch vi detetor TCD v ga tr dn nhit ca nit rt gn vi dn ca nhiu kh hoc hi cht hu
c nn c trng hp pic sc k s ngc. Trong phn tch nh, h s hiu chnh c trng s x dch rt mnh khi 1
khi dng kh mang c dn cao.

66

Khng kh v oxi: tinh khit ca oxi thng mi cng t yu cu cho sc k kh nhng cn phi sy kh
v rt d ln nc trong bom ng kh. Khng kh nn c th ly t bom kh bm nn kiu du. Nu dng bm nn th
phi ch khng cho hi du i vo thit b sc k.
Bng 3.1. Tng kt v tnh cht v kh nng s dng ca cc kh
TNH CHT V KH NNG S

KH MANG
Argon
Hli

Nit

Hidro

0,52
321
Rt rng
99,999%
t

4,08
270
Rt rng
99,999%
Rt t

0,73
246
Rng
99,999%
R

5,47
121
Hp
99,999%
R

Mc an ton

Cao

Cao

Cao

Rt thp

Mc ph bin

Rt t

Cao

DNG
dn in (104 cal / cms o .K )
nht ( 1atm; 200oC)
Khong nhit lm vic
Yu cu v tinh khit
Tnh kinh t

4. Detetor
Detetor l b phn c nh hng n nhy phng php. Detetor phi ph hp vi i tng v cu t cn
tch, c nhy v p ng cao. Detetor c nhim v chuyn ha mt i lng khng in (trong trng hp ny
l nng cc cht c tch ra khi ct sc k kh) thnh i lng in. Ngy nay c gn 30 loi detetor khc
nhau. Sau y l bng lit k mt s loi detetor thng dng vi cc tnh nng ca chng. Sau y l mt s Detetor
dng dng trong st k kh.
4.1. Detetor dn nhit (TCD thermal conductivity detetor)
Nguyn tc hot ng: So snh dn nhit ca kh mang tinh khit v ca kh mang c cht tan thng qua
vic o in tr ca mt dy dn c t nng. Mch so snh c lp theo kiu Wheattone.

Kh mang c cht
tan

Kh
mang

Hnh 4.1. S ca detetor dn nhit

nhy ca detetor ph thuc vo kh nng dn in ca kh mang, dng nui in tr
u im:p ng c vi tt c cc cht nhy cho nhiu cht cn phn tch (c 10ppm),Khong tuyn
tnh ca tn hiu kha tt rng (cch nhau n 104 ln),Cu to n gin Tn hiu kh bn ( lu tn hiu)
4.2. Detetor ion ha ngn la (FID Flame ionzation Detetor)

67

Nguyn tc hot ng: o s bin i dn in ca ngn la trong mt in trng khi c cht hu c i

qua. (Nh nhit cao ca ngn la m cc cht i ra khi ct c bit l cc cht hu c b ion ha v to thnh
dng ion trong in trng
gim th ca in trng).

ng gp
Ngn la

B phn d
tn hiu

Hnh 4.2. S ca detetor

FID:
nhy ca detetor ph thuc vo kh nng c dng in cc nh ca h vi mch in t ( c 10 12 A); nhit
ca ngn la ion ha; bn cht ca cc cu t b ion ha (d hoc kh b ion ha); khng nhy vi cc cht v c v
cc cht hu c v carbon b oxi ha mc cao nh cc nhm cacbonyl.
u im: chn lc cao (p ng tt cc hp cht hydrocacbon rt thun tin khi phn tch du khong)
- C nhy kh cao i vi nhiu cht (c th pht hin cht c nng c 100ppb)
- Khong tuyn tnh rt rng (hn km nhau n hng triu ln)
- bn tnh hiu cao, t b nh hng bi tc dng kh mang v cc thng s thc nghim khc
4.3. Detetor cng kt in t (EDC electron capture detetor)
Nguyn tc hot ng: da trn c tnh cc cht c kh nng cng kt (bt) in t t do trong pha kh.
Trong trng hp ny, cc ion c to ra bi mt ngun phng x nh 3 H hoc

63

Ni , chng pht ra cc ht

, cc ht ny ion ha cc cht (chng hn nh kh mang N 2 +

N 2+ + 2e ) v to ra mt dng trong mch.
Khi c cht c kh nng bt electron (l cc cht cha cc nguyn t c m in cao nh halogen, oxy, nit,
photpho, lu hunh) c a vo detetor th lm cho dng gim xung.
Ngun

Cht thi

Ct GC

B phn
o dng

Hnh 4.3. S detetor EDC

nhy ca detetor EDC ph thuc ch yu vo: ln ca dng nn,Mc nng lng ca electron pht ra
ca ngun bc x, m in ca nguyn t gn vo cht cn phn tch,in th t vo hai cc ca detetor. Detetor

68

ny rt nhy vi cc hp cht halogenua, gii hn tn hiu c th t n c 2.1014 g / s . Khong tuyn tnh kh tt

(hn km nhau c 1000 ln),Bn cht kh mang
u nhc im ca detetor EDC: Ch chn lc v c nhy cao vi cc hp cht c halogen (c 0,01ppb
i vi Lindan), khng nhy i vi cc cht khc. v th c dng ch yu trong vic phn tch cc PCB, Pesticides
c ngun gc c clo, cc Fluorocarbons v cc cht tng t
Khong tuyn tnh hp
4.4. Detetor khi ph (MSD mass spectroscopy detetor)
Nguyn tc hot ng: cc cht ra khi ct c chuyn hon ton thnh dng kh v tch cht tan khi kh mang,
sau dng cc ngun ion ha cc mnh ct cc phn t thnh cc mnh ion, cc mnh ion ny c tch ra khi
nhau theo mt trt t nht nh v khi lng v cui cng nhn bit cc ion ra khi b phn tch khi lng bng
thit b thch hp.
C th ni MSD c 4 b phn:

B phn
lc kh
mu

B phn
ion ha
mu

B phn
tch ion
theo khi
lng

B phn
pht hin cc
ion ra khi
b phn
phn tch
ion

Hnh 4.4. S detetor MSD

nhy ca detetor ph thuc vo hiu sut ct mnh ca b phn ion ha mu in trng ca t cc.
u nhc im: Ty theo dng sc m c th d on cng thc, cu trc ca cc phn t m khng cn cht
chun (qua catalog c sn trong phn mm), c nhy v chnh xc cao (c 10 1 n 10 3 ppm), khong tuyn
tnh tt (hn km nhau c 104 ln), thi gian phn tch nhanh.
4.5. Detector pht x nguyn t (Atomic emission detector: AED)
Cc nguyn t trong dng hi i ra t ct mao qun ca SKK. Sau khi ra khi ct, hi i vo bo quang in
vi sng - y cc thnh phn b phn hy v cc nguyn t ca chng b kch thch bi nng lng ca t bo quang
in. nh sng pht ra bi cc phn t b kch thch, c tch ra thnh cc tia ring bit i qua chui quang diod n
my tnh. My tnh chn ra cc tia ring bit v ghi sc di dng cc pic ch tng ng vi mt cu t c th ca
cc cht bay hi i ra t ct SKK.

69

Hnh 4.5. S cu to detector cng kt in t

4.6. Detector quang ha (chemiluminescence detector)

Detector quang ha c ni vi my SK lng hoc SKK v ng vai tr quan trng trong cc phng th
nghim phn tch. Trong ph quang ha, nng lng cn thit kch thch cht phn tch cao hn cc trng thi in
t quay vng quanh v dao ng v khng xut pht t ngun sng bn ngoi nh laser hoc n. Vn phn tn
ngun kch thch hon ton trnh c. Nng lng c to ra bng phn ng ha hc gia cht phn tch v thuc
th:
CH3 S CH3 + F2 sn phm + hv (nh sng)
Quang ha pha kh, s pht nh sng c to ra bi phn ng gia cht phn tch v kh oxy ha mnh .
Phn ng xy ra vo khong thi gian m sn phm ca nh sng to ra ngay lp tc. V vy h thng phn tch trn
cht phn tch vi thuc th ngay trong bnh th tch nh, trc tip pha trc ca khuch i nh sng. Khi cht
phn tch i ra t ct sc k kh, ui ct gn trc tip vo bnh phn ng. V vy nng lng ca phn ng c gii
phng ra bao nhiu th c dng by nhiu kch thch phn t cht phn tch. S mt nng lng thng qua s va
chm ca pha kh l khng mong mun. V vy p sut kh bnh phn ng phi c duy tr thp bng bm chn
khng hn ch ti a tc dng bt hot ca s va chm

Hnh 4.6.S cu to ca detector quang ha

4.7. Detector quang k ngn la (Flame photometric detector: FPD)
Detector quang k ngn la c s dng xc nh cc cht c cha lu hunh v phosphor. Detector ny
s dng cc phn ng quang ha ca lu hunh v phosphor trong ngn la hydro / khng kh nh mt ngun thng
tin tng i c bit ca cc hp cht c cha nguyn t lu hunh v nguyn t phosphor. Lama cc i dn truyn
ca cc ht lu hunh c kch thch (S2) vo khong 394nm. Lamda cc i cp i (duplet) ca cc ht phosphor
b kch thch (HPO): 510-526. c th pht hin mt cch chn lc hoc lu hunh hoc phosphor khi cc thnh

70

phn ny i ra t ct SKK, ngi ta s dng mt knh lc nhiu sng. Knh ny c t gia vng bc x kh kin
v t ngoi pht x bi ngn la. Khi khng c tng lng ln bc x hng ngoi, pht x bi ngn la chy, phn
ng t nng n PMT v lm tng tn hiu ca PMT.

Hnh 4.7 . S detector quang k ngn la

4.8. Detector nitrogen phosphor (Nitrogen phosphorus detector: NPD)
Cu to ca detector nitrogen phosphor tng t nh detector ion ha ngn la. S khc nhau chnh l ngn
la hydro / khng kh ca detector ion ha ngn la c thay bng ht rubidisilicat t nng trong detector nitrogen
phosphor. Cc cht t ct SKK i ra qua ht mui rubidi nng pht ra cc ion ca cac thnh phn c cha nitrogen
v phosphor i qua n. Cc ion c tp trung bnh thu pha trn ht nung nng, to ra dng in tng t nh det
ion ha ngn la.
4.9. detector quang ha ion (Photoionization detector:PID)
Detector quang ion ha c ng dng xc nh cc hydrocabua thm hoc cc hp cht c nguyn t d
vng. Detector quang ion ha s dng nh sng t ngoi ion ha cc cht b kch thch i ra t ct SKK. Cc ion
to ra c tp hp in cc. V vy dng in pht sinh l thc o nng cht phn tch.
Phn ng quang ion ha xy ra nh sau: R = hv R+ + eNu nng lng ca mt phonton i vo cao, th s quang kch c th xy ra, 1 in t hon ton tch ra
khi phn t qu o l s ion ha. Nu s ion ha c mt cht xy ra, th dng din sinh ra tp trung in cc ca
bnh phn ng ca detector quang ion ha, t l thun vi chnh cht i vo bnh phn ng. Ch c cc hydrocabua
thm v cc cht cc nguyn t d vng (tng t nh sunlfua hu c v phosphor hu c) mi c xc nh bng
detector quang ion ha, bi v ch c cc cht ny mi c kh nng ion ha trong phm vi ngun nh sng ca cc n
ngoi t ang c bn trn th trng. Nng lng ca cc n t ngoi t 8,3 n 11,7 ev, c ngha l lamda max trong
khong t 150nm n 106 nm. Hu ht cc detector quang ion ha bn trn th trng ch c 1 n t ngoi. Cc n
t ngoi c thay i ph thuc vo yu cu phn tch.
Pht hin chn lc bng detector quang ion ha: th d v s pht hin chn lc benzen trong s c mt ca
isopropanol. im si ca benzen 80,1 0C, kh nng ion ha = 9,24 ev - ng vi n t ngoi c nng lng bng 9,5
ev trong detector quang ion ha bi v nng lng ny cao hn kh nng ion ha ca benzen (9,24 ev). Isopropanol
c im si tng t nh benzen (82,5 0C) v 2 cht ny s ra khi ct SKK gn nh cng mt thi gian. Mc d vy,
isopropanol c kh nng ion ha cao hnw nng lng ca n t ngoi (10,15 ev) nn khng nhn thy hoc cm ng
rt yu detector quang ion ha v v th ch pht hin nn khng nhn thy hoc cm ng rt yu detector quang
ion ha v v th ch pht hin c mt cht l benzen.

71

Hnh 4.8. S cu to detector quang ion ha

Cu to ca detector quang ion ha c trnh by hnh 25: detector quang ion ha l detector SKK khng
phn hy v ch c mt phn rt nh ca cht phn tch b ion ha bung ca detector. V th c th ni ng x t
detector quang ion ha vi mt detector khc (nh detector ion ha ngn la hoc detector cng kt in t) c kt
qu phn tch ng thi bng 2 detector. Nhim v chnh y l thit k bung ion ha v ng ni vi detector th 2
c th tch nh nht (ng knh trong nh) cc pic khi tch bng ct SKK khng b m rng trc khi pht hin.

5. La chn cc iu kin cho php st k kh

chn quy trnh phn tch sc k kh, cn phi bit ngun gc v thnh phn mu, iu ny c bit quan
trng i vi phn tch nh lng. Ni chung, mi mt mu u c th c phn loi theo thnh phn hoc cc c
tnh ca cu t theo mt trong cc nhm sau:
Nhm A: cc cu t c hm lng tng t nhau trong mu
Nhm B: bn cnh cu t chnh, mu cn cha mt s cu t vi hm lng rt nh (thng gi l cu t vt)
Nhm C: cc cu t ca mu (thuc nhm A hoc B) c im si rt khc nhau (gi l hn hp vi khong si
rng)
Nhm D: cc cu t ca hn hp (nhm A, B hoc C) rt phn cc nh ancol vi s nguyn t cacbon thp,
axit bo t do, cc amin bc 1 v este ca axit bo.
Nhm E: mu (nhm A, B, C hoc D) tn ti dng dung dch
Nhm F: mu c cha mt s thnh phn khng bay hi hoc rt kh bay hi.
Trn c s thnh phn ca mu s quyt nh la chn nhng phng tin thit b v iu kin lm vic.
5.1. Chn ct tch.
5.1.1. Chn kiu ct tch
Nu mu thuc nhm A v C th ct mao qun s cho kt qu tt hn ct nhi.
Nu mu thuc nhm B th khng c php dng ct mao qun phim mng (WCOT) m phi dng ct mao
qun lp mng (SCOT) hoc ct nhi.
Mu thuc nhm D s c phn tch tt trn cc ct mao qun c b mt thy tinh c mui ha.
5.1.2. Chn kch thc ct tch
Chiu di ct tch c xc nh bi hiu sut phn gii cn thit. Song nu tng chiu di ct tch gp i th
phn gii ch tng thm khong 40%. Mt khc, vic tng chiu di ct tch li lm tng chnh lch p sut theo
phng trnh: p =

