Sac Ky

CHUYN : SC K
(C S L THUYT V NG DNG)
TS NGUYN NH LM
B MN CNG NGH HA HC V VT LIU, KHOA HA
TRNG I HC BCH KHOA I HC NNG
Sample Mobile phase
t
0
t
1
t
2
t
3
t
4
t
0
t
1
t
2
t
3
t
4
D
e
t
e
c
t
o
r
s
i
g
n
a
l
s
Time
Detector
Sample Mobile phase
t
0
t
1
t
2
t
3
t
4
t
0
t
1
t
2
t
3
t
4
t
0
t
1
t
2
t
3
t
4
t
0
t
1
t
2
t
3
t
4
D
e
t
e
c
t
o
r
s
i
g
n
a
l
s
Time
Detector
A. I CNG V PHNG PHP SC K
(Chromatography)
c pht minh bi nh sinh vt hc ngi Nga Mikhail Tswest
Tch Chlorophills v Xanthophylls bng CaCO
3

Ting Hy-lp: Chroma: mu
Graphein: ghi
Phng php sc k:
K thut tch (seperation) cc cu t trong mt h ng th (kh hoc
lng)
Cn bng nng ca cc cu t trong hai pha tip xc nhau: pha tnh
(stationary phase) v pha ng (mobile phase)
S phn tch da trn tc ko theo (elution) khc nhau ca cc cu t
trong ct (column)
Mt u d (detector) u ra ca ct cho php nh lng lin tc cc
cu t trong hn hp u
Sc k mng mng (planar chromatography), Sc k ct (Column chromatography)
I CNG V PHNG PHP SC K
(Chromatography)
T=0
T=10
T=20
Injector Injector
Detector Detector
Most Most Interaction with Stationary Phase Interaction with Stationary Phase Least Least
Flow of Mobile Phase Flow of Mobile Phase
T=0
T=10
T=20
Injector Injector
Detector Detector
Sc k phn tch
(Elution chromatography)
Phn tch sc k: Cc cht tan b ra qua mt pha tnh nh s chuyn ng
ca pha ng qua n
Mu Pha ng
Detector
t
0
t
1
t
2
t
3
t
4
t
0
t
1
t
2
t
3
t
4
T
n

h
i
u

d
e
t
e
c
t
o
r

Thi gian
Sc k phn tch
(Elution chromatography)
Phn tch sc k: Cc cht tan b ra qua mt pha tnh nh s chuyn ng
ca pha ng qua ct cha pha tnh
Pha ng
Sc k
(Chromatogrames)
iu kin thu c sc k :
- u d (Detector) c lp t im cui ca ct
- u d tng thch vi cc cht cn pht hin
Sc k : Biu din s bin thin ca tn
hiu ra theo thi gian hoc theo th tch
tiu hao ca pha ng
Cc peaks i xng (hoc khng i xng)
Phn tch nh tnh (qualitative) v nh lng (quantitative)
Sc k
(Chromatogrames)
S phn gii (resolution)
Vn tc di chuyn tng i (relative migration rates)
S gin peak (band broadening)
Vn tc di chuyn ca cc cht tan
(Migration rates of solutes)
Thi gian lu t
R

(Retention time)
Tc di chuyn trung
bnh ca cht tan
R
t
L
v =
Tc di chuyn trung
bnh pha ng
o
t
L
u =
M
s
c
c
K =
solute of moles total
phase mobile in solute of moles
u v =
Quan h gia tc di chuyn v h s phn b
H s phn b K
(Partition Ratios)
Cn bng phn b ca cht tan trong pha ng v pha tnh
A
mobile
A
stationary

