Sc k lng

1. Khi nim v k thut sc k lng

- Phng php tch
- Cc cu t c tch phn b gia
pha tnh v pha ng
Mobile phase
(Pha ng)
Stationary phase
(Pha tnh)

- Qu trnh tch da vo tnh cht ha hc, vt

l v ha l ca cc cht.
- Da trn 2 qu trnh:
Hp ph
Gii hp ph
- Xy ra lin tc gia 2 pha:
Pha tnh: cht rn hoc lng
Pha ng: cht lng (1 cht hoc hn hp
nhiu cht)

Pha ng: ha tan v di chuyn cht phn tch

Pha tnh: gi cht phn tch
SKL chia thnh 2 nhm
- SK lng p sut thng (sc k c in)
- SK lng p sut cao (SKL hiu nng cao:
HPLC)
(High Performance Liquid Chromatography)

Da vo bn cht ca qu trnh sc k, HPLC:

- SK phn b
- SK pha thng (normal phase chromatography
- SK pha o (reversed phase chromatography)
- Sk trao i ion (ion exchange chromatography)
- SK ghp cp ion (ion pair chromatography)

- Da vo trng thi pha tnh

Pha ng: Lng

SK lng lng (LLC)

Pha tnh: Lng

(Liquid liquid
chromatography)

Pha ng: lng

SK lng rn (LSC)

Pha tnh: Rn

(Liquid solid
chromatography)

Khi ni vi u do (detector), HPLC cho php:

- nh tnh: da vo thi gian lu
- nh lng: da vo chiu cao hoc din
tch peak

2. Nguyn tc cu to ca h thng my
HPLC
0

0: Ngun cung cp pha ng (mobile phase)

- Bnh cha pha ng

1: Bm cao p (h thng cung cp dung

mi)
- Bm pha ng vo ct tch
- iu khin tc dng, p sut ca pha
ng

2. Van bm mu (Injection valve):

- Bm mu PT vo ct tch theo nhng lng
mu nht nh
Tim mu bng tay

Tim mu t ng
(Auto sampler)

3: Ct tch (Column)
- Ct cha pha tnh
- Yu t quyt nh qu trnh tch sc k
- Ct tch c kch c
khc nhau
- Chiu di: 10 25cm
- ng knh: 2 5mm

4: u d (detector)
- Thit b pht hin cht phn tch (nh tnh
v nh lng)
- C nhiu loi khc nhau ty mc ch phn
tch: UV-VIS, Huynh Quang, o
Dan, ien Hoa, Khoi Pho,

5. H thng ghi nhn v x l tn hiu:

- Thu thp v x l kt qu
- Recorder, Computer + printer, software

Injector

H thng HPLC n gin

Column

Mobile phase

Detector

Start

tM

tR1

tR2

tR3

tR4

Detector
UV-Vis: detector ph hp thu phn t
Xv nh cc cht c kh nng hp thu quang
Hunh quang (Fluorescence detector): xc nh cc cht
c kh nng pht hunh quang
- Alflatoxin, Mycotoxin, Amino Acid, thuc tr su h
Carbamate,.
u d ch s khc x (Refractive Index Detector: RI)

 u d dn (Conductivity detector):
Xc nh cc ion v c, hu c
u d khi ph (MS: mass spectrometry)
Xc nh phn ln cc cht hu c

3. Cc qu trnh tch trong sc k lng

- Qu trnh quan trng nht trong phng php
sc k
- Nhng cn bng ng xy ra gia pha tnh v
pha ng trong ct sc k
- L s vn chuyn v phn b lin tc ca cht
PT t u ct n cui ct

- Cht phn tch lun phn b gia 2 pha, trong

pha ng lun chy qua ct tch vi mt tc

nht nh hoc gradient
- Hiu qu ca qu trnh tch ph thuc rt nhiu vo

tng tc gia cc cht trong pha tnh v pha ng

- Mc ch chnh ca sc k l tch v nh tnh cc

cht trong hn hp cht phc tp

- Thi gian cht PT b pha tnh lu gi (thi

gian lu) quyt nh bi:
Bn cht ca pha tnh, cu trc v tnh cht
ca cht PT
Bn cht v thnh phn ca pha ng
dng ra gii cht PT ra khi ct sc k
(pha tnh)

- Ghi li ton b qu trnh tch sc k ca hn

hp cht PT sc k gm nhiu peak.
- c im ca peak PT:
Cc peak c th tch ri nhau hon ton
Chp nhau mt phn
Chp nhau hon ton

- Sc k phn nh qu trnh tch sc k

trong ct tt hay khng tt.
- Tch tt: hn hp c bao nhiu cht c
by nhiu peak ring bit khng chp nhau
- Cht no b lu gi mnh s c ra
gii ra sau cng, cht lu gi km s ra
trc

4. u im ca phng php sc k
-

C th phn tch ng thi nhiu hp cht

Khng cn lm bay hi mu

- phn gii cao nh qu trnh tch trn ct

- nhy cao (ppm-ppb) nh u d
- Th tch mu phn tch nh (1-100L)

Hn hp cht tch khi

nhau th no ?
Flow

Pha tnh

Ti sao li c s khc nhau?