B0

.Lu

72

V vy khng th ko di ct tch mt cch ty . Ring v tit din ct tch s do dung lng cn thit ca
mu quyt nh. Trog sc k kh iu ch, ng knh trong ca ct tch phi ln hn 4mm.
Khi s dng detetor TCD phn tch nhng mu thuc nhm A hoc C cn phi dng ct c ng knh tht
nh, sao cho vi nhng lu lng kh mang thp khng lm gin pic. gi tc kh mang u trong ct c nh, i
vi ct c tit din nh phi lm gim lu lng dng kh. Trong trng hp , kh nng chu ti ca ct s gim t
l thun vi tit din ct, tc l lng mu phi gim. Song nng mu trong kh mang vn gi nguyn, do cc
tn hiu o c cng khng b gim.
Nu mu thuc nhm B (phn tch lng vt) m vn mun s dng detetor TCD ( phn tch cc kh vnh
cu, hydro, oxy, kh c cha nit nh: NO, H 2S l nhng cht khng th pht hin c bng detector FID) th phi
bm nhiu mu, ti trng ca ct tng nn bt buc ct phi c ng knh ln hn. Mc ln ty thuc vo hm
lng mu phn tch (thng ng knh trong ca ct tch khong 4,25 6,35mm). Tuy vy, khi phn tch lng vt
thng s dng detetor FID.
Khi vn hnh vi detetor ion ha (FID, ECD), cn phi to ra cc dng cht ln c th c bng cch tng
tc dng kh mang nhng vn nm trong vng ti u. Mt khc cng cn quan tm n cc phn ng xy ra ti
detetor (nu dng kh qu ln s lm tt ngn la hoc ngn la s chy vng khng ti u), thng thng ch s
dng ct c ng knh trong t 3 4mm. Vi ct mao qun cn la chn ct c ng knh trong nh t hiu
sut ti a. Nu ti trng ca ct mao qun khng p ng c yu cu tch th dng ct mao qun lp mng.
Khi dng ct mao qun, vn tit kim chiu di ct tch thng t c cp. Vi mu c hng s phn
b K kh ln th ct mao qun phim mng c chiu di 25m l thch hp
5.1.3. iu khin nhit ct tch
ng nhit : y l phng php n gin nht v c bit cn thit i vi vic xc nh cc thng s ha l. Nu
mu c cha t cu t vi cc hng s phn b tng t nhau th phng thc lm vic s cho s thng tin ti a. Mu
thuc nhm C th thi gian phn tch qu di cng nh t s gia chiu cao v rng pic ngy cng bt li cho phn
tch nh lng. Trng hp ny tt nht l s dng chng trnh nhit .
Chng trnh nhit : Chng trnh nhit gip phn tch hn hp nhm C ch cn mt lng bm mu.
u im ca phng php ny l:
- Rt ngn thi gian phn tch i vi cc mu c khong im si rng
- T l gia chiu cao pic v chiu rng pic lun n nh v th thun li cho phn tch nh lng. Thng thng, cc
mu hp cht thin nhin rt phc tp nn khng th bng mt phng trnh tuyn tnh thu c kt qu mong mun
m phi s dng a phng trnh, ngha l khong nhit gia chng c th gi ng nhit ct trong thi gian
thch ng ri li tng tip vi tc mong mun. Trc v sau chng trnh c th gi nhit c nh.
i vi mt s pha tnh, khi tng nhit ct tch th d b cun i bi dng kh, gy ra hin tng lch
ng nn gy kh khn cho vic s dng tch phn k in t. iu ny c khc phc bng cch s dng ct kp,
tn hiu ca hai ct c phn cc tri du nhau nn b tr c vic pha tnh bay hi. Tuy vy, khi s dng chng
trnh nhit cn lu n cc im sau y:
Nhit ca bung iu nhit phi c iu chnh vi tc thch ng nh chng trnh quy nh. Hin nay cha c
my sc k no lm vic chnh xc vi tc nh hn 0,3o C /pht v ln hn 24o C /pht. Thc t ct nhi thng
dng vi tc 5 20 o C /pht, ct mao qun l 0, 25 2,5 oC /pht.

73

Vic lm ngui bung iu nhit cn phi c tin hnh nhanh chng, v thng lm ngui su hn nhit
lm vic ban u qu trnh thc hin chng trnh mi nhanh chng n nh, v khi ca bung iu nhit nng,
nhit trong cc tm cch nhit li ph ra.
Nhit ca bung bay hi mu cn phi c a ln cao, c lp vi nhit ca bung iu nhit nhm lm s
bay hi mu n nh v lp li cho d nhit tng hay gim.
Kh mang s dng phi tinh khit, nu khng cc tp cht s tch t trc ht phn u ca ct tch. Khi nhit
ca ct tng ln n s vn chuyn qua ct gy ra pic l bt ng.
S dng ct nhi cn lu n dy ca thnh ct m bo truyn nhit tt v ng knh ct khng qu
ln (thng s dng ct c ng knh 3mm) m bo cho chnh lch nhit trong v ngoi ct tch l t
nht. Ct mao qun thch hp vi chng trnh nhit hn so vi ct nhi.
5.2. Chn pha tnh
Bao gi cng nn s dng pha tnh c chn lc cao nht. Song cn lu phi s dng loi pha tnh c
bn nhit thch hp, ngha l nhit lm vic ti a bao gi cng nh hn nhit chn trn ca pha tnh . Do ,
trong nhiu trng hp c nhng pha tnh rt chn lc cho mt i tng nhng v nhit chn trn qu thp (p
sut hi qu ln) nn khng s dng c. Trong thc tin phi s dng mt cch dung ha hai yu t va nu.
Ring trong trng hp pha tnh l tinh th lng, cn phi lu c nhit chn di bi v hiu qu tch cao
nht ch th hin trong trng hp pha tnh trng thi tinh th lng. V d: i vi metoxietoxiazo xibenzen l 96 1220C.
Vi pha tnh l cao su silicon, khi s dng detetor FID th nhit ct tch phi thp hn nhit chn vi
chc v trong trng hp ny mt phn pha tnh thot ra khi ct s chy trong ngn la hydro to thnh SiO 2 gy
bn hai bn in cc v lm tc u phun, iu s lm gim nhy v nhiu ng nn.
Trong trng hp sc k iu ch, pha tnh c th b ko theo vo bnh hng mu v ngng t lm bn
cc cht thu c nn phi tinh ch li.
5.3. Chn v phi hp detector
Vic la chn detetor l mt trong nhng vn rt kh v ph thuc vo nhiu thng s.
- i vi ct mao qun thng s dng detetor FID.
- Cc detetor dn nhit c nh cho n nay hu nh khng c s dng v chng qu nhy vi s thay i tc
. cng l mt kh khn khi mun pht hin mt s loi kh nh CO, CO 2, H2O nu dng ct mao qun.
i vi mu thuc nhm B hoc C th nn dng detetor FID v c phm vi tuyn tnh ln v khng nhy cm vi s
thay i lu lng dng kh nht l trong trng hp s dng chng trnh nhit . pht hin cc mu c th
phi s dng detetor c trng tng ng.
V d, pht hin hp cht cha halogen trong thuc tr su, cht c, cn phi s dng detetor ECD,
pht hin cc hp cht cha lu hunh v photpho phi dng detetor FPD.
Nhm pht hin mt cch hiu qu cc cht cn tch, i khi c th s dng kt hp hai hoc nhiu detetor
cng mt lc. V d, kt hp FID ci TCD, FID vi ECD, kt hp vi s gip ca b chia dng li ra v my t
ghi nhiu knh.
Trong sc k mao qun, thng s dng b chia dng li vo nhm khng ch lu lng kh mang v lng mu
bm vo ct.
5.4 Chn kh mang
Vic la chn kh mang trc ht ph thuc vo detetor s dng.

74

- Vi detetor TCD, tt nht l dng hli hoc hydro.

- Vi detetor FID th dng nit l .
Gn y, hydro c s dng nhiu lm kh mang v n c nht thp, c bit trong sc k mao qun.
Detetor ECD s dng nit s dng nit hoc argon c trn 5% metan..

6. C s l thuyt ca sc k kh mao qun

Sc k kh mao qun l mt hnh thi c bit ca phng php sc k kh, c c trng bi nng sut tch
v hiu sut phn gii rt cao. Nh vic s dng cc ct mao qun h vi chiu di kh ln (25m, 50m, 100m).
V nguyn tc khng c g khc v c tnh nguyn tc gia sc k kh mao qun vi sc k kh ct nhi. Tuy
nhin do vic khng s dng cht nhi ct (di dng cc ht cht mang c tm pha tnh), t to ra ng i t do
cho dng kh mang, nn c mt vi thay i trong phng trnh c bn nh gi ct tch so vi trng hp sc k
kh ct nhi.
V d : Phng trnh Van Deemter th trong sc k kh mao qun c thay bng phng trnh Golay: h=
B/u +(Cl + Cs).u
Cl,Cs c trng cho s cn tr i vi qu trnh vn chuyn cht t pha ng sang pha tnh v ngc li.
Tng t nh sc k kh ct nhi, trong sc k kh mao qun thng phi chn tc kh mang trung bnh
ln gp i so vi tc kh mang ti u (v nhnh bn phi ca ng cong Golay choi hn so vi nhnh bn tri )
Trong sc k kh mao qun chiu cao ca a l thuyt gim nn lm tng nng sut ct tch :
Gim dy ca lp phim pha tnh
Gim nhit ct tch
Gim ng knh ct tch
S dng kh mang vi nht thp

7. Phn tch bng phng php sc k kh

7.1. Phn tch nh tnh:
Thng thng ngi ta s snh thi gian lu ca cht cn xc nh vi thi gian lu ca cht chun c sn
hoc thm. iu ny ch c ngha khi tin hnh tch trong cng iu kin nhit . Thc t khng phi khi no cng
c sn cht chun nn c nhiu chng trnh phn mm ca my ci sn thi gian lu ca mt s cht iu kin
chy sc k no ngi phn tch tham kho.
'
Do t R ph thuc vo nhiu yu t (u, to, khi lng mu, pha tnh, Vs, tm, p) nn vic xc nh n so vi cc

d liu c trong phn mm c sn l cc k kh, v th ngi ta dng thi gian lu tng i ch s lu Kovats
(KRI) nh tnh. Theo phng php ny, dy ng ng n-parafin c chn lm cht chun.
Thi gian lu ca cc cht cn xc nh c tnh so vi mt cht no trong dy ng ng ca n-parafin. T
thi gian lu tng i ny tin hnh so snh cc cht cn phn tch vi cht chun c sn.
Theo Kovats, mi parafin c mt gi tr ch s l 100Z (Z ch s carbon v d CH 4 = 100; C2H6 = 200; C3H8 =
300) do mt cht phn tch c KRI l 540 tng ng vi 1 parafin thng thng cha t 4 n 5 cacbon. KRI
c th tnh c t sc da vo phng trnh:

75

KRI = 100.

lg t R' , X lg t R' , Z
lg t R' ,Z +1 lg tR' ,Z

+Z

t R' , X l thi gian lu hiu chnh ca cht X cn phn tch

t R' , Z ; t R' , Z +1 l thi gian lu hiu chnh ln lt ca hidrocacbon no cha Z nguyn t C c ra gii trc X v ca
hidrocacbon no cha Z+1 nguyn t cacbon c ra gii sau X.
Ch s lu Kovats c nhng u im sau:
- Rt t ph thuc vo thng s lm vic, do n l gia tr c trng cho mi cht ng vi mt pha tnh v mt
nhit nht nh. S ph thuc vo nhit ca chng rt nh v v nguyn tc ch khong 2 6 n v khi thay i
10oC.
- lp li ca vic xc nh ch s lu ca hai phng th nghim khc nhau ch lch 1 n v v thm ch vi
nhng iu kin thun li ch lch 0,05 n v.

7.2. Phn tch nh lng

T vic xc nh din tch hoc chiu cao ca pic, so snh vi mu chun (ni chun, ngoi chun hoc thm
chun) hoc t ng chun (ni hoc ngoi chun) tng ng, tnh ra hm lng ca cht cn phn tch trong mu
bm vo ct. Di y l th d v cch xc nh din tch ca mt pic: cch th nht tnh theo b rng ca chn pic;
cch th hai tnh theo b rng ca pic ng vi 1/2 chiu cao ca pic.

S pic

1
(w.a)
2

S pic w1/2 .h
Hnh 7.1: S pic sc k

H = K.Cx; S = K.Cx
- Qu trnh nh lng gm 4 giai on:
Giai on 1: Tch sc k (nghin cu tm iu kin tch ti u - h s tch gia
hai cu t lin k bng 1 l tt nht).
Giai on 2: Thu tn hiu detector v chuyn tn hiu thnh s liu (o din tch nh).

76

Giai on 3: nh lng bng cch so snh cc din tch nh.

Giai on 4: X l s liu v gii thch kt qu.
- Cc phng php ph bin nh lng:
a) Phng php ngoi chun
Tu thuc vo tnh cht ca mu: hm lng, yu t cn tr, tnh cht phc tp ca nn
cng nh chnh xc yu cu m ngi phn tch c th p dng cc cch nh lng khc
nhau: Chun ho mt im v chun ho nhiu im.

Chun ha mt im
- Nguyn tc: Chun ha mt im l s dng mt php o ca cht chun bit trc
nng , o c lp v song song vi php o cht cn phn tch.
- Phng php ny thng p dng:
+ i vi cht phn tch c hm lng ln hoc lm giu n mc cn thit.
+ nh gi khi khng yu cu chnh xc cao v y b qua cc yu t cn,
cc sai s ngu nhin c th lm gim tnh chnh xc ca php o.
+ Nng ca cht phn tch Cx phi nm trong khong tuyn tnh.
- Phng php: S dng hai php o c lp:
+ Php th nht: Chy sc k cht chun trong iu kin tng t cht phn tch
(nn v cc thng s khc ging nhau).
+ Php th hai: Chy sc k cht phn tch.
Kt qu tnh nh sau:

Hnh 7.2: Chun ha mt im

Ta c:

C x Cc
C
C
=
C x = c h x hay: C x = c Sx
hx hc
hc
Sc

Chun ha nhiu im (phng php ng chun)

- Chun b mt dy dung dch chun c nng khc nhau trong iu kin tng t mu
phn tch. Tin hnh phn tch sc k v tnh din tch ca pic i vi dy cht cht chun.
Sau dng ng chun hay ng hi quy tuyn tnh biu din s ph thuc chiu cao (din
tch) ca pic vo nng cht phn tch. xc nh nng cht phn tch Cx ta cng thc

77

hin php o i vi cht phn tch, c Sx (hx) nht nh, i chiu vi ng chun ta c nng
cht phn tch trong mu.

Hnh 7.3: ng chun nhiu im xc nh cht phn tch

Phng php ny c u im l sau khi dng ng chun ri th c th s dng
phn tch hng lot trong cng iu kin c kho st. Nu hm lng cht phn tch qu b
nm di gii hn ca ng chun th phi s dng phng php lm giu tng nng ca
cht phn tch n nm trong vng tuyn tnh. Trong nhiu trng hp mu phn tch c
thnh phn phc tp, chng ta cha bit chnh xc nn khng th chun b dy mu u tho
mn nhng quy nh cho phng php ny do khng xc nh chnh xc v tr ca ng
chun, v vy kt qu phn tch mc sai s ln. Trng hp ny khng nn s dng phng
php ng chun m tt nht l s dng phng php thm chun xc nh nng ca cht
phn tch trong mu.
b) Phng php thm chun
- Nguyn tc ca phng php ny l dng ngay mu phn tch lm nn chun b dy
mu u.
- Chun b mu: Ly mt lng mu phn tch nht nh (Cx) ri thm vo nhng lng
chnh xc cht cn xc nh vo C1, C2, C3, C4, C5 ta c dy mu u c hm lng cht xc
nh l:

Co = C x
C I = C x + C1
C II = C x + C 2
C III = C x + C3
C IV = C x + C 4
CV = C x + C5
- Tin hnh phn tch sc k v tnh din tch (chiu cao) ca pic i vi dy mu u.
Sau dng ng chun hay ng hi quy tuyn tnh biu din s ph thuc chiu cao (din
tch) ca pic vo nng cht phn tch.