M S
M M S S S S M M
M M
V KV
u v
V c V c
u
V c V c
V c
u v
+
=
+
=
+
=
1
1
1
1
V
S
v V
M
c th xc nh da
theo phng php chun b ct
M
M R
A
A M R
t
t t
k
k t
L
t
L
=
+
=
'
'
1
1
H s kh nng
(Capacity Factor)
Thng s thc nghim quan trng
M t tc di chuyn ca cht tan trong ct
i vi cht tan A, h s kh nng k
A
:
t
R
t
M
'
'
1
1
A M
S A
A
k
u v
V
V K
k
+
= =
A
B
K
K
= o
Tc di chuyn tng i: H s chn lc o
(Selectivity Factor)
B l cu t b gi mnh trn ct
A l cu t b hp ph yu hn trn ct
o > 1
( )
( )
M
A
R
M
B
R
A
B
t t
t t
k
k
= =
'
'
o
Hiu qu ca ct sc k
(Efficiency of chromatographic colunms)
S gin peaks (band
broadenning)
Dng hnh hc ca peak: phn b Gaussian
hoc ng cong sai s chun (normal error
curves)
Mt phn t chu hng ngn ln chuyn t pha
ng sang pha tnh
Cn trao i nng lng gia phn t v mi
trng xung quanh
Thi gian lu ca mt phn t trong mt pha
thng c sai lch ngu nhin so vi cc
phn t cng loi khc
Gin i xng (symmetric spread) xung quanh
mt gi tr chnh
Khong cch di chuyn thc t trong ct c
th khc nhau gia cc phn t
Hiu qu ca ct sc k
NH LNG HIU QU CA CT SC K
Chiu cao tng ng ca a (H)
(Plate height)
S a l thuyt (N)
(Number of theoritical plates)
N = L/H
lch chun (o)
Variance (o
2
)
L
H
2
o
=
W
2
16
|
.
|
\
|
=
W
t
N
R
t
R
= (t)
R
+t
o
Hiu qu ca ct sc k
Variance thi gian ca peak: t
2

R
t L
o
t =
2
16
|
.
|
\
|
=
W
t
N
R
Vi L/t
R
: Vn tc thng trung bnh (average linear velocity) ca cht phn tich
Xc nh t t thc nghim:
V 2 tip tuyn t cc im un
Din tch tam gic = 96% din tch peak
(sai lch 2t) v W= 4t
R
t
LW
4
= o
2
2
16
R
t
LW
H =
N = L/H
W
t
R
2o
Hiu qu ca ct sc k
CC YU T NH HNG N HIU QU CT SC K
Tc dng ca pha ng: H
minimum
(Hiu qu cao nht) xut hin
vng tc thp (0,1 0,2 m.s
-1
: LC v 1-2 m.s
-1
: GC)
Kch thc ht ca pha tnh i vi ct nhi (column packings)
Chiu dy mng hn ca lp c nh (immobilized film) khi pha tnh
l cht lng hp th trn cht rn (liquid adsorbed on a solide)
Tng nhit s lm gim s gin peak i vi tt c cc trng
hp
Gim nht ca pha ng
phn gii ca ct (R
s
)
(Column resolution)
phn gii ca ct (R
s
) cung cp cc gi tr nh lng c trng cho
kh nng tch ca hai cht cn phn tch
( ) ( ) | |
B A
A R B R
B A
s
W W
t t
W W
Z
R
+
=
+
A
=
2
2
R
S
= 0,75 phn gii v phn
tch peak km
R
S
= 1 Vng A cha khong 4% B
v vng B cha khong 4%
(overlap = 4%)
R
S
= 1,5 phn tch peak gn tuyt
i (overlap = 0,3%)
Tng phn gii:
Tng chiu di ct >< thi gian
phn gii ca ct (R
s
)
(Column resolution)
nh hng ca cc h s kh nng v chn lc n phn gii
S lng a (number of plates), N
H s kh nng (capacity factor), k
B

H s chon lc (selectivity factor), o
R
s

|
|
.
|
\
|
+
|
.
|
\
|

=
'
'
1
1
4
B
B
s
k
k N
R
o
o
Xt phn gii ca hai cht A v B:
2
'
'
2
2
1
1
16
|
|
.
|
\
| +
|
.
|
\
|
=
B
B
s
k
k
R N
o
o
S a cn thit (chiu cao ct sc
k t c mt phn gii
cho trc
phn gii ca ct (R
s
)
(Column resolution)
nh hng ca phn gii n thi gian lu
Mc ch ca mt qu trnh phn tch sc k
phn gii cao
Thi gian lu nh nht
Xc nh thi gian lu t
R

i vi cu t kh tch (t
R
)
B

( )
( )
( )
2
'
2
'
2
2
1
1
16
B
B s
B R
k
k
u
H R
t
+
|
.
|
\
|
=
o
o
u: Tc tuyn tnh ca pha ng
Tm tt cc cng thc
H s kh nng (capacity factor)
H s chn lc (selectivity factor)
Tc di chuyn trung
bnh ca cht tan
R
t
L
v =
Tc di chuyn trung
bnh ca cht tan
R
t
L
v =
Tc di chuyn trung
bnh pha ng
o
t
L
u =
Tc di chuyn trung
bnh pha ng
o
t
L
u =
M S
V KV
u v
+
=
1
1
'
'
1
1
A M
S A
A
k
u v
V
V K
k
+
= =
M
M R
A
t
t t
k

