SC K KH LNG CAO P

Trnh by: Hunh Khnh Duy Application Manager

TP. HCM 09/2013

(C) HKD 2013

I CNG
SC K LNG CAO P
Sc k v sc k lng (HPLC)
Cc loi HPLC
Pha thun
Pha nghch

Pha nghch cp ion

Trao i ion (IC)
Loi tr kch thc (SEC)

Tch ng phn quang hc

Cc phng thc chy HPLC
ng dng (isocratic)
Dng thay i theo thi gian (gradient)
(C) HKD 2013

SC K

Dung mi

Ct tch
t

0
(C) HKD 2013

NGUYN L CA SC K

Vt liu nhi ct 5-10 m

Ct tch
4

(C) HKD 2013

NGUYN L CA SC K

Hng chy ca pha ng

Pha tnh

(C) HKD 2013

THNH PHN C BN HPLC

(C) HKD 2013

THNH PHN C BN HPLC

CC B LC
Ct bo v
Van tim mu
B lc trc ct

Ct phn tch

B lc dung mi

B lc dung mi
 Thp khng r hoc thy tinh
vi kch thc l xp 10
micron.
 Loi cc ht c kch thc
nh khi dung mi.

B lc trc ct
 Nm gia van tim mu v ct bo v
 Kch thc l xp t 2 n 0.5 micron
 Loi cc ht c kch thc nh t mu
hoc van tim mu.
 c thit k chng li s khuch tn.

(C) HKD 2013

THNH PHN C BN HPLC

B PHN UI KH DUNG MI

(C) HKD 2013

THNH PHN C BN HPLC

BM CAO P
9 Chu p sut cao (n 415 bar)
9 Phn phi lu lng chnh xc (0.1-10 mL/min

9 p sut dng n nh

(C) HKD 2013

THNH PHN C BN HPLC

NH HNG CA DAO NG P SUT

Low pressure-fluctuation pump

S dao ng p sut ca dng nh

hng n tn hiu ca u d

P=0.5

High pressure-fluctuation pump

P=1.5

Methanol 1.0mL/min 40 kgf/cm2

(C) HKD 2013

10

THNH PHN C BN HPLC

BM CAO P

motor v cam

u bm

check
valve

10 -100L

plunger
plunger seal
11

(C) HKD 2013

THNH PHN C BN HPLC

BM CAO P HAI PISTON SONG SONG

Check

Thch hp cho:
9 u d chit sut
9 u d dn
9 u d khi ph
9 u d in ha

Valves

Nhiu linh kin bo tr

A

Piston 'A' tin

(C) HKD 2013

Piston B li
12

THNH PHN C BN HPLC

BM CAO P HAI PISTON NI TIP

Piston ht c chu k nhanh hn 2
ln piston y.
Thch hp cho:
9 u d UV / PDA
9 u d hunh quang

S linh kin bo tr t.

(C) HKD 2013

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THNH PHN C BN HPLC

CC B PHN CA BM

(C) HKD 2013

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THNH PHN C BN HPLC

B GIM XUNG CHO BM CAO P

Gim xung ~ 3% 16.6 bar
Hot ng 3.5 7 bar

p sut ngoi
(iu chnh bi h thng)

Gim xung < 0.1%

Gim xung < 0.1%

p sut

2%

15

(C) HKD 2013

THNH PHN C BN HPLC

CC H THNG BM

p sut thp

p sut cao
(C) HKD 2013

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THNH PHN C BN HPLC

VAN TIM MU

(C) HKD 2013

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THNH PHN C BN HPLC

VAN TIM MU BNG TAY

(C) HKD 2013

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THNH PHN C BN HPLC

VAN TIM MU T NG

Step 1

Step 2

Step 3

(C) HKD 2013

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THNH PHN C BN HPLC

CT TCH
Phn loi HPLC theo c ch phn tch
Sc k pha thun
Sc k pha o
Sc k trao i ion
Sc k loi tr kch thc ( GPC / GFC )
Sc k chn lc ng phn quang hc
L = 5- 30 cm.
ID = 50m 4 mm
Kch thc ht nhi < 10 m
(C) HKD 2013

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THNH PHN C BN HPLC

CT TCH SC K PHA THUN

Pha tnh: silicagel

PHN CC

(C) HKD 2013

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THNH PHN C BN HPLC

CT TCH SC K PHA THUN

Pha ng
DUNG MI KHNG PHN CC
1) Dung mi chnh
Hydrocarcbons: hexane, cyclohexane, n-pentane, n-heptane.
Hydrocarbons thm: benzene, toluene, xylene.
CH2Cl2, CHCl3, CCl4
2) Dung mi ph thm vo dung mi chnh iu chnh phn cc
Cc ether: methyl-t-butyl ether (MTBE), diethyl ether, tetrahydrofuran
(THF), dioxane.
Cc ru: 2-propanol, ethanol, methanol.
Pyridine, ethyl acetate, acetonitrile, acetone.