78

Hnh 7.4: ng chun ca phng php thm chun

Xc nh Cx bng phng php ngoi suy: Ko di ng chun v pha tri, ng ny ct
trc honh ti im Cx, on OCx chnh bng gi tr nng Cx cn tm. Cng c th xc nh Cx
bng cch t gc to O k mt ng thng song song vi ng chun, t im Ao k mt
ng thng song song vi trc honh, hai ng ny ct nhau ti im M, t M h ng vung
gc xung trc honh. im ct ny l Cx. on OCx chnh bng gi tr nng Cx cn tm.
Phng php ny c u im l qu trnh chun b mu d dng, khng cn c nhiu ha
cht c tinh khit cao chun b dy mu u. Mt khc li loi tr hon ton nh hng v
thnh phn ca mu cng nh cu trc vt l ca mu, nhng sai s ngu nhin do thao tc
Phng php ny xc nh c hm lng cht phn tch nng b m phng php ng
chun khng phn tch c v cho chnh xc cao, c nhiu ngi s dng. Nhng phi
ch rng, nng thm ca cht chun phi theo tng bc v khong cch ca cc bc phi
xp x bng Cx th phn ni suy tuyn tnh mi chnh xc.
c) Phng php ni chun
Trong mu, cc cht phn tch c th b nh hng ca nhiu yu t: nh hng nn, cc
iu kin khc gy sai s h thng. loi tr nh hng ny ngi ta thm vo mu cht ni
chun c tnh cht gn ging tnh cht ca cht phn tch, c nng cng gn bng nng
ca cht phn tch (Cx).
Tin hnh 3 php o sc k: mt php cho cht chun ni, mt php cho cht chun c
nng xc nh (t cc cht chun), t rt ra c h s nh hng Fx, php sc k th 3 cho
cht phn tch cha bit v t tnh ra nng ca n. H s Fx chnh l i lng a vo
loi tr nh hng ca nn cng nh cc iu kin o khc.

79

Hnh 7.5: Phng php chun ni xc nh cht phn tch

Cc Ci
C .h
: = F x F x= c i .
hc hi
h c .Ci

Cx Cc
C
=
C x = i .h x .Fx
h x hc
hi
d)Phng php vi sai
- Phng php vi sai nng ln:
Chun b mu:
Dung dch chun 1: C1
Dung dch chun 2: C2 (C2 > C1)
Dung dch phn tch: Cx (Cx > C1)
Tin hnh php tch sc k dung dch chun 2 v dung dch phn tch trn nn dung
dch chun 1 v tnh din tch (chiu cao) ca pic S21 = S2 S1 , Sx1 = Sx S1 . Ta c:

C x C1 Sx1
S
=
C x = x1 ( C 2 C1 ) + C1 .
C 2 C1 S21
S21
- Phng php vi sai nng b:
Chun b mu:
Dung dch chun 1: C1
Dung dch chun 2: C2 (C2 < C1)
Dung dch phn tch: Cx (Cx < C1)
Tin hnh php tch sc k dung dch chun 1 trn nn dung dch chun 2 v v dung
dch phn tch. Tnh din tch (chiu cao) ca pic S12 = S1 S2 , S1x = S1 Sx . Ta c:

C1 C x S1x
S
=
C x = 12 ( C1 C 2 ) + C1 .
C1 C2 S12
S1x
Phng php ny s dng xc nh cc cht c nng qu ln hoc qa b

8. Sc k kh ghp khi ph v mt s ng dng (GC/MS)

8.1. Sc k kh ghp khi ph
GC/MS (Gas Chromatography Mass Spectometry) l mt trong nhng phng php sc k hin i nht
hin nay vi nhy v c hiu cao v c s dng trong cc nghin cu v phn tch kt hp. Thit b GC/MS
c cu to thnh 2 phn: phn sc k kh (GC) dng phn tch hn hp cc cht v tm ra cht cn phn tch,
phn khi ph (MS) m t cc hp phn ring l bng cch m t s khi. Bng s kt hp 2 k thut ny
(GC/MS_Gas Chromatography Mass Spectometry), cc nh ho hc c th nh gi, phn tch nh tnh v nh
lng v c cch gii quyt i vi mt s ha cht. Ngy nay, ngi ta ng dng k thut GC/MS rt nhiu v s
dng rng ri trong cc nghnh nh y hc, mi trng, nng sn, kim nghim thc phm

Sc k kh (GC: Gas Chromatography)

80

Sc k kh c dng chia tch cc hn hp ca ha cht ra cc phn ring l, mi phn c mt gi tr ring bit.

Trong sc k kh (GC) chia tch xut hin khi mu bm vo pha ng. Trong sc k lng (LC) pha ng l mt dung
mi hu c, cn trong GC pha ng l mt kh tr gng nh helium. Pha ng mang hn hp mu i qua pha tnh,
pha tnh c s dng l cc ha cht, ha cht ny c nhy v hp th thnh phn hn hp trong mu.
Thnh phn hn hp trong pha ng tng tc vi pha tnh, mi hp cht trong hn hp tng tc vi mt t
l khc nhau, hp cht tng tc nhanh s thot ra khi ct trc v hp cht tng tc chm s ra khi ct sau. l
c trng c bn ca pha ng v pha tnh, hn na qu trnh chia tch c th xy ra bi s thay i nhit ca pha
tnh hoc l p sut ca pha ng.
Ct trong GC c lm bng thy tinh, inox hoc thp khng r c kch thc, kch c rt a dng. Ct ca
GC di c th l 25m, 30m, 50m, 100m v c ng knh rt nh, bn trong ng knh c trnh bng mt lp
polimer c bit nhphenyl 5% + dimetylsiloxane polymer 95%), ng knh ct thng rt nh ging nh l mt
ng mao dn. Thng thng ct c s dng l semivolatile, hp cht hu c khng phn cc nh PAHs, cc cht
trong hn hp c phn tch bng cch chy dc theo ct ny.
Mt cht chia tch, ra gii phng i ra khi ct v i vo u d. u d c kh nng to ra mt tn hiu bt
k lc no, khi pht hin ra cht cn phn tch. Tn hiu ny pht ra t my tnh, thi gian t khi bm mu n khi ra
gii gi l thi gian lu (TR).
Trong khi cc thit b chy, my s a ra cc biu t cc tn hiu nh hnh 8.1. y gi l sc , mi
mt peak trong sc s miu t mt tn hiu to nn khi cht gii hp t ct sc k v i vo u d detector, trc
honh biu din thi gian lu v trc tung biu din cng ca tn hiu, trong hnh 1 mi nh (peak) biu din mt
cht ring l, cht ny c tch t hn hp mu phn tch, peak c thi gian lu (T R) 4,97 pht l odecan , 6.36 pht
l biphenyl, 7.64 pht l chlolobiphenyl, 9.41 pht l hexadecanoic acid methyl ester.
Nu trong cng iu kin sc k nh nhit , loi ct gng nhau th cng cht lun c thi gian lu ging
nhau, khi bit thi gian lu ca hp cht th chng ta c th chp nhn c nhy ca n. Tuy nhin, cht c tnh
cht ging nhau th thng c thi gian lu ging nhau.

Hnh 8.1: Sc ca sc k kh
Khi ph:

- S xut hin ph khi lng:

Khi cho cc phn t trng thi kh va chm vi mt dng electron c nng lng cao th t cc phn t s bt
ra mt hay hai electron v n tr thnh cc ion c in tch +1 (chim t l ln) v +2.
Gi thit phn t cht nghin cu l ABC th:

ABC + e

ABC+ + 2e

(1)

ABC+2 + 3e

81
(2)

Loi ion ABC+ c gi l ion gc hay ion phn t.

Nu cc ion phn t ABC+ tip tc va chm vi chm electron c nng lng cao th chng s b ph v
thnh nhiu mnh ion, thnh cc gc hoc cc phn t trung ha khc nhau. Qu trnh ny c gi l qu trnh phn
mnh (gracmentation).

ABC+

.
A+ + BC

ABC+

.
AB+ + C
.
A+ +B

Nng lng ca qu trnh phn mnh ch vo khong 30-100eV, hn nhiu so vi nng lng ion ha ca phn t (815eV). Qu trnh bin cc phn t trung ha thnh cc ion c gi l s ion ha.
Trn th hnh di y:
I

AB+
ABC+

A(eV)
20

40

60

80

Hnh 8.2: Xc sut c mt ion mnh axeton.

100

Cho thy: xc sut c mt ca cc ion ph thuc vo nng lng va chm phn t. Nng lng khong
15eV th ion phn t ABC + t cc i nhng cc mnh ion AB + tip tc tng n nng lng70eV th tng chm v
t gi tr cc i.
Cc ion c khi lng ion v in tch e. T s m/l c gi l s khi z. Bng cch no , tch cc ion c s
khi khc nhau ra khi nhau v xc nh c xc sut c mt ca chng ri v th biu din mi quan h gia xc
sut c mt (hay cng ph khi lng I) v s khi z, tc l: I = (

m
) th th ny gi l ph khi lng.
e

%B

100
50
O

m/e
Hnh 8.3: Ph khi lng.

82

- Khi ph: c dng xc nh mt cht ha hc da trn cu trc ca n. Khi gii hp cc hp cht ring
l t ct sc k, chng i vo u d c dng in ion ha (mass spectrometry). Khi , chng s tn cng vo cc
lung, do chng b b thnh nhng mnh vn, nhng mnh vn ny c th ln hoc nh;
Nhng mnh vn thc t l cc vt mang in hay cn gi l in, iu ny l quan trng bi v cc ht cn
trng thi tch in th mi i qua c b lc. Cc khi nh chc chn, khi ca mnh v c chia bi cc vt mang
gi l t l vt mang khi (M/Z);
Hu ht cc mnh vn c in tch l +1, M/Z thng miu t cc phn t nng ca mnh vn.Nhm gm c
4 nam chm in gi l t cc (quadrapole), tiu im ca cc mnh vn i xuyn qua cc khe h v i vo u d
detector, t c c thnh lp bi phn mn chng trnh v hng cc mnh vn i vo cc khe ca khi ph.

Phn tch kt qu

My tnh s ghi li cc biu ca mi ln qut. Trc honh biu din t l m/z cn trc tung biu din cng
tn hiu ca mi mnh vn c qut bi u d detector. y l th ca s khi.
Trong hnh 19, hnh nh khi cao nht l dodecane, phn nm ca GC/MS n ging nh l mt th vin hnh
nh dng nhn ra cc cht cha bit tn ti trong hn hp mu. Th vin ny c th so snh hnh nh khi t thnh
phn ca mu vi hnh nh khi trong th vin ca my.

Hnh 8.4: Mass-spectrum

8.2. Mt s thnh cng trong phn tch lng vt
V d : Tinh du t r v thn cy pmu thu c bng phng php chng ct li cun hi nc. Cc ch
tiu ho - l ca tinh du c xc nh bng phng php phn tch ho hc. Thnh phn v cu trc ca mt s cu
t chnh c xc nh bng phng php phn tch sc k - khi ph (GC-MS). Kt qu thu c cho thy, hm
lng mt s cu chnh trong tinh du l E-Nerolidol (14.91% trong thn, 11.03% trong r), Elemol (13.56% trong
thn, 31.29% trong r), -Eudesmol (13.09% trong thn, 31.43% trong r), -Eudesmol (5.34% trong thn, 12.03%
trong r).
- Thnh phn ho hc ca tinh du pmu:
Ph GC-MS ca tinh du r v thn pmu c trnh by hnh 21 v 22.

83

Hnh 8.5: Ph GC-MS ca tinh du r pmu

Hnh 8.6: Ph GC-MS ca tinh du thn pmu

84

C. BI TP
Bi 1:
Tnh s a l thuyt hiu lc nef v chiu cao ca a l thuyt hiu lc Hef ca h sc k c tnh cht sau:
t RA = 280 s; t RB = 300 s; WA = 14s; WB = 15s. ,thi gian cht l tm=10s, ct c chiu di l 3,2m.
Gii:
Theo l thuyt ta c s a l thuyt hiu lc nef l:
k '2
n =n
; vi k l t s dung tch.
( k '+1) 2

t t
nef = 16 R M
W

280
nA = 16
= 6400
14
2

300
nB = 16
= 6400
15
T ta c:

t R t M 280 10
=
= 27;
tM 10

i vi pic A: k ' =

27
nef A = 6400
= 5951, 07
27 + 1
t t 300 10
i vi pic B: k ' = R M =
= 29;
tM 10
2

Vy,

29
nef B = 6400
= 5980, 4
29 + 1
300
nef = 5065, 7 ; H ef =
= 0, 053 .
5965, 7

Bi 2:
Pic sc k ca hp cht X c pht hin sau 15 pht sau khi a mu vo
(lc ca hp cht Y khng c gi bi vt liu ca ct xut hin qua 1,32 pht). Pic ca cht X c dng ng
phn b Gauss vi b rng ca y l 24,2s. di ca ct l 40,2cm.
1. Tnh s a l thuyt trong ct.
2. Tnh H ca ct.
3. Tnh T v ca ct.
4. Tnh ch s lu gi ca cht X.
5. T phng php chun b bit rng th tch ca cht lng c gi trn b mt ca cht mang ca ct bng
9,9ml. Th tch ca pha ng bng 12,3ml. Hy tnh h s phn b X.
6. Tiu chun phn chia X v Y l bao nhiu nu yu t phn chia i vi X v Y bng 1,011.
Gii:
1. S a l thuyt (n):
2

t
15.60
n X = 16 RX = 16
= 22130
24,2
WX
2. Tnh H:
L
L
40,2
H = =
= 1,82.10 3 (cm)
Ta c n =
H
n 22130
3. Tnh v T:
T n =

2
= n.H 2 = 22130.(1,82.10 3 ) 2 = 0,27(cm)
2
H
85

 .tR

Ta c: T =

15.0,27
= 0,101( phut )
40,2

4. Ch s lu gi ca cht X:
Ta c:

R=

tm 1,32
=
= 0,088
tR
15

5. H s phn b X (kX):
Ta c: R =

Vm
V (1 R ) 12,3(1 0,088)
kX = m
=
= 12,9
Vm + k .VS
R.VS
0,088.9,9

6. Tiu chun phn chia (Rs):

n
1
(1 ) =
4

Ta c: RS =

22130
1
(1
) = 0,4 vi
4
1,011

l yu t phn chia.

Bi 3: Cho h sc k c tnh cht sau:

t RA = 280 s; t RB = 300 s; WA = 14s; WB = 15s. Ct c chiu di l 3,2m. Thay i chiu di t 320 cm ln

400 cm. Tnh phn gii?

t RA = 280.

Gii:

400
= 350( s )
320
2

t RB = 300.
2

t
280
nA = 16. RA = 16
= 6400
14
WA

400
= 375( s)
320
2

t
300
nB = 16. RB = 16
= 6400
15
WB

400
t
n = 16. R = 6400.
= 8000
320
W
2

16.t 2R
tR
W=
n
W

Ta c: n = 16.

16.(350) 2
= 15, 65( s)
8000
2. ( 375 350 )
R=
= 1,54
15, 65 + 16, 77

WA =

WB =

16.(375) 2
= 16, 77( s)
8000

Bi 4:
Mt ct sc k mao qun di 30m, chy mt cht chun hai tc pha ng khc nhau, cho cc s liu sau:
Tc (cm/s)
S a l thuyt
0,5
150 000
120 000
Xc nh vn tc ti u,chiu cao a l thuyt cc tiu v s a l thuyt vn tc ti u ,cho A=0.
Gii
TN1
H1 = 3000/150000 = 0,02 cm
TN2
H2 = 3000/120000 = 0,025 cm.
Ta c phng trnh:

B
+ 0,5C
0,5
B
0, 025 = + C
1
0, 02 =

Gii phng trnh ta c : C= 0,02 s ; B = 0,005 cm2/s.

