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1/71

CHUYN : S C K (C S L THUY T V NG D NG)

TS NGUY N NH LM

B MN CNG NGH HA H C V V T LI U, KHOA HA

TR NG I H C BCH KHOA I H C N NG

Sample Mobile phase

t0 t1 t2 t3 t4

t0 t1 t2 t3 t4

Detector

signals

Time

Detector

Sample Mobile phase

t0 t1 t2 t3 t4t0 t1 t2 t3 t4

t0 t1 t2 t3 t4t0 t1 t2 t3 t4

Detector

signals

Time

Detector


2/71

A. I C NG V PH NG PHP S C K (Chromatography)

c pht minh b i nh sinh v t h c ng i Nga Mikhail TswestTch Chlorophills v Xanthophylls b ng CaCO 3

Ti ng Hy-l p: Chroma: mu

Graphein: ghi

Ph ng php s c k:

K thu t tch (seperation) cc c u t trong m t h ng th (kh ho c l ng)

Cn b ng n ng c a cc c u t trong hai pha ti p xc nhau: pha tnh (stationary phase) v pha ng ( mobile phase)

S phn tch d a trn t c ko theo ( elution) khc nhau c a cc c u t trong c t ( column)

M t u d (detector) u ra c a c t cho php nh l ng lin t c cc c u t trong h n h p u

S c k mng m ng ( planar chromatography), S c k c t ( Column chromatography)


3/71

I C NG V PH NG PHP S C K (Chromatography)

T= 0

T= 10

T= 20

I njectorI njector DetectorDetector

MostMost I nteraction w ith Stationary PhaseI nteraction w ith Stationary Phase LeastLeast

Flow of Mobile PhaseFlow of Mobile Phase

T= 0

T= 10

T= 20


MostMost I nteraction w ith Stationary PhaseI nteraction w ith Stationary Phase LeastLeast



4/71

S c k phn tch(Elution chromatography)

Phn tch s c k : Cc ch t tan b r a qua m t pha tnh nh s chuy n

ng c a pha ng qua n M u Pha ng

Detectort0 t1 t2 t3 t4

t0 t1 t2 t3 t4

Tnh

i

u

detector

Th i gian


5/71

S c k phn tch(Elution chromatography)

Phn tch s c k : Cc ch t tan b r a qua m t pha tnh nh s chuy n

ng c a pha ng qua c t ch a pha tnh

Pha ng


6/71

S c k (Chromatogrames)

i u ki n thu c s c k :

- u d (Detector) c l p t i m cu i c a c t

- u d t ng thch v i cc ch t c n pht hi n S c k : Bi u di n s bi n thin c a tn hi u ra theo th i gian ho c theo th tch tiu hao c a pha ng

Cc peaks i x ng (ho c khng i x ng)

Phn tch nh tnh (qualitative) v nh l ng (quantitative)


7/71

S c k (Chromatogrames)

S phn gi i (resolution)

V n t c di chuy n t ng i (relative migration rates) S gin peak (band broadening)


8/71

V n t c di chuy n c a cc ch t tan (Migration rates of solutes)

Th i gian l u t R

(Retention time)

T c di chuy n trung bnh c a ch t tan

RtLv =

T c di chuy n trung bnh pha ng

ot

Lu =


9/71


M

s

c

cK=

soluteofmolestotal

phasemobileinsoluteofmolesuv =

Quan h gi a t c di chuy n v h s phn b

H s phn b K

(Partition Ratios)

Cn b ng phn b c a ch t tan trong pha ng v pha tnh

Amobile Astationary

MS

MMSSSSMM

MM

VKVuv

VcVcu

VcVc

Vcuv

+=

+=

+=

1

1

1

1

VS v VM c th xc nh d a theo ph ng php chu n b c t


10/71


M

MRA

AMR t

ttk

kt

L

t

L =+

= ''1

1

H s kh nng

(Capacity Factor)

Thng s th c nghi m quan tr ng

M t t c di chuy n c a ch t tan trong c t

i v i ch t tan A, h s kh nng k A:

tR

tM

'

'

1

1

AM

SAA

kuv

VVKk

+==


11/71


A

B

K

K=

T c di chuy n t ng i: H s ch n l c (Selectivity Factor)

B l c u t b gi m nh trn c t

A l c u t b h p ph y u h n trn c t 1

( )

