HPLC

Hng dan: Ha Kim Thanh
Li cm n!
hon thnh chng trnh thc tp mn Ha Phn tch II ny, ngoi s n

lc ca tp th nhm I, cn c s gip , h tr, s hng dntn tnh ca qu
Cng ty. Tp th nhm I xin gi nhng li cm n chn thnh nht n tt c nhng
anh ch gip chng em.
Trc tin, tp th nhm xin gi nhng li cm n chn thnh nht n ch
Ha Kim Thanh, ngi trc tip hng dn, tn tnh gip , to iu kin thun
li cho nhm c thc hnh trc tip cc thit b trong sut qu trnh thc tp.
Thng xuyn quan tm, theo st nhm trong sut qu trnh hc tp, gii p nhng
vn m nhm cha hiu. c bit, vic ch mun nhm vit cng thc tp
trc khi thc tp v mi ngy vit bo co gip nhm lm vic c mc ch hn,
hiu c vn nhm sp phi lm v lm c g. Cng thm nhng li nhc
nh ca ch cho nhm l nhng bi hc qu bu nhm khng mc phi na trong
nhng ln sau, gip nhm lm vic k lut hn.
Nhm cng xin chn thnh cm n cc anh ch k s khc v qu cng ty
nhit tnh gip , hng dn, cung cp ha cht, dng c y , to mi iu kin
thun li nhm ni ring v tp th lp ni chung c thc tp ti cng ty, c
tip xc thc t nhiu hn.
Nhm cng chn thnh cm n qu thy, qu c thuc Khoa Ha hc ng
dng lun ng hnh cng chng em trong sut qu trnh hc.
Tuy nhin, trong qu trnh thc tp ti Cng ty, nhm phm phi rt nhiu
thiu xt, sai lm nh lm vic khng tp trung, phm nhiu li, lm nhng khng
lm n ni, khng hon thnh nhim v, lm ch khng vui, khng hi lng. Tp th
nhm hy vng s c ch b qua v lun mong nhn c nhng li nhc nh,
hng dn ca ch khc ngy cng hon thin hn v c kin thc ln k nng
ca mnh.
Trn trng
Tp th nhm I
Bao cao thc tap phan tch Nhom I
-1-
NHAN XET
-2-
MC LC
BI 1 KHO ST CU TRC PHN T CA HP CHT BNG FT- IR

SPECTROMETER ...................................................................................................................9
I.
Gii thiu chung .................................................................................................... 9
II.
L thuyt v thit b............................................................................................. 10

1.
Cu to............................................................................................................... 10
2.
Nguyn tc hot ng .................................................................................... 10
III.
Thc nghim .................................................................................................... 11
1.
M my .............................................................................................................. 11
2.
Chun b mu ................................................................................................... 12
2.1 i vi mu chy background ................................................................... 12
2.2
i vi mu cn phn tch ..................................................................... 13
3.
Thao tc o mu ............................................................................................. 13
3.1
3.2
3.3
Chy background..................................................................................... 13
Chun b mu............................................................................................ 13
o mu ....................................................................................................... 13
4.
X l v in kt qu........................................................................................... 14
5.
Tt my,v sinh v ghi nht k thit b........................................................ 14
IV.
Kt qu thc nghim ...................................................................................... 14
V.
Tho lun v kt qu thu c......................................................................... 25
VI.
Hng pht trin ............................................................................................. 26
VII.
Ti liu tham kho ........................................................................................... 26
-3-
Bi 2 - KIM TRA TINH KHIT CA NGUYN LIU BNG HPLC........................37

I. Gii thiu chung ...................................................................................................... 37
II. L thuyt v thit b ............................................................................................... 38
II.1 Cu to[1] ............................................................................................................. 38
II.2 Nguyn tc hot ng ca h thng HPLC ................................................. 47
II.3 Qu trnh sc k[2] ............................................................................................. 47
II.4 ng dng ca HPLC......................................................................................... 49
III. Thc nghim........................................................................................................... 51
III.1 Chun b mu.................................................................................................... 51
III.2 Cch o mu ..................................................................................................... 51
IV. Kt qu thc nghim ............................................................................................ 53
V. Tho lun kt qu ................................................................................................... 59
VI. Mt s vn trong qu trnh o mu.............................................................. 61
VII. Ti liu tham kho ................................................................................................ 62
-4-
DANH SCH T VIT TT
FTIR : Fourier Transform Infrared Spectrometer

HPLC : High Performance Liquid Chromatography
UV-Vis : Ultraviolet visible
RP: Reversible phase
PDA: Photodiode Array Detector
TMI: 2,3,3-trimethyl indolenine
TMBI: 1,1,2-trimethyl benzen[e]indole
MPT: Methyl-p-toluene-sulfonate
-5-
DANH SCH BNG BIU MINH HA

FTIR
Bng 1.1 Mt s dng c chun b mu ....................................................................11
Bng 1.2 Mt s ha cht s dng ..................................................................................12
Bng 1.3 Mi mu phn tch ph FTIR.........................................................................14
Bng 1.4 Cng thc cu to v ph chun cc mu phn tch..................................25
HPLC
Bng 2.1 Mt s u khuyt im ca pha tnh ..............................................................42
Bng 2.2 Thng s ct HPLC ............................................................................................45
Bng 2.3 Cng thc cu to ca cht phn tch ...........................................................60
-6-
DANH SCH HNH MINH HA

FTIR
Hnh 1.1 My ph FTIR 100 ca hng PerkinElmer .........................................................9
Hnh 1.2 S cu to my ph FTIR .............................................................................10
Hnh 1.3 S nguyn l hot ng my ph FTIR .....................................................11
Hnh 1.4 Ph ca 4-methoxypheno............................................................................15
Hnh 1.5 Ph ca 3,4-Dihydroxybenzaldehyde .......................................................16
Hnh 1.6 Ph ca 3-Nitrobenzaldehyde.....................................................................17
Hnh 1.7 Ph ca Xylene ..............................................................................................18
Hnh 1.8 Ph ca Methacrylamide ..............................................................................19
Hnh 1.9 Ph ca 2-hydroxybenzaldehyd..................................................................20
Hnh 1.10 Ph ca 4-carboxybenzaldehyde.............................................................21
Hnh 1.11 Ph ca 1-Iodopropane .............................................................................22
Hnh 1.12 Ph ca 1-Iodobutane ................................................................................23
Hnh 1.13 Ph ca 1,2-Diaminopropane ...................................................................24
HPLC
Hnh 2.1 H thng my HPLC ...........................................................................................37
Hnh 2.2 S h thng HPLC .........................................................................................38
Hnh 2.3 Pha tnh ca RP-HPLC ......................................................................................42
-7-