Mnh

Yu
Do lc tng tc khc nhau

Cc cn bng trong ct HPLC

Mu PT i qua ct: tn ti ng thi 3 thnh phn:
- SP khng chuyn ng
- Cht PT va chuyn ng t u ct n cui
ct, ng thi phn b li gia SP v MP:
B SP hp ph, ri ra gii bi MP
- MP: dung mi ra gii v chuyn ng
Tng tc vi cc cht, li ko v mang cht PT
ra khi ct

3 tng tc chnh:
- S tng tc v cn bng ca cht PT vi
SP
- S tng tc v cn bng ca cht PT vi
MP
- S tng tc ca SP v MP

PT
Fb

Fa

SP

Fc

MP

Ftot = Fa + Fb + Fc
- Cht no c lc tng tc ln nht s b
gi li lu trn ct
- Cht no c F nh b ra gii u tin
- Ftot khc nhau nhiu th qu trnh tch tt

 Qu trnh tch din ra trong ct sc k

column
Vt liu nhi ct 3- 5m

Qu trnh tch
mixed sample
Mobile phase

column

5. Sc k lng pha thng v pha o

Pha thng

Pha o

Pha tnh

Phn cc

Khng phn
cc

Pha ng

Khng phn
cc

Phn cc

Ct nhi trong sc k pha thng

- Ct silica trung tnh:

Si

Si

Si

O
Si

O
Si

-B mt c cha nhm
phn cc (a nc): -OH
-Xc nh cc cht khng
phn cc hoc t phn
cc

- Ct silica trn nn mch carbon:

-Si-CH2CH2CH2CN
-Si-CH2CH2CH2NH2
-Si-CH2CH2CH2OCH(OH)-CH2(OH)

+ Ct cyano: a mc ch
+ Ct amino: phn tch ng
+ Ct diol: protein

- Dung mi ch yu: khng phn cc

+ Hydrocarbon: hexan, pentan, octan
+ Aromatic hydrocarbon: benzen, toluen, xylen,
+ Chloroform CHCl3
+ Methylene chloride CH2Cl2
- Dung mi ph: phn cc hoc hi phn cc
+ Ethanol, methanol,.

Lin kt hydrogen v thi gian lu

Mnh
Phn
cc

HO

SiOH
SiOH

Yu
Rt yu
OH

Rt
2

Lin kt hydrogen
Mu phn tch c:
COOH: nhm carboxyl
OH

: nhm hydroxyl

NH2

: nhm amino

Lin kt
hydrogen mnh

Mu phn tch c nhm tert-butyl hoc cc

nhm khng phn cc ln
LK hydrogen s yu

Ct nhi trong sc k pha o

- Ct: khng phn cc
- Pha ng: phn cc
- Ct silica alkyl ha cc nhm OH trn
b mt silica trung tnh:
B mt khng
Ct C18 (ODS)
phn cc
Ct C8 (octyl)
hay t phn cc
Ct C4 (butyl)
Ct phenyl

(k nc)

 Xc nh cht phn cc, khng phn

cc v t phn cc

Thi gian lu v lin kt k nc

HO

C18 (ODS)

Yu

Mnh
OH

Dung mi trong HPLC pha o

- Dung mi phn cc:
Methanol

(CH3OH:

MEOH),

acetonitrile

(CH3CN: ACN)
- Dung dch m
Ti u ha t l dd m/ dung mi rt quan
trng qu trnh tch tt

So snh pha thng v pha o

ng dng ca HPLC
- Ch yu xc nh cc hp cht hu c kh
bay hi trong nhiu i tng khc nhau:
+ Amino acid
+ Acid hu c
+ Thuc tr su
+ .

Sc k ion (Ion Chromatography)

Ion Exchange
R

Lc ion

N+ R
R

SO3

Sample
+ + + +
+
+
Sample
+
+
+ + + + +

- Phn tch cc hp cht ion

- C 2 loi ct:
+ Ct trao i cation
Strong cation exchange

: (R-SO3-)

Weak cation exchange

: (R-COO-)

+ Ct trao i anion:
Strong anion exchange : (R4N+)
Weak anion exchange :diethyl aminoethyl)

Pha ng: thng l dung dch m trong

dung mi nc
Anion:
Carbonat/bicarbonate (Na2CO3/NaHCO3)
Potassium hydroxide (KOH)
Cation:
Nitric acid
Tartaric acid
Tartaric acid/dipicolinic acid
Tartaric acid/citric acid

n tp
1.
2.
3.
-

Khi nim v phn tch nh lng

Tnh ton v x l s liu phn tch
Phng php phn tch dng c
PP ph hp th phn t
PP ph nguyn t
PP in ha
PP sc k lng
4. Cc bi thc tp, tnh ton s liu t s o
thc nghim