=
'
( )
( )
o A R
o B R
A
B
t t
t t
k
k
= =
'
'
o
A
B
K
K
= o
( ) ( ) | |
B A
A R B R
B A
s
W W
t t
W W
Z
R
+
=
+
A
=
2
2
|
|
.
|
\
|
+
|
.
|
\
|

=
'
'
1
1
4
B
B
s
k
k N
R
o
o
( )
( )
( )
2
'
2
'
2
2
1
1
16
B
B s
B R
k
k
u
H R
t
+
|
.
|
\
|
=
o
o
2
'
'
2
2
1
1
16
|
|
.
|
\
| +
|
.
|
\
|
=
B
B
s
k
k
R N
o
o
2
16 |
.
|
\
|
=
W
t
N
R
L
H
2
o
=
2
2
16
R
t
LW
H =
N = L/H
Chiu cao a (plate height) v s a
(number of plates)
phn gii ca ct (column resolution)
p dng
S liu ban u:
(t
R
)
A
=16.4 pht, (t
R
)
B
=17.63 pht, (t
R
)
M
=1.3 pht, chiu di ct: L=30 cm
rng ca peak ti ng nn: W
A
=1.11 pht v W
B
=1.21 pht
Tnh ton: R
s
, N, H, Chiu di ca ct bo m R
s
=1.5 v (t
R
)
B
tng ng.
R
s
= 2(17.63 16.4)/(1.11+1.21) = 1.06

Gii:
N = 16(16.4/1.11)
2
= 3493 v N = 16(17.63/1.21)
2
= 3397
N = (3493+3397)/2= 3445
H = L/N = 30/3445 = 8.710
- 3
cm
Do k
B
v o khng thay i khi tng chiu cao ca ct, ta c:
( )
( )
cm H N L
N
N N
N
R
R
s
s
60 10 7 . 8 10 9 . 6
10 9 . 6
3445
5 . 1
06 . 1
3 3
3
2
2 2
1
2
1
= = =
= = =
( )
( )
( )
( )
( )
( ) phut t
t
R
R
t
t
R
R
s
s
R
R
35
5 . 1
06 . 1 63 . 17
2 2
2
2
2
2
2
1
2
1
= = =
|
|
.
|
\
|
+
|
.
|
\
|
=
'
'
1
1
4
B
B
s
k
k N
R
o
o
( )
( )
( )
2
'
2
'
2
2
1
1
16
B
B s
B R
k
k
u
H R
t
+
|
.
|
\
|
=
o
o
Cc ng dng ca sc k
Phn tch nh tnh

Phn tch nh lng

Phn tch da vo chiu cao peak

Phn tch da vo din tch peak

Xy dng ng chun (calibration with standards)

Phng php chun ni (internal-standard)
B. Sc k kh
(Gas-Liquid Chromatography)
Sc k kh
Gas-Liquid Chromatography (GLC) hoc l Gas Chromatography (GC)
Bc hi mu
Tch cc cu t trong ct nh vo s phn b trong pha ng v pha tnh
Pha ng: pha kh (N
2
, He, Ar)
Pha tnh: pha rn hoc pha lng ph ln pha rn c gi trong ct
Phng php cng c phn tch v xc nh cc hp cht ha hc
B. Sc k kh
Mu (sample) phn tch c
- a vo b phn np mu (heated injector)
- Di chuyn qua mt ct phn tch (seperating column) nh mt dng kh
mang tr (inert carrier gas)
- Pht hin v ghi li di dng cc peaks khi cc cu t i ra khi ct
B. Sc k kh
T=0
T=10
T=20
Injector Injector
Detector Detector
T=0
T=10
T=20
Injector Injector
Detector Detector
B. Sc k kh
Ngun cung cp kh mang (Carrier Supply)

F = 25 150 ml.min
-1
: Ct nhi (Packed column)
F = 1 25 ml.min
-1
: Ct mao qun (Open-tubular or
Capillary column)
- N
2
: chi ph thp, an ton
- H2: chi ph thp, nguy c chy n
- He: thng thng, t
- Ar:
Bnh cha p sut cao (pressurized tank)
- Dng c iu chnh p sut (pressure
regulator)
- iu khin lu lng dng kh (Flow
controller)
Two stages pressure regulator
B. Sc k kh
Ngun cung cp kh mang (Carrier Supply)