Hnh2.4 H thng bm cao p ..........................................................................................43
Hnh 5: H tim mu ..........................................................................................................44
Hnh 2.5 Valve tim mu ............................................................................................. 44
Hnh 2.6 Ct sc k .............................................................................................................45
Hnh 2.7 Waters 2996 Photodiode Array Detector ..................................................................46
Hnh 2.8 S hnh thnh peak .............................................................................................49
Hnh 2.10 S sc k pht hin cht ............................................................................50
Hnh 2.11 sc k ca 4-methoxyphenol....................................................................54
Hnh 2.12 sc k ca TMI ..............................................................................................55
Hnh 2.13 Sc k ca 3-hydroxy benzaldyhyde ......................................................56
Hnh 2.14 Sc k ca 4-hydroxy benzaldyhyde ......................................................57
Hnh 2.15 Sc k ca TMBI.................................................................................... 58
Hnh 2.16 sc k ca MPT ..................................................................................... 59
-8-
GII THIU
HPLC - High Performance Liquid Chromatography l phng php sc k
phn tch c tc nhanh v hiu sut cao, dng phn tch nh tnh (xc nh
thnh phn ca hn hp) v nh lng (xc nh nng ca cc cht) cc hp
cht hu c trong mt s lnh vc nh: ha cng nghip, ha thc phm, ha
dc,
Trong vic phn tch cc hp cht hu c, ngoi k thut HPLC th FTIR l
mt trong nhng k thut phn tch kh quan trng v ph bin.
FTIR - Fourier Transform Infrared Spectrocopy l k thut dng phn tch
nhng phn t c kh nng hp th bc x hng ngoi cho hiu ng ph dao
ng. K thut ny dng ng nht cc cht (so snh ph ca cht phn tch vi
ph chun) v dng xc nh cc nhm chc trong mt hp cht cha bit.
BI 1 KHO ST CU TRC PHN T CA HP CHT

BNG FT- IR SPECTROMETER
Hnh 1.1 My ph FTIR 100 ca hng PerkinElmer
I.
Gii thiu chung

Fourier Transform Infrared (FTIR) Spectrometer c ng dng rng ri trong
vic xc nh chnh xc cu trc phn t ca mt hp cht hay cc nhm chc c

mt trong hp cht . FTIR spectrometer cng c th dng nhn bit mt
-9-

hp cht hu c hay cng c th dng nh gi tinh khit ca mt hp
cht bng cch so snh vi ph chun.
FTIR spectrometer c u im vt tri so vi cc my ph hng ngoi thng
thng do vic dng giao thoa k cho php lm khe sng rng hn lm cho
lng nh sng thu c trn giao thoa k s ln hn nhiu so vi trn b to
n sc cc my ph th h c.Nh nguyn l hot ng mi, FTIR
spectrometer gim c nhiu, lm tng tn hiu.[1]
Ngoi ra, FTIR spectrometer c th ni vi my sc k nhn bit cc hp
phn tch ra sau ct sc k.[1]
II.
L thuyt v thit b
1. Cu to
Gm ngun sng, giao thoa k, mu, detector v computer.
Hnh 1.2 S cu to my ph FTIR

2. Nguyn tc hot ng
- 10 -
Hnh 1.3 S nguyn l hot ng my ph FTIR

Ngun bc x pht ra chm tia hng ngoi, chm tia ny qua b to giao thoa
gm t mt gng c nh, gng di ng v b phn chia chm sng. Bc x
hng ngoi sau khi ra khi giao thoa k s i qua mu ri n detector. Detector
ghi nhn s bin i cng ca bc x theo qung ng d m gng di
ng thc hin c ri chuyn thnh tn hiu in. Khi ta thu c tn hiu
di dng hm ca in th V theo qung ng, V=f(d). Computer s thc hin
php bin i Fourier chuyn hm V=f(d) thnh hm ca cng bc x I
theo nghch o ca qung ng d, I=(d-1). V d-1 chnh l s sng V , do
thc cht ta c hm s ph thuc ca cng bc x vo s sng.[1]
Bin i Fourier
V=f(d)
III.
I=(d-1)
Thc nghim
1. M my
Kim tra cht lm kh (decriccant): mu xanh v tr 15/20 l OK.
M cng tc sau my, mm hnh trn my hin th dng ch
PerkinElmer Spectrum 100 Series.

- 11 -
Chn biu tngSpectrum trn desktop ri login.
Nhp user name v password.
T ca s Spectrum login, chn Spectrum 100, chn OK kt ni
chng trnh vi my.

2. Chun b mu
Dng c
Bng 1.1 Mt s dng c chun b mu
STT
Tn dng c
S lng
Cuvet to mu rn
B ci v chy
Cha kha
Ha cht
Bng 1.2 Mt s ha cht s dng
STT
Tn ha cht
Trng thi vt l
KBr
Rn
4-methoxyphenol
3,4-dihydroxybenzaldehyde
3-nitrobenzaldehyde
Xylene
Methacrylamide
. 2-hydroxybenzaldehyde
4-carboxybenzaldehyde
1-Iodopropane
10
1-Iodobutane
11
1,2-Diaminopropane
2.1 i vi mu chy background
Ra sch v sy kh khun to mu.
- 12 -
C nh mt con c vo khun trc, sau cho khong 0.2g bt KBr
vo khun, vn con c cn li vo.
Dng 2 chic cha kha xit cht 2 con c to vin KBr sao cho vin
KBr nm khong gia khun. Vin KBr c to phi c b dy ng nht,

b mt lng bng, dng nh trong sut, ng vin phi u.
2.2 i vi mu cn phn tch
Mu lng: To vin KBr trc sau dng tampon chm dung dch
mu cn phn tch ln b mt vin KBr.
Mu rn: Ra sch v sy kh b to mu. Cho khong 0.2g bt KBr
v mt lng rt t mu cn phn tch vo ci, dng chy tn u hn hp ri cho

hn hp vo khun to vin tng t nh to vin KBr chy background.
Ch : Bt KBr c tnh ht m nn phi c bo qun trong bnh kn
c cha cc gi silicagel ht m v ch ly lng va cho mt ln to vin.
3. Thao tc o mu (gm 2 bc)
3.1 Chy background
3.2 Chun b mu
t cuvet cha vin KBr vo bung cha mu.
T mn hnh chnh chn biu tng Instrument trn thanh cng c,
mn hnh s xut hin ca s Scan & Instrument Setup.T ca s ny ta chn
v thay i cc thng tin trong cc ca s con nh Sample, Scan, Instrument.
Sau khi nhp, chn Apply ghi nhn s thay i v my s t ng
lu kt qu ny sau khi o.
Chn Background s xut hin ca s Scan progress, click Scan my
s chy background v th background s xut hin trn ca s Scan
progress trong sut qu trnh Scan.
3.3 o mu
t cuvet cha mu vo bung cha mu.
Tng t, t ca s Scan & Instrument Setup ta cng chn v thay
i cc thng tin trong cc ca s con nh Sample, Scan, Instrument ri Apply.
Sau thay v chn backgroud, ta chn Start. Mn hnh cng s xut
hin ca s Scan progress, click Scan my s tin hnh o mu v ph s
xut hin trn ca s Scan progress trong sut qu trnh Scan.
- 13 -

Sau khi Scan xong, my s t ng lu v hin th th mu va o
ln mn hnh chnh.
4. X l v in kt qu
Chn ph mun x l, click vo biu tng Label Peak hin th
tn s ti nh peak. Sau khi x l xong c th lu kt qu li bng cch vo

File/Save As vi tn mi.
Mun in th ch cn copy ph paste vo mu report ri print.Cn
mun in thng tin ca mu nh km th vo Status.My s chuyn th v

thng tin v mu vo bn bo co phn tch c sn, nhn print sau OK.
5. Tt my,v sinh v ghi nht k thit b
IV.
Vo File/Exit ngt kt ni.
Tt cng tt sau my.
V sinh v ghi nht k thit b.