( ) MAR

MBR

A

B

tt

tt

k

k

==

'

'


12/71

Hi u qu c a c t s c k (Efficiency of chromatographic colunms)

S gin peaks (bandbroadenning)

D ng hnh h c c a peak: phn b Gaussian ho c ng cong sai s chu n (normal error curves)

M t phn t ch u hng ngn l n chuy n t pha ng sang pha tnh

C n trao i nng l ng gi a phn t v mi tr ng xung quanh

Th i gian l u c a m t phn t trong m t pha th ng c sai l ch ng u nhin so v i cc phn t cng lo i khc

Gin i x ng (symmetric spread) xung quanh m t gi tr chnh

Kho ng cch di chuy n th c t trong c t c th khc nhau gi a cc phn t


13/71


NH L NG HI U QU C A C T S C K

Chi u cao t ng ng c a a (H) (Plate height)

S a l thuy t (N) (Number of theoritical plates)

N = L/H

l ch chu n ( )

Variance (2)

LH

2

=W

2

16

=W

tN R

tR = (t)R + to


14/71


Variance th i gian c a peak: 2

RtL

=

2

16

=W

tN R

V i L/t R: V n t c th ng trung bnh (average linear velocity) c a ch t phn tich

Xc nh t th c nghi m:

V 2 ti p tuy n t cc i m u n

Di n tch tam gic = 96% di n tch peak

(sai l ch 2 ) v W= 4

Rt

LW

4=

2

2

16 Rt

LWH =

N = L/H

W

tR

2


15/71


CC Y U T NH H NG N HI U QU C T S C K

T c dng c a pha ng: H minimum (Hi u qu cao nh t) xu t hi n vng t c th p ( 0,1 0,2 m.s-1: LC v 1-2 m.s-1: GC)

Kch th c h t c a pha tnh i v i c t nh i (column packings)

Chi u dy m ng h n c a l p c nh (immobilized film) khi pha tnh

l ch t l ng h p th trn ch t r n (liquid adsorbed on a solide)

Tng nhi t s lm gi m s gin peak i v i t t c cc tr ng h p

Gi m nh t c a pha ng


16/71

phn gi i c a c t (R s)(Column resolution)

phn gi i c a c t (R s) cung c p cc gi tr nh l ng c tr ng cho kh nng tch c a hai ch t c n phn tch

( ) ( )[ ]

BA

ARBR

BA

sWW

tt

WW

ZR

+

=

+

=

22

RS = 0,75 phn gi i v phn tch peak km

RS = 1 Vng A ch a kho ng 4% B v vng B ch a kho ng 4% (overlap = 4%)

RS = 1,5 phn tch peak g n tuy t i (overlap = 0,3%)

Tng phn gi i:

Tng chi u di c t >< th i gian


17/71


nh h ng c a cc h s kh nng v ch n l c n phn gi i

S l ng a (number of plates), N

H s kh nng (capacity factor), kB

H s chon l c (selectivity factor),

Rs

+

=

'

'

1

1

4B

Bs

k

kNR

Xt phn gi i c a hai ch t A v B:

2

'

'2

2 1

116

+

=

B

Bs

k

kRN

S a c n thi t (chi u cao c t

s c k t c m t phn gi i cho tr c


18/71


nh h ng c a phn gi i n th i gian l u

M c ch c a m t qu trnh phn tch s c k

phn gi i cao

Th i gian l u nh nh t

Xc nh th i gian l u t R

i v i c u t kh tch (t R)B

( )( )

( ) 2'

2'22 1

1

16

B

Bs

BR

k

k

u

HRt

+

=

u: T c tuy n tnh c a pha ng


19/71

Tm t t cc cng th c

H s kh nng (capacity factor)

H s ch n l c (selectivity factor)

Tc di chuyn trungbnh ca cht tan

Rt

Lv =

Tc di chuyn trungbnh ca cht tan

Rt

Lv =

Tc di chuyn trungbnh pha ng

ot

Lu =

Tc di chuyn trungbnh pha ng

ot

Lu =

MS VKVuv

+=

1

1

'

'

1

1

AM

SAA

kuv

V

VKk

+==

M

MRA

t

ttk

='

( )

( ) oAR

oBR

A

B

tt

tt

k

k

==

'

'

A

B

K

K=

( ) ( )[ ]