U ti u = (B/C)1/2 = 0,5 cm/s
Hmin = 2(B.C)1/2 = 0,02 cm.
S a l thuyt vn tc ti u l:

n=

3000
= 150000 .
0, 02

Bi 5:

86

Mt hn hp cha metyl xiclohexan,metyl xiclohexen v toluen c tch bng phng php sc k lng kh
trn ct nhi di 42 cm. Sc k cung cp thi gian lu ca chng ln lt l 10,1 ; 11,0 ; 13,5 v chiu rng pic ln
lt l 0,75 ; 0,80 ; 1,05 pht. Hy tnh:
4. S a l thuyt i vi mi pic, gi tr trung bnh, lch chun ca n v chiu cao a l thuyt ca ct.
5. phn gii gia metyl xiclohexan v metyl xiclohexen,gia metyl xiclohexen v toluen. Cho bit kh c
s dng l khng kh vi tR(KK) = 1,2 pht (khng kh c xem l cu t khng b lu gi bi ct).
6. Hy tnh chiu di ct phn gii gia cc cu t t yu cu ti thiu l 1,5.
Gii
K hiu A : metyl xiclohexan
B: metyl xiclohexen
C: toluen.
1. S dng phng trnh : n = 16(

tR 2
) tnh c s a l thuyt
w

A: n = 2902
B: n = 3025
C: n =2645
ntb = 2900

H=
n=

L
420
=
= 0, 015cm
ntb 2900

2
= n.H 2 = 2900.(0, 015) 2 = 0,81(cm)
2
H
2. phn gii gia metyl xiclohexan v metyl xiclohexen,gia metyl xiclohexen v toluen

Rs , A B =

2t R
= 1,16
wA + wB

Rs , B C =

2t R
= 2, 70
wA + wB

3.Chiu di ct phn gii gia cc cu t t yu cu ti thiu l 1,5.

V Rs,A-B = 2,70>1,5 nn ch cn tnh chiu di phn gii gia A-B t yu cu 1,5.
Gi n1,n2 l s a l thuyt ca ct c v ct mi,ta c:

Rs ,1
Rs ,2

n1
n2

1,16
2900
=
n2 = 4800
1,50
n2

Chiu di ct phn gii tng ln 1,5:

L2 = H.n2 = 0,015. 4800 = 73 cm.
Bi 6: Mt php sc k kh s dng ct di 36 m, chy cht chun 3 th nghim vi tc pha ng khc nhau cho
cc pic c thi gian lu v rng pic nh sau:
Th nghim
u (cm/s)
tR (s)
1
10
300
2
20
250
3
30
150
Xc nh vn tc ti u, s a l thuyt cho ct vn hnh vn tc ti u

w (s)
20
16
10

Gii :
Theo phng trnh Van-Deemter, mun xc nh c vn tc ti u, H min v s a l thuyt ta cn xc nh
cc h s A, B, C. Mun vy ta xc nh s a l thuyt v chiu cao a l thuyt cho tng th nghim :
Ta c : L=36 m = 3600 cm
2

TN 1:

300
n1 = 16
= 3600
20

TN 2:

250
n2 = 16
= 3906
16

H1 =

3600
= 1,00(cm)
3600

H2 =

3600
= 0,92(cm)
3906

87

150
n3 = 16
= 3600
10

TN 3:

H3 =

3600
= 1,00(cm)
3600

Thay cc s liu vo phng trnh trn ta c h phng trnh sau:

1,00 = A +

B
+ 10C
10

0,92 = A +

B
+ 20C (2)
20

1,00 = A +

B
+ 30C (3)
30

(1)

Gii h gm 3 phng trnh (1), (2) v (3) ta c:

A= 0,36 (cm), B = 4,8 (cm2/s) v C = 0,016 (s)
Vn tc ti u, Hmin v s a l thuyt c tnh l :
utoiuu =

B
=
C

4,8
= 17,32(cm / s )
0,016

H min = A + 2 B.C = 0,36 + 2 4,8.0,016 = 0,914(cm)

n=

L
3600
=
= 3939
H
0,914

88

D. KT LUN
Qua mt thi gian tm hiu, ti thu c mt s kt qu nh sau :
-

Tng quan c ti liu v a ra c nhng vn chung nht v c bn nht trong l thuyt ca phng

php sc k kh nh: nguyn tc ca sc k kh, thit b k thut sc k kh, mt s ng dng c bn ca sc k kh,

cc iu kin ti u nhm gip vic phn tch t hiu qu cao nht ... v trn c s a ra kt lun v u nhc
im ca sc k kh.
-

Su tm mt s bi tp c li gii v sc k.

T nhng g ti tm hiu c, mt ln na gip ti khng nh rng sc k kh tht s l mt phng tin u tay

cho cc nh khoa hc.
Mc d c gng nhiu nhng do thi gian v trnh c hn nn bi tiu lun ca ti khng trnh khi thiu
st. V vy ti rt mong nhn c s gp ca thy cng nh cc bn kin thc ca ti c hon chnh hn.

TI LIU THAM KHO

1. Trn T Hiu, T Vng Nghi, Nguyn Vn Ri, Nguyn Xun Trung (2007), Ha hc phn tch, Tp 2:
Cc phng php phn tch cng c, Nh xut bn Khoa hc v K thut.
2. Phm Hng Vit (2005), Sc k kh C s l thuyt v kh nng ng dng, Nh xut bn i hc Quc gia
H Ni.
3. Trn Vn Sung (2005), Cc phng php phn tch vt l trong ha hc, Nh xut bn i hc Quc gia H Ni.
4. o Hu Vinh, Nguyn Xun Dng, Trn Th M Linh, Phm Hng Vit (1985), Cc phng php sc k, NXB
Khoa hc v k thut.
5. TS. Nguyn Vn T, Bi ging chuyn ha phn tch hin i, Khoa Ha trng HSP Hu

Chng 4. SC K IN DI MAO QUN HIU NNG CAO

1. Gii thiu v phng php HPCEC

1.1. S ra i v pht trin
Nm 1937: Sc k in di c nghin cu ln u tin bi Tiselius (Thy in) khi ng tch c cc
cu t trong huyt thanh (, , globumin). Sau c ng pht trin trn c s nghin cu tch v phn tch

89

cc hn hp protein, amin, amino axit th thp (110 220V) v c gi l in di c in. Nh kt qu

nghin cu v xut ra k thut tch mi ny nn Tiselius nhn c gii thng Nobel nm 1949.
Nm 1953: Edstrom dng si mao qun ng knh 15 m tch c lng nh ADN.
Nm 1967: Hjerten nghin cu mt cch c h thng v m t chi tit, cng nh hon chnh c s l
thuyt sc k in di mao qun trong ng h. sau l cc nh khoa hc Later Virtamen, Mikkers nghin
cu v pht trin tip k thut ny trn ng mao qun bng thy tinh v teflon. Tip nh khoa hc Jorgen
son phn loi l thuyt v cc kiu CE v m t c im v cch hot ng ca mi loi.
Gia thp k 80: sc k in di mao qun hiu nng cao (HPCEC) c nghin cu v pht trin, n
l s kt hp gia in di c in vi tnh cht ca ct sc k kh mao qun v cc detector c nhy cao
ca k thut HPLC. Song mun thc hin in di c trong ng mao qun th phi p vo 2 u mao qun
th cao (15 50 kV), v vy c tn l sc k in di mao qun th cao.
Cui nm 1992: mt s hng nh ISCO, HP, Berkman bt u a ra th trng mt s h thng
my CEC u tin.
Nm 1994: thi k pht trin ca HPCEC.
Nm 1995: cc nc tin tin p dng k thut phn tch ny trn nhiu lnh vc khoa hc, kinh t,
nng cng nghip v y dc.
T nm 1995 n nay, k thut HPCEC c bc pht trin nhy vt. Hin nay k thut phn tch HPCEC
v ang pht trin mc cao v c ng dng rt hiu qu trong nhiu lnh vc khc nhau ca khoa hc
cng ngh, nht l y dc v sinh hc.

Hnh 1. S pht trin ca HPCEC (giai on t nm 1980 n 1997)

90

1.2. Phn loi cc kiu HPCEC

Sc k in di mao qun rt a dng, c nhiu kiu (mode) t n gin n hon chnh v phc tp.
Ty theo c ch, bn cht v c im ca s tch xy ra trong ng mao qun m ngi ta phn thnh cc
kiu khc nhau nh sau:
Bng 1. Phn loi cc kiu in di mao qun
Kiu in di

Bn cht ca s tch v c trng

- S in di dung dch t do theo vng mu.

in di mao qun vng

(Capillary Zone
Electrophoresis-CZE)

- S tch cc cht xut hin do s di chuyn ca cht tan

trong mao qun theo mi vng ca cht ring r, chng di
chuyn theo tc khc nhau v do dng EOF trong ng mao
qun l nhn t quyt nh di tc dng ca lc in trng
E.

- S tng tc ionic hay hydrophobic vi Micell.

Sc k in di mao qun
- Bn cht, trung tm ca s tch l s hnh thnh cc Micelle
in ng Micell (Micellary
trong mao qun, cc tiu phn ny s dn dt v ng gp
Capillary
Electro-Kenetic
thm kh nng ca qu trnh tch cc cht trn nn dung dch
Cromatoghraphy-MEKC)
m v cht in di trong ng mao qun.
Sc k in di mao qun
gel
(ry
phn
t)
((Capillary
Gel
Electrophoresis-Gel-CEC)

Tch theo kch thc v in tch cht tan.

- Da vo tnh cht ca im ng in pI v vng mu.

Sc k in di mao qun
- S tch sc k y l da trn c s ca s in di kt hp
hi t ng in ((Capillary
vi s khc nhau gi tr pI ca cht tan v s hi t ng in
Isoelectric
Focusing
trong mt iu kin nht nh hi t vng mu gn li, lm
Chromatography- CIEFC)
giu cht phn tch.
Sc k in di mao qun
ng tc (Capillary
Isotachophores
Chromatography-CITEC)

Da vo tnh cht ng tc nht thi.

1.3. c im ca sc k in di mao qun hiu nng cao

Phng php HPCEC c cc c im sau y:
1. C hiu lc tch cc cht rt cao trong ng mao qun thy tinh, ng teflon (ng knh trong 25100 m).
2. Th in di rt cao, thng t 10 50 kV ( 150-550 V/cm ) t hai u ng mao qun to in
trng E.
3. S a hiu lc ca ct tch l rt ln, thng t 105 n 106 a /m nn cho kt qu tch tt i vi
nhng cht phc tp.
4. Thi gian tch hn hp nhanh, khong 5 20 pht.

91

5. Vic pht hin hu ht cc cht bng detector UV hay UV-VIs (trc tip hoc gin tip) c thc
hin ngay trn mt on nh ca u cui ng mao qun nn khng cn b flowcell nh trong HPLC (Hnh
2).
6. Lng mu np vo ct tch rt nh, khong 5 - 20nL (nh hn lng mu ca HPLC trn 1000
ln) nn rt ph hp tch cc mu c khi lng rt nh, nh mu mu bnh nhn, vt liu qu him.
7. C nhiu kiu tch (bng 1) nn kh nng ng dng thc t rt rng ri, a dng v phong ph. c
bit thch hp cho nhiu i tng phc tp nh cc mu sinh hc.
8. S tch ch yu thc hin trong dung mi nc c cht m pH v cht in ly nn khng tn nhiu
dung mi t tin nh trong HPLC.
9. Vic vn hnh tch v phn tch khng phc tp, cng d dng tng t HPLC.
10. C th s dng nhiu trang b ca HPLC nh my t ghi, tch phn k. Do vy c s no c
HPLC th d dng pht trin v ng dng HPCEC.
11. T ng ha c trong qu trnh tch v phn tch hng lot mu.
12. Do ng knh trong ca mao qun nh nn n gii hn v lm kh khn dng chy, v th d xut
hin hiu ng nhit Jun khng c li. Do phi khng ch nhit ca mao qun m bo thu c kt
qu phn tch n nh.

Hnh 2. Mt on ng mao qun trong HPCEC c xem nh mt Fowcell

Vi nhng c im trn, k thut phn tch HPCEC c pht trin rt nhanh v ng dng nhiu
trong y hc, ha hc, sinh hc, dc, nng nghip vi tc cha tng thy trong cc k thut phn tch.

2. Mt s i lng trong HPCEC

2.1. Tc in di, in di, thi gian in di
2.1. 1. Tc in di
S tch cc cht bng in di da trn c s tc di chuyn khc nhau ca cc cht trong ng mao
qun trn nn dung dch m v cht in li thch hp di tc dng ca in trng E thch hp do th V
t vo 2 u ng mao qun sinh ra. Tc di chuyn ca cht i c tnh theo cng thc:

vi = iE = i.

V
L

(2.1)

Trong : vi l tc in di ca cht i trong ng mao qun;

i l in di ca cht i;
92

V l th t vo 2 u ng mao qun (V);

E l cng in trng gia 2 u ct mao qun do th V sinh ra;
L l chiu di tng ca ng mao qun (cm).
2.1.2. in di
Theo cng thc (2.1) ta c:

i =

vi
E

(2.2)

Trong iu kin tc dng n nh, lc in trng F E v lc cn Ff (lc ma st) cn bng nhau

(cng ln nhng ngc chiu), FE = Ff , do :

qE = 6R.vi

(2.3)

Trong : l nht ca dung dch; R l bn knh ca ion i.