Kt qu thc nghim
Bng 1.3 Mi mu phn tch ph FTIR

STT
Tn mu
4-methoxyphenol
3-nitrobenzaldehyde
Xylene
Methacrylamide
1-Iodopropane
1-Iodobutane
10
1,2-Diaminopropane
- 14 -
%T
5 .4
4 0 0 0 .0
10
15
20
25
30
35
40
45
50
55
60
65
70
75
7 8 .8
3600
3349
3200
2952
3034
2800
2834
2400
- 15 -
1860
1800
cm- 1
1600
1607
1633
Hnh 1.4 4-methoxyphenol
2000
2057
1511
1400
1454
1375
1299
1200
1233
1179
1102
1000
1032
800
824
734
600
510
4 5 0 .0
-15
-10
-5
10
15
20
25
30
- 2 0 .0
4 0 0 0 .0
%T
35
40
45
50
55
60
65
70
75
8 0 .0
3600
3324
3200
2824
2800
2875
- 16 -
2000
1800
cm- 1
1750
1595
1600
1646
1537
1400
1443
1422
1388
Hnh 1.5 3,4-Dihydroxybenzaldehyde
2400
1875
1297
1200
1167
1192
1119
1000
975
946
877
800
814 755
776
676
600
631
591
4 5 0 .0
5 .0
4 0 0 0 .0
10
15
20
25
30
35
40
45
50
% T 55
60
65
70
75
80
85
90
95
100
105
1 1 0 .0
3600
3681
3696
3392
3200
2923
2973
2800
2865
2844
2400
- 17 -
1800
cm-1
1701
1600
1535
1346
1400
1455
Hnh 1.6 3-Nitrobenzaldehyde
2000
2076
1200
1000
1055
1032
1015
800
733
600
4 5 0 .0
474
%T
9 7 .1
4 0 0 0 .0
98
99
100
101
102
103
104
105
106
107
108
109
110
111
112
113
1 1 4 .0
3600
3200
2938
3031
2800
2400
2000
1800
cm-1
- 18 -
1496
1400
Hnh 1.7 Xylene
1600
1612
1200
1000
741
800
768
795
690
600
4 5 0 .0
482
15
20
25
30
35
40
45
50
1 0 .0
4 0 0 0 .0
%T
55
60
65
70
75
80
85
90
9 5 .4
3600
3681
3696
3399
3200
2973
2923
2800
2866
2844
2400
2000
2076
- 19 -
cm-1
1600
1346
1400
1455
Hnh 1.8 Methacrylamide
1800
1671
1636
1200
1000
1032
1055
1015
800
693
600
573
4 5 0 .0
30
35
40
45
50
55
60
2 5 .0
4 0 0 0 .0
%T
65
70
75
80
85
90
95
9 8 .8
3600
3200
3063
2800
2845
2751
2400
- 20 -
1800
c m- 1
1600
1400
1459
Hnh 1.9 2-hydroxybenzaldehyde
2000
1665
1620
1486
1580
1386
1278
1150
1200
1227
1203
1114
1000
1029
883
800
765
716
666
600
562
538
4 5 0 .0
10
15
20
25
30
35
40
- 5 .4
4 0 0 0 .0
%T
45
50
55
60
65
70
75
80
85
9 0 .9
3600
3200
2992
2800
2834
2636
2557
- 21 -
2000
1800
cm-1
1687
1600
1574
1609
1393
1400
1505 1429
Hnh 1.10 4-carboxybenzaldehyde
2400
1954
1291
1200
1205
1247
1125
1108
1000
1015
931
851
800
761
789
685
600
542
522
4 5 0 .0
460
%T
9 0 .0
4 0 0 0 .0
92
94
96
98
100
102
104
106
108
110
112
114
116
118
120
122
1 2 5 .0
124
3600
3200
2975
2800
2400
- 22 -
1800
cm-1
1600
Hnh 1.11 1-Iodopropane
2000
1459
1400
1275
1200
1193
1000
800
763
600
4 5 0 .0
68
70
72
74
76
78
80
82
84
86
88
66
6 5 .0
4 0 0 0 .0
%T
90
92
94
96
98
100
102
104
106
108
1 0 9 .5
3749
3600
3200
2972
2800
2885
2400
- 23 -
1800
cm-1
1600
Hnh 1.12 1-Iodobutane
2000
1463
1400
1380
1191
1200
1247
1000
800
734
600
593
4 5 0 .0
50
52
54
56
58
60
62
64
66
68
70
72
4 8 .0
4 0 0 0 .0
%T
74
76
78
80
82
84
86
88
90
92
9 5 .0
94
3600
3200
3350
3274
2800
2870
2960
2926
2400
2325
- 24 -
1800
cm-1
1600
1376
1400
Hnh 1.13 1,2-Diaminopropane
2000
1594
1456
1302
1200
1144
1062
1000
950
800
866 810
600
620
508
4 5 0 .0

V.
Tho lun v kt qu thu c
So snh ph FTIR phn tch vi ph chun t ngun Sigma Aldrich[3 ] c kh

nhiu s sai khc. Nguyn nhn c th l do k thut chun b mu cn yu km,
thnh phm mu khng tinh khit hay do iu kin thc nghim khng ng nht.
Bng 1.4 Cng thc cu to v ph chun cc mu phn tch
STT
Tn hp cht
Cng thc cu to
Ph FTIR
chun
4-methoxyphenol
Ph lc 1
Ph lc 2
3-nitrobenzaldehyde
Ph lc 3
Xylene
Ph lc 4
Methacrylamide
Ph lc 5
Ph lc 6
Ph lc 7
1-Iodopropane
CH3CH2CH2I
Ph lc 8
1-Iodobutane
CH3CH2CH2 CH2I
Ph lc 9
- 25 -