(C) HKD 2013

22

THNH PHN C BN HPLC

CT TCH SC K PHA THUN

HO

SiOH
SiOH

OH

23

(C) HKD 2013

THNH PHN C BN HPLC

CT TCH SC K PHA O
C18 (ODS)
C8
phenyl
cyano
amino
diol

Pha tnh: silicagel bin tnh

KHNG
PHN CC
(C) HKD 2013

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THNH PHN C BN HPLC

CT TCH SC K PHA O
Pha ng
DUNG MI PHN CC
Nc hoc dung dch m + Dung mi hu c
9 Dung dch m: nng v pH l thng s quan trng.
9 Dung mi hu c: methanol (MeOH), acetonitrile, THF.
9 Phi ti u t l dung dch m v dung mi hu c.

25

(C) HKD 2013

THNH PHN C BN HPLC

CT TCH SC K PHA O

OH

C18 (ODS)
Mnh
OH

(C) HKD 2013

26

THNH PHN C BN HPLC

CT TCH SC K PHA O
nh hng ca pha tnh
C8
Trung bnh

C18 (ODS)

sample
Mnh

C4

sample
Yu
sample
(C) HKD 2013

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THNH PHN C BN HPLC

PHA THUN V PHA O

Pha thun
9 Phn tch tt cc ng phn quang hc.

9 Thi gian lu c th thay i.

Pha o
9 Lp li thi gian lu.
9 Mi hn hp cn phn tch yu cu mt loi pha tnh ring.

(C) HKD 2013

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THNH PHN C BN HPLC

TCH NG PHN QUANG HC

Tt c tnh cht vt l u nh nhau,

ngoi tr gc quay cc
(C) HKD 2013

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THNH PHN C BN HPLC

TCH NG PHN QUANG HC

Gc quay cc

(C) HKD 2013

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THNH PHN C BN HPLC

TCH NG PHN QUANG HC

Gc quay cc

(C) HKD 2013

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THNH PHN C BN HPLC

TCH NG PHN QUANG HC

Gc quay cc

(C) HKD 2013

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THNH PHN C BN HPLC

TCH NG PHN QUANG HC

Pha tnh c cha tm bt i xng
(L)

(R)

((D
(D))

(C) HKD 2013

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THNH PHN C BN HPLC

CT TCH SC K TRAO I ION

Ct trao i cation
" R-SO3- (sulfonate): trao i mnh (SCX).

" R-COO- (carboxylate): trao i yu (WCX).

Ct trao i anion

" R4N+ (ammonium bc 4): trao i mnh (SAX).

" diethylaminoethyl (DEAE): trao i yu (WAX).
(C) HKD 2013

34

THNH PHN C BN HPLC

CT TCH SC K TRAO I ION

H+

SO3-

Resin or gel

Resin or gel

SO3- H+

SO3- H+

SO3- H+

K+

SO3- H+

SO3- H+
35

(C) HKD 2013

THNH PHN C BN HPLC

CT TCH SC K TRAO I ION

# Thi gian lu tng khi tng bn knh, in tch v mc
phn cc ca ion
Ca2+

>

K+

Cl-

>

F-

SCN-

>

(C) HKD 2013

SO42-

36

THNH PHN C BN HPLC

CT TCH SC K TRAO I ION

Resin or gel

# Tng nng pha ng lm gim thi gian lu

SO3- K+

H+

SO3- H+

SO3- H+

(Mu cha ion K+ v pha ng cha ion H+ )

37

(C) HKD 2013

THNH PHN C BN HPLC

CT TCH SC K TRAO I ION

# Kh nng hot ng ca ct ph thuc vo pH v nhit

COOH

a) Undissociated weak
cation exchanger

COOCOOCOO-

b) Dissociated weak
cation exchanger
(C) HKD 2013

4 < pH < 8
Resin or gel

COO
H
COOH

pH > 8
Resin or gel

Resin or gel

pH < 4

COO
H
COOCOOH

c) Partly dissociated
weak cation exchanger
38

THNH PHN C BN HPLC

CT TCH SC K LOI TR KCH THC

Silica
9 Chu p, chu dung mi, cn bng
nhanh, n nh nhit cao.
9 Hp ph, xc tc, kch thc l
xp gii hn.
Vt liu polymer
9 Kch thc l xp a dng.
9 a nc v k nc.
9 Bin dng, p sut gii hn.

(C) HKD 2013

39

THNH PHN C BN HPLC

L CT
# Gi nhit ct n nh trong qu trnh phn tch.
m bo s n nh thi gian lu v lp li.