T (2.1), (2.2), (2.3) ta c biu thc in di i:

i =

q
6 R

(2.4)

Theo (2.4) ta thy:

ln ca in di i t l thun vi in tch ion q v t l nghch vi nht ca dung dch , bn
knh ca ion R, ngha l trong in trng E nht nh th:
Phn t no c in tch ln v kch thc nh s di chuyn nhanh.
Cc ion cng in tch th ion no c kch thc nh hn s di chuyn nhanh hn.
Cc ion cng kch thc th ion no c in tch ln hn s di chuyn nhanh hn.
in di i ca cc cht c xc nh bng thc nghim trong iu kin chun c gi in di
chun i0. Nhng trong thc t, cc dung dch trong ng mao qun khng phi trong iu kin chun hay ch
duy nht cht nghin cu m cn c cc cht khc. phn nh ng v ph hp cc iu kin thc nghim
trong ng mao qun ngi ta dng in di hiu lc ef. Gi tr ef ngoi ph thuc bn cht, kch thc,
in tch ca cht phn tch, in th V t vo 2 u ng mao qun th cn ph thuc:
Gi tr pH v nng ca dung dch m in gii trong mao qun;
Thnh phn v nng cht in gii trong pha ng;
Hng s in li K ca cht tan (axit, bazo, phc, );
Cht hot ng b mt, cht ph gia trung tnh trong pha ng;
Dung mi hu c thm vo pha ng in di hay mu phn tch,
2.1.3. Thi gian in di
Khi in di, thi gian cn thit cho mt cht tan di chuyn t lc c np vo u ng mao qun n khi
n n v tr trung tm flowcell ca detector, c pht hin v tr pic sc k cc i, c gi l thi gian in di
ca cht tan. Tng t nh trong HPLC, ngi ta cng gi l thi gian lu hay thi gian ra gii ca cht tan ti.
Thi gian in di c tnh theo cng thc:

ti =

l
L.l
=
vi V i

(2.5)

Trong :
93

l l chiu di hiu lc (cm) (tnh t im mu c np n v tr trung tm ca flowcell ca detector);

L l chiu di tng ca ng mao qun (cm), L > l;
V l in th t vo 2 u ng mao qun (V).
V cht tan di chuyn trong dng in di thm thu (EOF) nn thi gian in di v cc thng s thc
nghim khc c th c dng tnh ton in di phi l gi tr tng s tot ca cc phn t cht tan. Do
in di ton phn trong mao qun ca cht tan i l (E l cng in trng to bi in th V):

vi
l
L.l
=
=
E ti .E V .ti

tot = ( i + EOF ) =

(2.6)

Suy ra thi gian lu ca cht tan i l:

ti =

L.l
V tot

(2.7)

2.2. H s phn b v h s dung tch

2.2.1. H s phn b Ki
Trong qu trnh in di, nu ng mao qun c pha tnh (gi hoc tht) th lun c s phn b cht tan
gia 2 pha l pha tnh v pha ng. S phn b ny cng l mt qu trnh cn bng ng hc ca s in di.
c trng cho qu trnh phn b ny l hng s cn bng hay h s phn b Ki.
H s phn b Ki cho bit trong iu kin in di c th th cht tan i phn b nh th no gia 2 pha
trong ng mao qun:

Ki =

Ci ( SP )

(2.8)

Ci ( MP )

Trong : Ci(SP) v Ci(MP) ln lt l nng ca cht tan i trong pha tnh (SP) v pha ng (MP).
H s phn b Ki c xc nh bi:
Bn cht v cc c trng ca pha tnh;
Bn cht, thnh phn, cc cht c trong pha ng v gi tr pH ca n;
Lc in trng E, tc l th V t vo 2 u mao qun;
Nhit ca mao qun.
Theo quan im ca nhit ng hc, h s phn b K i

ln(Ki) =

H 0 S 0
+
RT
R

c xc nh bi cng thc sau:

(2.9)

Trong : H0, S0 ln lt l entapi chun, entropy chun ca cht tan;

R l hng s kh; T l nhit tuyt i trong ng mao qun.
2.2.2. H s dung tch ki
'
H s dung tch ki cho bit trong iu kin in di c th th cht tan i tn ti nh th no trong pha

tnh ca ng mao qun v c xc nh bng cng thc sau:

ki' =

(ti t0 )
[t0 .(1 ti / t0 )]

(2.10)
94

ki' = Ki .

Hay

VSP
VMP

(2.11)

Trong :

ti l thi gian lu ca cht tan i trong mao qun;

t0 l thi gian khng lu gi ca cht tan i trong mao qun;
Ki l h s phn b;
VSP,VMP l th tch ca pha tnh v pha ng;
VSP
c gi l t s pha ca h ct tch.
VMP
Vic iu chnh t s pha s lm thay i h s dung tch k i. Mt khc nu t s pha khng i th h
s dung tch ca cht tan coi nh l hm ca h s phn b K i ca chng. Chnh mi quan h ny to iu
kin ti u ha cc iu kin in di, t c c k i thch hp thu c kt qu sc k tt nht.
Ngoi ra ki cn ph thuc pH ca pha ng. Li dng iu ny ngi ta thay i iu kin pha ng nh
cht m, pH m, thnh phn v nng cht in gii c ki thch hp.
Nu trong ct tch l h pha MEKC th i lng ti / t0 c thay bng ti / tMC vi tMC l thi gian
khng lu gi ca pha Micell. T tnh c thi gian lu tng ca cht tan l:

t0 .t MC (1 + ki' )
ti =
tMC + t0 ki'

(2.12)

Nu tMC << t0ki th ta c:

t MC (1 + ki' )
ti =
ki'

(2.13)

T (2.12) v (2.13) ta thy: khi ki ln th thi gian in di ti s di v ngc li.

2.3. Chiu cao a, s a v s a hiu lc ca mao qun
S khc nhau cn thit v in di ef cho 2 vng mu ca 2 cht tan tch khi nhau ph thuc cc
iu kin in di v rng ca vng cht tan tn ti trong ng mao qun.
rng ny li ph thuc qu trnh phn tn ca cht tan trong mao qun khi n di chuyn di tc
dng ca in trng E. S phn tn thng gy ra do s m rng vng cht tan trong mao qun. N l kt
qu ca s di chuyn khng ng u ca cc cht tan trong vng mu , v c coi nh bng rng
ca pic sc k in di Wb, Wb = 4, l lch chun.
S a l thuyt N ca ng mao qun c tnh theo cng thc :

.V .l
l
N = ( ) 2 = ef

2 Di .L

vi

2 = 2 Di ti

Trong : l l chiu di hiu lc ca ng mao qun;

Di l h s khuch tn ca cht tan i trong pha ng;
ti l thi gian lu ca cht tan i.
T ta c chiu cao ca mt a l thuyt l:

95

(2.14)

H=

2 Di .L
l
=
N
i .V

(2.15)

Mt khc ta li c:

2 Di 1
ki'
d 2V
+ .
.
H=
V
16 (1 + ki' ) 2 Di
Trong :

(2.16)

ki' l h s dung tch ca cht tan i;

Di l h s khuch tn ca cht tan i trong pha ng;
d l ng knh trong ca ng mao qun;
V l in th t vo 2 u ng mao qun.

Tng t nh trong HPLC s a l thuyt ca ct mao qun cng c th tnh trc tip t sc theo
cng thc:

N = 5,54.(
Trong :

tR 2
)
W1/ 2

(2.17)

tR l thi gian lu ca cht tan i,

tR =

L.l
V .i

W1/2 l rng na chiu cao pic;

Trong thc t th ngi ta hay dng cng thc (2.17) xc nh s a l thuyt N hn l dng cng
thc (2.14).
c trng cho hiu lc ca s tch trong cc iu kin c th nht nh m c k n s m rng
vng mu th ngi ta dng khi nim s a l thuyt hiu lc Nef v tnh theo cng thc sau:

N ef = (

l 2
)
tot

(2.18)

Trong :
2
l l chiu di hiu lc; ( tot ) l lch chun c tnh n cc yu lm m rng vng mu, bao gm:

( D ) 2 - s khuych tn thng; ( ln J ) 2 - s np mu; ( Temp ) 2 - s gradient nhit v hiu ng nhit Jun;

( ad ) 2 - s hp ph ca thnh mao qun; ( det ) 2 - ca detector; ( elect ) 2 : do yu t in v in tch.
Nh vy:

( tot ) 2 = ( D ) 2 + ( ln J ) 2 + ( Temp ) 2 + ( ad ) 2 + ( det ) 2 + ( elect ) 2

2.4. Tc tuyn tnh

Tng t nh trong HPLC, trong cc h HPCEC, tc tuyn tnh u cho bit trong mt n v thi
gian, vng mu ca mt cht trong mao qun di chuyn c mt on l bao nhiu. n v ca u l
( mm/s).
Tc tuyn tnh u c xc nh bi cc yu t:
Th mt chiu V t vo 2 u ca mao qun;
Bn cht ca cc cht, kch thc v in tch ca n;
Bn cht, thnh phn v pH ca pha ng in di.
96

Ngoi ra u chu tc ng ca cc yu t:
Cc c trng b mt ca ng mao qun;
Cc cht ph gia, dung mi hu c,thm vo pha ng;
Nhit ca mao qun.
So vi HPLC, trong HPCEC th cc gi tr tc u 2 bn gi tr u 0 ti u thay i rt t. iu ny
cho php chn cc gi tr u mt vng rng xung quanh u 0 m vn cho kt qu tch in di tt. Cng v vy
m trong HPCEC vic tnh ton chnh xc tc tuyn tnh u khng yu cu nghim ngt nh trong HPLC.
Mt cch gn ng, i lng u c tnh theo cng thc:

u=

l
ti

(2.19)

Trong : l l chiu di hiu lc ca mao qun; ti l thi gian lu ca cht.

2.5. phn gii
phn gii R ca 2 cht i v j lin nhau (pic sc k cnh nhau) c nh ngha nh sau:

Rij =

2(ti t j )
(Wi + Wj )

(ti t j )

(2.20)

Trong : ti , t j l thi gian lu ca cht tan i v j ( giy hoc pht ) v ti > t j ;

wi , w j l rng y ca pic 2 cht i, j ( giy hoc pht );
l lch chun.
phn gii Rij ca 2 cht i v j trong HPCEC cng c th c biu din theo s a l thuyt hiu
lc Nef nh sau:

Rij = 0.25 N ef .

tb

vi

N ef =

ef .V l
.
2 Di L

(2.21)

Trong :
= i j l hiu in di hiu lc ca 2 cht i v j;

tb =

i + j
l trung bnh cng in di hiu lc ca 2 cht tan i v j.
2

Ta c th vit li biu thc phn gii Rij nh sau:

Rij = 0, 25.( N ef ).( ).

Hay:

Rij = 0, 25. ( i + EOF ).

V
Di .( tb + EOF )

(2.22)

V .l ij
.(
)
2 Di L tb

(2.23)

Theo (2.22) th Rij ph thuc vo cn bc 2 ca Nef v V nhng theo (2.23) th Rij ph thuc tuyn tnh
vo ij ca 2 cht tan k nhau. Cng theo (2.21) th R ij t cc i khi tb v EOF bng nhau nhng ngc
du, tc l tc di chuyn ca dng ion cng ln vi tc dng in di thm thu EOF nhng ngc
97

chiu nhau. Lc ny thi gian in di s rt di. Do vy cn cn i gia thi gian phn tch v phn gii
t hiu qu tch tt nht.
2.6. Hiu ng nhit v s gradient nhit
2.6.1. Hiu ng nhit Jun v nh hng ca n
* Khi nim: Hiu ng nhit Jun y l nhit c sinh ra (lm nhit ng mao qun tng) khi c
mt dng in i chy qua dung dch trong ng mao qun. S tng nhit ny c quyt nh bi cng
sut ca dng in qua ng mao qun (tch s i.V).
* nh hng ca hiu ng nhit Jun: hiu ng nhit Jun gy ra s gradien nhit trong mao qun,
dn n thay i nht tng vng trong mao qun. iu ny lm cho cc phn t ca ngay mt cht
cng di chuyn khng ng nht, kt qu lm dong chn pic sc k, gim s a hiu lc v lm gim hiu
qu tch.
S gradien nhit gia tm v thnh ng mao qun ph thuc vo ng knh ca ng mao qun,
dy thnh mao qun, dy ca lp polime ph ln thnh mao qun v h s truyn nhit ra mi trng
xung quanh. S ph thuc ny c m t gn ng theo cng thc:

W.(r1 ) 2 1
r3
r2
1
1 1
Ttot =
.
ln(
)
+
ln(
)
+
(
).( )

2
k
r
k
r
r
h

1
1
2
2
3

(2.24)

Trong :
W l cng sut sinh nhit ( w/mm2 );
r1, r2, r3 ln lt l bn knh mao qun trong, ngoi v c lp PVC ph ngoi (m);
ki l dn nhit vng i ( i=1,2,3);
h l tc truyn nhit t mao qun ra ngoi.
Bng 2. S chnh lch nhit gia tm v thnh mao qun
( vi V = 350V/1cm, thnh mao qun dy 250 m)
Nhit (K)

Bn knh trong ng
mao qun (m )

St thnh

tm

S chnh lch nhit

, T

25

299,00

299,55

0,55

50

301,20

302,59

1,39

75

304,40

307,64

3,24

100

307,00

312,58

5,58

125

311,60

320,32

8,72

150

324,80

335,90

11,10

Thc nghim cho thy khi ng knh trong ca mao qun cng ln th s chnh lch nhit bn
trong v bn ngoi mao qun cng ln, tc s gradien nhit cng mnh. Vi cc mao qun ng knh
trong 15 25 m th hiu ng nhit Jun hu nh khng ng k. Song khi dng mao qun nh nh th
( di 25 m) th lng mu np vo l qu nh (ch 2 3 nL) nn kh np mu chnh xc hoc vic pht
hin cht phn tch gp kh. V th nn dng mao qun c ng knh trong t 25 - 75 m.

98

2.6.2. Kim tra v khng ch hiu ng nhit

S gradien nhit do hiu ng nhit Jun gy ra l mt iu bt li v l nhc im cng nh l kh
khn ca k thut sc k in di mao qun. V vy vic kim tra v khng ch nhit ca mao qun hng
nh l iu rt cn thit. Sau y l mt s phng php:
1. iu ha bung mao qun bng dng khng kh mt c nhit mong mun, hay khng kh nhit
phng th nghim. Cch ny n gin, d thc hin nhng vic khng ch nhit ch t c trong
phm vi 20C.
2. iu nhit mao qun bng cht lng nh my iu nhit. C 2 cch thc hin:
iu ha mao qun bng dng nc sch mt c nhit c khng ch nh my iu nhit
(ng mao qun t trong hp nc c iu nhit). Cch ny ch p dng khi in th V t vo
2 u mao qun di 18 kV, cn khi V cao hn th rt nguy him.
iu ha mao qun bng dng du tr (khng dn in th cao) c khng ch nhit mong
mun (ng mao qun t trong hp du tr c iu nhit). Cch ny rt tt, c th dng khi th
n 40 kV.
2.7. S tng tc ca cht phn tch vi thnh mao qun
2.7.1. Khi nim v s tng tc
S tng tc ca cht phn tch vi thnh ng mao qun trong CEC l mt yu t lun xy ra v l bt li
cho s tch cc cht. S tng tc ny c th theo cc c ch sau:
Tng tc tnh in ca cc phn t c in tch tri du;
Tng tc hp ph vt l ca b mt;
Tng tc phn tn ca b mt xp ht pha tnh (khi mao qun c pha tnh).
Cc tng tc c th xy ra ng thi, hoc cng c th ch u tin mt trong ba c ch trn, ty
thuc cc iu kin in di, nh pha ng, pH, th V v loi mao qun, cng nh cc cht mu.
Cht phn tch c th b hp ph mt phn, hoc l b hp ph ton phn ln b mt thnh mao qun.
Nguyn nhn ch yu gy ra s hp ph ca thnh ng mao qun silica l tng tc ionic gia cation ca
cht tan vi b mt mao qun mang in tch m, k c s tng tc a nc hay k nc.
2.7.2. Kim tra v khng ch tng tc c hi
S tng tc ca cht phn tch vi thnh mao qun trong hu ht cc trng hp thng gy ra nh
hng xu n s in di ca cht phn tch, nh lm dong pic, gim hiu qu tch. V th kim tra v
khng ch s tng tc ca cht phn tch vi thnh mao qun l mt vic rt cn thit loi b hay hn
ch n mc ti thiu s tng tc . khc phc nh hng ny, ngi ta phi x l b mt mao qun
bng cch l hot ha v bin i b mt mao qun.
Hot ha v bin i b mt mao qun l lm thay i lp in tch trn b mt thnh mao qun,
gii hn, hay lm nh s hp ph, hay s tng tc ca n vi cc cht phn tch, cho qu trnh in di
xy ra tt, hn ch s dong pic do tng tc cht phn tch vi thnh mao qun gy ra. bin i b mt
thnh mao qun, hin nay c 2 kiu thc hin:
Kiu 1: Bin i vnh cu v c nh b mt thnh mao qun
Ngi ta trng hay ph ln thnh mao qun mt lp mng mng ca mt polime thch hp. C th
dng phng php trng ph vt l hay dng phng php lin kt ha hc ca cht polime ph vi silica

99

ca mao qun to thnh mt lp mng mng polime khng hay rt km tng tc vi cht phn tch. Lp
mng ny c gi l mng bm hay pha bm.
u im:
+ Khng yu cu nghim ngt s bo dng thng xuyn b mt ( ch cn ra sau mi ln th
nghim ).
+ Bn vi nhiu loi dung dch ra v dng chy.
Nhc im:
+ Ch s dng trong vng pH hp.
+ Lp mng ny mn dn ( khi pH >8 v t0 >400C ).
Kiu 2: Bin i b mt mao qun theo kiu ng hc
Thm vo pha ng in di mt cht ph gia c nng thch hp. Cht ny phi c i lc hp ph
mnh vi thnh mao qun, khi chy in di th cht ny s ph ln thnh mao qun ngn khng cho cht
phn tch tip xc v tc dng vi thnh mao qun.
Cc ng mao qun c hot ha ng hc b mt kh tnh hp ph ca b mt thnh mao qun c
tnh u vit hn cc ng mao qun c b mt c hot ha vnh cu. y l cch hot ha b mt mao
qun ch trong qu trnh chy sc k in di nn c gi l phng php hot ha ng hc. Trong phng
php ny ngi ta ch cn thm vo dung dch pha ng in di nhng cht thch hp trong qu trnh in
di n t to ra mt lp mng bm ln b mt thnh mao qun c tc dng lm gim hay kh b tc dng ca
s tng tc hp ph gia b mt mao qun v cht phn tch.
u im chnh ca phng php l bn v s n nh ca lp mng Lp mng hot ha lin tc
c cng c v bo v, chng li cc tc ng n mn trong qu trnh in di (V cc cht ph gia lun
ngay trong dung dch pha ng nn s hot ha, sa cha lp mng trn b mt mao qun l lin tc c
duy tr trong sut qu trnh in di).
Nhc im:
+ C khi cht tan cng nh b mt mao qun u c tc dng vi cht ph gia thm vo;
+ Thi gian cn bng cn thit c c b mt lp li n nh v dng chy in di thm thu EOF
n nh b ko di;
+ Cht ph gia b thnh mao qun hp ph ln To lp ngn cch gia cht phn tch vi thnh mao
qun.
+ Mt s trng hp lm nhy ca CEC gim xung, nht l khi thm ph gia vi nng cao.

