Trng i hc Si Gn

Khoa Khoa hc mi trng

Tiu lun mn hc: PHN TCH MI TRNG

ti: TM HIU MY HPLC

Nhm: 2 Lp: DKM1091

Thnh vin thc hin: M sinh vin:
Hng Vn 3109340144
Nguyn Th Nh Mai 3109340073
Hunh Trn Chin Cng 3109340015
Trng Th Tm Tm 3109340
GV Hng dn: GS.TS. Phm Nguyn Kim Tuyn
 1 TNG QUAN

2 CU TO

HPLC 3 NGUYN TC HOT NG

4 NG DNG CA MY HPLC
I. TNG QUAN
HPLC ( High Performance Liquid Chromatography) l
phng php sc k lng hiu nng cao. Trc kia gi l
phng php sc k lng cao p (High Pressure Liquid
Chromatography).
Phng php ny ra i t nm 1967-1968 trn c s
pht trin v ci tin t phng php sc k ct c in.
Hin nay n p dng rt ln trong nhiu ngnh kim
nghim c bit l ng dng cho nghnh kim nghim
thuc . V n hin l cng c c lc trong phn tch cc
thuc a thnh phn cho php nh tnh v nh lng .
Phng php sc k (c pht minh bi nh sinh vt hc ngi Nga Mikhail
Tswest ) :
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

Flow of Mobile Phase

Injector Detector
T=0

T=10

T=20

Most Interaction with Stationary Phase Least

 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
t0 t1 t2 t3 t4
detector
Tn hiu

t0 t1 t2 t3 t4 Thi gian
 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
 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)
 Khi nim v k thut sc k lng hiu nng cao

Sc k lng hiu nng cao l mt phng php chia tch trong

pha ng l cht lng v pha tnh cha trong ct l cht rn c
phn chia di dng tiu phn hoc mt cht lng ph ln mt cht
mang rn, hay mt cht mang c bin i bng lin kt ho
hc vi cc nhm chc hu c .

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, gi cht phn tch
Pha ng: cht lng (1 cht hoc hn hp nhiu cht), ha tan
v di chuyn cht phn tch
- Sc k lng chia thnh 2 nhm
Sc k lng p sut thng (sc k c in)
Sc k lng p sut cao (Sc k lng hiu nng cao:
HPLC)
Da vo bn cht ca qu trnh sc k, HPLC chia thnh:

SK
phn
b SK pha
thng
SK ghp
cp ion
HPLC
SK
pha
o
Sk trao
i ion
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
 II.CU TO MY HPLC

Trong :
1- Bnh cha dung mi pha ng
2- B phn kh kh
3- Bm cao p
4- B phn tim mu ( bng tay hay Autosample)
5- Ct sc k ( Pha tnh ) ( ngai mi trng hay trong b
iu nhit )
6- Detector ( nhn tn hiu )
7- H thng my tnh gn phn mm nhn,tn hiu v s l d
liu v iu khin h thng HPLC.
8- In d liu .
1 - Bnh ng dung mi
- Hin ti my HPLC NGUN CUNG CP PHA NG
thng c 04 ng dung mi vo (MOBILE PHASE): L BNH NG
u bm cao p .Cho php chng ta DUNG MI HOC DUNG MI
s dng 04 bnh cha dung mi CHY MY
cng 1 ln ra gii theo t l
mong mun v tng t l dung mi
ca 4 ng l 100 % .
- Tuy nhin theo kinh
nghim th chng ta t khi s dng 04
ng dung mi cng mt lc m
chng ta chi s dng ti a l 3 v 2
ng cho h pha ng lun
c pha trn ng nht hn,h pha
ng n gin hn qu trnh ra
gii n nh .
- Hin 04 ng dung mi
phc v ch yu cho vic ra gii
Gradial dung mi theo thi gian v
cng tc xy dng tiu chun .
2 B kh kh Degasse :
Mc ch ca b kh kh nhm loi tr cc bt nh
cn st li trong dung mi pha ng .
Nu nh trong qu trnh phn tch m dung mi
pha ng cn st cc bt kh th mt s hin tng sau
y s sy ra:
- T l pha ng ca cc ng dung mi ly
khng ng s lm cho thi gian lu ca Peak thay i .
- Trong trng hp bt qu nhiu b kh kh
khng th loi tr ht c th c th Pump s khng ht
c dung mi khi p sut khng ln v my sc k s
ngng hot ng .
3- Bm cao p (h thng cung cp dung mi):
- Bm pha ng vo ct tch
- iu khin tc dng, p sut ca pha ng
- My sc k lng ca chng ta hin nay thng c p sut ti
a 412 Bar (1at = 0.98Bar). Tc dng 0.1-9.999 ml/pht .