10
1,2-Diaminopropane
VI.
Ph lc 10
Hng pht trin
Thun li: Thao tc thc hnh trn FTIR spectrometer tng i n gin
v phn tch c kh nhiu mu.
Kh khn: Trong vic gii ph, nh mt chc li c nhiu khong hp th
v ng vi mt khong hp th li c nhiu chc,do s hiu bit cn hn ch nn

vic xc nh chnh xc mt chc gp nhiu kh khn.
xut: Mong nhn c s hng dn v vic gii ph v tt c cc
chc nng c trong phn mm Spectrum hon thin hn s hiu bit v thit b.
VII.
Ti liu tham kho
[1] Nguyn Hu nh, Trn Th , ng dng mt s phng php ph nghin

cu cu trc phn t, NXB Gio Dc, 1999.
[2] Nguyn nh Triu, Phng php ph nghim nghin cu cu trc phn t,
NXB Khoa hc-K thut, 2003.
[3] www.Sigmaaldrich.com, truy nhp cui cng ngy 18/08/09.
[4] www.thermonicolet.com, truy nhp cui cng ngy 18/08/09.
[5] www.perkinelmer.com, truy nhp cui cng ngy 18/08/09.
- 26 -
Ph lc 1
- 27 -
Ph lc 2
- 28 -
Ph lc 3
- 29 -
Ph lc 4
- 30 -
Ph lc 5
- 31 -
Ph lc 6
- 32 -
Ph lc 7
- 33 -
Ph lc 8
- 34 -
Ph lc 9
- 35 -
Ph lc 10
- 36 -
Bi 2 - KIM TRA TINH KHIT CA NGUYN LIU

BNG HPLC
I. Gii thiu chung
Hnh 2.1 H thng my HPLC
Sc k lng theo phng php c in l mt h thng dng ct sc k c

ng knh 1-5cm v chiu di 50-50cm, ht cht nhi ct (pha tnh) c ng knh
150-200mm. Nhng ct sc k ny kh to, pha ng chy qua ct l do trng lc,
tc chy khong vi phn mi ml/pht, do vy thi gian sc k di v hiu qu
tch khng tt. Cc phng php ny cng t c dng ch tr khi dng sc k
ch ha. n cui nhng nm 70 ngi ta sn xut c nhng cht nhi ct c
c ht khong 10 mm c hiu sut tch cao. C ht b i hi dng bm nn
dung mi qua ct, vi loi cht nhi ct ny v h thng my tnh, k thut sc k
lng hiu nng cao ra i.
HPLC c xem l phng tin nh tnh, nh lng c nhy cao,
chnh xc, gim thi gian sc k, t tn mu, c th p dng cho hu ht cc cht
- 37 -

nh: cc cht d b phn hu bi nhit , cc acid amin, protein, acid nucleic,
cacbohydrat, khng sinh, cc cht v c
Trong k thut HPLC tu theo bn cht ca pha tnh trong ct tch m ngi
ta chia thnh:
Sc k hp ph
Sc k trao i ion v cp ion
Sc k phn b (chit)
Sc k ry phn t
My sc k lng hiu nng cao chng ta ang s dng l sc k hp ph pha

o. Pha tnh khng phn cc, pha ng (dung mi) phn cc. Bn cht chnh ca
s tch trong loi sc k ny l da trn s hp ph b mt ca pha tnh. C ch
tch l c ch hp ph. V th tt c cc tnh cht ng hc hp ph ca b mt pha
tnh u c nh hng n kt qu ca s sc k mt hn hp cht mu.
II. L thuyt v thit b

II.1 Cu to[1]
Hnh 2.2 S h thng HPLC
- 38 -

II.1.1 Bnh cha dung mi v pha ng
Bnh ng dung mi thng lm bng thu tinh th tch thay i t 100ml
2lit. Bnh lm bng thu tinh mu nu cha nhng cht nhy vi nh sng, bnh phi
tht kn nu pha ng l nhng cht bay hi. V d nh aceton, methanol,Nu
khng nng s b thay i.
i khi phi c h thng kh kh v vi dung mi c th ha tan kh trong kh
quyn nht l oxygen, ngi ta cng c th kh kh bng siu m hay bng cch sc
kh Helium vo (He t tan trong dung mi nhng c th y cc kh khc ra khi dung
mi).
Pha ng l dung mi ra gii cht tan (cht phn tch) ra khi ct tch
thc hin qu trnh sc k. Pha ng trong HPLC c th l dung mi hu c n
nh methanol, acetonitrile, benzene, n-hexene, water, hay cng c th l hn hp
ca hai hoc ba dung mi c trn vo nhau theo nhng t l ph hp. V d nh:
hn hp methanol vi nc theo t l 70 methanol/30 nc, acetonitrile vi nc
theo t l 80/20N cng c th l dung mi nc c cha cc cht m pH, cht
to phc, cht lm chmvi nng nht nh.
Ni chung, vi mi loi sc k s c cc h dung mi (pha ng) ra gii
ring thch hp vi n, c th thu c kt qu tt nht. Sau pha tnh th pha
ng l yu t th hai quyt nh hiu sut tch sc k ca mt hn hp mu. Ngha
l trong mt h pha, th pha ng v pha tnh l hai yu t chnh ca qua trnh sc
k. Hai yu t ny gi vai tr quyt nh thi gian lu gi ca cc cht mu v hiu
qu s tch sc k.
Ni chung, pha ng c th nh hng n nhng vn sau ca s tch
sc k ca cc cht:
chn lc ca h pha.
Thi gian lu gi ca cc cht tan.
Hiu lc ca ct tch.
phn gii ca cht trong pha tnh.
rng ca peak sc k.
- 39 -

Do , vi mt pha tnh chn ri, nu nh chng ta li chn c mt
dung mi c thnh phn ph hp, th ta s c hiu tch sc k ca mt hn hp mu
l tt nht i vi mt pha tnh chn.
V vy, pha ng l mt yu t linh ng v phi tha mn mt s iu kin
sau y:
Pha ng phi tr i vi pha tnh, khng lm hng pha tnh theo bt k
mt tnh cht no trong qu trnh sc k. Khng c s tng tc ph vi pha tnh

sinh ra nhng sn phm khc lm phc tp cho qu trnh sc k.
Pha ng phi ha tan c cht mu. C nh th mi lm cho cht
mu c vn chuyn tt t u ct n cui ct tch khi thc hin qu trnh ra

gii.
Pha ng phi bn vng theo thi gian, ngha l khng b phn hy hay
thay i khng theo mun khi chy sc k.
Pha ng phi c tinh khit cao, m bo khng b nhim bn
phn tch gy sai s cho kt qu phn tch. Ngha l cc cht pha ch pha ng
phi c tinh khit cao.
Phi nhanh t cn bng trong qu trnh sc k, nh cn bng hp ph,
phn b , trao i ion, tu theo bn cht tng loi sc k.
Phi ph hp vi loi detector dng pht hin cc cht phn tch. V
d detector UV-Vis th pha ng phi trong sut vng ph , hay detector hunh
quang th pha ng phi khng c tnh cht hunh quang.
iu kin cui cng l pha ng phi c tnh kinh t, khng him, khng
qu t.
l yu cu cn thit i vi mt h pha ng trong k thut tch HPLC cho
phn tch hn hp mu nht nh.
- 40 -
II.1.2 Pha tnh