(C) HKD 2013

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THNH PHN C BN HPLC

U D
Ultraviolet / Visible detector

(UV/VIS)

Photodiode Array detector (PDA)

Fluorescence detector

(RF)

Conductivity detector

(CDD)

Refractive Index detector (RID)

Electrochemical detector (ECD)

Mass spectrometer detector

(MS)

(C) HKD 2013

41

THNH PHN C BN HPLC

U D UV-VIS

C : concentration

Cell
Iin

Iout

Lambert-Beer's law

ACl = - log (Iout / Iin)

(A : absorbance)
(C) HKD 2013

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THNH PHN C BN HPLC

U D UV-VIS

Sample Cell

Grating

Iin

Iout

Photodiode

Iin

Iin

Photodiode

Reference Cell
D2 / W lamp

(C) HKD 2013

43

THNH PHN C BN HPLC

U D UV-VIS

hp thu

A= eCl = - log (Iout / Iin)

2.5

Khong tuyn tnh

Nng
(C) HKD 2013

44

THNH PHN C BN HPLC

U D UV-VIS

Abs
208 nm

275 nm

275 nm
208 nm

45

(C) HKD 2013

THNH PHN C BN HPLC

U D PDA
Sample Cell

Grating
One element can
detect one absorbance
at one wavelength

D2 / W lamp

512 Elements Photodiode Array

(C) HKD 2013

46

THNH PHN C BN HPLC

U D PDA

47

(C) HKD 2013

1: Azelaic acid
30%

2: Benzoic acid
3: Nitrobenzoic acid

Acetonitrile
concentration

15%

min
(C) HKD 2013

48

Acetonitrile
30%

Elution sequence

15%

Peak 1

Peak 2

Peak 3

49

(C) HKD 2013

THNH PHN C BN HPLC

U D HUNH QUANG

Bc sng kch thch

+ h1

h2+

A*

Bc sng pht x

h2

h1

Hunh quang
A

(C) HKD 2013

50

THNH PHN C BN HPLC

U D HUNH QUANG
XeLamp

Emission grating

Photo-multiplier

Fluorescence

Excitation grating

Excitation Sample cell

(C) HKD 2013

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THNH PHN C BN HPLC

U D CHIT SUT

(C) HKD 2013

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THNH PHN C BN HPLC

U D CHIT SUT

Photo cell
Reference cell
Light

Sample cell

(C) HKD 2013

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THNH PHN C BN HPLC

U D DN
V
I

K ( dn) = I [A] / E [V]

=A [cm2] / L [cm] * k
(k : dn in ring, -1.cm-1)

electrode
(C) HKD 2013

54

THNH PHN C BN HPLC

U D IN HA

O + H+

R
e-

Electrode
Glassy Carbon (GC)
Pt, Ag, Au

A
GC
Pt
Ag
Au

: phenol compounds
general use
: H2O2
: halogen ion
: sugar analysis

S dng: nhy ( lng picogram)

vi catecholamines, ng kh,
glycoproteins.
) Thch hp vi HPLC pha o v
trao i ion.
(C) HKD 2013

55

THNH PHN C BN HPLC

U D IN HA

(C) HKD 2013

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THNH PHN C BN HPLC

U D KHI PH
Pneumatically Assisted ElectroSpray(ESI)
+
++- - + -+ +- + - +

Liquid Samples
Nebulizing gas

+
+

+
+-+-++

+
+-+-+++

+
+

High Voltage

Atmospheric Pressure Chemical Ionization(APCI)

Ion molecular reaction
Liquid Samples

Heater

Nebulizing gas

Corona Discharge
Needle

(C) HKD 2013

57

THNH PHN C BN HPLC

U D KHI PH

ESI
drugs and their metabolites
peptides
proteins
many kinds of natural product
(-OH, -NH2,-COOH, SO2, PO3 etc.)
APCI
pesticides
steroids

drugs

(C) HKD 2013

58

THNH PHN C BN HPLC

U D KHI PH

Atmosphere

High Vacuum

API Probe
MS
detector

RP TMP1 TMP2
(Vacuum pump system)
(C) HKD 2013

59

THNH PHN C BN HPLC

U D KHI PH

(C) HKD 2013

60

THNH PHN C BN HPLC

NG DNG U D KHI PH

# nh tnh
# nh lng vi chn lc cao
M/Z

M/Z

M/Z
61

(C) HKD 2013

THNH PHN C BN HPLC

NG DNG U D KHI PH

m/z=100
A

B
TIC

A:100
B:100
D:150
C:150

m/z=150

(C) HKD 2013

62

THNH PHN C BN HPLC

SO SNH CC U D

UV/ VIS
LOD 1 ppb
GC
Yes

RF
10 ppt
Yes

RID
100 ppb
No

CDD
1 ppb
No

ECD
10 ppt
No

MS
1 ppb
Yes

LOD : Gii hn pht hin (S/N=3)

GC : Tng thch vi ch gradient (Gradient
Compatibility)
u d PDA c nhy hm km hn u d UV
(C) HKD 2013

63

THNH PHN C BN HPLC

CH CHY HPLC

Isocratic : thnh phn pha ng khng thay i trong sut qu

trnh phn tch.
Gradient: thnh phn pha ng thay i trong qu trnh phn
tch.

(C) HKD 2013

64

THNH PHN C BN HPLC

CH CHY HPLC
MeOH / H2O = 6 / 4
Long Time Analysis

Bad Separation
MeOH / H2O = 8 / 2
( column : ODS type )
(C) HKD 2013

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THNH PHN C BN HPLC

CH CHY HPLC

(C) HKD 2013

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THNH PHN C BN HPLC

CH CHY HPLC

(C) HKD 2013

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