3. C s l thuyt ca sc k in di mao qun

3.1. Nguyn tc ca sc k in di mao qun
Nguyn tc ca s tch trong k thut sc k in mao qun hiu nng cao (HPCEC) l da trn c s
tnh cht in di ca cc phn t cht tan (cc ion cht tan, cht phn tch) trong ng mao qun (ng knh
trong 25 - 100 m) trn nn ca dung dch cht in gii v cht m pH thch hp, di tc dng ca mt
in trng E nht nh c cung cp bi ngun th cao mt chiu (10 - 40 kV) t vo hai u ng mao
qun.
C ch tch:
Da trn s di chuyn khc nhau ca cht tan di tc dng ca in trng E nht nh v tnh cht
c trng ca dng in di thm thu trong s ph thuc vo in tch v kch thc ca n.
100

Th V to ra in trng E t vo hai u ca ng mao qun c dng trong CEC l rt ln (thng

trong vng 10-40 kV/1m hay t 100-550V/1cm chiu di mao qun) to ra lc in F E (FE = qE). Chnh lc
in trng ny l ng lc lm cho cc phn t cht tan di chuyn theo hng nht nh. Cc cht khc
nhau c in tch v kch thc khc nhau s di chuyn vi cc tc khc nhau v to ra s tch sc k cc
cht.

Hnh 3. S tch cc cht trong HPCEC (kiu CZE)

3.2. Th in di v lc in di
Qu trnh in di trong mao qun ch xy ra khi c ngun th V mt chiu nht nh (th in di) t
vo 2 u ca mao qun. Th V ny to ra in trng E v dng in i trong mao qun, n iu khin v
duy tr s in di ca cc cht v l mt trong cc yu t chnh quyt nh kt qu ca s in di. V th
trong qu trnh sc k mt hn hp mu, c kt qu tt v n nh, phi chn gi tr th V thch hp nht,
khng ch v gi cho gi tr th V ny lun khng i, c c gi tr E v dng in i cng khng i.
Dng in i trong mao qun ph thuc vo:
in th mt chiu V t vo hai u mao qun;
Gi tr pH v thnh phn ca cht in gii trong pha ng in di;
Bn cht, in tch ca cc cht c trong mao qun.
Trong CEC, in th V mt chiu thng c dng t vo hai u mao qun t 15 40kV, hay l t
100 550V/1cm mao qun. Song th V c dng phi lm sao khng cho dng in i qu ln trong mao
qun, ni chung dng in i ny ch nn nm trong vng t 70 75 A. V khi dng in i qu ln s gy ra
hiu ng nhit Jun ln lm nng mao qun, gy ra s dong pic, tc l gim hiu qu tch. Vic chn th
in di V tu thuc vo nhiu yu t nh:
Bn cht ca cht phn tch v cht nn ca mu;
Bn cht ca mao qun v pha tnh trong mao qun (nu c);
Bn cht, thnh phn ca cc cht in gii v cht m trong pha ng;
Gi tr pH ca pha ng in di.
3.3. Dung dch m v pha ng trong HPCEC
3.3.1. Khi nim v pha ng trong HPCEC
Qu trnh in di trong k thut HPCEC c thc hin trong mi trng ca mt dung dch m pH
v cht in gii thch hp (c khi ch c cht m pH lm c 2 chc nng), dung dch ny gi l pha ng.
Gi tr pH, nng , loi cht m in gii c vai tr rt quan trng, n xc nh cc tnh cht v c
trng ca cc qu trnh xy ra trong mao qun, gm:
+ B mt trong ca ng mao qun Silica;
101

+ S tn ti v n nh ln ca lp in kp, th Zeta;
+ Xc nh s chuyn ng v tnh bn vng ca dng in thm thu EOF;
+ Duy tr v n nh dng in i trong mao qun.
Qua cc yu t cui cng s nh hng n hiu qu ca qu trnh sc k: gi tr H ef v Nef ca
mao qun, nt ca pic sc k,
Do ty vo cht phn tch ta phi chn pha ng c thnh phn, gi tr pH thch hp.
3.3.2. Cht m v gi tr pH m trong HPCEC
Cht m v gi tr pH ca cht m (pha ng) c nh hng trc tip n thnh mao qun:
+ Quyt nh s hnh thnh lp in kp, ln ca lp in kp v th Zeta;
+ Gi tr pH ca pha ng c th lm cho thnh mao qun tch in m (b anionic ha) hoc tch in
dng (b cationic ha) ty vo gi tr pH ca pha ng (v trn b mt mao qun silica c cc nhm OH,
nhm ny b ion ha theo s tng pH).
Do phi chn c dung dch m c pH ph hp v ng thi khng ch n khng i sut qu
trnh in di.
Cc h m dng trong HPCEC ch c hiu lc tt khi gi tr pH ca n nm trong vng:
1 hoc pH = pKb 1.

pH = pK a

Trong cc loi h m th h m ca cc poli axit hay poli baz c hiu lc tt hn h m ca cc

n axit-baz ( V cc poli axit hay poli baz do c nhiu nc nn c kh nng m trong vng pH rng v
do s c tnh linh hot cao). Song ni chung, cc h m dng trong HPCEC phi tha mn cc iu kin
sau:
Phi c dung lng m kh ln trong vng pH chn.
Phi c hp ph quang rt nh (khi dng detector UV hay Vis).
Cc ion ca cht m c in di khng ln (cc ion trong dung dch m phi c in tch
nh nhng c kch thc ln hn ch vic to ra dng in v ch hoc to ra hiu ng nhit
trong mao qun).
Cc cht m phi tan tt trong nc v bn vng trong qu trnh bo qun cng nh trong qu
trnh in di.
Hin nay cc h m sinh hc c s dng rt ph bin nh: TRIS, Borat, Histidin, Caps,V khi s
dng cc h m ny ta c th dng c nng cao m khng gy ra hiu ng nhit cao i cho ng
mao qun. Tuy nhin h m ny c nhc im l c hp th quang trong vng UV hay Vis mt mc
nht nh do m gy kh khn cho vic pht hin cc cht phn tch khi dng detector UV hay UV-Vis.
V vy chng ta cn phi qut ph cc cht m, xem xt v chn bc sng o thch hp cho cht phn
tch, chn nng thch hp hn ch nh hng ca cht m.
Bng 3. Mt s loi h m thng dng trong CEC
STT

Loi cht m

Vng pH hiu lc

Borat/NaOH

9-12

Borat/HCl

4-9

Axit boric/NaCl

4-9

Axit boric/NaOH/NaCl

4-9

Na2HPO4/ NaH2PO4

4-8

102

NaH2PO4/NaOH

7-12

CH3COOH/CH3COONa

4-6

TRIS/HCl

4-7

TRIS/NaOH

6-9

3.3.3. Cht in gii trong pha ng ca HPCEC

Cng vi cht m pH, trong pha ng cua HPCEC cng c cha cc cht in gii. Cc cht in gii
thng l cc mui tan tt trong nc cho cc ion c in tch nh nht nhng bn knh hidrat ca n li
ln, thng l cc mui halogen hay l nitrat ca cc ion kim loi kim v amoni nh: NaCl, LiCl, NH 4Cl,
Trong mt s trng hp th cht m pH ng thi ng vai tr cht in gii cho qu trnh in di, nh
CH3COONH4. Cc cht in gii ny c la chn cho vo pha ng vi nng ph hp vi php phn
tch.
Vai tr: Cht in gii rt cn thit duy tr v n nh dng in i trong mao qun nh lm gim
ln lp in kp.
3.4. Mao qun v pha tnh trong HPCEC
3.4.1. Mao qun trong HPCEC
Mao qun l mt b phn quan trng ca HPCEC, l mt trong cc yu t chnh quyt nh s tch sc
k hn hp cc cht.
Mao qun c ch to ch yu bng silica, c gi l mao qun silica. Trong mt s trng hp
ngi ta s dng mao qun teflon khi mao qun silica khng ph hp (v d mu c cha ion F - v tch
pH thp).
Mao qun thng c ng knh trong t 20-150 m, song tt nht l 25 -75 m. Mao qun c thnh
dy t 80-120 m.
Chiu di ca mao qun ty thuc vo hn hp cht cn phn tch. Nhng ni chung, hin nay c
dng cc ng mao qun c chiu di tng L t 30-120cm, nhng chiu di hiu dng l t 20-100cm.
Bn ngoi mao qun bao gi cng c bao bi lp polyme (PVC) c dy t 200-350 m, cho
mao qun c do, c th un li c m khng gy. Song chnh lp polyme ny li lm cho mao qun
c nhc im l s truyn nhit t trong mao qun ra mi trng bn ngoi km i.

103

Hnh 4. Mt loi mao qun trong HPCEC

Trong HPCEC, mao qun c th l ng silica trng, cng c th l ng silica c nhi pha tnh (thc
hoc gi) ty thuc kiu HPCEC v cht nghin cu.
3.4.2. Pha tnh trong HPCEC
Pha tnh thc
Pha tnh thc ch yu l c ch to trn nn ca silica c hot ha v bin tnh c tnh cht
sc k ca s hp ph, s trao i ion, hoc l ry phn t. N bao gm cc loi silica trung tnh trn b mt
c cc nhm OH, hay silica trung tnh c alkyl ha cc nhm OH trn b mt ht pha tnh bng cc
nhm alkyl loi mch cacbon C1, C3, C8, C18, hoc nhm phenyl (-C6H5) nh pha tnh ca HPLC.

Hnh 5. S ankyl ha b mt pha tnh

l cc ht pha tnh c l xp, chu nhit, chu p, c kch thc nh t 2-5 m. Cch nhi n vo
ng mao qun cng tng t nh ct mao qun ca sc k kh. V th n l mt lp mng pha tnh bm vo
thnh mao qun, c dy t 5-15 m, ty c ht ca pha tnh. Nh th, loi pha tnh ny l c gi c
nh trn thnh ca ng mao qun.
Trong kiu Gel-CE, pha tnh l cc cht cao phn t hu c, c ch to trn nn ca polyme hu c,
n thng l cc keo xp cao phn t, c xp thch hp, thc hin qu trnh tch ca cc cht phn tch
theo c ch in di ry phn t.
Pha tnh gi
i vi h MEKC, khi in di trong ng mao qun lun c xut hin pha tnh gi, l cc tiu phn
Micell. N c hnh thnh t cc cht hot ng b mt, nh cht SDS trong pha ng in di vi nng
thch hp (nng gii hn hnh thnh cc tiu phn Micell). Trong qu trnh in di, cc cht tan cng
104

phn b vo pha tnh ny. Ngha l gia pha tnh v pha ng c mt cn bng phn b cc cht tan v
chng c h s phn b Ki nht nh. Cc tiu phn micelle s dn dt v ng gp thm kh nng ca qu
trnh tch sc k in di trn cht nn ca dung dch m v cht in li trong mao qun.

Hnh 6. Micell v HPCEC vi mao qun c pha tnh loi micell

3.5. Lp in kp trn thnh mao qun
Trong qu trnh in di, lp in kp v th Zta st thnh mao qun lun xut hin, n ph thuc vo lc ion
ca dung dch pha ng in di. Yu t b mt mao qun, thnh phn v pH ca pha ng c lin quan n s hnh
thnh, ln v s tn ti ca lp in kp trn b mt mao qun v th Zta. Khi lc ion ca dung dch in di
tng th lp in kp s b nn li lm th Zta gim v nh hng n dng in di thm thu.

Hnh 7. Lp in kp trn thnh mao qun

(thnh mao qun tch in m do cc nhm SiO-)
ln ca th Zta (Vzet) c tnh gn ng theo cng thc:
105

VZet =

4 RD q

(3.1)

Trong :
l hng s in mi ca dung dch pha ng;
q l tng in tch dung dch theo mt n v th tch;
RD l bn knh hydrat ca ion (bn knh Debye) c xc nh theo biu thc:

RD =

kT
8 e2 I

(3.2)

Trong :
e l in tch ca electron; k l hng s Bolzoman; T l nhit tuyt i; I l lc ion ca dung dch
m in di.
Cng thc (3.2) cho thy: khi nng cht m in li ln, th gi tr lc ion I ln th R D gim dn n
th Zeta gim theo (3.1).
Bn cnh vic nh hng n lp in kp trn b mt mao qun, nng ca cht m, loi cht
in gii cng nh hng n s in di ca cht tan. Tc ng ny th hin qua s n nh v duy tr dng
in i v c dng in di thm thu EOF trong mao qun. T nh hng n in di hiu lc ef ca
cht phn tch.
3.6. Dng in di thm thu
3.6.1. Khi nim v dng in di thm thu
Bn cht chnh ca s hot ng trong ng mao qun ca HPCEC l dng chy in di thm thu
EOF, gi tt l dng in di thm thu.
Dng EOF l dng chy khi ca cht lng trong ng mao qun, n quan h mt thit vi lp in tch
trn thnh mao qun.
Dng EOF s xc nh thi gian tn ti ca cht tan trong mao qun. N bao trm ln dng di chuyn ca
cht tan, ngha l cht tan di chuyn trong dng EOF ny.
3.6.2. Ngun gc dng in di thm thu
Khi in di vi mao qun silica, cc nhm silanol (Si-OH) trn thnh mao qun s gii phng ra ion H +
v b mt mao qun mang in tch m. Do b mt mang in tch m nn cc cation do lc ht tnh in s
to thnh lp in kp cn bng in tch v to nn hiu th st thnh mao qun (th Zeta).