Thit b tch N
2
t khng kh nn (Pure Nitrogen Generator)
- Thm thu chn lc N
2

- 0.5 ppm O
2
, > 0.5 ppm H
2
O, > 2.0 ppb halocarbons hoc C
x
H
y
.
- Lu kng ti a ~ 1 l/min. p sut 3,5 7 atm.
Thit b cung cp kh H
2
t nc ct (Hydrogen Generators)
- Phng php in phn (Electrolysis)
- Cht in ly: polymer rn (solid polymer electrolyte)
- H
2
99.999%
- Kh nng lu tr H
2
: 4 litre
- p sut: 1,4 7 atm.
- Lu lng: 0 to 125 ml.min
-1
v c th t n 1200 ml.min
-1
.
B. Sc k kh
H thng np mu (Sample Injection system)

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

S nguyn l h thng np mu
Ct mao qun Ct nhi
B. Sc k kh

Ch np mu:
- Chia dng (split)
- Khng chia dng (splitless)
B. Sc k kh
Ct sc k (Column)

Ct nhi (packed column): ID: 2 4 mm, L: 2 3 m
- Pha tnh - Cht hp ph c nhi vo ct
- phn tch kh (gas analysis)
- Np mu n gin
- chnh xc cao
Ct mao qun (open-tubular or capillary column): ID: 0,25 0,5 mm, L: 25 50 m
- Np mu kh khn
- State of art column
- Pha tnh c ph vo mt trong ca ct (0,2 - 1m)
B. Sc k kh
Ct sc k (Column)

wall-coated open-tubular (WCOT) column
B. Sc k kh
Cu to ca ct nhi

V ct: thp khng g hoc thy tinh Pyrex
Cht hp ph (Adsorbents): hai loi cht c nhi vo ct :
- Cht hp ph.
- Cht mang (support) c ph pha tnh.

Cc cht hp ph thng s dng:
Alumina (Al
2
O
3
): Hot ha 200C 1h: tch kh v cc hyrocacbon
n C
5
, kch thc ht: 100/120 mesh, kch thc l xp:1 - 100 .
Silica (SiO
2
): tch cc kh c M nh v cc Hydrrocacbon nh
- B mt ring750 m
2
/g, kch thc l xp trung bnh: 22
- B mt ring 100m2/g kch thc l xp trung bnh: 300 .
Kch thc: - 30/40 -100/120 mesh
- ng nht
B. Sc k kh
Cu to ca ct nhi

Cc cht hp ph thng s dng:
Zeolith: Tch cc kh c M nh bng phng php loi tr (exclusion):
Ry phn t (molecular sieves)
- Cc zeolith k hiu: 5A v 13X: thng c s dng tch H2, O2,
N2, CH4, CO, Ar, Ne
Cacbon:
- Cacbon hot tnh: b mt ring ~ 1000 m
2
.g
-1

- Graphit: b mt ring 5 - 100 m
2
.g
-1
Cc hp cht cao phn t:
- Co-polymer ca polystyrene v divinylbenzene
- L xp: macropore v micropore
- B mt ring ln v xp cao
- Tng tc a dng vi cc dung mi v cht tan tip xc vi n.
B. Sc k kh
Cu to ca ct nhi

Cc cht mang s dng cho GLC:
- Celite (mt dng c bit ca khong diatomic), Celite nung, Celite nung
hot ha bi Ag hoc Au, cc ht vi cu thy tinh, polymer, teflon
Bin tnh Celite:
-Nung 900C vi Na
2
CO
3
v tr dung: silica cristobalite, cc vt Kim
loi tc dng vi Silica gy mu (hng) cho vt liu.
Chromosorb P, Chromosorb W, Chromosorb G v Chromosorb S
Chuyn cht hp ph ln cht mang:
S dung cc nhm Silanol (Si-OH)
Hexamethyldisilazane + Si-OH gc trimethylsilyl
Phng php tm (slurry method of coating)
Vi cu Polystyren
B. Sc k kh
Cu to ca ct mao qun (capillary or open-tubular column)