Pha tnh trong HPLC chnh l cht nhi ct lm nhim v tch sc k mt
hn hp cht phn tch.
N l nhng cht rn xp v c kch thc ht nh. Song d dng no th
pha tnh trong HPLC cng phi tho mn nhng yu cu c bn sau y:
Phi tr v bn vng vi cc iu kin ca mi trng sc k, khng c
phn ng ho hc ph vi dung mi ra gii hay vi cht phn tch, m bo c

ch ca s tch l ng theo bn cht chnh ca pha tnh, hay khng mt cht phn
tch cn tch.
C kh nng tch chn lc mt hn hp cht tan nht nh trong iu
kin sc k nht nh.
Tnh cht b mt phi n nh, c bit l c c trng xp ca n v
khng b thay i hay bin dng trong qu trnh sc k, khng b phn r di p

sut cao ca qu trnh sc k.
Cn bng ng ca qu trnh sc k phi xy ra nhanh v lp li tt. C
nh th mi m bo thu c kt qu tch tt.
C ht phi tng i ng nht, ngha l ng knh ca cc ht ln
nht so vi cc ht nh nht khng chnh lch nhau qu 10% v trn 85% s ht

phi nm trong vng c kch thc trung bnh. C nh th ct tch mi c hiu qu
cao.
l yu cu chung ca pha tnh trong k thut HPLC.
Trong sc k hp ph pha o, pha tnh thng l cc silica alkyl ho, do
n t phn cc hay khng phn cc. Pha ng l h dung mi phn cc.
- 41 -
Hnh 2.3 Pha tnh ca RP-HPLC

Bng 2.1 Mt s u khuyt im ca pha tnh
u im
Khuyt im
%C cng ln (cng nhiu C) th
Dy C qu di s km bn.
phm vi tch cng rng, nn qu trnh

tch nhanh v chnh xc hn.
Ht c kch thc cng ln th tch
cng tt.
Nu khong trng gia cc ht pha
tnh ln th tch c cht c phn t
ln.
Nu khong trng gia cc ht pha
tnh nh th din tch b mt tip xc
Ht c kch thc nh th peak

xut hin chm.
Din tch b mt tip xc gim dn
n gim kh nng tch.
Khng tch c cc cht c
phn t ln.
ln nn kh nng tch cao.
- 42 -
II.1.3 Bm
Bm c tc dng ht v y gm cc b phn nh sau:
u bm c hai van iu tit, mi van c mt l nh vi mt bi. Cc van
ny m bo cho vic ht pha ng t thn bm vo ct sc k c an ton.
Thn bm c mt pittng chuyn ng i li. Khi pittng tin ti, mt
bi ng l pha ct li v bi khc nhc ra pha ng i vo thn, khi pittng li li

mt bi ng bnh ng dung mi v m l pha ct pha ng vo ct sc k.
Hnh2.4 H thng bm cao p

II.1.4 B phn tim mu
II.1.4.1 Kiu bm trn
Trong cc h thng sc k c in ngi ta dng sypring bm mu vo
ct. Phng php ny c u im l:
Tin li
n gin
C hiu lc cao v mu c a trc tip vo ct
Hn ch c th tch ngoi ct
- 43 -

Tuy nhin cng c nhiu khuyt im l: lp li khng cao v th tch bm
mi ln khc nhau, kh iu chnh cho tht chnh xc. Khi a thng mu vo ct ht
ca cht nhi ct c th lm tt kim. Mi ln a mu vo ct phi lm cho ngng
dng chy, i n khi p sut bng p sut khng kh ri mi tim.
II.1.4.2 Kiu Vanloop
Kiu mu sau c dng nhiu nht, mu c a vo mt vng c th
tch khng i ri i vo h thng sc k sau khi quay valve. Kiu tim ny tt khi
dng vi p sut cao v kt qu phn tch lp li v c th t ng ho chng c.
Sau khi c lm y ton b th vng s a vo mt th tch khng i. Trong
nhng trng hp vi sc k hay sc k vi vn tc ln th ngi ta hay dng nhng
vng c th tch khong vi m
Hinh 2.5: H tim mu
Hnh 2.6 Valve tim mu
II.1.5 Ct sc k
Ct dng trong sc k lng thng l mt ng lm bng thp khng r hoc
lm bng thu tinh c bit ( ch dng khi p sut khong 60psi ).
- 44 -

Ct l tri tim ca mt qu trnh sc k, ct l mt yu t quang trng quyt
nh n kt qu ca qu trnh sc k ca mt hn hp mu. Ct tch c nhiu kch
thc khc nhau thng c chiu di t 10 25 cm; ng knh trong 2-5 mm.
Hnh 2.7 Ct sc k
Bng 2.2 Thng s ct
ng knh
Chiu di ct
trong ct
C ht nhi Loi sc k
1980s
15-30 cm
4- 10 mm
5-10 m
HPLC
1990s
9-10 cm
2,1-4 mm
3,5 m
HPLC
> 2004
3-7,5 cm
1-2 mm
1,7 m
UPLC
II.1.6 u d ( detector ) trong sc k lng hiu nng cao
- 45 -

Khi cho cht tan i ngang qua t bo o, u d s to nn nhng tn hiu
in. Mi s thay i v ln vt l ca cht tan s c chuyn i thnh xung
in v c th c c trn my ghi.
S gii p ca u d t l vi lng cht tan hay nng cht tan trong t
bo, lp li v c lp vi cht ra gii (tr trng hp ra gii vi gradient i vi
vi loi u d). u d tt l phi cho kt qu bn, nhanh, lp li. N cng phi c
nhy tt vi cng nng v khng bin i cht lng tch.
Da vo tnh cht ca mu cht cn phn tch m ta c cc loi detector sau:
Detector UV-Vis
Dectector hunh quang
Detector in ho
Detector khi ph
Detector diode pht quang v diode mng...

Nguyn l hot ng ca Waters 2996 Photodiode Array Detector
Hnh 2.8 Waters 2996 Photodiode Array Detector

u d ny o lng nh sng qua dy photodiode xc nh hp th
ca mu trong bung o (flowcell). N hot ng da trn c s tnh cht hp th
quang phn t ca cht mu trong pha ng ti mt bc sng no . Pha ng
chy lin tc qua bung o. nh sng UV-Vis t ngun (Deuterium lamp) i qua
bung mu v c chn li bi mt ci gng (spectrographic mirror). Chm nh
sng phn x t gng ny qua mt khe hp (aperture) 50m. Khe hp ny c tc
dng kim sot cng v bc sng nh sng ti photodiodes. Sau chm nh
sng b chn li bi mt lng knh (grating). Qua lng knh, chm sng ny b tn
sc thnh dy nh sng vi cc bc sng khc nhau tp trung ln dy photodiode.
- 46 -