BA

ARBR

BA

s

WW

tt

WW

ZR

+

=

+

=

22

+

=

'

'

1

1

4 B

Bs

k

kNR

( )( )

( )2

'

2'22 1

1

16

B

Bs

BR

k

k

u

HRt

+

=

2

'

'2

2 11

16

+

=

B

Bs

kkRN

2

16

=W

tN R

LH

2

= 22

16 Rt

LWH =

N = L/H

Chi u cao a (plate height) v s a (number of plates)

phn gi i c a c t (column resolution)


20/71

p d ngS li u ban u: (tR)A=16.4 pht, (tR)B=17.63 pht, (tR)M=1.3 pht, chi u di c t: L=30 cm

r ng c a peak t i ng n n: W A=1.11 pht v WB=1.21 phtTnh ton: Rs, N, H, Chi u di c a c t b o m R s=1.5 v (tR)B t ng ng.

Rs= 2(17.63 16.4)/(1.11+1.21) = 1.06

Gi i:

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 = 8.7 10- 3 cm

Do kB v khng thay i khi tng chi u cao c a c t, ta c: ( )

( )

cmHNL

NNN

N

R

R

s

s

60107.8109.6

109.63445

5.1

06.1

33

3

2

22

1

2

1

===

===

( )

( )

( )

( ) ( )( ) phutt

tR

R

t

tR

Rs

s

R

R35

5.1

06.163.1722

2

2

2

2

2

1

2

1 ===

+

=

'

'

1

1

4 B

Bs

k

kNR

( )( )

( )2'

2'22

1

1

16

B

Bs

BR

k

k

u

HRt

+

=


21/71

Cc ng d ng c a s c k

Phn tch nh tnh

Phn tch nh l ng

Phn tch d a vo chi u cao peak

Phn tch d a vo di n tch peak

Xy d ng ng chu n (calibration with standards)

Ph ng php chu n n i (internal-standard)


22/71

B. S c k kh(Gas-Liquid Chromatography)

S c k kh

Gas-Liquid Chromatography (GLC) ho c l Gas Chromatography (GC)

B c h i m u

Tch cc c u t trong c t nh vo s phn b trong pha ng v pha tnh

Pha ng: pha kh (N 2, He, Ar)

Pha tnh: pha r n ho c pha l ng ph ln pha r n c gi trong c t

Ph ng php cng c phn tch v xc nh cc h p ch t ha h c


23/71


M u (sample) phn tch c

- a vo b ph n n p m u (heated injector)

- Di chuy n qua m t c t phn tch (seperating column) nh m t dng khmang tr(inert carrier gas)

-Pht hi n v ghi l i d i d ng cc peaks khi cc c u t i ra kh i c t


24/71


T= 0

T= 10

T= 20


MostMost I nteraction with Stationary PhaseI nteraction with Stationary Phase LeastLeast


T= 0

T= 10

T= 20


MostMost I nteraction with Stationary PhaseI nteraction with Stationary Phase LeastLeast



25/71


Ngu n cung c p kh mang (Carrier Supply)

F = 25 150 ml.min-1: C t nh i (Packed column)F = 1 25 ml.min-1: C t mao qu n (Open-tubular orCapillary column)

- N2: chi ph th p, an ton

- H2: chi ph th p, nguy c chy n - He: thng th ng, t

- Ar:

Bnh ch a p su t cao (pressurized tank)

- D ng c i u ch nh p su t (pressure regulator)

- i u khi n l u l ng dng kh (Flow controller)

Two stages pressure regulator


26/71


Ngu n cung c p kh mang (Carrier Supply)

Thi t b tch N 2 t khng kh nn (Pure Nitrogen Generator)

- Th m th u ch n l c N 2

- 0.5 ppm O2, > 0.5 ppm H2O, > 2.0 ppb halocarbons ho c C xHy.

- L u k ng t i a 1 l/min. p su t 3,5 7 atm.

Thi t b cung c p kh H 2 t n c c t (Hydrogen Generators)

- Ph ng php i n phn (Electrolysis)

- Ch t i n ly: polymer r n (solid polymer electrolyte)

- H2 99.999%

- Kh nng l u tr H 2: 4 litre

- p su t: 1,4 7 atm.

- L u l ng: 0 to 125 ml.min -1 v c th t n 1200 ml.min -1.