Pht minh vo nhng nm 1950
Tc phn tch nhanh vi s lng a cc ln ~ 300.000 a
a vo p dng vo cui nhng nm 1970
Cu to t thy tinh hoc fused silica
ID = 0,25 0,5 mm
L = 25 50 m
B mt trong ca mao qun c ph mt lp mng
pha ng 0,25 1,5m
(WallCoated Open-Tubular - WCOT)
B. Sc k kh
Cu to ca ct mao qun (capillary or open-tubular column)

B. Sc k kh
Bin tnh b mt fused silica
phn cc (polar) ca cc gc Silanol trn b mt

Bin tnh b
mt Silica
Phn cc: -CN, -CO v OH
Khng phn cc:
Hydrrocacbon (dialkyl siloxane)
Phn cc ln: Polyester
B. Sc k kh
Cc pha tnh (ST) thng s dng trong GLC
Polar SP
Non-polar SP
nh hng ca phn cc ca pha
tnh n thi gian lu
B. Sc k kh
So snh ct nhi v ct mao qun
B. Sc k kh
n nhit ct sc k (Column Thermostating)
Mc ch: Bo m tnh lp li ca thi gian lu
L n nhit (thermostating oven)
T
opti.
= f(T
si
), T
opti
> T
si
vi RT= 2 30 pht
L n nhit
Ct sc k
Nhit chng trnh ha
(Temperature Programming)
Isothermal: mu n gin
Mu phc tp: Tch cc cu t ca
mu da vo s thay i ca T si
B. Sc k kh
So snh sc k hai ch : Isothermal
v chng trnh ha nhit
Chng trnh ha nhit
H s kh nng
phn gii
B. Sc k kh
Nhit chng trnh ha
Tng kh nng tch ca ct nh ngng t ri bc hi dung mi
Dung mi bc hi ngay khi
vo ct sc k
Dung mi ngng t trn ct
cng vi cc cu t khc,
sau bc hi, ti phn b
li cc cht cn phn tch
B. Sc k kh
Cc vng c gia nhit ca h sc k kh (GC)
B. Sc k kh
u d (Detectors)
Mt s yu cu:
Tn hu thu c tuyn tnh hoc gn tuyn tnh vi lng mu
Thi gian tr li nhanh
Pht hin a dng (universal detection)
Tn hiu ra khng ph thuc v nhit
Lm vic n nh t nhit thng n 400C (i vi GC)
Thermal Conductivity Detector (TCD)
Flame Ionization Detector (FID)
Electron Capture Detector (ECD)
Nitrogen-Phosphorous Detector (NPD)
Flame Photometric Detector (FPD): FID tweaked for S compounds
Photoionization Detector (PID)
B. Sc k kh
dn nhit ca khi mang (He hoc H2)
10 ln ln hn cc hp cht hu c
S nguyn l
Measures heat loss from a hot filament nearly universal
Filament heated to const T
When only carrier gas flows heat loss to metal block is constant, filament T
remains constant
When an analyte species flows past the filament generally thermal conductivity
goes down, T of filament will rise. (resistance of the filament will rise).
B. Sc k kh
B. Sc k kh
Flame Ionization Detector (FID)
Sensitive towards organics

Analyte is burned in H
2
/air, which produces CH and CHO
+
radicals
CHO
+
radicals are reduced at a cathode which produces a current
proportional to the radical quantity ~ 10
-12
A
Specific for organic carbon, insensitive to inorganics, CO
2
, SO
2
etc.
Generally DL 100x less than TCD about pg/s (flow rate dependent)
Response to specific organic depends on the number of organic carbons.
B. Sc k kh
Sensitive to electron withdrawing groups especially towards organics
containing F, -Cl, -Br, -I also, -CN, NO
2

