5/12/2014

TRNG I HC CNG NGHIP TP.HCM

KHOA CNG NGH HO HC

NI DUNG

PP PHN TCH HA L NG

DNG TRONG LC HA DU
CHNG 1: PHNG PHP

1.

L THUYT V HPLC

2.

CU TO CA HPLC

3.

LA CHN IU KIN PHN TCH

SC K SC K LNG
GV : ThS. KHU CHU QUANG

1. L THUYT V HPLC

1. L THUYT V HPLC

1.1. Khi nim

1.2. Phn loi

HPLC : High performance liquid chromatography,

Phn loi da trn bn cht cc tng tc nh:

gi l sc k lng hiu nng cao.

Sc k phn b: pha tnh l cht lng.

HPLC l phng php tch da trn cc qu

Sc k hp ph: pha tnh l cht hp ph.

trnh tng tc xy ra lin tc gia cht phn tch

Sc k trao i ion: pha tnh l cht trao i ion.

v hai pha:

Sc k ry phn t (FG HPLC).

Pha tnh: rn hoc lng

Pha ng: cht lng

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1. L THUYT V HPLC

1. L THUYT V HPLC

1.2. Phn loi

1.2. Phn loi

Sc k hp ph pha thun: NP - HPLC

Sc k hp ph pha o: RP - HPLC

(i nc).
(k nc).

hng

1. L THUYT V HPLC

1. L THUYT V HPLC

1.2. Phn loi

Sc k phn b: P - HPLC

1.2. Phn loi

Sc k trao i ion: IE - HPLC

nc,

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1. L THUYT V HPLC

1. L THUYT V HPLC

1.2. Phn loi

Sc k ry phn t: GEL HPLC hay F - HPLC

1.3. Cc qu trnh tch trong ct sc k

S tng tc X SP MP trong ct:

Xi : cht phn tch.

SP: pha tnh.

MP: pha ng.

Lc lu gi cc cht trong ct:

Ft = F1 + F2 + F3
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1. L THUYT V HPLC

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1. L THUYT V HPLC

1.3. Cc qu trnh tch trong ct sc k

Cn bng ng hc trong ct tch:

1.3. Cc qu trnh tch trong ct sc k

Cc dng cn bng ng hc:
+ Dng

tuyn tnh.
+ Dng
khng tuyn
tnh

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1. L THUYT V HPLC

1. L THUYT V HPLC

1.3. Cc qu trnh tch trong ct sc k

Qu trnh tch tt:

1.3. Cc qu trnh tch trong ct sc k

Cn bng tng tc trong ct tch:
Loi 1: Tng tc hp ph (rn lng)

MP: MeOH/ ACN

Loi 2: Tng tc chit (lng lng)

MP: MeOH/ THF

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1. L THUYT V HPLC

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1. L THUYT V HPLC

1.3. Cc qu trnh tch trong ct sc k

Cn bng tng tc trong ct tch:

1.3. Cc qu trnh tch trong ct sc k

Cn bng tng tc trong ct tch:

Loi 3: Tng tc tnh in.

Loi 4: Tng tc theo ln phn t, l tng

tc hp ph vo l xp ca ht SP v theo
ln ca cht Xi.

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1. L THUYT V HPLC

1. L THUYT V HPLC

1.4. Cc i lng c trng

1. Thi gian lu

1.4. Cc i lng c trng

nh hng ca SP vo thi gian lu

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1. L THUYT V HPLC

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1. L THUYT V HPLC

1.4. Cc i lng c trng

2. H s phn b (Kpb) l i lng i din cho

1.4. Cc i lng c trng

3. H s dung tch ki (t s phn b)

cn bng ng ca cht trong 2 pha .

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1. L THUYT V HPLC

1. L THUYT V HPLC

1.4. Cc i lng c trng

3. H s dung tch ki (t s phn b)

1.4. Cc i lng c trng

3. H s dung tch ki (t s phn b)

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1. L THUYT V HPLC

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1. L THUYT V HPLC

1.4. Cc i lng c trng

4. phn gii Rs

1.4. Cc i lng c trng

5. S a N ca ct tch

phn gii l i lng cho php nh gi hai

L i lng biu th tng s lp cht di chuyn

cht c th tch ra khi nhau khng.

trong ct tch.

t t
1,18( t t
R

0,5( w w ) w w


R2

R1

R2

R1

1
2

1
2

t
t
N 16 5.54
w

w

S mm l thuyt N :
2

S mm hiu dng
ca ct Neff:

Hay:

t t
N 16

w
R

1
2

eff

Hay tnh gn ng:

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2. CU TO HPLC

2. CU TO HPLC

H thng my ca HPLC

S nguyn l ca HPLC

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2. CU TO HPLC

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2. CU TO HPLC

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2. CU TO HPLC

2. CU TO HPLC

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2. CU TO HPLC

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2. CU TO HPLC

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2. CU TO HPLC

2. CU TO HPLC

2.1. Pha ng trong HPLC

2.1. Pha ng trong HPLC

Yu cu:

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2. CU TO HPLC

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2. CU TO HPLC

2.1. Pha ng trong HPLC

2.1. Pha ng trong HPLC

Pha ng IE HPLC:

Pha ng NP HPLC:

Pha ng RP HPLC:

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2.1. Pha ng trong HPLC

2. CU TO HPLC
2.1. Pha ng trong HPLC
phn cc ca dung mi trong HPLC:

Tnh
cht ca

Polar Solvents

mt
Water > Methanol > Acetonitrile > Ethanol

dung mi

Non-polar Solvents

trong

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

HPLC

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2.1. Pha
ng trong
HPLC

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2. CU TO HPLC
2.1. Pha ng trong HPLC
nh hng ca pha ng:

Tham
kho

ngun

Wiki

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2. CU TO HPLC

2. CU TO HPLC

2.1. Pha ng trong HPLC

nh hng ca pha ng:

2.1. Pha ng trong HPLC

nh hng ca pha ng:

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2. CU TO HPLC

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2. CU TO HPLC

2.2. Gradient ca pha ng

2.2. Gradient ca pha ng

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2. CU TO HPLC

2. CU TO HPLC

2.2. Gradient ca pha ng

Mc ch:

2.2. Gradient ca pha ng

+ Tng hiu qu tch ca cht.

+ Rt ngn thi gian lu.

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2. CU TO HPLC

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2. CU TO HPLC

2.2. Gradient ca pha ng

2.2. Gradient ca pha ng

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2. CU TO HPLC

2. CU TO HPLC

2.3. Bm cao p

2.3. Bm cao p

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2. CU TO HPLC

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2. CU TO HPLC

2.3. Bm cao p

2.4. B phn tim mu

a mu vo my theo phng php khng
ngng dng chy.

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2. CU TO HPLC

2. CU TO HPLC

2.4. B phn tim mu

2.4. B phn tim mu

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2. CU TO HPLC

2. CU TO HPLC

2.5. Ct sc k

2.5. Ct sc k
Thng thng:

Pha tnh-Pha o

Pha tnh bnh thng ca LC

(Stationary Phases for Reversed-Phase LC)

(Stationary Phases for Normal LC)

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

Gc R l C8 (n-octyl), C12 (n-octyl)

hoc C18 (n-octyldecyl).

100.000 a/m ct

Pha ng l H2O + dung mi ha tan

(acetonitrile,
methanol,
ethanol,
isopropanol).

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

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 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

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2. CU TO HPLC

2. CU TO HPLC

2.5. Ct sc k

2.5. Ct sc k

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

2. CU TO HPLC

2. CU TO HPLC

2.5. Ct sc k
nh hng ca bn cht pha tnh n cht lng tch

2.6. Detector
L b phn pht hin cc cht khi chng ra khi ct
v cho cc tn hiu ghi trn sc k nh tnh v

nh lng.
Tn hiu o c A:
A = k. C
Vi: C: nng cht phn tch.
k: hng s thc nghim ca detector.
Tn hiu A c th l hp thu quang, cng
Pha o

pht x, dn in, nhit, hiu in th, chit sut.

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2. CU TO HPLC

2. CU TO HPLC

2.6. Detector
Cc loi detector:

2.6. Detector
Detector UV, UV Vis
i lng o: hp thu quang ca cht phn tch
ti bc sng c nh.
Vi: mt cht v iu kin nht nh, v L l hng
s nn:

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2. CU TO HPLC

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2. CU TO HPLC

2.6. Detector
Detector UV, UV Vis

2.6. Detector
Detector Diode aray
o hp thu quang ca cc cht ti nhng gi tr

c nh ca mi cht trong cng mt lc (o ng

thi) theo dng khng gian.
a. Loi dng knh lc

Vi: mt cht v iu kin nht nh, v L l hng

s nn:

b. Loi 2 Flowcell

c. Loi 1 Flowcell

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2. CU TO HPLC

2. CU TO HPLC

2.6. Detector
Detector Diode aray

2.6. Detector
Detector hunh quang
i lng o: pht x hunh quang ca cht ti
Em nht nh khi c kch thch bng Ex nht

nh.
Vi: mt cht v iu kin nht nh, v Io l
hng s nn:
Detector mng diode vng UV ca Agilent

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2. CU TO HPLC

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2. CU TO HPLC

2.6. Detector
Detector hunh quang

2.6. Detector
Detector o chit sut
i lng o: ch s chit sut ca cht trong

pha ng. Chit sut dung dch ra gii t ct tch

i ra l:
a. H dng knh lc

hay:
Vi: no : chit sut pha ng.
noi: chit sut ca cht trong pha ng.
b. H c b phn gii ph cch t c. Detector HQ ca Agilent

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2. CU TO HPLC

3. LA CHN IU KIN PHN TCH

2.6. Detector
Detector o chit sut

3.1. Cc yu t nh hng trong HPLC

Pha tnh:

M hnh Detector chit sut

Loi pha tnh v nhm th R.