27/71


H th ng n p m u (Sample Injection system)

Cc yu c u:

- L ng m u thch h p

- T c n p m u ph i nhanh v m u n p khi vo c t tr ng thi kh

Gi m s gin peak (band broadenning) v tng phn gi i c a c t

Microsyringe chu n (calibrated)

Septum: mng b ng cao su silicone

Gia nhi t cho vng n p m u: T > 50C c a c u t c nhi t si cao nh t

Th tch n p m u: 20 l i v i c t nh i (packed column)

0,2 l ho c nh h m i v i mao qu n (open-tubular or

capillary column)


28/71



S nguyn l h th ng n p m u

C t mao qu n C t nh i

B S c k kh


29/71



Ch n p m u:

- Chia dng (split)

- Khng chia dng (splitless)

B S k kh
http://storage/tailieu/files/source/20100704/dinhvinh87/E:/Biblio/Chromato/GC.mov


30/71


C t s c k (Column)

C t nh i (packed column): ID: 2 4 mm, L: 2 3 m

- Pha tnh - Ch t h p ph c nh i vo c t

- phn tch kh (gas analysis)

- N p m u n gi n

- chnh xc cao

C t mao qu n (open-tubular or capillary column): ID: 0,25 0,5 mm, L: 25 50 m

- N p m u kh khn

- State of art column

- Pha tnh c ph vo m t trong c a c t (0,2 - 1 m)

B S k kh


31/71


C t s c k (Column)

wall-coated open-tubular (WCOT) column

B S k kh


32/71


C u t o c a c t nh i

V c t: thp khng g ho c th y tinh Pyrex

Ch t h p ph (Adsorbents): hai lo i ch t c nh i vo c t : - Ch t h p ph .

- Ch t mang (support) c ph pha tnh.

Cc ch t h p ph th ng s d ng:

Alumina (Al2O3): Ho t ha 200C 1h: tch kh v cc hyrocacbon n C 5, kch th c h t: 100/120 mesh, kch th c l x p:1 - 100 .

Silica (SiO2): tch cc kh c M nh v cc Hydrrocacbon nh

- B m t ring750 m 2/g, kch th c l x p trung bnh: 22

- B m t ring 100m2/g kch th c l x p trung bnh: 300 .

Kch th c: - 30/40 -100/120 mesh- ng nh t

B S k kh


33/71



Cc ch t h p ph th ng s d ng: Zeolith: Tch cc kh c M nh b ng ph ng php lo i tr (exclusion): Ry phn t ( molecular sieves)

- Cc zeolith k hi u: 5A v 13X: th ng c s d ng tch H2, O2, N2, CH4, CO, Ar, Ne

Cacbon:

- Cacbon ho t tnh: b m t ring 1000 m2.g-1

- Graphit: b m t ring 5 - 100 m 2.g-1

Cc h p ch t cao phn t : - Co-polymer c a polystyrene v divinylbenzene- L x p: macropore v micropore

- B m t ring l n v x p cao

- T ng tc a d ng v i cc dung mi v ch t tan ti p xc v i n.

B S k kh


34/71



Cc ch t mang s d ng cho GLC: - Celite (m t d ng c bi t c a khong diatomic), Celite nung, Celite nung ho t ha b i Ag ho c Au, cc h t vi c u th y tinh, polymer, teflon

Bi n tnh Celite:

-Nung 900C v i Na 2CO3 v tr dung: silica cristobalite, cc v t Kimlo i tc d ng v i Silica gy mu (h ng) cho v t li u.

Chromosorb P, Chromosorb W, Chromosorb G v Chromosorb S

Chuy n ch t h p ph ln ch t mang:

S dung cc nhm Silanol ( Si-OH)

Hexamethyldisilazane + Si-OH g c trimethylsilyl

Ph ng php t m (slurry method of coating)

Vi c u Polystyren

B S k kh


35/71


C u t o c a c t mao qu n (capillary or open-tubular column)

Pht minh vo nh ng nm 1950

T c phn tch nhanh v i s l ng a c c l n 300.000 a

a vo p d ng vo cu i nh ng nm 1970

C u t o t th y tinh ho c fused silica

ID = 0,25 0,5 mm

L = 25 50 m

B m t trong c a mao qu n c ph m t l p m ng pha ng 0,25 1,5 m

(WallCoated Open-Tubular - WCOT)