Mi photodiode ng vi mt bc sng ring v n s chuyn tn hiu in ln sc
k .
II.1.7 H thng thu nhn v x l s liu
l cc trm d liu hin i tip nhn v lu gi cc tn hiu pht ra t cc
detector v in cc sc k hon chnh vi chiu cao, din tch cc nh, cc thng
s nh tnh mu v cc bin s ca phng php. Chng cn c s dng lp
chng trnh sc k, kim sot hu ht cc bin s v theo di thng xuyn trong
thi gian vn hnh di khng c ngi trng coi.
Vi tin b ca cng ngh thng tin, hin nay b phn thu nhn v s l s
liu ca cc h thng HPLC thng l cc my vi tnh hin i c kh nng ghi
nhn, lu gi, bin tp, x l thng tin ht sc hiu qu.
Nh cc phn mm ci t tng thch vi cc chng trnh ca cc detector
khc nhau, cc my vi tnh h tr rt hiu qu cho vic nh tnh, nh lng cc cht
phn tch da vo cc c s d liu c ci sn.
II.2 Nguyn tc hot ng ca h thng HPLC
Dung mi (mobile phase) c h thng bm cao p bm lin tc qua ct vi
tc dng c iu chnh. Khi mu c tim vo loop v theo dng dung mi i
vo ct. Qu trnh tch sc k xy ra trong ct. Sau khi mu cht c tch ra khi
ct th c u d ghi nhn tn hiu v chuyn thnh tn hiu in th hin tng
peak ln sc k .
II.3 Qu trnh sc k[2]
Qu trnh tch trong k thut tch HPLC cng l nhng qu trnh t hp ca
nhiu qu trnh va c tnh cht ho hc, li va c tnh cht l hc. n l nhng cn
bng ng xy ra trong ct tch sc k gia pha tnh v pha ng. N l s vn
chuyn phn b li lin tc cc cht tan (hn hp mu phn tch) theo tng lp qua
cht nhi ct (pha tnh) t u ct tch n cui ct tch. Trong qu trnh cht tan
lun lun c phn b gia hai pha, trong khi pha ng lun lun chy qua ct tch
vi mt tc nht nh hay gradient. Tt nhin khi nim vn chuyn mu trong
tng lp trong pha tnh ch l tng i trong phm vi nht nh no ca qui lut
- 47 -

phn b. V thc t kh c th phn bit c dt khot tng vng, tng lp c lp
nhau trong hai pha trong qu trnh st k.
Song chc chn rng trong nhng iu kin st k nht nh. Th cht tan lun
lun phn b trong hai pha theo nhng cn bng ng hc t lc cht tan bt u
c np vo ct tch ( u ct cho n khi n c gii ra ra khi ct tch. Cho
nn, y nhng qui lut cn bng trong ho hc vn gip ta nh gi c phn
no ca mi cht tan i vo pha tnh v phn no i vo pha ng, cng nh s
tng tc v phn b ca chng ra sao. ng thi cng do tnh cht cn bng ng,
m lun lun c s vn chuyn ca cht tan t pha ny sang pha khc v ngc li,
khng ngng trong qu trnh sc k. Ngha l i vi mt phn t cht tan trong qu
trnh sc k, n lun lun nhy t pha ny sang pha kia nhiu ln t u ct n cui
ct sc k.
Mt khc, cng v tnh cht v tnh cht ca mi phn t cht tan l khc nhau
nn tc vn chuyn trung bnh ca mi cht l khc nhau trong qu trnh dch
chuyn . Khi trong pha ng, n dch chuyn theo tc ca pha ng, cn khi
pha trong pha tnh n li khng dch chuyn, m b pha tnh gi li. Nh vy l c
mt thi gian nht nh cht tan b gi li trong ct sc k. Thi gian cht tan b pha
tnh lu l c quuyt nh bi bn cht sc k ca pha tnh, cng nh cu trc v
tnh cht ca mi cht tan khc nhau. ng thi cng ph thuc vo bn cht v
thnh phn ca pha ng dng ra gii cht tan ra khi ct sc k. V th trong
qu trnh sc k c cht tan b lu gi trn ct, c cht tan t b lu gi.
iu dn n kt qu l, qu trnh tch ca cc cht ra khi ct sc k, khi
cht tan i t u ct n cui ct sc k. Nu ghi li qu trnh tch ca hn hp
cht mu phn tch, chng ta c mt sc k gm nhiu peak. Cc peak c tch
nhau hon ton, hoc cng c th chp vo nhau mt phn, hay cn hon ton chp
vo nhau. Sc phn nh qu trnh sc k trong ct c tt hay khng tt. Nu
qu trnh tch sc k l tt, th hn hp mu c bao nhiu cht, trong sc c by
nhiu peak ring khng chp vo nhau.Nh vy trong qu trnh sc k, cht no b
lu gi mnh nht s c ra gii sau cng, cht no b lu gi km nht s b ra
gii trc tin.
- 48 -
Hnh 2.9 S hnh thnh peak
II.4 ng dng ca HPLC

II.4.1 Phn tch nh tnh
II.4.1.1 Nguyn tc
Mt i lng c trng cho s tch sc k ca cc cht trong mt h pha l
thi gian lu (tr). Ngha l trong mt h pha v trong cc iu kin tch HPLC
chn nht nh, th mi cht tan trong hn hp mu s c thi lu khc nhau v c
nh.
Trong mt h pha HPLC nht nh th:
Da theo thi gian lu tr ca cht
So snh tr ca cht trong mu v cht chun
II.4.1.2 Phng php

V d pht hin cht X trong mu phi
Chy sc k v ghi sc ca cht chun X ta c Tx.
Chy sc k v ghi sc ca mu phn tch.
Tm trong sc mu cht no c tx nh cht chun X
Sau kt lun:
- 49 -
Nu c cht c tx suy ra c cht X trong mu
Khng c cht c tx suy ra khng c cht X trong mu.
Thi gian lu t (pht)

Hnh 2.10 S sc k pht hin cht
II.4.2 Phn tch nh lng

phn tch nh lng cc cht theo l thut HPLC, chng ta c th dng
mt trong hai phng php chun ho l phng php ng chun v phng
php thm tiu chun.
II.4.3 Phn tch bn nh lng
Trong trng hp khng cn chnh xc cao, ngi ta thng ch pht hin
bn nh lng cc cht c trong mu phn tch m khng cn c dy mu chun.
Nguyn tc ca cch ny l da trn c s tng din tch ca tt c cc peak sc k
ca cc cht trong mu c trong sc k pht hin c l 100%. Ngha l
chng ta c biu thc sau:
S= (S1+S2++Sn)=100%
(S1, S2Sn l din tch ca peak sc k ca cc cht 1,2n)
- 50 -