Nickel-63 source emits energetic electrons collides with N
2
(introduced as make-
up gas or can be used as carrier gas) producing more electrons:
Ni-63 e
-
, e
-
+ N
2
2e- + N
2
+
The result is a constant current that is detected by the electron collector (anode).
As an analyte flows through past the Ni-63 source, electron capture is possible by
electron-withdrawing species: A + e
-
A
-
Current decreases as a result of e- capture by analyte. This is one of the few
instances in which a signal is produced by a decrease in detectable phenomenon.
Very low DL for detected species 10
-15
g/ml for many halogenated substances
B. Sc k kh
Radioactive Ni-63 source
Easily contaminated with O2, H2O, sample overloading.
High maintenance device.
Highly variable response to halogenated substances
Sometimes complementary information from FID helps.
The bad
S nguyn l
B. Sc k kh
Mt s k thut chuyn valve
Back flushing Techniques
Mc ch: phn tch cc mu cha cc
cht tan tng tc mnh vi ct:
nhim bn ct hoc thi gian lu ko
di
Valve 8 cng
Chuyn valve v o chiu
chuyn ng ca pha ng
Hn ch s nhim bn ca ct
v thi gian phn tch
B. Sc k kh
Mt s k thut chuyn valve
Apparatus for Heart Cutting
Valve 6 ng
S dng i h thng vi hai ct v 2
detectors
Cc cu t vng gia ca mu c
phn tch trn ct 2
Cc cu t u v cui c phn
tch trn ct 1
B. Sc k kh
Thu thp v x l s liu (Data Acquisition and Processing )
Nguyn l
A/D Converter
B. Sc k kh
Mt s ng dng ca GC (GLC)
p dng i vi cc mu bc hi v n nh nhit n vi trm C
C kh nng pht hin v phn tch rt nhiu cht v hn hp
c ng dng rng ri tch v xc nh cc cu t trong cc mu t
nhiu chng loi khc nhau
Mt vi v d:
Ketones: polydimthyl siloxane
Alkalodes: 5% phenyl polydimethyl siloxane
Sterods: 50% phenyl polydimethyl siloxane
Chlorinated Aromatics: 50% Trifluoropropyl polydimethyl siloxane
Alcohols: Polyethylenglycol
Esters: 50% Cyanopropyl polydimethyl siloxane
Sc k kh kt hp khi ph
B. Sc k lng hiu qu cao
(Hight-Performance Liquid Chromatography - HPLC)
Phn loi HPLC da bn cht tng tc
Sc k phn b (partition chromatography)
Sc k hp ph hoc lng-rn (adsorption or liquid-
solid chromatography)
Sc k trao i ion (ion exchange chromatography)
Sc k loi tr kch thc (size exclusion
chromatography)
SO3
-
SO3
-
Na
+
COO
-
H3N
+
Na
+
COOH
H3N
+
pH2
pH4.5
Ion-exchange Resin
VD: nguyn l sc k trao i ion
(acide amine)
Sc k loi tr kch thc
c im ca HPLC
Pha tnh c nhi trong ct
Pha ng trng thi lng: Cc dung mi, hn hp dung mi hoc nc
Phn loi HPLC da vo vt liu nhi
Pha thng thng (Normal phase): vt liu nhi l silica n gin
Trao i ion: silica bin tnh (mdified silica)
Pha o (reverse-phase): silica bin tnh
Phn ln cc HPLC l pha o
Cht phn tch c gi trn pha tnh
phn cc nh hn cho n khi b ra tri
bi pha ng phn cc ln
Thao tc n gin
Hiu qu cao
Ct lm vic n nh
C th phn tch cho c hai loi cu t c
c tnh tng t hoc khc xa nhau
Mc du c nhiu l thuyt nghin cu v vic s dng pha o nhng phn
ln cc chng trnh HPLC pha o u thu c t phng php th v
sai (by trial and error).
Gc R l C8 (n-octyl), C12 (n-octyl)
hoc C18 (n-octyldecyl).
Pha ng l H2O + dung mi ha tan
(acetonitrile, methanol, ethanol,
isopropanol).
Cc cu t phn cc s b ra ra
nhanh nht, tng phn cc ca pha
ng s lm tng thi gian chy mu
Pha tnh-Pha o
(Stationary Phases for Reversed-Phase LC)
Pha tnh bnh thng ca LC
(Stationary Phases for Normal LC)
Pha ng tng i khng phn cc:
Hexane, Isopropyl eter, toluene
Cc cu t khng phn cc s b ra
ra nhanh nht, tng phn cc ca pha
ng s gim thi gian chy mu
nh hng ca bn cht pha tnh n cht lng tch
Pha o
S nguyn l ca HPLC
Cc yu cu i vi dung mi
p sut bm: vi trm atm (~6000psi), lu lng 0,1 10 ml.min
-1
vi E<0,5%
Vt liu bm bn n mn i vi nhiu loi dung mi khc nhau
Ch bm piston
C ht trong ct sc k: 3 - 10m
Mt hoc nhiu bnh cha dung mi (500 ml)
Loi b hon ton kh ha tan v cn trong dung mi gim rng ca peak
(band spreading) v nh hng n cht lng detector
ui kh ha tan trong dung mi bng kh tr (sparger)
La chn ch tch ra (elution) cho dung mi
Trang b cc loi valves t l (proportionating valves) cho php a dung mi t
hai bnh cha vi cc lu lng thay i lin tc
Hiu qu tch bng phng
php gradient elution
S dng mt dung mi n gin c thnh
phn khng i: isocratic
S dng hai hay nhiu hn cc h dung
mi c phn cc (polarity) khc nhau
nhiu: gradient elution
T l cc loi dung mi c chng trnh
ha lin tc hoc theo tng bc
Gradient elution: tng cht lng ca qu
trnh tch (improve seperation efficiency)
Qu trnh tch ra (Elution)
Polar Solvents