B S k kh


36/71


C u t o c a c t mao qu n (capillary or open-tubular column)

B S c k kh


37/71


Bi n tnh b m t fused silica

phn c c (polar) c a cc g c Silanol trn b m t

Bi n tnh b m t Silica

Phn c c: -CN, -CO v OH

Khng phn c c:Hydrrocacbon (dialkyl siloxane)

Phn c c l n: Polyester

B S c k kh


38/71


Cc pha tnh (ST) th ng s d ng trong GLC

Polar SP

Non-polar SP

nh h ng c a phn c c c a pha

tnh n th i gian l u

B S c k kh


39/71


So snh c t nh i v c t mao qu n

B S c k kh


40/71


n nhi t c t sc k (Column Thermostating)

M c ch: B o m tnh l p l i c a th i gian l u

L n nhi t (thermostating oven) Topti.= f(Tsi), Topti Tsiv i RT= 2 30 pht

L n nhi t

C t s c k

Nhi t ch ng trnh ha (Temperature Programming)

Isothermal: m u n gi n

M u ph c t p: Tch cc c u t c a m u d a vo s thay i c a T si

B S c k kh


41/71


So snh s c k hai ch : Isothermal v ch ng trnh ha nhi t

Ch ng trnh ha nhi t

H s kh nng

phn gi i

B S c k kh
http://storage/tailieu/files/source/20100704/dinhvinh87/E:/Biblio/Chromato/GCtemp.mov


42/71


Nhi t ch ng trnh ha

Tng kh nng tch c a c t nh ng ng t r i b c h i dung mi

Dung mi b c h i ngay khi vo c t s c k

Dung mi ng ng t trn c t cng v i cc c u t khc, sau b c h i, ti phn b l i cc ch t c n phn tch

B S c k kh
http://storage/tailieu/files/source/20100704/dinhvinh87/E:/Biblio/Chromato/focus2.mov


43/71


Cc vng c gia nhi t c a h s c k kh (GC)

B S c k kh


44/71


u d (Detectors)

M t s yu c u:

Tn h u thu c tuy n tnh ho c g n tuy n tnh v i l ng m u

Th i gian tr l i nhanh

Pht hi n a d ng (universal detection)

Tn hi u ra khng ph thu c v nhi t

Lm vi c n nh t nhi t th ng n 400C ( i v i 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. S c k kh


45/71



d n nhi t c a khi mang (He ho c H2)

10 l n l n h n cc h p ch t h u 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 Tremains constant

When an analyte species flows past the filament generally thermal conductivitygoes down, T of filament will rise. (resistance of the filament will rise).

B. S c k kh


46/71



B. S c k kh


47/71


Flame Ionization Detector (FID)

Sensitive towards organics Analyte is burned in H2/air, which produces CH and CHO

+ radicals

CHO+ radicals are reduced at a cathode which produces a currentproportional to the radical quantity 10-12 A

Specific for organic carbon, insensitive to inorganics, CO2, SO2 etc.

Generally DL 100x less than TCD about pg/s (flow rate dependent)

Response to specific organic depends on the number of organic carbons.

B. S c k kh
http://storage/tailieu/files/source/20100704/dinhvinh87/E:/Biblio/Chromato/FID.mov


48/71


Electron Capture Detector (ECD)

Sensitive to electron withdrawing groups especially towards organicscontaining F, -Cl, -Br, -I also, -CN, NO2

Nickel-63 source emits energetic electrons collides with N2 (introduced as make-

up gas or can be used as carrier gas) producing more electrons:

Ni-63 e-, e- + N22e- + N2+

The result is a constant current that is detected by the electron collector (anode).

As an analyte flows through past the Ni-63 source, electron capture is possible byelectron-withdrawing species: A + e-A-

Current decreases as a result of e- capture by analyte. This is one of the fewinstances in which a signal is produced by a decrease in detectable phenomenon.

Very low DL for detected species 10-15g/ml for many halogenated substances

B. S c k kh


49/71


Electron Capture Detector (ECD)

Radioactive Ni-63 source Easily contaminated with O2, H2O, sample overloading.

High maintenance device.

Highly variable response to halogenated substances

Sometimes complementary information from FID helps.