C ht pha tnh.
xp v din tch b mt ring ca ht pha
tnh.

a. Kiu khc x

Chiu di ct tch.

b. Kiu phn x

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3. LA CHN IU KIN PHN TCH

3. LA CHN IU KIN PHN TCH

3.1. Cc yu t nh hng trong HPLC

3.1. Cc yu t nh hng trong HPLC

Pha tnh:

C18 (ODS) type

C8 (octyl) type

C4 (butyl) type

Phenyl type

TMS type

Cyano type

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Pha tnh:

OH

Non-polar property
C18 (ODS)

-Si-C18H37

Weak

Strong
OH

Si

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3. LA CHN IU KIN PHN TCH

3. LA CHN IU KIN PHN TCH

3.1. Cc yu t nh hng trong HPLC

3.1. Cc yu t nh hng trong HPLC

Pha tnh:

Pha ng:
C8
Medium

C18 (ODS)

Sample

Strong

C4

Sample

Weak
Sample
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3. LA CHN IU KIN PHN TCH

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3. LA CHN IU KIN PHN TCH

3.1. Cc yu t nh hng trong HPLC

3.2. Ti u ha cc iu kin trong HPLC

Detector:

Mc ch:

hng

Cc yu t khc:

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3. LA CHN IU KIN PHN TCH

3. LA CHN IU KIN PHN TCH

3.2. Ti u ha cc iu kin trong HPLC

Qu trnh ti u ha:

3.2. Ti u ha cc iu kin trong HPLC

Qu trnh ti u ha:

1. Chn ct tch v pha tnh

2. Pha ng

3. Cc iu kin np mu
+ Vng mu, th tch mu.
+ Cch np mu.
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3. LA CHN IU KIN PHN TCH

3. LA CHN IU KIN PHN TCH

3.2. Ti u ha cc iu kin trong HPLC

Qu trnh ti u ha:

3.2. Ti u ha cc iu kin trong HPLC

Qu trnh ti u ha:

4. Detector

6. PP x l mu

+ Loi detector v thng s ca n.

+ K thut x l mu.

+ Phng php chun ha nh lng cht.

+ iu kin v mi trng x l.

+ Cc iu kin ghi sc k .

7. PP chun ha nh lng cht

5. Xem xt cc yu t nh hng

+ PP ng chun.

+ Nhit .

+ PP thm chun,..

+ Cht nn ca mu.

+ PP thm mt im chun.

+ Cc cht khc trong mu.

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3. LA CHN IU KIN PHN TCH

3.3. Qui trnh hiu chnh HPLC

3. LA CHN IU KIN PHN TCH

Chu k hiu chun: 6 hoc 12 thng.

3.3. Qui trnh hiu chnh HPLC

H thng phn phi pha ng (bm):

Cc b phn hiu chun:

n nh p sut y ca bm.

H thng phn phi pha ng (bm)

Chc nng gii hn p sut.

B phn tim mu t ng (nu c).

ng ca tc dng:
Thi gian o c = thi gian l thuyt 2%

Detector
Bung n nh nhit.

ng ca gradient dung mi:

Sai s cho php nng dung mi l 1%.

Ct sc k.

3. LA CHN IU KIN PHN TCH

3. LA CHN IU KIN PHN TCH

3.3. Qui trnh hiu chnh HPLC

B phn tim mu t ng (nu c):

3.3. Qui trnh hiu chnh HPLC

Detector:

chnh xc: lch chun tng i ca din

ng ca bc sng: Sai s cho php: 1

tch nh ca nhiu ln bm mu cng mt cht

khng c qu 2%.
Tnh tuyn tnh: H s tuyn tnh R2 0.999
Cht mang: hm lng cht mang khng qu

1%.

nm.
tuyn tnh ca detector: H s tuyn tnh R2
0.999
chnh xc:
Tri v nhiu ng nn:
-

Tri ng nn: < 2 x 10-4 AU/gi

Nhiu ng nn: < 1,0 x 10-4 AU

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3. LA CHN IU KIN PHN TCH

3.3. Qui trnh hiu chnh HPLC
Bung n nh nhit : ng ca nhit :

3. LA CHN IU KIN PHN TCH

3.4. So snh HPLC v GC
Cc c im chung:
Hiu qu, chn lc cao, ng dng rng ri

20C ca nhit ci t.

Th tich mu nh

Ct sc k:

C th khng ph hy mu (nondestructive of sample)

nh lng d dng

H s dung lng k > 1.

u im ca HPLC

S a l thuyt N.

p dng c vi cc mu khng
bay hi v khng bn nhit

phn gii R 1.

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

H s bt i xng T.

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