Khi dng phng php ny cn phi c my phn tch phn k. My ny s
gip ta ghi li peak sc k, thi gian lu, v tnh ton lun c din tch ca mi peak
sc k ca mi cht. Sau in kt qu ra.
III. Thc nghim
III.1 Chun b mu
Dng c: H bi, kim tim mu, bng gn lc mu, ng nh git
Tin hnh: mu sau khi ha tan vi dung mi acetonitrile, lc, sau
pha long khi tin hnh o, hp th khong t 0.8 1.0. Nu hp th qu
cao hoc qu thp th pha li.
III.2 Cch o mu
III.2.1 M my
Trc khi m my phi kim tra bnh dung mi v bnh
H2O(HPLC),Nu mc dung mi,H2O trong bnh thp hay gn ht th phi thm dung
mi,H2O vo,ng thi kim tra bnh nc thi,nu y th phi em
Ln lt m cng tc: my vi tnh, Waters In-line Degasser AF,
Waters 1525 Binary HPLC Pump, Waters 2996 Photo diodes Array Detector.
Chn biu tng Empower Login trn desktop m phn mm.
Sau xut hin ca s, t ca s ny, mc Project List chn HPLC_My lan 1;
mc System List chn ADS_HPLC, sau chn OK, chng trnh s t ng kt
ni vi my.
III.2.2 Chy ra ct v chy baseline (chy cn bng)
Chy ra
ct:
Trn
mn
hnh
iu
khin,
chn
mc
Run
Samples\Control Panel, nhp Flow A 0.0ml/min, Flow B 0.5ml/min Ramp for 1min
sau chn OK. i khi p sut u ct n nh (khong 447psi) th tng tc
Flow B 1.0ml/min, Ramp for 1min. i p sut n nh khong 941psi mi tin hnh
chy baseline.
- 51 -

Chy baseline (chy cn bng): Khi p sut n nh chn Monitor
chy cn bng. Chy cn bng khong 20 30 pht. Sau khi p sut n nh, dng
chy cn bng chun b o mu. dng chy cn bng nhp vo biu tng
th 3 trn View Acquisition (biu tng ny c tn press to abort the running
sample set or singlet injection)
III.2.3 Nhp tn mu chun b o
Vo File\new sample set\Using sample set method T nhng
phng php c sn chn 1 phng php ph hp nht i vi mu chy mu.
Sau nhp tn mu cn o trong mc Run Samples\Sample Queue.
Sau khi nhp tn mu o, nhp vo biu tng th 2 trn View
Acquisition (biu tng ny c tn Fill out the sample queue table to enable this
juntion). Sau mn hnh hin ca s Run Sample Set. ca s ny, trong mc
Name of this sample set nhp tn (v d Nhom 1) sau chn Run. Lc biu
tng th 4 trn View Acquisition hin ch: Sample set _ Waiting for Injection.
III.2.4 Inject mu
Ln lt ra kim v injector bng dung mi acetonitrile v mu sp chy
(injector v tr LOAD)
Dng ng tim 250l rt khong 100l n 150l dung dch mu, lu
trnh bt kh ngay trn u kim. Inject mu vo valve cha mu ti v tr LOAD. Khi
mn hnh xut hin dng ch Sample set _ Waiting for Injection mi gt valve
vo v tr LOAD sang v tr INJECT, mu bt u c phn tch. Phi m bo
khng c bt kh no c inject vo h thng.
Sau khi bm khong 02 pht, chuyn valve sang v tr LOAD ri ra
valve bng acetonitrile.
Sau khi o xong (xut hin ch System Idle Sample Set Complete)
chy cn bng vi tc dng tng ng vi tc dng ca phng php chun
b o mu sau (chy khong 2 pht). Sau lm tng t nh trn o mu tip
theo.
- 52 -
III.2.5 X l kt qu
Chn Browse Project\Injecting\Update, mu va o xong s xut hin
trn mng hnh. Nhp i vo mu mun phn tch, sau nhp i ln mu mt
ln na. Hnh ph ca mu s xut hin trn mn hnh. Bng % ca peak mu
nm ngay bn di ph th hin tinh khit ca mu. C th thao tc thay v tr
v s lng peak mong mun, xa bt nhng peak nhiu. Sau lu kt qu. Chn
File\Save\Results lu. (C th xut kt qu thnh file excel hoc file .pdf)
III.2.6 In kt qu phn tch
Chn Browse Project\Results\Update cp nht kt qu. Mun in kt
qu ca mu no th chn ngay tn mu , sau chn Print Review pha trn,
gc tri mn hnh. Sau chn OK.
Trn mn hnh xut hin bng bo co phn tch ca mu . Nhn
Print, sau chn OK.
III.2.6 Tt my
Chy tc dng Flow A 0.0ml.min; Flow B 1.0ml/min ra ct. Chy
khong 10 pht. i p sut n nh th lm li thao tc ny nhng chn c hai dng
l 0.0ml/min. Sau khi p sut u ct cn 0.0psi th ln lt tc cng tc cc my
Waters 2996 Photo diodes Array Detector, Waters 1525 Binary HPLC Pump, Waters
In-line Degasser AF, my vi tnh. (ngc li khi m my).
Ch : Khi tt my cn shutdown my tnh trc khi tt cc cng tc trn
thn my chnh trnh vic ngt kt ni t ngt vi thit b, d b li cho ln m
my tip theo.
IV. Kt qu thc nghim
Mu 4-methoxyphenol (M0000115) chy phng php M0000115
10A:90B (A: nc, B: acetonitrile).
Kt qu:
- 53 -

Thi gian lu:1.152 pht
tinh khit mu: 94%
Filename: 4-methoxyphenol
Hnh 2.11 sc k ca 4-methoxyphenol

Mu TMI (2,3,3-trimethyl indolenine) chy phng php M0000215
Kt qu:
tinh khit mu: 87.25%
Filename: TMI (1)
- 54 -
Hnh 2.12 sc k ca TMI

Mu 3-hydroxy benzaldyhyde (M0000097) chy phng php M0000097
Kt qu:
Filename: 3-hydroxy benzaldyhyde
- 55 -
Hnh 2.13 Sc k ca 3-hydroxy benzaldyhyde

Mu 4-hydroxy benzaldyhyde (M0000097) chy phng php M0000097
Kt qu:
Thi gian lu: 1.143 pht
tinh khit mu:83.71%
Filename: 4-hydroxy benzaldyhyde
- 56 -
Hnh 2.14 Sc k ca 4-hydroxy benzaldyhyde

Mu TMBI (M0000214) chy phng php M0000214 40A:60B (A: nc,
B: acetonitrile).
Kt qu:
Filename: Nhm1_5_8_2009
- 57 -
Hnh 2.15 Sc k ca TMBI

Mu MPT(M0000122) chy phng php M0000122 50A:50B (A: nc, B:
acetonitrile).
Kt qu:
Filename: Nhm1_3_8_2009
- 58 -
Hnh 2.16 sc k ca MPT
V. Tho lun kt qu
kt qu o c tt, chnh xc, sc k p, peak xut hin nhn, u
th vic chn t l dng dung mi (pha ng) l rt quan trng. Thit b s dng h
dung mi l acetonitril v nc. C hai u l dung mi phn cc nhng nc phn
cc hn. Pha tnh l cht khng phn cc v vy nhng cht mu c phn cc
yu hn s b gi lu hn trong ct. i vi nhng cht c phn cc thp th
dung mi ra di phi phn cc mnh, nu khng mu s kh c ra ra khi ct,
dn n kt qu phn tch khng tt v quan trng hn l ct b d. Tuy nhin, nu
dng dung mi (pha ng) qu phn cc s lm h ct. V vy, vic chn t l dng
dung mi cho tng cht phn tch l rt quan trng.
Cc cht c phn tch trn ln lt c cng thc phn t l:
- 59 -
Bng 2.3 Cng thc cu to ca cht phn tch