The badS nguyn l


50/71

B. S c k kh


51/71


M t s k thu t chuy n valve

Apparatus for Heart Cutting

Valve 6 ng

S d ng i h th ng v i hai c t v 2 detectors

Cc c u t vng gi a c a m u c phn tch trn c t 2

Cc c u t u v cu i c phn tch trn c t 1

B. S c k kh


52/71

(Gas-Liquid Chromatography)

Thu th p v x l s li u (Data Acquisition and Processing )

Nguyn lA/D Converter

B. S c k kh


53/71

(Gas-Liquid Chromatography)

M t s ng d ng c a GC (GLC)

p d ng i v i cc m u b c h i v n nh nhi t n vi trm C C kh nng pht hi n v phn tch r t nhi u ch t v h n h p

c ng d ng r ng ri tch v xc nh cc c u t trong cc m u t nhi u ch ng lo i khc nhau

M t 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

S c k kh k t h p kh i ph

B. S c k l ng hi u qu cao
http://storage/tailieu/files/source/20100704/dinhvinh87/E:/Biblio/Chromato/GC-MS.movhttp://storage/tailieu/files/source/20100704/dinhvinh87/E:/Biblio/Chromato/GC.mov


54/71

g q (Hight-Performance Liquid Chromatography - HPLC)

Phn lo i HPLC d a b n ch t t ng tc

S c k phn b (partition chromatography)

S c k h p ph ho c l ng-r n (adsorption or liquid-solid chromatography)

S c k trao i ion (ion exchange chromatography)

S c k lo i tr kch th c (size exclusion chromatography)

SO3-

SO3-

Na+COO

-H3N

+

Na+

COOH

H3N+

H2

H4.5

Ion-exchange Resin

VD: nguyn l s c k trao i ion (acide amine)

S c k lo i tr kch th c



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c i m c a HPLC Pha tnh c nh i trong c t Pha ng tr ng thi l ng: Cc dung mi, h n h p dung mi ho c n c

Phn lo i HPLC d a vo v t li u nh i

Pha thng th ng (Normal phase): v t li u nh i l silica n gi n

Trao i ion: silica bi n tnh (mdified silica) Pha o (reverse-phase): silica bi n tnh

Ph n l n cc HPLC l pha o

Ch t phn tch c gi trn pha tnh phn c c nh h n cho n khi b r a tri b i pha ng phn c c l n

Thao tc n gi n Hi u qu cao

C t lm vi c n nh

C th phn tch cho c hai lo i c u t c c tnh t ng t ho c khc xa nhau



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M c d u c nhi u l thuy t nghin c u v vi c s d ng pha o nh ng ph n l n cc ch ng trnh HPLC pha o u thu c t ph ng php th v sai (by trial and error).



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G c R l C8 (n-octyl), C12 (n-octyl)ho c C18 (n-octyldecyl).

Pha ng l H2O + dung mi ha tan(acetonitrile, methanol, ethanol,isopropanol).

Cc c u t phn c c s b r a ra nhanh nh t, tng phn c c c a pha ng s lm tng th i gian ch y m u

Pha tnh-Pha o(Stationary Phases for Reversed-Phase LC)

Pha tnh bnh th ng c a LC (Stationary Phases for Normal LC)

Pha ng t ng i khng phn c c: Hexane, Isopropyl eter, toluene

Cc c u t khng phn c c s b r a ra nhanh nh t, tng phn c c c a pha ng s gi m th i gian ch y m u



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nh h ng c a b n ch t pha tnh n ch t l ng tch

Pha o



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S nguyn l c a HPLC



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Cc yu c u i v i dung mi

p su t b m: vi trm atm ( 6000psi), l u l ng 0,1 10 ml.min -1 v i E


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g q(Hight-Performance Liquid Chromatography - HPLC)

Hi u qu tch b ng ph ng php gradient elution

S d ng m t dung mi n gi n c thnh ph n khng i: isocratic

S d ng hai hay nhi u h n cc h dung mi c phn c c (polarity) khc nhau nhi u: gradient elution

T l cc lo i dung mi c ch ng trnhha lin t c ho c theo t ng b c

Gradient elution: tng ch t l ng c a qu trnh tch (improve seperation efficiency)

Qu trnh tch r a (Elution)



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g q(Hight-Performance Liquid Chromatography - HPLC)