106

Hnh 8. Dng in di thm thu EOF

Khi p th V vo 2 u mao qun, cc cation trong lp in kp s i v catot. Nhng v cc cation ny
b sonvat ha nn ko c khi dung dch trong mao qun i v catot. S di chuyn ca khi dung dch trong
mao qun silica di tc dng ca in trng to nn dng in thm thu EOF.
Tc v ca dng EOF t l thun in trng E, vi th zeta V Zet v t l nghch vi nht ca
dung dch m in li theo cng thc:

Tc tuyn tnh u:

in di ca dng EOF:

vEOF =

EVZet

uEOF =

EVZet
4

(3.4)

VZet

(3.5)

EOF =

(3.3)

Dng EOF c quyt nh bi th V t vo 2 u mao qun, pH dung dch m in li, cht in

gii, lp in kp, nht ca dung dch pha ng, hng s in mi, xp, c ht. Nh c tnh ny
ngi ta chn c cc iu kin ti u c tc dng EOF ph hp cho qu trnh tch sc k.

107

Hnh 9. nh hng ca pH ln dng EOF

3.6.3. c im ca dng in di thm thu
Dng EOF c 2 c im chnh:
Th nht, dng EOF l dng chuyn khi phng trong mao qun. C c im ny v lc to ra s di
chuyn ca dng EOF phn b ng nht dc theo mao qun nn khng c st p trong mao qun. Trong
HPLC th dng chy c dng parabol do hiu ng thnh: cc phn t gia dng di chuyn nhanh, cc phn
t gn thnh di chuyn chm). V vy pic sc k trong HPCEC gn v sc nt hn HPLC.
Th hai, dng EOF a tt c cc tiu phn c mt trong dung dch in di, bt k c mang in hay
khng di chuyn theo cng mt hng nht nh t anot (cc +) v catot (cc -) (nu pH 4):
Cc cation di chuyn nhanh v catot vi tc khc nhau ty in tch v kch thc ca n;
Cc phn t trung ha c dng EOF mang theo cng vi tc ca dng EOF (nhng
chng khng tch ra c trong CEC);
Cc anion di chuyn v anot nu tc ca n ln hn tc dng EOF. Ngc li chng b
dng EOF mang theo v catot

108

P;l,

Hnh 10 . So snh dng chy trong HPLC v trong CEC

.

109

Hnh 11. S chuyn ca cc phn t trong CEC

Theo th t ra gii theo thi gian s l: cation in tch ln, kch thc nh ra u tin cc
phn t trung ha anion c in tch ln v kch thc nh ra sau cng (Hnh 12).

Hnh 12 . Th t ra gii trn in di

3.6.4. Kim tra v khng ch dng in di thm thu
Theo trn ta thy dng EOF l mt yu t thun li v c vai tr quan trng trong k thut CEC nn
cn kim tra v khng ch n mc ph hp cho mc ch phn tch. Cc cch kim tra, khng ch dng
EOF c tng kt bng 4.

Bng 4. Cc cch kim tra, khng ch EOF

Cch thc thay i

Kt qu

Nhn xt chung

in trng E (tc in S thay i t l vi EOF

th V)

+ phn gii v s a l thuyt

hiu lc gim.
+ Lm nng mao qun.

Gi tr pH ca dung dch EOF gim pH thp v tng + Cch tin li thay i EOF
110

m in di

pH cao

+ Thay i c in tch, cu
trc cht tan

Lc ion, nng dung Lm gim th zeta v EOF + Lc ion ln, to dng in ln v

dch m
khi tng lc ion, nng hiu ng Jun ln
+ nt pic sc k b nh hng
dung dch m
nu dn khc dn ca mu
+ Hn ch s np mu
Nhit

Thay i nht 2 3%/ C

Dng khi kim tra v khng ch

nhit t ng

Dng cc cht ph gia, Thay i th zeta, nht, + Thay i phc, xc nh d

dung mi hu c
EOF b gim
dng bng thc nghim
+ C th thay i chn lc ca
h pha
Cc cht hot ng
b mt

Hp ph ln thnh mao qun + Cht hot ng b mt l ation

qua tng tc ionic hay lm tng EOF, cation lm gim
EOF, c th i chiu EOF
hydrophopic
+ Thay i chn lc

Dng polymer
hydrophilic trung tnh

Hp ph ln thnh mao qun + Lm gim EOF qua thay i

qua tng tc hydrophopic
in tch b mt
+ Lm tng nht

Hot ha b mt bng To lin kt ha hc mi ln + C nhiu cch thay i:

cch cng hp
thnh mao qun
hydrophylicity, thay i in tch
+ Lm bn b mt mao qun

4. Ti u ha php phn tch sc k in di mao qun

Mun c kt qu sc k in di tt nht thit phi ti u ha cc iu kin cho s tch sc k. Ni dung
cng vic ny gm cc vn sau:
4.1. Chun b mu, np mu
4.1.1. Chun b mu
X l mu bng cc k thut nh: v c ha t, v c ha kh, kh t kt hp, cc k thut chit,
sc k, in phn,
Mu a vo ng mao qun ca HPCEC phi trng thi lng, do phi ha tan cht phn tch bng
dung mi thch hp. Dung mi ny thng l dung mi lm pha ng. Ty cht phn tch m chn dung mi
thch hp.
V d: x l mu xc nh cc v -caroten:
+ Mu xay thnh bt v bo qun -150C.
+ Ly 5g mu xay mn cho vo bnh chit, thm 15 20g Na 2SO4 khan, 1g MgCO3 khan, trn u,
thm 20 mL dung mi THF, khuy u trong 5 pht. Lc ht chn khng, ly pha hu c cha cc carotene vo
bnh ct quay chn khng, ct dn cn khong 1 mL, yn 1 pht cho kh ri ha tan v nh mc thnh 5 mL
bng THF. Li tm tch b cn. y l dung dch xc nh cc v -caroten bng HPCEC.
111

4.1.2. Np mu
Np mu vo ng mao qun ch c php ti a l 2% chiu di hiu lc l ca ng. C 3 phng php np
mu l: dng p sut, dng xi-phong v phng php in ng hc.

Hnh 13. Ba cch np mu trong CEC:

a) dng xi-phong; b) dng p sut; c) dng dng in
4.2. Chn cc iu kin ti u
4.2.1. Chn mao qun v pha tnh
Chn mao qun v pha tnh ty thuc mi kiu HPCEC v mu nghin cu:
+ Mao qun trng: dng cho CZE;
+ Mao qun c pha tnh: cc kiu cn li.
Chn chiu di mao qun: thng L = 30 120 cm, l = 20 100 cm.
Chn ng knh trong ca mao qun: thng khong 25 75 m.
4.2.2. Chn pha ng
Chn pha ng ph thuc mu phn tch. Pha ng thng l dung dch m pH v c cht
in li thch hp hoc ch c cht m pH lm c 2 chc nng.
Chn pha ng phi ch cc iu kin: thnh phn v gi tr pH m; nng cht in li;
cc cht ph gia nu c; dung mi hu c bin tnh pha ng in di.
4.2.3. Chn th in di
Chn th in di V da vo cc yu t:
Bn cht cht phn tch, cht nn ca mu;
Bn cht mao qun v pha tnh;
Bn cht, thnh phn cht in gii v cht m pH trong pha ng;
Gi tr pH m.
4.2.4. Chn etector
Ty bn cht cht phn tch chn detector ph hp theo bng 5.

112

4.3. Chn phng php phn tch thch hp

Da vo c im cht phn tch la chn phng php phn tch ph hp.
* Sc k in di mao qun vng (CZE): dng tch v phn tch cc amino axit, peptit, cc cht c cu
to ion, cc ng phn khng gian, cc loi hp cht c kh nng ion ha nh cc ion kim loi, mui ca
axit hay bazo hu c,
* Sc k in di mao qun kiu Micell (MEKC): s dng tch cc cht mang in v khng mang
in, cc hp cht a nc v k nc. N cng dng tch cc amino axit, cc h nucleotit, cc loi
vitamin, hidrocacbon thm, cc sn phm dc (thuc vin, thuc tim, thuc dng kem) v cc hp cht
sinh hc.
Bng 5. Mt s detector dng trong HPCEC
Detector

p dng cho

Gii hn pht
hin (M)

Hp th UV- Cht hp th trong vng UV- 10-5 10-8

Vis
Vis

Ghi ch

Phn tch nhiu

cht

Hunh quang

Cht c kh nng pht hunh 10-7 10-9

quang hoc to vi thuc th
cht c kh nng pht hunh
quang

dn in

Vn nng

10-7 10-8

o ampe

Cht c hot tnh in ha

10-10 10-11

Nhy, chn lc.

i hi vn hnh
cht ch

10-8 10-9

Nhy, cung cp
thng tin v cu
trc

Ph khi

Bc x k

10-10 10-12

Cht c hot tnh phng x

113

Nhy

Hnh 14. Tch cc cation trong CZE (dng tc nhn gin tip Cu2+)
iu kin: mao qun 50 cm x 50 m; V = 20 kV; detector UV, 215 nm.
Sb 3,6 ppm (1), K 7,8 ppm (2), Na 4,6 ppm (3), Ca 4 ppm (4), Mg 2,4 ppm (5), Sr 15 ppm (6), Li 0,69
ppm (7), Ba 27 ppm (8).

Hnh 15 . Tch cc heroin bng MEKC

iu kin: dung dch m (SDS 85 mM, borat 8,5 mM, photphat 8,5 mM, CH3CN 15%) pH = 8,5; l =
50 cm; ng knh trong 50 m; V = 30 kV; t0 = 500C;
detector UV, 210 nm
* Sc k in di gel (Gel-CE): dng tch cc cht c khi lng phn t ln nh axit nucleotit, protein,
da vo kch thc ca cc phn t cht tan.

Hnh 16.. Tch cc AND dng Gel-CE

iu kin: gradient in trng 400 100 V/cm; mao qun 27 cm x 100 m; m pH = 8,3 (TRIS
borat 100 mM, EDTA 2 mM)
M: 72 (1), 118 (2), 194 (3), 234 (4), 271 (5),
281 (6), 310 (7), 603 (8), 72 (9), 1078 (10), 1353 (11)

114

* Sc k in di mao qun hi t ng in (CIEFC): dng tch cc cht nh peptit, protein, cc ng phn

hnh hc, phn tch hemoglobin, cc dung dch sinh hc long

Hnh 17. Tch cc hemoglobin bng CIEFC

iu kin: mao qun: 12 cm x 25 m; V = 8 kV;
pH m 3 10; detector UV 280 nm
im ng in pI: A 7,1; F 7,15; S 7,25; C 7,5.

5. My sc k in di mao qun hiu nng cao

5.1. Cu to, trang b

115

Hnh 18. S nguyn tc cu to ca h HPCEC

thc hin s in di th h thng my HPCEC phi c cc b phn sau:
1. B bung in cc, bnh in di v cc in cc tr (Au hay Pt);
2. Ct tch sc k (ng mao qun);
3. Ngun cp th 1 chiu (10 40 kV) to in trng E;
4. B phn np mu vo mao qun;
5. B phn pht hin cc cht sau khi tch (detector);
6. B phn iu nhit cho ng mao qun;
7. B phn ghi nhn sc tch ca cc cht.

116

Hnh 19. S nguyn tc hot ng ca my HPCEC

5.2. Hot ng ca my HPCEC
Th 1 chiu V gy ra in trng E v pht sinh lc in lm cho cc cht tan di chuyn theo mt
hng nht nh. Cc cht khc nhau, c in tch v ln khc nhau s di chuyn vi tc nhanh, chm
khc nhau v to nn s tch sc k.
C th pht hin cc cht bng cch pht hin trc tip hoc gin tip ty thuc vo bn cht cc cht
phn tch hoc cc tnh cht l ha v vt l ca cht nh:
- S hp th quang phn t v nguyn t ca cc cht;
- S pht x hunh quang ca cc cht, khi b kch thch;
- Tnh cht in ha ca cc cht, nh phn ng in ha trn in cc;
- Ch s chit sut ca cc cht;
- dn nhit, hay s hp th nhit ca cc cht,
ng vi mi tnh cht ngi ta c mt lot detector tng ng:
+ Detector o ph hp th phn t vng UV- Vis (190 - 800 nm);
117

+ Detector o ph hunh quang phn t;

+ Detector o ph pht x v hp th phn t;
+ Detector in ha o dng hay o th;
+ Detector o ch s chit sut ca cht;
+ Detector o dn in, hay hp th nhit;
+ Detector khi ph;
+ Detector mng dit.

6. Cc phng php nh tnh, nh lng bng HPCEC

6.1. Phn tch nh tnh
Nguyn tc:
Mi cht phn tch trong cc iu kin tch CEC chn nht nh c mt thi gian lu tRi nht nh.
Thi gian lu tRi l thng s c trng dng pht hin nh tnh cc cht trong hn hp mu.
Cch tin hnh:
+ Chy sc k ca mu chun trong iu kin ti u chn xc nh thi gian lu ca tng cht.
+ Chy sc k mu phn tch trong cng iu kin nh mu chun v so snh thi gian lu kt lun
s c mt ca tng cht.

Nhn vo sc trn ta thy trong Chewing gum c cc cu t 3, 5, 6, 7.

6.2. Phn tch nh lng
6.2.1. Phng trnh c bn
Mi cht phn tch trong nhng iu kin ti u chn, vng nng nht nh th tn hiu phn
tch (din tch S, chiu cao H ca pic) lin h vi nng C ca cht phn tch qua biu thc:

S i = k1 C i

Hi = k 2 C i

(6.1)

Trong k1, k2 l cc hng s thc nghim.

Da trn phng trnh (6.1) ngi ta thng nh lng cc cht theo phng php ng chun v
phng php thm chun.
6.2.2. Phng php ng chun
* Cch tin hnh:
Chun b dy dung dch chun cha cht phn tch c nng C khc nhau trong iu kin tng t
mu phn tch. Tin hnh php tch sc k tnh chiu cao hay din tch i vi dy chun.
118

Dng ng chun biu din s ph thuc S hay H ca pic vo nng C.

Thc hin php phn tch vi cht nghin cu X trong cng iu kin xc nh S x hay Hx ri thay
vo phng trnh ng chun c Cx cn tm.
* u im: Phng php ng chun tin li cho vic phn tch hng lot mt loi mu cng i
tng v n n gin, nhanh v d thc hin.
* Nhc im: d mc sai s h thng v khng th chun b mt dy cc dung dch chun trong iu
kin hon ton ging nhau v ging vi mu tht. Lc ny phi dng phng php thm chun.

Hnh 20 . ng chun xc nh Cx
6.2.3. Phng php thm chun
* Cch tin hnh:
+ Pha dy chun cng chnh l dung dch nghin cu v c thm cht cn xc nh vi nhng nng
chnh xc Cti ri o S hoc H ca pic:
C

Cx

Cx + Ct1

Cx + Ct2

Cx + Ct3

Cx + Ct4

Cx + Ct5

S0

S1

S2

S3

S4

S5

+ V th Si Cti ri ngoi suy hoc tnh tin th v gc ta xc nh Cx.

Hnh 21.. Xc nh Cx bng phng php thm chun

* u im:

119

Phng php thm chun khc phc c hn ch ca phng php ng chun do loi tr c
mi nh hng ca nn (v mu no cng c nn ging nhau). Do phng php ny c chnh xc cao
hn phng php ng chun.