Water > Methanol > Acetonitrile > Ethanol > Oxydipropionitrile

Non-polar Solvents

N-Decane > N-Hexane > N-Pentane > Cyclohexane
phn cc ca mt s dung mi s dng trong HPLC
La chn pha ng v pha tnh
Ch yu da vo s phn cc ca cu t phn tch, pha ng, pha tnh
Quy tc chung: phn cc (polarity) ca cu t cn phn tch v pha tnh l
tng ng cn pha ng c phn cc sai bit
Khi phn cc ca cu t v pha tnh qu ging nhau: thng tc mnh gia
cu t cn phn tch v pha tnh thi gian phn tch ko di
Tnh cht mt s loi dung mi s dng trong HPLC
H thng np mu (Sample Injection Systems)
S dng valve 6 cng
Np mu qua vng ly mu (sampling loops) Sc k lng hin i
C th thay th sampling loops t 5 l n 500 l
Sai s ca lng mu np di 1%
Ct sc k HPLC
Thng thng:
L = 10 30 cm v c th ni tip 2 ct hoc nhiu hn
ID = 4 10 mm, kch thc ht nhi: 3, 5 v 10m
40.000 60.000 a/m ct
Ct tc cao v hiu qu hn
L = 3 - 7 cm v c th ni tip 2 ct hoc nhiu hn
ID = 1 4,6 mm, kch thc ht nhi: 3 hoc 5 m
100.000 a/m ct
Ct bo v (Guard Column)
c lp t trc ct phn tch ko di tui th ca ct
Thnh phn = thnh phn ca ct phn tch nhng c ht ln
hn gim tn tht p sut
n nh nhit ca ct (Column Thermostats)
Phn ln ng dng cua HPLC c thc hin nhit phng
Tuy vy cht lng ca sc k s tt hn nu duy tr nhit ca ct
khng thay i (sai s < 0,05C)
Thit b HPLC hin i c trang b thm l gia nhit cho ct (Column
heater) n nh nhit gn 150C vi sai s < 0,05C
Trang b h thng phun nc lm lnh (water jackets fed) t b n nhit
khng ch chnh xc nhit
u d (Detector) dng cho HPLC
Khng nhy v c kh nng phn tch a dng nh detector ca GC
Thng gp nht l Detector UV-Vis
LOC: Limit Of Detection
Mass LOD = concentration (mol/L) x inj. vol. (L) x FW (g/mol)
UV-Vis and Fluorescence Detector Electrochemical Detector
Amperometric detection = fixed potential and measure the current response.
= 200-400nm
s dng 254 nm
Refractive Index Detector
Cc phng php nng cao phn gii trong HPLC
Tng chiu di ca ct (Increase column length)
Gim ng knh ca ct (Decrease column diameter)
Gim lu lng pha ng (Decrease flow-rate)
Pha tnh (vt liu nhi ct) ng nht (Uniform stationary phase (packing))
Gim th tch bm mu (Decrease sample size)
La chn pha tnh sch hn (Select proper stationary phase)
La chon pha ng tinh khit hn (Select proper mobile phase)
S dng p sut n nh hn (Use proper pressure)
Thnh phn ca pha ng thay i hp l (Use gradient elution)
So snh HPLC v GC
(Comparison of HPLC and GLC)
Cc c im chung:
Hiu qu, chn lc cao, ng dng rng ri
Th tich mu nh
C th khng ph hy mu (nondestructive of sample)
nh lng d dng
u im ca HPLC
p dng c vi cc mu khng
bay hi v khng bn nhit
p dng c cho cc ion v c
u im ca GC
Thit b n gin v r
Nhanh chng
D dng kt ni vi ph khi

Sac Ky

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