Polar Solvents

Water > Methanol > Acetonitrile > Ethanol > Oxydipropionitrile

Non-polar Solvents

N-Decane > N-Hexane > N-Pentane > Cyclohexane

phn c c c a m t s dung mi s d ng trong HPLC

L a ch n pha ng v pha tnh

Ch y u d a vo s phn c c c a c u t phn tch, pha ng, pha tnh Quy t c chung: phn c c (polarity) c a c u t c n phn tch v pha tnh l t ng ng cn pha ng c phn c c sai bi t

Khi phn c c c a c u t v pha tnh qu gi ng nhau: th ng tc m nh gi a c u t c n phn tch v pha tnh th i gian phn tch ko di



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(Hight-Performance Liquid Chromatography - HPLC)

Tnh ch t m t s lo i dung mi s d ng trong HPLC

B. S c k l ng hi u qu cao (Hi h P f Li id Ch h HPLC)


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H th ng n p m u (Sample Injection Systems)

S d ng valve 6 c ng

N p m u qua vng l y m u (sampling loops) S c k l ng hi n i

C th thay th sampling loops t 5 l n 500 l

Sai s c a l ng m u n p d i 1%

B. S c k l ng hi u qu cao (Hi h P f Li id Ch h HPLC)


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C t s c k HPLC

Thng th ng:L = 10 30 cm v c th n i ti p 2 c t hoc nhi u h n

ID = 4 10 mm, kch th c h t nh i: 3, 5 v 10 m40.000 60.000 a/m c t C t t c cao v hi u qu h n

L = 3 - 7 cm v c th n i ti p 2 c t hoc nhi u h n

ID = 1 4,6 mm, kch th c h t nh i: 3 ho c 5 m

100.000 a/m c tC t b o v (Guard Column) c l p t tr c c t phn tch ko di tu i th c a c t

Thnh ph n = thnh ph n c a c t phn tch nh ng c h t l n h n gi m t n th t p su t

B. S c k l ng hi u qu cao (Hi ht P f Li id Ch t h HPLC)


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n nh nhi t c a c t (Column Thermostats)

Ph n l n ng d ng cua HPLC c th c hi n nhi t phng

Tuy v y ch t l ng c a s c k s t t h n n u duy tr nhi t c a c t khng thay i (sai s < 0,05C)

Thi t b HPLC hi n i c trang b thm l gia nhi t cho c t (Column heater) n nh nhi t g n 150C v i sai s < 0,05C

Trang b h th ng phun n c lm l nh (water jackets fed) t b n nhi t kh ng ch chnh xc nhi t



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u d (Detector) dng cho HPLC

Khng nh y v c kh nng phn tch a d ng nh detector c a GC Th ng g p nh t l Detector UV-Vis

LOC: Limit Of Detection

Mass LOD = concentration (mol/L) x inj. vol. (L) x FW (g/mol)



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UV-Vis and Fluorescence Detector Electrochemical Detector

Amperometric detection = fixed potential and measure the current response.

= 200-400nm= 200-400nm s d ng 254 nm254 nm

B. S c k l ng hi u qu cao (Hight P rf rm n Liq id Chr m t gr ph HPLC)


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Refractive Index Detector

B. S c k l ng hi u qu cao (Hight Performance Liquid Chromatography HPLC)


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Cc ph ng php nng cao phn gi i trong HPLC

Tng chi u di c a c t (Increase column length)

Gi m ng knh c a c t (Decrease column diameter)

Gi m l u l ng pha ng (Decrease flow-rate)

Pha tnh (v t li u nh i c t) ng nh t (Uniform stationary phase (packing))

Gi m th tch b m m u (Decrease sample size)

L a ch n pha tnh s ch h n (Select proper stationary phase)

L a chon pha ng tinh khi t h n (Select proper mobile phase)

S d ng p su t n nh h n (Use proper pressure)

Thnh ph n c a pha ng thay i h p l (Use gradient elution)

So snh HPLC v GC(C i f HPLC d GLC)


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(Comparison of HPLC and GLC)

Cc c i m chung:

Hi u qu , ch n l c cao, ng d ng r ng ri Th tich m u nh

C th khng ph h y m u (nondestructive of sample)

nh l ng d dng

u i m c a HPLC

p d ng c v i cc m u khng bay h i v khng b n nhi t

p d ng c cho cc ion v c

u i m c a GC

Thi t b n gi n v r

Nhanh chng

D dng k t n i v i ph kh i

chuyen de sac ky vn www 1 1 thuvien247 net 1046

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