Tn mu
Cng thc phn t

OH
4-methoxyphenol
OMe
TMI
N
CHO
3-hydroxybenzaldehyde
OH
CHO
4-hydroxybenzaldehyde
OH
TMBI
- 60 -

O
MPT
S
O
MPT l cht phn cc yu nht trong cc cht trn, v vy phng php

c s dng l 50A:50B (t l dng nc ln nht trong cc phng php dng
phn tch cc cht trn). Mc d dng l 50A:50B nhng thi gian lu cng kh ln
(2.691 pht) chng t n l cht phn cc kh yu.
Gia TMI v TMBI th TMBI l dn xut ca napthalene c phn cc yu
hn TMI l dn xut ca benzene. V vy pha ng cho TMBI cn phn cc hn
TMI. C th pha ng cho TMBI l 40A:60B v cho TMI l 25A:75B.
Gia 3-hydroxybezaldehyde v 4-hydroxybenzaldehyde dng cng t l pha
ng l 30A:70B v phn cc ca hai cht ny tng t nhau. Tuy nhin thi gian
lu ca 4-hydroxybenzaldehyde nh hn ca 3-hydroxybenzaldehyde mt cht
(1.074 so vi 1.143). V nhm ht in t CHO gn v tr para s nh hng ln
nhm OH nhiu hn v tr meta. M nhm ht in t s lm cho nhm OH phn
cc hn. V vy 4-hydroxybenzaldehyde phn cc hn 3-hydroxybenzaldehyde mt
cht dn n thi gian lu ca 4-hydroxybenzaldehyde nh hn ca 3hydroxybenzaldehyde mt cht.
4-methoxyphenol l cht phn cc nht trong cc cht trn nn dng
phng php vi t l dng 10A:90B. T l dng ca nc thp.
VI. Mt s vn trong qu trnh o mu
Khi chy cn bng thy p sut ct tng ln rt nhanh, thay i lin tc. Khi
thay i dng th p sut cng tng lin tc. Nguyn nhn c th l do ct b bn. Ta
c th ra ct bng nc hay l nhng dung mi phn cc. Sau khi ra ch p sut
n nh mi tin hnh o mu.
Trc khi o mu phi chy monitor khong 20 pht n nh p sut
trc khi o mu. Trc khi tt my ta cng chy monitor khong 30 pht ra
ct. trnh trng hp ct sc k b nhim bn.
- 61 -

Trong qu trnh o mu khng c thay i dng mt cch t ngt v nh
th s lm hng ct do p sut tng qu nhanh. trnh trng hp ny xy ra th
ta phi i dng t t trc khi o cc mu khc nhau.
Bnh dung mi phi c y kn trnh thot hi ra phng th nghim.
VII. Ti liu tham kho
[1] Chemical Analysis-Modern Instrumentation Methods and Techniques,
page 64 to 75, Francis Rouessac and Annick Rouessac, University of Le
Mans, France. Translated by Francis and Annick Rouessac and Steve
Brooks.
[2] L thuyt sc k, trang 11 n 43, Thc s Hunh Kim Lin, Khoa
Dc, i Hc Y Dc Cn Th.
[3] www.waters.com , truy cp cui cng ngy 10/8/2009.
[4] www.chemvn.net , truy cp cui cng ngy 12/8/2009.
- 62 -

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Hng dan: Ha Kim Thanh

hon thnh chng trnh thc tp mn Ha Phn tch II ny, ngoi s n

Bao cao thc tap phan tch Nhom I

Hng dan: Ha Kim Thanh

Bao cao thc tap phan tch Nhom I

Hng dan: Ha Kim Thanh

BI 1 KHO ST CU TRC PHN T CA HP CHT BNG FT- IR

Gii thiu chung .................................................................................................... 9

L thuyt v thit b............................................................................................. 10

Nguyn tc hot ng .................................................................................... 10

Thc nghim .................................................................................................... 11

Tt my,v sinh v ghi nht k thit b........................................................ 14

Kt qu thc nghim ...................................................................................... 14

Tho lun v kt qu thu c......................................................................... 25

Hng pht trin ............................................................................................. 26

Ti liu tham kho ........................................................................................... 26

Bao cao thc tap phan tch Nhom I

Hng dan: Ha Kim Thanh

Bi 2 - KIM TRA TINH KHIT CA NGUYN LIU BNG HPLC........................37

Bao cao thc tap phan tch Nhom I

Hng dan: Ha Kim Thanh

DANH SCH T VIT TT

FTIR : Fourier Transform Infrared Spectrometer

Bao cao thc tap phan tch Nhom I

Hng dan: Ha Kim Thanh

DANH SCH BNG BIU MINH HA

Bao cao thc tap phan tch Nhom I

Hng dan: Ha Kim Thanh

DANH SCH HNH MINH HA

Hng dan: Ha Kim Thanh

Bao cao thc tap phan tch Nhom I

Hng dan: Ha Kim Thanh

BI 1 KHO ST CU TRC PHN T CA HP CHT

Hnh 1.1 My ph FTIR 100 ca hng PerkinElmer

Gii thiu chung

vic xc nh chnh xc cu trc phn t ca mt hp cht hay cc nhm chc c

Hng dan: Ha Kim Thanh

Hnh 1.2 S cu to my ph FTIR

Bao cao thc tap phan tch Nhom I

Hng dan: Ha Kim Thanh

Hnh 1.3 S nguyn l hot ng my ph FTIR

Kim tra cht lm kh (decriccant): mu xanh v tr 15/20 l OK.

M cng tc sau my, mm hnh trn my hin th dng ch

PerkinElmer Spectrum 100 Series.

Hng dan: Ha Kim Thanh

Chn biu tngSpectrum trn desktop ri login.

Nhp user name v password.

T ca s Spectrum login, chn Spectrum 100, chn OK kt ni

chng trnh vi my.

2.1 i vi mu chy background

Ra sch v sy kh khun to mu.

Bao cao thc tap phan tch Nhom I

Hng dan: Ha Kim Thanh

C nh mt con c vo khun trc, sau cho khong 0.2g bt KBr

vo khun, vn con c cn li vo.

KBr nm khong gia khun. Vin KBr c to phi c b dy ng nht,

mu cn phn tch ln b mt vin KBr.

Mu rn: Ra sch v sy kh b to mu. Cho khong 0.2g bt KBr

v mt lng rt t mu cn phn tch vo ci, dng chy tn u hn hp ri cho

Hng dan: Ha Kim Thanh

Chn ph mun x l, click vo biu tng Label Peak hin th

tn s ti nh peak. Sau khi x l xong c th lu kt qu li bng cch vo

Mun in th ch cn copy ph paste vo mu report ri print.Cn

mun in thng tin ca mu nh km th vo Status.My s chuyn th v