H THNG IU KHIN P SUT

HOC TC DNG PHA NG,
CN GI L H THNG CUNG CP
DUNG MI: LC PUMP
4 B phn tim mu ( injection valve):
a mu vo ct phn tch theo phng php khng ngng
dng chy . Vi dung tch ca l bp l 5 - 100l .
C 02 cch ly mu vo trong ct : Bng tim mu th cng
(tim bng tay) v tim mu t ng (Autosample) .

BM MU BNG TAY

BM MU T NG
(AUTO SAMPLE)
5. Ct tch (Colume)
L CT (COLUME OVEN): DNG CT TCH: L NI XY RA CC
IU KHIN NHIT CT QU TRNH TCH CHT, GM
TCH CT PHN TCH V CT BO V

- Ct tch c kch c khc

nhau
- Chiu di: 10 25cm
- ng knh: 2 5mm
5 Ct tch :
Ct cha pha tnh c coi l tri tim ca h thng sc k lng hiu
nng cao.
- Ct pha tnh thng thng lm bng thp khng r ,chiu di ct
khong 10 -30cm ,ng knh trong 1-10mm ,ht cht nhi c = 5-10
m.(ngoi ra cn c mt s trng hp c bit v kch thc v kch c
ht....)
- Vi cht nhi ct c = 1.8 -5 m c th dng ct ngn ( 3-10 cm )
v nh (ng knh trong 1-4.6 mm) loi ct ny c hiu nng tch cao.
- Cht nhi ct ty theo lai ct v kiu sc k
- Thng thng cht nhi ct l Silicagel (pha thun)
hoc l Silicagel c Silan ha hoc c bao mt lp mng hu c (
pha o ) , ngoi ra ngi ta cng dng cc loi ht khc nh : Nhm
Oxit,Polyme xp,cht trao i ion.
* * i vi mt s phng php phn tch i hi phi c nhit cao
hoc thp hn nhit phng th ct c t trong b phn iu nhit
(Oven column)
6 .AU DO (DETECTOR):
CO NHIEU LOAI KHAC NHAU TUY THEO MUC CH
PHAN TCH.
DUNG E PHAT HIEN CHAT VA NH LNG :
UV-VIS, HUYNH QUANG, O DAN, IEN HOA, KHOI PHO

7. HE THONG GHI NHAN VA X LY TN HIEU

(INTERFACE, SOFTWARE, PC) : DUNG E THU THAP
VA TNH TOAN CAC KET QUA.
III. NGUYN TC HOT NG MY HPLC
- Mu cht lng c a vo bung bm mu sau c bm t
ng vo ct tch.
- Da trn s phn b lin tc cc cu t cht phn tch ln hai pha:
mt pha thng ng yn, c kh nng hp thu cht phn tch gi l pha
tnh, mt pha di chuyn qua pha tnh gi l pha ng. Do cc cu t cht
phn tch c i lc khc nhau vi pha tnh, chng di chuyn vi tc khc
nhau v tch ra khi nhau.
- 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 phn tch 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
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
 Qu trnh tch din ra trong ct sc k

column

Vt liu nhi ct 3- 5m
 Qu trnh tch

mixed sample
Mobile phase
column
Hn hp cht tch khi
nhau th no ?

Flow

Pha tnh
 - Sau khi cc cu t tch ra khi nhau s ln lt i vo
detector, ti chng c chuyn thnh tn hiu in. Tn hiu ny
c khuych i ri chuyn sang b ghi. Cc tn hiu c x l
ri chuyn sang b phn in v lu kt qu.
- Trn sc nhn c, s c cc tn hiu ng vi cc cu t
c tch gi l peak. Thi gian lu ca peak l i lng c trng
(nh tnh) cho cht cn tch. Cn din tch peak l thc o nh
lng cho cht cn phn tch.
-Thi gian cht phn tch b pha tnh lu gi (thi gian lu)
quyt nh bi:
Bn cht ca pha tnh, cu trc v tnh cht ca cht
phn tch.
Bn cht v thnh phn ca pha ng dng ra gii
cht phn tch ra khi ct sc k (pha tnh).
 - Ghi li ton b qu trnh tch sc k ca hn hp cht phn
tch sc k gm nhiu peak.
- c im ca peak phn tch :
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
Tu tng cht cn nghin cu v cc iu kin phn tch
chn cc detector khc nhau.
 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
+ .