BI TP
Bi 1. Trong ng mao qun c chiu di hiu lc 50 cm, di tng cng 58,5 cm t mt hiu th
25000 V. Sau qu trnh chy sc k, trn sc ngi ta thu c 3 pic vi thi gian lu ln lt l 38,4s,
50,7s, 93,1s ng vi 3 cht X, Y, Z. Tnh in di ton phn, in di hiu lc ca X, Y, Z. Bit Y l
cht trung tnh, t c nhn xt g v gi tr in di hiu lc ef ?
GII

Ta c cng thc tnh in di ton phn: tot = i + EOF =

tot ( X ) =

tot ( Z ) =

L.l
V .ti

58,5.50
= 3,05.10-3 (cm2.V-1.s-1)
38, 4.25000

58,5.50
= 1,26.10-3 (cm2.V-1.s-1)
93,1.25000

Cht Y trung tnh nn i = 0, do :

tot (Y ) = EOF =

58,5.50
= 2,31.10-3 (cm2.V-1.s-1)
50, 7.25000

in di hiu lc: ef = tot EOF

ef (X) = 3,05.10-3 - 2,31.10-3 = 7,40.10-3 (cm2.V-1.s-1)

ef (Z) = 1,26.10-3 - 2,31.10-3 = -1,05.10-3 (cm2.V-1.s-1)
ef (Y) = 2,31.10-3 (cm2.V-1.s-1)
Nhn xt: ef (Z) < 0 Z l mt anion; ef (X) > 0 X l mt cation.
Bi 2. nh lng NO3- trong mt b nui c bng phng php CZE vi cht ni chun IO 4-. Dung
dch chun NO3- 15,0 ppm v IO4- 10,0 ppm cho din tch pic ln lt l 95,0 v 100,1 n v. Mt mu
nc c pha long 100 ln ri thm cht ni chun n nng 10,0 ppm. Phn tch bng CZE thu c
cc din tch ln lt l 29,2 v 105,8 i vi NO 3- v IO4-. Tnh nng NO3- trong mu nc b nui c
theo ppm.
GII
p dng cng thc trong phng php ni chun:

SA
C
= k. A
S IS
CIS
Trong : SA, SIS ln lt l din tch pic ca cht chun v cht ni chun; C A, CIS ln lt l nng
ca cht chun v cht ni chun.
Theo bi ta c:

95
15
= k.
100,1
10

k = 0,6327
120

Th cc gi tr vo tnh i vi mu nc ta c:

29, 2
C
= 0, 6327. A
105,8
10

CA = 4,362 ppm

Nng NO3- trong mu nc b nui c: 100CA = 436,2 ppm.

Bi 3. Tch cc ankylpiridin bng CZE. Vic tch c thc hin trong ng mao qun c ng knh
trong 50 m, chiu di tng cng 57 cm, chiu di hiu lc 50 cm. Dung dch m pH = 2,5. S tch ti u
vi th in di l 15 kV. in di thm thu o c l 6,398.10 -5 cm2.V-1.s-1. H s phn b ca cc
ankylpyridin, D = 1,0.10-5 cm2.s-1.
a) Tnh in di ca 2-etylpyridin, bit thi gian lu ca n l 8,20 pht.
b) Tnh s a l thuyt i vi 2-etylpyridin.
c) in di ca 3-etylpyridin v 4-etylpyridin ln lt l 3,366.10 -4 cm2.V-1.s-1 v 3,397. 10-4 cm2.V1 -1
.s . Tnh phn gii ca 2 cht ny.
GII
a) p dng cng thc:

i =

L.l
EOF
V .ti

tot = i + EOF =
=

L.l
V .ti

57.50
- 6,398.10-5 = 3,22.10-4 (cm2.V-1.s-1)
15000.8, 2.60

b) p dng cng thc tnh s a l thuyt ta c:

N=

( i + EOF ).V (3, 22.104 + 6, 398.105 ).15000

=
= 28950
2D
2.1.105

c) tnh phn gii R trc ht tnh:

= 2 - 1 = 3,397.10-4 - 3,366.10-4 = 0,031.10-4 (cm2.V-1.s-1)
TB =

1 + 2
= (3,366.10-4 + 3,397.10-4)/2 = 3,3815.10-4 (cm2.V-1.s-1)
2

Ta c:
R = 0,177.

V
15000
= 0,177.0, 031.104.
4
( TB + EOF ).D
(3,3815.10 + 6,398.105 ).10 5

R = 1,1.
Bi 4. Tch cc protein bng HPCEC. Mao qun di 84 cm, th in di 25000 V. Mt phn t trung
ha c dng EOF mang theo phi mt ht 308s di chuyn on 64 cm t ch np mu n detector.
Cht A c thi gian lu l 343s. Tnh in di tng cng v in di hiu lc ca cht A.
GII
in di tng cng ca cht A:
Cng in trng E: E = V/L = 25000/84 = 298 V/cm
tot =

u A 64 / 343
=
= 6,26.10-4 (cm2.V-1.s-1)
E
298

Tc tuyn tnh ca dng EOF:

121

uEOF =

64
= 0,208 ( cm/s)
308

in di ca dng EOF:
T uEOF = EOF.E EOF = uEOF /E = 0,208 /298 = 6,98.10-4 (cm2.V-1.s-1)
in di hiu lc:

ef = tot EOF = 6,26.10-4 - 6,98.10-4 = -0,72.10-4 (cm2.V-1.s-1)

V ef < 0 nn c th suy ra A l mt anion.
Bi 5. Phn tch sc k in di mao qun vi k thut CZE. Mao qun c chiu di tng l 32cm, chiu
di hiu lc l 24,5cm th in di 30 kV. Di nhng iu kin ca th nghim ghi c trong mi trng
trung tnh xut hin cht c thi gian lu l 3 pht.
a) Tnh in di hiu lc ca mt cht c thi gian lu 2,5 pht.
b) Tnh h s khuych tn di nhng iu kin cho ca cht , bit s a l thuyt N = 80 000.
S:
a) 2,88.10-5 (cm2.V-1.s-1)
b) D = 2,5.10-5 (cm2.s-1)
Bi 6. Trong mt php tch HPCEC, mao qun c chiu di tng bng 1m, chiu di hiu lc bng
90cm. Th p t vo hai u ng mao qun l 30 kV. Detector c t pha trc catot vi cht m c
pH = 5. Trong mt dung dch tiu chun, cht tan c thi gian lu l tm = 10 pht.
a) Tnh in di tng cng ca cht .
b) Nu mt phn t nh khng mang in c thi gian lu bng 5 pht, hy xc nh in di ca
dng EOF.
c) Tnh in di hiu lc ca cht .
d) Tnh s a l thuyt N nu cht c h s khuch tan D = 2.10-5 cm2/s.
S:
-4

-1

-1

-3

-1

a) 7,5.10 (cm .V .s ) b) 1,5.10 (cm .V .s-1)

c) 7,5.10-4 (cm2.V-1.s-1) d) N = 337 500.
Bi 7. Cho mt ct mao qun di 50cm c p t th 30 000V, in di tnh c vi thi gian 10
pht l 2.10-8 m2s-1V-1. Tnh s a l thuyt cho ion Li+ (D = 1,0.10-9 m2s-1) v cho mt protein c phn t
khi 105 (D = 3.10-11m2s-1).
S: Li+: 3.105 a; Protein: 107 a.
Bi 8. nh lng vitamin B1 bng k thut CZE hoc MEKC s dng cht ni chun l oethoxybenzamide. Khi phn tch dung dch cha 100,0 ppm B 1 v 100,0 ppm o- ethoxybenzamide th din
tch pic ca B1 bng 71% din tch pic cht ni chun. Phn tch 0,125g thuc vin vitamin B cho din tch
pic ca B1 bng 1,82 ln so vi cht ni chun. Tnh s mg vitamin B 1 c trong thuc ny, bit th tch dung
dch ban u l 20 mL.
S: 5,1 mg
Bi 9. Mt cation v c c in di 4,31.10 -4 cm2.V-1.s-1 v h s khuch tn 9,8.10-6 cm2.s-1 c
phn tch bng k thut CZE vi mao qun di 50 cm v th in di 10 kV. Trong iu kin ny, dng in
di thm thu c tc 0,85 mm/s i v pha catot. Nu detector c t cch im np mu 40 cm th
cation mt bao lu n c detector ?
122

S: 3,9 pht.

TI LIU THAM KHO

1. Phm Lun (2002), C s l thuyt ca Sc k in di mao qun hiu nng cao (HPCEC), H
KHTN HQG H Ni.
2. David Harvey (2000), Modern analytical chemistry, Mc Graw Hill.
3. Daniel Harris (2007), Quantitative Chemical Analysis, W.H Freeman and Company, New
York.
4. Nicola Volpi (2005), Capillary Electrophoresis of Carbohydrates, Humana Press.
5. Robetr Weinberger (1999), Practical Capillary Electrophoresis, AP.
6. Ruth Freitag (2002), Modern Advances in Chromatography, University of California, USA.
7. Jack Cazes (2004), Encyclopedia of Chromatography.
8. Skoog, West, Holler & Crouch (2004), Fundamentals of analytical chemistry, Thomson
Brooks/Cole.
.

123

MC LC
C nhy kh cao i vi nhiu cht (c th pht hin cht c nng c 100ppb)..............................68
Khong tuyn tnh rt rng (hn km nhau n hng triu ln).................................................................68
bn tnh hiu cao, t b nh hng bi tc dng kh mang v cc thng s thc nghim khc........68
Nhm E: mu (nhm A, B, C hoc D) tn ti dng dung dch ..............................................................72
Nhm F: mu c cha mt s thnh phn khng bay hi hoc rt kh bay hi........................................72
Trn c s thnh phn ca mu s quyt nh la chn nhng phng tin thit b v iu kin lm vic.
......................................................................................................................................................................72
5.1. Chn ct tch........................................................................................................................................72
- Rt ngn thi gian phn tch i vi cc mu c khong im si rng..................................................73
- T l gia chiu cao pic v chiu rng pic lun n nh v th thun li cho phn tch nh lng. Thng
thng, cc mu hp cht thin nhin rt phc tp nn khng th bng mt phng trnh tuyn tnh thu
c kt qu mong mun m phi s dng a phng trnh, ngha l khong nhit gia chng c th
gi ng nhit ct trong thi gian thch ng ri li tng tip vi tc mong mun. Trc v sau chng
trnh c th gi nhit c nh...............................................................................................................73
Nhit ca bung iu nhit phi c iu chnh vi tc thch ng nh chng trnh quy nh.
Hin nay cha c my sc k no lm vic chnh xc vi tc nh hn /pht v ln hn /pht. Thc t
ct nhi thng dng vi tc /pht, ct mao qun l /pht.....................................................................73
Vic lm ngui bung iu nhit cn phi c tin hnh nhanh chng, v thng lm ngui su hn
nhit lm vic ban u qu trnh thc hin chng trnh mi nhanh chng n nh, v khi ca bung
iu nhit nng, nhit trong cc tm cch nhit li ph ra...........................................................................74
Nhit ca bung bay hi mu cn phi c a ln cao, c lp vi nhit ca bung iu nhit
nhm lm s bay hi mu n nh v lp li cho d nhit tng hay gim..............................................74
Kh mang s dng phi tinh khit, nu khng cc tp cht s tch t trc ht phn u ca ct tch.
Khi nhit ca ct tng ln n s vn chuyn qua ct gy ra pic l bt ng.............................................74
Gim ng knh ct tch .........................................................................................................................75
S dng kh mang vi nht thp ..........................................................................................................75
Rt t ph thuc vo thng s lm vic, do n l gia tr c trng cho mi cht ng vi mt pha tnh v
mt nhit nht nh. S ph thuc vo nhit ca chng rt nh v v nguyn tc ch khong 2 6
n v khi thay i 10oC..............................................................................................................................76
lp li ca vic xc nh ch s lu ca hai phng th nghim khc nhau ch lch 1 n v v thm ch
vi nhng iu kin thun li ch lch 0,05 n v......................................................................................76
1. Gii thiu v phng php HPCEC......................................................................................89
1.1. S ra i v pht trin..........................................................................................................................89
1.2. Phn loi cc kiu HPCEC ..................................................................................................................91
1.3. c im ca sc k in di mao qun hiu nng cao..........................................................................91
2. Mt s i lng trong HPCEC............................................................................................92
2.1. Tc in di, in di, thi gian in di.........................................................................................92
2.1. 1. Tc in di................................................................................................................................92
2.1.2. in di.......................................................................................................................................93
2.1.3. Thi gian in di............................................................................................................................93
2.2. H s phn b v h s dung tch.........................................................................................................94
2.2.1. H s phn b Ki............................................................................................................................94
2.2.2. H s dung tch ki.........................................................................................................................94
2.3. Chiu cao a, s a v s a hiu lc ca mao qun........................................................................95
2.4. Tc tuyn tnh .................................................................................................................................96
2.5. phn gii.........................................................................................................................................97
124

2.6. Hiu ng nhit v s gradient nhit ................................................................................................98

2.6.1. Hiu ng nhit Jun v nh hng ca n.......................................................................................98
2.6.2. Kim tra v khng ch hiu ng nhit...........................................................................................99
2.7. S tng tc ca cht phn tch vi thnh mao qun...........................................................................99
2.7.1. Khi nim v s tng tc..............................................................................................................99
2.7.2. Kim tra v khng ch tng tc c hi.........................................................................................99
3. C s l thuyt ca sc k in di mao qun.......................................................................100
3.1. Nguyn tc ca sc k in di mao qun............................................................................................100
3.2. Th in di v lc in di...................................................................................................................101
3.3. Dung dch m v pha ng trong HPCEC.......................................................................................101
3.3.1. Khi nim v pha ng trong HPCEC.........................................................................................101
3.3.2. Cht m v gi tr pH m trong HPCEC..................................................................................102
3.3.3. Cht in gii trong pha ng ca HPCEC..................................................................................103
3.4. Mao qun v pha tnh trong HPCEC..................................................................................................103
3.4.1. Mao qun trong HPCEC..............................................................................................................103
3.4.2. Pha tnh trong HPCEC.................................................................................................................104
3.5. Lp in kp trn thnh mao qun.....................................................................................................105
3.6. Dng in di thm thu......................................................................................................................106
3.6.1. Khi nim v dng in di thm thu...........................................................................................106
3.6.2. Ngun gc dng in di thm thu..............................................................................................106
3.6.3. c im ca dng in di thm thu..........................................................................................108
3.6.4. Kim tra v khng ch dng in di thm thu............................................................................110
4. Ti u ha php phn tch sc k in di mao qun............................................................111
4.1. Chun b mu, np mu......................................................................................................................111
4.1.1. Chun b mu................................................................................................................................111
4.1.2. Np mu.......................................................................................................................................112
4.2. Chn cc iu kin ti u...................................................................................................................112
4.2.1. Chn mao qun v pha tnh..........................................................................................................112
4.2.2. Chn pha ng.............................................................................................................................112
4.2.3. Chn th in di...........................................................................................................................112
4.2.4. Chn etector...............................................................................................................................112
4.3. Chn phng php phn tch thch hp.............................................................................................113
5. My sc k in di mao qun hiu nng cao.......................................................................115
5.1. Cu to, trang b.................................................................................................................................115
5.2. Hot ng ca my HPCEC...............................................................................................................117
6. Cc phng php nh tnh, nh lng bng HPCEC.......................................................118
6.1. Phn tch nh tnh..............................................................................................................................118
6.2. Phn tch nh lng..........................................................................................................................118
6.2.1. Phng trnh c bn.....................................................................................................................118
6.2.2. Phng php ng chun...........................................................................................................118
6.2.3. Phng php thm chun.............................................................................................................119
BI TP.................................................................................................................................120
TI LIU THAM KHO......................................................................................................123
125

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